Phospholipase A as a potent virulence factor of Vibrio vulnificus.

Vibrio vulnificus infection has attracted special interest because of its high mortality rate. However, the identification of its major pathogenic determinant still remains obscure. In this study, a cytolysin-negative mutant strain of V. vulnificus CVD707 was used to determine the role of phospholipase A (PLA) in the pathogenesis of this bacterial infection. The mutant strain caused the lysis of erythrocytes in vitro and elevated plasma hemoglobin during the infection in mice. Both the hemolytic and PLA activities were dependent on calcium. Inhibition of hemolysis by PLA inhibitors including tetracyclin and the PLA substrate phosphatidylcholine also supports the possibility of membranous PLA as a major hemolytic factor in the cytolysin-deficient mutant. To identify the role of PLA in the pathogenesis of V. vulnificus infection, the effects of tetracycline on bacteria-induced macrophage cytotoxicity and lethality were compared with those of penicillin, an antibiotic with no inhibitory effect on PLA. Both the macrophage cytotoxicity and the lethality of V. vulnificus CVD707 to mice were significantly attenuated by tetracycline, but not by penicillin. However, bacterial counts in culture medium and mouse blood revealed that penicillin was more effective than tetracycline in killing bacteria under our experimental conditions. These results indicate that PLA activity is important in V. vulnificus-induced cytotoxicity and lethality, suggesting a crucial role for PLA in the pathogenesis of V. vulnificus infection.

A calcium-calmodulin antagonist blocks experimental Vibrio vulnificus cytolysin-induced lethality in an experimental mouse model.

We demonstrated that trifluoperazine, a calcium-calmodulin antagonist, blocked the hyperpermeability induced by Vibrio vulnificus cytolysin in in vitro-modeled endothelium and prevented the deaths of mice. Furthermore, compared to tetracycline alone, tetracycline combined with trifluoperazine enhanced the survival rate of V. vulnificus-infected mice, indicating the role of the cytolysin as an important factor in pathogenesis.

Cytotoxic mechanism of Vibrio vulnificus cytolysin in CPAE cells.

Vibrio vulnificus is an estuarian bacterium that causes septicemia and serious wound infection. The cytolysin, one of the important virulence determinants in V. vulnificus infection, has been reported to have lethal activity primarily by increasing pulmonary vascular permeability. In the present study, we investigated the cytotoxic mechanism of V. vulnificus cytolysin in cultured pulmonary artery endothelial (CPAE) cells, which are possible target cells of cytolysin in vivo. V. vulnificus cytolysin caused the CPAE cell damages with elevation of the cytosolic free Ca2+, DNA fragmentation, and decrease of the cellular NAD+ and ATP level. These cytotoxic effects of V. vulnificus cytolysin were prevented by EGTA and aminobenzamide, but were not affected by verapamil or catalase. These results indicate that the elevation of cytosolic free Ca2+ induced by V. vulnificus cytolysin causes the increase of DNA fragmentation and the damaged DNA activates nuclear poly(ADP-ribose) synthetase, which depletes the cellular NAD+ and ATP, resulting in cell death.

Induction of nitric oxide synthase expression by Vibrio vulnificus cytolysin.

The pore-forming cytolysin of Vibrio vulnificus (VVC) causes severe hypotension and vasodilatation in vivo. Under the condition of bacterial sepsis, large amounts of nitric oxide (NO) produced by inducible NO synthase (iNOS) can contribute to host-induced tissue damage causing hypotension and septic shock. In this study, we investigated the effect of purified VVC on NO production in mouse peritoneal macrophages. VVC induced NO production in the presence of interferon-gamma. Increased NO production was not affected by polymyxin B, and heat inactivation of cytolysin abolished the NO-inducing capability. NO production was induced at the same concentration range of cytolysin for pore formation, as evidenced by the release of preloaded 2-deoxy-d-[(3)H]glucose. At the higher concentrations of cytolysin causing the depletion of cellular ATP, no NO production was observed. Increased expression of iNOS and activation of NFkappaB by VVC were confirmed by Western blotting and gel shift assay, respectively. These results suggest the role of cytolysin as an inducer of iNOS and NO production in macrophage and as a possible virulence determinant in V. vulnificus infection.

Induction of apoptosis by diallyl disulfide through activation of caspase-3 in human leukemia HL-60 cells.

Diallyl disulfide (DADS), a component of garlic (Allium sativum), has been known to exert potent chemopreventative activity against colon, lung, and skin cancers. However, its molecular mechanism of action is still obscure. The present study demonstrated that DADS induces apoptosis of human leukemia HL-60 cells in a concentration- and time-dependent manner with an IC50 for cell viability of less than 25 microM. DADS activated caspase-3 as evidenced by both the proteolytic cleavage of the proenzyme and increased protease activity. Activation of caspase-3 was maximal at 3 hr and led to the cleavage of 116 kDa poly(ADP-ribose) polymerase (PARP), resulting in the accumulation of an 85 kDa cleavage product. Both activation of caspase-3 and cleavage of PARP were blocked by pretreatment with either antioxidants or a caspase-3 inhibitor, but not a caspase-1 inhibitor. DADS increased the production of intracellular hydrogen peroxide, which was blocked by preincubation with catalase. These results indicate that DADS-induced apoptosis is triggered by the generation of hydrogen peroxide, activation of caspase-3, degradation of PARP, and fragmentation of DNA. The induction of apoptosis by DADS may be the pivotal mechanism by which its chemopreventative action against cancer is based.