*2.2.2 Other related El Tor hemolysin of Vibrio species*

Several studies have reported that other *Vibrio* species such as *V. mimicus*, *V. vulnificus*, and *V. fluvialis* also produce hemolysin that shares some common structural features with HlyA [100–102].

*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 highest sensitivity for the horse erythrocytes [100].

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 similar to β-prism lectin domain of HlyA.

*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* pathogenicity.

*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 of raw seafood, especially shellfish such as oyster contaminated by *V. vulnificus*, and it is reported that 95% of all seafood-related deaths are caused by *V. vulnificus* in the United States [121, 122]. Because most septicemia patients have an underlying disease such as hepatic cirrhosis, hepatitis, or diabetes, the septicemia by *V. vulnificus* is considered as an opportunistic infection [24]. Wound infections characterized clinical symptoms are edema, erythema, or necrosis and occurred after exposure to contaminated seawater or marine products. However, gastrointestinal symptom like diarrhea is very rare due to *V. vulnificus* infection [25, 26]. *V. vulnificus* produces various extracellular virulence factors such as hemolysin or protease [123, 124]. Hemolysin secreted by *V. vulnificus* called as *V. vulnificus* hemolysin (VVH) is also a toxin that can form pore on the target membranes of various mammalian cells. Purified VVH exhibits lytic activity against erythrocytes of various mammals and cultured cells such as CHO, mast, and pulmonary endothelial cells [101, 125–127]. In addition, it is reported that the sublytic doses of hemolysin can trigger apoptotic signaling pathway in human vascular endothelial cell line, ECV304 cells [128], and oligomerization of VVH is essential for the apoptotic activity in CHO cells [129].

VVH (VvhA) precursor has molecular weight of 51 kDa encoded by the structure gene *vvhA*, which constitutes an operon with *vvhB* gene. The *vvhB* gene is present upstream of *vvhA* and encodes 18 kDa protein VvhB. The VvhA precursor is composed of a signal peptide (20 amino acid residues) and cytolysin domain (Gln1 to Arg318) including a putative pre-stem and β-trefoil lectin-like domain (His319 to Leu451) (**Figure 1**); the pro-region and β-prism lectin domain are absent as compared with HlyA precursor [78, 130]. Although the function of VvhB is unknown, it might act as a chaperon in the absence of the pro-region like HlyA. This speculation is supported by the fact that even though VvhA is expressed in the absence of *vvhB* in vitro, the hemolytic activity cannot be detected [131]. Although VVH lacks β-prism lectin domain, the β-trefoil lectin domain has displayed binding capability for glycerol, N-acetyl-D-galactosamine, and N-acetyl-D-lactosamine unlike HlyA [92, 130]. In fact, VVH exhibits decreased ability to bind CHO cells when preincubated with methyl-beta-cyclodextrin, an oligosaccharide, and, thus, inhibition of its cytotoxic effect [132]. Similar to HlyA, it is believed that the VVH monomer binds to the cell membrane and forms oligomers [101, 133, 134] and the crystal structure of β-trefoil lectin domain of VVH reveals a heptameric ring arrangement [130]. It is strongly suggested that cholesterol is the receptor for VVH and facilitates conversion of monomer to oligomer [133, 135]. In addition, it is reported that Thr438 in the β-trefoil lectin domain is responsible for binding to cholesterol [131]. On the other hand, Phe334 in cytolysin domain that is located near the joint of two domains is essential for oligomerization of toxin monomer [136]. Moreover, it is shown that the mutation of Leu451 causes inhibition of hemolytic activity without reducing the membrane binding ability; this suggests that the Leu451 is essential for the oligomer formation [137]. Recently, a study showed that properties such as polarity and indole ring of amino acid Trp246 are essential for the binding of toxin to the target membrane [138]. It is assumed that hemolytic process of VVH is almost similar to HlyA though there are some differences in the function and structure of VVH.

It has been reported that a heat-labile hemolysin purified from *V. tubiashii*, a pathogen of juvenile bivalve, is similar to VVH [139]. Like VVH, this toxin has showed competitive inhibition by cholesterol and can lyse erythrocytes. In addition, the toxin exhibits cytotoxicity to CHO, Caco-2, and Atlantic menhaden liver cells in tissue culture.

*V. damsela* has been reported to cause wound infection by handling of fish, exposure to seawater and marine animals, and ingestion of raw seafood [21, 140–143]. It has been considered that there is not any other hemolysin in this bacterium, except of a hemolysin with phospholipase D activity known as damselysin [144].

**95**

*Hemolysin of* Vibrio *Species*

like domain (**Figure 1**) [145].

**3. Conclusion**

with these pathogens.

**Acknowledgements**

Development (AMED).

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

Recently, it is reported that this bacterium possesses HlyA-like hemolysin encoded within a new virulence plasmid pPHDD1. The characteristics of this new HlyA-like hemolysin from *V. damsela* are not yet identified, but the predicted amino acid sequences show 69% similarity with HlyA of *V. cholerae*, missing the β-prism lectin-

This chapter is focused on the hemolysins produced by *Vibrio* species, especially the human pathogens. Hemolysins are classified into two groups, namely, thermostable direct hemolysin (TDH) and El Tor hemolysin (HlyA). This chapter pays attention to Vp-TDH (*V. parahaemolyticus*), HlyA (*V. cholerae*), VMH (*V. mimicus*), and VVH (*V. vulnificus*) because these are well studied in terms of the toxin structure and their relation with the pathogenesis. The mechanism of action by HlyA and the essential amino acid residues have been clarified through the crystal structure of HlyA pro-toxin and the transmembrane heptameric oligomer over the past decade. Although the crystal structure has revealed the structural information about Vp-TDH and VVH, the exact mechanism of pore formation in the target membrane is yet to be studied. Several studies have indicated the involvement of novel virulence factors in pathogenesis like T3SS, but still Vp-TDH and Vp-TRH are considered to be the major virulence factors of *V. parahaemolyticus*, one of the important food poisoning bacteria in Japan and other eastern and Southeast Asian countries. HlyA is thought to be a major factor in CT-negative strains (e.g., *V. cholerae* non-O1/ non-O139) that can cause diarrhea because it can induce enterotoxicity as well as apoptosis. *V. mimicus* hemolysin, VMH, is just one of the many enterotoxic factors. Even though there is detailed information about structural composition and mode of action of some of the hemolysin such as Vp-TDH and HlyA, still there is a lack of information about other hemolysins. Therefore, it is necessary to further enhance our knowledge regarding these toxins in order to thoroughly understand the mechanism of pathogenesis for the prevention of endemic infectious diseases associated

This investigation was supported by a grant from the Program of the Japan

Technology in Japan (MEXT), and from the Japan Agency for Medical Research and

Initiative for Global Research Network on Infectious Diseases (J-GRID), JP19fm0108002, from Ministry of Education, Culture, Sport, Science and

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

Recently, it is reported that this bacterium possesses HlyA-like hemolysin encoded within a new virulence plasmid pPHDD1. The characteristics of this new HlyA-like hemolysin from *V. damsela* are not yet identified, but the predicted amino acid sequences show 69% similarity with HlyA of *V. cholerae*, missing the β-prism lectinlike domain (**Figure 1**) [145].
