*2.1.1 Thermostable direct hemolysin (TDH)*

*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 test [33, 40–42].

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 tetramer [33, 44, 52].

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 virulence factors including T3SS.
