FIG 4.

Invasion of polarized epithelial cells by Salmonella is highly cooperative. (A) Experimental design of sequential-invasion experiments. (B and C) Infections were performed with polarized MDCK cells (B) or nonpolarized HeLa cells (C). Single infections by WT, SPI1, and SPI4 strains were quantified by gentamicin protection assays (open bars) at an MOI of 1. For sequential infection experiments (filled bars), host cells were infected with WT Salmonella for 25 min at an MOI of 1. Noninternalized cells were removed by washing. A second infection was performed at an MOI of 5 with the WT, SPI1, or SPI4 strain, as indicated by the arrows. Single and sequential infections were performed without centrifugation. After incubation for 25 min, noninternalized bacteria were removed by washing, and the remaining cells were killed by the addition of gentamicin. Subsequently, host cells were lysed by addition of PBS containing 0.5% deoxycholate, and numbers of internalized bacteria were determined by plating on agar plates containing antibiotics selective for either the strain of the first infection or the strain of the second infection. Means and standard deviations for triplicates are shown, and the data are representative of three independent experiments. Statistical analyses were performed by one-way analysis of variance (ANOVA): n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.