**Part 2**

**Biology of** *Vibrio Cholera*

56 Cholera

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Sharma C, Thungapathra M, Ghosh A, Mukhopadhyay AK., Basu A, Mitra R, Basu I,

Toma C, Sisavath L, Higa N, and Iwanaga M. Characterization of *Vibrio cholerae* O1 isolated in Lao People's Democratic Republic. Jpn J. Trop Med. Hyg. 1997; 25: 85-87 Watanabe T, Miura T, Sasaki T, Nakamura S and Omura T. *Mekong ryu-iki ni okeru mizu-*

Yamai S, Okitsu T, Shimada T, and Katube Y. Distribution of serogroups of *Vibrio cholerae*

and addition of novel serogroups. *Kansenshogaku Zasshi* 1997; 10: 1037-1045 Yamamoto K, Ichinose Y, Nakasone N, Tanabe M, Nagahama M, Sakurai J, and Iwanaga M.

biotype El Tor. Infection and Immunity 1986; 51: 927-931

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Nakamura S, and Newton PN. Fatal bacteremia due to immotile *Vibrio cholerae* serogroup O21 in Vientiane, Laos – a case report. Annals of Clinical Microbiology

agents implicated in the etiology of diarrheal diseases among children in Lao People's Democratic Republic. Southeast Asian J. Trop. Med. Public Health vol. 30,

Epidemic cholera in West Africa: The role of food handling and high-risk foods.

assessment of food-borne disease relating with marine products consumption in Vientiane, Lao P.D.R. The Second Symposium on Southeast Asian Water

Bhattacharya SK, Shimada T, Ramamurthy T, Takeda T, Yamasaki S, Takeda Y, Nair GB. Molecular analysis of non-O1, non-O139 Vibrio cholerae associated with an unusual upsurge in the incidence of cholera-like disease in Calcutta, India. J Clin

*kyokyu sisutemu nichakumoku shita suikei-kansensho no risuku-hyoka*. [Risk evaluation for waterborne infectious diseases in the Mekong watershed considering water supply system] *Kankyo-Kogaku Kenkyu Ronbun-shu* [Environmental Engineering

non-O1 non-O139 with specific reference to their ability to produce cholera toxin

Identity of hemolysins produced by *Vibrio cholerae* Non-O1 and *V. cholerae* O1,

**4** 

 *USA* 

*Vibrio cholerae* **Flagellar** 

 **Synthesis and Virulence** 

Anastasia R. Rugel1,2 and Karl E. Klose1

*2Department of Microbiology and Immunology,* 

 *University of Texas at San Antonio, San Antonio, Texas* 

*1South Texas Center for Emerging Infectious Diseases, Department of Biology,* 

*University of Texas Health Science Center at San Antonio, San Antonio, Texas* 

*Vibrio cholerae* is a Gram-negative bacterium with a single sheathed polar flagellum (Fig. 1.). *V. cholerae* causes the severe diarrheal disease cholera in humans when it colonizes the small intestine and expresses various virulence factors, including cholera toxin (CT) and toxin coregulated pilus (TCP). *V. cholerae* is also a natural inhabitant of the marine environment, where it forms biofilms on chitinous surfaces. Motility contributes to both aspects of the *V. cholerae* lifecycle. The flagellum facilitates chemotactic-directed movement toward the preferred colonization site within the intestine (Camilli and Mekalanos 1995; Butler and Camilli 2004), and also contributes to biofilm formation within the environment (Watnick and Kolter 1999). *V. cholerae* strains defective for motility are less virulent than motile strains (Guentzel and Berry 1975; Freter and O'Brien 1981; Richardson 1991). As flagellar synthesis, motility, and chemotaxis have become better understood in *V. cholerae*, it has also become clear that motility is intimately integrated into all aspects of the lifestyle of this bacterium.

The flagellum is a motor-driven organelle present in many bacteria. Different flagellar placement and quantity are seen in different bacteria. Monotrichous bacteria have a single

**1. Introduction** 

Fig. 1. Vibrio cholerae

**2. Structure** 

## *Vibrio cholerae* **Flagellar Synthesis and Virulence**

Anastasia R. Rugel1,2 and Karl E. Klose1 *1South Texas Center for Emerging Infectious Diseases, Department of Biology, University of Texas at San Antonio, San Antonio, Texas 2Department of Microbiology and Immunology, University of Texas Health Science Center at San Antonio, San Antonio, Texas USA* 

## **1. Introduction**

*Vibrio cholerae* is a Gram-negative bacterium with a single sheathed polar flagellum (Fig. 1.). *V. cholerae* causes the severe diarrheal disease cholera in humans when it colonizes the small intestine and expresses various virulence factors, including cholera toxin (CT) and toxin coregulated pilus (TCP). *V. cholerae* is also a natural inhabitant of the marine environment, where it forms biofilms on chitinous surfaces. Motility contributes to both aspects of the *V. cholerae* lifecycle. The flagellum facilitates chemotactic-directed movement toward the preferred colonization site within the intestine (Camilli and Mekalanos 1995; Butler and Camilli 2004), and also contributes to biofilm formation within the environment (Watnick and Kolter 1999). *V. cholerae* strains defective for motility are less virulent than motile strains (Guentzel and Berry 1975; Freter and O'Brien 1981; Richardson 1991). As flagellar synthesis, motility, and chemotaxis have become better understood in *V. cholerae*, it has also become clear that motility is intimately integrated into all aspects of the lifestyle of this bacterium.

Fig. 1. Vibrio cholerae

#### **2. Structure**

The flagellum is a motor-driven organelle present in many bacteria. Different flagellar placement and quantity are seen in different bacteria. Monotrichous bacteria have a single

*Vibrio cholerae* Flagellar Synthesis and Virulence 61

*V. cholerae* flagellar synthesis rather than the filament protruding through the OM as in other

The bacterial flagellar filament is made up of thousands of flagellin subunits, with a cap protein (FliD) at the distal end (Ikeda, Asakura et al. 1985; Ikeda, Homma et al. 1987; Homma, DeRosier et al. 1990). The structure of the *Salmonella typhimurium* flagellin FliC has been solved by cryomicroscopy. FliC is composed of domains at its N- and C-termini that interact with each other: D0 (aa 1-45 and 456-495), D1 (aa 46-180 and 408-455), and D2 (aa 181-190 and 285-407). The D2 domains, along with the D3 domain (aa 191-284) form the antigenic variable region that are present on the filament surface, (Yonekura, Maki-Yonekura et al. 2003). Interaction of the D0 and D1 domains allows the flagellins to polymerize under the cap (FliD) protein into a hollow helical filament at the growing tip of

In contrast to most other bacteria which have filaments composed of a single flagellin subunit, *V. cholerae* has a filament composed of 5 different flagellins, FlaABCDE. These flagellins share a high degree of homology, yet only FlaA is essential for flagellar synthesis; the other four flagellins are not required for the synthesis of the filament (Klose and Mekalanos 1998). Alignment of FlaA with the other four *V. cholerae* flagellins, as well as with *S. typhimurium* FliC, reveals that the D0 and D1 domains are well-conserved, whereas the variable regions D2 and D3 are more divergent. Interestingly, the *V. cholerae* flagellins have a much shorter region corresponding to D2 and D3 (129 aa shorter) when compared to *S. typhimurium* FliC. Because this antigenic portion of the flagellins extends out from the hollow filament core, it may be that the presence of the flagellar sheath over the *V. cholerae*

The basal body contains the rod structure (FlgB, FlgC, FlgF and FlgG) with L (FlgH), P (FlgI), and MS rings (FliF) localized to the OM, periplasm (peptidoglycan), and cytoplasmic membranes, respectively. In *Vibrio* spp. an additional T ring is located immediately below the P ring, which is composed of the *Vibrio*-specific components MotX, MotY, and FlgT . The C-ring, which extends into the cytoplasm from the MS ring and is made up of FliG, FliM, and FliN, is difficult to preserve during microscopy and has not been visualized in its entirety in *Vibrio* spp.(Aizawa, Dean et al. 1985; Homma, Aizawa et al. 1987; Homma, Ohnishi et al. 1987; Homma, DeRosier et al. 1990; Homma, Kutsukake et al. 1990; Ueno, Oosawa et al. 1992; Francis, Sosinsky et al. 1994; Schoenhals and Macnab 1996; Terashima, Koike et al. 2010). The chemotaxis protein CheY relays information from the chemotaxis sensory system by binding to the C ring (FliM), causing the flagellum to switch rotation

In *S. typhimurium* and *E. coli* MotA and MotB are membrane proteins that compose the motor that utilizes H+ motive force to drive flagellar rotation (Lloyd, Tang et al. 1996; Zhou, Lloyd et al. 1998; Zhou, Sharp et al. 1998; Braun, Poulson et al. 1999; Blair 2003). *Vibrio spp.*  contain MotA and MotB homologues, alternately referred to as PomA and PomB (Dean, Macnab et al. 1984; Stader, Matsumura et al. 1986; Blair and Berg 1990; Stolz and Berg 1991; Asai, Kojima et al. 1997; Sato and Homma 2000; Sato and Homma 2000; Yorimitsu, Asai et al. 2000; Fukuoka, Yakushi et al. 2005), but they also contain *Vibrio-* specific motor proteins MotX and MotY, localized in the T ring (McCarter 1994; McCarter 1994; Okunishi, Kawagishi et al. 1996; Okabe, Yakushi et al. 2001; Okabe, Yakushi et al. 2002; Okabe,

filament restricts the size of the antigenic region protruding from the filament.

bacterial flagella is not understood.

the flagellum as they are being secreted.

from counterclockwise to clockwise.

polar flagellum (e.g. *V. cholerae*), lofotrichous bacteria have multiple flagella at a single pole (e.g. *Helicobacter pylori*), amphitrichous bacteria have flagella at two poles (e.g. *Campylobacter jejuni*), and peritrichous bacteria have multiple flagella emanating from the cell in all directions (e.g. *Escherichia coli)*.

The base of the bacterial flagellum is composed of a secretion system related to the Type III secretion system, which facilitates export of flagellar components from the cytoplasm to the periplasm and the exterior of the cell. The basic components of the flagellum are the basal body, which extends from the cytoplasmic membrane through the periplasm and into the outer membrane (OM), connected to the flexible hook (composed of FliE) found exterior to the cell, which in turn is connected to the flagellar filament (Kojima and Blair 2004; Terashima, Kojima et al. 2008). The motor components that drive flagellar rotation are found in the cytoplasmic membrane, and the switch components (FliG, FliM, FliN) that interact with the chemotaxis signaling system and the motor (Francis, Sosinsky et al. 1994) extend into the cytoplasm from the basal body (Fig. 2.).

Fig. 2. Flagellar Motor Complex

Unlike most other bacterial flagella, the *V. cholerae* flagellum has a sheath composed of OM that coats the entire filament (Allen and Baumann 1971; Sjoblad, Emala et al. 1983; Fuerst and Perry 1988). Sheathed flagella are found in *Vibrio spp.* and a few other Gram-negative bacteria (e.g. *H. pylori*). It is hypothesized that the sheath acts as a protective covering that shields the antigenic flagellins from recognition by the host's immune response (Yoon and Mekalanos 2008). The mechanism whereby the OM is extended to cover the filament during

polar flagellum (e.g. *V. cholerae*), lofotrichous bacteria have multiple flagella at a single pole (e.g. *Helicobacter pylori*), amphitrichous bacteria have flagella at two poles (e.g. *Campylobacter jejuni*), and peritrichous bacteria have multiple flagella emanating from the cell in all

The base of the bacterial flagellum is composed of a secretion system related to the Type III secretion system, which facilitates export of flagellar components from the cytoplasm to the periplasm and the exterior of the cell. The basic components of the flagellum are the basal body, which extends from the cytoplasmic membrane through the periplasm and into the outer membrane (OM), connected to the flexible hook (composed of FliE) found exterior to the cell, which in turn is connected to the flagellar filament (Kojima and Blair 2004; Terashima, Kojima et al. 2008). The motor components that drive flagellar rotation are found in the cytoplasmic membrane, and the switch components (FliG, FliM, FliN) that interact with the chemotaxis signaling system and the motor (Francis, Sosinsky et al. 1994) extend

Unlike most other bacterial flagella, the *V. cholerae* flagellum has a sheath composed of OM that coats the entire filament (Allen and Baumann 1971; Sjoblad, Emala et al. 1983; Fuerst and Perry 1988). Sheathed flagella are found in *Vibrio spp.* and a few other Gram-negative bacteria (e.g. *H. pylori*). It is hypothesized that the sheath acts as a protective covering that shields the antigenic flagellins from recognition by the host's immune response (Yoon and Mekalanos 2008). The mechanism whereby the OM is extended to cover the filament during

directions (e.g. *Escherichia coli)*.

Fig. 2. Flagellar Motor Complex

into the cytoplasm from the basal body (Fig. 2.).

*V. cholerae* flagellar synthesis rather than the filament protruding through the OM as in other bacterial flagella is not understood.

The bacterial flagellar filament is made up of thousands of flagellin subunits, with a cap protein (FliD) at the distal end (Ikeda, Asakura et al. 1985; Ikeda, Homma et al. 1987; Homma, DeRosier et al. 1990). The structure of the *Salmonella typhimurium* flagellin FliC has been solved by cryomicroscopy. FliC is composed of domains at its N- and C-termini that interact with each other: D0 (aa 1-45 and 456-495), D1 (aa 46-180 and 408-455), and D2 (aa 181-190 and 285-407). The D2 domains, along with the D3 domain (aa 191-284) form the antigenic variable region that are present on the filament surface, (Yonekura, Maki-Yonekura et al. 2003). Interaction of the D0 and D1 domains allows the flagellins to polymerize under the cap (FliD) protein into a hollow helical filament at the growing tip of the flagellum as they are being secreted.

In contrast to most other bacteria which have filaments composed of a single flagellin subunit, *V. cholerae* has a filament composed of 5 different flagellins, FlaABCDE. These flagellins share a high degree of homology, yet only FlaA is essential for flagellar synthesis; the other four flagellins are not required for the synthesis of the filament (Klose and Mekalanos 1998). Alignment of FlaA with the other four *V. cholerae* flagellins, as well as with *S. typhimurium* FliC, reveals that the D0 and D1 domains are well-conserved, whereas the variable regions D2 and D3 are more divergent. Interestingly, the *V. cholerae* flagellins have a much shorter region corresponding to D2 and D3 (129 aa shorter) when compared to *S. typhimurium* FliC. Because this antigenic portion of the flagellins extends out from the hollow filament core, it may be that the presence of the flagellar sheath over the *V. cholerae* filament restricts the size of the antigenic region protruding from the filament.

The basal body contains the rod structure (FlgB, FlgC, FlgF and FlgG) with L (FlgH), P (FlgI), and MS rings (FliF) localized to the OM, periplasm (peptidoglycan), and cytoplasmic membranes, respectively. In *Vibrio* spp. an additional T ring is located immediately below the P ring, which is composed of the *Vibrio*-specific components MotX, MotY, and FlgT . The C-ring, which extends into the cytoplasm from the MS ring and is made up of FliG, FliM, and FliN, is difficult to preserve during microscopy and has not been visualized in its entirety in *Vibrio* spp.(Aizawa, Dean et al. 1985; Homma, Aizawa et al. 1987; Homma, Ohnishi et al. 1987; Homma, DeRosier et al. 1990; Homma, Kutsukake et al. 1990; Ueno, Oosawa et al. 1992; Francis, Sosinsky et al. 1994; Schoenhals and Macnab 1996; Terashima, Koike et al. 2010). The chemotaxis protein CheY relays information from the chemotaxis sensory system by binding to the C ring (FliM), causing the flagellum to switch rotation from counterclockwise to clockwise.

In *S. typhimurium* and *E. coli* MotA and MotB are membrane proteins that compose the motor that utilizes H+ motive force to drive flagellar rotation (Lloyd, Tang et al. 1996; Zhou, Lloyd et al. 1998; Zhou, Sharp et al. 1998; Braun, Poulson et al. 1999; Blair 2003). *Vibrio spp.*  contain MotA and MotB homologues, alternately referred to as PomA and PomB (Dean, Macnab et al. 1984; Stader, Matsumura et al. 1986; Blair and Berg 1990; Stolz and Berg 1991; Asai, Kojima et al. 1997; Sato and Homma 2000; Sato and Homma 2000; Yorimitsu, Asai et al. 2000; Fukuoka, Yakushi et al. 2005), but they also contain *Vibrio-* specific motor proteins MotX and MotY, localized in the T ring (McCarter 1994; McCarter 1994; Okunishi, Kawagishi et al. 1996; Okabe, Yakushi et al. 2001; Okabe, Yakushi et al. 2002; Okabe,

*Vibrio cholerae* Flagellar Synthesis and Virulence 63

FleQ lacking the N-terminus, indicating it binds to the transcriptional activation/DNA binding domain, which shares high homology (63% identity) with *V. cholerae* FlrA. It is not yet known whether FlrA binds to and is modulated by cdGMP. *P. aeruginosa* FleQ also binds to FleN, the homologue of FlhG (Dasgupta and Ramphal 2001). FleN binding to FleQ does not inhibit DNA binding, but downregulates FleQ-dependent transcription, resulting in reduced (single) flagellar number. As mentioned above, FlhG has a negative effect on *flrA* transcription in *V. cholerae*, but it is not known whether it also binds to FlrA and negatively

FlrA positively regulates Class II flagellar genes. Both FlrA and σ54-containing RNA polymerase are required to activate transcription of the Class II flagellar genes (Klose and Mekalanos 1998; Klose, Novik et al. 1998; Prouty, Correa et al. 2001). The class II genes encode components of the MS ring-switch-export apparatus as well as chemotaxis and regulatory proteins. Two large flagellar operons (*fliEFGHIJ* and the *flhA* operon, which contains *flhFG*, mentioned above, as well as *fliA* (σ28) and a number of chemotaxis genes), and the regulatory genes *flrBC*, are activated by FlrA. The Class II flagellar genes are predicted to encode an export apparatus-basal body intermediate; it seems likely that this structure is required to be assembled prior to progression to Class III gene expression, as is the case in *Campylobacter jejuni* and *Helicobacter pylori*, which have similar classes of flagellar

The regulatory proteins FlrBC are a two-component system that controls Class III gene transcription (Prouty, Correa et al. 2001). FlrB undergoes autophosphorylation, and then activates FlrC activity by transferring a phosphate to the conserved aspartate-54 (D54) residue in the amino terminus of FlrC (FlrC-P) allowing it to activate the σ54-dependent transcription of Class III genes (Correa, Lauriano et al. 2000; Correa and Klose 2005). The class III genes encode the rest of the components of the hook-basal body, as well as the flagellin FlaA and the OM proteins FlgOP. FlrC binds to enhancer sites downstream of the σ54-dependent Class III promoters (Correa, Lauriano et al. 2000; Correa and Klose 2005).

affects its activity.

Fig. 3. Flagellar Transcription Regulatory Hierarchy

genes (Hendrixson and DiRita 2003; Niehus, Gressmann et al. 2004).

Yakushi et al. 2005; Koerdt, Paulick et al. 2009). The *Vibrio* MotA and MotB form a membrane complex that utilizes a Na+ gradient (instead of H+ gradient) to drive flagellar rotation. A Na+ gradient is required to allow MotA/MotB to associate with the flagellum (through MotX/MotY) and open the Na+ channel; flux of Na+ through the channel provides the torque to generate flagellar rotation (McCarter 1994; McCarter 1994; Yorimitsu, Kojima et al. 2004; Terashima, Fukuoka et al. 2006).

Two additional proteins control flagellar number and placement in *Vibrio* spp. FlhG contains an ATPase motif and controls flagellar number; *Vibrio* cells without *flhG* synthesize multiple polar flagella, instead of a single polar flagellum (Correa, Peng et al. 2005; Kusumoto, Kamisaka et al. 2006; Kusumoto, Shinohara et al. 2008). FlhF contains a GTP binding motif and localizes to the cell pole, thus dictating polar localization of the flagellum. *Vibrio* cells without *flhF* are largely non-flagellated; however a few cells will synthesize a flagellum at a site away from the pole (Carpenter, Hanlon et al. 1992; Zanen, Antelmann et al. 2004; Salvetti, Ghelardi et al. 2007; Green, Kahramanoglou et al. 2009; Kusumoto, Nishioka et al. 2009). FlhG interacts with FlhF, and a current model suggests that FlhG interacts with FlhF to prevent additional FlhF deposition at the pole (Kusumoto, Shinohara et al. 2008). A *V. alginolyticus* strain lacking both FlhF and FlhG is mostly lacking flagella (Kojima, Nishioka et al. 2011), but a few cells possess multiple peritrichous flagella (similar to *S. typhimurium*). An unidentified suppressor mutation can lead to virtually all *flhFG V. alginolyticus* cells possessing peritrichous flagella and being able to swim; the identification of this suppressor mutation should lead to greater insights into control of polar flagellar synthesis in *Vibrio* spp.

Two additional outer membrane proteins, FlgO and FlgP, contribute to flagellar stability. FlgP homologues are restricted to *Vibrio*, *Helicobacter*, and *Campylobacter* spp. *V. cholerae* FlgP is a lipoprotein that affects flagellar stability; *flgP* mutants synthesize fragile flagella and appear non-motile in motility agar, presumably due to breakage of flagella during swimming (Morris, Peng et al. 2008; Martinez, Dharmasena et al. 2009). FlgO homologues are only found in *Vibrio* spp. *V. cholerae* strains lacking *flgO* have a similar phenotype as *flgP* strains, namely they produce fragile flagella that break easily while swimming (Morris, Peng et al. 2008; Martinez, Dharmasena et al. 2009).

## **3. Regulation**

Transcription of the *V. cholerae* flagellar genes is controlled by a four-tiered transcription hierarchy (Fig. 3.) (Prouty, Correa et al. 2001). The *V. cholerae* flagellar transcription hierarchy is similar to that which controls flagellar transcription in *Pseudomonas aeruginosa*, another bacterium with a single polar flagellum (Dasgupta, Wolfgang et al. 2003). The master regulator, FlrA, is a σ54-dependent transcriptional activator. FlrA represents the sole Class I gene product, and it activates transcription of Class II flagellar genes (Klose and Mekalanos 1998). It is not clear whether environmental conditions regulate transcription of *flrA*, but *flhG* (which controls flagellar number) also negatively regulates *flrA* transcription (Correa, Peng et al. 2005).

The *P. aeruginosa* FlrA homologue, FleQ, has been shown to bind to cyclic-di-GMP (cdGMP)(Hickman and Harwood 2008). Binding of cdGMP to FleQ prevents DNA binding, resulting in the absence of flagellar synthesis and de-repression in *P. aeruginosa* of genes involved in biofilm formation normally repressed by FleQ. Interestingly, cdGMP binds to

Yakushi et al. 2005; Koerdt, Paulick et al. 2009). The *Vibrio* MotA and MotB form a membrane complex that utilizes a Na+ gradient (instead of H+ gradient) to drive flagellar rotation. A Na+ gradient is required to allow MotA/MotB to associate with the flagellum (through MotX/MotY) and open the Na+ channel; flux of Na+ through the channel provides the torque to generate flagellar rotation (McCarter 1994; McCarter 1994; Yorimitsu, Kojima

Two additional proteins control flagellar number and placement in *Vibrio* spp. FlhG contains an ATPase motif and controls flagellar number; *Vibrio* cells without *flhG* synthesize multiple polar flagella, instead of a single polar flagellum (Correa, Peng et al. 2005; Kusumoto, Kamisaka et al. 2006; Kusumoto, Shinohara et al. 2008). FlhF contains a GTP binding motif and localizes to the cell pole, thus dictating polar localization of the flagellum. *Vibrio* cells without *flhF* are largely non-flagellated; however a few cells will synthesize a flagellum at a site away from the pole (Carpenter, Hanlon et al. 1992; Zanen, Antelmann et al. 2004; Salvetti, Ghelardi et al. 2007; Green, Kahramanoglou et al. 2009; Kusumoto, Nishioka et al. 2009). FlhG interacts with FlhF, and a current model suggests that FlhG interacts with FlhF to prevent additional FlhF deposition at the pole (Kusumoto, Shinohara et al. 2008). A *V. alginolyticus* strain lacking both FlhF and FlhG is mostly lacking flagella (Kojima, Nishioka et al. 2011), but a few cells possess multiple peritrichous flagella (similar to *S. typhimurium*). An unidentified suppressor mutation can lead to virtually all *flhFG V. alginolyticus* cells possessing peritrichous flagella and being able to swim; the identification of this suppressor mutation should lead to greater

Two additional outer membrane proteins, FlgO and FlgP, contribute to flagellar stability. FlgP homologues are restricted to *Vibrio*, *Helicobacter*, and *Campylobacter* spp. *V. cholerae* FlgP is a lipoprotein that affects flagellar stability; *flgP* mutants synthesize fragile flagella and appear non-motile in motility agar, presumably due to breakage of flagella during swimming (Morris, Peng et al. 2008; Martinez, Dharmasena et al. 2009). FlgO homologues are only found in *Vibrio* spp. *V. cholerae* strains lacking *flgO* have a similar phenotype as *flgP* strains, namely they produce fragile flagella that break easily while swimming (Morris,

Transcription of the *V. cholerae* flagellar genes is controlled by a four-tiered transcription hierarchy (Fig. 3.) (Prouty, Correa et al. 2001). The *V. cholerae* flagellar transcription hierarchy is similar to that which controls flagellar transcription in *Pseudomonas aeruginosa*, another bacterium with a single polar flagellum (Dasgupta, Wolfgang et al. 2003). The master regulator, FlrA, is a σ54-dependent transcriptional activator. FlrA represents the sole Class I gene product, and it activates transcription of Class II flagellar genes (Klose and Mekalanos 1998). It is not clear whether environmental conditions regulate transcription of *flrA*, but *flhG* (which controls flagellar number) also negatively regulates *flrA* transcription

The *P. aeruginosa* FlrA homologue, FleQ, has been shown to bind to cyclic-di-GMP (cdGMP)(Hickman and Harwood 2008). Binding of cdGMP to FleQ prevents DNA binding, resulting in the absence of flagellar synthesis and de-repression in *P. aeruginosa* of genes involved in biofilm formation normally repressed by FleQ. Interestingly, cdGMP binds to

et al. 2004; Terashima, Fukuoka et al. 2006).

insights into control of polar flagellar synthesis in *Vibrio* spp.

Peng et al. 2008; Martinez, Dharmasena et al. 2009).

**3. Regulation** 

(Correa, Peng et al. 2005).

FleQ lacking the N-terminus, indicating it binds to the transcriptional activation/DNA binding domain, which shares high homology (63% identity) with *V. cholerae* FlrA. It is not yet known whether FlrA binds to and is modulated by cdGMP. *P. aeruginosa* FleQ also binds to FleN, the homologue of FlhG (Dasgupta and Ramphal 2001). FleN binding to FleQ does not inhibit DNA binding, but downregulates FleQ-dependent transcription, resulting in reduced (single) flagellar number. As mentioned above, FlhG has a negative effect on *flrA* transcription in *V. cholerae*, but it is not known whether it also binds to FlrA and negatively affects its activity.

Fig. 3. Flagellar Transcription Regulatory Hierarchy

FlrA positively regulates Class II flagellar genes. Both FlrA and σ54-containing RNA polymerase are required to activate transcription of the Class II flagellar genes (Klose and Mekalanos 1998; Klose, Novik et al. 1998; Prouty, Correa et al. 2001). The class II genes encode components of the MS ring-switch-export apparatus as well as chemotaxis and regulatory proteins. Two large flagellar operons (*fliEFGHIJ* and the *flhA* operon, which contains *flhFG*, mentioned above, as well as *fliA* (σ28) and a number of chemotaxis genes), and the regulatory genes *flrBC*, are activated by FlrA. The Class II flagellar genes are predicted to encode an export apparatus-basal body intermediate; it seems likely that this structure is required to be assembled prior to progression to Class III gene expression, as is the case in *Campylobacter jejuni* and *Helicobacter pylori*, which have similar classes of flagellar genes (Hendrixson and DiRita 2003; Niehus, Gressmann et al. 2004).

The regulatory proteins FlrBC are a two-component system that controls Class III gene transcription (Prouty, Correa et al. 2001). FlrB undergoes autophosphorylation, and then activates FlrC activity by transferring a phosphate to the conserved aspartate-54 (D54) residue in the amino terminus of FlrC (FlrC-P) allowing it to activate the σ54-dependent transcription of Class III genes (Correa, Lauriano et al. 2000; Correa and Klose 2005). The class III genes encode the rest of the components of the hook-basal body, as well as the flagellin FlaA and the OM proteins FlgOP. FlrC binds to enhancer sites downstream of the σ54-dependent Class III promoters (Correa, Lauriano et al. 2000; Correa and Klose 2005).

*Vibrio cholerae* Flagellar Synthesis and Virulence 65

Doyle-Huntzinger et al. 1982; Carsiotis, Weinstein et al. 1984; Weinstein, Carsiotis et al. 1984; Schmitt, Darnell et al. 1994; Kennedy, Rosey et al. 1997; Watnick, Lauriano et al. 2001; Syed, Beyhan et al. 2009). Non-motile live attenuated *V. cholerae* vaccine strains exhibit reduced reactogenicity (disease symptoms) in human volunteers, when compared to motile isogenic strains. (Coster, Killeen et al. 1995; Kenner, Coster et al. 1995). Using a newly-developed infant rabbit model of cholera, Rui *et al.* demonstrated that flagellin expression (whether in motile or non-motile vaccine strains) causes reactogenicity in rabbits by inducing

proinflammatory cytokines in the intestine (Rui, Ritchie et al. 2010).

Fig. 4. Proposed Model of Flagellar-dependent Virulence Modulation

An inverse relationship between motility and virulence had been suggested by the observation that spontaneous hypermotile mutants express almost no CT or TCP, while spontaneous non-motile mutants express increased levels of CT and TCP (Gardel and Mekalanos 1996). Utilizing whole genome transcription profiling of *V. cholerae* strains with mutations in the key flagellar regulatory genes (*rpoN, flrA, flrC,* and *fliA*)*,* it was observed that non-flagellated strains exhibit increased transcription of known (CT, TCP) and putative virulence factors (T6SS, hemolysins, etc)(Syed, Beyhan et al. 2009). The results suggest coordinate regulation by the flagellar regulatory hierarchy over a variety of virulence factors

It had been known that non-motile *V. cholerae* mutants exhibited enhanced hemagglutinating activity and decreased hemolytic activity, but the identity of the respective factors was unknown (Gardel and Mekalanos 1996). The transcriptional profiling of the flagellar regulatory mutants identified the flagellar-regulated hemolysin as TLH, which is encoded adjacent to HlyA, the "El Tor" hemolysin (Syed, Beyhan et al. 2009). Also identified was the flagellar-regulated hemagglutinin, FrhA, which is a large cadherin-

whose regulation was previously thought to be unlinked (Syed, Beyhan et al. 2009).

Most of the Class III gene products are only required in small amounts, but the FlaA flagellin is transcribed at very high levels. One mechanism for achieving these different levels of expression is the relative binding strength of the FlrC sites, which bind FlrC strongly at the *flaA* promoter, but only weakly at other Class III promoters, e.g. the *flgK* promoter (Correa and Klose 2005).

FlrC must be phosphorylated to activate σ54-dependent transcription, so presumably FlrB only phosphorylates FlrC upon assembly (not function) of the Class II export apparatusbasal body intermediate; a similar event controls expression of σ54-dependent Class III genes in *C. jejuni* (Joslin and Hendrixson 2009). Detection of an intermediate that is not secretion competent may explain why the genes encoding some of the components presumably required for secretion (e.g. *fliOPQ*) are Class III (i.e. activated by FlrC) rather than Class II genes. FlrB is a soluble protein and could thus directly interact with the apparatus intermediate in the cytoplasmic membrane and phosphorylate FlrC upon assembly. Deletion of *flhF* in *V. cholerae* specifically downregulates Class III gene expression (Correa, Peng et al. 2005), suggesting that FlhF regulates FlrC-dependent transcription in addition to regulating polar flagellar placement (as discussed above). An inner membrane protein, FlrD, is also a positive regulator of class III genes. Expression of FlrD is not regulated by the flagellar transcription hierarchy, but the protein possesses a HAMP domain, so it may interact with FlrB or FlrC to influence phosphorylation and Class III transcription (Moisi, Jenul et al. 2009)

The Class II gene *fliA* encodes σ28, which is required for transcription of Class IV flagellar genes (Klose and Mekalanos 1998). Similar to the checkpoint in *S. typhimurium* (Karlinsey, Tanaka et al. 2000; Chevance and Hughes 2008), the *V. cholerae* anti-sigma factor FlgM prevents σ28 transcriptional activity until it is secreted through a functional hook-basal body complex (Correa, Barker et al. 2004). The secretion of FlgM through the sheathed flagellum indicates that the sheath does not completely enclose the flagellum, at least at the tip. Secretion of FlgM frees σ28 to interact with RNA polymerase and activate Class IV flagellar genes, which encode the other four flagellins, FlaBCDE, as well as motor components (MotABX) and chemotaxis proteins (Klose and Mekalanos 1998). *V. cholerae* lacking *fliA* are non-motile and synthesize a truncated flagellum. The lack of expression of the four additional Class IV (σ28-dependent) flagellins (FlaBCDE) in the *fliA* strain is likely not the reason for the truncated flagellum and lack of motility, since strains lacking *flaBCDE* are still motile and synthesize a full length flagellum, whereas a strain lacking the Class III FlaA flagellin is non-motile and aflagellate (Klose and Mekalanos 1998). Rather, the lack of expression of other Class IV genes (e.g. motor genes) likely contributes to the *fliA* phenotype. The contribution of the four Class IV flagellins to flagellar synthesis and motility is mysterious, considering that only the Class III FlaA flagellin is essential for flagellar synthesis, but perhaps the other flagellins impart subtle differences to the flagellum and thus swimming behavior that are not obvious under laboratory growth conditions.

#### **4. Motility and virulence**

*V. cholerae* virulence has been linked to motility. Spontaneous non-motile *V. cholerae* strains were characterized as less virulent than motile strains in several *in vivo* and *in vitro* rabbit models of cholera. Mutations that adversely affect flagellar synthesis and motility generally lead to decreased intestinal colonization in infant mice (Guentzel and Berry 1975; Montie,

Most of the Class III gene products are only required in small amounts, but the FlaA flagellin is transcribed at very high levels. One mechanism for achieving these different levels of expression is the relative binding strength of the FlrC sites, which bind FlrC strongly at the *flaA* promoter, but only weakly at other Class III promoters, e.g. the *flgK*

FlrC must be phosphorylated to activate σ54-dependent transcription, so presumably FlrB only phosphorylates FlrC upon assembly (not function) of the Class II export apparatusbasal body intermediate; a similar event controls expression of σ54-dependent Class III genes in *C. jejuni* (Joslin and Hendrixson 2009). Detection of an intermediate that is not secretion competent may explain why the genes encoding some of the components presumably required for secretion (e.g. *fliOPQ*) are Class III (i.e. activated by FlrC) rather than Class II genes. FlrB is a soluble protein and could thus directly interact with the apparatus intermediate in the cytoplasmic membrane and phosphorylate FlrC upon assembly. Deletion of *flhF* in *V. cholerae* specifically downregulates Class III gene expression (Correa, Peng et al. 2005), suggesting that FlhF regulates FlrC-dependent transcription in addition to regulating polar flagellar placement (as discussed above). An inner membrane protein, FlrD, is also a positive regulator of class III genes. Expression of FlrD is not regulated by the flagellar transcription hierarchy, but the protein possesses a HAMP domain, so it may interact with FlrB or FlrC to influence phosphorylation and Class III transcription (Moisi,

The Class II gene *fliA* encodes σ28, which is required for transcription of Class IV flagellar genes (Klose and Mekalanos 1998). Similar to the checkpoint in *S. typhimurium* (Karlinsey, Tanaka et al. 2000; Chevance and Hughes 2008), the *V. cholerae* anti-sigma factor FlgM prevents σ28 transcriptional activity until it is secreted through a functional hook-basal body complex (Correa, Barker et al. 2004). The secretion of FlgM through the sheathed flagellum indicates that the sheath does not completely enclose the flagellum, at least at the tip. Secretion of FlgM frees σ28 to interact with RNA polymerase and activate Class IV flagellar genes, which encode the other four flagellins, FlaBCDE, as well as motor components (MotABX) and chemotaxis proteins (Klose and Mekalanos 1998). *V. cholerae* lacking *fliA* are non-motile and synthesize a truncated flagellum. The lack of expression of the four additional Class IV (σ28-dependent) flagellins (FlaBCDE) in the *fliA* strain is likely not the reason for the truncated flagellum and lack of motility, since strains lacking *flaBCDE* are still motile and synthesize a full length flagellum, whereas a strain lacking the Class III FlaA flagellin is non-motile and aflagellate (Klose and Mekalanos 1998). Rather, the lack of expression of other Class IV genes (e.g. motor genes) likely contributes to the *fliA* phenotype. The contribution of the four Class IV flagellins to flagellar synthesis and motility is mysterious, considering that only the Class III FlaA flagellin is essential for flagellar synthesis, but perhaps the other flagellins impart subtle differences to the flagellum and

thus swimming behavior that are not obvious under laboratory growth conditions.

*V. cholerae* virulence has been linked to motility. Spontaneous non-motile *V. cholerae* strains were characterized as less virulent than motile strains in several *in vivo* and *in vitro* rabbit models of cholera. Mutations that adversely affect flagellar synthesis and motility generally lead to decreased intestinal colonization in infant mice (Guentzel and Berry 1975; Montie,

promoter (Correa and Klose 2005).

Jenul et al. 2009)

**4. Motility and virulence** 

Doyle-Huntzinger et al. 1982; Carsiotis, Weinstein et al. 1984; Weinstein, Carsiotis et al. 1984; Schmitt, Darnell et al. 1994; Kennedy, Rosey et al. 1997; Watnick, Lauriano et al. 2001; Syed, Beyhan et al. 2009). Non-motile live attenuated *V. cholerae* vaccine strains exhibit reduced reactogenicity (disease symptoms) in human volunteers, when compared to motile isogenic strains. (Coster, Killeen et al. 1995; Kenner, Coster et al. 1995). Using a newly-developed infant rabbit model of cholera, Rui *et al.* demonstrated that flagellin expression (whether in motile or non-motile vaccine strains) causes reactogenicity in rabbits by inducing proinflammatory cytokines in the intestine (Rui, Ritchie et al. 2010).

Fig. 4. Proposed Model of Flagellar-dependent Virulence Modulation

An inverse relationship between motility and virulence had been suggested by the observation that spontaneous hypermotile mutants express almost no CT or TCP, while spontaneous non-motile mutants express increased levels of CT and TCP (Gardel and Mekalanos 1996). Utilizing whole genome transcription profiling of *V. cholerae* strains with mutations in the key flagellar regulatory genes (*rpoN, flrA, flrC,* and *fliA*)*,* it was observed that non-flagellated strains exhibit increased transcription of known (CT, TCP) and putative virulence factors (T6SS, hemolysins, etc)(Syed, Beyhan et al. 2009). The results suggest coordinate regulation by the flagellar regulatory hierarchy over a variety of virulence factors whose regulation was previously thought to be unlinked (Syed, Beyhan et al. 2009).

It had been known that non-motile *V. cholerae* mutants exhibited enhanced hemagglutinating activity and decreased hemolytic activity, but the identity of the respective factors was unknown (Gardel and Mekalanos 1996). The transcriptional profiling of the flagellar regulatory mutants identified the flagellar-regulated hemolysin as TLH, which is encoded adjacent to HlyA, the "El Tor" hemolysin (Syed, Beyhan et al. 2009). Also identified was the flagellar-regulated hemagglutinin, FrhA, which is a large cadherin-

*Vibrio cholerae* Flagellar Synthesis and Virulence 67

*V. cholerae* readily forms biofilms in the laboratory, and it is generally thought that *V. cholerae* predominantly exists as biofilms associated with various surfaces in the aquatic environment, including close associations with shellfish and zooplankton (Costerton, Lewandowski et al. 1995; Watnick and Kolter 1999; Faruque, Biswas et al. 2006; Yildiz and Visick 2009). Biofilm growth on chitinous surfaces induces competence in *V. cholerae,*  facilitating horizontal gene transfer and rapid evolution in the marine environment (Blokesch and Schoolnik 2007). *V. cholerae* biofilms are more resistant to environmental stresses such as antibiotics, chlorine, protozoan grazing, and bacteriophage infection (Vess, Anderson et al. 1993; Faruque, Albert et al. 1998; Watnick and Kolter 1999; Matz, McDougald et al. 2005). A significant amount of study has gone into understanding *V.* 

Biofilm formation requires an initial phase where the bacterium associates with a solid surface, followed by attachment, formation of microcolonies, and finally the formation of the mature three-dimensional biofilm structure with characteristic pillars and water channels (Costerton, Lewandowski et al. 1995; Watnick and Kolter 1999). Formation of the mature biofilm requires the expression of the *Vibrio* exopolysaccharide (VPS), which is the polysaccharide matrix that holds the structure together (Yildiz and Schoolnik 1999; Watnick, Lauriano et al. 2001; Lauriano, Ghosh et al. 2004). *V. cholerae* expressing the VPS results in obviously wrinkled ("rugose") colony morphology, and *V. cholerae* undergoes phase variation that leads to the rugose colony phenotype and enhanced biofilm formation ( Yildiz and Schoolnik 1999; Watnick, Lauriano et al. 2001; Lim, Beyhan et al. 2007). A number of regulatory factors are involved in VPS expression and biofilm formation, and one of the driving signals behind biofilm formation is increased expression of the signaling molecule cdi-GMP (Tischler and Camilli 2004; Beyhan, Tischler et al. 2006; Beyhan, Bilecen et al. 2007; Lim, Beyhan et al. 2007; Beyhan, Odell et al. 2008; Hickman and Harwood 2008; Syed,

In an initial screen for *V. cholerae* mutants unable to form biofilms, Watnick and Kolter identified motility as a major contributor to biofilm formation (Watnick and Kolter 1999). These results suggested that flagellar-mediated motility was important to approach and colonize a surface, and also to facilitate microcolony formation. Subsequently, it was determined that the flagellar motor itself controls VPS expression, at least in some *V. cholerae* strains, because non-flagellated mutants switch to the rugose phenotype, and this is dependent on a functional motor, suggesting that the motor acts as a sensor to induce mature biofilm formation (Lauriano, Ghosh et al. 2004). The *Vibrio* Na+-driven motor functioning to sense environmental conditions and drive altered gene expression is not unprecedented; the *V. parahaemolyticus* Na+-driven polar flagellar motor functions as a sensor to drive lateral flagellar

In general, elevated levels of cdGMP drive *V. cholerae* toward enhanced VPS expression and down-regulate motility and virulence gene expression (Tischler and Camilli 2004; Yildiz and Visick 2009). Elevated cdGMP levels cause a decrease in Class III and IV flagellar transcription, and noticeable decreases in motility in soft agar assays(Beyhan, Tischler et al. 2006). These results suggest that activity of the Class III regulator FlrC may be responsive to elevated cdGMP levels. The effect of specific cdGMP synthases/phosphodiesterases on motility is

synthesis (McCarter, Hilmen et al. 1988; Kawagishi, Imagawa et al. 1996).

**6. Biofilm formation** 

*cholerae* biofilm formation.

Beyhan et al. 2009; Yildiz and Visick 2009).

containing protein that enhances binding to epithelial cells *in vitro* and intestinal colonization in both infant and adult mice. The flagellar regulatory hierarchy positively regulates *frhA* transcription and negatively regulates *tlh* transcription. Regulation of *frhA* transcription by the flagellar hierarchy is mediated through an intermediate, CdgD, a cdGMP synthase. cdGMP is an important signaling molecule that modulates complex behaviors in bacteria, most notably biofilm formation (discussed below). The results demonstrate that the flagellar hierarchy controls the transcription of non-flagellar genes that contribute to other aspects of the *V. cholerae* lifecycle besides motility (Syed, Beyhan et al. 2009).

## **5. Chemotaxis and virulence**

Chemotaxis controls flagellar rotation in response to environmental factors, and thus is intimately tied to motility. Chemoattractants stimulate the chemotaxis machinery to cause increased clockwise (CW) rotation of the flagellum, while chemorepellants enable increased counter-clockwise (CCW) rotation (Armitage 1999; Butler and Camilli 2005). The net result of these effects on flagellar rotation is net swimming towards chemoattractants and away from chemorepellants (Falke, Bass et al. 1997; Armitage 1999). *V. cholerae* encodes three clusters of chemotaxis proteins (Heidelberg, Eisen et al. 2000), but the cluster that is embedded within the flagellar gene cluster (within the Class II *flhA* operon: *cheY3, cheZ, cheA2, cheB2*, and *cheW1*) appears to be the major chemotaxis machinery that controls flagellar rotation under most conditions (Camilli and Mekalanos 1995; Hyakutake, Homma et al. 2005). Methyl-accepting chemotaxis proteins (MCPs) in the cytoplasmic membrane interact with chemoattractant/repellants and the signal is transmitted through CheA to CheY via phosphorylation. Phospho-CheY then interacts with the C-ring of the flagellum, which causes a reversion from CCW to CW rotation, resulting in a change of swimming direction. CheB and CheW are involved in modulating the signal transduction pathway (Freter and O'Brien 1981; Alm and Manning 1990; Everiss, Hughes et al. 1994; Harkey, Everiss et al. 1994; Lee, Butler et al. 2001; Banerjee, Das et al. 2002; Hyakutake, Homma et al. 2005).

Interestingly, *V. cholerae* in stool exhibit a transient hyper-infectious phenotype predicted to facilitate epidemic spread of cholera, and transcription profiling revealed a transient repression of chemotaxis genes (specifically *cheW*) in these bacteria (Merrell, Butler et al. 2002). In the infant mouse model, non-chemotactic *V. cholerae* are able to outcompete chemotactic *V. cholerae* for intestinal colonization, indicating that the repression of chemotaxis in stool bacteria enhances epidemic spread (Butler and Camilli 2004; Butler, Nelson et al. 2006). Preventing phosphorylation of CheY prevents chemotactic signal transduction to the flagellum and biases it toward CCW flagellar rotation (and hence longer periods of swimming in a straight direction). The flagellum can also be biased toward CW flagellar rotation (and shorter periods of swimming in a straight direction) by the introduction of mutations into CheY that inhibit its dephosphorylation. Within the intestine, only the CCW-biased *V. cholerae* dramatically outcompete chemotactic *V. cholerae*, whereas the CW-biased bacteria are defective for intestinal colonization (Butler and Camilli 2004). Chemotactic *V. cholerae* colonize the distal end of the small intestine, whereas the CCWbiased non-chemotactic *V. cholerae* colonize the entire length of the small intestine. These results suggest that chemotaxis normally facilitates the recognition of chemoattractants within the distal small intestine or, alternatively, the recognition of chemorepellants within the proximal small intestine.

### **6. Biofilm formation**

66 Cholera

containing protein that enhances binding to epithelial cells *in vitro* and intestinal colonization in both infant and adult mice. The flagellar regulatory hierarchy positively regulates *frhA* transcription and negatively regulates *tlh* transcription. Regulation of *frhA* transcription by the flagellar hierarchy is mediated through an intermediate, CdgD, a cdGMP synthase. cdGMP is an important signaling molecule that modulates complex behaviors in bacteria, most notably biofilm formation (discussed below). The results demonstrate that the flagellar hierarchy controls the transcription of non-flagellar genes that contribute to other aspects of the *V.* 

Chemotaxis controls flagellar rotation in response to environmental factors, and thus is intimately tied to motility. Chemoattractants stimulate the chemotaxis machinery to cause increased clockwise (CW) rotation of the flagellum, while chemorepellants enable increased counter-clockwise (CCW) rotation (Armitage 1999; Butler and Camilli 2005). The net result of these effects on flagellar rotation is net swimming towards chemoattractants and away from chemorepellants (Falke, Bass et al. 1997; Armitage 1999). *V. cholerae* encodes three clusters of chemotaxis proteins (Heidelberg, Eisen et al. 2000), but the cluster that is embedded within the flagellar gene cluster (within the Class II *flhA* operon: *cheY3, cheZ, cheA2, cheB2*, and *cheW1*) appears to be the major chemotaxis machinery that controls flagellar rotation under most conditions (Camilli and Mekalanos 1995; Hyakutake, Homma et al. 2005). Methyl-accepting chemotaxis proteins (MCPs) in the cytoplasmic membrane interact with chemoattractant/repellants and the signal is transmitted through CheA to CheY via phosphorylation. Phospho-CheY then interacts with the C-ring of the flagellum, which causes a reversion from CCW to CW rotation, resulting in a change of swimming direction. CheB and CheW are involved in modulating the signal transduction pathway (Freter and O'Brien 1981; Alm and Manning 1990; Everiss, Hughes et al. 1994; Harkey, Everiss et al. 1994; Lee, Butler et

Interestingly, *V. cholerae* in stool exhibit a transient hyper-infectious phenotype predicted to facilitate epidemic spread of cholera, and transcription profiling revealed a transient repression of chemotaxis genes (specifically *cheW*) in these bacteria (Merrell, Butler et al. 2002). In the infant mouse model, non-chemotactic *V. cholerae* are able to outcompete chemotactic *V. cholerae* for intestinal colonization, indicating that the repression of chemotaxis in stool bacteria enhances epidemic spread (Butler and Camilli 2004; Butler, Nelson et al. 2006). Preventing phosphorylation of CheY prevents chemotactic signal transduction to the flagellum and biases it toward CCW flagellar rotation (and hence longer periods of swimming in a straight direction). The flagellum can also be biased toward CW flagellar rotation (and shorter periods of swimming in a straight direction) by the introduction of mutations into CheY that inhibit its dephosphorylation. Within the intestine, only the CCW-biased *V. cholerae* dramatically outcompete chemotactic *V. cholerae*, whereas the CW-biased bacteria are defective for intestinal colonization (Butler and Camilli 2004). Chemotactic *V. cholerae* colonize the distal end of the small intestine, whereas the CCWbiased non-chemotactic *V. cholerae* colonize the entire length of the small intestine. These results suggest that chemotaxis normally facilitates the recognition of chemoattractants within the distal small intestine or, alternatively, the recognition of chemorepellants within

*cholerae* lifecycle besides motility (Syed, Beyhan et al. 2009).

al. 2001; Banerjee, Das et al. 2002; Hyakutake, Homma et al. 2005).

**5. Chemotaxis and virulence** 

the proximal small intestine.

*V. cholerae* readily forms biofilms in the laboratory, and it is generally thought that *V. cholerae* predominantly exists as biofilms associated with various surfaces in the aquatic environment, including close associations with shellfish and zooplankton (Costerton, Lewandowski et al. 1995; Watnick and Kolter 1999; Faruque, Biswas et al. 2006; Yildiz and Visick 2009). Biofilm growth on chitinous surfaces induces competence in *V. cholerae,*  facilitating horizontal gene transfer and rapid evolution in the marine environment (Blokesch and Schoolnik 2007). *V. cholerae* biofilms are more resistant to environmental stresses such as antibiotics, chlorine, protozoan grazing, and bacteriophage infection (Vess, Anderson et al. 1993; Faruque, Albert et al. 1998; Watnick and Kolter 1999; Matz, McDougald et al. 2005). A significant amount of study has gone into understanding *V. cholerae* biofilm formation.

Biofilm formation requires an initial phase where the bacterium associates with a solid surface, followed by attachment, formation of microcolonies, and finally the formation of the mature three-dimensional biofilm structure with characteristic pillars and water channels (Costerton, Lewandowski et al. 1995; Watnick and Kolter 1999). Formation of the mature biofilm requires the expression of the *Vibrio* exopolysaccharide (VPS), which is the polysaccharide matrix that holds the structure together (Yildiz and Schoolnik 1999; Watnick, Lauriano et al. 2001; Lauriano, Ghosh et al. 2004). *V. cholerae* expressing the VPS results in obviously wrinkled ("rugose") colony morphology, and *V. cholerae* undergoes phase variation that leads to the rugose colony phenotype and enhanced biofilm formation ( Yildiz and Schoolnik 1999; Watnick, Lauriano et al. 2001; Lim, Beyhan et al. 2007). A number of regulatory factors are involved in VPS expression and biofilm formation, and one of the driving signals behind biofilm formation is increased expression of the signaling molecule cdi-GMP (Tischler and Camilli 2004; Beyhan, Tischler et al. 2006; Beyhan, Bilecen et al. 2007; Lim, Beyhan et al. 2007; Beyhan, Odell et al. 2008; Hickman and Harwood 2008; Syed, Beyhan et al. 2009; Yildiz and Visick 2009).

In an initial screen for *V. cholerae* mutants unable to form biofilms, Watnick and Kolter identified motility as a major contributor to biofilm formation (Watnick and Kolter 1999). These results suggested that flagellar-mediated motility was important to approach and colonize a surface, and also to facilitate microcolony formation. Subsequently, it was determined that the flagellar motor itself controls VPS expression, at least in some *V. cholerae* strains, because non-flagellated mutants switch to the rugose phenotype, and this is dependent on a functional motor, suggesting that the motor acts as a sensor to induce mature biofilm formation (Lauriano, Ghosh et al. 2004). The *Vibrio* Na+-driven motor functioning to sense environmental conditions and drive altered gene expression is not unprecedented; the *V. parahaemolyticus* Na+-driven polar flagellar motor functions as a sensor to drive lateral flagellar synthesis (McCarter, Hilmen et al. 1988; Kawagishi, Imagawa et al. 1996).

In general, elevated levels of cdGMP drive *V. cholerae* toward enhanced VPS expression and down-regulate motility and virulence gene expression (Tischler and Camilli 2004; Yildiz and Visick 2009). Elevated cdGMP levels cause a decrease in Class III and IV flagellar transcription, and noticeable decreases in motility in soft agar assays(Beyhan, Tischler et al. 2006). These results suggest that activity of the Class III regulator FlrC may be responsive to elevated cdGMP levels. The effect of specific cdGMP synthases/phosphodiesterases on motility is

*Vibrio cholerae* Flagellar Synthesis and Virulence 69

Blair, D. F. (2003). "Flagellar movement driven by proton translocation." FEBS Lett 545(1):

Blair, D. F. and H. C. Berg (1990). "The MotA protein of E. coli is a proton-conducting

Blokesch, M. and G. K. Schoolnik (2007). "Serogroup conversion of Vibrio cholerae in

Braun, T. F., S. Poulson, et al. (1999). "Function of proline residues of MotA in torque

Butler, S. M. and A. Camilli (2004). "Both chemotaxis and net motility greatly influence the infectivity of Vibrio cholerae." Proc Natl Acad Sci U S A 101(14): 5018-5023. Butler, S. M. and A. Camilli (2005). "Going against the grain: chemotaxis and infection in

Butler, S. M., E. J. Nelson, et al. (2006). "Cholera stool bacteria repress chemotaxis to increase

Camilli, A. and J. J. Mekalanos (1995). "Use of recombinase gene fusions to identify Vibrio cholerae genes induced during infection." Mol Microbiol 18(4): 671-683. Carpenter, P. B., D. W. Hanlon, et al. (1992). "flhF, a Bacillus subtilis flagellar gene that encodes a putative GTP-binding protein." Mol Microbiol 6(18): 2705-2713. Carsiotis, M., D. L. Weinstein, et al. (1984). "Flagella of Salmonella typhimurium are a virulence factor in infected C57BL/6J mice." Infect Immun 46(3): 814-818. Chevance, F. F. and K. T. Hughes (2008). "Coordinating assembly of a bacterial

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Dasgupta, N. and R. Ramphal (2001). "Interaction of the antiactivator FleN with the

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transcriptional activator FleQ regulates flagellar number in Pseudomonas

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sigma28 factor that is secreted through the sheathed polar flagellum." J Bacteriol

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186(14): 4613-4619.

711-745.

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aeruginosa." J Bacteriol 183(22): 6636-6644.

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86-95.

complicated by the presence of multiple paralogs of both types of enzymes in *V. cholerae* (Lim, Beyhan et al. 2006; Beyhan, Odell et al. 2008). Moreover, the flagellar hierarchy also regulates the expression of cdGMP modulating enzymes (mentioned above), so the effect of cdGMP on flagellar synthesis and motility is likely extremely complex, involving a large number of counteracting enzymes that regulate and are regulated by the flagellar hierarchy.

## **7. Conclusion**

The single polar flagellum of *V. cholerae* is assembled in a stepwise fashion of components that are tightly regulated by a flagellar transcriptional hierarchy. The study of some of the unique aspects of this flagellum are likely to yield further insight into the role of flagellar synthesis, motility, and chemotaxis on the virulence and environmental persistence of this important human pathogen. One of the most unique aspects is the sheath surrounding the flagellum, which is still mysterious. The presence and function of the multiple flagellins still needs to be elucidated. Regulation of the flagellar transcriptional hierarchy is still not understood, nor how this hierarchy regulates non-flagellar genes that influence virulence and biofilm formation. Clearly much remains to be illuminated in the study of the contribution of flagellar synthesis and motility to the lifecycle of *V. cholerae*.

## **8. Acknowledgement**

Funded by NIH AI43486

## **9. References**


complicated by the presence of multiple paralogs of both types of enzymes in *V. cholerae* (Lim, Beyhan et al. 2006; Beyhan, Odell et al. 2008). Moreover, the flagellar hierarchy also regulates the expression of cdGMP modulating enzymes (mentioned above), so the effect of cdGMP on flagellar synthesis and motility is likely extremely complex, involving a large number of

The single polar flagellum of *V. cholerae* is assembled in a stepwise fashion of components that are tightly regulated by a flagellar transcriptional hierarchy. The study of some of the unique aspects of this flagellum are likely to yield further insight into the role of flagellar synthesis, motility, and chemotaxis on the virulence and environmental persistence of this important human pathogen. One of the most unique aspects is the sheath surrounding the flagellum, which is still mysterious. The presence and function of the multiple flagellins still needs to be elucidated. Regulation of the flagellar transcriptional hierarchy is still not understood, nor how this hierarchy regulates non-flagellar genes that influence virulence and biofilm formation. Clearly much remains to be illuminated in the study of the

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**7. Conclusion** 

**8. Acknowledgement**  Funded by NIH AI43486

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**5** 

*Malaysia* 

**Genetic Analysis of CTX Prophage and** 

 **Atypical El Tor Biotype from Kelantan, Malaysia** 

Epidemic and global pandemic cholera have claimed millions of lives since the first pandemic in 1817 and it continues to exact a huge annual toll, with endemics established in approximately 50 countries worldwide (Ryan, 2011). More than 200 serogroups have been reported to date (Safa et al., 2009), but only two serogroups of *Vibrio cholerae*, the O1 and O139 serogroups, are known to have the potential for unleashing epidemic and pandemic cholera. The O1 serogroup of *V. cholerae* can be further divided into the classical or El Tor biotypes based on a number of phenotypic and genotypic characteristics (Sack et al., 2004). Seven pandemics of cholera have been recorded to date, where the first six pandemics were associated with *V. cholerae* serogroup O1 of the classical biotype, whereas the current ongoing seventh pandemic (1961 until present day) is caused by *V. cholerae* serogroup O1 of the El Tor biotype (Faruque et al., 1998). In 1992, the emergence and rapid spread of *V. cholerae* serogroup O139 from the Indian subcontinent to neighbouring countries was viewed as a possible threat that might initiate an eighth cholera pandemic. At the height of the O139 outbreak, the Indian subcontinent saw a dramatic displacement of the *V. cholerae* O1 El Tor as the dominant strain. However, rather than being driven to gradual extinction like the classical biotype, an unprecedented turn of events in 1994 saw *V. cholerae* O1 El Tor regain its predominance over the O139 serogroup and both serogroups continue to cause

The current seventh pandemic rein has reached its half a century mark, but several variants of the *V. cholerae* O1 El Tor biotype emerged cryptically during the late 1990s. These variants were untypable according to the conventional biotyping classification, because they possessed traits of both classical and El Tor biotypes (Nair et al., 2002; Ansaruzzaman et al., 2004). Given this dilemma, Safa et al. (2009) proposed the designation 'atypical El Tor' as an umbrella term to encompass all variants of the El Tor biotype. One such atypical El Tor was collectively known as the Matlab variants isolated from hospitalized patients in Matlab, Bangladesh between 1991 and 1994. Chronologically, the Matlab variants were the first to be characterized as having attributes of both the classical and El Tor biotypes, which meant

disease on the Indian subcontinent (Faruque et al., 2003).

**1. Introduction** 

*School of Medical Sciences, Health Campus, Universiti Sains Malaysia, Kelantan,* 

**Antibiotic Resistance Determinants in** 

*Vibrio cholerae* **O1 Belonging to the** 

Choo Yee Yu, Geik Yong Ang and Chan Yean Yean *Department of Medical Microbiology and Parasitology,* 


## **Genetic Analysis of CTX Prophage and Antibiotic Resistance Determinants in**  *Vibrio cholerae* **O1 Belonging to the Atypical El Tor Biotype from Kelantan, Malaysia**

Choo Yee Yu, Geik Yong Ang and Chan Yean Yean *Department of Medical Microbiology and Parasitology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, Kelantan,* 

*Malaysia* 

#### **1. Introduction**

74 Cholera

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cholerae biofilm formation." Mol Microbiol 53(3): 857-869.

within murine macrophages." Infect Immun 46(3): 819-825.

Epidemic and global pandemic cholera have claimed millions of lives since the first pandemic in 1817 and it continues to exact a huge annual toll, with endemics established in approximately 50 countries worldwide (Ryan, 2011). More than 200 serogroups have been reported to date (Safa et al., 2009), but only two serogroups of *Vibrio cholerae*, the O1 and O139 serogroups, are known to have the potential for unleashing epidemic and pandemic cholera. The O1 serogroup of *V. cholerae* can be further divided into the classical or El Tor biotypes based on a number of phenotypic and genotypic characteristics (Sack et al., 2004). Seven pandemics of cholera have been recorded to date, where the first six pandemics were associated with *V. cholerae* serogroup O1 of the classical biotype, whereas the current ongoing seventh pandemic (1961 until present day) is caused by *V. cholerae* serogroup O1 of the El Tor biotype (Faruque et al., 1998). In 1992, the emergence and rapid spread of *V. cholerae* serogroup O139 from the Indian subcontinent to neighbouring countries was viewed as a possible threat that might initiate an eighth cholera pandemic. At the height of the O139 outbreak, the Indian subcontinent saw a dramatic displacement of the *V. cholerae* O1 El Tor as the dominant strain. However, rather than being driven to gradual extinction like the classical biotype, an unprecedented turn of events in 1994 saw *V. cholerae* O1 El Tor regain its predominance over the O139 serogroup and both serogroups continue to cause disease on the Indian subcontinent (Faruque et al., 2003).

The current seventh pandemic rein has reached its half a century mark, but several variants of the *V. cholerae* O1 El Tor biotype emerged cryptically during the late 1990s. These variants were untypable according to the conventional biotyping classification, because they possessed traits of both classical and El Tor biotypes (Nair et al., 2002; Ansaruzzaman et al., 2004). Given this dilemma, Safa et al. (2009) proposed the designation 'atypical El Tor' as an umbrella term to encompass all variants of the El Tor biotype. One such atypical El Tor was collectively known as the Matlab variants isolated from hospitalized patients in Matlab, Bangladesh between 1991 and 1994. Chronologically, the Matlab variants were the first to be characterized as having attributes of both the classical and El Tor biotypes, which meant

Genetic Analysis of CTX Prophage and Antibiotic Resistance Determinants

**Primer Nucleotide sequence (5 to 3) References** 

**2.3** *rstR* **typing** 

presented in Table 1.

Table 1. Primers used in this study

in *Vibrio cholerae* O1 Belonging to the Atypical El Tor Biotype from Kelantan, Malaysia 77

The type of *rstR* gene in each isolate was determined using a set of allele-specific forward primers (*rstREl Tor*, *rstRClassical*, *rstRCalcutta* and *rstREnvironment*) and a common reverse primer, *rstRR* (Nusrin et al., 2004; Bhattacharya et al., 2006). A list of all primers used in this study is

*rstRClassical* CTTCTCATCAGCAAAGCCTCCATC Bhattacharya et al., 2006 *rstREl Tor* GCACCATGATTTAAGATGCTC Bhattacharya et al., 2006 *rstRCalcutta* CTGTAAATCTCTTCAATCCTAGG Bhattacharya et al., 2006 *rstREnvironment* GTTAACGCTTCAAGCCTG Nusrin et al., 2004 *rstRR* TCGAGTTGTAATTCATCAAGAGTG Bhattacharya et al., 2006 Ch1F GACCACTCAGGCCGCTGAAAT Nguyen et al., 2009 Ch1R CCGCGCTCAAGTGGTTATCGG Nguyen et al., 2009 Ch2F AACAACAGGTTGCAAGAGAGCATT Nguyen et al., 2009 Ch2R TATTGCTTTTTTAATGGCCGTT Nguyen et al., 2009 rstAR CCGTGAAAGTCATCAACG Nguyen et al., 2009 rstCF GATGTTTACGATAGCCTAGAAGACTT Nguyen et al., 2009 rstCR TACAGTGATGGCTCAGTCAATGC Nguyen et al., 2009 ctxBF AGATATTTTCGTATACAGAATCTCTAG Nguyen et al., 2009 cepR AAACAGCAAGAAAACCCCGAGT Nguyen et al., 2009 rstCF4 AAATCCGCAACTCAAGGCATTGA Nguyen et al., 2009 rstCR4 TAAGCGCCTGAACGCAGATATAAAG Nguyen et al., 2009 *rtxC-F* CGACGAAGATCATTGACGAC Chow et al., 2001 *rtxC-R* CATCGTCGTTATGTGGTTGC Chow et al., 2001 inDS-F CGGAATGGCCGAGCAGAT C Dalsgaard et al., 2001 inDS-B CAAGGTTCTGGACCAGTTGCG Dalsgaard et al., 2001 qacE∆1-F ATCGCAATAGTTGGCGAAGT Dalsgaard et al., 2001 su1-B GCAAGGCGGAAACCCGCGCGG Dalsgaard et al., 2001 in-F GGCATCCAAGCAGCAAGC Dalsgaard et al., 2001 in-B AAGCAGACT TGACCTGAT Dalsgaard et al., 2001 aadA-B ATTGCCCAGTCGGCAGCG Dalsgaard et al., 2001 INT1 GCTGGATAGGTTAAGGGCAG Hochhnut et al., 2001 INT2 CTCTATGGGCACTGTCCACATTG Hochhnut et al., 2001 Sul2-F AGG GGG CAG ATG TGATCGAC Hochhnut et al., 2001 Sul2-B TGTGCGGATGAAGTCAGCTCC Hochhnut et al., 2001 FLOR-F TTATCTCCCTGTCGTTCCAGCG Hochhnut et al., 2001 FLOR-2 CCTATG AGCACACGGGGAGC Iwanaga et al., 2004 strB-F GGCACCCATAAGCGTACGCC Iwanaga et al., 2004 strB-R TGCCGAGCACGGCGACTACC Iwanaga et al., 2004 DFR1-F CGAAGAATGGAGTTATCGGG Iwanaga et al., 2004 DFR1-B TGCTGGGGATTTCAGGAAAG Iwanaga et al., 2004

that they could not be differentiated into a specific biotype based on their phenotypic traits (Nair et al., 2002). In 2004, a second variant, designated as the Mozambique variant, was found to have typical El Tor phenotypic traits but it genetically harboured a tandem repeat of the classical CTX prophage on the small chromosome (Ansaruzzaman et al., 2004). The third, and perhaps the most significant atypical El Tor, was the altered El Tor that is uniquely recognized as carrying the classical cholera toxin while retaining almost all aspects of the prototypic seventh pandemic El Tor strain.

This altered El Tor was initially reported in Bangladesh and growing evidence suggests the wide spread of this variant around the world in recent years (Nguyen et al., 2009; Okada et al., 2010; Morita et al., 2010; Sithivong et al., 2010; Ceccarelli et al., 2011). The altered El Tor was reported to have fully displaced prototypic seventh pandemic El Tor strains in several countries, including India and Bangladesh (Nair et al., 2006; Raychoudhuri et al., 2009). Two different conjectures have been proposed for the emergence and global transmission of atypical El Tor (Alam et al., 2010). The emergence of altered El Tor was postulated to be the result of either clonal expansion of a single ancestral El Tor which had acquired the classical *ctxB* gene in a cholera endemic region or a multiclonal event occurring independently in each region from co-existing El Tor and classical strains. Transnational transmission of altered El Tor was exemplify by the recent 2010 Haiti outbreak which was thought to be introduced by human activity from South Asian countries (Chin et al., 2011) and subsequently spread to United States, Canada and Dominican Republic via importation by travellers from Haiti (CDC, 2010; Gilmour et al., 2011).

The emergence of atypical El Tor marks a significant event in the evolution of *V. cholerae* and the epidemiology of cholera. The 2009 cholera outbreak strain from Kelantan state on the east coast of peninsular Malaysia was characterized as belonging to the altered El Tor biotype and it carried the classical cholera toxin (*ctxB*) gene (Ang et al., 2010). The present study further investigated genetic aspects of the Kelantan altered El Tor strain using multiple PCR analysis to elucidate the structure of the CTX prophage and to detect the presence of class I integron and SXT element antibiotic determinants.

## **2. Materials and methods**

#### **2.1** *V. cholerae* **strains**

A total of 20 *V. cholerae* isolates belonging to serogroup O1 of the altered El Tor biotype were collected during the 2009 cholera outbreak in Kelantan, Malaysia as described earlier (Ang et al., 2010). All the *V. cholerae* isolates were revived from glycerol stock and identification was performed using standard biochemical methods (Kay et al., 1994). Serotyping was conducted using slide agglutination tests with polyvalent O1 and monospecific Ogawa and Inaba antisera (Denka Seikan, Japan). All isolates were routinely grown on Luria-Bertani (LB) agar throughout the study.

#### **2.2 Genomic DNA preparation**

The genomic DNA template for genetic analysis was purified using a NucleoSpin Tissue kit (Macherey-Nagel, Germany), according to the manufacturer's instructions. The purity and concentration of purified genomic DNA was determined using a Biophotometer (Eppendorf, Germany).

#### **2.3** *rstR* **typing**

76 Cholera

that they could not be differentiated into a specific biotype based on their phenotypic traits (Nair et al., 2002). In 2004, a second variant, designated as the Mozambique variant, was found to have typical El Tor phenotypic traits but it genetically harboured a tandem repeat of the classical CTX prophage on the small chromosome (Ansaruzzaman et al., 2004). The third, and perhaps the most significant atypical El Tor, was the altered El Tor that is uniquely recognized as carrying the classical cholera toxin while retaining almost all aspects

This altered El Tor was initially reported in Bangladesh and growing evidence suggests the wide spread of this variant around the world in recent years (Nguyen et al., 2009; Okada et al., 2010; Morita et al., 2010; Sithivong et al., 2010; Ceccarelli et al., 2011). The altered El Tor was reported to have fully displaced prototypic seventh pandemic El Tor strains in several countries, including India and Bangladesh (Nair et al., 2006; Raychoudhuri et al., 2009). Two different conjectures have been proposed for the emergence and global transmission of atypical El Tor (Alam et al., 2010). The emergence of altered El Tor was postulated to be the result of either clonal expansion of a single ancestral El Tor which had acquired the classical *ctxB* gene in a cholera endemic region or a multiclonal event occurring independently in each region from co-existing El Tor and classical strains. Transnational transmission of altered El Tor was exemplify by the recent 2010 Haiti outbreak which was thought to be introduced by human activity from South Asian countries (Chin et al., 2011) and subsequently spread to United States, Canada and Dominican Republic via importation by

The emergence of atypical El Tor marks a significant event in the evolution of *V. cholerae* and the epidemiology of cholera. The 2009 cholera outbreak strain from Kelantan state on the east coast of peninsular Malaysia was characterized as belonging to the altered El Tor biotype and it carried the classical cholera toxin (*ctxB*) gene (Ang et al., 2010). The present study further investigated genetic aspects of the Kelantan altered El Tor strain using multiple PCR analysis to elucidate the structure of the CTX prophage and to detect the

A total of 20 *V. cholerae* isolates belonging to serogroup O1 of the altered El Tor biotype were collected during the 2009 cholera outbreak in Kelantan, Malaysia as described earlier (Ang et al., 2010). All the *V. cholerae* isolates were revived from glycerol stock and identification was performed using standard biochemical methods (Kay et al., 1994). Serotyping was conducted using slide agglutination tests with polyvalent O1 and monospecific Ogawa and Inaba antisera (Denka Seikan, Japan). All isolates were routinely grown on Luria-Bertani

The genomic DNA template for genetic analysis was purified using a NucleoSpin Tissue kit (Macherey-Nagel, Germany), according to the manufacturer's instructions. The purity and concentration of purified genomic DNA was determined using a Biophotometer

of the prototypic seventh pandemic El Tor strain.

travellers from Haiti (CDC, 2010; Gilmour et al., 2011).

**2. Materials and methods** 

(LB) agar throughout the study.

**2.2 Genomic DNA preparation** 

(Eppendorf, Germany).

**2.1** *V. cholerae* **strains** 

presence of class I integron and SXT element antibiotic determinants.

The type of *rstR* gene in each isolate was determined using a set of allele-specific forward primers (*rstREl Tor*, *rstRClassical*, *rstRCalcutta* and *rstREnvironment*) and a common reverse primer, *rstRR* (Nusrin et al., 2004; Bhattacharya et al., 2006). A list of all primers used in this study is presented in Table 1.


Table 1. Primers used in this study

Genetic Analysis of CTX Prophage and Antibiotic Resistance Determinants

the absence of classical, Calcutta, and environmental type *rstR*.

Calcutta type *rstR*; *rstREnv*: environmental type *rstR*.

**3.2 Genetic analysis of the CTX prophage array** 

JN545750, respectively.

**3. Results** 

Ogawa serotype.

**3.1** *rstR* **typing** 

in *Vibrio cholerae* O1 Belonging to the Atypical El Tor Biotype from Kelantan, Malaysia 79

*R* were confirmed by sequencing reactions. Prior to being sequenced, all amplicons from positive PCR reactions were purified using Wizard SV Gel and PCR Clean-up System (Promega, Australia), according to the manufacturer's instructions. The complete nucleotide sequences of *Sui1* gene amplified using the primer pair Ch2F/Ch2R for isolates 03/09-KB and 27/09-KB were assigned the GenBank accession numbers JN545747 and JN545748. The partial nucleotide sequences of *rtxC*, SXT element, S*ulII*, *strB,* and *dfrA1* for isolate 03/09-KB were deposited under accession numbers JN545752, JN545751, JN545754, JN545753, and

All the revived isolates were identified as *V. cholerae* biotype El Tor using standard biochemical tests and slide agglutination tests showed they belonged to serogroup O1 of the

The *rstR* typing by PCR amplification of the 501 bp amplicon using the primer pair *rstREl Tor*/*rstRR* showed that all isolates possessed only the El Tor type *rstR* (Fig. 1). No amplicon was produced for other allele-specific primers among all isolates analyzed, which indicated

Fig. 1. Agarose gel electrophoresis products of *rstR* typing. The expected product size and types of *rstR* targeted by specific primer pairs are indicated below the gel. Lane M: 100 bp DNA ladder; lanes 1, 3, 5, and 7: representative isolate 03/09-KB; lanes 2, 4, 6, and 8: representative isolate 27/09-KB. *rstRCla*: classical type *rstR*; *rstRET*: El Tor type *rstR*; *rstCal*:

The presence of RS1 element was confirmed using the primer pair rstCF/rstCR to amplify a 197 bp region of *rstC* gene from all the isolates (Fig. 2a). The CTX prophage and RS1 element in each isolate was found to be arranged in the form of a RS1-CTX prophage array, as shown

## **2.4 Genetic analysis of CTX prophage array**

Genetic analysis of CTX prophage array was performed using several combinations of primer pairs, as described by Nguyen et al. (2009). The presence of a RS1 element was determined using the primer pair rstCF/rstCR. Investigations of the arrays for RS1 and CTX prophage were performed using two primer pairs: ctxBF/rstCR for the CTX prophage-RS1 array and rstCF4/rstAR for the RS1-CTX prophage array. The presence of tandem repeats of the RS1 element or CTX prophage was determined using the primer pair rstCF4/rstCR4 and ctxBF/cepR, respectively. The chromosomal localization of RS1 and CTX prophage was confirmed using the primer pairs Ch1F/rstRR and ctxBF/Ch1R for the large chromosome and Ch2F/Ch2R for the small chromosome.

## **2.5** *rtxC* **PCR**

Detection of the *rtxC* gene was performed using the primer pair *rtxC-F*/*rtxC-R* (Chow et al., 2001).

## **2.6 PCR detection of class I integrons**

Detection of class I integrons was performed using a set of primers described by Dalsgaard et al. (2001). Briefly, the primer pairs inDS-F/inDS-B and qacE∆1-F/su1-B were used for the amplification of the 5-CS and 3-CS of the class I integron. The primer pair in-F/in-B was used to amplify gene cassettes inserted in the integron, while the primer pair in-F/aadA-B was used for the amplification of the gene cassette *aad1A* that encodes streptomycin resistance.

## **2.7 PCR detection of SXT constins**

The isolates were screened for the presence of SXT constins (large conjugative elements) using the primer pair INT1/INT2. Presence of the antibiotic resistance genes *floR* that encodes resistance to chloramphenicol, *sulII* for resistance to sulfamethoxazole, *strB* for resistance to streptomycin, and *dfrA1* for resistance to trimethoprim, were determined using the primer pairs FLOR-F/FLOR-2, Sul2-F/Sul2-B, strB-F/strB-R, and DFR1-F/DFR1-B, respectively (Hochhut et al., 2001).

#### **2.8 Sequencing of tandem repeats of RS1 elements and upstream regions of the CTX prophage**

The DNA sequence spanning tandem repeats of RS1 elements and upstream regions of the CTX prophage (~8.5 kb) was generated with the primer pairs Ch1F/rstCR, rstCF4/rstCR4, and rstCF/cepR. Amplicons from each PCR reaction were cloned into the pCR4-TOPO vector (Invitrogen, CA) and sequenced by First Base Laboratories Sdn. Bhd. (Malaysia). The sequencing data was assembled and complete nucleotide sequences of the RS1-RS1-CTX prophage arrays for isolates 03/09-KB and 27/09-KB were deposited in GenBank under the accession numbers JN545744 and JN545745.

### **2.9 Sequencing of PCR amplicons**

The nucleotide sequences of the PCR amplicons generated by each of the primer pairs Ch2F/Ch2R, INT1/INT2, Sul2-F/Sul2-B, strB-F/strB-R, DFR1-F/DFR1-B, and *rtxC-F*/*rtxC-*

*R* were confirmed by sequencing reactions. Prior to being sequenced, all amplicons from positive PCR reactions were purified using Wizard SV Gel and PCR Clean-up System (Promega, Australia), according to the manufacturer's instructions. The complete nucleotide sequences of *Sui1* gene amplified using the primer pair Ch2F/Ch2R for isolates 03/09-KB and 27/09-KB were assigned the GenBank accession numbers JN545747 and JN545748. The partial nucleotide sequences of *rtxC*, SXT element, S*ulII*, *strB,* and *dfrA1* for isolate 03/09-KB were deposited under accession numbers JN545752, JN545751, JN545754, JN545753, and JN545750, respectively.

## **3. Results**

78 Cholera

Genetic analysis of CTX prophage array was performed using several combinations of primer pairs, as described by Nguyen et al. (2009). The presence of a RS1 element was determined using the primer pair rstCF/rstCR. Investigations of the arrays for RS1 and CTX prophage were performed using two primer pairs: ctxBF/rstCR for the CTX prophage-RS1 array and rstCF4/rstAR for the RS1-CTX prophage array. The presence of tandem repeats of the RS1 element or CTX prophage was determined using the primer pair rstCF4/rstCR4 and ctxBF/cepR, respectively. The chromosomal localization of RS1 and CTX prophage was confirmed using the primer pairs Ch1F/rstRR and ctxBF/Ch1R for the large chromosome

Detection of the *rtxC* gene was performed using the primer pair *rtxC-F*/*rtxC-R* (Chow et al.,

Detection of class I integrons was performed using a set of primers described by Dalsgaard et al. (2001). Briefly, the primer pairs inDS-F/inDS-B and qacE∆1-F/su1-B were used for the amplification of the 5-CS and 3-CS of the class I integron. The primer pair in-F/in-B was used to amplify gene cassettes inserted in the integron, while the primer pair in-F/aadA-B was used

The isolates were screened for the presence of SXT constins (large conjugative elements) using the primer pair INT1/INT2. Presence of the antibiotic resistance genes *floR* that encodes resistance to chloramphenicol, *sulII* for resistance to sulfamethoxazole, *strB* for resistance to streptomycin, and *dfrA1* for resistance to trimethoprim, were determined using the primer pairs FLOR-F/FLOR-2, Sul2-F/Sul2-B, strB-F/strB-R, and DFR1-F/DFR1-B,

**2.8 Sequencing of tandem repeats of RS1 elements and upstream regions of the CTX** 

The DNA sequence spanning tandem repeats of RS1 elements and upstream regions of the CTX prophage (~8.5 kb) was generated with the primer pairs Ch1F/rstCR, rstCF4/rstCR4, and rstCF/cepR. Amplicons from each PCR reaction were cloned into the pCR4-TOPO vector (Invitrogen, CA) and sequenced by First Base Laboratories Sdn. Bhd. (Malaysia). The sequencing data was assembled and complete nucleotide sequences of the RS1-RS1-CTX prophage arrays for isolates 03/09-KB and 27/09-KB were deposited in GenBank under the

The nucleotide sequences of the PCR amplicons generated by each of the primer pairs Ch2F/Ch2R, INT1/INT2, Sul2-F/Sul2-B, strB-F/strB-R, DFR1-F/DFR1-B, and *rtxC-F*/*rtxC-*

for the amplification of the gene cassette *aad1A* that encodes streptomycin resistance.

**2.4 Genetic analysis of CTX prophage array** 

and Ch2F/Ch2R for the small chromosome.

**2.6 PCR detection of class I integrons** 

**2.7 PCR detection of SXT constins** 

respectively (Hochhut et al., 2001).

accession numbers JN545744 and JN545745.

**2.9 Sequencing of PCR amplicons** 

**2.5** *rtxC* **PCR** 

2001).

**prophage** 

All the revived isolates were identified as *V. cholerae* biotype El Tor using standard biochemical tests and slide agglutination tests showed they belonged to serogroup O1 of the Ogawa serotype.

## **3.1** *rstR* **typing**

The *rstR* typing by PCR amplification of the 501 bp amplicon using the primer pair *rstREl Tor*/*rstRR* showed that all isolates possessed only the El Tor type *rstR* (Fig. 1). No amplicon was produced for other allele-specific primers among all isolates analyzed, which indicated the absence of classical, Calcutta, and environmental type *rstR*.

Fig. 1. Agarose gel electrophoresis products of *rstR* typing. The expected product size and types of *rstR* targeted by specific primer pairs are indicated below the gel. Lane M: 100 bp DNA ladder; lanes 1, 3, 5, and 7: representative isolate 03/09-KB; lanes 2, 4, 6, and 8: representative isolate 27/09-KB. *rstRCla*: classical type *rstR*; *rstRET*: El Tor type *rstR*; *rstCal*: Calcutta type *rstR*; *rstREnv*: environmental type *rstR*.

### **3.2 Genetic analysis of the CTX prophage array**

The presence of RS1 element was confirmed using the primer pair rstCF/rstCR to amplify a 197 bp region of *rstC* gene from all the isolates (Fig. 2a). The CTX prophage and RS1 element in each isolate was found to be arranged in the form of a RS1-CTX prophage array, as shown

Genetic Analysis of CTX Prophage and Antibiotic Resistance Determinants

**3.3** *rtxC* **PCR** 

(Fig. 3).

27/09-KB.

**4. Discussion** 

**3.4 PCR detection of class I integrons** 

obtained with all the primer pairs tested.

**3.5 PCR detection of SXT constins** 

(Nguyen et al., 2009; Lee et al., 2009).

in *Vibrio cholerae* O1 Belonging to the Atypical El Tor Biotype from Kelantan, Malaysia 81

PCR analysis of *rtxC* showed that all the isolates yielded a 263 bp amplicon of the *rtxC* gene

Fig. 3. Agarose gel electrophoresis products from the detection of the *rtxC* gene and the analysis of the SXT constin. The combinations of different primer pairs and their expected product sizes are indicated below the gel. Lane M: 100 bp Plus DNA ladder; lanes 1, 3, 5, 7, 9, and 11: representative isolate 03/09-KB; lanes 2, 4, 6, 8, 10, and 12: representative isolate

None of the isolates were positive for class I integrons, because no PCR amplicons were

All the isolates were shown to be positive for SXT element by the amplification of a 592 bp amplicon with the primer pair INT1/INT2 (Fig. 3). These isolates also had positive PCR results for the *SulII*, *strB,* and *dfrA1* genes, because 626 bp, 470 bp, and 372 bp amplicons were generated for the respective genes. None of the isolates were positive for *floR* genes.

In November 2009, Kelantan was struck by a cholera outbreak that marked the reemergence of this secretory diarrheal disease in the eastern state after years of absence. The aetiological agent was later found to be *V. cholerae* O1 of the altered El Tor biotype and this discovery indicated the first reported appearance of the atypical El Tor strain on Malaysian soil (Ang et al., 2010). Various genetic studies have been undertaken to gain insights into the evolution of this predominant atypical strain and research into the altered El Tor strain has primarily been directed towards the CTX prophage encoding the classical cholera toxin gene

by the positive amplification of a 1551 bp amplicon with the primer pair rstCF4/rstAR (Fig. 2b). However, no additional RS1 element was found downstream of the CTX prophage because no amplicon was generated by the primer pair ctxBF/rstCR. The primer pair rstCF4/rstCR4 indicated the tandem arrangement of the RS1 element through the amplification of a 2629 bp amplicon, whereas no amplicon was produced for the primer pair ctxBF/cepR which indicated the presence of only a single CTX prophage. The location of the RS1-RS1-CTX prophage array on the large chromosome was verified using the primer pairs Ch1F/rstRR and ctxBF/Ch1R which amplify fragments corresponding to the upstream and downstream regions of the RS1-RS1-CTX prophage array found on the large chromosome. The absence of an RS1 element or CTX prophage on the small chromosome was confirmed through the amplification of a 910 bp amplicon using the primer pair Ch2F/Ch2R.

Fig. 2. (a) Agarose gel electrophoresis products in the detection of RS1 elements from representative isolates. Lane M: 100 bp Plus DNA ladder; lane 1: isolate 03/09-KB; lane 2: isolate 11/09-KB; lane 3: isolate 27/09-KB; lane 4: isolate 29/09-KB. (b) Agarose gel electrophoresis products from the analysis of the CTX prophage array. The combinations of different primer pairs and their expected product sizes are indicated below the gel. Lane M: 1 kb DNA ladder; lanes 1, 3, 5, 7, 9, 11, and 13: representative isolate 03/09-KB; lanes 2, 4, 6, 8, 10, 12, and 14: representative isolate 27/09-KB.

## **3.3** *rtxC* **PCR**

(a)

(b)

80 Cholera

by the positive amplification of a 1551 bp amplicon with the primer pair rstCF4/rstAR (Fig. 2b). However, no additional RS1 element was found downstream of the CTX prophage because no amplicon was generated by the primer pair ctxBF/rstCR. The primer pair rstCF4/rstCR4 indicated the tandem arrangement of the RS1 element through the amplification of a 2629 bp amplicon, whereas no amplicon was produced for the primer pair ctxBF/cepR which indicated the presence of only a single CTX prophage. The location of the RS1-RS1-CTX prophage array on the large chromosome was verified using the primer pairs Ch1F/rstRR and ctxBF/Ch1R which amplify fragments corresponding to the upstream and downstream regions of the RS1-RS1-CTX prophage array found on the large chromosome. The absence of an RS1 element or CTX prophage on the small chromosome was confirmed through the amplification of a 910 bp amplicon using the

Fig. 2. (a) Agarose gel electrophoresis products in the detection of RS1 elements from representative isolates. Lane M: 100 bp Plus DNA ladder; lane 1: isolate 03/09-KB; lane 2: isolate 11/09-KB; lane 3: isolate 27/09-KB; lane 4: isolate 29/09-KB. (b) Agarose gel

8, 10, 12, and 14: representative isolate 27/09-KB.

electrophoresis products from the analysis of the CTX prophage array. The combinations of different primer pairs and their expected product sizes are indicated below the gel. Lane M: 1 kb DNA ladder; lanes 1, 3, 5, 7, 9, 11, and 13: representative isolate 03/09-KB; lanes 2, 4, 6,

primer pair Ch2F/Ch2R.

PCR analysis of *rtxC* showed that all the isolates yielded a 263 bp amplicon of the *rtxC* gene (Fig. 3).

Fig. 3. Agarose gel electrophoresis products from the detection of the *rtxC* gene and the analysis of the SXT constin. The combinations of different primer pairs and their expected product sizes are indicated below the gel. Lane M: 100 bp Plus DNA ladder; lanes 1, 3, 5, 7, 9, and 11: representative isolate 03/09-KB; lanes 2, 4, 6, 8, 10, and 12: representative isolate 27/09-KB.

## **3.4 PCR detection of class I integrons**

None of the isolates were positive for class I integrons, because no PCR amplicons were obtained with all the primer pairs tested.

#### **3.5 PCR detection of SXT constins**

All the isolates were shown to be positive for SXT element by the amplification of a 592 bp amplicon with the primer pair INT1/INT2 (Fig. 3). These isolates also had positive PCR results for the *SulII*, *strB,* and *dfrA1* genes, because 626 bp, 470 bp, and 372 bp amplicons were generated for the respective genes. None of the isolates were positive for *floR* genes.

## **4. Discussion**

In November 2009, Kelantan was struck by a cholera outbreak that marked the reemergence of this secretory diarrheal disease in the eastern state after years of absence. The aetiological agent was later found to be *V. cholerae* O1 of the altered El Tor biotype and this discovery indicated the first reported appearance of the atypical El Tor strain on Malaysian soil (Ang et al., 2010). Various genetic studies have been undertaken to gain insights into the evolution of this predominant atypical strain and research into the altered El Tor strain has primarily been directed towards the CTX prophage encoding the classical cholera toxin gene (Nguyen et al., 2009; Lee et al., 2009).

Genetic Analysis of CTX Prophage and Antibiotic Resistance Determinants

in *Vibrio cholerae* O1 Belonging to the Atypical El Tor Biotype from Kelantan, Malaysia 83

Fig. 4. Genetic map comparison of the CTX prophage arrays found in the classical reference strain (O395), El Tor reference strain (N16961), Vietnam altered El Tor, and the Kelantan altered El Tor characterized in this study. The transcription direction of each gene is indicated by arrows and each gene is shaded in different colours. Chr I: chromosome I; Chr II: chromosome II; *rstRET*: El Tor type *rstR*; *rstRCla*: classical type *rstR*; *ctxBET*: El Tor type *ctxB*;

*ctxBCla*: classical type *ctxB*, TLC: toxin-linked cryptic. The map is not drawn to scale.

The CTX prophage found in the genome of pathogenic *V. cholerae* strains is actually an integrated form of a lysogenic filamentous bacteriophage known as the CTX phage (CTX). CTX is approximately 7 kb in length and it is composed of a 4.6 kb core region with a 2.4 kb RS2 region (Waldor & Mekalanos, 1996). The core region contains genes that encode for proteins involved in phage morphogenesis, specifically the core-encoded pilin (*cep*), pIIICTX (previously known as *orfU*), accessory cholera enterotoxin (*ace*), and zonula occludens (*zot*) genes. This core region also contains genes encoding for cholera toxin, so the acquisition of CTX is viewed as virulence acquisition by a host cell. The RS2 region complements the core, because it contains genes that enable the replication (*rstA*), integration (*rstB*), and regulation (*rstR*) of CTX. The RS1 element is another RS2-like element that is frequently found adjacent to the CTX prophage. The RS1 element is a 2.7 kb satellite phage that only differs from RS2 by an additional gene designated *rstC,* which encodes for a novel antirepressor to the RstR protein (Waldor et al., 1997; Heilpern & Waldor, 2003). The RS1 element provides a dual function by promoting the transcription of phage genes via an interaction between RstC and RstR, as well as enabling the replication of an adjacent CTX prophage to produce infective phage particles (Davis et al., 2002).

The *rstR* regulatory gene sequence found in the RS2 region also determined the type of CTX prophage carried by a *V. cholerae* strain. Three types of CTX prophage has been established to date, i.e., the classical CTX prophage and El Tor CTX prophage that were first detected in the *V. cholerae* serogroup O1 of the respective biotypes, and the Calcutta CTX prophage from the epidemic-causing serogroup O139 (Kimsey et al., 1998). A fourth type, the Mozambique CTX prophage, was proposed by Choi et al. (2010) and described based on the inclusion of other genetic features of the CTX prophage, including intergenic sequences and the *rstA* gene, although the CTX prophage contained a classical *rstR* gene. In the present study, the CTX prophage from the 2009 Kelantan cholera outbreak strain was found to be regulated by an El Tor type *rstR* repressor gene, but it carried a classical type *ctxB* gene. A CTX prophage with this combination of El Tor type *rstR* and classical cholera toxin gene was also designated as a hybrid CTX prophage by Grim et al. (2010). Further genetic analysis of the CTX prophage structure revealed that all isolates harboured a RS1-RS1-CTX prophage array, which was integrated on the large chromosome. As found in the prototypic seventh pandemic El Tor strains, no RS1 element or CTX prophage was integrated on the small chromosome. The RS1- RS1-CTX prophage array of the Kelantan variant represents a novel arrangement for these genetic elements among atypical El Tor strains. To the best of our knowledge, no RS1-RS1-CTX prophage array with an El Tor type *rstR* on the large chromosome has been demonstrated or reported elsewhere among the altered El Tor biotypes.

In 2009, Nguyen et al. were the first to characterize and report the CTX prophage array of altered El Tor strains isolated during cholera outbreaks that occurred in Vietnam between 2007 and 2008. All the Vietnamese isolates were found to contain the RS1-CTX prophage array with an El Tor type *rstR* on the large chromosome. Similarly, 400 *V. cholerae* isolates obtained between 2003 and 2007 from Kolkata, India were also characterized as having the RS1-CTX prophage array (Nguyen et al., 2009). Recently, the same RS1-CTX prophage array was identified in the altered El Tor isolates from Angola, Africa in 2006 (Ceccarelli et al., 2011) and from Hyderabad, India in 2009 (Goel et al., 2011). A study conducted by Goel et al. (2011) found that one of the altered El Tor isolates (VCH35) from Hyderabad, India harboured a tandem repeat of the CTX prophage in the small chromosome in addition to a RS1-CTX

The CTX prophage found in the genome of pathogenic *V. cholerae* strains is actually an integrated form of a lysogenic filamentous bacteriophage known as the CTX phage (CTX). CTX is approximately 7 kb in length and it is composed of a 4.6 kb core region with a 2.4 kb RS2 region (Waldor & Mekalanos, 1996). The core region contains genes that encode for proteins involved in phage morphogenesis, specifically the core-encoded pilin (*cep*), pIIICTX (previously known as *orfU*), accessory cholera enterotoxin (*ace*), and zonula occludens (*zot*) genes. This core region also contains genes encoding for cholera toxin, so the acquisition of CTX is viewed as virulence acquisition by a host cell. The RS2 region complements the core, because it contains genes that enable the replication (*rstA*), integration (*rstB*), and regulation (*rstR*) of CTX. The RS1 element is another RS2-like element that is frequently found adjacent to the CTX prophage. The RS1 element is a 2.7 kb satellite phage that only differs from RS2 by an additional gene designated *rstC,* which encodes for a novel antirepressor to the RstR protein (Waldor et al., 1997; Heilpern & Waldor, 2003). The RS1 element provides a dual function by promoting the transcription of phage genes via an interaction between RstC and RstR, as well as enabling the replication of an adjacent CTX

The *rstR* regulatory gene sequence found in the RS2 region also determined the type of CTX prophage carried by a *V. cholerae* strain. Three types of CTX prophage has been established to date, i.e., the classical CTX prophage and El Tor CTX prophage that were first detected in the *V. cholerae* serogroup O1 of the respective biotypes, and the Calcutta CTX prophage from the epidemic-causing serogroup O139 (Kimsey et al., 1998). A fourth type, the Mozambique CTX prophage, was proposed by Choi et al. (2010) and described based on the inclusion of other genetic features of the CTX prophage, including intergenic sequences and the *rstA* gene, although the CTX prophage contained a classical *rstR* gene. In the present study, the CTX prophage from the 2009 Kelantan cholera outbreak strain was found to be regulated by an El Tor type *rstR* repressor gene, but it carried a classical type *ctxB* gene. A CTX prophage with this combination of El Tor type *rstR* and classical cholera toxin gene was also designated as a hybrid CTX prophage by Grim et al. (2010). Further genetic analysis of the CTX prophage structure revealed that all isolates harboured a RS1-RS1-CTX prophage array, which was integrated on the large chromosome. As found in the prototypic seventh pandemic El Tor strains, no RS1 element or CTX prophage was integrated on the small chromosome. The RS1- RS1-CTX prophage array of the Kelantan variant represents a novel arrangement for these genetic elements among atypical El Tor strains. To the best of our knowledge, no RS1-RS1-CTX prophage array with an El Tor type *rstR* on the large chromosome has been demonstrated or

In 2009, Nguyen et al. were the first to characterize and report the CTX prophage array of altered El Tor strains isolated during cholera outbreaks that occurred in Vietnam between 2007 and 2008. All the Vietnamese isolates were found to contain the RS1-CTX prophage array with an El Tor type *rstR* on the large chromosome. Similarly, 400 *V. cholerae* isolates obtained between 2003 and 2007 from Kolkata, India were also characterized as having the RS1-CTX prophage array (Nguyen et al., 2009). Recently, the same RS1-CTX prophage array was identified in the altered El Tor isolates from Angola, Africa in 2006 (Ceccarelli et al., 2011) and from Hyderabad, India in 2009 (Goel et al., 2011). A study conducted by Goel et al. (2011) found that one of the altered El Tor isolates (VCH35) from Hyderabad, India harboured a tandem repeat of the CTX prophage in the small chromosome in addition to a RS1-CTX

prophage to produce infective phage particles (Davis et al., 2002).

reported elsewhere among the altered El Tor biotypes.

Fig. 4. Genetic map comparison of the CTX prophage arrays found in the classical reference strain (O395), El Tor reference strain (N16961), Vietnam altered El Tor, and the Kelantan altered El Tor characterized in this study. The transcription direction of each gene is indicated by arrows and each gene is shaded in different colours. Chr I: chromosome I; Chr II: chromosome II; *rstRET*: El Tor type *rstR*; *rstRCla*: classical type *rstR*; *ctxBET*: El Tor type *ctxB*; *ctxBCla*: classical type *ctxB*, TLC: toxin-linked cryptic. The map is not drawn to scale.

Genetic Analysis of CTX Prophage and Antibiotic Resistance Determinants

backbone was preserved in the Kelantan altered El Tor strain.

streptomycin and spectinomycin.

in *Vibrio cholerae* O1 Belonging to the Atypical El Tor Biotype from Kelantan, Malaysia 85

for biotyping *V. cholerae* serogroup O1. We used this biotyping assay and found that all the isolates possessed the *rtxC* gene. This provided further evidence that the El Tor genomic

The emergence of multidrug-resistant *V. cholerae* strains has been documented frequently in recent years (Kiiru et al., 2009; Jain et al., 2011) and this phenomenon has led to repeated calls for the more prudent use of antibiotics by the global community. Multidrug-resistant *V. cholerae* have been reported from Malaysia and the antibiogram profile of the Kelantan outbreak strain showed that the isolates were resistant to various antibiotics, including, tetracycline, erythromycin, sulfamethoxazole-trimethoprim, streptomycin, penicillin G, and polymyxin B. However, they were susceptible to ciprofloxacin, norfloxacin, chloramphenicol, gentamicin, and kanamycin (Ang et al., 2010). Therefore, we investigated the phenotypes of the outbreak strain by characterizing the corresponding genes encoding for antibiotic resistance. Amita et al. (2003) studied antibiotic resistance genes in *V. cholerae* O1 and showed that the class I integron carrying the *aadA1* gene cassette was prevalent in strains isolated before 1992, whereas the SXT element was prevalent in strains isolated after 1992. Integrons are characterized by the presence of an integrase gene (*intI*) that mediates recombination between the *attI* site found on the integron and the *attC* site on the gene cassette. The insertion of a gene cassette into the integron results in the expression of functional proteins using a promoter found in the integron (Recchia & Hall, 1995). In agreement with Amita et al. (2003), PCR analysis of the Kelantan outbreak strain showed it was negative for the class I integron and the gene cassette *aadA1* encoding resistance to

In one of the most remarkable events in the recorded history of cholera, a novel serogroup of *V. cholerae* emerged in 1992 that was designated O139 and it replaced the O1 El Tor biotype in Bangladesh and the Indian subcontinent where it became the dominant strain, although its reign was short-lived (Faruque et al., 2003). The *V. cholerae* serogroup O139 differed from serogroup O1 in having a different somatic antigen and it was also uniquely characterized by its antibiotic resistance to sulfamethoxazole, trimethoprim, chloramphenicol, and streptomycin. Interestingly, a distinctive pattern of antibiotic resistance was found after the re-emergence of serogroup O1 El Tor in 1994 where all the El Tor strains were found to be resistant to these four antibiotics, which strikingly resembled the profile of serogroup O139 (Waldor et al., 1996). The corresponding antibiotic resistance genes in serogroup O1 El Tor were collectively referred to as ICE*Vch*Ind1 (previously known as SXTET) and they were genetically closely related to the SXTMO10, which encodes for antibiotic resistance to the same four antibiotics mentioned above by serogroup O139. Based on a comparative DNA analysis, both the ICE*Vch*Ind1 and the SXTMO10 elements were considered to be derived from a common precursor (Hochhut et al., 2001; Burrus et al., 2006). The results of PCR conducted on the SXT constin in this study showed that all the Kelantan outbreak strains contained the SXT element. Genes conferring resistance to sulfamethoxazole (*SulII*), trimethoprim (*dfrA1*), and streptomycin (*strB*) were detected in all isolates, with the exception of the chloramphenicol resistance gene (*floR*). The detection of the antibiotic genes was consistent with the findings in the phenotypic antibiotic susceptibility testing and the SXT constin detected in the present outbreak strain appeared to have a deletion of the *floR* gene, when compared with the STXET reported from elsewhere (Hochhut et al., 2001). The SXT constin without the *floR* gene represented a variant of the SXTET constin and, other than this study,

prophage array in the large chromosome. Although *rstR* typing of VCH35 revealed the presence of both El Tor and classical type *rstR*, the localization of these *rstR* alleles in the multiple CTX prophages was not confirmed. Various combinations of CTX prophage arrays have been documented, but the RS1-CTX prophage array appears to be the most frequently reported arrangement in altered El Tor strains associated with cholera outbreaks (Lee et al., 2009). Fig. 4 provides a diagrammatic comparison of various CTX prophage arrays, including the classical, prototypic seventh pandemic El Tor, and altered El Tor.

One of the main issues accompanying the emergence of atypical El Tor is standardization of the nomenclature and the classification scheme used when referring to these variants. This is further complicated by the fact that the current genotypic and phenotypic diversity reported among atypical El Tor strains is only the tip of the iceberg, because there are frequent new reports. In 2009, Lee et al. proposed the classification of atypical El Tor strains into two groups based on genetic differences in their RS1 element and the CTX prophage structure on each chromosome. Group I represents atypical El Tor strains with a tandem repeat of classical CTX prophage on the small chromosome, while Group II represents those possessing the RS1 and CTX prophage with El Tor type *rstR* and classical *ctxB* on the large chromosome. Based on these criteria, the Matlab and Mozambique variants were classified into Group I, while altered El Tor, such as those described by Nguyen et al. (2009), fell into Group II. This classification system was also used by Goel et al. (2011) to categorize the VCH35 isolate from Hyderabad, India into Group I, because it carried a tandem repeat of CTX prophage on the small chromosome. The type of *rstR* gene determines the type of corresponding CTX prophage, so a minor discrepancy when adhering to this classification system arises when both El Tor and classical type *rstR* are present, as is the case with VCH35. Therefore, the exact nature of the CTX prophages in tandem arrangements needs to be elucidated to ascertain whether VCH35 truly belonged to Group I. The existence of VCH35 also questions the possible need for a subgroup within Group I, should further analysis of VCH35 reveal the presence of both an El Tor and classical type CTX prophage in a tandem arrangement. In-depth genetic analysis of VCH35 is highly warranted before any conclusions can be drawn on its classification. In contrast, only one array of the RS1-CTX prophage has been reported in Group II (Lee et al., 2009). Therefore, we were able to describe a new type of array belonging to Group II based on the findings of this study. An arrangement of RS1 in tandem repeats followed by CTX prophage with an El Tor type *rstR* and classical *ctxB* was characterized in this study. This suggests that more varieties of the CTX prophage array may exist among the altered El Tor than are currently known.

The current study revealed an El Tor type *rstR* in the CTX prophage, but we also reported in our previous study that the 2009 Kelantan outbreak strain carried the El Tor type *tcpA* gene allele. In order to substantiate the El Tor lineage of this strain, we performed additional PCR analysis on the repeat in the toxin (RTX) gene cluster. The RTX gene cluster in *V. cholerae* was first identified and characterized in 1999 and it was found to be physically linked to the downstream region of the CTX prophage. The RTX gene cluster consists of *rtxA*, *rtxB*, *rtxC,* and *rtxD* genes, and it is responsible for the cytotoxic activity of *V. cholerae* in mammalian cells *in vitro*. However, gene deletions in the RTX gene cluster (specifically the *rtxC* gene and the downstream region of the *rtxA* gene) were noted among the classical biotype of *V. cholerae* O1, which resulted in defective production of cytotoxic activity (Lin et al., 1999). Based on this observation, Chow et al. (2001) developed a PCR assay targeting the *rtxC* gene

prophage array in the large chromosome. Although *rstR* typing of VCH35 revealed the presence of both El Tor and classical type *rstR*, the localization of these *rstR* alleles in the multiple CTX prophages was not confirmed. Various combinations of CTX prophage arrays have been documented, but the RS1-CTX prophage array appears to be the most frequently reported arrangement in altered El Tor strains associated with cholera outbreaks (Lee et al., 2009). Fig. 4 provides a diagrammatic comparison of various CTX prophage arrays,

One of the main issues accompanying the emergence of atypical El Tor is standardization of the nomenclature and the classification scheme used when referring to these variants. This is further complicated by the fact that the current genotypic and phenotypic diversity reported among atypical El Tor strains is only the tip of the iceberg, because there are frequent new reports. In 2009, Lee et al. proposed the classification of atypical El Tor strains into two groups based on genetic differences in their RS1 element and the CTX prophage structure on each chromosome. Group I represents atypical El Tor strains with a tandem repeat of classical CTX prophage on the small chromosome, while Group II represents those possessing the RS1 and CTX prophage with El Tor type *rstR* and classical *ctxB* on the large chromosome. Based on these criteria, the Matlab and Mozambique variants were classified into Group I, while altered El Tor, such as those described by Nguyen et al. (2009), fell into Group II. This classification system was also used by Goel et al. (2011) to categorize the VCH35 isolate from Hyderabad, India into Group I, because it carried a tandem repeat of CTX prophage on the small chromosome. The type of *rstR* gene determines the type of corresponding CTX prophage, so a minor discrepancy when adhering to this classification system arises when both El Tor and classical type *rstR* are present, as is the case with VCH35. Therefore, the exact nature of the CTX prophages in tandem arrangements needs to be elucidated to ascertain whether VCH35 truly belonged to Group I. The existence of VCH35 also questions the possible need for a subgroup within Group I, should further analysis of VCH35 reveal the presence of both an El Tor and classical type CTX prophage in a tandem arrangement. In-depth genetic analysis of VCH35 is highly warranted before any conclusions can be drawn on its classification. In contrast, only one array of the RS1-CTX prophage has been reported in Group II (Lee et al., 2009). Therefore, we were able to describe a new type of array belonging to Group II based on the findings of this study. An arrangement of RS1 in tandem repeats followed by CTX prophage with an El Tor type *rstR* and classical *ctxB* was characterized in this study. This suggests that more varieties of the

including the classical, prototypic seventh pandemic El Tor, and altered El Tor.

CTX prophage array may exist among the altered El Tor than are currently known.

The current study revealed an El Tor type *rstR* in the CTX prophage, but we also reported in our previous study that the 2009 Kelantan outbreak strain carried the El Tor type *tcpA* gene allele. In order to substantiate the El Tor lineage of this strain, we performed additional PCR analysis on the repeat in the toxin (RTX) gene cluster. The RTX gene cluster in *V. cholerae* was first identified and characterized in 1999 and it was found to be physically linked to the downstream region of the CTX prophage. The RTX gene cluster consists of *rtxA*, *rtxB*, *rtxC,* and *rtxD* genes, and it is responsible for the cytotoxic activity of *V. cholerae* in mammalian cells *in vitro*. However, gene deletions in the RTX gene cluster (specifically the *rtxC* gene and the downstream region of the *rtxA* gene) were noted among the classical biotype of *V. cholerae* O1, which resulted in defective production of cytotoxic activity (Lin et al., 1999). Based on this observation, Chow et al. (2001) developed a PCR assay targeting the *rtxC* gene for biotyping *V. cholerae* serogroup O1. We used this biotyping assay and found that all the isolates possessed the *rtxC* gene. This provided further evidence that the El Tor genomic backbone was preserved in the Kelantan altered El Tor strain.

The emergence of multidrug-resistant *V. cholerae* strains has been documented frequently in recent years (Kiiru et al., 2009; Jain et al., 2011) and this phenomenon has led to repeated calls for the more prudent use of antibiotics by the global community. Multidrug-resistant *V. cholerae* have been reported from Malaysia and the antibiogram profile of the Kelantan outbreak strain showed that the isolates were resistant to various antibiotics, including, tetracycline, erythromycin, sulfamethoxazole-trimethoprim, streptomycin, penicillin G, and polymyxin B. However, they were susceptible to ciprofloxacin, norfloxacin, chloramphenicol, gentamicin, and kanamycin (Ang et al., 2010). Therefore, we investigated the phenotypes of the outbreak strain by characterizing the corresponding genes encoding for antibiotic resistance. Amita et al. (2003) studied antibiotic resistance genes in *V. cholerae* O1 and showed that the class I integron carrying the *aadA1* gene cassette was prevalent in strains isolated before 1992, whereas the SXT element was prevalent in strains isolated after 1992. Integrons are characterized by the presence of an integrase gene (*intI*) that mediates recombination between the *attI* site found on the integron and the *attC* site on the gene cassette. The insertion of a gene cassette into the integron results in the expression of functional proteins using a promoter found in the integron (Recchia & Hall, 1995). In agreement with Amita et al. (2003), PCR analysis of the Kelantan outbreak strain showed it was negative for the class I integron and the gene cassette *aadA1* encoding resistance to streptomycin and spectinomycin.

In one of the most remarkable events in the recorded history of cholera, a novel serogroup of *V. cholerae* emerged in 1992 that was designated O139 and it replaced the O1 El Tor biotype in Bangladesh and the Indian subcontinent where it became the dominant strain, although its reign was short-lived (Faruque et al., 2003). The *V. cholerae* serogroup O139 differed from serogroup O1 in having a different somatic antigen and it was also uniquely characterized by its antibiotic resistance to sulfamethoxazole, trimethoprim, chloramphenicol, and streptomycin. Interestingly, a distinctive pattern of antibiotic resistance was found after the re-emergence of serogroup O1 El Tor in 1994 where all the El Tor strains were found to be resistant to these four antibiotics, which strikingly resembled the profile of serogroup O139 (Waldor et al., 1996). The corresponding antibiotic resistance genes in serogroup O1 El Tor were collectively referred to as ICE*Vch*Ind1 (previously known as SXTET) and they were genetically closely related to the SXTMO10, which encodes for antibiotic resistance to the same four antibiotics mentioned above by serogroup O139. Based on a comparative DNA analysis, both the ICE*Vch*Ind1 and the SXTMO10 elements were considered to be derived from a common precursor (Hochhut et al., 2001; Burrus et al., 2006). The results of PCR conducted on the SXT constin in this study showed that all the Kelantan outbreak strains contained the SXT element. Genes conferring resistance to sulfamethoxazole (*SulII*), trimethoprim (*dfrA1*), and streptomycin (*strB*) were detected in all isolates, with the exception of the chloramphenicol resistance gene (*floR*). The detection of the antibiotic genes was consistent with the findings in the phenotypic antibiotic susceptibility testing and the SXT constin detected in the present outbreak strain appeared to have a deletion of the *floR* gene, when compared with the STXET reported from elsewhere (Hochhut et al., 2001). The SXT constin without the *floR* gene represented a variant of the SXTET constin and, other than this study,

Genetic Analysis of CTX Prophage and Antibiotic Resistance Determinants

O139 strains. *Environ Microbiol,* 8 (3), 526-634.

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Chow, K. H., Ng, T. K., Yuen, K. Y. & Yam, W. C. (2001). Detection of RTX toxin gene in

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*Vibrio cholerae* by PCR. *J Clin Microbiol,* 39 (7), 2594-7.

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(2003). Class I integrons and SXT elements in El Tor strains isolated before and after 1992 *Vibrio cholerae* O139 outbreak, Calcutta, India. *Emerg Infect Dis,* 9 (4), 500-2. Ang, G. Y., Yu, C. Y., Balqis, K., Elina, H. T., Azura, H., Hani, M. H. & Yean, C. Y. (2010).

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K. (2006). Molecular analysis of the rstR and orfU genes of the CTX prophages integrated in the small chromosomes of environmental *Vibrio cholerae* non-O1, non-

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outbreak isolates in Mozambique and South Africa in 1998 are multiple-drug resistant, contain the SXT element and the aadA2 gene located on class 1 integrons.

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the SXT variant has also been found among altered El Tor strains from India (Goel & Jiang, 2010). It was recently reported that an altered El Tor strain carrying both an integron and an SXT element had been identified among outbreak strains from Solapur, India (Jain et al., 2011). These findings are important, because the management of cholera patients usually entails fluid replacement therapy to replace the electrolytes lost during profuse diarrheal bouts. However, antibiotic therapy serves as an adjunct to fluid replacement therapy to reduce the duration of the disease and the excretion of the bacterium (Lindenbaum et al., 1967). Thus, continuous monitoring of changes in antibiotic resistance patterns is highly recommended, because the SXT constin harbouring various antibiotic resistance genes can be acquired easily via lateral gene transfer (Iwanaga et al., 2004).

## **5. Conclusion**

Genetic analysis performed on the Kelantan altered El Tor strain isolated during a cholera outbreak in 2009 revealed a novel CTX prophage array where a tandem repeat of the RS1 element was found upstream of the CTX prophage on the large chromosome. This is the first report of a RS1-RS1-CTX prophage array among altered El Tor strains that fit into Group II according to the classification system of Lee et al. (2009). All isolates carried the SXT constin and we identified genes conferring resistance to sulfamethoxazole, trimethoprim and chloramphenicol, which correlated with their phenotypic expression in the antibiogram profile.

The emergence of the altered El Tor is viewed as an evolutionary optimization of *V. cholerae* strains in the development of a successor to the current cholera pandemic. The altered El Tor strains are poised to become epidemiologically dominant and they might hold the key to sustaining the current seventh pandemic. Equipped with the unique characteristics of the classical and El Tor biotypes, the altered El Tor has already been associated with more severe cases of cholera (Siddique et al., 2010) and it appears to be widely disseminated around the globe. The research community should actively unravel the wealth of knowledge that lies within these atypical El Tor strains and gain a better understanding that can be translated into measures to combat and conquer cholera.

### **6. Acknowledgements**

This study was funded by USM Research University Grant 1001/PPSP/813045, Postgraduate Research Grant Schemes (1001/PPSP/8144014 and 1001/PPSP/8144013) and eScienceFund 305/PPSP/6113214. The first and second authors gratefully acknowledge the financial support provided by USM through the Vice-Chancellor Award and USM Fellowship Scheme, respectively.

#### **7. References**

Alam, M., Nusrin, S., Islam, A., Bhuiyan, N. A., Rahim, N., Delgado, G., Morales, R., Mendez, J. L., Navarro, A., Gil, A. I., Watanabe, H., Morita, M., Nair, G. B. & Cravioto, A. (2010). Cholera between 1991 and 1997 in Mexico was associated with infection by classical, El Tor, and El Tor variants of *Vibrio cholerae*. *J Clin Microbiol*, 48 (10), 3666-74.

the SXT variant has also been found among altered El Tor strains from India (Goel & Jiang, 2010). It was recently reported that an altered El Tor strain carrying both an integron and an SXT element had been identified among outbreak strains from Solapur, India (Jain et al., 2011). These findings are important, because the management of cholera patients usually entails fluid replacement therapy to replace the electrolytes lost during profuse diarrheal bouts. However, antibiotic therapy serves as an adjunct to fluid replacement therapy to reduce the duration of the disease and the excretion of the bacterium (Lindenbaum et al., 1967). Thus, continuous monitoring of changes in antibiotic resistance patterns is highly recommended, because the SXT constin harbouring various antibiotic resistance genes can

Genetic analysis performed on the Kelantan altered El Tor strain isolated during a cholera outbreak in 2009 revealed a novel CTX prophage array where a tandem repeat of the RS1 element was found upstream of the CTX prophage on the large chromosome. This is the first report of a RS1-RS1-CTX prophage array among altered El Tor strains that fit into Group II according to the classification system of Lee et al. (2009). All isolates carried the SXT constin and we identified genes conferring resistance to sulfamethoxazole, trimethoprim and chloramphenicol, which correlated with their phenotypic expression in the antibiogram

The emergence of the altered El Tor is viewed as an evolutionary optimization of *V. cholerae* strains in the development of a successor to the current cholera pandemic. The altered El Tor strains are poised to become epidemiologically dominant and they might hold the key to sustaining the current seventh pandemic. Equipped with the unique characteristics of the classical and El Tor biotypes, the altered El Tor has already been associated with more severe cases of cholera (Siddique et al., 2010) and it appears to be widely disseminated around the globe. The research community should actively unravel the wealth of knowledge that lies within these atypical El Tor strains and gain a better understanding that can be

This study was funded by USM Research University Grant 1001/PPSP/813045, Postgraduate Research Grant Schemes (1001/PPSP/8144014 and 1001/PPSP/8144013) and eScienceFund 305/PPSP/6113214. The first and second authors gratefully acknowledge the financial support provided by USM through the Vice-Chancellor Award and USM

Alam, M., Nusrin, S., Islam, A., Bhuiyan, N. A., Rahim, N., Delgado, G., Morales, R.,

Mendez, J. L., Navarro, A., Gil, A. I., Watanabe, H., Morita, M., Nair, G. B. & Cravioto, A. (2010). Cholera between 1991 and 1997 in Mexico was associated with infection by classical, El Tor, and El Tor variants of *Vibrio cholerae*. *J Clin Microbiol*,

be acquired easily via lateral gene transfer (Iwanaga et al., 2004).

translated into measures to combat and conquer cholera.

**6. Acknowledgements** 

**7. References** 

Fellowship Scheme, respectively.

48 (10), 3666-74.

**5. Conclusion** 

profile.


Genetic Analysis of CTX Prophage and Antibiotic Resistance Determinants

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**6** 

*USA* 

**Integration of Global** 

*Vibrio Cholerae* **Behavior** 

Jorge A. Benitez1 and Anisia J. Silva2

 *Biochemistry & Immunology, Atlanta, Georgia* 

 **Regulatory Mechanisms Controlling** 

*2Morehouse School of Medicine Department of Microbiology,* 

*1Southern Research Institute Drug Discovery Division, Birmingham, Alabama* 

Cholera is an acute water-borne diarrheal disease caused by the facultative Gram-negative bacterium *Vibrio cholerae* of serogroup O1 of the classical and El Tor biotypes and serogroup O139. Characteristics of this bacterium are its comma-shaped morphology, expression of a fast-rotating polar flagellum, and production of cholera toxin (CT). The O1 *V. cholerae* serogroup contains a common A antigen and can be subdivided in Ogawa and Inaba serotypes on the basis of serotype-specific antigens B and C, respectively (Kaper et al., 1995). Mankind has experienced seven recorded cholera pandemics. The seventh and current pandemic is characterized by the predominance of the O1 serogroup El Tor biotype, with periodic emergence of O139 strains, which exhibit a new lipopolysaccharide (LPS) and a capsule (Albert, 1994). Cholera, which continues to be a major public health concern in endemic areas of South Asia and Africa, is estimated to cause 5.5 million cases of disease and 130,000 deaths per year. The disease, which commonly occurs as rapidly spreading and difficult to contain outbreaks in low-income countries, is a common sequel of natural and human disasters. The typical cholera symptoms include a profuse ricewatery diarrhea and vomiting. If untreated, this condition can lead to severe dehydration, electrolyte imbalances, and death. Cholera infections can be effectively treated with oral rehydration and, in cases of severe illness, with antibiotics. Antibiotic treatment lessens the duration of illness and reduces the excretion of highly infective *Vibrios* (Nelson et al., 2011). The downside however, is the emergence of multiple-antibiotic resistant O1 and O139 strains (Das & Kaur, 2008; Roychowdhury et al., 2008; Okeke et al., 2007; Mwansa et

As illustrated in Fig. 1, the cholera bacterium is fundamentally an organism adapted to the aquatic environment, which has evolved to maximize the benefit of being casually ingested by humans. The goal of this chapter is to examine the global regulatory mechanisms that assist the cholera bacterium in colonizing the small bowel of humans and persisting in the

**1. Introduction** 

al., 2007; Faruque et al., 2007).

aquatic environment.

Waldor, M. K., Tschape, H. & Mekalanos, J. J. (1996). A new type of conjugative transposon encodes resistance to sulfamethoxazole, trimethoprim, and streptomycin in *Vibrio cholerae* O139. *J Bacteriol,* 178 (14), 4157-65.

## **Integration of Global Regulatory Mechanisms Controlling**  *Vibrio Cholerae* **Behavior**

Jorge A. Benitez1 and Anisia J. Silva2

*1Southern Research Institute Drug Discovery Division, Birmingham, Alabama 2Morehouse School of Medicine Department of Microbiology, Biochemistry & Immunology, Atlanta, Georgia USA* 

#### **1. Introduction**

90 Cholera

Waldor, M. K., Tschape, H. & Mekalanos, J. J. (1996). A new type of conjugative transposon

*cholerae* O139. *J Bacteriol,* 178 (14), 4157-65.

encodes resistance to sulfamethoxazole, trimethoprim, and streptomycin in *Vibrio* 

Cholera is an acute water-borne diarrheal disease caused by the facultative Gram-negative bacterium *Vibrio cholerae* of serogroup O1 of the classical and El Tor biotypes and serogroup O139. Characteristics of this bacterium are its comma-shaped morphology, expression of a fast-rotating polar flagellum, and production of cholera toxin (CT). The O1 *V. cholerae* serogroup contains a common A antigen and can be subdivided in Ogawa and Inaba serotypes on the basis of serotype-specific antigens B and C, respectively (Kaper et al., 1995). Mankind has experienced seven recorded cholera pandemics. The seventh and current pandemic is characterized by the predominance of the O1 serogroup El Tor biotype, with periodic emergence of O139 strains, which exhibit a new lipopolysaccharide (LPS) and a capsule (Albert, 1994). Cholera, which continues to be a major public health concern in endemic areas of South Asia and Africa, is estimated to cause 5.5 million cases of disease and 130,000 deaths per year. The disease, which commonly occurs as rapidly spreading and difficult to contain outbreaks in low-income countries, is a common sequel of natural and human disasters. The typical cholera symptoms include a profuse ricewatery diarrhea and vomiting. If untreated, this condition can lead to severe dehydration, electrolyte imbalances, and death. Cholera infections can be effectively treated with oral rehydration and, in cases of severe illness, with antibiotics. Antibiotic treatment lessens the duration of illness and reduces the excretion of highly infective *Vibrios* (Nelson et al., 2011). The downside however, is the emergence of multiple-antibiotic resistant O1 and O139 strains (Das & Kaur, 2008; Roychowdhury et al., 2008; Okeke et al., 2007; Mwansa et al., 2007; Faruque et al., 2007).

As illustrated in Fig. 1, the cholera bacterium is fundamentally an organism adapted to the aquatic environment, which has evolved to maximize the benefit of being casually ingested by humans. The goal of this chapter is to examine the global regulatory mechanisms that assist the cholera bacterium in colonizing the small bowel of humans and persisting in the aquatic environment.

Integration of Global Regulatory Mechanisms Controlling *Vibrio Cholerae* Behavior 93

Fig. 2. Confocal microscopy three-dimensional image of a *V. cholerae* biofilm stained with the

attachment, followed by changes in gene expression patterns conducive to more permanent

**2.2 Transition of Vibrio cholerae between the aquatic environment and the human** 

A cholera infection starts with human ingestion of *V. cholerae* present in contaminated water or food (Fig. 1). Infecting *Vibrios* that survive passage through the acidic stomach compartment progress to the nutrient-rich environment of the human small intestine. It has been suggested that *V. cholerae* biofilm aggregates are more resistant to the initial low pH stress (Zhu & Mekalanos, 2003). Mutations in *vps* genes that block biofilm matrix exopolysaccharide biosynthesis impair colonization in the suckling mouse model (Fong et al., 2010). In addition, deletion of genes encoding the alternative stress-related sigma factors S and E inhibit bacterial colonization (Merrel et al., 2000; Kovacikova & Skorupski, 2002). *Vibrios* use their fastrotating polar flagellum to swim toward and bind to the mucus layer through their LPS (Benitez et al., 1997), the GbpA adhesin (Bhowmick et al., 2008; Jude et al., 2009), and other factors. Subsequent colonization requires expression of the toxin co-regulated pilus (TCP) (Herrington et al., 1988). Intestinal fluid secretion results from production by colonizing *Vibrios* of CT, which acts by increasing the cAMP content of host cells. Dissemination of the infection throughout the small bowel most likely involves detachment of *Vibrios* in a motile stage that could swim toward and adhere to other sites along the small intestine. *Vibrio* detachment and adherence could create new infective foci and enhance the severity of the disease. In the course of this process, however, *Vibrios* that detach but fail to adhere and establish new infection foci can be cleared from the small intestine by peristalsis (Walker & Owen, 1990) and excreted in the rice-watery diarrhea. As the overall population of *Vibrios* increases, and nutrients become in short supply, detachment predominates over re-colonization, a process also known as mucosal escape (Nielsen et al., 2006). Late in infection and, in preparation for their extraintestinal life, *Vibrios* associate into biofilm aggregates prior to exiting the host (Faruque et al., 2006). Such biofilms, formed *in vivo*, are in a stage of transient hyperinfectivity (Tamayo et al., 2010) that enhances their dissemination through the fecal-oral route (Merrel et al., 2002) (Fig. 1). This view of the time course of a cholera infection is consistent with the presence of highly

fluorescent dye, SYRO-9, and imaged using 485- and 498-nm excitation and emission wavelength, respectively. Biofilm developmental is initiated by a reversible surface

adherence, synthesis of exopolysaccharide matrix material, and building of three-

motile planktonic *Vibrios* and biofilm aggregates in freshly shed cholera stools.

dimensional columnar aggregates and channels.

**intestine** 

Fig. 1. The *V. cholerae* life cycle

## **2. The Vibrio cholerae dual life cycle**

### **2.1** *Vibrio cholerae* **persistence in the environment**

*V. cholerae* occur globally in most estuaries and coastal ecosystems, where their concentrations range from 101 to 104 cells per mL and can reach 106 cells per g of sediment (Urakawa & Rivera, 2006). In nature, *Vibrios* are subject to various physical and chemical environmental stresses, which include nutrient limitation, extreme temperatures, and oxidative stress. The persistence of *Vibrios* in the aquatic environment is additionally challenged by protozoan grazing and bacteriophage infection (Matz et al., 2005; Jensen et al., 2006; Faruque et al. 2005, 2005a). The bacterium can be found in the form of planktonic free-swimming cells or as sessile biofilm communities associated with phytoplankton and zooplankton (Watnick & Kolter, 1999; Kierek & Watnick, 2003; Huq et al., 1983; Islam et al., 1990; Kaper et al., 1979). The capacity of *V*. *cholerae* to form biofilm communities has been proposed to be involved in bacterial survival in the aquatic environment (Faruque et al., 2006: Joelsson et al., 2006: Matz et al., 2005: Schoolnik & Yildiz, 2000). Biofilm formation and adoption of a rugose colonial morphology correlate with the production of *V. cholerae* exopolysaccharide (*vps*) (Yildiz & Schoolnik, 1999). The *V. cholerae* rugose colonial variant described by White (1938) is more resistant to chlorinated water (Morris et al., 1996: Rice et al., 1992) and to osmotic and oxidative stresses (Wai et al., 1998: Yildiz & Schoolnik, 1999). In aquatic ecosystems, *V. cholerae* can also be found in the form of large biofilm aggregates of partially dormant cells that resist cultivation in conventional media but can be recovered as virulent *Vibrios* by animal passage (Faruque et al., 2006). These biofilm aggregates, named conditionally viable environmental cells (CVEC), appear to be similar to previously described viable but not culturable cells (Xu et al. 1982). The role of these biofilm aggregates in infection is considered below.

*V. cholerae* occur globally in most estuaries and coastal ecosystems, where their concentrations range from 101 to 104 cells per mL and can reach 106 cells per g of sediment (Urakawa & Rivera, 2006). In nature, *Vibrios* are subject to various physical and chemical environmental stresses, which include nutrient limitation, extreme temperatures, and oxidative stress. The persistence of *Vibrios* in the aquatic environment is additionally challenged by protozoan grazing and bacteriophage infection (Matz et al., 2005; Jensen et al., 2006; Faruque et al. 2005, 2005a). The bacterium can be found in the form of planktonic free-swimming cells or as sessile biofilm communities associated with phytoplankton and zooplankton (Watnick & Kolter, 1999; Kierek & Watnick, 2003; Huq et al., 1983; Islam et al., 1990; Kaper et al., 1979). The capacity of *V*. *cholerae* to form biofilm communities has been proposed to be involved in bacterial survival in the aquatic environment (Faruque et al., 2006: Joelsson et al., 2006: Matz et al., 2005: Schoolnik & Yildiz, 2000). Biofilm formation and adoption of a rugose colonial morphology correlate with the production of *V. cholerae* exopolysaccharide (*vps*) (Yildiz & Schoolnik, 1999). The *V. cholerae* rugose colonial variant described by White (1938) is more resistant to chlorinated water (Morris et al., 1996: Rice et al., 1992) and to osmotic and oxidative stresses (Wai et al., 1998: Yildiz & Schoolnik, 1999). In aquatic ecosystems, *V. cholerae* can also be found in the form of large biofilm aggregates of partially dormant cells that resist cultivation in conventional media but can be recovered as virulent *Vibrios* by animal passage (Faruque et al., 2006). These biofilm aggregates, named conditionally viable environmental cells (CVEC), appear to be similar to previously described viable but not culturable cells (Xu et

al. 1982). The role of these biofilm aggregates in infection is considered below.

Fig. 1. The *V. cholerae* life cycle

**2. The Vibrio cholerae dual life cycle** 

**2.1** *Vibrio cholerae* **persistence in the environment**

Fig. 2. Confocal microscopy three-dimensional image of a *V. cholerae* biofilm stained with the fluorescent dye, SYRO-9, and imaged using 485- and 498-nm excitation and emission wavelength, respectively. Biofilm developmental is initiated by a reversible surface attachment, followed by changes in gene expression patterns conducive to more permanent adherence, synthesis of exopolysaccharide matrix material, and building of threedimensional columnar aggregates and channels.

#### **2.2 Transition of Vibrio cholerae between the aquatic environment and the human intestine**

A cholera infection starts with human ingestion of *V. cholerae* present in contaminated water or food (Fig. 1). Infecting *Vibrios* that survive passage through the acidic stomach compartment progress to the nutrient-rich environment of the human small intestine. It has been suggested that *V. cholerae* biofilm aggregates are more resistant to the initial low pH stress (Zhu & Mekalanos, 2003). Mutations in *vps* genes that block biofilm matrix exopolysaccharide biosynthesis impair colonization in the suckling mouse model (Fong et al., 2010). In addition, deletion of genes encoding the alternative stress-related sigma factors S and E inhibit bacterial colonization (Merrel et al., 2000; Kovacikova & Skorupski, 2002). *Vibrios* use their fastrotating polar flagellum to swim toward and bind to the mucus layer through their LPS (Benitez et al., 1997), the GbpA adhesin (Bhowmick et al., 2008; Jude et al., 2009), and other factors. Subsequent colonization requires expression of the toxin co-regulated pilus (TCP) (Herrington et al., 1988). Intestinal fluid secretion results from production by colonizing *Vibrios* of CT, which acts by increasing the cAMP content of host cells. Dissemination of the infection throughout the small bowel most likely involves detachment of *Vibrios* in a motile stage that could swim toward and adhere to other sites along the small intestine. *Vibrio* detachment and adherence could create new infective foci and enhance the severity of the disease. In the course of this process, however, *Vibrios* that detach but fail to adhere and establish new infection foci can be cleared from the small intestine by peristalsis (Walker & Owen, 1990) and excreted in the rice-watery diarrhea. As the overall population of *Vibrios* increases, and nutrients become in short supply, detachment predominates over re-colonization, a process also known as mucosal escape (Nielsen et al., 2006). Late in infection and, in preparation for their extraintestinal life, *Vibrios* associate into biofilm aggregates prior to exiting the host (Faruque et al., 2006). Such biofilms, formed *in vivo*, are in a stage of transient hyperinfectivity (Tamayo et al., 2010) that enhances their dissemination through the fecal-oral route (Merrel et al., 2002) (Fig. 1). This view of the time course of a cholera infection is consistent with the presence of highly motile planktonic *Vibrios* and biofilm aggregates in freshly shed cholera stools.

Integration of Global Regulatory Mechanisms Controlling *Vibrio Cholerae* Behavior 95

host by interacting with Toll-like receptor V to induce the production of pro-inflammatory interleukin-8 (Harrison et al., 2008; Rui et al., 2010; Xicohtencalt-Cortes, et al. 2006). Flagellar motility also influences the expression of CT and TCP (Gardel & Mekalanos, 1996; Hase,

Motility is a complex phenotype that requires (a) the synthesis and export of the flagellum and its motor, (b) coupling of the flagellum motor to an energy source and (c) coupling of flagellum rotation to numerous chemosensory pathways. The *V. cholerae* genome encodes multiple flagellin genes (*fllaABCDE*), but only flagellin mutants lacking FlaA are nonflagellated (Klose & Mekalanos, 1998; Klose et al., 1998; Klose & Mekalanos, 1998a). The expression of motility requires a hierarchical regulatory cascade involving the alternative RNA polymerase (RNAP) subunits, 54 and 28 and the 54-dependent transcriptional activators, FlrA and FlrC (Correa et al., 2004; Correa & Klose, 2005; Correa et al., 2000;

The *V. cholerae* polar flagellum is powered by sodium motive force (Kojima et al., 1999). *V. cholerae* expresses Na+ pumps such as the Na+-translocating NADH: quinone oxidoreductase and multiple Na+/H+ antiporters responsible for maintaining the inward Na+ gradient that drives flagellum rotation (Hase et al., 2001). Genes required for flagellum rotation include *pomA* (*motA*), *pomB* (*motB*), *motX, motY, fliG, fliM and fliN*. Inactivation of *motA motB*, *motY* or *motX* by mutation abolish motility but does not prevent flagellum assembly (Boles & McCarter, 2000; Kim & McCarter, 2000; McCarter, 2001). MotA and MotB translocate Na+ by forming the Na+ conducting channel; MotX and MotY are required for torque generation (Asai et al., 1997). FliG, FliM and FliN, also required for torque generation, form the switch complex at the base of the flagellum basal body (Boles & McCarter, 2000; McCarter, 2001). The direction of flagellum rotation (clockwise or counterclockwise) is dictated by the interaction between the response regulator CheY3 and the FliM component of the motor

2001; Hase & Mekalanos, 1999: Hase et al., 2001; Silva et al., 2006; Syed et al., 2009).

Fig. 3. Regulation of CT and TCP expression

Correa et al., 2005; Prouty et al., 2001; Syed et al., 2009).

The timing of events that occur during infection is difficult to ascertain, since current models likely yield average data from *Vibrio* subpopulations at different stages of the infective process. A promising approach in this direction has been the development of a recombination-based *in vivo* expression technology (RIVET) (Camilli & Mekalanos, 1995; Lee et al., 1999). With this approach, it has been reported that *tcpA* and *ctxA* are expressed within the first 6 h of infection in the infant mouse intestine (Lee et al., 1999). Nevertheless, much remains to be learned about the events, occurring later in infection, that are involved in bacterial dissemination within the host and their exit to the environment.

## **3. Major virulence factors**

*V. cholerae* O1 and O139 strains, which cause epidemic cholera, exhibit three major characteristics: (a) production of CT, (b) expression of TCP, and (c) expression of a sheathed polar flagellum. *V. cholerae* produces additional potentially toxic factors, such hemagglutinin (HA)/protease (Hase & Finkelstein, 1991), hemolysin (Nagamune et al., 1996), the repeat toxin (RTX) (Lin et al., 1999), the zonula occludens toxin (Fasano et al., 1991), and the accessory cholera enterotoxin (Trucksis et al., 1993). The potential contributions of these secondary factors to the infective process has been reviewed by Fullner (2003). The regulatory pathways that control virulence, motility, and biofilm formation have been extensively studied and reviewed elsewhere (Childers & Klose, 2007; Matson et al., 2007). In this chapter, we discuss how these regulatory pathways are interconnected.

### **3.1 Cholera toxin and the toxin co-regulated pilus**

CT is an ADP-ribosyl transferase responsible for the profuse rice-watery diarrhea typical of this disease (Finkelstein, 1992; Kaper et al., 1995). It is composed of one A subunit (CTA) which catalyzes NAD-dependent ADP-ribosylation of host adenylate cyclase and five B subunits (CTB) that carry the ganglioside GM1 receptor binding site (Finkelstein, 1992). The genes encoding CTA (*ctxA*) and CTB (*ctxB*) are located in the genome of the filamentous phage CTX (Waldor & Mekalanos, 1996). The CTX receptor is the type IV pilus and colonization factor TCP (Waldor & Mekalanos, 1996). The expression of CT and TCP is coregulated by a complex regulatory network. At the top of the regulatory cascade, the regulator AphA enhances transcription of the transmembrane regulators TcpP and TcpH (Hase & Mekalanos, 1998; Kovacikova & Skorupski, 2001). AphA alone cannot activate transcription of *tcpPH*, but requires interaction with the LysR-type regulator, AphB which binds downstream of the AphA binding site to the *tcpPH* promoter (Kovacikova & Skorupski, 2001). TcpPH, in concert with the transmembrane regulators ToxR and ToxS (Miller & Mekalanos, 1985: Miller et al., 1989) (Fig. 3), activates expression of the soluble regulator, ToxT (DiRita et al., 1991). Finally, ToxT interacts with the *ctxA* and *tcpA*  promoters to activate production of CT and TCP (DiRita et al., 1991).

#### **3.2 Motility**

Motility is necessary for *V. cholerae* to establish infections, for colonization of the small intestine, to detach and spread along the small intestine, and/or to exit the host and return to the environment (Butler & Camilli, 2004; Lee et al., 2001; Nielsen et al., 2006; Silva et al., 2006). In addition, shedding of *V. cholerae* flagellins induce an inflammatory response in the host by interacting with Toll-like receptor V to induce the production of pro-inflammatory interleukin-8 (Harrison et al., 2008; Rui et al., 2010; Xicohtencalt-Cortes, et al. 2006). Flagellar motility also influences the expression of CT and TCP (Gardel & Mekalanos, 1996; Hase, 2001; Hase & Mekalanos, 1999: Hase et al., 2001; Silva et al., 2006; Syed et al., 2009).

Fig. 3. Regulation of CT and TCP expression

94 Cholera

The timing of events that occur during infection is difficult to ascertain, since current models likely yield average data from *Vibrio* subpopulations at different stages of the infective process. A promising approach in this direction has been the development of a recombination-based *in vivo* expression technology (RIVET) (Camilli & Mekalanos, 1995; Lee et al., 1999). With this approach, it has been reported that *tcpA* and *ctxA* are expressed within the first 6 h of infection in the infant mouse intestine (Lee et al., 1999). Nevertheless, much remains to be learned about the events, occurring later in infection, that are involved

*V. cholerae* O1 and O139 strains, which cause epidemic cholera, exhibit three major characteristics: (a) production of CT, (b) expression of TCP, and (c) expression of a sheathed polar flagellum. *V. cholerae* produces additional potentially toxic factors, such hemagglutinin (HA)/protease (Hase & Finkelstein, 1991), hemolysin (Nagamune et al., 1996), the repeat toxin (RTX) (Lin et al., 1999), the zonula occludens toxin (Fasano et al., 1991), and the accessory cholera enterotoxin (Trucksis et al., 1993). The potential contributions of these secondary factors to the infective process has been reviewed by Fullner (2003). The regulatory pathways that control virulence, motility, and biofilm formation have been extensively studied and reviewed elsewhere (Childers & Klose, 2007; Matson et al., 2007). In

CT is an ADP-ribosyl transferase responsible for the profuse rice-watery diarrhea typical of this disease (Finkelstein, 1992; Kaper et al., 1995). It is composed of one A subunit (CTA) which catalyzes NAD-dependent ADP-ribosylation of host adenylate cyclase and five B subunits (CTB) that carry the ganglioside GM1 receptor binding site (Finkelstein, 1992). The genes encoding CTA (*ctxA*) and CTB (*ctxB*) are located in the genome of the filamentous phage CTX (Waldor & Mekalanos, 1996). The CTX receptor is the type IV pilus and colonization factor TCP (Waldor & Mekalanos, 1996). The expression of CT and TCP is coregulated by a complex regulatory network. At the top of the regulatory cascade, the regulator AphA enhances transcription of the transmembrane regulators TcpP and TcpH (Hase & Mekalanos, 1998; Kovacikova & Skorupski, 2001). AphA alone cannot activate transcription of *tcpPH*, but requires interaction with the LysR-type regulator, AphB which binds downstream of the AphA binding site to the *tcpPH* promoter (Kovacikova & Skorupski, 2001). TcpPH, in concert with the transmembrane regulators ToxR and ToxS (Miller & Mekalanos, 1985: Miller et al., 1989) (Fig. 3), activates expression of the soluble regulator, ToxT (DiRita et al., 1991). Finally, ToxT interacts with the *ctxA* and *tcpA* 

Motility is necessary for *V. cholerae* to establish infections, for colonization of the small intestine, to detach and spread along the small intestine, and/or to exit the host and return to the environment (Butler & Camilli, 2004; Lee et al., 2001; Nielsen et al., 2006; Silva et al., 2006). In addition, shedding of *V. cholerae* flagellins induce an inflammatory response in the

in bacterial dissemination within the host and their exit to the environment.

this chapter, we discuss how these regulatory pathways are interconnected.

promoters to activate production of CT and TCP (DiRita et al., 1991).

**3.1 Cholera toxin and the toxin co-regulated pilus** 

**3. Major virulence factors** 

**3.2 Motility** 

Motility is a complex phenotype that requires (a) the synthesis and export of the flagellum and its motor, (b) coupling of the flagellum motor to an energy source and (c) coupling of flagellum rotation to numerous chemosensory pathways. The *V. cholerae* genome encodes multiple flagellin genes (*fllaABCDE*), but only flagellin mutants lacking FlaA are nonflagellated (Klose & Mekalanos, 1998; Klose et al., 1998; Klose & Mekalanos, 1998a). The expression of motility requires a hierarchical regulatory cascade involving the alternative RNA polymerase (RNAP) subunits, 54 and 28 and the 54-dependent transcriptional activators, FlrA and FlrC (Correa et al., 2004; Correa & Klose, 2005; Correa et al., 2000; Correa et al., 2005; Prouty et al., 2001; Syed et al., 2009).

The *V. cholerae* polar flagellum is powered by sodium motive force (Kojima et al., 1999). *V. cholerae* expresses Na+ pumps such as the Na+-translocating NADH: quinone oxidoreductase and multiple Na+/H+ antiporters responsible for maintaining the inward Na+ gradient that drives flagellum rotation (Hase et al., 2001). Genes required for flagellum rotation include *pomA* (*motA*), *pomB* (*motB*), *motX, motY, fliG, fliM and fliN*. Inactivation of *motA motB*, *motY* or *motX* by mutation abolish motility but does not prevent flagellum assembly (Boles & McCarter, 2000; Kim & McCarter, 2000; McCarter, 2001). MotA and MotB translocate Na+ by forming the Na+ conducting channel; MotX and MotY are required for torque generation (Asai et al., 1997). FliG, FliM and FliN, also required for torque generation, form the switch complex at the base of the flagellum basal body (Boles & McCarter, 2000; McCarter, 2001). The direction of flagellum rotation (clockwise or counterclockwise) is dictated by the interaction between the response regulator CheY3 and the FliM component of the motor

Integration of Global Regulatory Mechanisms Controlling *Vibrio Cholerae* Behavior 97

proposed role for HA/protease is to facilitate *V. cholerae* detachment from the intestinal mucosa when infecting *Vibrios* reach a high cell density (Finkelstein et al., 1992; Benitez et al., 1997; Silva et al., 2003; Silva et al., 2006; Robert et al., 1996). Consistently, inactivation of *hapA* encoding HA/protease enhances adherence to mucin-coated polystyrene plates (Silva et al., 2006), adherence to mucin-secreting differentiated HT29-18N2 cultured cells (Benitez et al., 1997), and colonization of the suckling mouse intestine (Robert et al., 1996; Silva et al., 2006). The mucinase activity of HA/protease (Finkelstein et al., 1983), together with its capacity to cleave the mucin-binding adhesin GbpA (Jude et al., 2009) at high cell density, has provided a mechanism supporting the "detachase" function attributed to this protein. The high viscosity of the mucus layer promotes breakage and loss of the polar flagellum (Liu et al., 2008). We have proposed that production of extracellular proteases facilitates preservation of the flagellum of *V. cholerae* during detachment by decreasing the viscosity of the medium (Silva et al., 2003). This could result from HA/protease degradation of preexisting mucin (Finkelstein et al., 1983) and cleavage of the GbpA adhesin, which

enhances the production of intestinal mucins (Bhowmick et al., 2008).

**4.1 Adenylate cyclase and cAMP signaling** 

**4.2 Cyclic diguanylate** 

**4. Global regulatory networks controlling** *Vibrio cholerae* **behavior** 

In a dynamic environment, the capacity of *V. cholerae* to switch between planktonic and sessile life styles or from virulence to detachment mode in response to environmental changes is essential. In the following sections, we discuss our current understanding of how *V. cholerae* integrates overlapping extracellular stimuli to adopt one or the other lifestyle.

Cyclic AMP (cAMP) is synthesized from ATP by the activity of adenylate cyclase. *V. cholerae* possesses only one adenylate cyclase, which belongs to the type-I (enterobacterial) class (Danchin, 1993: Baker et al., 2004). This enzyme is monomeric and consists of an N-terminal catalytic domain and a C-terminal regulatory domain. The C-terminal regulatory domain contains the His residue suggested to be phosphorylated by the phospho-EIIAglc component of the phosphoenolpyruvate phosphotransferase system (PTS), leading to its activation (Baker et al., 2004). The PTS is a phosphoryl cascade that allows the transport and phosphorylation of sugars (Deutscher et al., 2006; Deutscher, 2008). It acts as sensory system, feeding information to adenylate cyclase to regulate bacterial behavior in response to the availability of sugars in the medium and the energy state of the cell (Lengeler et al., 2009). In the PTS, phosphate is transferred from phosphoenolpyruvate to a sugar by a pathway that sequentially involves enzyme I (EI), the protein HPr, and a sugar-specific enzyme II (EII) complex. The different EII complexes are characterized by their domains (A, B, C) present either on a single or distinct polypeptide chains. In the presence of a rapidly metabolizable sugar (i.e., D-glucose) phospho-EIIAglc donates its phosphate to the sugar, leading to lower adenylate cyclase activity and lower intracellular concentrations of cAMP.

In a broad spectrum of bacterial species, the second messenger, cyclic diguanylic acid (c-di-GMP), regulates the transition between sessile and motile lifestyle by activating biofilm formation and inhibiting motility (D'Argenio & Miller, 2004; Hengge 2009; Simm et al., 2004;

(Berg, 2003; Boin et al., 2008; Hyakutake et al., 2005). The *V. cholerae* genome contains numerous chemotaxis-related genes, including multiple methyl-accepting chemotaxis proteins (MCP), methyltransferases (CheR), methylesterases (CheB), linker proteins (CheW), histidine kinases (CheA), and response regulators (CheY), mostly located in three clusters (Boin et al., 2008). Only a limited number of genes, however, have been demonstrated to be essential for chemotaxis. These genes include *cheA*-2 (Gosink et al., 2002), *cheR*-2 (Boin et al., 2004) and *cheY*-3 (Hyakutake et al., 2005). The function of other chemotaxis genes and the reason for their redundancy are not understood.


Table 1. Transcriptional organization of motility genes (adapted from Syed et al., 2009; Prouty et al., 2001).

We recently developed and validated a high-throughput screening assay for inhibitors of *V. cholerae* motility (Rasmussen et al., 2010). A new inhibitor consisting of a quinazoline 2,4 diamino analog (Q24DA) induced a flagellated, non-motile phenotype and was specific for the Na+-dependent polar flagellum motor of pathogenic *Vibrios* (Rasmussen et al., 2010). While some motility mutants express more CT and TCP (Silva et al., 2006, Syed et al. 2009), blocking motility with Q24DA diminished CT and TCP expression. Thus, the relationship between motility and CT expression could be more complex than anticipated by genetic studies. Identification of the molecular target of Q24DA and other inhibitors is required to clarify the disconnection between the genetic and chemical approaches.

#### **3.3 Hemagglutinin/protease**

Numerous *V. cholerae* strains of the El Tor biotype express a Zn-dependent metalloprotease (mucinase) known as hemagglutinin (HA)/protease (Finkelstein et al., 1983; Hase & Finkelstein, 1991). HA/protease enhances enterotoxicity in the rabbit ileal loop model of cholera (Ichinose et al., 1994; Silva et al., 2006) and contributes to live vaccine candidates' reactogenicity in humans (Benitez et al., 1999; Garcia et al., 2005). In cell culture, HA/protease perturbs the paracellular barrier of intestinal epithelial cells (Mel et al., 2000: Wu et al., 1996) by acting on tight junction-associated proteins (Wu et al., 2000). A second

(Berg, 2003; Boin et al., 2008; Hyakutake et al., 2005). The *V. cholerae* genome contains numerous chemotaxis-related genes, including multiple methyl-accepting chemotaxis proteins (MCP), methyltransferases (CheR), methylesterases (CheB), linker proteins (CheW), histidine kinases (CheA), and response regulators (CheY), mostly located in three clusters (Boin et al., 2008). Only a limited number of genes, however, have been demonstrated to be essential for chemotaxis. These genes include *cheA*-2 (Gosink et al., 2002), *cheR*-2 (Boin et al., 2004) and *cheY*-3 (Hyakutake et al., 2005). The function of other chemotaxis genes and the

**hierarchy class Class I Class II Class III Class IV** 

FlrA

*flrBC fliEFGHIJ flhA operon* 

*fliA*

Regulatory factors, MS ring-switch and

export components

Table 1. Transcriptional organization of motility genes (adapted from Syed et al., 2009;

clarify the disconnection between the genetic and chemical approaches.

We recently developed and validated a high-throughput screening assay for inhibitors of *V. cholerae* motility (Rasmussen et al., 2010). A new inhibitor consisting of a quinazoline 2,4 diamino analog (Q24DA) induced a flagellated, non-motile phenotype and was specific for the Na+-dependent polar flagellum motor of pathogenic *Vibrios* (Rasmussen et al., 2010). While some motility mutants express more CT and TCP (Silva et al., 2006, Syed et al. 2009), blocking motility with Q24DA diminished CT and TCP expression. Thus, the relationship between motility and CT expression could be more complex than anticipated by genetic studies. Identification of the molecular target of Q24DA and other inhibitors is required to

Numerous *V. cholerae* strains of the El Tor biotype express a Zn-dependent metalloprotease (mucinase) known as hemagglutinin (HA)/protease (Finkelstein et al., 1983; Hase & Finkelstein, 1991). HA/protease enhances enterotoxicity in the rabbit ileal loop model of cholera (Ichinose et al., 1994; Silva et al., 2006) and contributes to live vaccine candidates' reactogenicity in humans (Benitez et al., 1999; Garcia et al., 2005). In cell culture, HA/protease perturbs the paracellular barrier of intestinal epithelial cells (Mel et al., 2000: Wu et al., 1996) by acting on tight junction-associated proteins (Wu et al., 2000). A second

RpoN (54) and

Basal body-hook, major flagellin,

*flgBCDEFGHIJ fliKLMNOPQ* 

*motY flaA flhB flgKLOPT* 

motor component

FlrC FliA (28)

*motAB motX flaBCDE* 

Alternative flagellins, anti-sigma factor FlgM, motor components

reason for their redundancy are not understood.

**Genes** *flrA* 

**Upstream regulator** - RpoN (54) and

54-

dependent activator

**Transcription** 

**Function** 

Prouty et al., 2001).

**3.3 Hemagglutinin/protease** 

proposed role for HA/protease is to facilitate *V. cholerae* detachment from the intestinal mucosa when infecting *Vibrios* reach a high cell density (Finkelstein et al., 1992; Benitez et al., 1997; Silva et al., 2003; Silva et al., 2006; Robert et al., 1996). Consistently, inactivation of *hapA* encoding HA/protease enhances adherence to mucin-coated polystyrene plates (Silva et al., 2006), adherence to mucin-secreting differentiated HT29-18N2 cultured cells (Benitez et al., 1997), and colonization of the suckling mouse intestine (Robert et al., 1996; Silva et al., 2006). The mucinase activity of HA/protease (Finkelstein et al., 1983), together with its capacity to cleave the mucin-binding adhesin GbpA (Jude et al., 2009) at high cell density, has provided a mechanism supporting the "detachase" function attributed to this protein. The high viscosity of the mucus layer promotes breakage and loss of the polar flagellum (Liu et al., 2008). We have proposed that production of extracellular proteases facilitates preservation of the flagellum of *V. cholerae* during detachment by decreasing the viscosity of the medium (Silva et al., 2003). This could result from HA/protease degradation of preexisting mucin (Finkelstein et al., 1983) and cleavage of the GbpA adhesin, which enhances the production of intestinal mucins (Bhowmick et al., 2008).

## **4. Global regulatory networks controlling** *Vibrio cholerae* **behavior**

In a dynamic environment, the capacity of *V. cholerae* to switch between planktonic and sessile life styles or from virulence to detachment mode in response to environmental changes is essential. In the following sections, we discuss our current understanding of how *V. cholerae* integrates overlapping extracellular stimuli to adopt one or the other lifestyle.

#### **4.1 Adenylate cyclase and cAMP signaling**

Cyclic AMP (cAMP) is synthesized from ATP by the activity of adenylate cyclase. *V. cholerae* possesses only one adenylate cyclase, which belongs to the type-I (enterobacterial) class (Danchin, 1993: Baker et al., 2004). This enzyme is monomeric and consists of an N-terminal catalytic domain and a C-terminal regulatory domain. The C-terminal regulatory domain contains the His residue suggested to be phosphorylated by the phospho-EIIAglc component of the phosphoenolpyruvate phosphotransferase system (PTS), leading to its activation (Baker et al., 2004). The PTS is a phosphoryl cascade that allows the transport and phosphorylation of sugars (Deutscher et al., 2006; Deutscher, 2008). It acts as sensory system, feeding information to adenylate cyclase to regulate bacterial behavior in response to the availability of sugars in the medium and the energy state of the cell (Lengeler et al., 2009). In the PTS, phosphate is transferred from phosphoenolpyruvate to a sugar by a pathway that sequentially involves enzyme I (EI), the protein HPr, and a sugar-specific enzyme II (EII) complex. The different EII complexes are characterized by their domains (A, B, C) present either on a single or distinct polypeptide chains. In the presence of a rapidly metabolizable sugar (i.e., D-glucose) phospho-EIIAglc donates its phosphate to the sugar, leading to lower adenylate cyclase activity and lower intracellular concentrations of cAMP.

#### **4.2 Cyclic diguanylate**

In a broad spectrum of bacterial species, the second messenger, cyclic diguanylic acid (c-di-GMP), regulates the transition between sessile and motile lifestyle by activating biofilm formation and inhibiting motility (D'Argenio & Miller, 2004; Hengge 2009; Simm et al., 2004;

Integration of Global Regulatory Mechanisms Controlling *Vibrio Cholerae* Behavior 99

HapR binds to and represses its own promoter (Lin et al., 2005). Conversely, upon dilution of a high density culture, HapR activates the transcription of *qrr* sRNAs to promote rapid degradation of its own mRNA (Svenningsen et al., 2008). Further, at low cell density, the LuxR-type regulator, VqmA, enhances *hapR* transcription (Liu et al., 2006). In conclusion, at low cell density, *V. cholerae* expresses CT, TCP, and synthesizes matrix exopolysaccharide (*vps*); at high cell density, these functions are repressed, and production of HA/protease is

activated (Fig. 4).

Fig. 4. Quorum sensing in *Vibrio cholerae*

**4.4 Quorum sensing regulation of biofilm formation** 

The formation of a three-dimensional, mature biofilms involves a complex genetic program that includes the expression of motility and mannose-sensitive hemagglutinin for surface attachment and monolayer formation, as well as the biosynthesis of an exopolysaccharide matrix (Watnick & Kolter, 1999). Biofilm formation precedes adoption of the conditionally viable environmental cell stage described in section 1.3 (Kamruzzaman et al., 2010). In *V. cholerae*, biofilm formation is repressed by the master quorum sensing-regulator, HapR (Zhu & Mekalanos, 2003; Hammer & Bassler, 2003; Yildiz et al., 2004). The genes responsible for *vps* biosynthesis are clustered in two operons in which *vpsA* and *vpsL* are the first genes of operon I and II, respectively (Yildiz & Schoolnik, 1999). The expression of *vps* genes is regulated by a complex network involving a growing number of factors. For instance, the second messenger, c-di-GMP, enhances *vps* expression (Fong & Yildiz, 2008; Beyhan et al., 2006; Beyhan et al., 2007; Lim et al., 2006; Lim et al., 2007; Tischler & Camilli, 2004). Biofilm

Tamayo et al., 2007). Cyclic di-GMP is synthesized from GTP by GGDEF domain family proteins that exhibit diguanylate cyclase (DGC) activity. On the other hand, proteins of the EAL and HD-GYP families exhibit a phosphodiesterase (PDE) activity degrading c-di-GMP to GMP (Galperin, 2004). The *V. cholerae* genome contains 31 genes encoding GGDEF domain family proteins; 10 genes encoding proteins with GGDEF and EAL domains; 12 genes encoding proteins with only EAL domains; and 9 genes encoding proteins with HD-GYP domains (Galperin, 2004). In *V. cholerae*, over-expression of the DGC, VCA0956, abolishes swimming, whereas expression of the PDE, VieA, enhances it (Tischler & Camilli, 2004). Transcriptional profiling has revealed that genes involved in flagellum biosynthesis, motility, and chemotaxis are repressed in response to an increase in intracellular c-di-GMP (Beyhan et al., 2006). The signaling pathways responsible for the phenotypic consequences of increasing the c-di-GMP pool are not fully understood. Potential c-di-GMP binding proteins include those containing the PilZ domain (Pratt et al., 2007). The *V. cholerae* genome contains five PilZ domain proteins (Pratt et al., 2007). Of these, PlzA and PlzE appear to be essential; PlzB, PlzC, and PlzD affect *V. cholerae* motility; and PlzC and PlzD bind to c-di-GMP *in vitro*. The positive regulator of biofilm formation, VpsT, can directly senses c-di-GMP to modulate motility and biofilm formation (Krasteva et al., 2010). Finally, there are two riboswitches responsive to c-di-GMP changes in the *V. cholerae* genome (Sudarsan et al., 2008). The function of these riboswitches in cholera infections is currently unknown.

#### **4.3 Quorum sensing**

Quorum sensing is a process by which bacteria communicate with one another by secreting extracellular signaling molecules termed autoinducers. In *V. cholerae*, two autoinducer/sensor systems have been identified. System 1 consists of cholera autoinducer 1 (CAI-1, 3-hydroxytridecane-4-one), synthesized by the activity of CqsA, and its cognate receptor, CqsS (Higgins et al., 2006: Miller et al., 2002). System 2 consists of an AI-2 molecule (a furanosyl borate diester), synthesized by the activity of LuxS, and its cognate receptor, LuxPQ (Chen et al., 2002; Miller et al., 2002). Sensory information is fed through a phosphorelay system to LuxO. At low cell density, the autokinase domains of CqsS and LuxPQ become phosphorylated, and phosphate is transferred to LuxU and then LuxO (Miller et al., 2002). Phospho-LuxO then activates expression of multiple, redundant small regulatory RNAs (sRNAs or *qrr*), which promotes translation of the mRNA encoding AphA and destabilize the *hapR* mRNA (Lenz et al., 2004; Rutherford et al., 2011). In addition, the global regulator, CsrA, and the small nucleoid protein factor for inversion stimulation (Fis) enhance phospho-LuxO activity to promote degradation of *hapR* mRNA at low cell density (Lenz et al., 2005; Lenz et al., 2007). When the amount of CAI-1 and AI-2 produced by growing bacteria reaches a threshold value, CqsS and LuxPQ switch from kinase activity to phosphatase. The flow of phosphate is reversed, and phospho-LuxO becomes dephosphorylated and inactive (Miller et al., 2002). At this stage (high cell density), HapR is expressed (Zhu et al., 2002). The consequences of HapR expression include (a) diminished expression of CT and TCP due to transcriptional repression of *aphA* (Kovacikova and Skorupski, 2002a; Lin et al., 2007), (b) inhibition of *vps* expression (Waters et al., 2008), and (c) activation of *hapA* encoding HA/protease (Jobling & Holmes, 1997). The transition into or out from the quorum-sensing mode appears to be finely regulated by additional mechanisms. For instance, upon transiting into quorum-sensing mode (high cell density),

Tamayo et al., 2007). Cyclic di-GMP is synthesized from GTP by GGDEF domain family proteins that exhibit diguanylate cyclase (DGC) activity. On the other hand, proteins of the EAL and HD-GYP families exhibit a phosphodiesterase (PDE) activity degrading c-di-GMP to GMP (Galperin, 2004). The *V. cholerae* genome contains 31 genes encoding GGDEF domain family proteins; 10 genes encoding proteins with GGDEF and EAL domains; 12 genes encoding proteins with only EAL domains; and 9 genes encoding proteins with HD-GYP domains (Galperin, 2004). In *V. cholerae*, over-expression of the DGC, VCA0956, abolishes swimming, whereas expression of the PDE, VieA, enhances it (Tischler & Camilli, 2004). Transcriptional profiling has revealed that genes involved in flagellum biosynthesis, motility, and chemotaxis are repressed in response to an increase in intracellular c-di-GMP (Beyhan et al., 2006). The signaling pathways responsible for the phenotypic consequences of increasing the c-di-GMP pool are not fully understood. Potential c-di-GMP binding proteins include those containing the PilZ domain (Pratt et al., 2007). The *V. cholerae* genome contains five PilZ domain proteins (Pratt et al., 2007). Of these, PlzA and PlzE appear to be essential; PlzB, PlzC, and PlzD affect *V. cholerae* motility; and PlzC and PlzD bind to c-di-GMP *in vitro*. The positive regulator of biofilm formation, VpsT, can directly senses c-di-GMP to modulate motility and biofilm formation (Krasteva et al., 2010). Finally, there are two riboswitches responsive to c-di-GMP changes in the *V. cholerae* genome (Sudarsan et al.,

2008). The function of these riboswitches in cholera infections is currently unknown.

Quorum sensing is a process by which bacteria communicate with one another by secreting extracellular signaling molecules termed autoinducers. In *V. cholerae*, two autoinducer/sensor systems have been identified. System 1 consists of cholera autoinducer 1 (CAI-1, 3-hydroxytridecane-4-one), synthesized by the activity of CqsA, and its cognate receptor, CqsS (Higgins et al., 2006: Miller et al., 2002). System 2 consists of an AI-2 molecule (a furanosyl borate diester), synthesized by the activity of LuxS, and its cognate receptor, LuxPQ (Chen et al., 2002; Miller et al., 2002). Sensory information is fed through a phosphorelay system to LuxO. At low cell density, the autokinase domains of CqsS and LuxPQ become phosphorylated, and phosphate is transferred to LuxU and then LuxO (Miller et al., 2002). Phospho-LuxO then activates expression of multiple, redundant small regulatory RNAs (sRNAs or *qrr*), which promotes translation of the mRNA encoding AphA and destabilize the *hapR* mRNA (Lenz et al., 2004; Rutherford et al., 2011). In addition, the global regulator, CsrA, and the small nucleoid protein factor for inversion stimulation (Fis) enhance phospho-LuxO activity to promote degradation of *hapR* mRNA at low cell density (Lenz et al., 2005; Lenz et al., 2007). When the amount of CAI-1 and AI-2 produced by growing bacteria reaches a threshold value, CqsS and LuxPQ switch from kinase activity to phosphatase. The flow of phosphate is reversed, and phospho-LuxO becomes dephosphorylated and inactive (Miller et al., 2002). At this stage (high cell density), HapR is expressed (Zhu et al., 2002). The consequences of HapR expression include (a) diminished expression of CT and TCP due to transcriptional repression of *aphA* (Kovacikova and Skorupski, 2002a; Lin et al., 2007), (b) inhibition of *vps* expression (Waters et al., 2008), and (c) activation of *hapA* encoding HA/protease (Jobling & Holmes, 1997). The transition into or out from the quorum-sensing mode appears to be finely regulated by additional mechanisms. For instance, upon transiting into quorum-sensing mode (high cell density),

**4.3 Quorum sensing** 

HapR binds to and represses its own promoter (Lin et al., 2005). Conversely, upon dilution of a high density culture, HapR activates the transcription of *qrr* sRNAs to promote rapid degradation of its own mRNA (Svenningsen et al., 2008). Further, at low cell density, the LuxR-type regulator, VqmA, enhances *hapR* transcription (Liu et al., 2006). In conclusion, at low cell density, *V. cholerae* expresses CT, TCP, and synthesizes matrix exopolysaccharide (*vps*); at high cell density, these functions are repressed, and production of HA/protease is activated (Fig. 4).

Fig. 4. Quorum sensing in *Vibrio cholerae*

## **4.4 Quorum sensing regulation of biofilm formation**

The formation of a three-dimensional, mature biofilms involves a complex genetic program that includes the expression of motility and mannose-sensitive hemagglutinin for surface attachment and monolayer formation, as well as the biosynthesis of an exopolysaccharide matrix (Watnick & Kolter, 1999). Biofilm formation precedes adoption of the conditionally viable environmental cell stage described in section 1.3 (Kamruzzaman et al., 2010). In *V. cholerae*, biofilm formation is repressed by the master quorum sensing-regulator, HapR (Zhu & Mekalanos, 2003; Hammer & Bassler, 2003; Yildiz et al., 2004). The genes responsible for *vps* biosynthesis are clustered in two operons in which *vpsA* and *vpsL* are the first genes of operon I and II, respectively (Yildiz & Schoolnik, 1999). The expression of *vps* genes is regulated by a complex network involving a growing number of factors. For instance, the second messenger, c-di-GMP, enhances *vps* expression (Fong & Yildiz, 2008; Beyhan et al., 2006; Beyhan et al., 2007; Lim et al., 2006; Lim et al., 2007; Tischler & Camilli, 2004). Biofilm

Integration of Global Regulatory Mechanisms Controlling *Vibrio Cholerae* Behavior 101

Fig. 5. Relative expression measured by quantitative, real-time reverse transcription PCR of global regulators *fis*, *rpoS,* and *hns* in a *V. cholerae crp* deletion mutant standardized by *recA*

Fig. 6. Global regulation by the cAMP receptor protein.

mRNA levels.

formation is also modulated by interplay between the positive transcription regulators, VpsT (Casper-Lindley & Yildiz, 2004) and VpsR (Yildiz et al., 2001), and the negative regulator, CytR (Haugo & Watnick, 2002). In addition, *vps* expression is modulated by the PhoBR two-component regulatory system (Pratts et al., 2009, 2010; Sultan et al., 2010) and by components of the PTS phosphoryl cascade (Houot et al., 2008; 2010, 2010a).

#### **4.5 The cAMP receptor protein (CRP)**

CRP is a member of the CRP/FNR family of transcriptional regulators known for its role in carbon catabolite repression, a process in which the presence of a favorable carbon source in the medium inhibits expression of enzymes involved in the catabolism of other carbon sources (Brückner & Titgemeyer, 2002; Stülke and Hillen, 1999). Activation of adenylate cyclase leads to high intracellular levels of cAMP. Then, cAMP binds to CRP to form a complex that acts at responsive promoters to activate or repress transcription (Brückner & Titgemeyer, 2002; Stülke & Hillen, 1999) The cAMP-CRP complex binds as a dimer to the consensus sequence TGTGA-(N6)-TCACAA which can be found within, adjacent to or upstream from responsive promoters. The complex is believed to assist in binding of RNAP to the promoter by bending the DNA molecule. *V. cholerae crp* mutants form small colonies, are less motile, do not express *hapA* (Benitez et al., 2001) and are defective in colonization of the suckling mouse intestine (Skorupski and Taylor, 1997). The cAMP-CRP complex negatively affects CT and TCP expression by directly repressing the *tcpPH* promoter (Skorupski & Taylor, 1997; Kovacikova & Skorupski, 2001). The fact that *crp* mutants show reduced colonization in the suckling mouse, although expressing elevated TCP, suggests that CRP is required for the expression of additional colonization factors.

As a global regulator, CRP indirectly affects the expression of many genes by controlling the expression of a broad range of transcriptional factors. As an example, an isogenic *crp* mutant of *V. cholerae* strain C7258 expressed elevated *fis* mRNA and lower levels of mRNAs encoding the general stress response regulator, RpoS, and the histone-like nucleoid structuring protein (H-NS) (Fig. 5) (Silva and Benitez, 2004; Liang et al., 2007). Gene expression profiling of a *crp* deletion mutant revealed 174 differentially expressed genes. With the exception of conserved hypothetical proteins, most differentially expressed genes fell into the functional categories of energy metabolism, transport and binding protein, and cellular processes (Fig. 6) (Liang et al., 2007). Furthermore, 77 % of the differentially expressed genes were down-regulated, suggesting that CRP most frequently acts as a positive regulator in *V. cholerae*. The *crp* mutant exhibited diminished expression of genes involved in motility and chemotaxis, outer membrane protein expression, genes specifically induced in rabbit ileal loops, and *rpoE* encoding E (Liang et al., 2007). These data explain the colonization defect exhibited by *crp* mutants. Among the differentially expressed genes, *cqsA* (VCA0523) encoding CAI-1 synthase and *hapR*, encoding the master quorum sensing regulator, HapR, were diminished. Another gene (VC0291), annotated as coding for a NifR3/Smm1 family protein was up-regulated in the *crp* mutant. Tn5 insertions in this locus reduced the expression of the small nucleoid protein, Fis (Lenz & Bassler, 2007), a regulator that enhances degradation of *hapR* mRNA at low cell density (Lenz & Bassler, 2007). Since VC0291 and *fis* are predicted to be part of an operon (Osuna et al., 1995), this finding is consistent with CRP being a repressor of Fis (Fig. 5).

formation is also modulated by interplay between the positive transcription regulators, VpsT (Casper-Lindley & Yildiz, 2004) and VpsR (Yildiz et al., 2001), and the negative regulator, CytR (Haugo & Watnick, 2002). In addition, *vps* expression is modulated by the PhoBR two-component regulatory system (Pratts et al., 2009, 2010; Sultan et al., 2010) and by

CRP is a member of the CRP/FNR family of transcriptional regulators known for its role in carbon catabolite repression, a process in which the presence of a favorable carbon source in the medium inhibits expression of enzymes involved in the catabolism of other carbon sources (Brückner & Titgemeyer, 2002; Stülke and Hillen, 1999). Activation of adenylate cyclase leads to high intracellular levels of cAMP. Then, cAMP binds to CRP to form a complex that acts at responsive promoters to activate or repress transcription (Brückner & Titgemeyer, 2002; Stülke & Hillen, 1999) The cAMP-CRP complex binds as a dimer to the consensus sequence TGTGA-(N6)-TCACAA which can be found within, adjacent to or upstream from responsive promoters. The complex is believed to assist in binding of RNAP to the promoter by bending the DNA molecule. *V. cholerae crp* mutants form small colonies, are less motile, do not express *hapA* (Benitez et al., 2001) and are defective in colonization of the suckling mouse intestine (Skorupski and Taylor, 1997). The cAMP-CRP complex negatively affects CT and TCP expression by directly repressing the *tcpPH* promoter (Skorupski & Taylor, 1997; Kovacikova & Skorupski, 2001). The fact that *crp* mutants show reduced colonization in the suckling mouse, although expressing elevated TCP, suggests

As a global regulator, CRP indirectly affects the expression of many genes by controlling the expression of a broad range of transcriptional factors. As an example, an isogenic *crp* mutant of *V. cholerae* strain C7258 expressed elevated *fis* mRNA and lower levels of mRNAs encoding the general stress response regulator, RpoS, and the histone-like nucleoid structuring protein (H-NS) (Fig. 5) (Silva and Benitez, 2004; Liang et al., 2007). Gene expression profiling of a *crp* deletion mutant revealed 174 differentially expressed genes. With the exception of conserved hypothetical proteins, most differentially expressed genes fell into the functional categories of energy metabolism, transport and binding protein, and cellular processes (Fig. 6) (Liang et al., 2007). Furthermore, 77 % of the differentially expressed genes were down-regulated, suggesting that CRP most frequently acts as a positive regulator in *V. cholerae*. The *crp* mutant exhibited diminished expression of genes involved in motility and chemotaxis, outer membrane protein expression, genes specifically induced in rabbit ileal loops, and *rpoE* encoding E (Liang et al., 2007). These data explain the colonization defect exhibited by *crp* mutants. Among the differentially expressed genes, *cqsA* (VCA0523) encoding CAI-1 synthase and *hapR*, encoding the master quorum sensing regulator, HapR, were diminished. Another gene (VC0291), annotated as coding for a NifR3/Smm1 family protein was up-regulated in the *crp* mutant. Tn5 insertions in this locus reduced the expression of the small nucleoid protein, Fis (Lenz & Bassler, 2007), a regulator that enhances degradation of *hapR* mRNA at low cell density (Lenz & Bassler, 2007). Since VC0291 and *fis* are predicted to be part of an operon (Osuna et al., 1995), this finding is

components of the PTS phosphoryl cascade (Houot et al., 2008; 2010, 2010a).

that CRP is required for the expression of additional colonization factors.

consistent with CRP being a repressor of Fis (Fig. 5).

**4.5 The cAMP receptor protein (CRP)** 

Fig. 5. Relative expression measured by quantitative, real-time reverse transcription PCR of global regulators *fis*, *rpoS,* and *hns* in a *V. cholerae crp* deletion mutant standardized by *recA* mRNA levels.

Fig. 6. Global regulation by the cAMP receptor protein.

Integration of Global Regulatory Mechanisms Controlling *Vibrio Cholerae* Behavior 103

*cholerae* switches its metabolism to the quorum-sensing mode. As a consequence, commitment of the bacterial population to enter the quorum sensing-mode and turn on the HapR transcriptional program is placed in context with other features of the environment. This principle is illustrated in Fig. 8. Consistent with this scheme, the cell density at which *V. cholerae* enters the quorum-sensing mode is increased by addition of glucose to the medium (to lower the cAMP pool) and diminished by addition of cAMP (Liang et al., 2008).

Fig. 8. Quorum modulation. When a culture at high cell density is diluted in broth, quorum sensing (i.e., light) is turned off. As the population increases, a threshold cell density is attained (quorum), at which quorum sensing is activated to generate a U-shape curve (b). The rate of autoinducer biosynthesis determines the cell density at which the bacterial population enters the quorum-sensing mode. A condition that enhances autoinducer biosynthesis lowers the threshold (a). A condition that inhibits autoinducer biosynthesis

Fig. 9. Effect of a nutritional downshift on the cell density required for expression of a quorum sensing-regulated *hapA-lacZ* promoter fusion measured as -galactosidase activity

increases the threshold (c).

(Miller units) (Silva and Benitez, 2004).

#### **4.6 Quorum modulation: Integration of cell density and carbon source sensory information**

Since the *crp* mutant expresses reduced *cqsA* and *hapR* mRNA, we used a bioassay to compare the production of CAI-1 in wild-type and mutant backgrounds using a *V. cholerae cqsAluxP* reporter containing the *V. harveyi lux* operon on a cosmid (Miller et al., 2002). This reporter strain does not make its own CAI-1 nor does it respond to AI-2. Exogenous CAI-1 from a cellfree culture supernatant activates expression of HapR, which in turn induces the *lux* operon to make light. As shown in Fig. 7, no CAI-1 can be detected in culture supernatants of *crp* and *cya* (adenylate cyclase). The *crpc* allele containing the amino acid substitutions T127L/S128A encodes a CRP protein that activates transcription in the absence of cAMP (Krueger et al., 1998; Shi et al., 1999; Wang et al., 2000). As shown in Fig. 7, introduction of this constitutive allele into a *cya* mutant restored expression of HapR and light production. Furthermore, quorum sensing was restored in *crp* and *cya* mutants by introducing the corresponding genes on a plasmid vector and, in the case of a *cya* mutant, by adding cAMP or the cAMP analog, 7 deaza-cAMP, to the culture medium (Liang et al., 2008). The mechanism by which the cAMP-CRP complex regulates *cqsA* expression is not known, although there is evidence suggesting a posttranscriptional regulation (Liang et al., 2008).

Fig. 7. The cAMP receptor protein is required for the biosynthesis of the *Vibrio cholerae* major autoinducer.

In section 4.5., we showed that cAMP-CRP controls quorum sensing by activating *cqsA* (Fig. 7) and repressing *fis* (Fig. 5). These findings suggest that the intensity of bacterial cell-to-cell communication is modulated by environmental signals other than population density, such as the type and availability of carbon sources. Thus, we propose a new level of regulation, termed quorum modulation, mediated in this case by cAMP. Quorum modulation functions in the following way. Under environmental conditions conducive to low intracellular cAMP levels (i.e., high glucose), the amount of CAI-1 produced per cell is diminished. The *V. cholerae* population would require a higher quorum (i.e., cells/ml) to activate HapR. Conversely, under conditions conducive to high cAMP levels (i.e., low glucose) the production of CAI-1 per cell is enhanced, and the bacterial population requires a lower quorum to activate HapR. Thus, quorum modulation controls the cell density at which *V.* 

Since the *crp* mutant expresses reduced *cqsA* and *hapR* mRNA, we used a bioassay to compare the production of CAI-1 in wild-type and mutant backgrounds using a *V. cholerae cqsAluxP* reporter containing the *V. harveyi lux* operon on a cosmid (Miller et al., 2002). This reporter strain does not make its own CAI-1 nor does it respond to AI-2. Exogenous CAI-1 from a cellfree culture supernatant activates expression of HapR, which in turn induces the *lux* operon to make light. As shown in Fig. 7, no CAI-1 can be detected in culture supernatants of *crp* and *cya* (adenylate cyclase). The *crpc* allele containing the amino acid substitutions T127L/S128A encodes a CRP protein that activates transcription in the absence of cAMP (Krueger et al., 1998; Shi et al., 1999; Wang et al., 2000). As shown in Fig. 7, introduction of this constitutive allele into a *cya* mutant restored expression of HapR and light production. Furthermore, quorum sensing was restored in *crp* and *cya* mutants by introducing the corresponding genes on a plasmid vector and, in the case of a *cya* mutant, by adding cAMP or the cAMP analog, 7 deaza-cAMP, to the culture medium (Liang et al., 2008). The mechanism by which the cAMP-CRP complex regulates *cqsA* expression is not known, although there is evidence suggesting a

Fig. 7. The cAMP receptor protein is required for the biosynthesis of the *Vibrio cholerae* major

In section 4.5., we showed that cAMP-CRP controls quorum sensing by activating *cqsA* (Fig. 7) and repressing *fis* (Fig. 5). These findings suggest that the intensity of bacterial cell-to-cell communication is modulated by environmental signals other than population density, such as the type and availability of carbon sources. Thus, we propose a new level of regulation, termed quorum modulation, mediated in this case by cAMP. Quorum modulation functions in the following way. Under environmental conditions conducive to low intracellular cAMP levels (i.e., high glucose), the amount of CAI-1 produced per cell is diminished. The *V. cholerae* population would require a higher quorum (i.e., cells/ml) to activate HapR. Conversely, under conditions conducive to high cAMP levels (i.e., low glucose) the production of CAI-1 per cell is enhanced, and the bacterial population requires a lower quorum to activate HapR. Thus, quorum modulation controls the cell density at which *V.* 

**4.6 Quorum modulation: Integration of cell density and carbon source sensory** 

**information** 

autoinducer.

posttranscriptional regulation (Liang et al., 2008).

*cholerae* switches its metabolism to the quorum-sensing mode. As a consequence, commitment of the bacterial population to enter the quorum sensing-mode and turn on the HapR transcriptional program is placed in context with other features of the environment. This principle is illustrated in Fig. 8. Consistent with this scheme, the cell density at which *V. cholerae* enters the quorum-sensing mode is increased by addition of glucose to the medium (to lower the cAMP pool) and diminished by addition of cAMP (Liang et al., 2008).

Fig. 8. Quorum modulation. When a culture at high cell density is diluted in broth, quorum sensing (i.e., light) is turned off. As the population increases, a threshold cell density is attained (quorum), at which quorum sensing is activated to generate a U-shape curve (b). The rate of autoinducer biosynthesis determines the cell density at which the bacterial population enters the quorum-sensing mode. A condition that enhances autoinducer biosynthesis lowers the threshold (a). A condition that inhibits autoinducer biosynthesis increases the threshold (c).

Fig. 9. Effect of a nutritional downshift on the cell density required for expression of a quorum sensing-regulated *hapA-lacZ* promoter fusion measured as -galactosidase activity (Miller units) (Silva and Benitez, 2004).

Integration of Global Regulatory Mechanisms Controlling *Vibrio Cholerae* Behavior 105

regulator HapR. As a result, ∆*crp* mutants express elevated *vpsA* and *vpsL* compared to the wild-type strain, but this increase is not large enough to induce rugose colonial morphology. Maximal exopolysaccharide expression requires inactivation of HapR (repressor) but an active *crp* allele for enhancing VpsR (activator). In a different strain, however, CRP repressed VpsR, suggesting that there is strain variability in the regulation of this protein

In addition to the abovementioned regulatory effects of *crp* and *cya* on *vps* expression, several components of the PTS that function upstream of adenylate cyclase have their own regulatory input on *vps* gene expression. For instance, in minimal medium, phosphoryl transfer from EI to HPr and FPr represses *vps* expression (Houot et al., 2008); in LB medium, glucose-specific EIIAglc and nitrogen-specific EIIANtr1 and EIIANtr2 activate and repress *vps*

Fig. 11. Model for the dual regulatory input of the cAMP-CRP complex on biosynthesis of *V.* 

Since freshwater and estuarine ecosystems where *Vibrios* survive and persist outside the human host are limited in phosphate content (Benitez-Nelson, 2000; Correl, 1999), *V. cholerae* build stores of intracellular polyphosphate (poly-P) (Ogawa et al., 2000). The enzyme polyphosphate kinase (PPK) is responsible for the synthesis of poly-P from ATP (Ahn and Kornberg, 1990). A *V. cholerae ppk* mutant exhibits a reduced capacity to withstand conditions of low pH, high salinity, and oxidative stress in low- phosphate medium (Jahid et al., 2006). These findings underline the importance of phosphate homeostasis as well as sensing and responding to changes in extracellular phosphate in the *V. cholerae* life cycle. In *E. coli*, deprivation of phosphate induces the expression of the PhoB regulon (Lamarche et

**4.8 Effect of extracellular phosphate on vps expression and biofilm formation** 

(Fong et al., 2008).

expression, respectively (Houot et al., 2010).

*cholerae* matrix exopolysaccharide.

A practical example of how quorum modulation works is provided in Fig. 9. In this case, a *V. cholerae* strain containing a chromosomally integrated *hapA*-*lacZ* promoter fusion is grown in rich medium, and, at an optical density of 0.5, half of the culture is nutritionally downshifted by centrifugation and reconstitution in a medium of diminished strength. This experiment shows that *hapA* expression can be detected at a lower cell density in nutritionally downshifted cells (Silva and Benitez, 2004). Quorum modulation is not restricted to carbon regulation of cellular cAMP levels, as other environmental conditions might influence autoinducer biosynthesis or even autoinducer stability in the medium.

#### **4.7 Interplay between quorum sensing and cAMP in the fine regulation of matrix exopolysaccharide expression**

Over-expression of *vps* in *hapR* mutants gives rise to the rugose colonial morphology (Yildiz et al., 2004). Although HapR is not detected in *crp* and *cya* mutants, these strains still produce the smooth colonial variant (Liang et al., 2007, 2007a). As shown in Fig. 10, the rugose colonial morphology of a *hapR* mutant is turned to smooth by deletion of *crp* or *cya,* and the resulting smooth strains can be converted back to rugose by introduction of the *crp* and *cya* genes on a plasmid vector. These results suggest that formation of the cAMP-CRP complex has a dual effect on *vps* expression by activating quorum sensing (a negative effect) and by enhancing the expression of a positive factor. In our strain, the positive factor was the regulator, VpsR (Liang et al., 2007a).

Fig. 10. Scanning electron microscopy of *V. cholerae crp*, *cya*, and *hapR* colonies. A, *crp*; B, *hapR*; C, *hapRcrp*; D, *hapRcrp* transformed with *crp* plasmid; E, *hapRcya*; F, *hapRcya* transformed with *cya* plasmid

Fig. 11 schematically illustrates the dual input of adenylate cyclase and CRP on *V. cholerae* expression of *vps.* In this model, deletion of *crp* results in diminished expression of the positive regulator, VpsR, which is required for expression of rugose colonial morphology. This event, however, is partially compensated for by reduced expression of the negative

A practical example of how quorum modulation works is provided in Fig. 9. In this case, a *V. cholerae* strain containing a chromosomally integrated *hapA*-*lacZ* promoter fusion is grown in rich medium, and, at an optical density of 0.5, half of the culture is nutritionally downshifted by centrifugation and reconstitution in a medium of diminished strength. This experiment shows that *hapA* expression can be detected at a lower cell density in nutritionally downshifted cells (Silva and Benitez, 2004). Quorum modulation is not restricted to carbon regulation of cellular cAMP levels, as other environmental conditions might influence autoinducer biosynthesis or even autoinducer stability in the medium.

**4.7 Interplay between quorum sensing and cAMP in the fine regulation of matrix** 

Over-expression of *vps* in *hapR* mutants gives rise to the rugose colonial morphology (Yildiz et al., 2004). Although HapR is not detected in *crp* and *cya* mutants, these strains still produce the smooth colonial variant (Liang et al., 2007, 2007a). As shown in Fig. 10, the rugose colonial morphology of a *hapR* mutant is turned to smooth by deletion of *crp* or *cya,* and the resulting smooth strains can be converted back to rugose by introduction of the *crp* and *cya* genes on a plasmid vector. These results suggest that formation of the cAMP-CRP complex has a dual effect on *vps* expression by activating quorum sensing (a negative effect) and by enhancing the expression of a positive factor. In our strain, the positive factor was

Fig. 10. Scanning electron microscopy of *V. cholerae crp*, *cya*, and *hapR* colonies. A, *crp*; B, *hapR*; C, *hapRcrp*; D, *hapRcrp* transformed with *crp* plasmid; E, *hapRcya*; F,

Fig. 11 schematically illustrates the dual input of adenylate cyclase and CRP on *V. cholerae* expression of *vps.* In this model, deletion of *crp* results in diminished expression of the positive regulator, VpsR, which is required for expression of rugose colonial morphology. This event, however, is partially compensated for by reduced expression of the negative

**exopolysaccharide expression** 

the regulator, VpsR (Liang et al., 2007a).

*hapRcya* transformed with *cya* plasmid

regulator HapR. As a result, ∆*crp* mutants express elevated *vpsA* and *vpsL* compared to the wild-type strain, but this increase is not large enough to induce rugose colonial morphology. Maximal exopolysaccharide expression requires inactivation of HapR (repressor) but an active *crp* allele for enhancing VpsR (activator). In a different strain, however, CRP repressed VpsR, suggesting that there is strain variability in the regulation of this protein (Fong et al., 2008).

In addition to the abovementioned regulatory effects of *crp* and *cya* on *vps* expression, several components of the PTS that function upstream of adenylate cyclase have their own regulatory input on *vps* gene expression. For instance, in minimal medium, phosphoryl transfer from EI to HPr and FPr represses *vps* expression (Houot et al., 2008); in LB medium, glucose-specific EIIAglc and nitrogen-specific EIIANtr1 and EIIANtr2 activate and repress *vps* expression, respectively (Houot et al., 2010).

Fig. 11. Model for the dual regulatory input of the cAMP-CRP complex on biosynthesis of *V. cholerae* matrix exopolysaccharide.

#### **4.8 Effect of extracellular phosphate on vps expression and biofilm formation**

Since freshwater and estuarine ecosystems where *Vibrios* survive and persist outside the human host are limited in phosphate content (Benitez-Nelson, 2000; Correl, 1999), *V. cholerae* build stores of intracellular polyphosphate (poly-P) (Ogawa et al., 2000). The enzyme polyphosphate kinase (PPK) is responsible for the synthesis of poly-P from ATP (Ahn and Kornberg, 1990). A *V. cholerae ppk* mutant exhibits a reduced capacity to withstand conditions of low pH, high salinity, and oxidative stress in low- phosphate medium (Jahid et al., 2006). These findings underline the importance of phosphate homeostasis as well as sensing and responding to changes in extracellular phosphate in the *V. cholerae* life cycle. In *E. coli*, deprivation of phosphate induces the expression of the PhoB regulon (Lamarche et

Integration of Global Regulatory Mechanisms Controlling *Vibrio Cholerae* Behavior 107

bacteria to adjust their response quantitatively to chemical and physical changes in the extracellular milieu. For instance, in defined chemical media, maximal VpsR expression would be expected to occur under conditions of low glucose concentration (high cellular cAMP) and high phosphate (PhoB inactive). Furthermore, according to the scheme presented in Fig. 12, the regulator VpsT acts as a receiver of population density and carbon sensory information; VpsR acts as a receiver of carbon and phosphate sensory information

As shown in Fig. 5, CRP enhances expression of the general response regulator, RpoS. The *rpoS* gene encodes the RNAP S subunit, which regulates expression of more than 100 genes in response to starvation and other stresses such as osmotic shock, acid shock, and temperature changes (Hengge-Aronis, 2002). It is not clear what makes a given promoter selective for transcription by RpoS. Several promoter elements have been described including an upstream (UP) element, a more degenerate -35 region, a cytosine at -13, and a AT-rich region downstream from -10 (Hengge-Aronis, 2002a; Typas et al., 2007). It also has been suggested that histone-like proteins such as H-NS, the Leucine-responsive protein (Lrp), or the integration host factor (IHF) can contribute to S promoter selectivity (Hengge-Aronis, 2002a; Hengge-Aronis, 1999; Typas et al., 2007). For instance, many S-dependent genes are repressed by H-NS, and association of RNAP with S on these promoters may overcome H-NS transcriptional repression (Barth et al., 1995; Bouvier et al., 1998; Hengge-Aronis, 1999). In *E. coli*, the intracellular level of S is controlled at the levels of transcription, translation, and protein stability (Hengge-Aronis, 2002; Nogueira & Springer, 2000; Vicente et al., 1999). The regulation of RpoS expression in *V. cholerae* is less understood but, relative to *E. coli*, there are differences that likely reflect adaptation to distinct environments. In contrast to *E. coli*, *V. cholerae* mutants that produce diminished guanosine tetraphosphate (ppGpp) and poly-P are not affected in *rpoS* expression (Jahid et al., 2006, Silva and Benitez, 2006). Moreover, deletion of Hfq, a factor that enhances *rpoS* translation in *E. coli*, has no effect on expression of *V. cholerae* RpoS (Ding et al., 2005). Additionally, we have shown that H-NS, which negatively influences *rpoS* translation in *E. coli*, has the opposite effect in *V. cholerae* (Silva et al., 2008). We have constructed an RpoS reporter strain expressing an RpoS-FLAG protein from native *rpoS* transcription and translation signals (Wang et al., 2010). In rich tryptone soy broth, RpoS was detected in the late logarithmic phase and after the population entered quorum-sensing mode (Wang et al., 2010). The quorum-sensing

**hierarchy Increase in c-di-GMP Deletion of** *rpoS*

Class IV *flaCD, flgM flaBCDE* 

Table 2. Comparison of motility genes differentially expressed in response to an increase in c-di-GMP and to deletion of *rpoS* (adapted from Beyhan et al., 2006; Nielsen et al., 2007).

Class III *flaAGI, flgBCDEFGI flgBCDEFGHIJL, fliMNOPQ,* 

*28) flhAF, fliFGJ* 

*flaA* 

to regulate the transition between the planktonic and biofilm life styles.

**regulator, RpoS** 

regulator, HapR, enhanced RpoS expression.

Class II *flhF, fliFGHIN, fliA (*

**Transcription** 

**4.9 Enhancement of motility and detachment by the general stress response** 

al., 2008). PhoB is part of the PhoR/PhoB two-component regulatory system. PhoR is an inner membrane histidine kinase that responds to periplasmic orthophosphate through its interaction with the phosphate transport system. Under conditions of phosphate limitation, phosphorus is transferred from phospho-PhoR to the response regulator PhoB. Phospho-PhoB then binds to DNA pho boxes to activate or repress the transcription of target genes (Lamarche et al., 2008). A proteomic comparison of wild-type and *phoB V. cholerae* strains revealed 140 differentially expressed proteins (von Kruger et al., 2006). A *V. cholerae phoB* mutant colonized rabbit ileal loops to a lesser extent suggesting a role for this regulator in intestinal colonization and pathogenesis (von Kruger et al., 1999). More recently, it was shown that PhoB negatively affects CT and TCP expression by repressing the *tcpPH* promoter (Pratt et al., 2010). In addition, PhoB negatively regulates biofilm formation in *V. cholerae* of classical and El Tor biotypes (Pratt et al., 2009; Sultan et al., 2010). A comparison of the levels of expression of known regulators of *vps* and biofilm between wild-type and *phoB V. cholerae* of the El Tor biotype revealed that VpsR, is negatively regulated by PhoB (Sultan et al., 2010).

Fig. 12. Model for the integration of cell density, carbon and phosphorus sensory information in the regulation of biofilm formation.

Since VpsR is positively modulated by CRP (Fig. 11), we propose that VpsR has an essential function in biofilm formation by acting as a receiver of external carbon and phosphorus sensory information to modulate biosynthesis of the exopolysaccharide matrix. PhoB and CRP exhibit antagonistic effects on VpsR (Fig. 12). The parallel function of multiple signaling pathways with opposing and/or re-enforcing effects appears to be a common theme in metabolic regulation. We suggest that such a regulatory architecture allows

al., 2008). PhoB is part of the PhoR/PhoB two-component regulatory system. PhoR is an inner membrane histidine kinase that responds to periplasmic orthophosphate through its interaction with the phosphate transport system. Under conditions of phosphate limitation, phosphorus is transferred from phospho-PhoR to the response regulator PhoB. Phospho-PhoB then binds to DNA pho boxes to activate or repress the transcription of target genes (Lamarche et al., 2008). A proteomic comparison of wild-type and *phoB V. cholerae* strains revealed 140 differentially expressed proteins (von Kruger et al., 2006). A *V. cholerae phoB* mutant colonized rabbit ileal loops to a lesser extent suggesting a role for this regulator in intestinal colonization and pathogenesis (von Kruger et al., 1999). More recently, it was shown that PhoB negatively affects CT and TCP expression by repressing the *tcpPH* promoter (Pratt et al., 2010). In addition, PhoB negatively regulates biofilm formation in *V. cholerae* of classical and El Tor biotypes (Pratt et al., 2009; Sultan et al., 2010). A comparison of the levels of expression of known regulators of *vps* and biofilm between wild-type and *phoB V. cholerae* of the El Tor biotype revealed that VpsR, is negatively regulated by PhoB

Fig. 12. Model for the integration of cell density, carbon and phosphorus sensory

Since VpsR is positively modulated by CRP (Fig. 11), we propose that VpsR has an essential function in biofilm formation by acting as a receiver of external carbon and phosphorus sensory information to modulate biosynthesis of the exopolysaccharide matrix. PhoB and CRP exhibit antagonistic effects on VpsR (Fig. 12). The parallel function of multiple signaling pathways with opposing and/or re-enforcing effects appears to be a common theme in metabolic regulation. We suggest that such a regulatory architecture allows

information in the regulation of biofilm formation.

(Sultan et al., 2010).

bacteria to adjust their response quantitatively to chemical and physical changes in the extracellular milieu. For instance, in defined chemical media, maximal VpsR expression would be expected to occur under conditions of low glucose concentration (high cellular cAMP) and high phosphate (PhoB inactive). Furthermore, according to the scheme presented in Fig. 12, the regulator VpsT acts as a receiver of population density and carbon sensory information; VpsR acts as a receiver of carbon and phosphate sensory information to regulate the transition between the planktonic and biofilm life styles.

#### **4.9 Enhancement of motility and detachment by the general stress response regulator, RpoS**

As shown in Fig. 5, CRP enhances expression of the general response regulator, RpoS. The *rpoS* gene encodes the RNAP S subunit, which regulates expression of more than 100 genes in response to starvation and other stresses such as osmotic shock, acid shock, and temperature changes (Hengge-Aronis, 2002). It is not clear what makes a given promoter selective for transcription by RpoS. Several promoter elements have been described including an upstream (UP) element, a more degenerate -35 region, a cytosine at -13, and a AT-rich region downstream from -10 (Hengge-Aronis, 2002a; Typas et al., 2007). It also has been suggested that histone-like proteins such as H-NS, the Leucine-responsive protein (Lrp), or the integration host factor (IHF) can contribute to S promoter selectivity (Hengge-Aronis, 2002a; Hengge-Aronis, 1999; Typas et al., 2007). For instance, many S-dependent genes are repressed by H-NS, and association of RNAP with S on these promoters may overcome H-NS transcriptional repression (Barth et al., 1995; Bouvier et al., 1998; Hengge-Aronis, 1999). In *E. coli*, the intracellular level of S is controlled at the levels of transcription, translation, and protein stability (Hengge-Aronis, 2002; Nogueira & Springer, 2000; Vicente et al., 1999). The regulation of RpoS expression in *V. cholerae* is less understood but, relative to *E. coli*, there are differences that likely reflect adaptation to distinct environments. In contrast to *E. coli*, *V. cholerae* mutants that produce diminished guanosine tetraphosphate (ppGpp) and poly-P are not affected in *rpoS* expression (Jahid et al., 2006, Silva and Benitez, 2006). Moreover, deletion of Hfq, a factor that enhances *rpoS* translation in *E. coli*, has no effect on expression of *V. cholerae* RpoS (Ding et al., 2005). Additionally, we have shown that H-NS, which negatively influences *rpoS* translation in *E. coli*, has the opposite effect in *V. cholerae* (Silva et al., 2008). We have constructed an RpoS reporter strain expressing an RpoS-FLAG protein from native *rpoS* transcription and translation signals (Wang et al., 2010). In rich tryptone soy broth, RpoS was detected in the late logarithmic phase and after the population entered quorum-sensing mode (Wang et al., 2010). The quorum-sensing regulator, HapR, enhanced RpoS expression.


Table 2. Comparison of motility genes differentially expressed in response to an increase in c-di-GMP and to deletion of *rpoS* (adapted from Beyhan et al., 2006; Nielsen et al., 2007).

Integration of Global Regulatory Mechanisms Controlling *Vibrio Cholerae* Behavior 109

Fig. 14. Coordinate regulation of HA/protease and motility by c-di-GMP, HapR, VpsT and

**4.10 The histone-like nucleoid structuring protein (H-NS) represses the expression of** 

H-NS belongs to a family of small nucleoid-associated proteins that include its paralog StpA; Fis, the heat-unstable protein (HU); and IHF (Dorman, 2004; Dorman & Deighan, 2003). In *E. coli,* H-NS is a 15-kDa, highly abundant protein present at about 20,000 copies per cell; it was initially characterized for its capacity to mediate DNA condensation (Dorman, 2004; Dorman & Deighan, 2003). Mutations that inactivate *hns* are pleiotropic, suggesting that H-NS influences a broad spectrum of physiological processes (Atlung & Hansen, 2002; Atlung & Ingmer, 1997; Hommais et al., 2001). The H-NS proteins of *E. coli* and *V. cholerae* contain an N-terminal oligomerization domain connected by a flexible linker to a nucleic acid binding domain (Atlung & Ingmer, 1997; Cerdan et al., 2003; Dorman, 2004; Nye & Taylor, 2003). Both oligomerization and DNA binding are essential for the biological activities of H-NS, which include DNA condensation and regulation of transcription (Dame et al., 2001; Spurio et al., 1997). In regulation of transcription, H-NS most commonly negatively affects gene expression by binding to promoters exhibiting AT-rich, highly curved DNA regions that contain clusters of the more conserved 10 bp motif, TCGATAAATT (Lang et al., 2007; Owen-Hughes et al., 1992; Uegushi & Mizuno, 1993). In *V. cholerae*, *hns* mutants form small colonies, are incapable of using -glucosides as a carbon source, exhibit diminished motility (Fig. 12) and intestinal colonization capacity, and show altered responses to environmental stresses (Ghosh et al., 2006; Krishnan et al., 2004; Silva et al., 2008; Tending et al., 2000; Silva et al. 2008). An emerging function of H-NS is the transcriptional silencing of horizontally acquired genes (Lucchini et al., 2006; Navarre et al., 2006; Oshima et al., 2006). Consistent with this role, H-NS silences expression of virulence genes in *V. cholerae* by acting at different levels of the ToxR regulatory cascade (Fig. 3), which include the *toxT*, *tcpA* and *ctxA* promoters (Nye et al., 2000). Binding of H-NS to a promoter apparently inhibits transcription by a bridging mechanism consisting of crosslinking DNA segments in a manner that traps RNAP (Dorman & Kane, 2009). There is

RpoS.

**virulence genes** 

Fig. 13. Motility of *V. cholerae rpoS* and *hns* (*hns*::km) mutants in swarm agar plates

*V. cholerae rpoS* mutants are more sensitive to starvation, high osmolarity, and oxidative stresses*,* are less motile than their wild-type precursors (Fig. 13), and do not express *hapA* (Nielsen et al., 2006; Silva et al., 2008; Yildiz & Schoolnik, 1998; Silva & Benitez, 2004). We have recently shown that, similar to HapR, expression of RpoS acts to diminish the c-di-GMP pool (Wang et al., 2011). Gene profiling experiments have shown that RpoS positively controls the expression of several proteins putatively identified as PDEs (Nielsen et al., 2006). As shown in Table 2, both deletion of *rpoS* and artificial enhancement of the c-di-GMP pool modulate the expression of class II through IV hierarchy motility genes. The data suggest that RpoS enhances motility by diminishing the c-di-GMP pool and by acting at an early step of the motility transcription hierarchy, such as RpoN and/or FlrA.

A common approach to investigate the phenotypic consequences of changes in intracellular c-di-GMP content is to increase or diminish the c-di-GMP pool by over-expressing a DGC or PDE, respectively. Using this method, we have investigated the effect of artificially altering the c-di-GMP pool on HapR and HA/protease expression. These studies revealed a complex interplay between c-di-GMP, HapR, VpsT, and RpoS that favors detachment of *V. cholerae* at high cell density (Fig. 13). Increasing the c-di-GMP pool enhances the expression of the c-di-GMP sensing protein, VpsT (Beyhan et al., 2006), which acts as a repressor of HapR (Yildiz et al., 2004). In the model shown in Fig. 14, expression of HapR at high cell density results in lower c-di-GMP content (Waters et al., 2008); lowering of c-di-GMP further enhances HapR, generating a double-negative regulatory loop that requires VpsT; HapR positively enhances RpoS expression (Joelsson et al., 2007); and the elevated expression of RpoS feeds into the regulatory loop by diminishing the intracellular concentration of c-di-GMP. The concurrent activation of HapR and RpoS results in elevated expression of HA/protease and motility, which promotes detachment. By promoting multiple cycles of detachment and recolonization, the coordinate expression of HA/protease and motility could contribute to the dissemination of colonizing *Vibrios* along the small intestine.

Fig. 13. Motility of *V. cholerae rpoS* and *hns* (*hns*::km) mutants in swarm agar plates

early step of the motility transcription hierarchy, such as RpoN and/or FlrA.

dissemination of colonizing *Vibrios* along the small intestine.

*V. cholerae rpoS* mutants are more sensitive to starvation, high osmolarity, and oxidative stresses*,* are less motile than their wild-type precursors (Fig. 13), and do not express *hapA* (Nielsen et al., 2006; Silva et al., 2008; Yildiz & Schoolnik, 1998; Silva & Benitez, 2004). We have recently shown that, similar to HapR, expression of RpoS acts to diminish the c-di-GMP pool (Wang et al., 2011). Gene profiling experiments have shown that RpoS positively controls the expression of several proteins putatively identified as PDEs (Nielsen et al., 2006). As shown in Table 2, both deletion of *rpoS* and artificial enhancement of the c-di-GMP pool modulate the expression of class II through IV hierarchy motility genes. The data suggest that RpoS enhances motility by diminishing the c-di-GMP pool and by acting at an

A common approach to investigate the phenotypic consequences of changes in intracellular c-di-GMP content is to increase or diminish the c-di-GMP pool by over-expressing a DGC or PDE, respectively. Using this method, we have investigated the effect of artificially altering the c-di-GMP pool on HapR and HA/protease expression. These studies revealed a complex interplay between c-di-GMP, HapR, VpsT, and RpoS that favors detachment of *V. cholerae* at high cell density (Fig. 13). Increasing the c-di-GMP pool enhances the expression of the c-di-GMP sensing protein, VpsT (Beyhan et al., 2006), which acts as a repressor of HapR (Yildiz et al., 2004). In the model shown in Fig. 14, expression of HapR at high cell density results in lower c-di-GMP content (Waters et al., 2008); lowering of c-di-GMP further enhances HapR, generating a double-negative regulatory loop that requires VpsT; HapR positively enhances RpoS expression (Joelsson et al., 2007); and the elevated expression of RpoS feeds into the regulatory loop by diminishing the intracellular concentration of c-di-GMP. The concurrent activation of HapR and RpoS results in elevated expression of HA/protease and motility, which promotes detachment. By promoting multiple cycles of detachment and recolonization, the coordinate expression of HA/protease and motility could contribute to the

Fig. 14. Coordinate regulation of HA/protease and motility by c-di-GMP, HapR, VpsT and RpoS.

#### **4.10 The histone-like nucleoid structuring protein (H-NS) represses the expression of virulence genes**

H-NS belongs to a family of small nucleoid-associated proteins that include its paralog StpA; Fis, the heat-unstable protein (HU); and IHF (Dorman, 2004; Dorman & Deighan, 2003). In *E. coli,* H-NS is a 15-kDa, highly abundant protein present at about 20,000 copies per cell; it was initially characterized for its capacity to mediate DNA condensation (Dorman, 2004; Dorman & Deighan, 2003). Mutations that inactivate *hns* are pleiotropic, suggesting that H-NS influences a broad spectrum of physiological processes (Atlung & Hansen, 2002; Atlung & Ingmer, 1997; Hommais et al., 2001). The H-NS proteins of *E. coli* and *V. cholerae* contain an N-terminal oligomerization domain connected by a flexible linker to a nucleic acid binding domain (Atlung & Ingmer, 1997; Cerdan et al., 2003; Dorman, 2004; Nye & Taylor, 2003). Both oligomerization and DNA binding are essential for the biological activities of H-NS, which include DNA condensation and regulation of transcription (Dame et al., 2001; Spurio et al., 1997). In regulation of transcription, H-NS most commonly negatively affects gene expression by binding to promoters exhibiting AT-rich, highly curved DNA regions that contain clusters of the more conserved 10 bp motif, TCGATAAATT (Lang et al., 2007; Owen-Hughes et al., 1992; Uegushi & Mizuno, 1993). In *V. cholerae*, *hns* mutants form small colonies, are incapable of using -glucosides as a carbon source, exhibit diminished motility (Fig. 12) and intestinal colonization capacity, and show altered responses to environmental stresses (Ghosh et al., 2006; Krishnan et al., 2004; Silva et al., 2008; Tending et al., 2000; Silva et al. 2008). An emerging function of H-NS is the transcriptional silencing of horizontally acquired genes (Lucchini et al., 2006; Navarre et al., 2006; Oshima et al., 2006). Consistent with this role, H-NS silences expression of virulence genes in *V. cholerae* by acting at different levels of the ToxR regulatory cascade (Fig. 3), which include the *toxT*, *tcpA* and *ctxA* promoters (Nye et al., 2000). Binding of H-NS to a promoter apparently inhibits transcription by a bridging mechanism consisting of crosslinking DNA segments in a manner that traps RNAP (Dorman & Kane, 2009). There is

Integration of Global Regulatory Mechanisms Controlling *Vibrio Cholerae* Behavior 111

expression of other nucleoid-associated proteins, such as IHF to attenuate H-NS repression

As described in the preceding sections, CRP acts as an upstream master regulator modulating quorum sensing, virulence, stress response, motility, and biofilm development. Thus, in Fig. 16, we provide an integrative model for CRP regulation of *V. cholerae* behavior. The CRP protein is activated when the intracellular concentration of cAMP is increased due to PTS activation of adenylate cyclase. Thus, the PTS system acts as the primary carbon source sensing mechanism. The state of activity of CRP controls the execution of secondary genetic programs mediated by HapR, RpoS and H-NS that modulate *V. cholerae* switching from planktonic to sessile life styles or from virulence to detachment. For instance, in its active state, CRP enhances the expression of (a) HapR by activating CAI-1 biosynthesis and repressing Fis, (b) RpoS, and (c) H-NS. The expression of HapR and RpoS enhances motility and activates HA/protease, favoring detachment. Simultaneous activation of HapR and H-NS leads to quorum-sensing repression of *aphA* and to H-NS-mediated transcriptional silencing of *toxT, ctxA,* and *tcpA* to diminish expression of virulence genes. By enhancing HapR, RpoS and H-NS, formation of the cAMP-CRP complex favors the motile planktonic

by an anti-repressor (anti-bridging) mechanism (Dorman & Kane, 2009).

**5. CRP as a master regulator of** *V. cholerae* **behavior** 

stage; the opposite is true for biofilm formation.

Fig. 16. Multilevel regulation of *V. cholerae* behavior by CRP.

The increase in our understanding of the regulatory pathways that control *V. cholerae* behavior creates the possibility of identifying new drugs to block infection and to prevent biofilm development. This is particularly relevant due to the emergence of antibioticresistant *V. cholerae* (Das & Kaur, 2008; Roychowdhury et al., 2008; Okeke et al., 2007; Mwansa et al., 2000). Consequently, development of anti-virulence drugs is proceeding

**6. Significance to anti-virulence drug discovery** 

considerable evidence indicating that H-NS-mediated repression can be antagonized in response to environmental stimuli that activate the expression of other regulators whose binding sites overlap that of H-NS (anti-bridging) (Dorman & Kane, 2009). For instance, the small nucleoid protein, Fis opposes the repression activity of H-NS at a various promoters (Dorman & Kane, 2009). The IHF alleviates H-NS silencing of *S. enterica hilA* (Queiroz et al., 2011)*, E. coli csgD* (Ogasawara et al., 2010)*, Shigella flexneri vir* genes (Porter & Dorman, 1997), and bacteriophage Mu early promoter (Van Ulsen et al., 1996). In the case of *V. cholerae*, transcriptional silencing of the *tcpA* and *ctxA* promoters by H-NS is antagonized by the AraC-like transcriptional regulator, ToxT, and by IHF (Stonehouse et al., 2008, 2010; Yu & DiRita, 2002). In theory, other small nucleoid proteins, such as Lrp and HU, can exhibit the anti-bridging activity required to attenuate H-NS repression (Dorman & Kane, 2009).

Fig. 15. Hypothetical model for the interaction of RpoS and H-NS in the regulation of *V. cholerae* motility. RpoS could enhance the transcription of motility by inducing the expression of an antirepressor (I), and by binding to core RNAP to promote transcription initiation that is H-NS-resistant.

The effect of H-NS on motility of *V. cholerae* is not fully understood. On one hand, H-NS could enhance motility by positively affecting the expression of RpoS (Silva et al., 2008). This effect, however, is modest compared to the reduction in motility observed for *hns* mutants (Fig. 13). Bright-field microscopy has revealed that *hns* mutants are flagellated nut nonmotile. In *E. coli*, H-NS binds to the switch protein, FliG, to enhance motility, and *hns* mutants express a paralyzed flagellum (Donato & Kawula, 1998). A similar mechanism may function in *V. cholerae*. Moreover, in the absence of RpoS, H-NS appears to function as a repressor of the motility genes, *flaA*, *flaC* and *motY* (Silva et al., 2008). This suggests that, in addition to lowering the c-di-GMP pool, RpoS enhances motility by attenuating H-NS repression. A speculative model for the anti-repressor function of RpoS is shown in Fig. 15. Based on data derived by transcriptional profiling, we suggest that RpoS is most likely to act at the *rpoN* and/or *flrA* promoters to enhance expression of downstream class II-IV motility genes (Table 2). In the stationary phase, transcription initiation at the *rpoN* and *flrA* promoters by RNAP containing *S* could be more resistant to H-NS repression (Barth et al., 1995; Bouvier et al., 1998; Hengge-Aronis, 1999). In addition, RpoS could enhance the expression of other nucleoid-associated proteins, such as IHF to attenuate H-NS repression by an anti-repressor (anti-bridging) mechanism (Dorman & Kane, 2009).

## **5. CRP as a master regulator of** *V. cholerae* **behavior**

110 Cholera

considerable evidence indicating that H-NS-mediated repression can be antagonized in response to environmental stimuli that activate the expression of other regulators whose binding sites overlap that of H-NS (anti-bridging) (Dorman & Kane, 2009). For instance, the small nucleoid protein, Fis opposes the repression activity of H-NS at a various promoters (Dorman & Kane, 2009). The IHF alleviates H-NS silencing of *S. enterica hilA* (Queiroz et al., 2011)*, E. coli csgD* (Ogasawara et al., 2010)*, Shigella flexneri vir* genes (Porter & Dorman, 1997), and bacteriophage Mu early promoter (Van Ulsen et al., 1996). In the case of *V. cholerae*, transcriptional silencing of the *tcpA* and *ctxA* promoters by H-NS is antagonized by the AraC-like transcriptional regulator, ToxT, and by IHF (Stonehouse et al., 2008, 2010; Yu & DiRita, 2002). In theory, other small nucleoid proteins, such as Lrp and HU, can exhibit the anti-bridging activity required to attenuate H-NS repression (Dorman & Kane, 2009).

Fig. 15. Hypothetical model for the interaction of RpoS and H-NS in the regulation of *V. cholerae* motility. RpoS could enhance the transcription of motility by inducing the expression of an antirepressor (I), and by binding to core RNAP to promote transcription

The effect of H-NS on motility of *V. cholerae* is not fully understood. On one hand, H-NS could enhance motility by positively affecting the expression of RpoS (Silva et al., 2008). This effect, however, is modest compared to the reduction in motility observed for *hns* mutants (Fig. 13). Bright-field microscopy has revealed that *hns* mutants are flagellated nut nonmotile. In *E. coli*, H-NS binds to the switch protein, FliG, to enhance motility, and *hns* mutants express a paralyzed flagellum (Donato & Kawula, 1998). A similar mechanism may function in *V. cholerae*. Moreover, in the absence of RpoS, H-NS appears to function as a repressor of the motility genes, *flaA*, *flaC* and *motY* (Silva et al., 2008). This suggests that, in addition to lowering the c-di-GMP pool, RpoS enhances motility by attenuating H-NS repression. A speculative model for the anti-repressor function of RpoS is shown in Fig. 15. Based on data derived by transcriptional profiling, we suggest that RpoS is most likely to act at the *rpoN* and/or *flrA* promoters to enhance expression of downstream class II-IV motility genes (Table 2). In the stationary phase, transcription initiation at the *rpoN* and *flrA* promoters by RNAP containing *S* could be more resistant to H-NS repression (Barth et al., 1995; Bouvier et al., 1998; Hengge-Aronis, 1999). In addition, RpoS could enhance the

initiation that is H-NS-resistant.

As described in the preceding sections, CRP acts as an upstream master regulator modulating quorum sensing, virulence, stress response, motility, and biofilm development. Thus, in Fig. 16, we provide an integrative model for CRP regulation of *V. cholerae* behavior. The CRP protein is activated when the intracellular concentration of cAMP is increased due to PTS activation of adenylate cyclase. Thus, the PTS system acts as the primary carbon source sensing mechanism. The state of activity of CRP controls the execution of secondary genetic programs mediated by HapR, RpoS and H-NS that modulate *V. cholerae* switching from planktonic to sessile life styles or from virulence to detachment. For instance, in its active state, CRP enhances the expression of (a) HapR by activating CAI-1 biosynthesis and repressing Fis, (b) RpoS, and (c) H-NS. The expression of HapR and RpoS enhances motility and activates HA/protease, favoring detachment. Simultaneous activation of HapR and H-NS leads to quorum-sensing repression of *aphA* and to H-NS-mediated transcriptional silencing of *toxT, ctxA,* and *tcpA* to diminish expression of virulence genes. By enhancing HapR, RpoS and H-NS, formation of the cAMP-CRP complex favors the motile planktonic stage; the opposite is true for biofilm formation.

Fig. 16. Multilevel regulation of *V. cholerae* behavior by CRP.

#### **6. Significance to anti-virulence drug discovery**

The increase in our understanding of the regulatory pathways that control *V. cholerae* behavior creates the possibility of identifying new drugs to block infection and to prevent biofilm development. This is particularly relevant due to the emergence of antibioticresistant *V. cholerae* (Das & Kaur, 2008; Roychowdhury et al., 2008; Okeke et al., 2007; Mwansa et al., 2000). Consequently, development of anti-virulence drugs is proceeding

Integration of Global Regulatory Mechanisms Controlling *Vibrio Cholerae* Behavior 113

d. A salient feature of this complex global regulatory network is the occurrence of parallel or overlapping regulatory outputs with opposing or re-enforcing effects to fine-tune bacterial responses to environmental stresses. In this chapter, this principle is illustrated by the dual effect of CRP on *vps* expression involving HapR and VpsR, and the

e. As we continue to develop a better understanding of the *V. cholerae* regulatory landscape, it should become possible to identify small molecules capable of shifting

We wish to acknowledge the contributions of postdoctoral associates Weili Liang, Zafar S. Sultan, Hongxia Wang and Julio C. Ayala to results included in this chapter. In addition, studies from our laboratory included in this chapter were supported by research grants

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Asai, Y., Kojima, S., Kato, H., Nishioka, N., Kawagishi, I., & Homma, M. (1997). Putative

Atlung, T., & Hansen, F.G. (2002). Effect of different concentrations of H-NS protein on

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and many σS-dependent genes in *Escherichia coli*. *J. Bacteriol*. 177: 3455-3464. Benitez, J. A., Garcia, L. Silva, A.J., Garcia, H., Fando, R., Cedre, B., Perez, A., Campos, J.,

Benitez, J.A., Silva, A. & Finkelstein, R.A. (2001). Environmental signals controlling

histone-like protein H-NS in growth phase-dependent and osmotic regulation of σ<sup>S</sup>

Rodriguez, B.L., Perez, J.L., Valmaseda, T., Perez, O., Perez, A., Ramirez, M., Ledon, T., Diaz, M., Lastre, M., Bravo, L., & Sierra, G. (1999). Preliminary assessment of the safety and immunogenicity of a new CTX\_- negative hemagglutinin/protease-defective El Tor strain as a cholera vaccine candidate.

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bacterial behavior from pathogenic to commensal.

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**8. Acknowledgements** 

265:11734–11739.

**9. References** 

1850.

6553.

expression as well as the interactions between RpoS and H-NS repression in the

(Waldor, 2006). An example is the small molecule inhibitor of *V. cholerae* intestinal colonization, virstatin [N-(1, 8-(naphthalimide)-n-butyric acid], which was identified in a high-throughput phenotypic screen (Hung et al., 2005; Shakhnovich et al., 2007, 2007a). This molecule inhibits *ctxAB* and *tcpA* transcription by preventing dimerization of their positive regulator, ToxT (Shakhnovich et al., 2007a). Another example is the newly identified inhibitor of the Na+-dependent flagellar motor, Q24DA, which could prevent infection dissemination by blocking motility and indirectly diminishing CT and TCP secretion (Rasmussen et al., 2010. An attractive target to block infection is the quorum- sensing phosphoryl cascade leading to the expression of HapR (Higgins et al., 2007). Pretreatment of mice with commensal bacteria engineered to express CAI-1 affords protection against a cholera challenge (Duan and March, 2010). A second set of attractive targets are the PTS components that modulate biofilm formation (Houot et al., 2008, 2010, 2010a). A comparative genomic analysis of 202 fully sequenced genomes (174 bacterial, 19 archaeal, and 9 eukaryotic) did not reveal components of the PTS system in eukaryotic cells (Barabote et al., 2005). A third potential target is CRP, whose cyclic nucleotide binding (CNB) domain is substantially different from eukaryotic cAMP binding proteins, with substitutions in highly conserved positions within the phosphate-binding cassette that determine ligand specificity (Kannan et al., 2007). These differences can be exploited to identify and/or synthesize CRP ligands selective for the bacterial CNB domain. Based on the results described in this chapter, CRP agonists would be expected to inhibit expression of virulence genes and biofilm formation while favoring motility and detachment. An advantage of targeting the PTS and CRP is their broad range. For instance, CRP modulates the expression of virulence factors in bacterial pathogens such as *V. vulnificus*, *Salmonella enterica* serovar Typhimurium, *Yersinia enterocolitica*, *Y. pestis,* and *Pseudomonas aeruginosa*. The genes encoding global regulators, such as CRP, and the virulence factors under its control are generally not essential. Thus, contrary to anti-bacterial drugs, inhibitors of expression of virulence genes are not expected to exert a high selective pressure for the dissemination of resistance or to impact the commensal flora. Finally, targeting global regulators such as CRP, RpoS and H-NS could significantly impair bacterial *in vivo* stress response allowing the host to clear the infection and diminish the use of antibiotics. The use of anti-virulence versus anti-bacterial therapies is still a matter of debate, particularly in regard to pathogens capable of long-term persistence in the host, which is not the case in cholera.

## **7. Conclusions**

The bacterial pathogen *V. cholerae* has evolved to colonize the human small bowel efficiently and to persist in aquatic environments. A sophisticated regulatory network allows the cholera bacterium to modify its behavior in response to the environmental changes dictated by its dual life cycle. Studies conducted in the last few decades have revealed that:


expression as well as the interactions between RpoS and H-NS repression in the regulation of expression of motility genes.


## **8. Acknowledgements**

We wish to acknowledge the contributions of postdoctoral associates Weili Liang, Zafar S. Sultan, Hongxia Wang and Julio C. Ayala to results included in this chapter. In addition, studies from our laboratory included in this chapter were supported by research grants from the National Institutes of Health (Bethesda, Maryland).

### **9. References**

112 Cholera

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capable of long-term persistence in the host, which is not the case in cholera.

by its dual life cycle. Studies conducted in the last few decades have revealed that:

(biofilm) life styles to maximize fitness.

(HapR), RpoS, and H-NS.

The bacterial pathogen *V. cholerae* has evolved to colonize the human small bowel efficiently and to persist in aquatic environments. A sophisticated regulatory network allows the cholera bacterium to modify its behavior in response to the environmental changes dictated

a. *V. cholerae* can switch from virulence to detachment modes and from motile to sessile

b. The cAMP receptor protein acts as an upstream master regulator of *V. cholerae* behavior by controlling the execution of secondary regulatory pathways, such as quorum sensing

c. Efficient regulation requires substantial molecular cross-talking between regulatory modules, as exemplified by the interplays between quorum sensing (HapR) and RpoS

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**Part 3** 

**Cholera Toxin and Antagonists** 


**Part 3** 

**Cholera Toxin and Antagonists** 

126 Cholera

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Quorum-sensing regulators control virulence gene expression in *Vibrio cholerae*.

**7** 

*Argentina* 

**The Cholera Toxin as a Biotechnological Tool** 

It was as early as 1886 when Robert Koch proposed that the symptoms caused by *Vibrio cholerae* were initiated by a "poison" produced by the pathogen. However, it was not until 1959 that this postulate could be demonstrated by reproducing the disease in an animal model [De, 1959]. Today, cholera toxin (CT) is known to exhibit toxic effects in human cells and produces dehydrating diarrhea in humans. It is produced almost exclusively by few serogroups of *V. cholera*, however, sometimes may be naturally produced by other organisms, as the opportunistic pathogen *V. mimicus* [Nishibuchi and Seidler, 1983; Spira

CT has important immunological properties and for that reason it has been extensively used as a systemic and mucosal adjuvant because it enhances the immunogenicity of most

The aim of this chapter will be to describe the biotechnological utilities of CT, with special attention to its adjuvant effect as well as its application in the treatment of autoimmune

CT belongs to the family of AB5-type toxins, since it is composed of two subunits in a 1:5 ratio. The A subunit (CTA), of 28 kDa, is a heterodimer associated non-covalently to a homopentamer formed by the subunits B (CTB) of 56 kDa [Merritt et al., 1994; Vanden Broeck et al., 2007]. CTA is responsible for the biological activity and CTB binds to the cell

CTA comprises 240 amino acids, and the 11.6 kDa B subunit monomers each have 103 amino acids. CTA is synthesized as a single polypeptide chain and is post-translationally modified through the action of a *V. cholerae* protease at position R192 [Mekalanos et al., 1979]. The cleavage of this amino acid, found in an exposed loop that extends from C187 to C199 residues, generates two fragments named CTA1 and CTA2, which remain linked by a disulfide bridge [Lencer and Tsai, 2003; Tsai et al., 2001]. The toxic activity (enzymatic ADP-ribosylating) activity of CTA resides in CTA1, whereas CTA2 serves to insert CTA into the CTB pentamer [Sanchez and Holmgren, 2011]. The C-terminal hydrophobic region including residues 162-192 of CTA1, plays a key role in toxicity. It triggers the ERassociated degradation (ERAD) mechanism (see section 3) and facilitates interaction with

membrane receptor [Holmgren et al., 1973; Lonnroth and Holmgren, 1973] (Fig. 1.).

antigens fused or co-administered with the toxin [Sanchez and Holmgren, 2008].

diseases through its ability to generate oral tolerance.

**1. Introduction** 

and Fedorka-Cray, 1984].

**2. Structure** 

Noelia Olivera, Maia Cédola and Ricardo M Gómez *Instituto de Biotecnología y Biología Molecular, CONICET La Plata* 

## **The Cholera Toxin as a Biotechnological Tool**

Noelia Olivera, Maia Cédola and Ricardo M Gómez *Instituto de Biotecnología y Biología Molecular, CONICET La Plata Argentina* 

## **1. Introduction**

It was as early as 1886 when Robert Koch proposed that the symptoms caused by *Vibrio cholerae* were initiated by a "poison" produced by the pathogen. However, it was not until 1959 that this postulate could be demonstrated by reproducing the disease in an animal model [De, 1959]. Today, cholera toxin (CT) is known to exhibit toxic effects in human cells and produces dehydrating diarrhea in humans. It is produced almost exclusively by few serogroups of *V. cholera*, however, sometimes may be naturally produced by other organisms, as the opportunistic pathogen *V. mimicus* [Nishibuchi and Seidler, 1983; Spira and Fedorka-Cray, 1984].

CT has important immunological properties and for that reason it has been extensively used as a systemic and mucosal adjuvant because it enhances the immunogenicity of most antigens fused or co-administered with the toxin [Sanchez and Holmgren, 2008].

The aim of this chapter will be to describe the biotechnological utilities of CT, with special attention to its adjuvant effect as well as its application in the treatment of autoimmune diseases through its ability to generate oral tolerance.

## **2. Structure**

CT belongs to the family of AB5-type toxins, since it is composed of two subunits in a 1:5 ratio. The A subunit (CTA), of 28 kDa, is a heterodimer associated non-covalently to a homopentamer formed by the subunits B (CTB) of 56 kDa [Merritt et al., 1994; Vanden Broeck et al., 2007]. CTA is responsible for the biological activity and CTB binds to the cell membrane receptor [Holmgren et al., 1973; Lonnroth and Holmgren, 1973] (Fig. 1.).

CTA comprises 240 amino acids, and the 11.6 kDa B subunit monomers each have 103 amino acids. CTA is synthesized as a single polypeptide chain and is post-translationally modified through the action of a *V. cholerae* protease at position R192 [Mekalanos et al., 1979]. The cleavage of this amino acid, found in an exposed loop that extends from C187 to C199 residues, generates two fragments named CTA1 and CTA2, which remain linked by a disulfide bridge [Lencer and Tsai, 2003; Tsai et al., 2001]. The toxic activity (enzymatic ADP-ribosylating) activity of CTA resides in CTA1, whereas CTA2 serves to insert CTA into the CTB pentamer [Sanchez and Holmgren, 2011]. The C-terminal hydrophobic region including residues 162-192 of CTA1, plays a key role in toxicity. It triggers the ERassociated degradation (ERAD) mechanism (see section 3) and facilitates interaction with

The Cholera Toxin as a Biotechnological Tool 131

ribose unit from NAD+ oxidizing agent to an arginine residue of Gs protein. This covalent modification leads to the loss of GTPase activity of the Gs protein, which remains attached to GTP, keeping the adenylate cyclase (AC) enzyme active that will produce increasing amounts of cAMP. Over 100 times the normal concentration of cAMP, the intestinal mucosa

water to the gut lumen that causes the characteristic acute diarrhea of cholera [Spangler, 1992]. As little as 5 µg of purified CT administered orally is sufficient to induce significant diarrhea in human volunteers while ingestion of 25 µg of CT elicits a full 20 litres cholera

Adjuvants are substances that have the ability to enhance the immune response when coadministered with poor immunogenic molecules. CT is a bacterial immunogen with a great function as an adjuvant to a variety of antigens when given by systemic and mucosal route whether these are linked to or simply mixed with the toxin, generating a long-term immune

These properties may be explained by three main characteristics of the molecule. First, CT is remarkably stable to proteases, bile salts and other compounds in the intestine. Secondly, its high affinity to GM1 ganglioside receptor, which is present on most mammalian cells including the M cells covering the Peyers patches, as well as all antigen-presenting cells (APC), facilitates the uptake and presentation of the toxin to the gut mucosal immune system. Finally, CT has strong inherent adjuvant and immunomodulating activities that depend both on its cell binding capability and its enzymatic ADP-ribosylating function

Pioneer studies carried out in 1972 showed that CT delivered by the intravenous route with a foreign antigen behaved as an adjuvant [Northrup and Fauci, 1972], a fact confirmed later by several groups using a number of unrelated antigens of little immunogenicity [Bianchi et al., 1990; Elson and Ealding, 1984]. Additional studies revealed that upon co-administration of CT and antigen through parenteral, mucosal, and transcutaneous routes resulted in substantial enhancement of mucosal immunoglobulin A (IgA) and serum IgG responses to the co-administered antigen [Chen and Strober, 1990; Drew et al., 1992; Reuman et al., 1991]. In addition to enhancing humoral immune responses, CT also augmented cellular immune responses to co-administered antigens enhancing induction of CD4+ T helper (Th) and class I-restricted cytolitic T lymphocyte responses [Nurkkala et al.; Simmons et al., 1999]. In most cases, CT induced a Th2 bias response [Lavelle et al., 2004; Okahashi et al., 1996]. However, other studies have reported Th1 [Sasaki et al., 2003; Taniguchi et al., 2008] or mixed Th1/Th2 responses following oral, sublingual and intranasal immunization with antigens in the presence of CT [Cuburu et al., 2007; Fecek et al., 2010]. More importantly, subsequent studies showed that CT elicited a long-term memory response and thus was detectable long

after the initial immune response [Soenawan et al., 2004; Vajdy and Lycke, 1992].

CT also acts as mucosal adjuvant against a variety of pathogens. Examples include, tetanus toxoid [Jackson et al., 1993], *Helicobacter felis* [Jiang et al., 2003], *Schistosoma japonicum* [Kohama et al., 2010], *Helicobacter pylori* [Raghavan et al., 2002], and *Sendai virus* [Liang et al., 1988]. There are many other examples where it was shown that CT has significant potential

channels in the cytoplasmic membrane, resulting in an influx of ions and

cells open a Cl-

purge [Levine et al., 1983].

**4. Immune properties** 

(Sanchez and Holmgren 2008).

response (Elson 1989; Vajdy and Lycke 1992).

the cytosolic ADP-ribosylation factors (ARFs) that serve as allosteric activators of CTA1 [Teter et al., 2006].

The remarkable stability of pentameric CTB is attributed to non-covalent interactions including 130 hydrogen bonds, 20 salt bridges, as well as tight packing of subunits via hydrophobic and pentamer-pentamer interactions. Consequently, the CTB pentamer is held together and remains as a complex unless boiled or monomerized by acidification at pH below 3 [Sanchez and Holmgren, 2008].

Fig. 1. Cholera toxin structure. A) Schematic model of cholera toxin. A subunit contains the toxic activity while B subunits bind to cells. B) Model based on X-ray crystallography analysis. Each subunit is represented by a different color. Adapted from Zhang et al 2005.

#### **3. Binding and mechanism of action**

CT is secreted through the outer membrane of *V. cholerae* and its toxic action begins when its B subunit binds to the high-affinity monoganglioside GM1 receptor. GM1 is a glycolipid commonly found in caveolae, organized membrane structures enriched in glycolipids, cholesterol and caveolin, involved in endocytosis and transcytosis, cellular transport and signal transduction [Shin and Abraham, 2001]. These membrane structures are present in various cell types, including immune cells [Thomas et al., 2004]. Each B subunit monomer has a binding site for GM1, however, the CTB pentamer has a much higher binding affinity for the receptor due to the important role played by a single amino acid from an adjacent B subunit that enhances this action [Merritt et al., 1994]. After binding to the receptor, CT enters human intestinal cells through endocytosis and is transported from early endosomes to the Golgi. Endocytosis of CT may follow one of three pathways: (i) lipid raft/caveolae mediated endocytic pathway, (ii) clathrin mediated endocytic pathway, or (iii) noncaveolar clathrin-independent pathway [Chinnapen et al., 2007]. GM1 is the vehicle for retrograde transport of the CT holotoxin from the plasma membrane to the ER [Fujinaga et al., 2003]. In the ER, the disulfide bond that links CTA1 and CTA2 to CTB is reduced and a protein disulfide isomerase mediates the dissociation of CTA1 from CTA2/CTB. CTA1 moves from the ER to the cytosol by the ERAD dislocation mechanism, wich recognizes misfolded proteins in the ER and exports them to the cytosol for degradation by the 26S proteasome [Massey et al., 2009]. Once inside the host cells, CTA1 catalyzes the transfer of an ADP-

ribose unit from NAD+ oxidizing agent to an arginine residue of Gs protein. This covalent modification leads to the loss of GTPase activity of the Gs protein, which remains attached to GTP, keeping the adenylate cyclase (AC) enzyme active that will produce increasing amounts of cAMP. Over 100 times the normal concentration of cAMP, the intestinal mucosa cells open a Cl channels in the cytoplasmic membrane, resulting in an influx of ions and water to the gut lumen that causes the characteristic acute diarrhea of cholera [Spangler, 1992]. As little as 5 µg of purified CT administered orally is sufficient to induce significant diarrhea in human volunteers while ingestion of 25 µg of CT elicits a full 20 litres cholera purge [Levine et al., 1983].

## **4. Immune properties**

130 Cholera

the cytosolic ADP-ribosylation factors (ARFs) that serve as allosteric activators of CTA1

The remarkable stability of pentameric CTB is attributed to non-covalent interactions including 130 hydrogen bonds, 20 salt bridges, as well as tight packing of subunits via hydrophobic and pentamer-pentamer interactions. Consequently, the CTB pentamer is held together and remains as a complex unless boiled or monomerized by acidification at pH

Fig. 1. Cholera toxin structure. A) Schematic model of cholera toxin. A subunit contains the toxic activity while B subunits bind to cells. B) Model based on X-ray crystallography analysis. Each subunit is represented by a different color. Adapted from Zhang et al 2005.

CT is secreted through the outer membrane of *V. cholerae* and its toxic action begins when its B subunit binds to the high-affinity monoganglioside GM1 receptor. GM1 is a glycolipid commonly found in caveolae, organized membrane structures enriched in glycolipids, cholesterol and caveolin, involved in endocytosis and transcytosis, cellular transport and signal transduction [Shin and Abraham, 2001]. These membrane structures are present in various cell types, including immune cells [Thomas et al., 2004]. Each B subunit monomer has a binding site for GM1, however, the CTB pentamer has a much higher binding affinity for the receptor due to the important role played by a single amino acid from an adjacent B subunit that enhances this action [Merritt et al., 1994]. After binding to the receptor, CT enters human intestinal cells through endocytosis and is transported from early endosomes to the Golgi. Endocytosis of CT may follow one of three pathways: (i) lipid raft/caveolae mediated endocytic pathway, (ii) clathrin mediated endocytic pathway, or (iii) noncaveolar clathrin-independent pathway [Chinnapen et al., 2007]. GM1 is the vehicle for retrograde transport of the CT holotoxin from the plasma membrane to the ER [Fujinaga et al., 2003]. In the ER, the disulfide bond that links CTA1 and CTA2 to CTB is reduced and a protein disulfide isomerase mediates the dissociation of CTA1 from CTA2/CTB. CTA1 moves from the ER to the cytosol by the ERAD dislocation mechanism, wich recognizes misfolded proteins in the ER and exports them to the cytosol for degradation by the 26S proteasome [Massey et al., 2009]. Once inside the host cells, CTA1 catalyzes the transfer of an ADP-

[Teter et al., 2006].

below 3 [Sanchez and Holmgren, 2008].

**3. Binding and mechanism of action** 

Adjuvants are substances that have the ability to enhance the immune response when coadministered with poor immunogenic molecules. CT is a bacterial immunogen with a great function as an adjuvant to a variety of antigens when given by systemic and mucosal route whether these are linked to or simply mixed with the toxin, generating a long-term immune response (Elson 1989; Vajdy and Lycke 1992).

These properties may be explained by three main characteristics of the molecule. First, CT is remarkably stable to proteases, bile salts and other compounds in the intestine. Secondly, its high affinity to GM1 ganglioside receptor, which is present on most mammalian cells including the M cells covering the Peyers patches, as well as all antigen-presenting cells (APC), facilitates the uptake and presentation of the toxin to the gut mucosal immune system. Finally, CT has strong inherent adjuvant and immunomodulating activities that depend both on its cell binding capability and its enzymatic ADP-ribosylating function (Sanchez and Holmgren 2008).

Pioneer studies carried out in 1972 showed that CT delivered by the intravenous route with a foreign antigen behaved as an adjuvant [Northrup and Fauci, 1972], a fact confirmed later by several groups using a number of unrelated antigens of little immunogenicity [Bianchi et al., 1990; Elson and Ealding, 1984]. Additional studies revealed that upon co-administration of CT and antigen through parenteral, mucosal, and transcutaneous routes resulted in substantial enhancement of mucosal immunoglobulin A (IgA) and serum IgG responses to the co-administered antigen [Chen and Strober, 1990; Drew et al., 1992; Reuman et al., 1991]. In addition to enhancing humoral immune responses, CT also augmented cellular immune responses to co-administered antigens enhancing induction of CD4+ T helper (Th) and class I-restricted cytolitic T lymphocyte responses [Nurkkala et al.; Simmons et al., 1999]. In most cases, CT induced a Th2 bias response [Lavelle et al., 2004; Okahashi et al., 1996]. However, other studies have reported Th1 [Sasaki et al., 2003; Taniguchi et al., 2008] or mixed Th1/Th2 responses following oral, sublingual and intranasal immunization with antigens in the presence of CT [Cuburu et al., 2007; Fecek et al., 2010]. More importantly, subsequent studies showed that CT elicited a long-term memory response and thus was detectable long after the initial immune response [Soenawan et al., 2004; Vajdy and Lycke, 1992].

CT also acts as mucosal adjuvant against a variety of pathogens. Examples include, tetanus toxoid [Jackson et al., 1993], *Helicobacter felis* [Jiang et al., 2003], *Schistosoma japonicum* [Kohama et al., 2010], *Helicobacter pylori* [Raghavan et al., 2002], and *Sendai virus* [Liang et al., 1988]. There are many other examples where it was shown that CT has significant potential

The Cholera Toxin as a Biotechnological Tool 133

Fig. 2. Proposed mechanism of action by CT as a mucosal adjuvant. CT induces increased permeability of the intestinal epithelium leading to 1) enhanced uptake of co-administered antigens and 2) enhanced antigen-presentation by various APC. 3) It causes the depletion of

CD8+ lymphocyte population that may produce inhibitory cytokines, and 4) induces maturation and mobilization of DC. In addition, 5) CT promotes a strong Th2 dominant response to bystander antigens, and can either 6) induce or inhibit a Th1 response. Moreover, 7) CT induces strong Th17-type responses. Furthermore, 8) mucosal epithelial cells contribute to the adjuvant activity of CT by secreting a number of chemokines and acting on polymorphonuclear leukocytes, macrophages, eosinophils and T cells.

for use as adjuvant for mucosally administered antigens [Clapp et al., 2010; Jhon Carlos Castaño Osorio, 2002].

## **5. Mechanism of adjuvant activity**

The mechanism of adjuvanticity of CT is still unclear but is has been related to: (i) the induction of increased permeability of the intestinal epithelium leading to enhanced uptake of co-administered antigens; (ii) the induction of enhanced antigen presentation by various APC; (iii) the promotion of isotype differentiation in B cells leading to increased IgA formation; and (iv) exhibition of complex stimulatory as well as inhibitory effects on T cell proliferation and cytokine production. Among these many effects, those leading to enhanced antigen presentation by various APC are probably of the greatest importance [Sanchez and Holmgren, 2011].

As mentioned before, the polarity of the immune response generated by CT is a matter of debate. Some studies indicate that CT primes naïve T cells *in vitro* and drives them towards a Th2 phenotype, with production of interleukins IL-4 (a cytokine needed for B cell differentiation), IL-5, IL-6 and IL-10, but little IFN-γ (a cytokine needed to evoke Th1 responses) and suppression of IL-12 production by dendritic cells (DC) [Braun et al., 1999; Klimpel et al., 1995; Wilson et al., 1991]. Moreover, after immunization of animals with CT co-administered antigens, IL-4 levels were significantly elevated in gut-associated tissues and in spleen, while the levels of IFN-γ either decreased or remained static [Akhiani et al., 1997; Marinaro et al., 1995]. These results are supported by evidence of increased secretory IgA, serum IgA and IgE levels [Adel-Patient et al., 2005; Bourguin et al., 1991], and higher titers of IgG1 than IgG2a [Glenn et al., 1998; Lycke et al., 1990].

In contrast, others have reported that CT induces a mixed Th1/Th2 type of immune response with the production of IFN-γ and IL-4 [Fromantin et al., 2001; Imaoka et al., 1998]. In addition, it has been shown that CT induces strong Th17-type responses after intranasal delivery [Datta et al.; Lee et al., 2009].

Furthermore, CT markedly increased antigen-presentation by DC, macrophages, and B cells [Bromander et al., 1991; George-Chandy et al., 2001]. Also, CT upregulates the expression of MHC/HLA-DR molecules, CD80/B7.1 and CD86/B7.2 co-stimulatory molecules, as well as chemokine receptors CCR7 and CXCR4, on both murine and human DC, among other APC [Cong et al., 1997; Gagliardi et al., 2000]. Importantly, CT also induced the secretion of IL-1β from both DC and macrophages. IL-1β not only induces the maturation of DC, but also acts as an efficient mucosal adjuvant when co-administered with protein antigens and might mediate a significant part of the adjuvant activity of CT [Staats and Ennis, 1999]. Treatment with CT has been demonstrated to induce maturation and mobilization of DC [Lavelle et al., 2003]. Also, CT interferes with the differentiation of monocytes into DC, giving rise to a distinct population (Ma-DC), which displays an activated macrophage-like phenotype, induces a strong allogeneic and antigen specific response, and promotes the polarization of naïve CD4+ T lymphocytes toward a Th2 profile [Raghavan et al., 2010]. In additon, CT enhanced IL-6 secretion by peritoneal mast cell [Leal-Berumen et al., 1996] and production of IL-1β, IL-6, and IL-10 together with inhibition of IL-12, TNF-α, and nitric oxid in macrophages [Cong et al., 2001], depleted the CD8+ intraepithelial lymphocyte population [Flach et al., 2005], and induced isotype differentiation of B cells acting synergistically with IL-4 [Salmond et al., 2002]. Recent studies show that CT enhances STAT3 gene expression

for use as adjuvant for mucosally administered antigens [Clapp et al., 2010; Jhon Carlos

The mechanism of adjuvanticity of CT is still unclear but is has been related to: (i) the induction of increased permeability of the intestinal epithelium leading to enhanced uptake of co-administered antigens; (ii) the induction of enhanced antigen presentation by various APC; (iii) the promotion of isotype differentiation in B cells leading to increased IgA formation; and (iv) exhibition of complex stimulatory as well as inhibitory effects on T cell proliferation and cytokine production. Among these many effects, those leading to enhanced antigen presentation by various APC are probably of the greatest importance

As mentioned before, the polarity of the immune response generated by CT is a matter of debate. Some studies indicate that CT primes naïve T cells *in vitro* and drives them towards a Th2 phenotype, with production of interleukins IL-4 (a cytokine needed for B cell differentiation), IL-5, IL-6 and IL-10, but little IFN-γ (a cytokine needed to evoke Th1 responses) and suppression of IL-12 production by dendritic cells (DC) [Braun et al., 1999; Klimpel et al., 1995; Wilson et al., 1991]. Moreover, after immunization of animals with CT co-administered antigens, IL-4 levels were significantly elevated in gut-associated tissues and in spleen, while the levels of IFN-γ either decreased or remained static [Akhiani et al., 1997; Marinaro et al., 1995]. These results are supported by evidence of increased secretory IgA, serum IgA and IgE levels [Adel-Patient et al., 2005; Bourguin et al., 1991], and higher

In contrast, others have reported that CT induces a mixed Th1/Th2 type of immune response with the production of IFN-γ and IL-4 [Fromantin et al., 2001; Imaoka et al., 1998]. In addition, it has been shown that CT induces strong Th17-type responses after intranasal

Furthermore, CT markedly increased antigen-presentation by DC, macrophages, and B cells [Bromander et al., 1991; George-Chandy et al., 2001]. Also, CT upregulates the expression of MHC/HLA-DR molecules, CD80/B7.1 and CD86/B7.2 co-stimulatory molecules, as well as chemokine receptors CCR7 and CXCR4, on both murine and human DC, among other APC [Cong et al., 1997; Gagliardi et al., 2000]. Importantly, CT also induced the secretion of IL-1β from both DC and macrophages. IL-1β not only induces the maturation of DC, but also acts as an efficient mucosal adjuvant when co-administered with protein antigens and might mediate a significant part of the adjuvant activity of CT [Staats and Ennis, 1999]. Treatment with CT has been demonstrated to induce maturation and mobilization of DC [Lavelle et al., 2003]. Also, CT interferes with the differentiation of monocytes into DC, giving rise to a distinct population (Ma-DC), which displays an activated macrophage-like phenotype, induces a strong allogeneic and antigen specific response, and promotes the polarization of naïve CD4+ T lymphocytes toward a Th2 profile [Raghavan et al., 2010]. In additon, CT enhanced IL-6 secretion by peritoneal mast cell [Leal-Berumen et al., 1996] and production of IL-1β, IL-6, and IL-10 together with inhibition of IL-12, TNF-α, and nitric oxid in macrophages [Cong et al., 2001], depleted the CD8+ intraepithelial lymphocyte population [Flach et al., 2005], and induced isotype differentiation of B cells acting synergistically with IL-4 [Salmond et al., 2002]. Recent studies show that CT enhances STAT3 gene expression

titers of IgG1 than IgG2a [Glenn et al., 1998; Lycke et al., 1990].

Castaño Osorio, 2002].

**5. Mechanism of adjuvant activity** 

[Sanchez and Holmgren, 2011].

delivery [Datta et al.; Lee et al., 2009].

Fig. 2. Proposed mechanism of action by CT as a mucosal adjuvant. CT induces increased permeability of the intestinal epithelium leading to 1) enhanced uptake of co-administered antigens and 2) enhanced antigen-presentation by various APC. 3) It causes the depletion of CD8+ lymphocyte population that may produce inhibitory cytokines, and 4) induces maturation and mobilization of DC. In addition, 5) CT promotes a strong Th2 dominant response to bystander antigens, and can either 6) induce or inhibit a Th1 response. Moreover, 7) CT induces strong Th17-type responses. Furthermore, 8) mucosal epithelial cells contribute to the adjuvant activity of CT by secreting a number of chemokines and acting on polymorphonuclear leukocytes, macrophages, eosinophils and T cells.

The Cholera Toxin as a Biotechnological Tool 135

Several studies using different conditions and routes of administration have described that CTB has several immunomodulatory properties opening many perspectives for future therapeutic and biotechnological applications. In this regard, intranasal immunization of women with CTB resulted in the production of long-lasting IgG and IgA anti-CTB in serum,

However, its capacity as mucosal adjuvant has proven to be much less than that of the toxin when given together with non-coupled antigens by the oral route [Sanchez and Holmgren, 2008]. Recombinant CTB has been successfully used as a mucosal adjuvant in vaccines for human use such as the cholera vaccine itself [Quiding et al., 1991], and the vaccine against enterotoxigenic *E. coli* that causes diarrhea [Peltola et al., 1991; Qadri et al., 2000]. Analogously, CTB proved to be good adjuvant for a *Streptococcus pneumoniae* cellular vaccine [Malley et al., 2004] and a severe acute respiratory syndrome-associated coronavirus

Given the potential of CTB as a regulator of the immune response, this subunit has been produced in various biological systems such as *Vibrio cholerae* [Sanchez and Holmgren, 1989], *Escherichia coli* [Arimitsu et al., 2009], *Bacillus brevis* [Goto et al., 2000], *Lactobacillus paracasei* and *plantarum* [Slos et al., 1998], in the yeasts *Hansenula polymorpha* [Song et al., 2004] and *Saccharomyces cerevisiae* [Mohsen and Rezae, 2005], and in silkworm [Gong et al., 2005]. In addition, CTB has been expressed successfully in tomato [Jani et al., 2002], lettuce [Young-Sook Kim, 2006], rice [Oszvald et al., 2008], tobacco [Hein et al., 1996], carrots [Kim et al., 2009], banana [Renuga et al., 2010] and potato transgenic plants, [Arakawa et al., 1997] where ubiquitin fusion enhances CTB expression [Mishra et al., 2006]. CTB may induce systemic immune responses in mice after gavage of the animals with the transgenic vegetal [Jiang et al., 2007]. The advantage of this approach is that plants present a low-cost agricultural-based effective production system. Different formulations, such as encapsulation in liposomes or microspheres with antigens [Seo et al., 2002] or combined with vesicles or liposomes containing antigens [Harokopakis et al., 1998; Lian et al., 1999]

CTB is a useful carrier protein for induction of mucosal IgA antibodies against chemically coupled antigens. In this regard, mice immunized intraduodenally with the horseradish peroxidase (HRP) covalently coupled to CTB showed a 33–120 fold higher level of IgA anti-HRP in intestinal washes as well as increased levels of serum IgG anti-HRP [McKenzie and Halsey, 1984]. In addition, CTB chemically conjugated to the protein I/II of *Streptococcus mutans* when administered in mice by oral [Russell and Wu, 1991], intranasal [Wu and Russell, 1998], and intragastric routes [Wu and Russell, 1993] results in the production of antistreptococcal IgG and IgA in serum and mucosa, as well as the presence of large numbers of antibody-secreting cells in salivary glands, mesenteric lymph nodes, and spleens. Similar results were found with CTB conjugated to human gamma globulin (HGG) and the recombinant *Neisseria gonorrhoeae* transferrin binding proteins, TbpA and TbpB. Vaginal and intranasal immunizations with CTB-HGG resulted in high levels of anti-HGG antibodies [Johansson et al., 1998], while rCTB-TbpA and rCTB-TbpB administered intranasally induced antibody responses in the serum and genital tract [Price et al., 2005]. Moreover, CTB was chemically conjugated to type III capsular polysaccharide from

nasal and vaginal secretions in a dose-dependent manner [Bergquist et al., 1997].

**7. Immunological and adjuvant properties of CTB** 

vaccine [Qu et al., 2005] when administered intranasally in mice.

were also successfully tested.

in murine B cells, and may critically modulate immune responses in both a proinflammatory and anti-inflammatory direction, depending on the circumstances and the types of cells involved Sjoblom-Hallen et al., (2010).

It has been suggested that mucosal epithelial cells may also play a role in adjuvanticity. Human epithelial cells express and secrete high levels of the chemoattractant cytokines IL-8, GROα, GROβ, GROγ, and ENA-78 in response to stimulation with TNF-α, IL-1β, or infection with enteroinvasive microorganisms. These chemokines attract and activate polymorphonuclear leukocytes. Activated epithelial cells also secret MCP-1, MIP-1β, MIP-1α, and RANTES, which variably act on monocytes/macrophages, eosinophils, and subpopulations of T-cells [Freytag and Clements, 2005]. One possibility is that CT interacts with epithelial cells triggering expression of one or more immunomodulatory factors that recruite APC and immune effector cells or activate those cells, or both [Lopes et al., 2000; Soriani et al., 2002].

A proposed mechanism of action of CT as adjuvant is shown in Fig. 2.

## **6. Genetic modifications of CT**

The inherent enterotoxicity of CT has limited its widespread use as a vaccine component and adjuvant. In dogs, protection due to CT occurred only with doses that caused transient, sometimes severe, diarrhea [Pierce et al., 1982]. Moreover, murine models demonstrated that intranasal sensitization with CT as adjuvant led to increased lung inflammation with a massive recruitment of macrophages as well as accumulation in the olfactory nerves, epithelium and the olfactory bulbs of mice after binding to GM1 gangliosides [Fischer et al., 2005]. These limitations have led to mucosal strategies involving nontoxic mutants and purified B subunits.

Although early reports showed that mutants without the ADP-ribosyltransferase activity lack their adjuvant properties [Lycke et al., 1992], later studies showed that non-toxic mutants retained their adjuvant and immunogenic properties [Douce et al., 1997; Yamamoto et al., 1997] without central nervous system (CNS) toxicity [Hagiwara et al., 2006]. This suggests that the ADP-ribosyltransferase activity is not essential for its immunogenic properties, though it contributes to the adjuvant effect.

In a different approach, the CTA1 fragment linked to a synthetic analogue of *Staphylococcus aureus* protein A, the D fragment with affinity for APC, [Agren et al., 1997], proved to be non-toxic [Eriksson et al., 2004]. The fusion protein CTA1-DD binds specifically to immunoglobulins on the surface of antigen-presenting B cells through the DD polypeptide, and induces the ADP ribosylation by CTA1. Although this produces a good immune response when administered intranasally, it has been shown not to work as well after oral administration. This limitation was overcome by fusing CTA1-DD with immunostimulating complexes, such as ISCOMs (lipophilic immune stimulating complexes), producing both Th1/Th2 responses at systemic and mucosal levels [Andersen et al., 2007]. A recent report showed that CTA1 potently enhances a GeneGun-delivered DNA prime for human and simian immunodeficiency viruses antigens boost in macaques and mice [Bagley et al., 2011].

in murine B cells, and may critically modulate immune responses in both a proinflammatory and anti-inflammatory direction, depending on the circumstances and the

It has been suggested that mucosal epithelial cells may also play a role in adjuvanticity. Human epithelial cells express and secrete high levels of the chemoattractant cytokines IL-8, GROα, GROβ, GROγ, and ENA-78 in response to stimulation with TNF-α, IL-1β, or infection with enteroinvasive microorganisms. These chemokines attract and activate polymorphonuclear leukocytes. Activated epithelial cells also secret MCP-1, MIP-1β, MIP-1α, and RANTES, which variably act on monocytes/macrophages, eosinophils, and subpopulations of T-cells [Freytag and Clements, 2005]. One possibility is that CT interacts with epithelial cells triggering expression of one or more immunomodulatory factors that recruite APC and immune effector cells or activate those cells, or both [Lopes et al., 2000;

The inherent enterotoxicity of CT has limited its widespread use as a vaccine component and adjuvant. In dogs, protection due to CT occurred only with doses that caused transient, sometimes severe, diarrhea [Pierce et al., 1982]. Moreover, murine models demonstrated that intranasal sensitization with CT as adjuvant led to increased lung inflammation with a massive recruitment of macrophages as well as accumulation in the olfactory nerves, epithelium and the olfactory bulbs of mice after binding to GM1 gangliosides [Fischer et al., 2005]. These limitations have led to mucosal strategies involving nontoxic mutants and

Although early reports showed that mutants without the ADP-ribosyltransferase activity lack their adjuvant properties [Lycke et al., 1992], later studies showed that non-toxic mutants retained their adjuvant and immunogenic properties [Douce et al., 1997; Yamamoto et al., 1997] without central nervous system (CNS) toxicity [Hagiwara et al., 2006]. This suggests that the ADP-ribosyltransferase activity is not essential for its immunogenic

In a different approach, the CTA1 fragment linked to a synthetic analogue of *Staphylococcus aureus* protein A, the D fragment with affinity for APC, [Agren et al., 1997], proved to be non-toxic [Eriksson et al., 2004]. The fusion protein CTA1-DD binds specifically to immunoglobulins on the surface of antigen-presenting B cells through the DD polypeptide, and induces the ADP ribosylation by CTA1. Although this produces a good immune response when administered intranasally, it has been shown not to work as well after oral administration. This limitation was overcome by fusing CTA1-DD with immunostimulating complexes, such as ISCOMs (lipophilic immune stimulating complexes), producing both Th1/Th2 responses at systemic and mucosal levels [Andersen et al., 2007]. A recent report showed that CTA1 potently enhances a GeneGun-delivered DNA prime for human and simian immunodeficiency viruses antigens boost in macaques

types of cells involved Sjoblom-Hallen et al., (2010).

A proposed mechanism of action of CT as adjuvant is shown in Fig. 2.

properties, though it contributes to the adjuvant effect.

Soriani et al., 2002].

purified B subunits.

and mice [Bagley et al., 2011].

**6. Genetic modifications of CT** 

## **7. Immunological and adjuvant properties of CTB**

Several studies using different conditions and routes of administration have described that CTB has several immunomodulatory properties opening many perspectives for future therapeutic and biotechnological applications. In this regard, intranasal immunization of women with CTB resulted in the production of long-lasting IgG and IgA anti-CTB in serum, nasal and vaginal secretions in a dose-dependent manner [Bergquist et al., 1997].

However, its capacity as mucosal adjuvant has proven to be much less than that of the toxin when given together with non-coupled antigens by the oral route [Sanchez and Holmgren, 2008]. Recombinant CTB has been successfully used as a mucosal adjuvant in vaccines for human use such as the cholera vaccine itself [Quiding et al., 1991], and the vaccine against enterotoxigenic *E. coli* that causes diarrhea [Peltola et al., 1991; Qadri et al., 2000]. Analogously, CTB proved to be good adjuvant for a *Streptococcus pneumoniae* cellular vaccine [Malley et al., 2004] and a severe acute respiratory syndrome-associated coronavirus vaccine [Qu et al., 2005] when administered intranasally in mice.

Given the potential of CTB as a regulator of the immune response, this subunit has been produced in various biological systems such as *Vibrio cholerae* [Sanchez and Holmgren, 1989], *Escherichia coli* [Arimitsu et al., 2009], *Bacillus brevis* [Goto et al., 2000], *Lactobacillus paracasei* and *plantarum* [Slos et al., 1998], in the yeasts *Hansenula polymorpha* [Song et al., 2004] and *Saccharomyces cerevisiae* [Mohsen and Rezae, 2005], and in silkworm [Gong et al., 2005]. In addition, CTB has been expressed successfully in tomato [Jani et al., 2002], lettuce [Young-Sook Kim, 2006], rice [Oszvald et al., 2008], tobacco [Hein et al., 1996], carrots [Kim et al., 2009], banana [Renuga et al., 2010] and potato transgenic plants, [Arakawa et al., 1997] where ubiquitin fusion enhances CTB expression [Mishra et al., 2006]. CTB may induce systemic immune responses in mice after gavage of the animals with the transgenic vegetal [Jiang et al., 2007]. The advantage of this approach is that plants present a low-cost agricultural-based effective production system. Different formulations, such as encapsulation in liposomes or microspheres with antigens [Seo et al., 2002] or combined with vesicles or liposomes containing antigens [Harokopakis et al., 1998; Lian et al., 1999] were also successfully tested.

CTB is a useful carrier protein for induction of mucosal IgA antibodies against chemically coupled antigens. In this regard, mice immunized intraduodenally with the horseradish peroxidase (HRP) covalently coupled to CTB showed a 33–120 fold higher level of IgA anti-HRP in intestinal washes as well as increased levels of serum IgG anti-HRP [McKenzie and Halsey, 1984]. In addition, CTB chemically conjugated to the protein I/II of *Streptococcus mutans* when administered in mice by oral [Russell and Wu, 1991], intranasal [Wu and Russell, 1998], and intragastric routes [Wu and Russell, 1993] results in the production of antistreptococcal IgG and IgA in serum and mucosa, as well as the presence of large numbers of antibody-secreting cells in salivary glands, mesenteric lymph nodes, and spleens. Similar results were found with CTB conjugated to human gamma globulin (HGG) and the recombinant *Neisseria gonorrhoeae* transferrin binding proteins, TbpA and TbpB. Vaginal and intranasal immunizations with CTB-HGG resulted in high levels of anti-HGG antibodies [Johansson et al., 1998], while rCTB-TbpA and rCTB-TbpB administered intranasally induced antibody responses in the serum and genital tract [Price et al., 2005]. Moreover, CTB was chemically conjugated to type III capsular polysaccharide from

The Cholera Toxin as a Biotechnological Tool 137

[Dertzbaugh and Elson, 1993]. Some examples of genetic incorporation of epitopes to CTB include triple glutamic acid decarboxylase [Gong et al., 2009], dodecapeptide repeat of the serine-rich *Entamoeba histolytica* protein [Zhang et al., 1995] and human insulin B-chain [Sadeghi et al., 2002]. There are many studies showing the induction of immune responses through immunization of mice with CTB fused to soluble antigens expressed both in bacteria [Larsson et al., 2004; Lee et al., 2003; Sun et al., 1999; Tsuji et al., 2003] and in transgenic plants [Jani et al., 2004; Matsumoto et al., 2009]. In all cases there was generation of IgG and IgA antigen-specific antibodies and, in some cases, protection. Some examples of

One of the strategies for using CTB as an adjuvant genetically fused to antigens has been described by Arêas *et al*. and is based on the expression vector called pAEctxB (Fig. 3.). In the generation of the vector, the gene ctxB was modified to ensure that the codons were those most frequently used by *E. coli, L. casei* and *S. typhimurium* [Areas et al., 2002]. The genetically engineered ORF was then cloned into the expression vector pAE [Ramos et al., 2004] and includes two consecutive restriction sites Mlu*I* and Hind*III*. The resulting vector allows expression, under the control of a T7 promoter, of proteins fused to the C-terminus of CTB with 6 histidine residues at the N terminus, which facilitate protein purification by

The pAE-ctxB plasmid was used to clone the pneumococcal surface adhesin A (PspA) [Areas et al., 2004], the *Leptospira interrogans* protein LipL32 [Habarta et al., 2010], the fatty-acid binding protein from *Schistosoma mansoni* S14 [Henrique Roman Ramos, 2010], and the *Bordetella pertussis* type III secretion system effector protein Bsp22 (Olivera et al., unpublished results). Intradermal immunization with CTB-PspA induced high titers of anti-PspA IgG and partially protected mice after challenge with *S. pneumonia* [Areas et al., 2005]. Moreover, intranasal immunization with CTB-PsaA protected mice against colonization with *S. pneumoniae* without alteration of the natural oral or nasopharyngeal microbiota of mice [Pimenta et al., 2006]. CTB-Sm14 itself was not able to reduce *Schistosoma mansoni* worm burden on intranasally immunized BALB/c mice, but reduced the hepatic granulomas around trapped eggs. CTB-LipL32 generated higher specific titers in mice immunized without external adjuvant than co-administration of CTB with LipL32, supporting CTB-LipL32 as a promising

Mucosal administration by the oral, sublingual or nasal routes of many antigens can induce peripheral tolerance. Mucosal-induced tolerance has been recognized for a long time as a promising approach to prevent or treat allergic or autoimmune disorders and is characterized by a decreased immune response to systemic immunization with the same antigen [Sun et al., 2009; Sun et al., 1994]. In this regard, promising results have been obtained with auto-antigen coupled to CTB in order to induce oral tolerance. Although not known the mechanism by which CTB conjugated to antigens has the ability to potentiate the induction of oral tolerance, it is believed that in addition to the processes already mentioned before for CT, it may result in selected DC subsets with increased ability to induce different types of TGF-β-expressing suppressor T cells including CD4+ CD25+ Tr cells [Holmgren et al., 2005] and a direct depletion of effector T cells since CTB induces CD4+ and CD8+ T cell

the adjuvant action of CTB are shown in Table 1.

immobilized metal ion affinity chromatography.

antigen for use in the control and study of leptospirosis.

**8. CTB for mucosal immunotherapy** 

apoptosis [Christelle Basset, 2010].

*Streptococcus* group B [Shen et al., 2000] or to protein-polysaccharide conjugates [Bergquist et al., 1995] and in both cases, after subcutaneous administration, high levels of specific antibodies were detected. In addition to generating humoral response, simian immunodeficiency virus (SIV) virus-like particles (VLP) chemically conjugated to CTB showed higher levels of cytokine IFN-γ-producing splenocytes and cytotoxic-T-lymphocyte activities of immune cells than VLPs plus CTB, indicating a generation of a Th1 response in mice by CTB-VLP [Kang et al., 2003]. Finally, CTB chemically conjugated to the *Plasmodium vivax* ookinete surface protein, Pvs25, proved to be a potent transmission-blocking antigen in both intranasal and subcutaneal routes in mice [Miyata et al., 2010], and to protect against pharyngeal colonization by group A *streptococcus* when conjugated to the widely shared C repeat region of M6 protein [Bessen and Fischetti, 1990].


Table 1. Antigens towards which CT has adjuvant activity. in: intranasal, im: intramuscular, sl: sublingual, sc: subcutaneous, ig: intragastric.

Another way of using CTB as an adjuvant is in genetic constructions based on the toxin and heterologous antigens. In general, these hybrid molecules are composed of antigens fused to the amino [Laloi et al., 1996; Song et al., 2004] or carboxyl [Kim et al., 2004; Wang et al., 2010] terminus of CTB, being GM1-binding much more efficient in the latter case [Liljeqvist et al., 1997], but also protein epitopes have been introduced at internal positions in CTB

*Streptococcus* group B [Shen et al., 2000] or to protein-polysaccharide conjugates [Bergquist et al., 1995] and in both cases, after subcutaneous administration, high levels of specific antibodies were detected. In addition to generating humoral response, simian immunodeficiency virus (SIV) virus-like particles (VLP) chemically conjugated to CTB showed higher levels of cytokine IFN-γ-producing splenocytes and cytotoxic-T-lymphocyte activities of immune cells than VLPs plus CTB, indicating a generation of a Th1 response in mice by CTB-VLP [Kang et al., 2003]. Finally, CTB chemically conjugated to the *Plasmodium vivax* ookinete surface protein, Pvs25, proved to be a potent transmission-blocking antigen in both intranasal and subcutaneal routes in mice [Miyata et al., 2010], and to protect against pharyngeal colonization by group A *streptococcus* when conjugated to the widely shared C

**Antigen Route CTB administration Reference** 

virus in co-administered [Guo et al., 2010]

antigen in co-administered [Isaka et al., 2001]

OVA im co-administered [Rolland-Turner

in genetically fused [Lebens et al.,

administered [Shen et al., 2000]

chemically coupled/co-

HIV-1 gp41 sl chemically coupled [Hervouet et al.,

2003]

et al., 2004]

2010]

2003]

1998]

Robinson, 1991]

1995]

1999]

MSP4 5 malaria protein Oral co-administered [Wang et al.,

in, oral, rectal, and vaginal

*cholerae* O1, serotype Inaba sc chemically coupled [Gupta et al.,

polysaccharide Oral co-administered [Abraham and

Measles virus in, ig co-administered [Muller et al.,

Influenza virus in co-administered [Yang et al.] Pneumocystis carinii in co-administered [Pascale et al.,

Table 1. Antigens towards which CT has adjuvant activity. in: intranasal, im: intramuscular,

Another way of using CTB as an adjuvant is in genetic constructions based on the toxin and heterologous antigens. In general, these hybrid molecules are composed of antigens fused to the amino [Laloi et al., 1996; Song et al., 2004] or carboxyl [Kim et al., 2004; Wang et al., 2010] terminus of CTB, being GM1-binding much more efficient in the latter case [Liljeqvist et al., 1997], but also protein epitopes have been introduced at internal positions in CTB

repeat region of M6 protein [Bessen and Fischetti, 1990].

Nucleoprotein of Influenza A

Hepatitis B virus surface

Epitopes from *Schistosoma mansoni* glutathione-Stransferase

Group B Streptococcus Type III Capsular Polysaccharide

Lipopolysaccharide from *V.* 

*Pseudomonas aeruginosa*

sl: sublingual, sc: subcutaneous, ig: intragastric.

Proteins

Polysacharide

Micro-

or

ganisms

[Dertzbaugh and Elson, 1993]. Some examples of genetic incorporation of epitopes to CTB include triple glutamic acid decarboxylase [Gong et al., 2009], dodecapeptide repeat of the serine-rich *Entamoeba histolytica* protein [Zhang et al., 1995] and human insulin B-chain [Sadeghi et al., 2002]. There are many studies showing the induction of immune responses through immunization of mice with CTB fused to soluble antigens expressed both in bacteria [Larsson et al., 2004; Lee et al., 2003; Sun et al., 1999; Tsuji et al., 2003] and in transgenic plants [Jani et al., 2004; Matsumoto et al., 2009]. In all cases there was generation of IgG and IgA antigen-specific antibodies and, in some cases, protection. Some examples of the adjuvant action of CTB are shown in Table 1.

One of the strategies for using CTB as an adjuvant genetically fused to antigens has been described by Arêas *et al*. and is based on the expression vector called pAEctxB (Fig. 3.). In the generation of the vector, the gene ctxB was modified to ensure that the codons were those most frequently used by *E. coli, L. casei* and *S. typhimurium* [Areas et al., 2002]. The genetically engineered ORF was then cloned into the expression vector pAE [Ramos et al., 2004] and includes two consecutive restriction sites Mlu*I* and Hind*III*. The resulting vector allows expression, under the control of a T7 promoter, of proteins fused to the C-terminus of CTB with 6 histidine residues at the N terminus, which facilitate protein purification by immobilized metal ion affinity chromatography.

The pAE-ctxB plasmid was used to clone the pneumococcal surface adhesin A (PspA) [Areas et al., 2004], the *Leptospira interrogans* protein LipL32 [Habarta et al., 2010], the fatty-acid binding protein from *Schistosoma mansoni* S14 [Henrique Roman Ramos, 2010], and the *Bordetella pertussis* type III secretion system effector protein Bsp22 (Olivera et al., unpublished results). Intradermal immunization with CTB-PspA induced high titers of anti-PspA IgG and partially protected mice after challenge with *S. pneumonia* [Areas et al., 2005]. Moreover, intranasal immunization with CTB-PsaA protected mice against colonization with *S. pneumoniae* without alteration of the natural oral or nasopharyngeal microbiota of mice [Pimenta et al., 2006]. CTB-Sm14 itself was not able to reduce *Schistosoma mansoni* worm burden on intranasally immunized BALB/c mice, but reduced the hepatic granulomas around trapped eggs. CTB-LipL32 generated higher specific titers in mice immunized without external adjuvant than co-administration of CTB with LipL32, supporting CTB-LipL32 as a promising antigen for use in the control and study of leptospirosis.

## **8. CTB for mucosal immunotherapy**

Mucosal administration by the oral, sublingual or nasal routes of many antigens can induce peripheral tolerance. Mucosal-induced tolerance has been recognized for a long time as a promising approach to prevent or treat allergic or autoimmune disorders and is characterized by a decreased immune response to systemic immunization with the same antigen [Sun et al., 2009; Sun et al., 1994]. In this regard, promising results have been obtained with auto-antigen coupled to CTB in order to induce oral tolerance. Although not known the mechanism by which CTB conjugated to antigens has the ability to potentiate the induction of oral tolerance, it is believed that in addition to the processes already mentioned before for CT, it may result in selected DC subsets with increased ability to induce different types of TGF-β-expressing suppressor T cells including CD4+ CD25+ Tr cells [Holmgren et al., 2005] and a direct depletion of effector T cells since CTB induces CD4+ and CD8+ T cell apoptosis [Christelle Basset, 2010].

The Cholera Toxin as a Biotechnological Tool 139

CTB conjugates were also effective in the induction of tolerance to type II collagen, leading to a suppression of chondritis in a model of autoimmune ear disease [Kim et al., 2001]. Oral administration of allogeneic antigen linked to CTB induced immunological tolerance against allograft rejection [Sun et al., 2000a]. Finally, transconjunctival immunotherapy using CTB could suppress clinical effects for experimental allergic conjunctivitis in guinea pigs

CT has been studied for over 40 years. Both CT and its non-toxic derivatives or its B subunit, have shown to be excellent mucosal adjuvants. The possibility to use them as biotechnological tools in the development of new vaccines is being intensively studied in the present. In recent years, the prospect to use CTB fused to different protein antigens became relevant because these proteins can be expressed in high levels in a soluble form and directly purified in their active form, requiring only one fermentation step. In addition, several reports have shown that CTB can generate oral tolerance to different conjugated antigens, opening ways for the treatment of autoimmune diseases. Hopefully, future studies

This work was supported by grants from Agencia Nacional de Promoción Científica y

Abraham E, Robinson A. 1991. Oral immunization with bacterial polysaccharide and

Adel-Patient K, Bernard H, Ah-Leung S, Creminon C, Wal JM. 2005. Peanut- and cow's

Agren LC, Ekman L, Lowenadler B, Lycke NY. 1997. Genetically engineered nontoxic

Akhiani AA, Nilsson LA, Ouchterlony O. 1997. Intranasal administration of Schistosoma

Andersen CS, Dietrich J, Agger EM, Lycke NY, Lovgren K, Andersen P. 2007. The combined

Arakawa T, Chong DK, Merritt JL, Langridge WH. 1997. Expression of cholera toxin B subunit oligomers in transgenic potato plants. Transgenic Res 6(6):403-413.

mice orally sensitized with cholera toxin. Allergy 60(5):658-664.

cholera toxin A1 subunit. J Immunol 158(8):3936-3946.

response. Parasite Immunol 19(4):183-190.

Immun 75(1):408-416.

adjuvant enhances antigen-specific pulmonary secretory antibody response and

milk-specific IgE, Th2 cells and local anaphylactic reaction are induced in Balb/c

vaccine adjuvant that combines B cell targeting with immunomodulation by

mansoni adult worm antigen in combination with cholera toxin induces a Th2 cell

CTA1-DD/ISCOMs vector is an effective intranasal adjuvant for boosting prior Mycobacterium bovis BCG immunity to Mycobacterium tuberculosis. Infect

[Oikawa et al., 2011].

**10. Acknowledgements** 

**11. References** 

will focus on the use of CTB in such important issues.

Tecnológica (ANPCyT) PICT 07-00642 and PICT 07-00028 (RMG).

resistance to pneumonia. Vaccine 9(10):757-764.

**9. Conclusion** 

Fig. 3. Cloning strategy into pAEctxB plasmid

Oral delivery of CTB conjugated to myelin basic protein protected mice [Sun et al., 1996; Yuki et al., 2001] and rats [Sun et al., 2000b] against the development of experimental autoimmune encephalomyelitis. It was proposed that the inhibitory effect was a result of both the induction of TGF-β-producing Tr cells and down-regulation of IFNγ, IL-12, TNFα, MCP-1 and RANTES in the CNS [Wang et al., 2009].

Oral administration of a CTB-insulin conjugate prevented diabetes in non-obese diabetic (NOD) mice [Arakawa et al., 1998; Bergerot et al., 1997; Gong et al., 2007; Petersen et al., 2003; Ploix et al., 1999], which was associated with a reduction in IFNγ production and Tr cell migration into pancreatic islets [Aspord et al., 2002; Sobel et al., 1998]. On the other hand, oral administration of CTB-proinsulin fusion protein showed an increased expression of IL-4 and IL-10 in the pancreas of NOD-treated mice, suggesting that Th2 lymphocytemediated oral tolerance is a likely mechanism for the prevention of pancreatic insulitis [Ruhlman et al., 2007].

Oral delivery of CTB conjugated to a 60 kDa heat-shock protein derived peptide prevented mucosal induced uveitis in rats, an effect that was associated with enhanced IL-10 and TGFβ, and reduced IL-12 and IFN-γ production [Phipps et al., 2003]. Furthermore, a I/II phase clinical trial of the same peptide conjugated to CTB administered orally to 8 patients allowed the withdrawal of all immunosuppressive drugs in 5 of the 8 patients without a relapse of uveitis [Stanford et al., 2004].

In addition, oral administration of CTB in mice inhibits the induction of trinitrobenzene sulfonic acid-induced colitis and reverses such colitis after it has been established. This inhibition is associated with suppression of IL-12 and IFN-γ production [Boirivant et al., 2001; Coccia et al., 2005]. In a recent clinical trial, 40% of patients with active Crohn's disease responded to treatment with CTB [Stal et al., 2010].

CTB conjugates were also effective in the induction of tolerance to type II collagen, leading to a suppression of chondritis in a model of autoimmune ear disease [Kim et al., 2001]. Oral administration of allogeneic antigen linked to CTB induced immunological tolerance against allograft rejection [Sun et al., 2000a]. Finally, transconjunctival immunotherapy using CTB could suppress clinical effects for experimental allergic conjunctivitis in guinea pigs [Oikawa et al., 2011].

## **9. Conclusion**

138 Cholera

Oral delivery of CTB conjugated to myelin basic protein protected mice [Sun et al., 1996; Yuki et al., 2001] and rats [Sun et al., 2000b] against the development of experimental autoimmune encephalomyelitis. It was proposed that the inhibitory effect was a result of both the induction of TGF-β-producing Tr cells and down-regulation of IFNγ, IL-12, TNFα,

Oral administration of a CTB-insulin conjugate prevented diabetes in non-obese diabetic (NOD) mice [Arakawa et al., 1998; Bergerot et al., 1997; Gong et al., 2007; Petersen et al., 2003; Ploix et al., 1999], which was associated with a reduction in IFNγ production and Tr cell migration into pancreatic islets [Aspord et al., 2002; Sobel et al., 1998]. On the other hand, oral administration of CTB-proinsulin fusion protein showed an increased expression of IL-4 and IL-10 in the pancreas of NOD-treated mice, suggesting that Th2 lymphocytemediated oral tolerance is a likely mechanism for the prevention of pancreatic insulitis

Oral delivery of CTB conjugated to a 60 kDa heat-shock protein derived peptide prevented mucosal induced uveitis in rats, an effect that was associated with enhanced IL-10 and TGFβ, and reduced IL-12 and IFN-γ production [Phipps et al., 2003]. Furthermore, a I/II phase clinical trial of the same peptide conjugated to CTB administered orally to 8 patients allowed the withdrawal of all immunosuppressive drugs in 5 of the 8 patients without a

In addition, oral administration of CTB in mice inhibits the induction of trinitrobenzene sulfonic acid-induced colitis and reverses such colitis after it has been established. This inhibition is associated with suppression of IL-12 and IFN-γ production [Boirivant et al., 2001; Coccia et al., 2005]. In a recent clinical trial, 40% of patients with active Crohn's disease

Fig. 3. Cloning strategy into pAEctxB plasmid

MCP-1 and RANTES in the CNS [Wang et al., 2009].

[Ruhlman et al., 2007].

relapse of uveitis [Stanford et al., 2004].

responded to treatment with CTB [Stal et al., 2010].

CT has been studied for over 40 years. Both CT and its non-toxic derivatives or its B subunit, have shown to be excellent mucosal adjuvants. The possibility to use them as biotechnological tools in the development of new vaccines is being intensively studied in the present. In recent years, the prospect to use CTB fused to different protein antigens became relevant because these proteins can be expressed in high levels in a soluble form and directly purified in their active form, requiring only one fermentation step. In addition, several reports have shown that CTB can generate oral tolerance to different conjugated antigens, opening ways for the treatment of autoimmune diseases. Hopefully, future studies will focus on the use of CTB in such important issues.

## **10. Acknowledgements**

This work was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) PICT 07-00642 and PICT 07-00028 (RMG).

### **11. References**


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**8** 

 *Belgium* 

**Brefeldin A and Exo1 Completely Releave the** 

**Block of Cholera Toxin Action by a Dipeptide** 

Cholera toxin (CT), the enterotoxin secreted by *Vibrio cholerae* classical as well as *El Tor* biotypes, is the major causative agent of the acute diarrheal disease of humans. CT and the *Escherichia coli* heat labile enterotoxin (LT),are structurally and immunologically highly homologous,seeing that they belong to the same enterotoxin family (de Haan and Hirst, 2004; Spangler, 1992; Vanden Broeck et al., 2007). Both are oligomeric proteins of the A-B type. CT is composed of one A or activating subunit (CT-A Mr 27,400), which consists of two distinct polypeptide chains CT-A1 (Mr 22,000) and CT-A2 (Mr 5,400), linked by a single disulfide bridge, and 5 identical B subunits (Mr 11,600) arranged in a ring like configuration

The subunits are arranged in such a manner that CT-A occupies the central channel of the CT-B pentamer extending well above the plane of the pentameric ring (Sixma et al., 1991; Zhang et al., 1995). The CT-A2peptide goes through the pore in the doughnut-like structure of the CT-B pentamer, and protrudes on the side, which binds cell surface receptors with its COOH-terminal KDEL sequence exposed. CT elicits a secretory response from intestinal epithelia by binding to the apical cell membrane through interaction between CT-B and the monosialoganglioside GM1, followed by entry of polypeptide A1 into the cell, where it is able to stimulate the basolateral adenylatecyclase by catalyzing the ADP-ribosylation of Arg201 of the Gs subunit of the stimulatory GTP-binding regulatory protein (de Haan and

There is a distinct lag period between toxin binding and the activation of adenylatecyclase, during which the toxin must be internalized and processed.At the end of this lag period small amounts of CT-A1 appear in the cells parallel to activation of the cyclase(Kassis et al., 1982).

Early morphologic studies showed that CT is preferentially clustered into non-coated membrane invaginations characteristic of caveolae and enters several cell types via smooth,

Studies using cholesterol perturbing agents and chimeric toxins have shown that GM1 mediated association with detergent-resistant membrane fractions (DRMS) or lipid rafts is

required for toxic entry of CT (Orlandi and Fishman, 1998; Wolf et al., 1998, 2002).

Hirst, 2004; Spangler, 1992; Vanden Broeck et al., 2007a; Sixma et al., 1991).

non clathrin coated vesicles (Lencer et al., 1999).

**1. Introduction** 

(CT-B).

**Metalloendoprotease Substrate** 

*International Centre for Reproductive Health, Ghent University, Ghent,* 

Davy Vanden Broeck and Marc J.S. De Wolf


## **Brefeldin A and Exo1 Completely Releave the Block of Cholera Toxin Action by a Dipeptide Metalloendoprotease Substrate**

Davy Vanden Broeck and Marc J.S. De Wolf *International Centre for Reproductive Health, Ghent University, Ghent, Belgium* 

## **1. Introduction**

152 Cholera

Young-Sook Kim B-GK, Tae-Geum Kim, Tae-Jin Kang, Moon-Sik Yang. 2006. Expression of

Yuki Y, Byun Y, Fujita M, Izutani W, Suzuki T, Udaka S, Fujihashi K, McGhee JR, Kiyono H.

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210.

Biotechnol Bioeng 74(1):62-69.

Immun 63(4):1349-1355.

a cholera toxin B subunit in transgenic lettuce (Lactuca sativa L.) using Agrobacterium-mediated transformation system. Plant Cell Tiss Organ Cult 87:203-

2001. Production of a recombinant hybrid molecule of cholera toxin-B-subunit and proteolipid-protein-peptide for the treatment of experimental encephalomyelitis.

serine-rich Entamoeba histolytica protein (SREHP) fused to the cholera toxin B subunit induces a mucosal and systemic anti-SREHP antibody response. Infect

> Cholera toxin (CT), the enterotoxin secreted by *Vibrio cholerae* classical as well as *El Tor* biotypes, is the major causative agent of the acute diarrheal disease of humans. CT and the *Escherichia coli* heat labile enterotoxin (LT),are structurally and immunologically highly homologous,seeing that they belong to the same enterotoxin family (de Haan and Hirst, 2004; Spangler, 1992; Vanden Broeck et al., 2007). Both are oligomeric proteins of the A-B type. CT is composed of one A or activating subunit (CT-A Mr 27,400), which consists of two distinct polypeptide chains CT-A1 (Mr 22,000) and CT-A2 (Mr 5,400), linked by a single disulfide bridge, and 5 identical B subunits (Mr 11,600) arranged in a ring like configuration (CT-B).

> The subunits are arranged in such a manner that CT-A occupies the central channel of the CT-B pentamer extending well above the plane of the pentameric ring (Sixma et al., 1991; Zhang et al., 1995). The CT-A2peptide goes through the pore in the doughnut-like structure of the CT-B pentamer, and protrudes on the side, which binds cell surface receptors with its COOH-terminal KDEL sequence exposed. CT elicits a secretory response from intestinal epithelia by binding to the apical cell membrane through interaction between CT-B and the monosialoganglioside GM1, followed by entry of polypeptide A1 into the cell, where it is able to stimulate the basolateral adenylatecyclase by catalyzing the ADP-ribosylation of Arg201 of the Gs subunit of the stimulatory GTP-binding regulatory protein (de Haan and Hirst, 2004; Spangler, 1992; Vanden Broeck et al., 2007a; Sixma et al., 1991).

> There is a distinct lag period between toxin binding and the activation of adenylatecyclase, during which the toxin must be internalized and processed.At the end of this lag period small amounts of CT-A1 appear in the cells parallel to activation of the cyclase(Kassis et al., 1982).

> Early morphologic studies showed that CT is preferentially clustered into non-coated membrane invaginations characteristic of caveolae and enters several cell types via smooth, non clathrin coated vesicles (Lencer et al., 1999).

> Studies using cholesterol perturbing agents and chimeric toxins have shown that GM1 mediated association with detergent-resistant membrane fractions (DRMS) or lipid rafts is required for toxic entry of CT (Orlandi and Fishman, 1998; Wolf et al., 1998, 2002).

Brefeldin A and Exo1 Completely Releave the Block of

**2.2.Cell culture** 

**homogenate** 

counted in a hemocytometer.

**2.4 Gradient centrifugation** 

Cholera Toxin Action by a Dipeptide Metalloendoprotease Substrate 155

Unreacted 125I-Na was removed using gel filtration on a Sephadex G-50 mini-column using the centrifugation procedure of Tuszynski et al. (1980)*.*Cbz-Gly-Phe-NH2, brefeldinA, 3 isobutylmethylxanthine, iodixanol (OptiprepTM), 1,9-dideoxyforskolin, nocodazole and 2 deoxy-D-glucose were from Sigma. 2-(4-Fluorobenzoylamino)-benzoic acid methylester (Exo1) was from Calbiochem.1,3-Cyclohexane-bis(methylamine) (CBM) was purchased from Acros Organics. Na125I was obtained from MP Biochemicals& Reagents (formerly ICN).

Vero cells originally obtained from Flow Laboratories were cultured in Medium 199 with Earle's salts supplemented with 5% fetal calf serum (FCS).Human intestinal epithelial T84 cells (obtained from ATCC) were propagated in a 1:1 mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium with 2.5mM L-glutamine and 5% fetal bovine serum.Madin-Darby canine kidney (MDCK) cells (obtained from ATCC) were grown in Eagle's Minimum Essential Medium with 2mM L-glutamine and Earle's BSS adjusted to contain 1.5g/l sodium bicarbonate, 0.1mM non-essential amino acids, and 1.0mM sodium pyruvate with 10% fetal bovine serum. Growth medium was changed twice a week and cells were passed weekly (at confluency) using a 0.05% (w/v) trypsin solution (Gibco). Cells were

**2.3 Preparation of membranes from post nuclear supernatant of a Vero cell** 

x g for 1 h at 4°C. The membrane pellet was resuspended in Tris-sucrose buffer.

Subcellular fractionation was always performed on freshly prepared post nuclear membranes or PNS from cells labeled with 125I-CT. Membrane pellets prepared from PNS (post nuclear membranes),resuspended in 3 ml of homogenization buffer (50mM Tris-HCl, pH 7.4 containing 0.25M sucrose) or equal volumes of PNS,werelayered on top of two layers of respectively 4 ml of 25% iodixanol and 4 ml of 50% iodixanol each in isoosmotic

Vero cells were grown to confluency in 175cm2 culture flasks and maintained in culture for at least 14 days before harvesting. Cells were washed once with serum free medium to remove serum and harvested by short trypsinisation with a 0.05% trysin, EDTA solution in HBSS (Gibco). After 1 min the trypsinisation solution was removed by aspiration and the culture flasks were put at 37°C until the cells detached from the bottom. Cells needed for one gradient centrifugation experiment (5 culture flasks for each time interval) were collected in an Eppendorf tube, washed twice and suspended in 2ml of serum-free medium buffered with 25mM HEPES containing 0.1% BSA and chilled. Cells were then labeled with 125I-CT (106cpm /2 ml 10nM) in the same medium containing 0.01 % bovine serum albumin for 30 min at 4°C to allow binding without endocytosis. Subsequently, cells were washed and harvested immediately (t=0) or washed and incubated for 10,30,60 or 120 min in serum-free medium at 37°C and harvested.Cells were pelleted by low speed centrifugation. The pellets were resuspended in 50mM tris-HCl pH 7.4 containing 250mM sucrose (Tris-sucrose) buffer and homogenized on ice with 4 x 4 strokes of a Potter Elvejhem homogenizer (position 9). The homogenates were spun at 3,000 x g for 10 min to pellet unbroken cells, cell debris and nuclei to yield the PNS. Membranes from PNS were obtained by centrifugation of the PNS at 100,000

Although CT is currently used as a marker for endocytosis without utilising clathrin-coated pits,it appears to be endocytosed simultaneously through both clathrin -dependent andindependent routes (Orlandi and Fishman, 1998; Nichols et al., 2001; Shogomori and Futerman, 2001; Torgersen et al., 2001; Vanden Broeck et al., 2007b).

In a recent study using fluorescence microscopy (Massol et al., 2004)it has been shown that apart from clathrin-, caveolin-endocytic pathways, CT also enters cells via a pathway that is regulated by the small GTPase Arf6 and possibly a fourth pathway that is dynamin and Arf6-independent. However, after blocking all three known endocytic payhways simultaneously by over expression of negative dominant mutants of dynamin and Arf6, fluorescent CT in the Golgi and ER became undetectable, although CT induced toxicity was hardly affected (Massol et al., 2004).These findings illustrate the difficulty in correlating morphologic data with the functional entry of a potent toxin such as CT.

Consistent with the multiple ports of entry into the cell, CT can be found in early and recycling endosomes (Tran et al., 1987; Nichols, 2002)and in caveolin-1 containing endocytic intermediates (Nichols, 2002),which have been proposed to be responsible for the functional transport of CT. For CT to be toxic it must be transported through the Golgi to the ER. Brefeldin A (BFA), a fungal metabolite that disrupts the structural and functional integrity of the Golgi apparatus (Klausner et al., 1992),renders cells resistant to CT cytotoxicity and blocks intracellular formation of CT-A1(Orlandi et al., 1993; Nambiar et al., 1993; Lencer et al., 1993).

Movement into the Golgi can also be inhibited by blockage of COPI- and COPII- mediated vesicular transport, and this affects toxin function further implicating trafficking through the Golgi apparatus as a necessary step in toxin action (Richards et al., 2002; Majoul et al., 1998).

On reaching the ER, the reduced form of the lumenal chaperone protein disulfideisomerase binds to the A1 chain, dissociates it from the B subunit and unfolds it(Tsai et al., 2001; Tsai and Rapoport, 2002; Fujinaga et al., 2003). Subsequent oxidation of PDI by the ER lumenal ERO1, the A1 chain is released (Tsai and Rapoport, 2002)and is translocated to the cytosol probably via the Sec61 channel, identifying the rough ER as the compartment from which translocation occurs (Schmitz et al., 2000). It has been suggested that the rapid refolding (Tsai and Rapoport, 2002)may render the A1 chain resistant to poly ubiquitination and provide the driving force for retro translocation to the cytosol (Rodighiero et al., 2002).

We previously reported that the metalloendoprotease substrate N-benzoyloxycarbonyl-Gly-Phe-NH2(Cbz-Gly-Phe-NH2) completely blocked the response of different celltypes in culture to CT. The effect was reversible, dose-and time-dependent. The dipeptide had no effect on the binding of CT to the cell surface and did not decrease its internalization but appeared to affect a later step in toxin action (De Wolf, 2000).

In this study we further investigated the mechanism by which Cbz-Gly-Phe-NH2 blocks CT action.

## **2. Materials and methods**

#### **2.1.Materials**

Highly purified CT was obtained from List Biological Laboratories (Campbell, Ca.). CT was radiolabeled with 125I using the Iodo-gen method as described by Fraker and Speck (1978). Unreacted 125I-Na was removed using gel filtration on a Sephadex G-50 mini-column using the centrifugation procedure of Tuszynski et al. (1980)*.*Cbz-Gly-Phe-NH2, brefeldinA, 3 isobutylmethylxanthine, iodixanol (OptiprepTM), 1,9-dideoxyforskolin, nocodazole and 2 deoxy-D-glucose were from Sigma. 2-(4-Fluorobenzoylamino)-benzoic acid methylester (Exo1) was from Calbiochem.1,3-Cyclohexane-bis(methylamine) (CBM) was purchased from Acros Organics. Na125I was obtained from MP Biochemicals& Reagents (formerly ICN).

## **2.2.Cell culture**

154 Cholera

Although CT is currently used as a marker for endocytosis without utilising clathrin-coated pits,it appears to be endocytosed simultaneously through both clathrin -dependent andindependent routes (Orlandi and Fishman, 1998; Nichols et al., 2001; Shogomori and

In a recent study using fluorescence microscopy (Massol et al., 2004)it has been shown that apart from clathrin-, caveolin-endocytic pathways, CT also enters cells via a pathway that is regulated by the small GTPase Arf6 and possibly a fourth pathway that is dynamin and Arf6-independent. However, after blocking all three known endocytic payhways simultaneously by over expression of negative dominant mutants of dynamin and Arf6, fluorescent CT in the Golgi and ER became undetectable, although CT induced toxicity was hardly affected (Massol et al., 2004).These findings illustrate the difficulty in correlating

Consistent with the multiple ports of entry into the cell, CT can be found in early and recycling endosomes (Tran et al., 1987; Nichols, 2002)and in caveolin-1 containing endocytic intermediates (Nichols, 2002),which have been proposed to be responsible for the functional transport of CT. For CT to be toxic it must be transported through the Golgi to the ER. Brefeldin A (BFA), a fungal metabolite that disrupts the structural and functional integrity of the Golgi apparatus (Klausner et al., 1992),renders cells resistant to CT cytotoxicity and blocks intracellular formation of CT-A1(Orlandi et al., 1993; Nambiar et al., 1993; Lencer et al., 1993). Movement into the Golgi can also be inhibited by blockage of COPI- and COPII- mediated vesicular transport, and this affects toxin function further implicating trafficking through the Golgi apparatus as a necessary step in toxin action (Richards et al., 2002; Majoul et al., 1998). On reaching the ER, the reduced form of the lumenal chaperone protein disulfideisomerase binds to the A1 chain, dissociates it from the B subunit and unfolds it(Tsai et al., 2001; Tsai and Rapoport, 2002; Fujinaga et al., 2003). Subsequent oxidation of PDI by the ER lumenal ERO1, the A1 chain is released (Tsai and Rapoport, 2002)and is translocated to the cytosol probably via the Sec61 channel, identifying the rough ER as the compartment from which translocation occurs (Schmitz et al., 2000). It has been suggested that the rapid refolding (Tsai and Rapoport, 2002)may render the A1 chain resistant to poly ubiquitination and provide the driving force for retro translocation to the cytosol (Rodighiero et al., 2002).

We previously reported that the metalloendoprotease substrate N-benzoyloxycarbonyl-Gly-Phe-NH2(Cbz-Gly-Phe-NH2) completely blocked the response of different celltypes in culture to CT. The effect was reversible, dose-and time-dependent. The dipeptide had no effect on the binding of CT to the cell surface and did not decrease its internalization but

In this study we further investigated the mechanism by which Cbz-Gly-Phe-NH2 blocks CT

Highly purified CT was obtained from List Biological Laboratories (Campbell, Ca.). CT was radiolabeled with 125I using the Iodo-gen method as described by Fraker and Speck (1978).

appeared to affect a later step in toxin action (De Wolf, 2000).

action.

**2.1.Materials** 

**2. Materials and methods** 

Futerman, 2001; Torgersen et al., 2001; Vanden Broeck et al., 2007b).

morphologic data with the functional entry of a potent toxin such as CT.

Vero cells originally obtained from Flow Laboratories were cultured in Medium 199 with Earle's salts supplemented with 5% fetal calf serum (FCS).Human intestinal epithelial T84 cells (obtained from ATCC) were propagated in a 1:1 mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium with 2.5mM L-glutamine and 5% fetal bovine serum.Madin-Darby canine kidney (MDCK) cells (obtained from ATCC) were grown in Eagle's Minimum Essential Medium with 2mM L-glutamine and Earle's BSS adjusted to contain 1.5g/l sodium bicarbonate, 0.1mM non-essential amino acids, and 1.0mM sodium pyruvate with 10% fetal bovine serum. Growth medium was changed twice a week and cells were passed weekly (at confluency) using a 0.05% (w/v) trypsin solution (Gibco). Cells were counted in a hemocytometer.

#### **2.3 Preparation of membranes from post nuclear supernatant of a Vero cell homogenate**

Vero cells were grown to confluency in 175cm2 culture flasks and maintained in culture for at least 14 days before harvesting. Cells were washed once with serum free medium to remove serum and harvested by short trypsinisation with a 0.05% trysin, EDTA solution in HBSS (Gibco). After 1 min the trypsinisation solution was removed by aspiration and the culture flasks were put at 37°C until the cells detached from the bottom. Cells needed for one gradient centrifugation experiment (5 culture flasks for each time interval) were collected in an Eppendorf tube, washed twice and suspended in 2ml of serum-free medium buffered with 25mM HEPES containing 0.1% BSA and chilled. Cells were then labeled with 125I-CT (106cpm /2 ml 10nM) in the same medium containing 0.01 % bovine serum albumin for 30 min at 4°C to allow binding without endocytosis. Subsequently, cells were washed and harvested immediately (t=0) or washed and incubated for 10,30,60 or 120 min in serum-free medium at 37°C and harvested.Cells were pelleted by low speed centrifugation. The pellets were resuspended in 50mM tris-HCl pH 7.4 containing 250mM sucrose (Tris-sucrose) buffer and homogenized on ice with 4 x 4 strokes of a Potter Elvejhem homogenizer (position 9). The homogenates were spun at 3,000 x g for 10 min to pellet unbroken cells, cell debris and nuclei to yield the PNS. Membranes from PNS were obtained by centrifugation of the PNS at 100,000 x g for 1 h at 4°C. The membrane pellet was resuspended in Tris-sucrose buffer.

## **2.4 Gradient centrifugation**

Subcellular fractionation was always performed on freshly prepared post nuclear membranes or PNS from cells labeled with 125I-CT. Membrane pellets prepared from PNS (post nuclear membranes),resuspended in 3 ml of homogenization buffer (50mM Tris-HCl, pH 7.4 containing 0.25M sucrose) or equal volumes of PNS,werelayered on top of two layers of respectively 4 ml of 25% iodixanol and 4 ml of 50% iodixanol each in isoosmotic

Brefeldin A and Exo1 Completely Releave the Block of

blue, warmed at 37°C for 10 min and run on each slab gel.

re-culture time was 120 min this amount was less than 10%.

were not affected upon increasing the re-culture time (data not shown).

**3. Results** 

Cholera Toxin Action by a Dipeptide Metalloendoprotease Substrate 157

Each sample (10,000 cpm) was separated on 16% tris-glycine gel (Novex pre-cast gels). After the gels were run and stained with coomassie blue, protein bands corresponding to CT-A1 were cut from the gel, which was subsequently dissolved in lumasolveand, after addition of lipoluma (Lumac, LSC), counted in a liquid scintillation counter. CT reduced with dithiothreitol and dialysed against 10mM tris/HCl pH 7.4 containing 10mM NEM, to remove the excess of reducing agent, was adjusted to 1% SDS, 20% glycerol and 0.02% bromphenol

**3.1 Effect of Cbz-Gly-Phe-NH2 on the intracellular retrograde transport of CT** 

experiments on post-nuclear membranes of Vero cells with prebound125I-CT.

In order to assess whether the metalloendoprotease substrate Cbz-Gly-Phe-NH2 perturbs the uptake and intracellular trafficking of CT, we performed subcellular fractionation

The distribution profiles of marker enzymes after isopycnic gradient centrifugation of these membranes, using self-generating gradients of iodixanol, showed a reasonable separation of Golgi fractions (as represented by UDP-galactosyltransferase) from the ER fractions (rotenoneinsensitive NADPH cytochrome c reductase) and plasma membranes (alkaline phosphatase) (Fig.1,C). Membrane proteins were distributed all over the gradient (profile not shown).

At the outset,125I-CT was allowed to bind to Vero cells in suspension for 30 min at 4°C, minimizing endocytosis.Cells were washed and homogenized immediately (t=0) or they were washed and re-cultured for an additional 10 min (t=10); 30 min (t=30); 60 min (t=60) and 120 min (t=120) at 37°C prior to preparation of membranes from PNS. It was noticed that during re-culture, radioactivity was progressively released into the culture medium (after 10 h more than 60%, mostly (80%) as non-precipitable material).However, when the

As shown in Fig.1 panels A and C, the radioactivity profile at zero time corresponded to that of alkaline phosphatase, a cell-surface membrane marker. Upon increasing the re-culture time,ra-dioactivity shifted to higher densities. After 30 min most of the radioactivity equilibrated at densities (1.127g/ml) corresponding to Golgi-derived membranes, as evidenced by the distribution of the Golgi marker UDP-galactosyltransferase.At still later time points (60–120min),125I-CT further moved to higher densities corresponding to those of membranes from the rough ER, which is in agreement with the generally accepted retrograde transport of the toxin to the ER. The distribution profiles of marker enzymes

As shown in Fig.1,B, pre-treatment of Vero cells with Cbz-Gly-Phe-NH2 (3mM) for 30 min at 37° C, before binding and internalization of 125I-CT, markedly affected the distribution profiles of radioactivity upon increasing re-culture times. Whereas the internalization,in agreement with our previous results (Schmitz et al., 2000), was not affected, further transport of the toxin appeared to be strongly perturbed. After re-culture the toxin still moved to densities corresponding to those of Golgi-derived membranes, but at an apparently lower rate, and further transport to the ER appeared to be blocked. Pre-exposure of cells to Cbz-Gly-Phe-NH2 (3mM) did not significantly affect the distribution profiles of marker enzymes. Only the Golgi marker equilibrated at a somewhat higher density

homogenization buffer in a 11.5 ml centrifugation tube (Sorvall). The tubes were centrifuged in a (Sorvall TV-850) vertical rotor at 35,000 rpm for 18 h at 4°C using a Kontron centrifuge Centricon T2060. Fractions of 0.3 ml were collected from the bottom of the gradient using a device with a perforation needle and a density gradient fractionator. A 15 µl portion of each gradient fraction was used to determine its refractive index () as measured by an Abbe refractometer. From the refractive indices the densities of individual gradient fractions were calculated using the equation =3.4911-3.6664 as reported by Graham et al. (1994). Fractions were further analyzed for protein by the method of Bradford (1976) and for the trans-Golgi marker UDP-galactosyltransferase(Verdon and Berger, 1983) as well as the classical subcellular organelle markers rotenone-insensitive NADPH cytochrome creductase, alkaline phosphatase, acid phosphatase and cytochromecoxidase as described before (De Wolf et al., 1985).

### **2.5 Assay of cellular cyclic AMP content**

Monolayer cultures of cells were washed twice with Earle's balanced salt solution without Ca2+ and Mg2+ and treated with 0.05% (w/v) trypsin solution containing 0.02% EDTA in HBSS. After 1 min the trypsin solution was removed and 10-15 min later cells were suspended in culture medium. Cells were collected by centrifugation at 750 rpm for 5 min and resuspended in 1 ml serum-free medium containing 25mM Hepes, 0.01% (w/v) bovine serum albumin and 1mM 3-isobutyl-1-methylxanthine. 1 ml aliquots of cell suspension (105 cells/ml were divided over Eppendorf tubes and incubated with CT (1µg/ml) and the required effectors were added at the indicated times as described in the legend of figures. Afterwards, cell suspensions were put on ice, centrifuged for 6 min at 1000 rpm and resuspended in 0.1 ml 0.5M sodium acetate buffer (pH 6.2). The suspensions were boiled for 10 min and then sonicated for 30 s. After centrifugation for 10 min at 10000 rpm, 20 µl of cell extract was taken for cyclic AMP (cAMP) assay. cAMP was assayed using a cAMP assay kit (Pharmacia Amersham) based on a competitive protein-binding method. Results for cAMP represent the mean of values from duplicate samples each assayed in triplicate.

#### **2.6 Generation of CT-A1 and analysis by SDS-polyacrylamide gel electrophoresis**

Vero cells were grown to confluency in small (=3cm) petri dishes (250.106 cells) and maintained in culture for at least one week before use. Cells were washed once with serumfree medium buffered with 25mM Hepes and incubated with and without Cbz-Gly-Phe-NH2(3mM) for 30 min at 37°C.The medium was replaced with ice-cold serum-free medium/Hepes (total volume=3 ml) containing 125I-CT (106cpm/ml;1nM CT) and 0.01% BSA and the cells were further incubated at 4°C for 30 min.The cells were then washed with icecold serum-free medium/Hepes and incubated at 37°C for 60 min by replacing the medium with warm serum-free medium-/Hepes. With each medium change, Cbz-Gly-Phe-NH2(3mM) wasadded as required. The cell incubations were either stopped immediately or after the addition of BFA (1µg/ml), further incubated for 30 min at 37°C and then stopped. Incubations were stopped by adding 1 ml of ice-cold N-ethylmaleimide (NEM) (1mM) in phosphate-buffer saline to prevent any further reduction of CT (Kassis et al., 1982), scraped in PBS, and pelleted by low speed centrifugation. The cells were lysed and solubilised by addition of a small volume of 0.125M Tris/HCl (pH 8.0), 2mM phenyl-methyl sulfonylfluoride and 1 % SDS. After 10 min at 37°C the solubilised material was adjusted to 20% glycerol and 0.02% bromphenol blue and applied to the gels. The amount of CT-A1 generated was determined by SDS-PAGE. Each sample (10,000 cpm) was separated on 16% tris-glycine gel (Novex pre-cast gels). After the gels were run and stained with coomassie blue, protein bands corresponding to CT-A1 were cut from the gel, which was subsequently dissolved in lumasolveand, after addition of lipoluma (Lumac, LSC), counted in a liquid scintillation counter. CT reduced with dithiothreitol and dialysed against 10mM tris/HCl pH 7.4 containing 10mM NEM, to remove the excess of reducing agent, was adjusted to 1% SDS, 20% glycerol and 0.02% bromphenol blue, warmed at 37°C for 10 min and run on each slab gel.

## **3. Results**

156 Cholera

homogenization buffer in a 11.5 ml centrifugation tube (Sorvall). The tubes were centrifuged in a (Sorvall TV-850) vertical rotor at 35,000 rpm for 18 h at 4°C using a Kontron centrifuge Centricon T2060. Fractions of 0.3 ml were collected from the bottom of the gradient using a device with a perforation needle and a density gradient fractionator. A 15 µl portion of each gradient fraction was used to determine its refractive index () as measured by an Abbe refractometer. From the refractive indices the densities of individual gradient fractions were calculated using the equation =3.4911-3.6664 as reported by Graham et al. (1994). Fractions were further analyzed for protein by the method of Bradford (1976) and for the trans-Golgi marker UDP-galactosyltransferase(Verdon and Berger, 1983) as well as the classical subcellular organelle markers rotenone-insensitive NADPH cytochrome creductase, alkaline phosphatase,

acid phosphatase and cytochromecoxidase as described before (De Wolf et al., 1985).

represent the mean of values from duplicate samples each assayed in triplicate.

**2.6 Generation of CT-A1 and analysis by SDS-polyacrylamide gel electrophoresis** 

Vero cells were grown to confluency in small (=3cm) petri dishes (250.106 cells) and maintained in culture for at least one week before use. Cells were washed once with serumfree medium buffered with 25mM Hepes and incubated with and without Cbz-Gly-Phe-NH2(3mM) for 30 min at 37°C.The medium was replaced with ice-cold serum-free medium/Hepes (total volume=3 ml) containing 125I-CT (106cpm/ml;1nM CT) and 0.01% BSA and the cells were further incubated at 4°C for 30 min.The cells were then washed with icecold serum-free medium/Hepes and incubated at 37°C for 60 min by replacing the medium with warm serum-free medium-/Hepes. With each medium change, Cbz-Gly-Phe-NH2(3mM) wasadded as required. The cell incubations were either stopped immediately or after the addition of BFA (1µg/ml), further incubated for 30 min at 37°C and then stopped. Incubations were stopped by adding 1 ml of ice-cold N-ethylmaleimide (NEM) (1mM) in phosphate-buffer saline to prevent any further reduction of CT (Kassis et al., 1982), scraped in PBS, and pelleted by low speed centrifugation. The cells were lysed and solubilised by addition of a small volume of 0.125M Tris/HCl (pH 8.0), 2mM phenyl-methyl sulfonylfluoride and 1 % SDS. After 10 min at 37°C the solubilised material was adjusted to 20% glycerol and 0.02% bromphenol blue and applied to the gels. The amount of CT-A1 generated was determined by SDS-PAGE.

Monolayer cultures of cells were washed twice with Earle's balanced salt solution without Ca2+ and Mg2+ and treated with 0.05% (w/v) trypsin solution containing 0.02% EDTA in HBSS. After 1 min the trypsin solution was removed and 10-15 min later cells were suspended in culture medium. Cells were collected by centrifugation at 750 rpm for 5 min and resuspended in 1 ml serum-free medium containing 25mM Hepes, 0.01% (w/v) bovine serum albumin and 1mM 3-isobutyl-1-methylxanthine. 1 ml aliquots of cell suspension (105 cells/ml were divided over Eppendorf tubes and incubated with CT (1µg/ml) and the required effectors were added at the indicated times as described in the legend of figures. Afterwards, cell suspensions were put on ice, centrifuged for 6 min at 1000 rpm and resuspended in 0.1 ml 0.5M sodium acetate buffer (pH 6.2). The suspensions were boiled for 10 min and then sonicated for 30 s. After centrifugation for 10 min at 10000 rpm, 20 µl of cell extract was taken for cyclic AMP (cAMP) assay. cAMP was assayed using a cAMP assay kit (Pharmacia Amersham) based on a competitive protein-binding method. Results for cAMP

**2.5 Assay of cellular cyclic AMP content** 

## **3.1 Effect of Cbz-Gly-Phe-NH2 on the intracellular retrograde transport of CT**

In order to assess whether the metalloendoprotease substrate Cbz-Gly-Phe-NH2 perturbs the uptake and intracellular trafficking of CT, we performed subcellular fractionation experiments on post-nuclear membranes of Vero cells with prebound125I-CT.

The distribution profiles of marker enzymes after isopycnic gradient centrifugation of these membranes, using self-generating gradients of iodixanol, showed a reasonable separation of Golgi fractions (as represented by UDP-galactosyltransferase) from the ER fractions (rotenoneinsensitive NADPH cytochrome c reductase) and plasma membranes (alkaline phosphatase) (Fig.1,C). Membrane proteins were distributed all over the gradient (profile not shown).

At the outset,125I-CT was allowed to bind to Vero cells in suspension for 30 min at 4°C, minimizing endocytosis.Cells were washed and homogenized immediately (t=0) or they were washed and re-cultured for an additional 10 min (t=10); 30 min (t=30); 60 min (t=60) and 120 min (t=120) at 37°C prior to preparation of membranes from PNS. It was noticed that during re-culture, radioactivity was progressively released into the culture medium (after 10 h more than 60%, mostly (80%) as non-precipitable material).However, when the re-culture time was 120 min this amount was less than 10%.

As shown in Fig.1 panels A and C, the radioactivity profile at zero time corresponded to that of alkaline phosphatase, a cell-surface membrane marker. Upon increasing the re-culture time,ra-dioactivity shifted to higher densities. After 30 min most of the radioactivity equilibrated at densities (1.127g/ml) corresponding to Golgi-derived membranes, as evidenced by the distribution of the Golgi marker UDP-galactosyltransferase.At still later time points (60–120min),125I-CT further moved to higher densities corresponding to those of membranes from the rough ER, which is in agreement with the generally accepted retrograde transport of the toxin to the ER. The distribution profiles of marker enzymes were not affected upon increasing the re-culture time (data not shown).

As shown in Fig.1,B, pre-treatment of Vero cells with Cbz-Gly-Phe-NH2 (3mM) for 30 min at 37° C, before binding and internalization of 125I-CT, markedly affected the distribution profiles of radioactivity upon increasing re-culture times. Whereas the internalization,in agreement with our previous results (Schmitz et al., 2000), was not affected, further transport of the toxin appeared to be strongly perturbed. After re-culture the toxin still moved to densities corresponding to those of Golgi-derived membranes, but at an apparently lower rate, and further transport to the ER appeared to be blocked. Pre-exposure of cells to Cbz-Gly-Phe-NH2 (3mM) did not significantly affect the distribution profiles of marker enzymes. Only the Golgi marker equilibrated at a somewhat higher density

Brefeldin A and Exo1 Completely Releave the Block of

Phe-NH2-mediated inhibition of CT action.

CT action in a dose-dependent way(EC500.5µg/ml) ( Fig.2,B,D).

ARF1 from Golgi membranes in BSC1 fibroblasts (Feng et al., 2003).

CT ac-tion in a similar dose-dependent way (Fig.2,B,-D,F).

**inhibition of CT action** 

Cholera Toxin Action by a Dipeptide Metalloendoprotease Substrate 159

From the density gradient centrifugation experiments it is clear that the metalloendoprotease substrate Cbz-Gly-Phe-NH2 affects the intracellular transport of CT and that in its presence the toxin appears to be trapped in an intracellular compartment, which cofractionated with a marker of the Golgi apparatus.In order to find out whether in the presence of Cbz-Gly-Phe-NH2 the toxin travels beyond the trans-Golgi network (TGN) and reaches the cisternae of the Golgi complex, we explored whether drugs that are able to redistibute Golgi membranes and its content into the ER also caused a reversal of Cbz-Gly-

As shown in Fig.2,A,C and in agreement with previous results (De Wolf, 2000),prior incubation of Vero cells and T84 cells with Cbz-Gly-Phe-NH2 or with BFA for 30 min at 37°C resulted in respectively a complete or strong inhibition of CT action in a dose-dependent way. However, when these cells were preincubated with Cbz-Gly-Phe-NH2 (3mM) for 30 min at 37 °C and then incubated in the presence of CT (1µg/ml) for an additional 60 min time period, a time at which CT - as evidenced by the gradient centrifugation experiments - becomes trapped in a compartment where it is unable to reach the cytosol and raise intracellular cAMP levels, subsequent addition of BFA completely reversed the Cbz-Gly-Phe-NH2-mediated inhibition of

The concentrations at which reversal of the inhibition occurred, corresponded to the concentrations needed for inhibition of CT action and induction of the redistribution of Golgi

We next determined the effect of 2-(4-fluoro-benzoylamino)-benzoic acid methylester (Exo1), a novel chemical inhibitor of the exocytotic pathway (Feng et al., 2003).Like BFA, Exo1 induces the release of ADP-ribosylation factor (ARF)1 from Golgi membranes, inducing/generating/stimulating a rapid collapse of the Golgi into the endoplasmic reticulum in different cell types. However, unlike BFA this drug has less effect on the organization of the trans-Golgi network (Feng et al., 2003). As shown in Fig.2,A,C,E, prior exposure to Exo1 blocked the CT induced cAMP accumulation in all cell types tested. The effect was dose-dependent with an IC50 value of 0.35µM. This value is almost two orders of magnitude lower than that reported for its inhibitory effect on the anterograde movement of the viral glycoprotein VSVG from the ER to the Golgi and its stimulation of the release or

As expected, Exo1 also completely reversed the Cbz-Gly-Phe-NH2-mediated inhibition of

We previously showed (De Wolf, 2000)that there are some similarities in the inhibitory effects of Cbz-Gly-Phe-NH2 and BFA on CT action. However, Madin-Darby canine kidney epithelial (MDCK) cells, of which the Golgi structure is BFA resistant (Hunziker et al., 1991)and as a consequence are insensitive to the inhibitory effect of BFA on CT action, were sensitive to the inhibitory effect of Cbz-Gly-Phe-NH2.In agreement with our previous results (De Wolf, 2000),prior exposure of these cells toCbz-Gly-Phe-NH2(Fig.2,E) completely suppressed (IC50=0.5mM ) CT action. Therefore, it was of interest to determine whether BFA is also able to reverse the inhibitory effect of Cbz-Gly-Phe-NH2 on CT action in MDCK cells.

membranes into the ER (Fig.2,A,C) (Doms et al., 1989; Lippincott-Schwartz et al., 1989).

**3.2 Effect of drugs perturbing the Golgi structure on theCbz-Gly-Phe-NH2-mediated** 

(1.132g/ml versus 1.127g/ml) but clearly did not shift to densities corresponding to the ER, as was the case after pretreatment of cells with BFA (data not shown).

Fig. 1. Subcellular fractionation of post-nuclear membranes from Vero cells prelabeled with 125I-CT.Vero cells prelabeled with 125I-CT at low temperature to block endocytosis were, after washing, incubated at 37°C for 0 time (), 15 min (), 30 min (), 60 min () or 120 min () and post-nuclear membranes were prepared and centrifuged through a self-generating gradient of iodixanol as described under Materials and Methods. A. Subcellular distribution profiles of membrane-bound 125I-CT after increasing re-culture periods.B. Subcellular distribution profiles of membrane-bound 125I-CT after pretreatment of Vero cells with Cbz-Gly-Phe-NH2 and different re-culture periods. Vero cells in suspension were pretreated with Cbz-Gly-Phe-NH2 (3mM) for 30 min at 37°C. The cells were chilled and after binding of 125I-CT at 4°C, washed and re-cultured for different periods of time in the presence of Cbz-Gly-Phe-NH2 (3mM) at 37°C.C. Distribution profiles of subcellular marker enzymes at zero time (no-reculture),() Alkaline phosphatase, () UDP- galactosyltransferase, () NADPH cytochrome creductase, () Acid phosphatase, () cytcoxidase. The distribution profiles are representative for three similar experiments.

(1.132g/ml versus 1.127g/ml) but clearly did not shift to densities corresponding to the ER,

Fig. 1. Subcellular fractionation of post-nuclear membranes from Vero cells prelabeled with 125I-CT.Vero cells prelabeled with 125I-CT at low temperature to block endocytosis were, after washing, incubated at 37°C for 0 time (), 15 min (), 30 min (), 60 min () or 120 min () and post-nuclear membranes were prepared and centrifuged through a self-generating gradient of iodixanol as described under Materials and Methods. A. Subcellular distribution profiles of membrane-bound 125I-CT after increasing re-culture periods.B. Subcellular distribution profiles of membrane-bound 125I-CT after pretreatment of Vero cells with Cbz-Gly-Phe-NH2 and different re-culture periods. Vero cells in suspension were pretreated with Cbz-Gly-Phe-NH2 (3mM) for 30 min at 37°C. The cells were chilled and after binding of 125I-CT at 4°C, washed and re-cultured for different periods of time in the presence of Cbz-Gly-Phe-NH2 (3mM) at 37°C.C. Distribution profiles of subcellular marker enzymes at zero time (no-reculture),() Alkaline phosphatase, () UDP- galactosyltransferase, () NADPH cytochrome creductase, () Acid phosphatase, () cytcoxidase. The distribution profiles are

representative for three similar experiments.

as was the case after pretreatment of cells with BFA (data not shown).

#### **3.2 Effect of drugs perturbing the Golgi structure on theCbz-Gly-Phe-NH2-mediated inhibition of CT action**

From the density gradient centrifugation experiments it is clear that the metalloendoprotease substrate Cbz-Gly-Phe-NH2 affects the intracellular transport of CT and that in its presence the toxin appears to be trapped in an intracellular compartment, which cofractionated with a marker of the Golgi apparatus.In order to find out whether in the presence of Cbz-Gly-Phe-NH2 the toxin travels beyond the trans-Golgi network (TGN) and reaches the cisternae of the Golgi complex, we explored whether drugs that are able to redistibute Golgi membranes and its content into the ER also caused a reversal of Cbz-Gly-Phe-NH2-mediated inhibition of CT action.

As shown in Fig.2,A,C and in agreement with previous results (De Wolf, 2000),prior incubation of Vero cells and T84 cells with Cbz-Gly-Phe-NH2 or with BFA for 30 min at 37°C resulted in respectively a complete or strong inhibition of CT action in a dose-dependent way.

However, when these cells were preincubated with Cbz-Gly-Phe-NH2 (3mM) for 30 min at 37 °C and then incubated in the presence of CT (1µg/ml) for an additional 60 min time period, a time at which CT - as evidenced by the gradient centrifugation experiments - becomes trapped in a compartment where it is unable to reach the cytosol and raise intracellular cAMP levels, subsequent addition of BFA completely reversed the Cbz-Gly-Phe-NH2-mediated inhibition of CT action in a dose-dependent way(EC500.5µg/ml) ( Fig.2,B,D).

The concentrations at which reversal of the inhibition occurred, corresponded to the concentrations needed for inhibition of CT action and induction of the redistribution of Golgi membranes into the ER (Fig.2,A,C) (Doms et al., 1989; Lippincott-Schwartz et al., 1989).

We next determined the effect of 2-(4-fluoro-benzoylamino)-benzoic acid methylester (Exo1), a novel chemical inhibitor of the exocytotic pathway (Feng et al., 2003).Like BFA, Exo1 induces the release of ADP-ribosylation factor (ARF)1 from Golgi membranes, inducing/generating/stimulating a rapid collapse of the Golgi into the endoplasmic reticulum in different cell types. However, unlike BFA this drug has less effect on the organization of the trans-Golgi network (Feng et al., 2003). As shown in Fig.2,A,C,E, prior exposure to Exo1 blocked the CT induced cAMP accumulation in all cell types tested. The effect was dose-dependent with an IC50 value of 0.35µM. This value is almost two orders of magnitude lower than that reported for its inhibitory effect on the anterograde movement of the viral glycoprotein VSVG from the ER to the Golgi and its stimulation of the release or ARF1 from Golgi membranes in BSC1 fibroblasts (Feng et al., 2003).

As expected, Exo1 also completely reversed the Cbz-Gly-Phe-NH2-mediated inhibition of CT ac-tion in a similar dose-dependent way (Fig.2,B,-D,F).

We previously showed (De Wolf, 2000)that there are some similarities in the inhibitory effects of Cbz-Gly-Phe-NH2 and BFA on CT action. However, Madin-Darby canine kidney epithelial (MDCK) cells, of which the Golgi structure is BFA resistant (Hunziker et al., 1991)and as a consequence are insensitive to the inhibitory effect of BFA on CT action, were sensitive to the inhibitory effect of Cbz-Gly-Phe-NH2.In agreement with our previous results (De Wolf, 2000),prior exposure of these cells toCbz-Gly-Phe-NH2(Fig.2,E) completely suppressed (IC50=0.5mM ) CT action. Therefore, it was of interest to determine whether BFA is also able to reverse the inhibitory effect of Cbz-Gly-Phe-NH2 on CT action in MDCK cells.

Brefeldin A and Exo1 Completely Releave the Block of

GAP activity(Feng et al., 2003).

strongly reduced cell viability.

ribosylation.

Cbz-Gly-Phe-NH2 in a similar dose-dependent way (Fig.2,-E,F).

Cholera Toxin Action by a Dipeptide Metalloendoprotease Substrate 161

As shown in Fig.2,E,F, BFA did neither prevent CT induced cAMP accumulation nor reverse the inhibitory effect of Cbz-Gly-Phe-NH2 on CT action in MDCK cells. In contrast, Exo1, which is able to prevent CT action in these cells, was also able to reverse the inhibition by

This is in line with the proposal (Feng et al., 2003)that although Exo1 and BFA are exerting similar effects they probably have different protein targets. Whereas BFA blocks GDP to GTP exchange on ARF1 and therefore reduces the concentration of ARF1-GTP on Golgi membranes, it is believed that Exo1 also reduces the concentration of ARF1-GTP on Golgi membranes by accelerating the hydrolysis of GTP bound to ARF1 by an activation of ARF1-

By causing the release of ARF1 and COPI from membranes, BFA and Exo1 directly interfere with the Golgi-ER retrograde trafficking machinery and this likely perturbs normal recycling from the Golgi to the ER. Therefore it was of interest to see whether a treatment of cells, which affects the Golgi structure without directly interfering with the retrograde transport, also caused a reversal of the inhibition of the CT action by Cbz-Gly-Phe-NH2.

A constant influx of membrane from the ER is required to maintain the Golgi structure. Microtubule disruption prevents this influx by blocking the peripherial pre-Golgi intermediates from tracking into the Golgi region (Cole et al., 1996; Storrie et al., 1998). The microtubule depolymerizing agent nocodazole, which blocks the forward traffic into the Golgi complex without a corresponding effect on recycling, leads to the fragmentation of the Golgi complex and redistribution of its material to the site of perturbation (Cole et al., 1996). Whereas prior exposure of cells to nocodazole did not affect CT action (Fig.2,A,C,E), it was able to reverse the Cbz-Gly-Phe-NH2-mediated inhibition of CT action in a dose-dependent way. Maximal reversal was observed at a concentration of 0.5µg/ml. The effect was most pronounced in Vero cells (Fig.2,B), whereas in T84 and MDCK cells the effect was minimal. Increasing the concentration of nocodazole above 2µg/ml impairedthe effect because of

**3.3 Time dependence of the BFA-, Exo1-and nocodazole-induced reversal of the** 

As depicted in Fig.3 BFA (2µg/ml), Exo1 (2µM) and nocodazole (1µg/ml) caused a rapid increase in the cAMP concentration after CT had accumulated in an intracellular compartment in the presence of Cbz-Gly-Phe-NH2 (3mM). Already within 1 min after the addition of each drug a significant increase in cAMP accumulation could be observed and after a 5 to 10 min time period the cAMP concentration reached its maximal value. This time course is similar to that observed for the redistribution of Golgi membranes into the ER (Lippincott-Schwartz et al., 1989; Feng et al., 2003; Sciaky et al., 1997)and indicates that once CT has reached the Golgi, the BFA-, Exo1-or nocodazole-induced redistribution of Golgi membranes and content into the ER is followed by a very fast activation of the adenylyl cyclase.Thisimplies that within approximately one minute a fraction of the toxin reaches the ER, becomes activated by reduction, translocates into the cytosol and gains access to its substrate the GSsubunit of GS, which finally activates the cyclase after mono-ADP

**inhibitory effect of Cbz-Gly-Phe-NH2on CTaction in Vero cells** 

Fig. 2. Inhibition of CT action by different concentrations of BFA, Exo1 and nocodazole and their effect on the Cbz-Gly-Phe-NH2 - mediated inhibition of CT action in several cell types. A, Vero cells; C, T84 human intestinal epithelial cells and E, MDCK cells in suspension were preincubated at 37°C for 30 min in the presence of the indicated amounts of BFA (), Exo1 (), nocodazole () or Cbz-Gly-Phe-NH2 (). After the addition of CT (1µg/ml) cells were further incubated for 90 min at 37°C and the cAMP accumulation measured. B, Vero cells; D, T84 human, intestinal epithelial cells and F, MDCK cells were preincubated with Cbz-Gly-Phe-NH2 (3mM) for 30 min at 37°C. Following the addition of CT (1µg/ml) cells were further incubated for 60 min at 37°C. Finally, the indicated amounts of BFA (),Exo1 () and nocodazole ()were added and the cells incubated for an additional 30 min at 37°C and cAMP measured. Values are the means SD of triplicate assays from one of three similar experiments. Error bars are indicated when they were more than 5% of the mean.

Fig. 2. Inhibition of CT action by different concentrations of BFA, Exo1 and nocodazole and their effect on the Cbz-Gly-Phe-NH2 - mediated inhibition of CT action in several cell types. A, Vero cells; C, T84 human intestinal epithelial cells and E, MDCK cells in suspension were preincubated at 37°C for 30 min in the presence of the indicated amounts of BFA (), Exo1 (), nocodazole () or Cbz-Gly-Phe-NH2 (). After the addition of CT (1µg/ml) cells were further incubated for 90 min at 37°C and the cAMP accumulation measured. B, Vero cells; D, T84 human, intestinal epithelial cells and F, MDCK cells were preincubated with Cbz-Gly-Phe-NH2 (3mM) for 30 min at 37°C. Following the addition of CT (1µg/ml) cells were further incubated for 60 min at 37°C. Finally, the indicated amounts of BFA (),Exo1 () and nocodazole ()were added and the cells incubated for an additional 30 min at 37°C and cAMP measured. Values are the means SD of triplicate assays from one of three similar experiments. Error bars are indicated when they were more than 5% of the mean.

As shown in Fig.2,E,F, BFA did neither prevent CT induced cAMP accumulation nor reverse the inhibitory effect of Cbz-Gly-Phe-NH2 on CT action in MDCK cells. In contrast, Exo1, which is able to prevent CT action in these cells, was also able to reverse the inhibition by Cbz-Gly-Phe-NH2 in a similar dose-dependent way (Fig.2,-E,F).

This is in line with the proposal (Feng et al., 2003)that although Exo1 and BFA are exerting similar effects they probably have different protein targets. Whereas BFA blocks GDP to GTP exchange on ARF1 and therefore reduces the concentration of ARF1-GTP on Golgi membranes, it is believed that Exo1 also reduces the concentration of ARF1-GTP on Golgi membranes by accelerating the hydrolysis of GTP bound to ARF1 by an activation of ARF1- GAP activity(Feng et al., 2003).

By causing the release of ARF1 and COPI from membranes, BFA and Exo1 directly interfere with the Golgi-ER retrograde trafficking machinery and this likely perturbs normal recycling from the Golgi to the ER. Therefore it was of interest to see whether a treatment of cells, which affects the Golgi structure without directly interfering with the retrograde transport, also caused a reversal of the inhibition of the CT action by Cbz-Gly-Phe-NH2.

A constant influx of membrane from the ER is required to maintain the Golgi structure. Microtubule disruption prevents this influx by blocking the peripherial pre-Golgi intermediates from tracking into the Golgi region (Cole et al., 1996; Storrie et al., 1998). The microtubule depolymerizing agent nocodazole, which blocks the forward traffic into the Golgi complex without a corresponding effect on recycling, leads to the fragmentation of the Golgi complex and redistribution of its material to the site of perturbation (Cole et al., 1996).

Whereas prior exposure of cells to nocodazole did not affect CT action (Fig.2,A,C,E), it was able to reverse the Cbz-Gly-Phe-NH2-mediated inhibition of CT action in a dose-dependent way. Maximal reversal was observed at a concentration of 0.5µg/ml. The effect was most pronounced in Vero cells (Fig.2,B), whereas in T84 and MDCK cells the effect was minimal. Increasing the concentration of nocodazole above 2µg/ml impairedthe effect because of strongly reduced cell viability.

### **3.3 Time dependence of the BFA-, Exo1-and nocodazole-induced reversal of the inhibitory effect of Cbz-Gly-Phe-NH2on CTaction in Vero cells**

As depicted in Fig.3 BFA (2µg/ml), Exo1 (2µM) and nocodazole (1µg/ml) caused a rapid increase in the cAMP concentration after CT had accumulated in an intracellular compartment in the presence of Cbz-Gly-Phe-NH2 (3mM). Already within 1 min after the addition of each drug a significant increase in cAMP accumulation could be observed and after a 5 to 10 min time period the cAMP concentration reached its maximal value. This time course is similar to that observed for the redistribution of Golgi membranes into the ER (Lippincott-Schwartz et al., 1989; Feng et al., 2003; Sciaky et al., 1997)and indicates that once CT has reached the Golgi, the BFA-, Exo1-or nocodazole-induced redistribution of Golgi membranes and content into the ER is followed by a very fast activation of the adenylyl cyclase.Thisimplies that within approximately one minute a fraction of the toxin reaches the ER, becomes activated by reduction, translocates into the cytosol and gains access to its substrate the GSsubunit of GS, which finally activates the cyclase after mono-ADP ribosylation.

Brefeldin A and Exo1 Completely Releave the Block of

continued for 30 min at 37°C.

**that redistributes into the ER upon addition of BFA** 

min and cAMP accumulation measured.

addition of BFA.

Cholera Toxin Action by a Dipeptide Metalloendoprotease Substrate 163

(Tsai and Rapoport, 2002). As shown in Table I, incubation of cells with 5mM dithiothreitol (DTT), a reducing agent known to permeate into the ER of intact cells (Braakman et al., 1992)or the oxidant diamide (0.5mM),did almost not influence the BFA-induced reversal of the inhibitory effect ofCbz-Gly-Phe-NH2. Also, the membrane-permeant sulfhydryl blocker NEM, which at a concentration of 10µM has been shown to completely inhibit the reduction of CT to CT-A1 by intact CaCo-2 cells (Orlandi, 1997),had only a minor effect on the BFA-

induced restoration of CT toxicity in the presence of Cbz-Gly-Phe-NH2 (Table I).

Control 7.5 0.3 DTT (5mM) 6.7 0.3 Diamide (0.5mM) 7.5 0.3 NEM (0.01mM) 7.4 0.2 NEM (0.1mM) 5.8 0.2 2-deoxy-D-glucose (50mM) + 0.02 % sodium azide 2.0 0.2 Nocodazole (2µM) 7.4 0.3 1,3-cyclohexanebis(methylamine) (2mM) 7.5 0.2 All treatments no BFA added 0.4 0.2

Treatment Accumulation of cAMP pmoles/105 cells

Table 1. Effect of several treatments on the BFA-induced reversal of the inhibitory effect of Cbz-Gly-Phe-NH2 on CT cytotoxicity. Vero cells in suspension were preincubated for 30 min at 37°C with Cbz-Gly-Phe-NH2 (3mM). After addition of CT (1µg/ml) cells were further incubated for 60 min at 37°C. The indicated amounts of agents were added and the cells incubated for 15 min at 37°C. Finally, BFA (1µg/ml) wad added and the incubation

**3.4 Kinetics of CT transport in the presence of Cbz-Gly-Phe-NH2 to a compartment** 

compartment that redistributes into the ER following the addition of BFA.

immunofluorescence studies (Majoul et al., 1996) on Vero cells.

min) redistribution of the Golgi lipids and proteins into the ER.

The rapidBFA-induced reversal of the inhibition of CT action by Cbz-Gly-Phe-NH2 allowed us to estimate the time it takes for CT to reach, in the presence of Cbz-Gly-Phe-NH2,a

In these experiments, cells (Vero,T84 and MDCK) were preincubated with Cbz-Gly-Phe-NH2 (3mM) for 30 min at 37°C. CT (1µg/ml) was added and the cells further incubated for the indicated times. Finally, BFA (5µg/ml) was added and the incubation continued for 90

As shown in Fig.4, in the presence of Cbz-Gly-Phe-NH2 substantial amounts of CTreached, within approximately 5 to10 min, a compartment that redistributes into the ER following the

This time period corresponds to the time needed for CT to reach the Golgi complex, as evidenced by our subcellular fractionation experiments (Fig.1) and previous

In these experiments theBFA concentration was kept high to induce rapid (within less than 5

Fig. 3. Time dependence of the BFA-, Exo1- and nocodazole-induced reversal of the inhibitory effect of Cbz-Gly-Phe-NH2 on the CT-induced accumulation of cAMP in Vero cells. Vero cells in suspension were preincubated with Cbz-Gly-Phe-NH2 (3mM) for 30 min at 37°C. After the addition of CT (1µg/ml) cells were further incubated for 60 min at 37°C. Subsequently, BFA (2µg/ml) (), Exo1 (2µM) () and nocodazole (1µg/ml) () were added and the cells incubated for the indicated times followed by a determination of cAMP. Data points are the means of triplicate assays from one of at least three similar experiments.

Therefore we assume that, at least in the presence of Cbz-Gly-Phe-NH2,CT also becomes activated at the level of the Golgi apparatus, and that in the ER the A1 fragment is rapidly translocated into the cytosol. A generation of CT-A1 at the level of the Golgi complex is in line with our previous observation (De Wolf, 2000)that in the presence of Cbz-Gly-Phe-NH2 (3mM), CT-A1 can still be formed. Furthermore, in this study we found that upon direct quantitation of the amount of CT-A1 generated, the BFA-induced redistribution of Golgi membranes into the ER did not increase the fraction of CT-A that became reduced in cells pretreated with Cbz-Gly-Phe-NH2 (3mM) (data not shown).

Likewise, the BFA-induced reversal of the inhibitory effect ofCbz-Gly-Phe-NH2on the CT action was hardly affected by the addition of agents that are able to change the redox potential of the ER and affect the unfolding of CT and the generation of CT-A1 fragment

Fig. 3. Time dependence of the BFA-, Exo1- and nocodazole-induced reversal of the inhibitory effect of Cbz-Gly-Phe-NH2 on the CT-induced accumulation of cAMP in Vero cells. Vero cells in suspension were preincubated with Cbz-Gly-Phe-NH2 (3mM) for 30 min at 37°C. After the addition of CT (1µg/ml) cells were further incubated for 60 min at 37°C. Subsequently, BFA (2µg/ml) (), Exo1 (2µM) () and nocodazole (1µg/ml) () were added and the cells incubated for the indicated times followed by a determination of cAMP. Data points are the means of triplicate assays from one of at least three similar experiments.

pretreated with Cbz-Gly-Phe-NH2 (3mM) (data not shown).

Therefore we assume that, at least in the presence of Cbz-Gly-Phe-NH2,CT also becomes activated at the level of the Golgi apparatus, and that in the ER the A1 fragment is rapidly translocated into the cytosol. A generation of CT-A1 at the level of the Golgi complex is in line with our previous observation (De Wolf, 2000)that in the presence of Cbz-Gly-Phe-NH2 (3mM), CT-A1 can still be formed. Furthermore, in this study we found that upon direct quantitation of the amount of CT-A1 generated, the BFA-induced redistribution of Golgi membranes into the ER did not increase the fraction of CT-A that became reduced in cells

Likewise, the BFA-induced reversal of the inhibitory effect ofCbz-Gly-Phe-NH2on the CT action was hardly affected by the addition of agents that are able to change the redox potential of the ER and affect the unfolding of CT and the generation of CT-A1 fragment (Tsai and Rapoport, 2002). As shown in Table I, incubation of cells with 5mM dithiothreitol (DTT), a reducing agent known to permeate into the ER of intact cells (Braakman et al., 1992)or the oxidant diamide (0.5mM),did almost not influence the BFA-induced reversal of the inhibitory effect ofCbz-Gly-Phe-NH2. Also, the membrane-permeant sulfhydryl blocker NEM, which at a concentration of 10µM has been shown to completely inhibit the reduction of CT to CT-A1 by intact CaCo-2 cells (Orlandi, 1997),had only a minor effect on the BFAinduced restoration of CT toxicity in the presence of Cbz-Gly-Phe-NH2 (Table I).


Table 1. Effect of several treatments on the BFA-induced reversal of the inhibitory effect of Cbz-Gly-Phe-NH2 on CT cytotoxicity. Vero cells in suspension were preincubated for 30 min at 37°C with Cbz-Gly-Phe-NH2 (3mM). After addition of CT (1µg/ml) cells were further incubated for 60 min at 37°C. The indicated amounts of agents were added and the cells incubated for 15 min at 37°C. Finally, BFA (1µg/ml) wad added and the incubation continued for 30 min at 37°C.

### **3.4 Kinetics of CT transport in the presence of Cbz-Gly-Phe-NH2 to a compartment that redistributes into the ER upon addition of BFA**

The rapidBFA-induced reversal of the inhibition of CT action by Cbz-Gly-Phe-NH2 allowed us to estimate the time it takes for CT to reach, in the presence of Cbz-Gly-Phe-NH2,a compartment that redistributes into the ER following the addition of BFA.

In these experiments, cells (Vero,T84 and MDCK) were preincubated with Cbz-Gly-Phe-NH2 (3mM) for 30 min at 37°C. CT (1µg/ml) was added and the cells further incubated for the indicated times. Finally, BFA (5µg/ml) was added and the incubation continued for 90 min and cAMP accumulation measured.

As shown in Fig.4, in the presence of Cbz-Gly-Phe-NH2 substantial amounts of CTreached, within approximately 5 to10 min, a compartment that redistributes into the ER following the addition of BFA.

This time period corresponds to the time needed for CT to reach the Golgi complex, as evidenced by our subcellular fractionation experiments (Fig.1) and previous immunofluorescence studies (Majoul et al., 1996) on Vero cells.

In these experiments theBFA concentration was kept high to induce rapid (within less than 5 min) redistribution of the Golgi lipids and proteins into the ER.

Brefeldin A and Exo1 Completely Releave the Block of

1997; Majoul et al., 1996; Lippincott-Schwartz, 1990).

dissociation(Donaldson et al., 1991a; Finazzi et al., 1994).

GTP hydrolysis (Rittinger et al., 1997; Scheffzek et al., 1997).

**action by Cbz-Gly-Phe-NH2**

thecAMP content determined.

**action by Cbz-Gly-Phe-NH2**

**-**

is that AlF4-locksARF1 in a GDP.AlF4-

Phe-NH2. From Fig.5,A it is clear that AlF4-


reverse the effect of Cbz-Gly-Phe-NH2.

activation of GS,since addition of AlF4

**3.5.1 Effect of AlF4**

cells (data not shown).

It has been reported that AlF4

However, the effect of AlF4

Cholera Toxin Action by a Dipeptide Metalloendoprotease Substrate 165

It has been shown previously (Lippincott-Schwartz, 1990)that within minutes of adding BFA to most cells, the Golgi apparatus disassembles, giving rise to long, uncoated tubules that extend along microtubules fusing uniquely with the ER. Several treatments have been

forskolin and some of its derivatives, reduced temperature and energy depletion (Orlandi,

In order to further examine whether the BFA- and Exo1-induced reversal of the inhibition of CT action by Cbz-Gly-Phe-NH2 is related to tubulation and disassembly of the Golgi

To this end, Vero cells in suspension were preincubated for 30 min at 37°C with Cbz-Gly-Phe-NH2 (3mM). Subsequently, CT (1µg/ml) was added and the cells further incubated for 60 min at 37°C. The cells were then treated with the different reagents and finally BFA (1µg/ml) or Exo1 (2µM) wasadded and the incubation continued for 30 min at 37°C and

*A*lF4- stabilizes coatomer binding to Golgi membranes in vitro and in vivo and renders COPI highly resistant to removal by BFA, apparently by inhibition of COPI

The mechanism by which AlF4- stabilizes COPI binding is not well understood but probably in-volvestrimeric G-proteins(Donaldson et al., 1991b; Helms et al., 1998). Another possibility

cofactor GAP, as shown for other small GTPases,and in this way reduces the overall rate of

As shown in Fig.5,A, treatment of Vero cells with 30mM NaF plus 50µM AlCl3 elicted, however, only a minor inhibitory effect on the BFA induced reversal of the inhibition of CT action by Cbz-Gly-Phe-NH2. This slight reduction of the reversal of the inhibition of CT action by Cbz-Gly-Phe-NH2 appearedto be cell specific, since it was not observed with T84

bound ARFwt-GTP from Golgi membranes (Feng et al., 2003). Therefore, we also determined the effect of AlF4-on the Exo1-induced reversal of the inhibition of CT action by Cbz-Gly-

AlF4- is able to directly interact with heterotrimeric G-proteins and, as described above, with

Cbz-Gly-Phe-NH2 wasmost likely not influenced by an increase of the cAMP level by a direct

small GTP binding proteins complexed with specific GTPase activating proteins.


**on the BFA- and Exo1- induced reversal of the inhibition of CT** 

binding transition state together with a limiting

almostcompletely blocked the ability of Exo1 to


on the BFA-and Exo1-induced reversal of the inhibitory action of

to intact cells hadonly a minor effect on the cAMP level

, nocodazole,

**3.5 Characteristics of the BFA-and Exo1- induced reversal of the inhibition of CT** 

shown to inhibit this BFA-induced tubule formation. These included AlF4-

apparatus, similar treatments affecting Golgi tubule formation were applied.

Fig. 4. Kinetics of CT transport in the presence of Cbz-Gly-Phe-NH2 to a compartment which is redistributed to the ER following the addition of BFA. Vero cells (A,B), T84 (C,D) and MDCK cells (E,F) were preincubated with (,) and without (,) Cbz-Gly-Phe-NH2 (3mM) for 30 min at 37°C. Subsequently, CT (1µg/ml) was added and the cells incubated at 37°C for the indicated times. Finally, cells were incubated in the presence (,) and absence (,) of BFA (2µg/ml) for 90 min at 37°C and the cAMP accumulation determined. Values are the means SD of triplicate assays from one of three similar experiments. Error bars are indicated when they were more than 5% of the mean.

Fig. 4. Kinetics of CT transport in the presence of Cbz-Gly-Phe-NH2 to a compartment which is redistributed to the ER following the addition of BFA. Vero cells (A,B), T84 (C,D) and MDCK cells (E,F) were preincubated with (,) and without (,) Cbz-Gly-Phe-NH2 (3mM) for 30 min at 37°C. Subsequently, CT (1µg/ml) was added and the cells incubated at 37°C for the indicated times. Finally, cells were incubated in the presence (,) and absence (,) of BFA (2µg/ml) for 90 min at 37°C and the cAMP accumulation determined. Values are the means SD of triplicate assays from one of three similar experiments. Error bars are

indicated when they were more than 5% of the mean.

### **3.5 Characteristics of the BFA-and Exo1- induced reversal of the inhibition of CT action by Cbz-Gly-Phe-NH2**

It has been shown previously (Lippincott-Schwartz, 1990)that within minutes of adding BFA to most cells, the Golgi apparatus disassembles, giving rise to long, uncoated tubules that extend along microtubules fusing uniquely with the ER. Several treatments have been shown to inhibit this BFA-induced tubule formation. These included AlF4 - , nocodazole, forskolin and some of its derivatives, reduced temperature and energy depletion (Orlandi, 1997; Majoul et al., 1996; Lippincott-Schwartz, 1990).

In order to further examine whether the BFA- and Exo1-induced reversal of the inhibition of CT action by Cbz-Gly-Phe-NH2 is related to tubulation and disassembly of the Golgi apparatus, similar treatments affecting Golgi tubule formation were applied.

To this end, Vero cells in suspension were preincubated for 30 min at 37°C with Cbz-Gly-Phe-NH2 (3mM). Subsequently, CT (1µg/ml) was added and the cells further incubated for 60 min at 37°C. The cells were then treated with the different reagents and finally BFA (1µg/ml) or Exo1 (2µM) wasadded and the incubation continued for 30 min at 37°C and thecAMP content determined.

#### **3.5.1 Effect of AlF4 on the BFA- and Exo1- induced reversal of the inhibition of CT action by Cbz-Gly-Phe-NH2**

*A*lF4 - stabilizes coatomer binding to Golgi membranes in vitro and in vivo and renders COPI highly resistant to removal by BFA, apparently by inhibition of COPI dissociation(Donaldson et al., 1991a; Finazzi et al., 1994).

The mechanism by which AlF4- stabilizes COPI binding is not well understood but probably in-volvestrimeric G-proteins(Donaldson et al., 1991b; Helms et al., 1998). Another possibility is that AlF4-locksARF1 in a GDP.AlF4 binding transition state together with a limiting cofactor GAP, as shown for other small GTPases,and in this way reduces the overall rate of GTP hydrolysis (Rittinger et al., 1997; Scheffzek et al., 1997).

As shown in Fig.5,A, treatment of Vero cells with 30mM NaF plus 50µM AlCl3 elicted, however, only a minor inhibitory effect on the BFA induced reversal of the inhibition of CT action by Cbz-Gly-Phe-NH2. This slight reduction of the reversal of the inhibition of CT action by Cbz-Gly-Phe-NH2 appearedto be cell specific, since it was not observed with T84 cells (data not shown).

It has been reported that AlF4-blocks the ability of Exo1 to induce dissociation of membranebound ARFwt-GTP from Golgi membranes (Feng et al., 2003). Therefore, we also determined the effect of AlF4-on the Exo1-induced reversal of the inhibition of CT action by Cbz-Gly-Phe-NH2. From Fig.5,A it is clear that AlF4 almostcompletely blocked the ability of Exo1 to reverse the effect of Cbz-Gly-Phe-NH2.

AlF4- is able to directly interact with heterotrimeric G-proteins and, as described above, with small GTP binding proteins complexed with specific GTPase activating proteins.

However, the effect of AlF4 on the BFA-and Exo1-induced reversal of the inhibitory action of Cbz-Gly-Phe-NH2 wasmost likely not influenced by an increase of the cAMP level by a direct activation of GS,since addition of AlF4 to intact cells hadonly a minor effect on the cAMP level

Brefeldin A and Exo1 Completely Releave the Block of

al., 1999).

Cholera Toxin Action by a Dipeptide Metalloendoprotease Substrate 167

reversal of the inhibition of CT action by Cbz-Gly-Phe-NH2.This dibasic compound itself did also not reverse the inhibitory effect of Cbz-Gly-Phe-NH2 (data not shown), which is consistent with its inability to cause a redistribution of Golgi membranes into the ER (Hu et

In agreement with previous results (Chen et al., 2002),pre-treatment of Vero cells with CBM

These experiments indicate that there is no straightforward relationship between COPI dissociation and redistribution of Golgi membranes into the ER and also argue against

Fig. 6. Effect of 1,9-dideoxyforskolin on the BFA- or Exo1-induced reversal of the inhibition of CT action by Cbz-Gly-Phe-NH2. A. Effect of 1,9-dideoxyforskolin concentration.Vero cells in suspension were preincubated for 30 min at 37°C with Cbz-Gly-Phe-NH2 (3mM). CT (1µg/ml) was added and the cells further incubated for 60 min at 37°C. The indicated amounts of 1,9-dideoxyforskolin were added and after 15 min BFA (1 µg/ml) () or Exo1 (2µM) () were added and the cells finally incubated for an additional 30 min at 37°C and cAMP accumulation measured. B. Effect of 1,9-dideoxyforskolin on the time courses of the BFA or Exo1 induced reversal of the inhibition of CT action by Cbz-Gly-Phe-NH2.Cells were preincubated as described under A. After CT treatment cells were incubated in the presence and absence of 1,9 dideoxyforskolin (150µM) for 15 min and BFA (1µg/ml) (,) or Exo1 (2µM) (,) were added and the cells further incubated at 37°C for the indicated times. Values are the means SD of triplicate assays from one of three similar experiments. Error

**3.5.2 Effect of 1,9-dideoxyforskolin on the BFA- and Exo1-induced reversal of the** 

It has previously been shown that forskolin inhibits and reverses the effects of BFA on Golgi morphology(Lippincott-Schwartz, 1991a).Also, 1,9-dideoxyforskolin, a naturally occurring

(2mM) did not affect the CT-induced elevation of the cAMP level (data not shown).

COPI-and KDEL-dependent functional retrograde transport of CT.

bars are indicated when they were more than 5% of the mean.

**inhibition of CT action byCbz-Gly-Phe-NH2** 

(Fig.5,B). This is in contrast to its effect on the adenylyl cyclase activity of membrane preparations or crude cell lysates. In addition, CT- induced mono ADP ribosylation of GS depends on active ARF1 as a cofactor, therefore, an effect of AlF4 at the level of ARF1 should also be considered. A previous study (Kahn, 1991), however , has demonstrated that AlF4 - does not activate ARF1 and does not affect the CT induced mono ADP ribosylation of GS.

Fig. 5. Effect of AlF4 on the BFA- and Exo1-induced reversal of the inhibition of CT action by Cbz-Gly-Phe-NH2.A. Vero cells in suspension were incubated with Cbz-Gly-Phe-NH2 (3mM) for 30 min at 37°C. CT (1µg/ml) was added and the cells were incubated for an additional 60 min at 37°C. Then cells were further incubated for 15 min at 37°C with and without NaF (30mM) and AlCl3 (50µM). Finally cells were treated with BFA (1 µg/ml) or Exo1 (2µM) for 30 min at 37°C and the intracellular concentration of cAMP measured. B. In parallel experiments Vero cells were preincubated with and without NaF (30mM) and AlCl3 (50µM) for 15 min at 37°C and further incubated for 60 min at 37°C with or without CT (1mg/ml) and the intracellular concentration of cAMP determined. Values are the means SD of triplicate assays from one of three similar experiments. Error bars are indicated when they were more than 5% of the mean.

Furthermore, as shown in Fig.5,B, prior exposure of Vero cells to AlF4- did not enhance but rather slightly reduced the CT-induced cAMP accumulation. A similar reduction was observed in the BFA-induced reversal of the inhibition of CT action by Cbz-Gly-Phe-NH2. Wealso looked whether1,3-cyclohexanebis-(methylamine)(CBM), a drug that interacts with COPI coatomer and inhibits coatomer binding to Golgi membranes (Hu et al., 1999), interferes with the BFA- or Exo1-induced reversal of the inhibition of CT action byCbz-Gly-Phe-NH2. As shown in Table I, CBM (2mM) had no effect on the BFA- or Exo1-induced

(Fig.5,B). This is in contrast to its effect on the adenylyl cyclase activity of membrane preparations or crude cell lysates. In addition, CT- induced mono ADP ribosylation of GS

Fig. 5. Effect of AlF4 on the BFA- and Exo1-induced reversal of the inhibition of CT action by Cbz-Gly-Phe-NH2.A. Vero cells in suspension were incubated with Cbz-Gly-Phe-NH2 (3mM) for 30 min at 37°C. CT (1µg/ml) was added and the cells were incubated for an additional 60 min at 37°C. Then cells were further incubated for 15 min at 37°C with and without NaF (30mM) and AlCl3 (50µM). Finally cells were treated with BFA (1 µg/ml) or Exo1 (2µM) for 30 min at 37°C and the intracellular concentration of cAMP measured. B. In parallel experiments Vero cells were preincubated with and without NaF (30mM) and AlCl3 (50µM) for 15 min at 37°C and further incubated for 60 min at 37°C with or without CT (1mg/ml) and the intracellular concentration of cAMP determined. Values are the means SD of triplicate assays from one of three similar experiments. Error bars are indicated when

Furthermore, as shown in Fig.5,B, prior exposure of Vero cells to AlF4- did not enhance but rather slightly reduced the CT-induced cAMP accumulation. A similar reduction was observed in the BFA-induced reversal of the inhibition of CT action by Cbz-Gly-Phe-NH2. Wealso looked whether1,3-cyclohexanebis-(methylamine)(CBM), a drug that interacts with COPI coatomer and inhibits coatomer binding to Golgi membranes (Hu et al., 1999), interferes with the BFA- or Exo1-induced reversal of the inhibition of CT action byCbz-Gly-Phe-NH2. As shown in Table I, CBM (2mM) had no effect on the BFA- or Exo1-induced

they were more than 5% of the mean.

also be considered. A previous study (Kahn, 1991), however , has demonstrated that AlF4

not activate ARF1 and does not affect the CT induced mono ADP ribosylation of GS.


at the level of ARF1 should


depends on active ARF1 as a cofactor, therefore, an effect of AlF4

reversal of the inhibition of CT action by Cbz-Gly-Phe-NH2.This dibasic compound itself did also not reverse the inhibitory effect of Cbz-Gly-Phe-NH2 (data not shown), which is consistent with its inability to cause a redistribution of Golgi membranes into the ER (Hu et al., 1999).

In agreement with previous results (Chen et al., 2002),pre-treatment of Vero cells with CBM (2mM) did not affect the CT-induced elevation of the cAMP level (data not shown).

These experiments indicate that there is no straightforward relationship between COPI dissociation and redistribution of Golgi membranes into the ER and also argue against COPI-and KDEL-dependent functional retrograde transport of CT.

Fig. 6. Effect of 1,9-dideoxyforskolin on the BFA- or Exo1-induced reversal of the inhibition of CT action by Cbz-Gly-Phe-NH2. A. Effect of 1,9-dideoxyforskolin concentration.Vero cells in suspension were preincubated for 30 min at 37°C with Cbz-Gly-Phe-NH2 (3mM). CT (1µg/ml) was added and the cells further incubated for 60 min at 37°C. The indicated amounts of 1,9-dideoxyforskolin were added and after 15 min BFA (1 µg/ml) () or Exo1 (2µM) () were added and the cells finally incubated for an additional 30 min at 37°C and cAMP accumulation measured. B. Effect of 1,9-dideoxyforskolin on the time courses of the BFA or Exo1 induced reversal of the inhibition of CT action by Cbz-Gly-Phe-NH2.Cells were preincubated as described under A. After CT treatment cells were incubated in the presence and absence of 1,9 dideoxyforskolin (150µM) for 15 min and BFA (1µg/ml) (,) or Exo1 (2µM) (,) were added and the cells further incubated at 37°C for the indicated times. Values are the means SD of triplicate assays from one of three similar experiments. Error bars are indicated when they were more than 5% of the mean.

### **3.5.2 Effect of 1,9-dideoxyforskolin on the BFA- and Exo1-induced reversal of the inhibition of CT action byCbz-Gly-Phe-NH2**

It has previously been shown that forskolin inhibits and reverses the effects of BFA on Golgi morphology(Lippincott-Schwartz, 1991a).Also, 1,9-dideoxyforskolin, a naturally occurring

Brefeldin A and Exo1 Completely Releave the Block of

**4. Discussion** 

vesicular transport.

apparatus.

toxin to the ER.

effect of AlF4-. Whereas AlF4

showing that, whereas AlF4

Cholera Toxin Action by a Dipeptide Metalloendoprotease Substrate 169

The metalloendoprotease substrate Cbz-Gly-Phe-NH2, but not its inactive analogue Cbz-Gly-Gly-NH2, renders cells completely resistant to the action of CT without apparently

In this study we further examined the Cbz-Gly-Phe-NH2-induced inhibition of CT action by looking at the effect of thisdipeptide on the intracellular trafficking of the toxin by using gradient centrifugation experiments and treatment of cells with agents affecting intracellular

Density gradient centrifugation experiments of post nuclear membranes or supernatants of Vero cells prelabeled with 125I-CT revealed that Cbz-Gly-Phe-NH2 does not affect the internalization of the toxin but blocks its transport to the ER. Following pretreatment of cells with Cbz-Gly-Phe-NH2 (3mM) the toxin appears to be trapped in an intracellular compartment, which cofractionates with UDP-galactosyltransferase a marker of the Golgi

To further explore whether in the presence of Cbz-Gly-Phe-NH2 the toxin travels beyond the trans Golgi-network (TGN) and actually reaches cisternae of the Golgi complex, we looked whether drugs known to redistribute Golgi membranes into the ER are also able to cause a

The idea for such an approach came from our previous experiments on the time dependence of the BFA effect on the CT-induced cAMP accumulation in Vero cells (De Wolf, 2000).In these experiments we noticed that addition of BFA 15 min after the addition of CT did not inhibit but rather enhanced the CT-induced increase in the cAMP level. As an explanation we proposed that once the toxin has reached the Golgi complex, the BFAmediatedredistribution of Golgi membranes into the ER increases the rate of delivery of the

In this study we showed that the fungal metabolite brefeldin A (BFA) and a novel chemical inhibitor of anterograde vesicular transport Exo1 are able to completely reverse the Cbz-Gly-Phe-NH2-induced inhibition of CT action. Both drugs have been shown to cause a rapid release of ADP-ribosylation factor (ARF)1 and COPI from Golgi membranes into the cytosol, followed by massive tubulation and collapse of the Golgi apparatus into the endoplasmic reticulum (Feng et al., 2003; Sciaky et al., 1997; Lippincott-Schwartz et al., 1991a). BFA and to a much lesser extent Exo1 also cause tubulation of the trans-Golgi network (TGN), however, no redistribution of TGN membranes into the ER occurs, instead theyfuse with early and recycling endosomes (Feng et al., 2003; Lippincott-Schwartz et al., 1991b).Therefore, our results indicated that, in the presence of Cbz-Gly-Phe-NH2, CT is able to travel to the Golgi,

The modes of action of BFA and Exo1, however, appear to be different, as illustrated by the

the BFA-induced reversal of inhibition of CT action by Cbz-Gly-Phe-NH2, it completely preventsthe reversal of inhibition by Exo1. This is consistent with earlier observations

ARF1 from Golgi membranes (Feng et al., 2003),it completely blocks the Exo1-induced



affecting the binding and internalization of the toxin (De Wolf, 2000).

reversal of Cbz-Gly-Phe-NH2-induced inhibition of CT action.

however, further retrograde transport to the ER is blocked.

dissociation of ARF1 from these membranes (Feng et al., 2003).

analogue of forskolin that does not activate adenylyl cyclase and reproduces many of the cAMP independent effects of forskolin(Laurenza et al., 1989),exerted a similar effect (Lippincott-Schwartz, 1991a). Therefore, it was of interest to look at the effect of 1,9 dideoxyforskolin on the BFA- and Exo1-induced reversal of the inhibition of CT action by Cbz-Gly-Phe-NH2.

As illustrated in Fig.6,A, the forskolin analogue completely antagonized the effect of BFA or Exo1 on the Cbz-Gly-Phe-NH2-induced inhibition of CT action in a dose-dependent (IC50 60µM) way. The effect of 1,9-dideoxyforskolin was very fast,since after its additionno raise in cAMP level could be observed one minute after the further addition of BFA or Exo1 (Fig.6,B).

#### **3.5.3 Effect of reduced temperature and energy depletion on the BFA-induced reversal of the inhibition of CT action byCbz-Gly-Phe-NH2**

As shownin Table I, reduction in cellular ATP levels using the metabolic inhibitors 2-deoxy-D-glucose (50mM) and sodium azide (0.02%) strongly suppressed the BFA-induced reversal of the inhibition of CT action by Cbz-Gly-Phe-NH2. Also, lowering the temperature to below 15°C abolished (Fig.7) the BFA-induced restoration of toxicity. The inhibitory effectsof these treatments are in line with their influence on the BFA-induced disassembly of the Golgi apparatus (Lippincott-Schwartz et al., 1990).

Fig. 7. Temperature dependence of the kinetics of the BFA-induced reversal of the Cbz-Gly-Phe-NH2-mediated inhibition of CT action on Vero cells. Vero cells in suspension were preincubated with Cbz-Gly-Phe-NH2 (3mM) for 30 min at 37°C. CT (1µg/ml) was added and the cells further incubated for 60 min at 37°C. Cells were subsequently cooled to the indicated temperatures and after the addition of BFA (5µg/ml) incubated for the indicated times and cAMP measured.Values are the means SD of triplicate assays from one of three similar experiments. Error bars are indicated when they were more than 5% of the mean.

## **4. Discussion**

168 Cholera

analogue of forskolin that does not activate adenylyl cyclase and reproduces many of the cAMP independent effects of forskolin(Laurenza et al., 1989),exerted a similar effect (Lippincott-Schwartz, 1991a). Therefore, it was of interest to look at the effect of 1,9 dideoxyforskolin on the BFA- and Exo1-induced reversal of the inhibition of CT action by

As illustrated in Fig.6,A, the forskolin analogue completely antagonized the effect of BFA or Exo1 on the Cbz-Gly-Phe-NH2-induced inhibition of CT action in a dose-dependent (IC50 60µM) way. The effect of 1,9-dideoxyforskolin was very fast,since after its additionno raise in cAMP level could be observed one minute after the further addition of BFA or Exo1 (Fig.6,B).

As shownin Table I, reduction in cellular ATP levels using the metabolic inhibitors 2-deoxy-D-glucose (50mM) and sodium azide (0.02%) strongly suppressed the BFA-induced reversal of the inhibition of CT action by Cbz-Gly-Phe-NH2. Also, lowering the temperature to below 15°C abolished (Fig.7) the BFA-induced restoration of toxicity. The inhibitory effectsof these treatments are in line with their influence on the BFA-induced disassembly of the Golgi

Fig. 7. Temperature dependence of the kinetics of the BFA-induced reversal of the Cbz-Gly-Phe-NH2-mediated inhibition of CT action on Vero cells. Vero cells in suspension were preincubated with Cbz-Gly-Phe-NH2 (3mM) for 30 min at 37°C. CT (1µg/ml) was added and the cells further incubated for 60 min at 37°C. Cells were subsequently cooled to the indicated temperatures and after the addition of BFA (5µg/ml) incubated for the indicated times and cAMP measured.Values are the means SD of triplicate assays from one of three similar experiments. Error bars are indicated when they were more than 5% of the mean.

**3.5.3 Effect of reduced temperature and energy depletion on the BFA-induced** 

**reversal of the inhibition of CT action byCbz-Gly-Phe-NH2**

apparatus (Lippincott-Schwartz et al., 1990).

Cbz-Gly-Phe-NH2.

The metalloendoprotease substrate Cbz-Gly-Phe-NH2, but not its inactive analogue Cbz-Gly-Gly-NH2, renders cells completely resistant to the action of CT without apparently affecting the binding and internalization of the toxin (De Wolf, 2000).

In this study we further examined the Cbz-Gly-Phe-NH2-induced inhibition of CT action by looking at the effect of thisdipeptide on the intracellular trafficking of the toxin by using gradient centrifugation experiments and treatment of cells with agents affecting intracellular vesicular transport.

Density gradient centrifugation experiments of post nuclear membranes or supernatants of Vero cells prelabeled with 125I-CT revealed that Cbz-Gly-Phe-NH2 does not affect the internalization of the toxin but blocks its transport to the ER. Following pretreatment of cells with Cbz-Gly-Phe-NH2 (3mM) the toxin appears to be trapped in an intracellular compartment, which cofractionates with UDP-galactosyltransferase a marker of the Golgi apparatus.

To further explore whether in the presence of Cbz-Gly-Phe-NH2 the toxin travels beyond the trans Golgi-network (TGN) and actually reaches cisternae of the Golgi complex, we looked whether drugs known to redistribute Golgi membranes into the ER are also able to cause a reversal of Cbz-Gly-Phe-NH2-induced inhibition of CT action.

The idea for such an approach came from our previous experiments on the time dependence of the BFA effect on the CT-induced cAMP accumulation in Vero cells (De Wolf, 2000).In these experiments we noticed that addition of BFA 15 min after the addition of CT did not inhibit but rather enhanced the CT-induced increase in the cAMP level. As an explanation we proposed that once the toxin has reached the Golgi complex, the BFAmediatedredistribution of Golgi membranes into the ER increases the rate of delivery of the toxin to the ER.

In this study we showed that the fungal metabolite brefeldin A (BFA) and a novel chemical inhibitor of anterograde vesicular transport Exo1 are able to completely reverse the Cbz-Gly-Phe-NH2-induced inhibition of CT action. Both drugs have been shown to cause a rapid release of ADP-ribosylation factor (ARF)1 and COPI from Golgi membranes into the cytosol, followed by massive tubulation and collapse of the Golgi apparatus into the endoplasmic reticulum (Feng et al., 2003; Sciaky et al., 1997; Lippincott-Schwartz et al., 1991a). BFA and to a much lesser extent Exo1 also cause tubulation of the trans-Golgi network (TGN), however, no redistribution of TGN membranes into the ER occurs, instead theyfuse with early and recycling endosomes (Feng et al., 2003; Lippincott-Schwartz et al., 1991b).Therefore, our results indicated that, in the presence of Cbz-Gly-Phe-NH2, CT is able to travel to the Golgi, however, further retrograde transport to the ER is blocked.

The modes of action of BFA and Exo1, however, appear to be different, as illustrated by the effect of AlF4 -. Whereas AlF4- has only a minor or no effect (depending on the cell type) on the BFA-induced reversal of inhibition of CT action by Cbz-Gly-Phe-NH2, it completely preventsthe reversal of inhibition by Exo1. This is consistent with earlier observations showing that, whereas AlF4 - slows but does not prevent the BFA-induced dissociation of ARF1 from Golgi membranes (Feng et al., 2003),it completely blocks the Exo1-induced dissociation of ARF1 from these membranes (Feng et al., 2003).

Brefeldin A and Exo1 Completely Releave the Block of

phenomenon therefore remains to be defined.

I thank R. Goossens for skilled technical assistance.

**5. Acknowledgment** 

Cholera Toxin Action by a Dipeptide Metalloendoprotease Substrate 171

form to the ER via a KDEL-dependent mechanism. Reduction of CT in an intermediate compartment before reaching the Golgi is unlikely, since in the presence of BFA, which as Cbz-Gly-Phe-NH2 does not impair the binding and internalization of CT, no reduction of CT-A occurs. Conditions prevailing in the Golgi (lower pH, less oxidizing) may also be favourable for the reduction of CT-A. For instance, it has been shown that in vitro the PDI

Previous studies (Lippincott-Schwartz et al., 1991a)have reported that forskolin inhibits and even reverses the effects of BFA on Golgi structure. These effects arealso reproduced by 1,9 dideoxyforskolin, a naturally occurring analogue of forskolin that does not activate adenylyl cyclase(Laurenza et al., 1989). In this study we demonstrated that 1,9-dideoxyforskolin also antagonizes the effect of BFA on the Cbz-Gly-Phe-NH2-induced inhibition of CT action in a dose-dependent way. It has been speculated that forskolin interferes with the action of BFA by (competition)/competing for the binding of BFA to its target protein, the Golgi-localized nucleotide exchange factor specific for ARF1(Lippincott-Schwartz et al., 1991a). A subsequent study (Nickel et al., 1996), however, showed that in vitro forskolin does not prevent inhibition of Golgi-catalyzed nucleotide exchange by BFA. Therefore, it was concluded that it is unlikely that forskolin and BFA bind to the same target protein. Forskolin treatment of CHO cells, however, resultsin increased levels of Cys-BFA, the major BFA conjugate secreted by CHO cells, in the medium, which led to the suggestion that the effect of forskolin on BFA-induced disassembly of the Golgi apparatus might be due to an enhanced detoxification of the drug. The present results do not favor this hypothesis, since 1,9-dideoxyforskolin also blocks the Exo1-induced reversal of the inhibition of CT action by Cbz-Gly-Phe-NH2. In addition,the fast action of 1,9-dideoxyforskolin is difficult to reconcile with the proposed enhanced detoxification and removal of BFA. In this light it is also interesting to note that forskolin also prevents the redistribution of Golgi membranesinto the ER, induced by the Epidermal-cell differentiation inhibitor (EDIN), an exoenzyme (ADPribosyltransferase) produced by Staphylococcus aureus with a substrate specificity of the rho protein (Sugai et al., 1992). Therefore, it is clear that forskolin inhibits the action of structurally totally unrelated compounds, which all cause disassembly of the Golgi complex. The exact target and mechanism of action of forskolin and its derivatives in this

Taken together, the results of this study are in agreement with the view that intoxication by CT requires retrograde transport of CT from the plasma membrane to the ER, involving passage through the TGN and Golgi apparatus. A direct transport from the TGN to the ER as recently proposed (Feng et al., 2004)is unlikely since Cbz-Gly-Phe-NH2 and Exo1 completely abolish toxicity,whereastransport of the toxin to the TGN still occurs. We also demonstrated that the metalloendoprotease substrate blocks the retrograde transport of CT from the Golgi complex to the ER. Therefore,metalloendoproteases may not only play a role in vesicular transport and secretion of newly synthesized proteins as previously proposed (Lennarz and Strittmatter, 1991),but may also be involved in retrograde transport between the Golgi and the ER. Finally, we also showed that successive addition of two strong inhibitors of CT action at the appropriate time points can annihilate their inhibitory effects.

mediated reduction of CT-A is maximalat a pH 5.5-6.0 (Tsai et al., 2001).

This agrees with the proposal (Feng et al., 2003)that BFA and Exo1 have different targets. In contrast with BFA, Exo1 appears not to interfere with the activity of guanine nucleotide exchange factors specific for Golgi associated ARF's, but probably acts at a step downstream from the ARF1-GTP loading step, most likely by increasing the rate of GTP hydrolysis through the activation of an ARF-GAP-dependent step (Feng et al., 2003).

The difference in the mechanisms of action of BFA and Exo1 is also apparent from their divergent effects on the action of CT in MDCK cells and reversal of the Cbz-Gly-Phe-NH2 induced inhibition of CT action in these cells. MDCK cells have a Golgi complex which is resistant to the action of BFA (Hunziker et al., 1991),but apparently,as shown in this study,not to that of Exo1. Kinetic analysis of the BFA- or Exo1-induced reversal of the inhibition of CT action by Cbz-Gly-Phe-NH2 revealed that the redistribution of Golgi membranes into the ER is a fast process. Furthermore, sincethe toxin-induced increase in cAMP content reached 50%of its maximal levelonly two minutes after the addition of BFA or Exo1, the subsequent steps in toxin action must also be taken very rapidly. Golgi tubules are known to mediate retrograde traffic in cells treated with BFA. In BFA treated cells, Golgi tubules resembling those of untreated cells are observed, but they are formed at a more rapid rate. Moreover, the tubules fail to detach from Golgi structures. This leads to the formation of a dynamic Golgi tubule network within 5-8 min of adding the drug. When one or more of the Golgi tubules fuses with the ER, Golgi membranes redistribute rapidly (within 30 sec) in the ER, leaving no Golgi structure behind (Sciaky et al., 1997).

Since BFA and Exo1, by perturbing ARF1 function and COPI binding, directly interfere with the Golgi to ER retrograde trafficking machinery, we also investigated the effect of the microtubule depolimerizing drug nocodazole. Microtubule disruption by nocodazole is known to block the inward translocation of pre-Golgi intermediates along microtubules without significant effects on the Golgi to ER traffic (Cole et al., 1996),causing a more naturalrecyclingof Golgi components to the ER. As shown in this study,nocodazole also partially reverses the Cbz-Gly-Phe-NH2 induced inhibition of CT action. The extent of reversal, however, appears to be cell type dependent.

We previously reported (De Wolf, 2000)that whereas Vero cells pretreated with BFA areunable to reduce CT to the CT-A1 peptide and arecompletely resistant to CT action, treatment of the same cells with Cbz-Gly-Phe-NH2 only partially reduced their ability to generate the CT-A1 peptide, although they also become completely insensitive to toxin action. To account for this observation we argued that the total amount of reduced toxin may not reflect toxin that can be translocated to the cytosol, for instance, in the presence of Cbz-Gly-Phe-NH2, reduced toxin may be trapped in a compartment where translocation to the cytosol is impossible or much less efficient. Several lines of evidence have indicated that a protein-disulfideisomerase (PDI; EC 5.34.1) mediates the reduction of CT-A (Tsai et al., 2001; Orlandi, 1997). This enzyme is found predominantly as a resident soluble protein within the lumen of the ER (Freedman et al., 1989),but has also been ascribed to the Golgi apparatus (Taylor and Varandani, 1985), the trans-Golgi network and the plasma membrane of mammalian cells (Varandani et al., 1978). Our results indicated that at least in the presence of Cbz-Gly-Phe-NH2, CT can be reduced and thus activated at the level of the Golgi complex. Modifications of CT structure at the level of the Golgi complex have been reported previously (Bastiaens et al., 1996). In this study evidence waspresented indicating that CT-A dissociates from CT-B in the Golgi, after which CT-A is transported in oxidized

This agrees with the proposal (Feng et al., 2003)that BFA and Exo1 have different targets. In contrast with BFA, Exo1 appears not to interfere with the activity of guanine nucleotide exchange factors specific for Golgi associated ARF's, but probably acts at a step downstream from the ARF1-GTP loading step, most likely by increasing the rate of GTP hydrolysis

The difference in the mechanisms of action of BFA and Exo1 is also apparent from their divergent effects on the action of CT in MDCK cells and reversal of the Cbz-Gly-Phe-NH2 induced inhibition of CT action in these cells. MDCK cells have a Golgi complex which is resistant to the action of BFA (Hunziker et al., 1991),but apparently,as shown in this study,not to that of Exo1. Kinetic analysis of the BFA- or Exo1-induced reversal of the inhibition of CT action by Cbz-Gly-Phe-NH2 revealed that the redistribution of Golgi membranes into the ER is a fast process. Furthermore, sincethe toxin-induced increase in cAMP content reached 50%of its maximal levelonly two minutes after the addition of BFA or Exo1, the subsequent steps in toxin action must also be taken very rapidly. Golgi tubules are known to mediate retrograde traffic in cells treated with BFA. In BFA treated cells, Golgi tubules resembling those of untreated cells are observed, but they are formed at a more rapid rate. Moreover, the tubules fail to detach from Golgi structures. This leads to the formation of a dynamic Golgi tubule network within 5-8 min of adding the drug. When one or more of the Golgi tubules fuses with the ER, Golgi membranes redistribute rapidly

through the activation of an ARF-GAP-dependent step (Feng et al., 2003).

(within 30 sec) in the ER, leaving no Golgi structure behind (Sciaky et al., 1997).

reversal, however, appears to be cell type dependent.

Since BFA and Exo1, by perturbing ARF1 function and COPI binding, directly interfere with the Golgi to ER retrograde trafficking machinery, we also investigated the effect of the microtubule depolimerizing drug nocodazole. Microtubule disruption by nocodazole is known to block the inward translocation of pre-Golgi intermediates along microtubules without significant effects on the Golgi to ER traffic (Cole et al., 1996),causing a more naturalrecyclingof Golgi components to the ER. As shown in this study,nocodazole also partially reverses the Cbz-Gly-Phe-NH2 induced inhibition of CT action. The extent of

We previously reported (De Wolf, 2000)that whereas Vero cells pretreated with BFA areunable to reduce CT to the CT-A1 peptide and arecompletely resistant to CT action, treatment of the same cells with Cbz-Gly-Phe-NH2 only partially reduced their ability to generate the CT-A1 peptide, although they also become completely insensitive to toxin action. To account for this observation we argued that the total amount of reduced toxin may not reflect toxin that can be translocated to the cytosol, for instance, in the presence of Cbz-Gly-Phe-NH2, reduced toxin may be trapped in a compartment where translocation to the cytosol is impossible or much less efficient. Several lines of evidence have indicated that a protein-disulfideisomerase (PDI; EC 5.34.1) mediates the reduction of CT-A (Tsai et al., 2001; Orlandi, 1997). This enzyme is found predominantly as a resident soluble protein within the lumen of the ER (Freedman et al., 1989),but has also been ascribed to the Golgi apparatus (Taylor and Varandani, 1985), the trans-Golgi network and the plasma membrane of mammalian cells (Varandani et al., 1978). Our results indicated that at least in the presence of Cbz-Gly-Phe-NH2, CT can be reduced and thus activated at the level of the Golgi complex. Modifications of CT structure at the level of the Golgi complex have been reported previously (Bastiaens et al., 1996). In this study evidence waspresented indicating that CT-A dissociates from CT-B in the Golgi, after which CT-A is transported in oxidized form to the ER via a KDEL-dependent mechanism. Reduction of CT in an intermediate compartment before reaching the Golgi is unlikely, since in the presence of BFA, which as Cbz-Gly-Phe-NH2 does not impair the binding and internalization of CT, no reduction of CT-A occurs. Conditions prevailing in the Golgi (lower pH, less oxidizing) may also be favourable for the reduction of CT-A. For instance, it has been shown that in vitro the PDI mediated reduction of CT-A is maximalat a pH 5.5-6.0 (Tsai et al., 2001).

Previous studies (Lippincott-Schwartz et al., 1991a)have reported that forskolin inhibits and even reverses the effects of BFA on Golgi structure. These effects arealso reproduced by 1,9 dideoxyforskolin, a naturally occurring analogue of forskolin that does not activate adenylyl cyclase(Laurenza et al., 1989). In this study we demonstrated that 1,9-dideoxyforskolin also antagonizes the effect of BFA on the Cbz-Gly-Phe-NH2-induced inhibition of CT action in a dose-dependent way. It has been speculated that forskolin interferes with the action of BFA by (competition)/competing for the binding of BFA to its target protein, the Golgi-localized nucleotide exchange factor specific for ARF1(Lippincott-Schwartz et al., 1991a). A subsequent study (Nickel et al., 1996), however, showed that in vitro forskolin does not prevent inhibition of Golgi-catalyzed nucleotide exchange by BFA. Therefore, it was concluded that it is unlikely that forskolin and BFA bind to the same target protein. Forskolin treatment of CHO cells, however, resultsin increased levels of Cys-BFA, the major BFA conjugate secreted by CHO cells, in the medium, which led to the suggestion that the effect of forskolin on BFA-induced disassembly of the Golgi apparatus might be due to an enhanced detoxification of the drug. The present results do not favor this hypothesis, since 1,9-dideoxyforskolin also blocks the Exo1-induced reversal of the inhibition of CT action by Cbz-Gly-Phe-NH2. In addition,the fast action of 1,9-dideoxyforskolin is difficult to reconcile with the proposed enhanced detoxification and removal of BFA. In this light it is also interesting to note that forskolin also prevents the redistribution of Golgi membranesinto the ER, induced by the Epidermal-cell differentiation inhibitor (EDIN), an exoenzyme (ADPribosyltransferase) produced by Staphylococcus aureus with a substrate specificity of the rho protein (Sugai et al., 1992). Therefore, it is clear that forskolin inhibits the action of structurally totally unrelated compounds, which all cause disassembly of the Golgi complex. The exact target and mechanism of action of forskolin and its derivatives in this phenomenon therefore remains to be defined.

Taken together, the results of this study are in agreement with the view that intoxication by CT requires retrograde transport of CT from the plasma membrane to the ER, involving passage through the TGN and Golgi apparatus. A direct transport from the TGN to the ER as recently proposed (Feng et al., 2004)is unlikely since Cbz-Gly-Phe-NH2 and Exo1 completely abolish toxicity,whereastransport of the toxin to the TGN still occurs. We also demonstrated that the metalloendoprotease substrate blocks the retrograde transport of CT from the Golgi complex to the ER. Therefore,metalloendoproteases may not only play a role in vesicular transport and secretion of newly synthesized proteins as previously proposed (Lennarz and Strittmatter, 1991),but may also be involved in retrograde transport between the Golgi and the ER. Finally, we also showed that successive addition of two strong inhibitors of CT action at the appropriate time points can annihilate their inhibitory effects.

## **5. Acknowledgment**

I thank R. Goossens for skilled technical assistance.

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A. The Ras-RasGAP complex: structural basis for GTPase activation and its loss in


**9** 

*1Slovenia 2Italy* 

**Structure Based Design** 

Črtomir Podlipnik1 and Jose J. Reina2

**of Cholera Toxin Antagonists** 

*1Faculty of Chemistry and Chemical Technology, University of Ljubljana 2Department of Organic and IndustrialChemistry, University of Milano* 

Cholera is an acute enteric infection, with huge pandemic potential, caused by ingestion of food or water contaminated with the bacterium *Vibrio cholerae*, the gram negative bacteria (Sack, Sack et al. 2004). The etiologic agent responsible for cholera was identified in 1883 when Robert Koch demonstrated that comma(-shaped) bacteria, later designated as *V. cholerae*, causes cholera infection (Koch 1884). Since Koch's discovery of cholera virulent factor, different specific strain variants of *V. cholerae* have been identified. For the majority of cholera's disease outbreaks two biotypes of *V. cholerae's*serogroup O1 are responsible: Clasical and "El Tor"*,* as well serogroup O139 that was responsible for a large epidemic in Bangladesh and India (Ramamurthy, Garg et al. 1993). Non-O1 and non-O139 *V. cholerae* can cause mild diarrhoea but do not generate epidemics (Ramamurthy, Bag et al. 1993). Cholera transmission is closely related to inadequate environmental conditions that can be find in suburban slums where the basic infrastructure is not available, as well as in camps for internally displaced people or refugees, where minimum requirements of clean water and sanitation are not met. A typical example of such non-promising situation has induced an outbreak of Cholera after earthquake in Haiti in January 2010 (Andrews and Basu 2011). The short incubation period of two hours to five days, enhances the potentially explosive pattern of outbreaks. Intensive efforts for the identification of the basis of Cholera disease at a molecular level were done by different research groups during the 1960s, until Finkelstein and co-workers recognized a protein toxin as a major virulent factor that causes the massive fluid release in Cholera infection (Finkelstein, Atthasampunna et al. 1966). The efforts to solve a complete structure of Cholera toxin by X-ray diffraction analysis were concluded during the 1990s (Spangler 1992; Zhang, Scott et al. 1995). Up to date, 27 X-ray structures related to Cholera toxin (CT) are deposited in the Protein Databank. The structure and function of CT at the molecular level will be the subject of our review. In this work we will also show some examples of structure based design of various types of CT inhibitors; we will introduce catechin-like compounds as inhibitors of the enzymatic A unit of CT; mimics of oGM1 as inhibitors of the non-toxic pentamer of B subunits of CT (CTB); as well as multivalent inhibitors that very effectively prevent adhesion of CTB to GM1 receptors at the surface of epithelial cells. At the end, we will also describe a new strategy for developing

inhibitors via targeting binding site for blood group antigens in Cholera Toxin.

**1. Introduction** 


## **Structure Based Design of Cholera Toxin Antagonists**

Črtomir Podlipnik1 and Jose J. Reina2 *1Faculty of Chemistry and Chemical Technology, University of Ljubljana 2Department of Organic and IndustrialChemistry, University of Milano 1Slovenia 2Italy* 

#### **1. Introduction**

176 Cholera

Wolf AA, Jobling MG, Wimer-Mackin S, Ferguson-Maltzman M, Madara JL, Holmes RK,

Zhang R-G, Scott DL,Westbrook ML, Nance S, Spangler BD, Shipley GG. Westbrook EM.The three-dimensional crystal structure of cholera toxin. J Mol Biol*.*1995; 251:563- 73.

Cell Biol*.*1998; 141:917-27.

Lencer WI.Ganglioside Structure Dictates Signal Transduction by Cholera Toxin and Association with Caveolae-like Membrane Domains in Polarized Epithelia. J

> Cholera is an acute enteric infection, with huge pandemic potential, caused by ingestion of food or water contaminated with the bacterium *Vibrio cholerae*, the gram negative bacteria (Sack, Sack et al. 2004). The etiologic agent responsible for cholera was identified in 1883 when Robert Koch demonstrated that comma(-shaped) bacteria, later designated as *V. cholerae*, causes cholera infection (Koch 1884). Since Koch's discovery of cholera virulent factor, different specific strain variants of *V. cholerae* have been identified. For the majority of cholera's disease outbreaks two biotypes of *V. cholerae's*serogroup O1 are responsible: Clasical and "El Tor"*,* as well serogroup O139 that was responsible for a large epidemic in Bangladesh and India (Ramamurthy, Garg et al. 1993). Non-O1 and non-O139 *V. cholerae* can cause mild diarrhoea but do not generate epidemics (Ramamurthy, Bag et al. 1993). Cholera transmission is closely related to inadequate environmental conditions that can be find in suburban slums where the basic infrastructure is not available, as well as in camps for internally displaced people or refugees, where minimum requirements of clean water and sanitation are not met. A typical example of such non-promising situation has induced an outbreak of Cholera after earthquake in Haiti in January 2010 (Andrews and Basu 2011). The short incubation period of two hours to five days, enhances the potentially explosive pattern of outbreaks. Intensive efforts for the identification of the basis of Cholera disease at a molecular level were done by different research groups during the 1960s, until Finkelstein and co-workers recognized a protein toxin as a major virulent factor that causes the massive fluid release in Cholera infection (Finkelstein, Atthasampunna et al. 1966). The efforts to solve a complete structure of Cholera toxin by X-ray diffraction analysis were concluded during the 1990s (Spangler 1992; Zhang, Scott et al. 1995). Up to date, 27 X-ray structures related to Cholera toxin (CT) are deposited in the Protein Databank. The structure and function of CT at the molecular level will be the subject of our review. In this work we will also show some examples of structure based design of various types of CT inhibitors; we will introduce catechin-like compounds as inhibitors of the enzymatic A unit of CT; mimics of oGM1 as inhibitors of the non-toxic pentamer of B subunits of CT (CTB); as well as multivalent inhibitors that very effectively prevent adhesion of CTB to GM1 receptors at the surface of epithelial cells. At the end, we will also describe a new strategy for developing inhibitors via targeting binding site for blood group antigens in Cholera Toxin.

Structure Based Design of Cholera Toxin Antagonists 179

strongest known protein-carbohydrate interaction. It has been also observed that all of the mono- or disaccharide fragments of oGM1 bind to CTB much more weakly; for example, the terminal galactose residue displays Kd=15 mM, which is in the case of the Gal-GalNAc

Fig. 2. (A) *Structure of ganglioside head groups. (B)* Close view of CTB:oGM1 interaction.

carboxyl group forms water-mediated hydrogen bond with Trp88.

The other important binding determinant, Neu5Ac, binds even more weakly to the protein (Kd ≈ 200 mM). The analysis of results obtained by dissecting of the oGM1:CTB interaction by ITC has shown that high affinity and selectivity of oGM1:CTB interaction originates mainly from the conformational pre-organisation of the branched GM1 pentasaccharide rather than through the effect of cooperativity of terminal moieties galactose and sialic acid. The terminal galactose residue in the "forefinger" binds to the CTB binding site very specifically. The pyranose ring of this galactose is stacked on top of Trp88 (CH/pi interaction) and forms an extensive hydrogen bond network involving Asn90, Lys91, Glu51 and Gln61 residues from CTB5. The terminal galactose is docked in a deep cavity that is shielded from the solvent, on the other hand the rest of the toxin's binding site is shallow and solvent exposed. The sialic acid (Neu5Ac) represents the second important moiety of GM1 required for recognition of Cholera toxin. The sugar ring of sialic acid makes hydrophobic interaction with Tyr12, whereas hydroxyl, and N-acetyl substituents form hydrogen bonds with CTB residues, while

The action of CT is initiated by binding of CT via the B5 pentamer to GM1s receptors that are part of the external membrane of intestinal epithelial cells. When CT binds to the cell, the whole toxin is transferred into the cell via receptor-mediated endocytosis. The toxin is then transported to the endoplasmic reticulum (ER), where A subunit dissociates from the rest of the protein assembly. Subunit A is then split to subunits A1 and A2 by peptide-disulfide isomerase in the ER. Subunit A1 is then translocated to the cytosol of the host cell, where it catalyses the covalent transfer of an ADP-ribose moiety from NAD+ to Arg201 of the signalling protein Gs<sup>α</sup> (a component of an adenylate cyclase system). Adenylate cyclase system is normally activated by a regulatory protein Gs and GTP; however the activation is normally brief, because another regulatory protein (Gi) hydrolyses GTP. The normal situation is described in Fig 3a. Cholera toxin catalyses transfer of ADP to adenyl cyclase

forefinger improved by only a factor of two.

## **2. Cholera toxin – structure and mode of action**

Cholera toxin (CT) belongs to the family of AB5 bacterial toxins, which includes CT itself and the *Escherichia coli* heat-labile toxins (LTs) LT-I and LT-II, among others. This family of bacterial toxins is named after the characteristic architecture comprising a single catalytically active component, A, and a non-toxic pentamer of B subunits (B5) (Fig. 1a). The structure and function of AB5 toxins have been reviewed in detail on several occasions (Bernardi, Podlipnik et al. 2006; Hol, Fan et al. 2004; Hayes, Turnbull 2011).

Fig. 1. (A) Holotoxin CT and (B) pentamer B complexed with oGM1.

The A subunit of CT is composed of two distinct parts A1 and A2. The A1 component is responsible for the toxic enzyme activity, while the A2 component serves as non-covalent linker of subunit A to subunit B. Each of five CT's B-subunits is composed from two α helices and two three-stranded β sheets, that form together a doughnut-shaped structure, which has a central pore into which the C-terminal of A2 subunit extends. The B pentamer is responsible for binding CT to ganglioside GM1 on the external membrane of intestinal epithelial cells. This binding is recognized as a key event for initiation of the threatening action of CT. The interaction of the oligosaccharidic head groups of ganglioside GM1 (Galβ1-3GalNAcβ1-4(NeuAcα2-3)Galβ1-4Glcβ1-OH,oGM1) with B5 unit of CT is depicted in Fig. 1b. It is interesting that the binding ability of the B-pentamer to cell surface receptors is retained even in absence of the A-subunit. However, the complete AB5 holotoxin is needed for actual intoxication.

The B pentamer of CT (CTB) interacts with the soluble, monovalent oligosaccharide portion of GM1 (oGM1) with strong affinity, the binding process is weakly cooperative. The close view of the interaction based on 1.25 Å resolution structure of oGM1:CTB complex (Merritt, Sarfaty et al. 1994) is shown in Fig 2b. We may observe that branched oGM1 (Fig 3) is attached to CTB binding site with two fingers: the first one is a sialic acid "thumb" and the second one a GalB(1->3)GalNAc "forefinger" (two-fingered grip). Most of the contacts are given by the "finger" tips: in terms of buried protein surface, the terminal Gal and Neu5Ac residues contribute 39% and 43%, the rest and minor part of protein surface is buried with GalNAc. The most recent value of the dissociation constant for interaction between one oGM1 and one CTB binding site has been evaluated by Isothermal Titration Calorimetry (ITC) and it was found to be 43 nM (Turnbull, Precious et al. 2004), this is one of the

Cholera toxin (CT) belongs to the family of AB5 bacterial toxins, which includes CT itself and the *Escherichia coli* heat-labile toxins (LTs) LT-I and LT-II, among others. This family of bacterial toxins is named after the characteristic architecture comprising a single catalytically active component, A, and a non-toxic pentamer of B subunits (B5) (Fig. 1a). The structure and function of AB5 toxins have been reviewed in detail on several occasions

The A subunit of CT is composed of two distinct parts A1 and A2. The A1 component is responsible for the toxic enzyme activity, while the A2 component serves as non-covalent linker of subunit A to subunit B. Each of five CT's B-subunits is composed from two α helices and two three-stranded β sheets, that form together a doughnut-shaped structure, which has a central pore into which the C-terminal of A2 subunit extends. The B pentamer is responsible for binding CT to ganglioside GM1 on the external membrane of intestinal epithelial cells. This binding is recognized as a key event for initiation of the threatening action of CT. The interaction of the oligosaccharidic head groups of ganglioside GM1 (Galβ1-3GalNAcβ1-4(NeuAcα2-3)Galβ1-4Glcβ1-OH,oGM1) with B5 unit of CT is depicted in Fig. 1b. It is interesting that the binding ability of the B-pentamer to cell surface receptors is retained even in absence of the A-subunit. However, the complete AB5 holotoxin is needed

The B pentamer of CT (CTB) interacts with the soluble, monovalent oligosaccharide portion of GM1 (oGM1) with strong affinity, the binding process is weakly cooperative. The close view of the interaction based on 1.25 Å resolution structure of oGM1:CTB complex (Merritt, Sarfaty et al. 1994) is shown in Fig 2b. We may observe that branched oGM1 (Fig 3) is attached to CTB binding site with two fingers: the first one is a sialic acid "thumb" and the second one a GalB(1->3)GalNAc "forefinger" (two-fingered grip). Most of the contacts are given by the "finger" tips: in terms of buried protein surface, the terminal Gal and Neu5Ac residues contribute 39% and 43%, the rest and minor part of protein surface is buried with GalNAc. The most recent value of the dissociation constant for interaction between one oGM1 and one CTB binding site has been evaluated by Isothermal Titration Calorimetry (ITC) and it was found to be 43 nM (Turnbull, Precious et al. 2004), this is one of the

(Bernardi, Podlipnik et al. 2006; Hol, Fan et al. 2004; Hayes, Turnbull 2011).

Fig. 1. (A) Holotoxin CT and (B) pentamer B complexed with oGM1.

for actual intoxication.

**2. Cholera toxin – structure and mode of action** 

strongest known protein-carbohydrate interaction. It has been also observed that all of the mono- or disaccharide fragments of oGM1 bind to CTB much more weakly; for example, the terminal galactose residue displays Kd=15 mM, which is in the case of the Gal-GalNAc forefinger improved by only a factor of two.

Fig. 2. (A) *Structure of ganglioside head groups. (B)* Close view of CTB:oGM1 interaction.

The other important binding determinant, Neu5Ac, binds even more weakly to the protein (Kd ≈ 200 mM). The analysis of results obtained by dissecting of the oGM1:CTB interaction by ITC has shown that high affinity and selectivity of oGM1:CTB interaction originates mainly from the conformational pre-organisation of the branched GM1 pentasaccharide rather than through the effect of cooperativity of terminal moieties galactose and sialic acid. The terminal galactose residue in the "forefinger" binds to the CTB binding site very specifically. The pyranose ring of this galactose is stacked on top of Trp88 (CH/pi interaction) and forms an extensive hydrogen bond network involving Asn90, Lys91, Glu51 and Gln61 residues from CTB5. The terminal galactose is docked in a deep cavity that is shielded from the solvent, on the other hand the rest of the toxin's binding site is shallow and solvent exposed. The sialic acid (Neu5Ac) represents the second important moiety of GM1 required for recognition of Cholera toxin. The sugar ring of sialic acid makes hydrophobic interaction with Tyr12, whereas hydroxyl, and N-acetyl substituents form hydrogen bonds with CTB residues, while carboxyl group forms water-mediated hydrogen bond with Trp88.

The action of CT is initiated by binding of CT via the B5 pentamer to GM1s receptors that are part of the external membrane of intestinal epithelial cells. When CT binds to the cell, the whole toxin is transferred into the cell via receptor-mediated endocytosis. The toxin is then transported to the endoplasmic reticulum (ER), where A subunit dissociates from the rest of the protein assembly. Subunit A is then split to subunits A1 and A2 by peptide-disulfide isomerase in the ER. Subunit A1 is then translocated to the cytosol of the host cell, where it catalyses the covalent transfer of an ADP-ribose moiety from NAD+ to Arg201 of the signalling protein Gs<sup>α</sup> (a component of an adenylate cyclase system). Adenylate cyclase system is normally activated by a regulatory protein Gs and GTP; however the activation is normally brief, because another regulatory protein (Gi) hydrolyses GTP. The normal situation is described in Fig 3a. Cholera toxin catalyses transfer of ADP to adenyl cyclase

Structure Based Design of Cholera Toxin Antagonists 181

of numerous natural products. The active substances from medicinal plants can treat Cholera via different pharmacological mechanisms; from the direct antimicrobial against *V. cholerae*, prevention of adhesion of CT to the GM1 receptors at surface of epithelial cells, to direct inhibition of ADP-ribosylation of active unit of CT. The improved understanding of the CT toxicity at the molecular level and the further set up of modern biological assays, has allowed in recent years the identification of different classes of bioactive natural products. An important class of such products are polyphenols from green and black tea, green apples, hop bract and the Chinese rhubarb rhizome. Garlic extract is another example of traditional cure against diarrhoea diseases such as cholera. Recent researches have recognized a galactan polysaccharide as a major anti-choleric component of garlic (Politi, Alvaro-Blanco et al. 2006). Some interesting natural inhibitors of CT are shown in Fig 4.

Toda *et al*. (1992) reported that polyphenol catechins (EGC, 3, ECG, 4, and EGCG, 5) isolated from green tea have protective function against infection with *V. cholerae* O1. EGCG and ECG also protect against hemolysin (another toxin from *V. cholerae* that causes red blood cell rupture) in a dose dependent manner–the more green tea catechins, the better the protection. Animal studies also showed that these catechins reduced the fluid accumulation

Toda *et al.* (1991) have also suggested that extracts of black tea have anti-bactericidal function against *V. cholerae* O1. The major active components of black tea that could be responsible for protective activity against *V. cholerae* O1 are theaflavin-3,3'-digallate, 9 and

Saito *et al.* (2002) have shown the anti-choleric activity of natural polyphenols extracted from immature apples. They described that the inhibitory effect of apple polyphenols extract (APE) on CT-catalyzed ADP-ribosylation of agmantine is dose-dependent and it is due to the inhibition of the enzymatic activity of the A subunit of CT. The concentration of APE at which 50% of the enzymatic activity of CT (15 μg/ml) is inhibited was approximately 8.7 μg/ml. Bioassay oriented fractionation of APE indicated that the highly polymerized catechins, also named procyanidine polymers, are the major inhibitory components of this apple extract. Other constituents like the non-catechin-type polyphenols (chlorogenic acid, phloridzin, phloretin, caffeic acid, and p-coumaric acid) and the monomeric catechins (catechin and epicatechin) have shown week inhibitory activity. The results indicate that APE disturbs the biological activity of CT *in vivo*, also but not only via inhibition of the enzymatic activity of A-subunit. An additional explanation for the *in vivo* reduction of secretory activity of APE can also be the protection of the intestine's mucosa with polymerized catechins. Procyanidins B1, 6, C2, 7 and tetracatechin, 8 representative

Hor *et al.* (1995) also reported *in vivo* CTA inhibitory activity of proantho-cyanidines extracted from *Guazuma ulimfolia,* a medicinal plant used in Mexico for traditional treatment

Oi *et al.* (2002) studied the bioactivity of rhubarb galloyl tannin (RG-tannin), a compound isolated from *Rhei rhizome,* against CT activities including ADP-ribosylation and fluid accumulation. This kind of heterologuos polyphenol gallate inhibits fluid accumulation in mouse and rabbit ileal loops that is induced by CT action, as well as catalytic activity of

(the primary cause of cholera fatality) from CT (Toda, Okubo et al. 1992).

structures from Saito study are shown in Fig. 4.

thearubigin, 10.

of diarrhoea.

cycle. Ribosylated form of Gs stabilizes the GTP bound form of protein, which stays continually activated (Cassel and Pfeuffer 1978). This situation is shown in Fig 3b.

Fig. 3. Action of enzymatic unit of Cholera Toxin. (A) Normal action. (B) Permanent activation of Adenylate cyclase system.

This modification of the adenylate cyclase system results in an elevated level of AMP which causes the activation of the sodium pumps in the lumen of the cells through an AMPdependent kinase pathway, forcing the Na+ ions out. The electrochemical imbalance is then compensated by driving Cl- and H2O out of the cells. The process of Cholera Toxin action is followed by enormous loss of water from the epithelial cells into the intestinal lumen, causing water diarrhoea characteristic for cholera.

It has been shown that A1 by itself has relatively low enzymatic activity in vitro. The interaction of A1 with ADP-ribosylation factor (ARF) protein from human host increases the enzymatic activity of A1. Numerous studies *in vitro* and *in vivo* have indicated the importance of tight interaction between A1 fragment and ARF. Recent structural investigations of a CTA1 :ARF6-GTP complex pointed out that binding of ARF6-GTP causes dramatic changes in the CTA1 loop regions that open the binding site for NAD+ (Hol, O'Neal et al. 2005).

Taking into account structural information and given mechanism of action of CT, three different strategies are possible to design a prophylactic cure against Cholera:


In further writing, the first two strategies of the development the Cholera toxin inhibitors will be reviewed, with special attention to experience based on our recent work in this field of medicinal chemistry.

## **3. Natural products as Cholera toxin inhibitors**

During the history, traditional healers have prepared medicaments for the treatment (prevention) of Cholera infections from various medicinal plants. The most common ways to administer such natural remedies are infusions or decoctions that are usually compositions

cycle. Ribosylated form of Gs stabilizes the GTP bound form of protein, which stays

continually activated (Cassel and Pfeuffer 1978). This situation is shown in Fig 3b.

Fig. 3. Action of enzymatic unit of Cholera Toxin. (A) Normal action. (B) Permanent

This modification of the adenylate cyclase system results in an elevated level of AMP which causes the activation of the sodium pumps in the lumen of the cells through an AMPdependent kinase pathway, forcing the Na+ ions out. The electrochemical imbalance is then compensated by driving Cl- and H2O out of the cells. The process of Cholera Toxin action is followed by enormous loss of water from the epithelial cells into the intestinal lumen,

It has been shown that A1 by itself has relatively low enzymatic activity in vitro. The interaction of A1 with ADP-ribosylation factor (ARF) protein from human host increases the enzymatic activity of A1. Numerous studies *in vitro* and *in vivo* have indicated the importance of tight interaction between A1 fragment and ARF. Recent structural investigations of a CTA1 :ARF6-GTP complex pointed out that binding of ARF6-GTP causes dramatic changes in the

Taking into account structural information and given mechanism of action of CT, three

Design of small molecules that act as a decoys for the toxin's GM1 binding site and thus

In further writing, the first two strategies of the development the Cholera toxin inhibitors will be reviewed, with special attention to experience based on our recent work in this field

During the history, traditional healers have prepared medicaments for the treatment (prevention) of Cholera infections from various medicinal plants. The most common ways to administer such natural remedies are infusions or decoctions that are usually compositions

CTA1 loop regions that open the binding site for NAD+ (Hol, O'Neal et al. 2005).

different strategies are possible to design a prophylactic cure against Cholera:

Inhibition of the action of the catalytically active unit A of CT.

**3. Natural products as Cholera toxin inhibitors** 

prevent adhesion of CT to cell membranes of epithelial cells. Prevention of assembly of AB5 complex that takes place in the cytosol.

activation of Adenylate cyclase system.

of medicinal chemistry.

causing water diarrhoea characteristic for cholera.

of numerous natural products. The active substances from medicinal plants can treat Cholera via different pharmacological mechanisms; from the direct antimicrobial against *V. cholerae*, prevention of adhesion of CT to the GM1 receptors at surface of epithelial cells, to direct inhibition of ADP-ribosylation of active unit of CT. The improved understanding of the CT toxicity at the molecular level and the further set up of modern biological assays, has allowed in recent years the identification of different classes of bioactive natural products. An important class of such products are polyphenols from green and black tea, green apples, hop bract and the Chinese rhubarb rhizome. Garlic extract is another example of traditional cure against diarrhoea diseases such as cholera. Recent researches have recognized a galactan polysaccharide as a major anti-choleric component of garlic (Politi, Alvaro-Blanco et al. 2006). Some interesting natural inhibitors of CT are shown in Fig 4.

Toda *et al*. (1992) reported that polyphenol catechins (EGC, 3, ECG, 4, and EGCG, 5) isolated from green tea have protective function against infection with *V. cholerae* O1. EGCG and ECG also protect against hemolysin (another toxin from *V. cholerae* that causes red blood cell rupture) in a dose dependent manner–the more green tea catechins, the better the protection. Animal studies also showed that these catechins reduced the fluid accumulation (the primary cause of cholera fatality) from CT (Toda, Okubo et al. 1992).

Toda *et al.* (1991) have also suggested that extracts of black tea have anti-bactericidal function against *V. cholerae* O1. The major active components of black tea that could be responsible for protective activity against *V. cholerae* O1 are theaflavin-3,3'-digallate, 9 and thearubigin, 10.

Saito *et al.* (2002) have shown the anti-choleric activity of natural polyphenols extracted from immature apples. They described that the inhibitory effect of apple polyphenols extract (APE) on CT-catalyzed ADP-ribosylation of agmantine is dose-dependent and it is due to the inhibition of the enzymatic activity of the A subunit of CT. The concentration of APE at which 50% of the enzymatic activity of CT (15 μg/ml) is inhibited was approximately 8.7 μg/ml. Bioassay oriented fractionation of APE indicated that the highly polymerized catechins, also named procyanidine polymers, are the major inhibitory components of this apple extract. Other constituents like the non-catechin-type polyphenols (chlorogenic acid, phloridzin, phloretin, caffeic acid, and p-coumaric acid) and the monomeric catechins (catechin and epicatechin) have shown week inhibitory activity. The results indicate that APE disturbs the biological activity of CT *in vivo*, also but not only via inhibition of the enzymatic activity of A-subunit. An additional explanation for the *in vivo* reduction of secretory activity of APE can also be the protection of the intestine's mucosa with polymerized catechins. Procyanidins B1, 6, C2, 7 and tetracatechin, 8 representative structures from Saito study are shown in Fig. 4.

Hor *et al.* (1995) also reported *in vivo* CTA inhibitory activity of proantho-cyanidines extracted from *Guazuma ulimfolia,* a medicinal plant used in Mexico for traditional treatment of diarrhoea.

Oi *et al.* (2002) studied the bioactivity of rhubarb galloyl tannin (RG-tannin), a compound isolated from *Rhei rhizome,* against CT activities including ADP-ribosylation and fluid accumulation. This kind of heterologuos polyphenol gallate inhibits fluid accumulation in mouse and rabbit ileal loops that is induced by CT action, as well as catalytic activity of

Structure Based Design of Cholera Toxin Antagonists 183

hand, oligocatechins can not penetrate into the binding site in the whole extension, such compounds could additionally form numerous non-specific contacts with the protein surface, and hinder NAD+ to access the binding site (Fig. 5c). Nice fits to CTA binding site have been also observed for theaflavin-3,3 digallate (Fig. 5d), 9 from black tea and Oi's synthetic gallate (Fig. 5b), 12, the results of molecular docking (Glide XP) indicate that these

two compounds could act as strong inhibitors of CT´s ADP-ribosylation activity .

Fig. 5. Docking poses (GlideXP) for selected polyphenols. (A) epigallocatechin gallate; (B) 1,2,3,6-tetra-O-galloyl-D-glucose; (C) tetrameric catechin; (D) theaflavin-3,3-digalate. Yellow

Many other plants have been used for centuries around the world in traditional medicine as natural remedies for cholera and other diarrhoeal infections. Most of the current pharmacological studies are oriented to test antibacterial activity of some of these medicinal species from plant extracts against *V. Cholerae*. On the other hand, direct investigations of natural products as potential CT inhibitors are very rare, this field of research is still open and some additional founding of direct biological action of natural compounds to cholera

The synthesis of ganglioside GM1 itself is very complex (Sugimoto, Numata et al. 1986), therefore one of the strategies how to prevent adhesion of CTB to the cell surface involves

NMR and theoretical studies of the conformational behavior of GM1 and of other ganglioside head groups (e.g. GM2, GM3, and asialo GM1) have shown that the 3,4-

coloured NAD+ is appeared in each CTA:ligand complex as a reference.

**4. Rational design of GM1 mimics as Cholera toxin inhibitors** 

design and synthesis of functional and structural mimics of oGM1 (Fig 6).

toxin and other AB5 toxins are more than welcome.

CTA. It was also observed that RG-tannin, had no effect on the binding of CTB to the ganglioside GM1 or an endogenous ADP-ribosylation of membrane proteins. The authors prepared and tested a small library of synthetic gallates (sugar moieties esterified with galloyl groups) against CTA's ADP-rybosylation activity a small library of synthetic gallates. Some of compounds (12 for example) from their library exhibited strong inhibitory activity of CTAs ADP-ribosylation.

Fig. 4. Structures of natural products that could serve as CT inhibitors.

Politi *et al.* (2006) reported binding activity between a high molecular weight polysaccharide, galactan from garlic water extract and the B subunit of CT (CTB). The interaction was confirmed with Saturation Transfer Difference (STD) experiment, one of the NMR methods used to measure interaction between ligands and target receptor, and with fluorometric binding assays. This study indicates that one galactan polymer could bind with large number of CTB protein monomers. The ability of galactan to form high molecular weight aggregates with CTB and thus prevent adhesion to cell-surface could be the main reason for its inhibitory activity. A fragment of galactan 13 is shown in Fig. 4.

Podlipnik (2009) has collected polyphenol structures from different sources and described a structure-based model of inhibition of ADP-ribosyltransferase activity of Cholera toxin by polyphenol's. Compounds 1-12 (Fig 4) are members of the polyphenol´s library used for virtual screening against CTA1 . For docking purposes a model based on a crystallographic structure of CTA1:ARF6-GTP complex (Hol, O'Neal et al. 2005) was prepared. From docking (Glide XP) results it is evident that mono catechines can penetrate deeply into the binding site of CTA (Fig. 5a). The inhibitory activity of polyphenols generally increases with their complexity, measured by number of hydroxyl groups attached to the scaffold. On the other

CTA. It was also observed that RG-tannin, had no effect on the binding of CTB to the ganglioside GM1 or an endogenous ADP-ribosylation of membrane proteins. The authors prepared and tested a small library of synthetic gallates (sugar moieties esterified with galloyl groups) against CTA's ADP-rybosylation activity a small library of synthetic gallates. Some of compounds (12 for example) from their library exhibited strong inhibitory activity

Fig. 4. Structures of natural products that could serve as CT inhibitors.

reason for its inhibitory activity. A fragment of galactan 13 is shown in Fig. 4.

Politi *et al.* (2006) reported binding activity between a high molecular weight polysaccharide, galactan from garlic water extract and the B subunit of CT (CTB). The interaction was confirmed with Saturation Transfer Difference (STD) experiment, one of the NMR methods used to measure interaction between ligands and target receptor, and with fluorometric binding assays. This study indicates that one galactan polymer could bind with large number of CTB protein monomers. The ability of galactan to form high molecular weight aggregates with CTB and thus prevent adhesion to cell-surface could be the main

Podlipnik (2009) has collected polyphenol structures from different sources and described a structure-based model of inhibition of ADP-ribosyltransferase activity of Cholera toxin by polyphenol's. Compounds 1-12 (Fig 4) are members of the polyphenol´s library used for virtual screening against CTA1 . For docking purposes a model based on a crystallographic structure of CTA1:ARF6-GTP complex (Hol, O'Neal et al. 2005) was prepared. From docking (Glide XP) results it is evident that mono catechines can penetrate deeply into the binding site of CTA (Fig. 5a). The inhibitory activity of polyphenols generally increases with their complexity, measured by number of hydroxyl groups attached to the scaffold. On the other

of CTAs ADP-ribosylation.

hand, oligocatechins can not penetrate into the binding site in the whole extension, such compounds could additionally form numerous non-specific contacts with the protein surface, and hinder NAD+ to access the binding site (Fig. 5c). Nice fits to CTA binding site have been also observed for theaflavin-3,3 digallate (Fig. 5d), 9 from black tea and Oi's synthetic gallate (Fig. 5b), 12, the results of molecular docking (Glide XP) indicate that these two compounds could act as strong inhibitors of CT´s ADP-ribosylation activity .

Fig. 5. Docking poses (GlideXP) for selected polyphenols. (A) epigallocatechin gallate; (B) 1,2,3,6-tetra-O-galloyl-D-glucose; (C) tetrameric catechin; (D) theaflavin-3,3-digalate. Yellow coloured NAD+ is appeared in each CTA:ligand complex as a reference.

Many other plants have been used for centuries around the world in traditional medicine as natural remedies for cholera and other diarrhoeal infections. Most of the current pharmacological studies are oriented to test antibacterial activity of some of these medicinal species from plant extracts against *V. Cholerae*. On the other hand, direct investigations of natural products as potential CT inhibitors are very rare, this field of research is still open and some additional founding of direct biological action of natural compounds to cholera toxin and other AB5 toxins are more than welcome.

## **4. Rational design of GM1 mimics as Cholera toxin inhibitors**

The synthesis of ganglioside GM1 itself is very complex (Sugimoto, Numata et al. 1986), therefore one of the strategies how to prevent adhesion of CTB to the cell surface involves design and synthesis of functional and structural mimics of oGM1 (Fig 6).

NMR and theoretical studies of the conformational behavior of GM1 and of other ganglioside head groups (e.g. GM2, GM3, and asialo GM1) have shown that the 3,4-

Structure Based Design of Cholera Toxin Antagonists 185

galactoside (MNPG), 21. Its affinity for CTB (IC50 = 720 μM) is two orders of magnitude higher than it is found for galactose. (Minke, Roach et al. 1999) (Fan, Merritt et al. 2001) Further crystallographic studies have shown the displacement of a water molecule that is structurally bound to CTB by the meta nitro group of the MNPG's phenyl ring. The mentioned displacement leads to an increase in the entropy of the system and creates tight hydrogen bond interactions between the nitro group of MNPG and CTB could, which may be the reason for an increased CT inhibitory activity (Fan, Merritt et al. 2001). It has been also observed that m-carboyxphenyl α-D-galactoside (MCPG), 22, binds to the target with a different binding mode. Poses of MCPG and MNPG extracted from crystallographic

structures are shown in Figs. 9a and 9b, respectively (Fan, Merritt et al. 2001).

Fig. 9. Poses of (A) MNPG, 21 and (B) MCPG, 22 to CTB. Poses are extracted from

Fan *et al.* designed a library of CTB antagonists where rigid hydrophobic rings were linked with different short and flexible aliphatic linkers to the meta position of phenyl ring of α-Dgalactoside (Fan, Pickens et al. 2002). Some compounds from the mentioned libraries are shown in Fig. 9. This modification of MNPG allows to explore different regions of CTB binding site. Minke *et al.* (1999) explored a hydrophobic pocket in the bottom of the LT-II binding with series of galactosides that have large hydrophobic moieties, and found that compound 24 had the lowest IC50=40 μM, which is more than three orders of magnitude

Fig. 8. Some examples from MNPG (MCPG) library.

crystallographic data.

branching at Gal-II residue is the main reason for rigidity of oGM1 structure, so Gal-II residue appears to act as the scaffold that holds together the two terminal Gal-IV and Neu5Ac moieties at the proper orientation for optimal interaction with CT (Bernardi and Raimondi 1995; Bernardi, Arosio et al. 2002). Bernardi *et al.* have used the above structural hypothesis to develop and design ligands (14 for example) using conformationally restricted *cis*-1,2-cyclohexanediol, 15, as a replacement of Gal-II residue in oGM1 (Bernardi, Checchia et al. 1999; Bernardi, Arosio et al. 2001). The experimental results obtained by ELISA assays and fluorescence titration have shown that CT inhibition activities of Bernardi's mimics and oGM1 are more or less in the same range. The major problem in the synthesis of the "first generation" of the Bernardi's mimics is the stereoselective syalilation of *cis*-1,2 cyclohexanediol, this step represents the bottleneck of the synthesis. Therefore, a further simplification of oGM1 structure that has been based on the replacement of the sialic acid residue with simple α-hydroxyl acids, 16-20 was proposed by Bernardi's research group (Bernardi, Carrettoni et al. 2000).

Fig. 6. Structures of Bernardi's GM1 mimics.

Fig. 7. Poses (Glide XP) of Bernardi's GM1 mimics: (A) 18 and (B) 20 to CTB .

The next widely used approach to design CTB inhibitors is to use the terminal galactose as an anchor to which various pharmacophores can be attached. Minke *et al.* (1999) have used fluorescence titrations and ELISA assays to screen a series of commercially available galactose derivates. The most active compound from this series was meta nitrophenyl α-D-

branching at Gal-II residue is the main reason for rigidity of oGM1 structure, so Gal-II residue appears to act as the scaffold that holds together the two terminal Gal-IV and Neu5Ac moieties at the proper orientation for optimal interaction with CT (Bernardi and Raimondi 1995; Bernardi, Arosio et al. 2002). Bernardi *et al.* have used the above structural hypothesis to develop and design ligands (14 for example) using conformationally restricted *cis*-1,2-cyclohexanediol, 15, as a replacement of Gal-II residue in oGM1 (Bernardi, Checchia et al. 1999; Bernardi, Arosio et al. 2001). The experimental results obtained by ELISA assays and fluorescence titration have shown that CT inhibition activities of Bernardi's mimics and oGM1 are more or less in the same range. The major problem in the synthesis of the "first generation" of the Bernardi's mimics is the stereoselective syalilation of *cis*-1,2 cyclohexanediol, this step represents the bottleneck of the synthesis. Therefore, a further simplification of oGM1 structure that has been based on the replacement of the sialic acid residue with simple α-hydroxyl acids, 16-20 was proposed by Bernardi's research group

(Bernardi, Carrettoni et al. 2000).

Fig. 6. Structures of Bernardi's GM1 mimics.

Fig. 7. Poses (Glide XP) of Bernardi's GM1 mimics: (A) 18 and (B) 20 to CTB .

The next widely used approach to design CTB inhibitors is to use the terminal galactose as an anchor to which various pharmacophores can be attached. Minke *et al.* (1999) have used fluorescence titrations and ELISA assays to screen a series of commercially available galactose derivates. The most active compound from this series was meta nitrophenyl α-D-

galactoside (MNPG), 21. Its affinity for CTB (IC50 = 720 μM) is two orders of magnitude higher than it is found for galactose. (Minke, Roach et al. 1999) (Fan, Merritt et al. 2001) Further crystallographic studies have shown the displacement of a water molecule that is structurally bound to CTB by the meta nitro group of the MNPG's phenyl ring. The mentioned displacement leads to an increase in the entropy of the system and creates tight hydrogen bond interactions between the nitro group of MNPG and CTB could, which may be the reason for an increased CT inhibitory activity (Fan, Merritt et al. 2001). It has been also observed that m-carboyxphenyl α-D-galactoside (MCPG), 22, binds to the target with a different binding mode. Poses of MCPG and MNPG extracted from crystallographic structures are shown in Figs. 9a and 9b, respectively (Fan, Merritt et al. 2001).

Fig. 8. Some examples from MNPG (MCPG) library.

Fig. 9. Poses of (A) MNPG, 21 and (B) MCPG, 22 to CTB. Poses are extracted from crystallographic data.

Fan *et al.* designed a library of CTB antagonists where rigid hydrophobic rings were linked with different short and flexible aliphatic linkers to the meta position of phenyl ring of α-Dgalactoside (Fan, Pickens et al. 2002). Some compounds from the mentioned libraries are shown in Fig. 9. This modification of MNPG allows to explore different regions of CTB binding site. Minke *et al.* (1999) explored a hydrophobic pocket in the bottom of the LT-II binding with series of galactosides that have large hydrophobic moieties, and found that compound 24 had the lowest IC50=40 μM, which is more than three orders of magnitude

Structure Based Design of Cholera Toxin Antagonists 187

for the selection of the scaffold. The approach that has been adopted to identify CT inhibitors involves the following steps: the development and validation of a docking/scoring protocol based on a set of known pseudo-GM1 ligands; the design of a focused library of C-galactosides; the synthesis and affinity evaluation (by SPR) of selected elements of the library. The authors have tried to design relatively rigid ligands with α configuration on Galactose anomeric center that could fit CTB binding site. Cinnamic acids and their derivatives have been found as an ideal solution for conjugation functionalized Cgalactosides (compounds 29-33 from Fig. 11). The best value of IC50 (125 μM) has been observed for compound 30. The pose of this compound within CTB binding site (Glide XP

Fig. 12. Docking (Glide XP) poses of (A) Verlinde's ligand 24 and ligand 30 from cinnamic acid galactoconjugates library to CTB. Comparison of two galactoconjugates with

Fig. 13. General scheme for design of non-hydrolysabile Cheshev's bidentate CT inhibitors.

Cheshev *et al.* (2010) have used click chemistry to design a library of non-hydrolyzable bidentate CTB ligand, where two binding determinants, galactose and sialic acid, are connected to one other as they are in oGM1. All compounds from their library were

docking) is shown in Fig. 12b.

configuration on anomeric centre.

lower from the IC50 of galactose . Docking (Glide XP) pose of 24, an α galactoside with large rigid hydrophobic moiety, to CT is shown in Fig. 12a.

Pieters *et al.* (2001; 2002) synthesized monovalent lactose-derived ligands for Cholera toxin, an example of such ligand is 25, where a thiourea moiety served as a spacer between lactose and aromatic system. A 72 fold enhancement of binding affinity of the compound 25 (Kd=248 μM) versus lactose (Kd=18 mM) to CTB determined by fluorescence titration was observed. The next step to improve Pieters' ligands was to increase the rigidity of a spacer between the lactose and a aryl group. The fluorescence study of 26 (Kd=23 μM) revealed one order of magnitude enhancement in the affinity of 25 for the CTB. Two examples from the library of Pieters' ligands based on lactose scaffold are shown in Fig. 10.

Mari *et al.* (2004; 2006) designed and synthesized a galactose-derived bi-cyclic scaffold, the rigid framework and possibility of functionalization at appended side-chain made these compounds interesting for further combinatorial development. NMR and conformational search analysis showed, however, that these compounds were more flexible than expected and did not fit the cholera toxin's binding site. An example, 27 from their library is shown in Fig. 11.

Fig. 11. Cholera toxin inhibitors: bi-cyclic inhibitor, 27 and cinnamic acid galactoconjugates 28-33.

Podlipnik *et al.* (2007) designed a small focused library of functionalized C-galactosides that could serve as non-hydolysable inhibitors of Cholera toxin. The fact that C-galactoside anchors (compound 28 from Fig. 11) can be synthesized in a few steps from galactose, thus avoiding the need for protecting groups, and their metabolical stability are the main reasons

lower from the IC50 of galactose . Docking (Glide XP) pose of 24, an α galactoside with large

Pieters *et al.* (2001; 2002) synthesized monovalent lactose-derived ligands for Cholera toxin, an example of such ligand is 25, where a thiourea moiety served as a spacer between lactose and aromatic system. A 72 fold enhancement of binding affinity of the compound 25 (Kd=248 μM) versus lactose (Kd=18 mM) to CTB determined by fluorescence titration was observed. The next step to improve Pieters' ligands was to increase the rigidity of a spacer between the lactose and a aryl group. The fluorescence study of 26 (Kd=23 μM) revealed one order of magnitude enhancement in the affinity of 25 for the CTB. Two examples from the

Mari *et al.* (2004; 2006) designed and synthesized a galactose-derived bi-cyclic scaffold, the rigid framework and possibility of functionalization at appended side-chain made these compounds interesting for further combinatorial development. NMR and conformational search analysis showed, however, that these compounds were more flexible than expected and did not fit the

Fig. 11. Cholera toxin inhibitors: bi-cyclic inhibitor, 27 and cinnamic acid galactoconjugates

Podlipnik *et al.* (2007) designed a small focused library of functionalized C-galactosides that could serve as non-hydolysable inhibitors of Cholera toxin. The fact that C-galactoside anchors (compound 28 from Fig. 11) can be synthesized in a few steps from galactose, thus avoiding the need for protecting groups, and their metabolical stability are the main reasons

cholera toxin's binding site. An example, 27 from their library is shown in Fig. 11.

rigid hydrophobic moiety, to CT is shown in Fig. 12a.

Fig. 10. Pieters' lactose derived ligands.

28-33.

library of Pieters' ligands based on lactose scaffold are shown in Fig. 10.

for the selection of the scaffold. The approach that has been adopted to identify CT inhibitors involves the following steps: the development and validation of a docking/scoring protocol based on a set of known pseudo-GM1 ligands; the design of a focused library of C-galactosides; the synthesis and affinity evaluation (by SPR) of selected elements of the library. The authors have tried to design relatively rigid ligands with α configuration on Galactose anomeric center that could fit CTB binding site. Cinnamic acids and their derivatives have been found as an ideal solution for conjugation functionalized Cgalactosides (compounds 29-33 from Fig. 11). The best value of IC50 (125 μM) has been observed for compound 30. The pose of this compound within CTB binding site (Glide XP docking) is shown in Fig. 12b.

Fig. 12. Docking (Glide XP) poses of (A) Verlinde's ligand 24 and ligand 30 from cinnamic acid galactoconjugates library to CTB. Comparison of two galactoconjugates with configuration on anomeric centre.

Fig. 13. General scheme for design of non-hydrolysabile Cheshev's bidentate CT inhibitors.

Cheshev *et al.* (2010) have used click chemistry to design a library of non-hydrolyzable bidentate CTB ligand, where two binding determinants, galactose and sialic acid, are connected to one other as they are in oGM1. All compounds from their library were

Structure Based Design of Cholera Toxin Antagonists 189

Fan *et al.* (2002) have designed a multivalent receptor-binding antagonists against CT and LT-II with particular focus on exploiting the 5-fold symmetry of the binding sites on the toxin B pentamer. A conceptual design of such symmetric pentavalent ligand where monovalent "fingers" that block the toxin receptor binding site are attached to symmetric core via modular linker units is shown in Fig. 15. Multivalent inhibitor 36 (Fig. 16) is shown as an example of symmetric pentavalent inhibitor with using MNPGs as monovalent fingers. The affinity of 36 for CTB was investigated using enzyme-linked addhesion assay, from which ED50=0.9 μM was determined. This represents more than 250-fold enhancement of the activity found for MNPG, 21. Crystallographic studies of complex between 36 and CTB brought additional support for a 1:1 association model between the ligand and the toxin, the canonical water is displaced also in

this case (Minke, Pickens et al. 2000; Hol, Zhang et al. 2002).

Fig. 15. General scheme of Fan's symmetric multivalent inhibitor design.

Fig. 16. An example of multivalent inhibitor with pent-fold symmetry designed by Fan.

Another strategy for the design of multivalent inhibitors involves the use of dendrimers. A typical example of such design was shown by Pieters and cooworkers (Pieters, Vrasidas, et al. 2001; Pieters, Vrasidas, et al. 2002), who derived denrimers from 3,5-di-(2-

synthesized from readily available precursors using high performance reactions, including click chemistry protocols, and avoiding glycosidic bonds. The general strategy of Cheshev's design is shown in Fig. 13. The affinity of bidentate ligands to CT measured by weak affinity chromatography could be enhanced up to one or two orders of magnitude relative to the individual pharmacological sugar residues. A further enhancement of CT inhibition could be accessed by conjugation of some of the compounds from the library with polyvalent aglycons. Two examples from Cheshev's library are shown in figure 14. Nice fit computed with Glide XP docking software of *R*-epimeric form of ligand 34 to CTB is shown in Fig. 14.

Fig. 14. Two examples from Cheshev´s design and docking (Glide XP) pose of R epimer of compound 35 to CTB.

In this section we shortly described a rational design of CTB antagonists derived from structural simplification of oGM1 – the natural receptor for CT. The presented structures are in the range of very close structurally related mimics of oGM1, such as psGM1, 14 and αhydroxylacid derivatives, 16-20 with Kd that is close to value found for oGM1 itself, to very simple galactoconjugates MNPG, 21, cinnamic acid galactoconjugates, 29-33 for example. The affinity data for binding of simple galactoconjugates to CTB are still far from ideal, but by conjugation of these compounds with polyvalent aglycons the affinity could be enhanced by several orders of magnitude. The design of multivalent inhibitors will be described in next section of the review.

## **5. Design of multivalent inhibitors**

A very effective way to enhance ligand's binding affinity toward its receptor target is to use the ligand in multivalent presentation. The Cholera toxin B-pentamer is an ideal target for studying multivalency and developing multivalent ligands against its action. Due to its high five-fold symmetry and the fact that it has five identical binding sites for GM1. A first attempt to improve oGM1 affinity using multivalency was reported by Schwarzmann *et al.*  (1978)*,* who designed and synthesized divalent oGM1 that has better affinity to CTB than oGM1 by itself. More recently, Schengrund *et al.* (1989) prepared highly active multivalent o-GM1 ligands by linking them to a polymer (poly-L-lisine) or to a dendrimer (octapropyleneimine) that serves as a core.

synthesized from readily available precursors using high performance reactions, including click chemistry protocols, and avoiding glycosidic bonds. The general strategy of Cheshev's design is shown in Fig. 13. The affinity of bidentate ligands to CT measured by weak affinity chromatography could be enhanced up to one or two orders of magnitude relative to the individual pharmacological sugar residues. A further enhancement of CT inhibition could be accessed by conjugation of some of the compounds from the library with polyvalent aglycons. Two examples from Cheshev's library are shown in figure 14. Nice fit computed with Glide XP docking software of *R*-epimeric form of ligand 34 to CTB is shown in Fig. 14.

Fig. 14. Two examples from Cheshev´s design and docking (Glide XP) pose of R epimer of

In this section we shortly described a rational design of CTB antagonists derived from structural simplification of oGM1 – the natural receptor for CT. The presented structures are in the range of very close structurally related mimics of oGM1, such as psGM1, 14 and αhydroxylacid derivatives, 16-20 with Kd that is close to value found for oGM1 itself, to very simple galactoconjugates MNPG, 21, cinnamic acid galactoconjugates, 29-33 for example. The affinity data for binding of simple galactoconjugates to CTB are still far from ideal, but by conjugation of these compounds with polyvalent aglycons the affinity could be enhanced by several orders of magnitude. The design of multivalent inhibitors will be described in

A very effective way to enhance ligand's binding affinity toward its receptor target is to use the ligand in multivalent presentation. The Cholera toxin B-pentamer is an ideal target for studying multivalency and developing multivalent ligands against its action. Due to its high five-fold symmetry and the fact that it has five identical binding sites for GM1. A first attempt to improve oGM1 affinity using multivalency was reported by Schwarzmann *et al.*  (1978)*,* who designed and synthesized divalent oGM1 that has better affinity to CTB than oGM1 by itself. More recently, Schengrund *et al.* (1989) prepared highly active multivalent o-GM1 ligands by linking them to a polymer (poly-L-lisine) or to a dendrimer

compound 35 to CTB.

next section of the review.

**5. Design of multivalent inhibitors** 

(octapropyleneimine) that serves as a core.

Fan *et al.* (2002) have designed a multivalent receptor-binding antagonists against CT and LT-II with particular focus on exploiting the 5-fold symmetry of the binding sites on the toxin B pentamer. A conceptual design of such symmetric pentavalent ligand where monovalent "fingers" that block the toxin receptor binding site are attached to symmetric core via modular linker units is shown in Fig. 15. Multivalent inhibitor 36 (Fig. 16) is shown as an example of symmetric pentavalent inhibitor with using MNPGs as monovalent fingers. The affinity of 36 for CTB was investigated using enzyme-linked addhesion assay, from which ED50=0.9 μM was determined. This represents more than 250-fold enhancement of the activity found for MNPG, 21. Crystallographic studies of complex between 36 and CTB brought additional support for a 1:1 association model between the ligand and the toxin, the canonical water is displaced also in this case (Minke, Pickens et al. 2000; Hol, Zhang et al. 2002).

Fig. 15. General scheme of Fan's symmetric multivalent inhibitor design.

Fig. 16. An example of multivalent inhibitor with pent-fold symmetry designed by Fan.

Another strategy for the design of multivalent inhibitors involves the use of dendrimers. A typical example of such design was shown by Pieters and cooworkers (Pieters, Vrasidas, et al. 2001; Pieters, Vrasidas, et al. 2002), who derived denrimers from 3,5-di-(2-

Structure Based Design of Cholera Toxin Antagonists 191

Fig. 18. A scheme of multivalent Bernardi-Pieters' inhibitor based on dendridic structure and

The most recent example of using multivalent strategy in the design of Cholera toxin inibitors presented in this chapter is based on the work of Tran and collaborators (Tran, Kitov et al. 2011). They are intensively working on designing a bidentate multivalent ligands. In their recent work they describe the synthesis and activities of a series of galactose conjugates on polyacryl amide and dextran. Nanomolar affinity of inhibitors against CT was obtained by conjugation of a second fragment (corresponding Neu5Ac's mimic), while galactose-only progenitors showed no detectable activity. The general idea of such

Fig. 19. Scheme for general design of multivalent Tran-Kitov's bidentate inhibitors.

Bernardi-Casnati's inhibitor based on Calix-[4]-arene core.

inhibitor's design is shown in Fig. 19.

aminoethoxy)benzoic acid repeating units with 2,4 and 8 end groups to which lactose isothiocyanate units, 25, were attached, providing thioureea-linked glycodendrimers 37 (Fig. 17). Analysis of fluorescence titration data showed that the affinity of these compounds for CT was increased by one order of magnitude relative to the monovalent ligands. It has been also shown that the branching of dendrimer provided only a modest increase in the potency of the ligand. The authors have also reported same indications that ligands are able to bind to multiple toxin molecules, rather than to single B pentamer.

Fig. 17. A scheme of Pieters' multivalent inhibitor based on dendridic structure.

A further improvement of multivalent ligand's affinity for the toxin has been obtained attaching Bernardi's monovalent GM1 mimic 25 to Pieters' dendrimeric core (Arosio, Vrasidas et al. 2004). For further improvement, the polysaccharide scaffold was provided with elongated spacer arms. The analysis of surface plasmon resonance data revealed EC50=0.5 μM for 38.

Another option of using Bernardi's GM1 mimics for multivalent inhibitor's design has been presented by Bernardi, Casnati and cooworkers (Bernardi, Arosio et al. 2005). They prepared a bivalent ligand 39 by hooking two units of GM1 mimic 18 to a functionalized calix-[4] arene core. The size of affinity enhancement measured by fluorescence titration was found to be 3800-fold (1900-fold per sugar mimic). Recently, the huge affinity enhancement of 39 versus 18 to CT were confirmed in our laboratory with isothermal calorimetry titration (Prislan, et al. 2011).

The best known multivalent inhibitors up-to date have been reported by Pukin *et al.* (2007). In this study, GM1 containing compound was synthetized enzimatically starting from ωazidoundecyl lactoside, that was then coupled onto Pieters' linker-extended dendrimers by cooper-catalysed azide-alkyne cycloaddition. Dendrimers bearing two, four and eight GM1 sugars were evaluated by ELISA, the IC50 values for these compounds were 2 nM, 0.2 nM and 50 pM, respectively.

aminoethoxy)benzoic acid repeating units with 2,4 and 8 end groups to which lactose isothiocyanate units, 25, were attached, providing thioureea-linked glycodendrimers 37 (Fig. 17). Analysis of fluorescence titration data showed that the affinity of these compounds for CT was increased by one order of magnitude relative to the monovalent ligands. It has been also shown that the branching of dendrimer provided only a modest increase in the potency of the ligand. The authors have also reported same indications that ligands are able to bind

to multiple toxin molecules, rather than to single B pentamer.

Fig. 17. A scheme of Pieters' multivalent inhibitor based on dendridic structure.

EC50=0.5 μM for 38.

(Prislan, et al. 2011).

and 50 pM, respectively.

A further improvement of multivalent ligand's affinity for the toxin has been obtained attaching Bernardi's monovalent GM1 mimic 25 to Pieters' dendrimeric core (Arosio, Vrasidas et al. 2004). For further improvement, the polysaccharide scaffold was provided with elongated spacer arms. The analysis of surface plasmon resonance data revealed

Another option of using Bernardi's GM1 mimics for multivalent inhibitor's design has been presented by Bernardi, Casnati and cooworkers (Bernardi, Arosio et al. 2005). They prepared a bivalent ligand 39 by hooking two units of GM1 mimic 18 to a functionalized calix-[4] arene core. The size of affinity enhancement measured by fluorescence titration was found to be 3800-fold (1900-fold per sugar mimic). Recently, the huge affinity enhancement of 39 versus 18 to CT were confirmed in our laboratory with isothermal calorimetry titration

The best known multivalent inhibitors up-to date have been reported by Pukin *et al.* (2007). In this study, GM1 containing compound was synthetized enzimatically starting from ωazidoundecyl lactoside, that was then coupled onto Pieters' linker-extended dendrimers by cooper-catalysed azide-alkyne cycloaddition. Dendrimers bearing two, four and eight GM1 sugars were evaluated by ELISA, the IC50 values for these compounds were 2 nM, 0.2 nM

Fig. 18. A scheme of multivalent Bernardi-Pieters' inhibitor based on dendridic structure and Bernardi-Casnati's inhibitor based on Calix-[4]-arene core.

The most recent example of using multivalent strategy in the design of Cholera toxin inibitors presented in this chapter is based on the work of Tran and collaborators (Tran, Kitov et al. 2011). They are intensively working on designing a bidentate multivalent ligands. In their recent work they describe the synthesis and activities of a series of galactose conjugates on polyacryl amide and dextran. Nanomolar affinity of inhibitors against CT was obtained by conjugation of a second fragment (corresponding Neu5Ac's mimic), while galactose-only progenitors showed no detectable activity. The general idea of such inhibitor's design is shown in Fig. 19.

Fig. 19. Scheme for general design of multivalent Tran-Kitov's bidentate inhibitors.

Structure Based Design of Cholera Toxin Antagonists 193

Following the discovery of the relation between blood group phenotype and cholera susceptibility, many studies have been conducted in order to investigate the ability of cholera toxin and of the highly homologous heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli (ETEC), to recognize blood group antigens of the ABO system (Bennun 1989, Monferran 1990, Barra 1992, Balanzino 1994, Balanzino 1999). It has been hypothesized that blood group antigens, with a preference for A and B epitopes, might disturb the action of the toxin by interfering with binding to GM1 ganglioside in the small intestine. However, the well-conserved GM1 binding site of CT is believed to be ganglioside-specific and cannot accommodate the fucosylated blood group antigens according to computer modelling. Consequently, the basis for the recognition of blood group antigens by Cholera Toxin at a molecular level is still unclear. In a recent investigation, Teneberg and co-workers have discovered a novel carbohydrate binding site studying a chimera between the B subunits of cholera toxin and the E. coli LT. This CTB/LTB chimera was shown to bind blood group A or B antigens on type 2 chains (Ångström 2000), and was subsequently characterized in complex with a blood group A analogue using protein crystallography by Krengel and co-workers (Holmner 2004). The structure of such complex is shown in Fig 21a. A follow-up study showed that native LTB, despite binding blood group antigens with lower affinity, also display the same mode of binding as the CTB/LTB chimera (Holmner 2007). In both cases, this binding site for blood group ligands is clearly distinct from the primary GM1 binding site. The blood group recognition site is located at the interface of two B-subunits, with one of the 2 subunits providing the majority of the contacts to the ligand. Based on the two crystal structures, it was possible to explain how the toxins discriminate between different ABH epitopes. The GalNAc3 residue characteristic of blood group antigens binds with the toxin via several hydrogen bonds, including one involving its acetamido nitrogen (Fig 21b). The blood group B antigen is characterized by a terminal galactose residue and only differ from the A antigen at the 2-position, i.e. the acetamido group is replaced by a hydroxyl group. This hydroxyl group should preserve most of the interactions with the toxin and explains why the toxin does not discriminate notably between A and B epitopes. The fucose residue on the ABH antigens is also an important contributor to receptor recognition, however, blood group H determinants lack the entire terminal saccharide residue compared to blood group A and B determinant, and would therefore be expected to have significantly reduced binding affinities to cholera toxin. This assumption is substantiated by the finding that the loss of a single water-mediated hydrogen bond to the terminal GalNAc3 residue results in a pronounced decrease in binding affinity (Holmner 2004), confirming the importance of the terminal GalNAc residue characteristic of blood group A antigens in molecular recognition. All these new contributions to understand the molecular basis of the interaction between blood group antigens and cholera toxin were reviewed in more detail by Krengel and co-

In conclusion, the new information on the molecular recognition of blood-group antigens by Cholera Toxin should encourage medicinal chemist to development improved drug design strategies to prepare new pharmacological agents that inactivate cholera toxin. Inhibitors of the interaction of the cholera toxin with its primary receptor, the GM1 ganglioside, are especially attractive. Development of antagonists for the blood group

workers (Holmner 2010).

A variety of multivalent inhibitors were described in this chapter. Basically, the multivalent inhibitors are designed using linkers to connect a galactose anchor to polymeric or dendrimeric cores or symmetric cores (5-fold symmetry). Recently, bidentate multivalent inhibitors were designed with conjugation of second fragment of that corresponds to Neu5Ac mimic. Generally, the above results showed that strategy of designing multivalent presentations of monovalent ligands can bring affinity closer to what is required for practical application against CT.

### **6. Novel binding site for blood group antigens in Cholera toxin: Potential target for the design of new Inhibitors?**

At the end of the 1970s, two epidemiological studies established a dependency between the severity of Cholera infections and the blood group phenotype (Baura, Paguio 1977; Chaudhuri, De 1997). In these studies it was reported that people with blood group O were more prone to develop severe symptoms in comparison with people of blood group A, B or AB phenotype. Also, it was found that this dependency is strain specific, for example, in *V. cholerae* O1 "El Tor" (responsible of the seventh (current) pandemic) and *V. cholerae* O139 infections a connection with blood group phenotype of individuals was found (Glass et al. 1985, Swerdlow et al. 1994, Farruque et al. 1994, Tacket et al. 1995, Harris et al. 2005, Harris et al. 2008). On the other hand, for infection with classical *V. Cholerae* strains, no such association was observed.

The blood group phenotype of an individual is determinated by the presence or absence of antigenic substances on the surface of red blood cells. The ABO antigens are fucosylated oligosaccharide structures, carried on both glycolipids and glycoproteins. (Fig. 20) These antigens are not only on the surface of red blood cells, but are widely distributed throughout body fluids and tissues and are found also in the small intestine, the site of Cholera and ETEC infections. In this tissue, blood group antigens are presented on the intestinal epithelium cell surface (Finne 1989, Breimer 1984, Björk 1987), close to GM1 gangliosides. Structurally, ABO antigens are very similar, the H antigen (responsible of the O phenotype) is a tetrasaccharide characterized by a terminal fucose residue. The A and B antigens are pentasaccharides with a core similar to the H antigen, but each contain and additional saccharide residue- a terminal 2'-*N*-acetyl galactosamine (GalNAc) in the A antigen, or a terminal galactose for the blood group B antigen.

Fig. 20. Schematic representation of the Blood Group antigens H, A and B.

A variety of multivalent inhibitors were described in this chapter. Basically, the multivalent inhibitors are designed using linkers to connect a galactose anchor to polymeric or dendrimeric cores or symmetric cores (5-fold symmetry). Recently, bidentate multivalent inhibitors were designed with conjugation of second fragment of that corresponds to Neu5Ac mimic. Generally, the above results showed that strategy of designing multivalent presentations of monovalent ligands can bring affinity closer to what is required for

**6. Novel binding site for blood group antigens in Cholera toxin: Potential** 

At the end of the 1970s, two epidemiological studies established a dependency between the severity of Cholera infections and the blood group phenotype (Baura, Paguio 1977; Chaudhuri, De 1997). In these studies it was reported that people with blood group O were more prone to develop severe symptoms in comparison with people of blood group A, B or AB phenotype. Also, it was found that this dependency is strain specific, for example, in *V. cholerae* O1 "El Tor" (responsible of the seventh (current) pandemic) and *V. cholerae* O139 infections a connection with blood group phenotype of individuals was found (Glass et al. 1985, Swerdlow et al. 1994, Farruque et al. 1994, Tacket et al. 1995, Harris et al. 2005, Harris et al. 2008). On the other hand, for infection with classical *V. Cholerae* strains, no such

The blood group phenotype of an individual is determinated by the presence or absence of antigenic substances on the surface of red blood cells. The ABO antigens are fucosylated oligosaccharide structures, carried on both glycolipids and glycoproteins. (Fig. 20) These antigens are not only on the surface of red blood cells, but are widely distributed throughout body fluids and tissues and are found also in the small intestine, the site of Cholera and ETEC infections. In this tissue, blood group antigens are presented on the intestinal epithelium cell surface (Finne 1989, Breimer 1984, Björk 1987), close to GM1 gangliosides. Structurally, ABO antigens are very similar, the H antigen (responsible of the O phenotype) is a tetrasaccharide characterized by a terminal fucose residue. The A and B antigens are pentasaccharides with a core similar to the H antigen, but each contain and additional saccharide residue- a terminal 2'-*N*-acetyl galactosamine (GalNAc) in the A

> O HO OH

O

HO

O OH OH

O O

OH

O OH

NHAc OR

O HO OH

O

HO

O OH OH

O O

OH

O OH

NHAc OR

O OHOH

B-penta

O

O HO OH

HO HO

O OHOH

A-penta

Fig. 20. Schematic representation of the Blood Group antigens H, A and B.

O

O HO OH

practical application against CT.

association was observed.

O HO OH

O

HO

O OH OH

O O

OH

O OH

NHAc OR

O OHOH

H-Tetra

HO

**target for the design of new Inhibitors?** 

antigen, or a terminal galactose for the blood group B antigen.

HO AcHN Following the discovery of the relation between blood group phenotype and cholera susceptibility, many studies have been conducted in order to investigate the ability of cholera toxin and of the highly homologous heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli (ETEC), to recognize blood group antigens of the ABO system (Bennun 1989, Monferran 1990, Barra 1992, Balanzino 1994, Balanzino 1999). It has been hypothesized that blood group antigens, with a preference for A and B epitopes, might disturb the action of the toxin by interfering with binding to GM1 ganglioside in the small intestine. However, the well-conserved GM1 binding site of CT is believed to be ganglioside-specific and cannot accommodate the fucosylated blood group antigens according to computer modelling. Consequently, the basis for the recognition of blood group antigens by Cholera Toxin at a molecular level is still unclear. In a recent investigation, Teneberg and co-workers have discovered a novel carbohydrate binding site studying a chimera between the B subunits of cholera toxin and the E. coli LT. This CTB/LTB chimera was shown to bind blood group A or B antigens on type 2 chains (Ångström 2000), and was subsequently characterized in complex with a blood group A analogue using protein crystallography by Krengel and co-workers (Holmner 2004). The structure of such complex is shown in Fig 21a. A follow-up study showed that native LTB, despite binding blood group antigens with lower affinity, also display the same mode of binding as the CTB/LTB chimera (Holmner 2007). In both cases, this binding site for blood group ligands is clearly distinct from the primary GM1 binding site. The blood group recognition site is located at the interface of two B-subunits, with one of the 2 subunits providing the majority of the contacts to the ligand. Based on the two crystal structures, it was possible to explain how the toxins discriminate between different ABH epitopes. The GalNAc3 residue characteristic of blood group antigens binds with the toxin via several hydrogen bonds, including one involving its acetamido nitrogen (Fig 21b). The blood group B antigen is characterized by a terminal galactose residue and only differ from the A antigen at the 2-position, i.e. the acetamido group is replaced by a hydroxyl group. This hydroxyl group should preserve most of the interactions with the toxin and explains why the toxin does not discriminate notably between A and B epitopes. The fucose residue on the ABH antigens is also an important contributor to receptor recognition, however, blood group H determinants lack the entire terminal saccharide residue compared to blood group A and B determinant, and would therefore be expected to have significantly reduced binding affinities to cholera toxin. This assumption is substantiated by the finding that the loss of a single water-mediated hydrogen bond to the terminal GalNAc3 residue results in a pronounced decrease in binding affinity (Holmner 2004), confirming the importance of the terminal GalNAc residue characteristic of blood group A antigens in molecular recognition. All these new contributions to understand the molecular basis of the interaction between blood group antigens and cholera toxin were reviewed in more detail by Krengel and coworkers (Holmner 2010).

In conclusion, the new information on the molecular recognition of blood-group antigens by Cholera Toxin should encourage medicinal chemist to development improved drug design strategies to prepare new pharmacological agents that inactivate cholera toxin. Inhibitors of the interaction of the cholera toxin with its primary receptor, the GM1 ganglioside, are especially attractive. Development of antagonists for the blood group

Structure Based Design of Cholera Toxin Antagonists 195

(NAD+ in case of CT's A-Site; oGM1 in case of B-Site). All ligands described in Sections 3 and 4 are prepared using Schrodinger's Ligprep. Glide XP (Murphy, Repasky et al. 2011) has been used for docking. The figures 1,2,5,7,9,12,14 and 21 representing poses were perpared

We reviewed different strategies to design an effective cure against cholera infections. The first strategy is based on exploring natural ligands as potential inhibitors of the ADPribosylation function of CT A subunit. The data collected from various sources indicate that catechin derivatives found in different natural sources could limit enzymatic activity of CT. Maybe this is one of the major reasons why during the centuries cholera pandemies have spared China and Japan, the catechin-consumig countries. The second approach is to design mimics in a mono- and/or multivalent presentation that could bind to the GM1 binding site in the B-pentamer, and thus prevent binding to GM1 receptors at the surface of epithelial cells, the first act necessary for Cholera toxin intoxication. The rational design of GM1 mimics is one of the most representative example of using structural information supported by molecular modelling methods in task to get an effective inhibitor. Nice examples of how multivalent presentation of single ligands can enhance affinity to CT by several orders of magnitude, and thus reach the levels of affinity required for practical applications against CT were presented. In addition we introduced a new strategy for developing CT inhibitors by targeting a newly identified binding site for blood group antigens in CT. This chapter describes examples of some successful application of knowledge that is connected with molecular structures and processes at the molecular level to design inhibitors toward Cholera toxin. The challenge to transfer the knowledge described in our review to achieve the practical, economic and scalable preparation of CT

Financial support (to Č.P.) from the Slovenian Research Agency (P1 0201) is greatly appreciated. J.J.R is supported by a Marie Curie Intra-European fellowship within the 7th EU Framework Programme (PIEF-2009-GA- 251763). The authors would like to thank Prof. Anna Bernardi (UNIMI) and Dr. Miha Lukšič (UNILJ) for helpful discussion and critical

Andrews, J. R. and S. Basu (2011). "Transmission dynamics and control of cholera in Haiti:

Ångström, J., Bäckström, M., et al.(2000) "Novel carbohydrate binding site recognizing

Arosio, D., I. Vrasidas et al. (2004) "Synthesis and Cholera Toxin Binding Properties of

heat-labile enterotoxin B-subunits." J. Biol. Chem. 275, 3231–3238.

Multivalent GM1 Mimics." Org. Biomol. Chem, 2, 2113-2124.

blood group A and B determinants in a hybrid of cholera toxin and Escherichia coli

an epidemic model." Lancet 377(9773): 1248-1255.

with YASARA (http://www.yasara.com).

**8. Conclusions** 

inhibitors remains still open.

**9. Acknowledgments** 

reading of manuscript.

**10. References** 

binding site of cholera toxin could enable a more effective combined therapy together with GM1 antagonists and, furthermore, could be used as a tool to understand the variability of susceptibility to Cholera infection with the blood group phenotype of individuals.

Fig. 21. (A) A crystallographic representation of CTB/LT chimera that shows the comparison between the binding site of blood group A pentasaccharide analog (green sticks) and GM1 ganglioside (sky-blue sticks) superimposed for comparison. (B) Interactions between blood group A and CTB/LT chimera – close view (Holmner 2010).

## **7. Methods and software**

The methodology of structure based design of CT is described in several occasions (Podlipnik and Bernardi 2007; Zhang 2009). Structure based design starts with preparing a model of the protein receptor site. Models described in our review are based on receptor:ligand complex. In case of exploring of A-site ligands (Section 3) we have used a model based on crystallographic structure of CTA1:ARF6-GTP complex (PDB-ID: 2A5F)(Hol, O'Neal et al. 2005). A high resolution crystallographic structure (1.25 Å) of Cholera toxin B pentamer complexed with oGM1 (PDB-ID:3CHB) (Merritt, Kuhn et al. 1998) has been used as a template for generating a model in the case of exploring GM1 mimics (Section 4). The raw crystallographic structures were in both cases optimized with protein preparation wizard provided as part of the Schrodinger Suite 2011 (http://www.schrodinger.com). The interaction field grids that were used for docking were centered at the center of the ligand

## **8. Conclusions**

194 Cholera

binding site of cholera toxin could enable a more effective combined therapy together with GM1 antagonists and, furthermore, could be used as a tool to understand the variability of susceptibility to Cholera infection with the blood group phenotype of

Fig. 21. (A) A crystallographic representation of CTB/LT chimera that shows the comparison between the binding site of blood group A pentasaccharide analog (green sticks) and GM1 ganglioside (sky-blue sticks) superimposed for comparison. (B) Interactions

The methodology of structure based design of CT is described in several occasions (Podlipnik and Bernardi 2007; Zhang 2009). Structure based design starts with preparing a model of the protein receptor site. Models described in our review are based on receptor:ligand complex. In case of exploring of A-site ligands (Section 3) we have used a model based on crystallographic structure of CTA1:ARF6-GTP complex (PDB-ID: 2A5F)(Hol, O'Neal et al. 2005). A high resolution crystallographic structure (1.25 Å) of Cholera toxin B pentamer complexed with oGM1 (PDB-ID:3CHB) (Merritt, Kuhn et al. 1998) has been used as a template for generating a model in the case of exploring GM1 mimics (Section 4). The raw crystallographic structures were in both cases optimized with protein preparation wizard provided as part of the Schrodinger Suite 2011 (http://www.schrodinger.com). The interaction field grids that were used for docking were centered at the center of the ligand

between blood group A and CTB/LT chimera – close view (Holmner 2010).

**7. Methods and software** 

individuals.

We reviewed different strategies to design an effective cure against cholera infections. The first strategy is based on exploring natural ligands as potential inhibitors of the ADPribosylation function of CT A subunit. The data collected from various sources indicate that catechin derivatives found in different natural sources could limit enzymatic activity of CT. Maybe this is one of the major reasons why during the centuries cholera pandemies have spared China and Japan, the catechin-consumig countries. The second approach is to design mimics in a mono- and/or multivalent presentation that could bind to the GM1 binding site in the B-pentamer, and thus prevent binding to GM1 receptors at the surface of epithelial cells, the first act necessary for Cholera toxin intoxication. The rational design of GM1 mimics is one of the most representative example of using structural information supported by molecular modelling methods in task to get an effective inhibitor. Nice examples of how multivalent presentation of single ligands can enhance affinity to CT by several orders of magnitude, and thus reach the levels of affinity required for practical applications against CT were presented. In addition we introduced a new strategy for developing CT inhibitors by targeting a newly identified binding site for blood group antigens in CT. This chapter describes examples of some successful application of knowledge that is connected with molecular structures and processes at the molecular level to design inhibitors toward Cholera toxin. The challenge to transfer the knowledge described in our review to achieve the practical, economic and scalable preparation of CT inhibitors remains still open.

## **9. Acknowledgments**

Financial support (to Č.P.) from the Slovenian Research Agency (P1 0201) is greatly appreciated. J.J.R is supported by a Marie Curie Intra-European fellowship within the 7th EU Framework Programme (PIEF-2009-GA- 251763). The authors would like to thank Prof. Anna Bernardi (UNIMI) and Dr. Miha Lukšič (UNILJ) for helpful discussion and critical reading of manuscript.

## **10. References**


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**Part 4** 

**Treatment** 

