Close Encounter of V. vulnificus with Host Cells is a Prerequisite to the Cytotoxicity of yet Unidentified New Virulence Factor

ABSTRACT

Live Vibrio vulnificus is highly cytotoxic to host cells in vivo and in vitro. The two most representative cytotoxins, cytolytic hemolysin and elastolytic protease, have been regarded to play major roles in the cytotoxicity of V. vulnificus. To further determine roles of the two cytotoxins in V. vulnificus pathogenesis, we constructed a double mutant of vvhA and vvpE genes, encoding a hemolysin and a protease, respectively. However, the cytotoxicity and the LD50 of a vvhA/vvpE double mutant showed no significant difference from those of the isogenic wild type strain. From these results, we came to speculate that yet unidentified, key cytotoxic factors should play a major role in the cytotoxic activity of V. vulnificus. The HeLa cells encountered with V. vulnificus became rounded, following detachment from the bottom of culture plate, and were killed eventually. However, the bacterial culture supernatant did not show any effect on the morphology and viability of HeLa cells. Also, no cytotoxicity could be noted when V. vulnificus was not allowed to contact with HeLa cells in the TranswellR system. Chloramphenicol, at lower concentration than minimum inhibitory concentration (MIC), decreased the cytotoxicity of a vvhA/vvpE double mutant to HeLa cells in a dose dependent manner. These results suggest that close encounter of V. vulnificus with host cells is a prerequisite to the cytotoxicity and that a yet unidentified virulence factor (s) should play an important role in the contact-dependent cytotoxicity.