*2.1.2 TDH-related hemolysin (Vp-TRH) and others*

Vp-TRH is identified as a new hemolysin found in KP-negative strains from clinical samples, named TDH-related hemolysin (Vp-TRH) [45]. Vp-TRH protein has a conserved domain of Vp-TDH and immunologically similar to Vp-TDH. But unlike Vp-TDH, it is heat-labile and lost its activity when heated at 60°C for 10 minutes. It is reported that there are significant nucleotide differences that exist within the *trh* family of two subgroups (*trh1* and *trh2*), sharing 84% sequence identity, as opposed to the less diversity (<3.3%) of five *tdh* genes (*tdh1* to *tdh5*) [38, 56–58]. Vp-TRH also induces chloride ion secretion in human colonic epithelial cells like Vp-TDH; therefore, it is considered as one of the important virulence factors among KP-negative strains of *V. parahaemolyticus* [59].

TDH-like toxins have also been found in *V. cholerae* non-O1/non-O139, *V. mimicus*, and *V. hollisae* known as NAG-TDH, Vm-TDH, and Vh-TDH, respectively [47–49]. It is reported that all clinical isolates of *V. hollisae* possess *tdh* gene [60], whereas only some clinical strains of *V. cholerae* non-O1/non-O139 and *V. mimicus* contain *tdh* gene [46, 61]. The molecular weight of these toxins is similar to Vp-TDH and shows immunological cross-reactivity with Vp-TDH. Both NAG-TDH and Vm-TDH are stable on heating at 100°C for 10 minutes, and the hemolytic activity against erythrocytes of most animals is almost similar to Vp-TDH [47, 48]. On the other hand, Vh-TDH is a heat-labile toxin that gets inactivated by heating at 70°C for 10 minutes, unlike Vp-TDH [49]. Moreover, it is reported that *V. alginolyticus* also produce TDH-like toxin, and it shows toxicity for mouse and fish [62].

#### **2.2 HlyA (El Tor hemolysin) and related toxins**

#### *2.2.1 HlyA of V. cholerae*

*V. cholerae* O1/O139 is the causative agent of cholera, and its main virulence factors are cholera toxin (CT) and toxin-coregulated pilus (TcpA) [11, 12]. *V. cholerae* produces some other virulence factors such as hemolysin, hemagglutinin/protease (HA/protease), T3SS, etc., which can also serve as important elements for the pathogenesis, especially in the strains devoid of CT and TcpA [63–65]. The watersoluble cytolytic toxin produced by *V. cholerae* El Tor O1 and non-O1/non-O139 strains is known as El Tor hemolysin (HlyA)/*V. cholerae* cytolysin (VCC) [66, 67]. HlyA can facilitate lysis of erythrocytes from various animals and other mammalian cells [66, 68]. It can also exhibit potent enterotoxicity as measured by fluid accumulation in the rabbit ileal loop test. Thus, HlyA has been considered to play a crucial role in the pathogenesis of gastroenteritis caused by *V. cholerae* strains [63].

The HlyA encoded by *hlyA* gene is produced in the form of 82 kDa inactive precursor, termed pre-pro-HlyA [69, 70]. This 82 kDa precursor consists of 25 amino acid long signal peptide at the N-terminal, a pro-region of 14 kDa and mature region of 65 kDa at the C-terminal. The mature form of HlyA is generated via a two-step process [71]. In the first step, the 82 kDa precursor is converted to 79 kDa pro-HlyA by cleavage of the signal peptide during its translocation through the inner membrane and then secreted extracellularly in an inactive form. In the second step, the inactive pro-HlyA is converted to active HlyA through the proteolytic removal of pro-region, usually at the bond between Ala157 and Asn158 (**Figure 1**; Proteolytic cleavage site). It has been found that the pro-HlyA can be activated by extracellular metalloprotease (HA/protease), a major protease of *V. cholerae* and also by other exogenous or endogenous proteases. However, the exact proteolytic cleavage site depends on the specificity of the protease, which is different compared to native processing [72]. Moreover, it is reported that pro-HlyA can

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*Hemolysin of* Vibrio *Species*

pro-toxin [74].

*hemolysin without β-prism lectin domain.*

**Figure 1.**

*DOI: http://dx.doi.org/10.5772/intechopen.88920*

bind as a monomer to eukaryotic cell membrane, and then this bound pro-HlyA can be activated by exogenous, endogenous, extracellular, and even by cell-bound proteases [73]. It is well known that the pro-region can act as an intramolecular chaperone, an essential role of pro-region that governs the proper folding of HlyA

*Comparison between V. cholerae hemolysin (HlyA) and other Vibrio spp. hemolysins. HlyA of V. cholerae consists of pro-region (light blue), cytolysin domain (blue), β-trefoil lectin domain (pink), and β-prism lectin domain (yellow). There is a proteolytic cleavage site (gray) between pro-region and cytolysin domain for the conversion of pro-HlyA to mature HlyA. The hemolysins from V. mimicus and V. fluvialis have no significant differences in domain construction. V. vulnificus hemolysin lacks β-prism lectin domain and pro-region; instead, V. vulnificus produces VvhB that might act as chaperon-like pro-region. V. damsela produces HlyA-like* 

HlyA belongs to bacterial β-barrel pore-forming toxins (β-PFTs) family that includes α-hemolysin of *Staphylococcus aureus* and aerolysin of *Aeromonas hydrophila* [75–77]. Consistent with generalized mode of action by β-PFT, the pore formation mechanism of HlyA has been proposed to follow three distinct steps (**Figure 2**); binding as a water-soluble monomer onto the target cell membrane, formation of pre-pore oligomeric intermediates by the self-assembly of toxin monomer, and finally insertion of the pore-forming stem-loop into the membrane, resulting into the formation transmembrane heptameric β-barrel pores on the cell membrane [78–80]. HlyA causes colloid osmotic lysis of mammalian cells by forming transmembrane pores on the target cell membranes [81, 82], which causes not only hemolysis but also potent cytotoxic effect such as vacuolation [83] and

The PFTs show affinity for a wide range of cell surface molecules such as cholesterol [86], glycosylphosphatidylinositol-anchored glycoproteins [87], and the human complement receptor CD59 [88]. In case of human erythrocyte membrane, glycophorin B has been reported to be a receptor for HlyA [89]. The hemolysis of rabbit erythrocytes by HlyA is competitively inhibited by asialofetuin and glycoproteins with multiple β1-galactosyl residues [90]; this provides an evidence that cell surface carbohydrates are acting as functional receptors. *V. cholerae* cytolysin also shows strong preference for cholesterol- and sphingolipid-rich vesicles [91]. So, it can be said like other PFTs, HlyA also shows

The mature HlyA is composed of three distinct domains: a central cytolysin domain and two lectin-like domains with β-trefoil and β-prism folds. The β-trefoil and β-prism domains exhibit structural homology to the carbohydrate-binding

apoptosis [84, 85] of epithelial and immune cells.

affinity for multiple cell surface receptor.

*Hemolysin of* Vibrio *Species DOI: http://dx.doi.org/10.5772/intechopen.88920*

#### **Figure 1.**

*Microorganisms*

*2.1.2 TDH-related hemolysin (Vp-TRH) and others*

KP-negative strains of *V. parahaemolyticus* [59].

**2.2 HlyA (El Tor hemolysin) and related toxins**

*2.2.1 HlyA of V. cholerae*

Vp-TRH is identified as a new hemolysin found in KP-negative strains from clinical samples, named TDH-related hemolysin (Vp-TRH) [45]. Vp-TRH protein has a conserved domain of Vp-TDH and immunologically similar to Vp-TDH. But unlike Vp-TDH, it is heat-labile and lost its activity when heated at 60°C for 10 minutes. It is reported that there are significant nucleotide differences that exist within the *trh* family of two subgroups (*trh1* and *trh2*), sharing 84% sequence identity, as opposed to the less diversity (<3.3%) of five *tdh* genes (*tdh1* to *tdh5*) [38, 56–58]. Vp-TRH also induces chloride ion secretion in human colonic epithelial cells like Vp-TDH; therefore, it is considered as one of the important virulence factors among

TDH-like toxins have also been found in *V. cholerae* non-O1/non-O139, *V. mimicus*, and *V. hollisae* known as NAG-TDH, Vm-TDH, and Vh-TDH, respectively [47–49]. It is reported that all clinical isolates of *V. hollisae* possess *tdh* gene [60], whereas only some clinical strains of *V. cholerae* non-O1/non-O139 and *V. mimicus* contain *tdh* gene [46, 61]. The molecular weight of these toxins is similar to Vp-TDH and shows immunological cross-reactivity with Vp-TDH. Both NAG-TDH and Vm-TDH are stable on heating at 100°C for 10 minutes, and the hemolytic activity against erythrocytes of most animals is almost similar to Vp-TDH [47, 48]. On the other hand, Vh-TDH is a heat-labile toxin that gets inactivated by heating at 70°C for 10 minutes, unlike Vp-TDH [49]. Moreover, it is reported that *V. alginolyticus* also produce TDH-like toxin, and it shows toxicity for mouse and fish [62].

*V. cholerae* O1/O139 is the causative agent of cholera, and its main virulence factors are cholera toxin (CT) and toxin-coregulated pilus (TcpA) [11, 12]. *V. cholerae* produces some other virulence factors such as hemolysin, hemagglutinin/protease (HA/protease), T3SS, etc., which can also serve as important elements for the pathogenesis, especially in the strains devoid of CT and TcpA [63–65]. The watersoluble cytolytic toxin produced by *V. cholerae* El Tor O1 and non-O1/non-O139 strains is known as El Tor hemolysin (HlyA)/*V. cholerae* cytolysin (VCC) [66, 67]. HlyA can facilitate lysis of erythrocytes from various animals and other mammalian cells [66, 68]. It can also exhibit potent enterotoxicity as measured by fluid accumulation in the rabbit ileal loop test. Thus, HlyA has been considered to play a crucial

role in the pathogenesis of gastroenteritis caused by *V. cholerae* strains [63].

The HlyA encoded by *hlyA* gene is produced in the form of 82 kDa inactive precursor, termed pre-pro-HlyA [69, 70]. This 82 kDa precursor consists of 25 amino acid long signal peptide at the N-terminal, a pro-region of 14 kDa and mature region of 65 kDa at the C-terminal. The mature form of HlyA is generated via a two-step process [71]. In the first step, the 82 kDa precursor is converted to 79 kDa pro-HlyA by cleavage of the signal peptide during its translocation through the inner membrane and then secreted extracellularly in an inactive form. In the second step, the inactive pro-HlyA is converted to active HlyA through the proteolytic removal of pro-region, usually at the bond between Ala157 and Asn158 (**Figure 1**; Proteolytic cleavage site). It has been found that the pro-HlyA can be activated by extracellular metalloprotease (HA/protease), a major protease of *V. cholerae* and also by other exogenous or endogenous proteases. However, the exact proteolytic cleavage site depends on the specificity of the protease, which is different compared to native processing [72]. Moreover, it is reported that pro-HlyA can

**90**

*Comparison between V. cholerae hemolysin (HlyA) and other Vibrio spp. hemolysins. HlyA of V. cholerae consists of pro-region (light blue), cytolysin domain (blue), β-trefoil lectin domain (pink), and β-prism lectin domain (yellow). There is a proteolytic cleavage site (gray) between pro-region and cytolysin domain for the conversion of pro-HlyA to mature HlyA. The hemolysins from V. mimicus and V. fluvialis have no significant differences in domain construction. V. vulnificus hemolysin lacks β-prism lectin domain and pro-region; instead, V. vulnificus produces VvhB that might act as chaperon-like pro-region. V. damsela produces HlyA-like hemolysin without β-prism lectin domain.*

bind as a monomer to eukaryotic cell membrane, and then this bound pro-HlyA can be activated by exogenous, endogenous, extracellular, and even by cell-bound proteases [73]. It is well known that the pro-region can act as an intramolecular chaperone, an essential role of pro-region that governs the proper folding of HlyA pro-toxin [74].

HlyA belongs to bacterial β-barrel pore-forming toxins (β-PFTs) family that includes α-hemolysin of *Staphylococcus aureus* and aerolysin of *Aeromonas hydrophila* [75–77]. Consistent with generalized mode of action by β-PFT, the pore formation mechanism of HlyA has been proposed to follow three distinct steps (**Figure 2**); binding as a water-soluble monomer onto the target cell membrane, formation of pre-pore oligomeric intermediates by the self-assembly of toxin monomer, and finally insertion of the pore-forming stem-loop into the membrane, resulting into the formation transmembrane heptameric β-barrel pores on the cell membrane [78–80]. HlyA causes colloid osmotic lysis of mammalian cells by forming transmembrane pores on the target cell membranes [81, 82], which causes not only hemolysis but also potent cytotoxic effect such as vacuolation [83] and apoptosis [84, 85] of epithelial and immune cells.

The PFTs show affinity for a wide range of cell surface molecules such as cholesterol [86], glycosylphosphatidylinositol-anchored glycoproteins [87], and the human complement receptor CD59 [88]. In case of human erythrocyte membrane, glycophorin B has been reported to be a receptor for HlyA [89]. The hemolysis of rabbit erythrocytes by HlyA is competitively inhibited by asialofetuin and glycoproteins with multiple β1-galactosyl residues [90]; this provides an evidence that cell surface carbohydrates are acting as functional receptors. *V. cholerae* cytolysin also shows strong preference for cholesterol- and sphingolipid-rich vesicles [91]. So, it can be said like other PFTs, HlyA also shows affinity for multiple cell surface receptor.

The mature HlyA is composed of three distinct domains: a central cytolysin domain and two lectin-like domains with β-trefoil and β-prism folds. The β-trefoil and β-prism domains exhibit structural homology to the carbohydrate-binding

#### **Figure 2.**

*Mechanism of transmembrane heptameric pore formation by HlyA. (a) Pro-HlyA structure. (b) Secreted pro-HlyA is activated through the removal of pro-region by protease. (c) HlyA monomer binds to the target membrane by using a rim region and/or β-prism lectin domain with membrane component such as cholesterol and carbohydrate receptor, respectively. (d) HlyA monomer assembles to heptameric pre-pore oligomeric intermediates. (e) The pre-stem of HlyA is inserted into the membrane, resulting into the formation of transmembrane heptameric β-barrel pores.*

domain of the plant lectin ricin and jacalin, respectively [78]. In fact, the 15 kDa β-prism lectin domain has carbohydrate-binding activity [92], and the deletion of 15 kDa β-prism lectin domain generates a 50 kDa variant (HlyA50) with no effect on the global conformation of the monomer, but the hemolytic activity reduced by approximately 1000-fold [93, 94]. The β-prism domain has been shown to promote self-assembly of the toxin monomer in carbohydrate-independent manner, suggesting the hemolytic activity of HlyA50 is compromised due to reduction in pre-pore oligomeric intermediates [95]. Another study proposed the role of β-trefoil domain and showed that it is critical for the folding of cytolysin domain to its active conformation [96]. Recently, it is reported that the three loop sequences located in the bottom tip of the cytolysin domain play a critical role in the initial interaction with membrane lipid bilayer. This study showed that the replacement of the amino acid residues in the three loop sequences designated as "rim region" compromises the specific interaction of HlyA monomer with membrane lipid bilayer and blocks the pore formation process. Thus, it leads to repression in the lysis of human erythrocytes and reduced cytotoxic activity for HT-29 human colorectal adenocarcinoma cells [97]. In the next step that is pre-pore oligomerization, it has been shown that alteration of key amino acids affects not only the formation of oligomeric intermediates but also the subsequent formation of functional transmembrane pore [98]. Finally, pre-pore oligomeric intermediates lead to the formation of transmembrane β-barrel pore. Paul et al. confirmed that the transmembrane stem region plays a significant role in the functional pore formation. However, the deletion of "pre-stem" loop of cytolysin domain does not affect the membrane binding and pre-pore heptamer formation [99]. Therefore, it is considered that each step of HlyA pore formation mechanism plays an indispensable role in the generation of functional transmembrane pore in the target cell and thus enhances the virulence of *V. cholerae*.

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pathogenicity.

*Hemolysin of* Vibrio *Species*

*DOI: http://dx.doi.org/10.5772/intechopen.88920*

tural features with HlyA [100–102].

*2.2.2 Other related El Tor hemolysin of Vibrio species*

est sensitivity for the horse erythrocytes [100].

similar to β-prism lectin domain of HlyA.

Several studies have reported that other *Vibrio* species such as *V. mimicus*, *V. vulnificus*, and *V. fluvialis* also produce hemolysin that shares some common struc-

*V. mimicus,* a species closely related to *V. cholerae*, is a causative agent of human gastroenteritis [103]. Pathogenic strains of *V. mimicus* exhibit various clinical symptoms from watery to dysentery-like diarrhea [104]. This pathogen produces many kinds of virulence factors such as CT-like enterotoxin and heat-stable enterotoxin [105–108], with Vm-TDH as a causative factor in some clinical strains. However, most clinical strains lack the ability to produce any of these toxins. The heat-labile hemolysin/cytolysin (*V. mimicus* hemolysin; VMH) is thought to be the most common virulent enteropathogenic factor [109, 110]. In fact, VMH induces FA in a ligated rabbit ileal loop in dose-dependent manner, and the antibody against VMH apparently reduces enterotoxicity by *V. mimicus* in the living cells [100, 111]. These findings indicate that VMH is potently related to pathogenesis of this pathogen. The enterotoxic activity of VMH might be due to intestinal Cl<sup>−</sup> secretion caused by the activation of both Ca2+-dependent and cyclic AMP-dependent Cl<sup>−</sup> secretion systems [111, 112]. Similar to HlyA, it has been indicated that VMH is also a pore-forming toxin. This toxin can disrupt various mammalian erythrocytes including bovine, rabbit, sheep, human, and mouse in colloid osmotic manner, and it shows the high-

VMH encoded by *vmhA* gene is predicted to be of 83 kDa with 82% similarity with *V. cholerae* HlyA. VMH is also secreted as 80 kDa precursor known as pro-VMH [113], which is then converted to 66 kDa mature toxin through the removal of N-terminal propeptide by trypsin-like protease of *V. mimicus* between the amino acid residues Arg151 and Ser152 [114, 115]. It has been assumed that VMH might be processed in a two-step reaction just like HlyA and pro-toxin can be activated by various proteases such as trypsin, chymotrypsin, and metalloprotease [115, 116]. Similar to 50 kDa variant of HlyA, mature VMH can be converted to 51 kDa of VMH (designated VMH51) through the removal of 15 kDa from C-terminal end by metalloprotease of *V. mimicus*. VMH51 almost showed no lytic activity toward horse erythrocytes because it lost the binding affinity toward erythrocyte membrane [116]. However, the VMH51 can associate with sheep erythrocyte membranes though the affinity is reduced as compared with intact VMH, suggesting that the truncated toxin interacts with other components in sheep erythrocyte membrane. It might be concluded that the 15 kDa C-terminal domain of VMH is functionally

*V. fluvialis* is one of the foodborne pathogens which can cause clinical symptoms similar to *V. cholerae* [117–119]. *V. fluvialis* secrets El Tor-like hemolysin, designed as *V. fluvialis* hemolysin (VFH), which can elicit lysis of erythrocytes from various animal. In addition to hemolytic activity, VFH can also trigger cytotoxicity toward Chinese hamster ovary (CHO) cells and induction of fluid accumulation in suckling mouse [102]. The purified VFH has molecular weight of 63 kDa, whose N-terminal amino acid sequence shares homology to HlyA from *V. cholerae* and VMH from *V. mimicus* [102]. It is suspected that VFH might play an important role in *V. fluvialis*

*V. vulnificus* was first isolated from a leg ulcer, and it was wrongly reported as *V. parahaemolyticus* [120]. Later, it was found that some characters were different from *V. parahaemolyticus* such as positive lactose fermentation, so subsequently it was termed as *V. vulnificus* [20]. *V. vulnificus* can cause two types of illness, the primary septicemia and the wound infection [24]. The former is remarkable for its high fatality rate (over 50%). The primary septicemia is caused by the consumption *Microorganisms*

**Figure 2.**

*transmembrane heptameric β-barrel pores.*

domain of the plant lectin ricin and jacalin, respectively [78]. In fact, the 15 kDa β-prism lectin domain has carbohydrate-binding activity [92], and the deletion of 15 kDa β-prism lectin domain generates a 50 kDa variant (HlyA50) with no effect on the global conformation of the monomer, but the hemolytic activity reduced by approximately 1000-fold [93, 94]. The β-prism domain has been shown to promote self-assembly of the toxin monomer in carbohydrate-independent manner, suggesting the hemolytic activity of HlyA50 is compromised due to reduction in pre-pore oligomeric intermediates [95]. Another study proposed the role of β-trefoil domain and showed that it is critical for the folding of cytolysin domain to its active conformation [96]. Recently, it is reported that the three loop sequences located in the bottom tip of the cytolysin domain play a critical role in the initial interaction with membrane lipid bilayer. This study showed that the replacement of the amino acid residues in the three loop sequences designated as "rim region" compromises the specific interaction of HlyA monomer with membrane lipid bilayer and blocks the pore formation process. Thus, it leads to repression in the lysis of human erythrocytes and reduced cytotoxic activity for HT-29 human colorectal adenocarcinoma cells [97]. In the next step that is pre-pore oligomerization, it has been shown that alteration of key amino acids affects not only the formation of oligomeric intermediates but also the subsequent formation of functional transmembrane pore [98]. Finally, pre-pore oligomeric intermediates lead to the formation of transmembrane β-barrel pore. Paul et al. confirmed that the transmembrane stem region plays a significant role in the functional pore formation. However, the deletion of "pre-stem" loop of cytolysin domain does not affect the membrane binding and pre-pore heptamer formation [99]. Therefore, it is considered that each step of HlyA pore formation mechanism plays an indispensable role in the generation of functional transmembrane pore in the target cell

*Mechanism of transmembrane heptameric pore formation by HlyA. (a) Pro-HlyA structure. (b) Secreted pro-HlyA is activated through the removal of pro-region by protease. (c) HlyA monomer binds to the target membrane by using a rim region and/or β-prism lectin domain with membrane component such as cholesterol and carbohydrate receptor, respectively. (d) HlyA monomer assembles to heptameric pre-pore oligomeric intermediates. (e) The pre-stem of HlyA is inserted into the membrane, resulting into the formation of* 

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and thus enhances the virulence of *V. cholerae*.
