Effects of <i>ipaC</i> mutations on <i>Shigella</i>-induced contact hemolysis.

aBacterial strains were grown to mid-exponential phase in the presence of Congo Red, and ipaC transformants were induced with IPTG to allow the expression and the secretion of the various IpaC constructs (Materials and Methods). Secretion was determined by analyzing the presence of the IpaC derivative in the bacterial culture supernatant by anti-IpaC Western-blot analysis. − : no detectable IpaC in the bacterial culture supernatant; + : detection of the IpaC derivative. No significant variation in the efficiency of secretion could be observed between the various constructs (not shown), with the exception of the CΔ1–100 construct that does not show significant levels of secretion. bHorse blood erythrocytes were challenged with the various bacterial strains, and the extent of hemolysis was quantitated by reading the absorbance at 595 nm (Materials and Methods). The relative percentage of hemolysis corresponds to the ratio of the hemolysis determined for the sample over that of total hemolysis, determined for samples treated with 0.1% SDS. The values correspond to the mean of three independent experiments performed in triplicates. The ipaC mutant strain shows residual hemolytic activity that was previously shown to depend on IpaB [12]. The ipaC transformants expressing the C57, C351, or C1myc derivatives show a hemolytic activity that is comparable to that conferred by full-length IpaC. cHeLa cells were challenged with bacterial strains for 15 min. at 37°C. Samples were fixed and processed for fluorescent staining of F-actin and bacterial LPS. Bacterial induced actin foci were scored microscopically, and the numbers were normalized to the number of cells analyzed. The values are representative of at least 40 cells analyzed in a representative experiment. dBacterial internalization was measured using the gentamicin assay. The values correspond to representative experiments performed in triplicates.