**2. Hemolysins**

Hemolysin is a toxin that attacks membranes of mammalian erythrocyte and causes cell lysis called hemolysis. It is reported that hemolysins are produced by different species of bacteria like *Escherichia coli*, *Staphylococcus aureus*, and *Vibrio* [31–34]. In most cases, the evidence based either on in vivo experiments or clinical reports that suggests the involvement of hemolysins in the pathogenicity is reported [33, 35]. This toxin plays certainly an important role in the infection process initiated by *Vibrio* spp. Hemolysin from *Vibrio* spp. can be classified mainly into two groups, thermostable direct hemolysin (TDH) from *V. parahaemolyticus* and El Tor hemolysin (HlyA) from *V. cholerae*. Even though these toxins partially share the sequence homology, the essential amino acids for the activity, structural features, and function are different between TDH and HlyA.

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*Hemolysin of* Vibrio *Species*

test [33, 40–42].

tetramer [33, 44, 52].

virulence factors including T3SS.

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

*2.1.1 Thermostable direct hemolysin (TDH)*

**2.1 Thermostable direct hemolysin (TDH), TRH, and others**

*V. parahaemolyticus* was first isolated by Fujino et al*.* as a causative agent of food poisoning in Osaka, Japan [14]. The pathogenicity of *V. parahaemolyticus* is determined by multiple virulence factors including adhesins, thermostable direct hemolysin (Vp-TDH), TDH-related hemolysin (Vp-TRH), and two type III secretion systems (T3SS), T3SS1 and T3SS2 [28]. It has been reported that the clinical isolates of *V. parahaemolyticus* show β-hemolysis activity on Wagatsuma blood agar medium [36], whereas almost all non-clinical isolates are non-hemolytic. This hemolytic activity has been given a specific term known as Kanagawa phenomenon (KP), and it is due to Vp-TDH encoded by the *tdh* gene [37, 38]. Thus, Vp-TDH has been considered as an important virulence factor in gastroenteritis cases and KP reaction as a good marker for the identification of pathogenic strains. Thermostable direct hemolysin, Vp-TDH, was named so because of its characteristics. These characters include persistence of activity even after heating at 100°C for 10 minutes and the ability to act directly on erythrocytes with no enhancement in activity level even by the addition of lecithin [39]. This purified toxin has numerous biological activities such as hemolytic activity for erythrocytes of various species, cytotoxic activity for some mammalian cells, and enterotoxic activity measured by fluid accumulation (FA) in the rabbit ileal loop

The mature form of Vp-TDH consists of 165 amino acids and is approximately of 19 kDa. It exists as a tetramer in solution, which is responsible for the membrane disruption [43, 44]. This is a pore-forming toxin, but it has no similarities with other bacterial pore formers except Vp-TDH homologs like Vp-TRH and TDH-like toxins from *V. cholerae* non-O1/non-O139, *V. mimicus,* and *V. hollisae* [45–49]. Vp-TDH forms pores of approximately 2 nm in diameter on erythrocyte membrane that results into colloidal osmotic lysis [50]; however, the exact mechanism of pore formation is not yet identified. The reactivity of Vp-TDH against erythrocytes from various animal species showed variability; for example, it causes hemolysis of erythrocytes from rat, human, rabbit, and sheep but not horse [51]. It is reported that the amino acid residues, Arg46, Gly62, Trp65, and Gly90, are critical for the hemolysis; in fact, the substitution of

residue Arg46 by site-directed mutagenesis inhibits the formation of

Enterotoxicity, which is another feature of Vp-TDH, has been evaluated by increase of FA in the rabbit ileal loop due to intestinal Cl<sup>−</sup> secretion as a manifestation of diarrhea induced by *V. parahaemolyticus*. The Cl<sup>−</sup> secretion from human colonic epithelial cells by Vp-TDH is caused by stimulation of Ca2+-activated chloride ion channel not by pore formation on the cells [53]. Evidence suggests that Vp-TDH acts in three sequential steps: receptor binding on the epithelial cells, followed by increase in intracellular Ca2+ concentration due to protein kinase C activation, and finally, stimulation of Ca2+-activated Cl<sup>−</sup> channel. However, it is reported that the deletion of *tdh* only leads to partial decrease in enterotoxicity against rabbit intestinal cells, whereas cytotoxicity to Hela cells was not affected at all [54]. Moreover, a recent study provides a new evidence that Vp-TDH can also engage as an effector of T3SS and implicated to elevate FA in animal model [55]. Therefore, the reason behind pathogenicity of *V. parahaemolyticus* is perhaps not only because of Vp-TDH but also because it involves a synergistic action of multiple *Microorganisms*

*V. cholerae*

**Table 1.**

to high lethal rates [24–26].

studies till date.

**2. Hemolysins**

The other species such as *V. cholerae* non-O1/non-O139, *V. mimicus*, and *V. fluvialis* are known as agents of foodborne illness [3, 16–19]. On the other hand, *V. vulnificus* is the most studied among *Vibrio* spp. as a causative bacterium of wound infections, though the clinical cases by *V. damsela* and *V. alginolyticus* are also reported [3, 20–24]. *V. vulnificus* is an opportunistic pathogen and poses a threat to individuals with compromised immunity because it can also cause septicemia, which leads

**Species Diseases Hemolysin family**

*V. alginolyticus* Wound infection, otitis media TDH

O1/O139 Cholera HlyA non-O1/non-O139 Gastroenteritis, wound infection HlyA, TDH *V. damsela* Wound infection HlyA *V. fluvialis* Gastroenteritis HlyA

*V. hollisae* Gastroenteritis, septicemia TDH

*V. mimicus* Gastroenteritis HlyA, TDH *V. parahaemolyticus* Gastroenteritis, wound infection TDH *V. vulnificus* Septicemia, wound infection HlyA

*V. carchariae* Wound infection *V. cincinnatiensis* Meningitis

*V. furnissii* Gastroenteritis

*V. metschnikovii* Cholecystitis

*HlyA, El Tor hemolysin group; TDH, thermostable direct hemolysin family.*

*Pathogenic Vibrio species for human and hemolysins produced by them [1, 3].*

These pathogenic *Vibrio* have been reported to produce various virulence factors, including enterotoxin such as CT produced by *V. cholerae* O1/O139 [12, 27], hemolysin, and Type III secretion system (T3SS) in *V. parahaemolyticus* [28, 29] and extracellular protease in *V. vulnificus* [30]. This chapter has mainly summarized how hemolysins play an important role in the pathogenicity of Vibrio spp. based on

Hemolysin is a toxin that attacks membranes of mammalian erythrocyte and causes cell lysis called hemolysis. It is reported that hemolysins are produced by different species of bacteria like *Escherichia coli*, *Staphylococcus aureus*, and *Vibrio* [31–34]. In most cases, the evidence based either on in vivo experiments or clinical reports that suggests the involvement of hemolysins in the pathogenicity is reported [33, 35]. This toxin plays certainly an important role in the infection process initiated by *Vibrio* spp. Hemolysin from *Vibrio* spp. can be classified mainly into two groups, thermostable direct hemolysin (TDH) from *V. parahaemolyticus* and El Tor hemolysin (HlyA) from *V. cholerae*. Even though these toxins partially share the sequence homology, the essential amino acids for the activity, structural features,

and function are different between TDH and HlyA.

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