FIG. 4.

Effect of altered OAg length on adherence to and invasion of epithelial cells. WT Salmonella enterica serovar Typhimurium (black bars), various mutant strains with a defect of the SPI1-T3SS (invC; white bars) or genes for OAg biosynthesis (gray bars), and complemented mutant strains (hatched bars) were used to infect epithelial cells. Infections were performed with nonpolarized cells (HeLa) (left) or polarized cells (MDCK) (right). (A) For quantification of adhesion, bacteria were allowed to adhere for 30 min at 4°C, and nonadherent bacteria were removed by washing. Subsequently, the cells were lysed and serial dilutions of the suspensions were plated onto LB plates for the quantification of adherent bacteria. (B) For quantification of invasion, bacteria were grown to late log phase and added to the cells at an MOI of 10 or 5 for HeLa or MDCK cells, respectively, followed by an incubation of 30 min at 37°C to allow invasion. Noninternalized bacteria were removed by washing, and the remaining extracellular bacteria were killed by addition of medium containing 100 μg ml⁻¹ gentamicin for 1 h. Subsequently, the cells were washed and lysed to release internalized bacteria, and serial dilutions of the suspensions were plated onto agar plates for the quantification of intracellular bacteria. The numbers of adherent or invading bacteria are expressed as percentages of the inoculum, and means and standard deviations for triplicate experiments are displayed. (C) Overexpression of V-OAg and VL-OAg negatively affects invasion of epithelial cells. HeLa cells were infected with WT S. enterica serovar Typhimurium, the invC strain, or the wzz_ST wzz_fepE mutant strain harboring plasmid pBAD as a control or plasmid p3456 for the overexpression of wzz_ST and wzz_fepE. The bacteria were cultured in LB containing 0.2% glucose, as a noninducing control, or 0.2% arabinose, to induce overexpression of wzz_ST wzz_fepE, as indicated. Statistical analysis was performed as described in the legend for Fig. 2.