FIG. 8.

Role of the OmpU protein of Vibrio vulnificus in bacterial cytotoxicity to HEp-2 cells. (A) Determination of cytotoxicity of various V. vulnificus strains by estimating the activity of LDH. Using a CytoTox96 assay kit, LDH released from the HEp-2 cells was measured upon incubation with wild-type V. vulnificus (open bars), ompU mutant DK1 (black bars), DK1 carrying pJH0311 (gray bars), or DK1 carrying pSM1 (hatched bars). HEp-2 cells (1 × 10⁵) were incubated with V. vulnificus cells for 30 min at two different MOIs, 50 and 100. Each assay was performed in quadruplicate and repeated three times. The data are shown with error bars, which are standard deviations of three independent experiments. Asterisks indicate enzyme activities that were significantly different (P < 0.01) from that of wild-type V. vulnificus by Student's t test. (B) Determination of the cytotoxicities of various V. vulnificus strains by staining with PI. The percentages of dead HEp-2 cells during incubation with wild-type V. vulnificus (open bars), ompU mutant strain DK1 (black bars), DK1 carrying pJH0311 (gray bars), or DK1 carrying pSM1 (hatched bars) were measured at two different MOIs, 10 and 20. HEp-2 cells not exposed to V. vulnificus were also stained with PI (dotted bars). Error bars represent the means ± standard deviations from three independent experiments. Asterisks indicate percentages of PI-stained cells that were significantly different (P < 0.01) from that of wild-type V. vulnificus cells by Student's t test.