*2.1.3 Secreted proteins*

In *V. cholerae*, the T2SS ensures the transportation of more than 20 proteins with extracellular activities such as enzymes, toxins, virulence and colonization effectors [6]. The T2SS is essential for survival in the aquatic environment and to infect the human gut. An essential process to colonize both environments is the ability to adhere to abiotic and biotic surfaces such as copepods and zooplankton exoskeletons and epithelial cells, respectively [17–19]. The surface-exposed GlcNAc binding protein (GbpA - VCA0811) is secreted by the T2SS and is an adhesion factor used by *V. cholerae* to bind chitinous surfaces, intestinal epithelial cells and mucin [18–21]. Chitin is the second most abundant polymer in nature and consists on N-acetyl-D-glucosamine (GlcNAc) monomers linked in β-1,4 and the main component of copepods and zooplankton exoskeleton [22]. Attachment to marine crustacea and zooplankton is an advantage for nutrients acquisition, survival and dispersion in aquatic environment. GlcNAc-containing glycoconjugates are often beared by some glycoproteins at the surface of the intestinal epithelial cells and might insure adhesion to the epithelial cells [17, 23, 24]. GbpA possesses 4 domains, the 1 and 4 being the most important for chitin binding, while only the 1 is needed for mucin binding [18]. As a secreted protein, GbpA must be able to interact with *V. cholerae* to allow its adhesion to the substrate. This function is assumed by the domains 3 and 4 of GbpA, that bind to the bacterial surface [18]. GbpA is regulated by the quorum sensing and produced at low cell density [25]. At high cell density, HA/P and PrtV digest GbpA to allow cell detachment and propagation [25, 26]. Also, higher temperatures increase the production of GbpA, promoting cell adhesion [27]. GbpA induces mucin secretion by intestinal epithelium, and mucin increases expression of GbpA [21]. Studies also determined that GbpA can induce necrosis of intestinal cells by increasing their membrane permeability [28]. Recently, a chitin cleavage activity under copper saturation has been described for GbpA and would therefore make it a lytic polysaccharide monooxygenase, a metalloenzyme copper-dependant capable of polysaccharide cleavage by oxidation [29]. Taken together, these findings suggest that GbpA is not only an early adhesion factor but might also have a more important role in pathogenesis.

In the aquatic environment, after binding to zooplankton, copepods and insect egg masses, *V. cholerae* can use chitin as source of carbon and nitrogen [14, 30]. To do so, *V. cholerae* secretes at least 2 chitinases: ChiA-1 and ChiA-2. The chitinase-1 (ChiA-1 - VC1952) and 2 (ChiA-2 – VC0027) are secreted by the T2SS and synergistically hydrolyzes the β1,4 bond between the GlcNAc monomers in the extracellular milieu [6, 14, 31]. The expression and activity of *V. cholerae* chitinases are influenced by environmental factors such as pH, salinity or temperature [32]. In the extracellular milieu, ChiA-1 expression is induced by chitin via the sensor kinase of the orphan two-component system ChiS [31]. In the intestine, the expression of the chitinases is constitutive and a role for ChiA-2 in mucin degradation and in virulence has been reported [33]. Besides ChiA-1 and ChiA-2, other proteins might have a role in chitin utilization including the VCA0140 gene that encodes for the spindolin-related protein, the VC0769 gene product and the chitin oligosaccharide deacetylase (COD - VC1280) [6, 31, 34]. All of them are secreted by the T2SS [6]. Regarding COD, Xibing Li *and coll*. demonstrated that it removes the GlcNAc from chitin oligosaccharides [34].

Besides chitin, collagen can also be used as carbon source by *V. cholerae*. Collagen is one of the most abundant components of host tissues and aquatic animals, and can therefore be found in aquatic environments in association with marine life and sedimentation of decomposing animals [35]. Its degradation provides a nitrogen source, giving a growth advantage to collagenases producing bacteria [35]. The collagenase (VchC - VC1650) is a metalloprotease that degrades type I collagen, providing another carbon source for *V. cholerae* [36]. VchC has been recognized as a T2SS dependant extracellular protein [36]. In other *Vibrio* species, collagenases are recognized as virulence factors as it facilitates their dispersion by degradation of the cellular basal lamina, but this role has not been attributed to VchC yet [36].

After the ingestion, *V. cholerae* navigates through the digestive tract, where it survives many physical and chemical barriers such as gastric acid, peristaltic movement, bile, mucin and microbiota. In the small intestine, it crosses mucin using its flagellum and the mucinase complex, that includes the vibriolysin, a zinc dependant metalloprotease hemagglutinin/protease (HA/P - VCA0865) secreted *via* the T2SS as a free protease or in a cell associated form [26, 37]. The structure, regulation, secretion mechanism and functions of HA/P have been reviewed recently [37]. Briefly, HA/P is expressed when cell density is high or when there is nutrient limitation through the HapR and RpoS regulators [38, 39]. It is translated as an inactive protein and chaperones ensure its inactive state inside the cytoplasm, then the secretion occurs in 2 steps; (i) HA/P is translocated *via* Sec into the periplasm, (ii) the T2SS exports the protease in the extracellular space where an autocatalytic event activates HA/P [37]. HA/P has multiple targets to facilitate spreading of *Vibrio* and increases its virulence [37]. *V. cholerae* gains access to the intestinal epithelial cells by degradation of the mucus layer, lactoferrin and fibronectin by HA/P in order to release toxic effectors into the epithelial cells [26]. In addition, HA/P can cleave toxins such as CT and lactoferrin to activate or increase their activity [26, 40, 41] and disrupts tight junctions between intestinal epithelial cells by occludin cleavage [42]. HA/P participates in *V. cholerae*'s release into the stool by degradation of mucin to detach bacteria from epithelial cells [43].

A second important component of the mucinase complex is the neuramidase (VCNA - VC1784). The sialidase, or neuraminidase, is encoded on the pathogenicity island of every toxigenic *V. cholerae* strains and is secreted by the T2SS [44]. VCNA removes the sialic acid that hides the ganglioside GM1, which is the receptor of the CT, on the surface of epithelial cells [45]. It binds to sialic acid to modify it by its N-terminal lectin domain [46]. Multiple enzymes (VesA, HA/P, and VCNA) appear to work synergistically and with redundancy, ensuring access to the receptor and the activation of the toxin immediately after its secretion [6].

The CT (VC1456-57) is an AB5 toxin secreted by the T2SS in the intestinal lumen, which represents the main virulence factor of *V. cholerae* found in O1 and O139 strains [3, 47]. The subunits are individually translocated by Sec into the periplasm, and the assembled toxin is translocated to the extracellular milieu by the T2SS [7]. The toxin is secreted in an inactive form and must be cleaved by human or bacterial protease to be activated [40]. CT is composed of five B subunits linked in a ring shape that bind to the ganglioside membrane receptor GM1 on the apical surface of intestinal epithelial cells [3]. CT is internalised and transits to the endoplasmic reticulum. The A subunit is heterodimeric with the A2 as the linker between B and A1 subunits, and A1 as a mono-ADP-rybosyltransferase [3]. A1 is released in the endoplasmic reticulum by disulfide isomerase and translocated to the cytosol where it activates the adenylate cyclase G protein by addition of an ADP. Thereby, the activated adenylate cyclase increases the intracellular levels of cyclic AMP, which in turn, activates the protein kinase A (PKA). Finally, PKA activates the chloride anion (Cl− ) excretion by phosphorylation of the chloride channel, that leads to major water secretion by osmose [3]. *ctxA* and *ctxB* genes are organized as

#### *The Secretome of* Vibrio cholerae *DOI: http://dx.doi.org/10.5772/intechopen.96803*

an operon on the integrated CTXφ lysogenic phage [48]. The secretin complex of the T2SS is required by CTXφ to exit the bacteria, which makes the T2SS essential for virulence and horizontal transfer of CT [49]. CT is expressed when the cell density is low, inversely to HA/P, which is why it has been suggested that HA/P could cleave the remaining non-activated CT when the cell density rises [50].

Prior to GM1 binding, the CT must be processed by extracellular proteases to be activated. These proteases are therefore important for virulence and colonization. Besides their capacity to activate the toxin, they have a role in finding a substrate (modification of integrin) and nutrients, and in deactivating host defense mechanisms. Among the T2SS secreted proteins, 3 serine proteases with 30% homology between them have been identified, the *Vibrio* extracellular serine proteases (VesA - VCA0803; VesB - VC1200; VesC - VC1649) [6]. All three proteins have a N-terminal protease domain [6, 51]. VesB has a similar structure and specificity to trypsin [51]. Mutation of *vesABC* allowed to identify that VesB is the main responsible of the proteolytic activity, while VesA and VesC are responsible of 20% of the total proteolytic activity [6]. VesABC do not require bivalent ions for their enzymatic activity [6]. Under laboratory growth conditions, VesA, and in a lesser extend VesB and HA/P, activate the CT in the extracellular milieu [6, 40]. VesC induces a hemorrhagic response in rabbit ileal loop model, which might also reflect a role in virulence [52]. VesB and VesC are expressed at low cell density while VesA is expressed at high cell density [4].

Other virulence factors secreted by the T2SS have been identified in *V. cholerae*. The extracellular metalloprotease (PrtV) is a Zn-dependant metalloprotease [53, 54]. Its activity depends on several autocatalytic events occurring inside and outside the cell for activation [55]. PrtV uses two mechanisms of secretion, in association with membrane vesicles (MV) and *via* the T2SS [6, 56]. PrtV cleaves host proteins such as extracellular matrix and substrate proteins, inducing a change in host cell conformation leading to cell death [53]. In addition, PrtV is necessary for killing of *Caenorhabditis elegans* and protection against predators [57]. To do so, PrtV has many substrates such as, but not limited to, fibronectin, fibrinogen and plasminogen [53]. PrtV is composed of two domains usually known to allow protein-protein or protein-carbohydrates interactions [55, 58]. Thus, PrtV is important for the colonization of ecological niches and in pathogenesis.

The cytolytic toxin cytolysin/hemolysin A (VCC or HlyA - VCA0219), is secreted by the T2SS [6]. All *V. cholerae* strains produce VCC, an iron-dependant secondary toxin activated by cleavage [59]. VCC leads to cell death by vacuolization of the target cell, after production of anions channels in the membrane [60]. Since VCC leads to chloride efflux in intestinal cells, and subsequently to sodium and water by osmosis, it has been suggested that VCC is the major factor responsible of diarrhea in non-producing CT strains.

Leucine aminopeptidase (Lap - VCA0812) and aminopeptidase (LapX - VCA0813) are other secreted proteases using T2SS [6]. Lap is a zinc dependant metallo-exopeptidase that cleaves leucin in N-terminal position, while the role of LapX remains unknown [61]. Both Lap and LapX have no role in virulence in a *C. elegan*s model [57]. While the TagA-related protein (Tarp - VCA0148), the unidentified VCA0583 and VCA0738 proteins, as well as the putative lipoprotein VC2298, have been recognized as T2SS secreted proteins, their role in *V. cholerae* is still unknown [6].

Finally, several proteins involved in biofilm formation and dissemination are also secreted by the T2SS in *V. cholerae*. Biofilm protects bacteria from antibiotics, immune system and poor environmental conditions, thus allows their survival in diversified range of ecological niches. Many components are secreted into the extracellular milieu to form the matrix. Among them, Biofilm associated protein 1 (Bap1 - VC1888) and rugosity and biofilm structure modulator A (RbmA - VC0928) and C (RbmC - VC0930) are the matrix proteins and are secreted by the T2SS [9]. In addition, the DNase Xds, an exonuclease would also be secreted by the T2SS [62]. Xds is expressed at the late stage of infection, can contribute to survival against neutrophiles NET traps, to acquisition of new DNA and dispersion of the biofilm [62, 63]. More details about the roles of the matrix proteins and nucleases in biofilm formation are presented in the Biofilms and Flagella section.

#### **2.2 Type VI secretion system for competition and DNA acquisition**

The type VI secretion system (T6SS) is a versatile syringe-like apparatus with homology to the phage T4 and produced by more than 25% Gram-negative bacteria that, upon contact with a target cell, punctures its cell wall, allowing translocation of toxic effectors directly into the neighboring cells [64, 65]. The cellular targets of these effectors are multiple; peptidoglycan, actin, cellular membrane, nucleic acids and immune system components, for instance [66]. As the target cells release their DNA into the extracellular milieu upon lysis, another function of the T6SS is to capture the extracellular DNA (eDNA) in order to acquire new features such as antibiotic resistance factors and new effectors or immunity proteins [67]. Bacteria use this device as a competition effector to take over the environmental niche and a single bacterium can possess as much as 6 different types of T6SS [65]. In *V. cholerae*, the T6SS is as efficient at killing bacterial competitors as it is at delivering toxic effectors to eukaryotic host cells, making it an important colonization and virulence factor [68].

#### *2.2.1 Structure and secretion through the T6SS*

The T6SS is anchored in the cell membrane and contains 4 distinct domains; (i) the membrane complex, (ii) the baseplate, (iii) the contractile sheath and (iv) the syringe. The current knowledge on the structure of the T6SS have been reviewed elsewhere [64].

Valine glycine rich proteins G 1, 2 and 3 (VgrG1-3) and a single proline-alaninealanine-arginine repeated motif protein (PAAR) form the tip of the syringe [69]. There are multiple PAAR proteins in *V. cholerae*'s genome but only one binds and folds in order to form a sharpened tip and it has been shown to be essential for an efficient secretion by the T6SS [69]. The PAAR proteins also have toxic effector functions. The syringe is a tube composed of multiple hexameric rings of hemolysin-coregulated protein (Hcp) [70]. Almost simultaneously, the syringe is wrapped by the helical contractile sheath made of VipA and VipB [71] which polymerizes in an extended conformation. This high-energy conformation provides enough energy, upon contraction signal, to propel the Hcp syringe, the VgrG-PAAR tip and the associated effectors into the extracellular milieu or directly into a near target cell by contraction and rotation of VipB [71]. VipA would function as a chaperone for the VipB subunits [71]. The contracted arrangement of VipB would expose the ClpV binding sites on VipB, which are hidden in the extended conformation. ClpV is the ATPase responsible for recycling the sheath components that can be reused for further effectors translocation [72, 73]. Adaptor proteins are required to load the effectors on the tip of the syringe; however, they are not secreted by the T6SS [74].

#### *2.2.2 Genes and regulation*

In *V. cholerae*, the core genes are organized in a main cluster operon that includes *vipAB*, *hsiF*, *vasA* to *vasM* and *clpV*, on the small chromosome [68]. It contains most of the structural components of the T6SS, except for Hcp, in addition to the regulator VasH and recycling protease ClpV. At least 2 auxiliary clusters (Hcp –1 and –2), harbouring Hcp, VgrGs, adaptor and effector/immunity proteins, are distributed in the genome [75]. Some strains, including pandemic strains, have an additional

#### *The Secretome of* Vibrio cholerae *DOI: http://dx.doi.org/10.5772/intechopen.96803*

auxiliary cluster (Aux –3) coding for a second PAAR protein and extra effector/ immunity module set [75]. Recently, two other auxiliary clusters, Aux –4, coding for the predicted Tse4, and Aux –5, coding for Hcp, a VgrG protein, an adaptor protein and effector/immunity module set, have been identified [75, 76]. While the genes from the main cluster are highly conserved, the auxiliary clusters, even from the pandemic strains harbouring the same effectors/immunity module sets, only share about 30 % homology between them [77].

The complexity of the apparatus and its organization require a fine regulation to insure its efficiency and recycling. The transcriptional regulation of the T6SS in *V. cholerae* is strain dependant [78, 79], complex and not entirely understood yet. As the environmental strains constitutively express the T6SS to control the surrounding bacterial populations and survive predators of the ecological niche, the pathogenic strains tightly regulate their T6SS [79, 80]. Quorum sensing, the chitin and bacterial competence pathways, osmolarity and other environmental conditions are involved in the regulation of the T6SS (for a more detailed review of the T6SS see: [64]).

#### *2.2.3 Secreted proteins*

As mentioned before, the T6SS apparatus carries toxic effectors directly into the target bacterial or eukaryotic cells. A single contraction event allows the translocation of many of these effectors at the same time into the target cell [69]. The cellular targets for these effectors are multiple; they go from peptidoglycan to cellular membrane, actin and nucleic acids [64]. To protect themselves against the toxic effectors they produce, bacterial cells express immunity proteins, which brings the notion of strains compatibility (for more information see: [81]). The secreted effectors and structure components can be reused by recipient cells to form a new T6SS [82].

Hcp is one of the proteins transported by the T6SS into the target cell, in addition to be part of its structure by forming the inner tube and serving as a chaperone to the effector molecules [83]. Hcp is encoded by two different yet functional genes (VC1415; VCA0017) producing the same protein [68, 84]. Both genes must be knocked out to suppress the T6SS activity [68]. Hcp is co-expressed with HlyA, and its secretion was observed before the discovery of the T6SS [84].

Similarly to Hcp, the VgrG proteins (VC1416; VCA0018; VCA0123) are part of the T6SS structure and are secreted into the target cell upon contraction of the T6SS [68, 85]. VgrG-1 has an actin cross-linking activity in eukaryotic cells, thus preventing cytoskeleton reorganization and phagocytosis [86]. VgrG-2 is homologous to VgrG-1, but without a functional C-terminal effector domain [85]. Both appear to be essential for secretion of other T6SS components as a mutational inactivation of one of these gene makes the mutant unable to secrete any T6SS-dependant effectors [85]. Since its toxicity is exclusive to eukaryotic cells, no immunity coupled protein is required against VgrG-1. The VgrG-3 protein is known to be active against other bacteria by hydrolyzing peptidoglycan with its lysozyme-like domain, after a translocation to the periplasm [85, 87]. It might also have a muramidase activity, which could be useful in its aquatic niche to gain access to chitin or in infection to cross mucin [88]. TsiV3 (VCA0124) acts as the antitoxin for VgrG-3 by biding to it and prevents the degradation of the cell wall in the predator bacteria [87]. Thus, VgrG-3 might be important for infection by killing gut microbiota and by hydrolysing mucin.

The PAAR proteins (VCA0284; VCA0105), along with VgrGs, form the tip of the syringe of T6SS, bind the effectors and are therefore essential for T6SS effectors' secretion. There are two proteins with a PAAR domain in *V. cholerae*'s genome with enzymatic activities that could be toxic for eukaryotic or prokaryotic cells, thus acting as effectors [69]. PAAR proteins are secreted by the T6SS by caping the tip of the syringe, the PAAR domain allowing the bond with the VgrG trimeric tip. As Hcp, VgrG

#### *Infections and Sepsis Development*

and PAAR proteins can bind and load effectors, the multiple effector translocation VgrGs (MERV) model has been proposed, suggesting that the T6SS spike (Hcp-VgrG-PAAR) can deliver different cargo effectors at the same time into the targeted cell [69].

The cargo effector VasX (VCA0020) acts as a colicin and targets the inner bacterial membrane or eukaryotic membrane in which it is believed to form pores, increase permeability and lead to its disruption [89]. It is encoded downstream of Hcp-2 and VgrG-2 and is regulated by VasH [89]. Its immunity coupled protein is TsiV2 (VCA0021) [88]. The VasW (VCA0019) protein encoded right upstream VasX is an adaptor protein that plays a role in secretion of VasX and an accessory role to VasX bactericidal activity [90].

The type six effector Lipase (TseL - VC1418) is another cargo effector and its secretion depends on the presence of VgrG-3. It carries a phospholipase domain that is believed to cause damage to cell membranes in both eukaryotic and prokaryotic cells [88, 91]. Its immunity coupled protein is TsiV1 (VC1419).

The type six effector Hydrolase (TseH - VCA0285) is encoded next to the PAAR protein and its secretion is dependant of the T6SS [92]. It has been shown that TseH is able to degrade peptidoglycan, a main component of the bacterial cell wall, by hydrolysis and would therefore make it an important effector as for interbacterial competition. Its immunity coupled protein is the type six immunity hydrolase (TsiH - VCA0286), which prevents cell wall degradation.

Recently, another lipase, the Type VI lipase effector *Vibrio* (TleV1) has been discovered in environmental *V. cholerae*'s genome [75]. TleV1 has a toxic activity in bacteria, mainly in periplasm, by targeting phospholipids and destabilizing the cellular membrane. Two immunity coupled proteins are associated with TleV1, TliV1a and TliV1b (type VI lipase immunity Vibrio 1a and 1b), but only TliV1a has an effective neutralizing effect against TleV1.

It is most likely that, as genome analysis of more *V. cholerae* strains will occur, new effector/immunity modules will be identified as they can be transferred between strains by genetic transfer or acquisition of eDNA upon target cell lysis [67, 75, 76, 93]. All the pandemic strains encode the same effector/immunity module sets (TseL/TsiV1, VasX/TsiV2 and VgrG3/TsiV3, called AAA), as a result of intraspecific competition [93]. Some strains harbour immunity genes without the coupled effector that they acquired from gene transfer, named orphan immunity proteins, allowing their survival from multiple toxic effectors [80]. The modules found within strains may differ from each other, however, their diversity and their omnipresence testify of their value for virulence and competition of the niche.

#### **2.3 Type I secretion system, a tool for auxiliary toxins secretion**

The type I secretion system (T1SS) is used by Gram-negative bacteria to secrete, in a one-step process using ATP, proteins directly into the extracellular milieu.

#### *2.3.1 Structure and secretion through the T1SS*

The most studied T1SS is the hemolysin A associated T1SS (HlyA-T1SS) from *E. coli* and its general structure has been reviewed elsewhere [94]. Briefly, the T1SS are composed of 3 proteins encoded on the same operon, next to their associated secreted protein and activator; (i) an outer membrane protein (TolC), (ii) an ATP binding cassette (ABC) transporter in the inner membrane (HlyB), and (iii) a linker protein (HlyD) anchored in the inner membrane, linking the two other components.

The secreted proteins carry a C-terminal secretion signal sensed by the inner membrane proteins upon binding [95]. The porin TolC is then recruited to the complex, and the proteins pass through the HlyB and HlyD channel. The binding

#### *The Secretome of* Vibrio cholerae *DOI: http://dx.doi.org/10.5772/intechopen.96803*

of TolC to the inner membrane complex allows its opening and the secretion of the protein to the extracellular milieu, whereafter TolC leaves the complex [94]. As the inner membrane proteins bind to specific substrates, the TolC can be used by multiple T1SS within a cell [96]. The secreted proteins have a functional domain in N-terminal and are secreted shortly after their translation in their unfolded state. In *V. cholerae*, the T1SS structure is atypical and is composed of 4 components: the periplasmic linker RtxD, the outer membrane protein TolC, and 2 ATPases RtxB and RtxE, which most probably form heterodimers in the inner membrane instead of the conventional homodimer ATPase [97].
