**3. Conclusion**

*Microorganisms*

(Gln1

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

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

*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].

**94**

tissue culture.

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 with these pathogens.
