Biol. Pharm. Bull. 28(7) 1294—1297 (2005)

causes fatal septicemia and necrotizing wound infections, especially in patients with hepatic disease, heavy alcohol drinking habits and hemochromatosis. V. vulnificus septicemia is characterized by rapid and fulminant progression, and results in a high mortality rate of over 50%. Several bacterial components have been suggested to be virulence factors of V. vulnificus. Of these, an extracellular hemolysin or cytolysin (VvhA) and an extracellular protease (VvpE) have been the most extensively studied factors. VvhA, the most potent exotoxin, kills mice and shows a variety of biological activities including hemolysis or cytolysis, apoptosis, vasodilatation, and so on. In animal studies, the injection of purified VvhA reproduces the same pathological manifestations of septicemia as caused by the injection of live bacteria. VvpE also exhibits a host of biological activities including dermonecrosis, edema, and ulceration, and increased vascular permeability. However, the pathogenetic significance of both VvhA and VvpE has been brought into serious doubt by mouse-lethality studies of VvhAand/or VvpE-deficient mutants. The inactivation of vvhA gene does not affect the mouse-lethality. This raises the possibility that only very small amounts of VvhA may be produced in vivo, and the produced VvhA may be rapidly inactivated by host factors such as cholesterol and bacterial factors such as VvpE. Therefore, in order to evidently determine the pathogenetic roles of VvhA, further detailed studies regarding the in vitro and in vivo production and inactivation of VvhA are necessary. Physiologically, VvhA is produced in the early growth phase, and becomes abruptly inactivated in the late growth phase with the concomitant production of VvpE. Accordingly, it has been classically believed that the inactivation of VvhA is attributable to the destruction of VvhA by VvpE. Recently, it has also been reported that the activity of VvhA in the culture supernatant of a VvpE-deficient mutant was twice that of the wild-type strain, and persisted for a much longer period, suggesting that VvhA might be a substrate of VvpE. However, direct evidence for the destruction of VvhA by VvpE has never been presented. From the standpoint of evolution, some doubt also exists as to whether VvhA is destroyed by VvpE or other proteases. In addition, if VvpE or other proteases can destroy and inactivate VvhA, the routine functional assay measuring hemolytic activity using red blood cells (RBC) may not reflect the actual production of VvhA. In this study, therefore, we attempted to obtain direct evidence for the inactivation of VvhA in the late growth phase. Surprisingly, we observed that the inactivation of VvhA was due to the novel oligomerization of VvhA by unknown mechanism, but not to the destruction of VvhA by VvpE.