FIG. 3.

(A) Flagellin expression in LB broth culture supernatant of WT and mutant EPEC and WT C. rodentium. Flagellin expression was assessed in WT EPEC, flagellin-deficient EPEC (ΔfliC EPEC), T3SS-deficient EPEC (ΔescN EPEC), and C. rodentium (CR) grown overnight in LB broth as described in the text. The bacterial culture supernatants were filter sterilized, and 100 μg of protein was subjected to SDS-PAGE in Western blots. The blots were probed with a mouse monoclonal antibody raised against E. coli flagellin and subsequently developed in enhanced chemiluminescence. (B) A/E bacterial motility assay in 0.3% agar. LB agar (0.3%) plates were inoculated with WT EPEC, FliC-deficient (ΔfliC) EPEC, T3SS-deficient (ΔescN) EPEC, and C. rodentium (CR) and incubated for 48 h at 37°C. (C) Bacterial adherence assay in Caco-2 cells. WT EPEC, ΔfliC EPEC, and C. rodentium (CR) were used to infect Caco-2 cells for 3 to 4 h. After infection, the cells were washed with DMEM three times and scraped into sterile warm PBS. Serial dilutions were prepared, plated on agar plates, and incubated overnight. y axis, CFU; x axis, bacterial strains plated at a dilution of 10⁻⁵. The error bars indicate standard errors of the mean. *, P < 0.05 versus ΔfliC EPEC.