FIG. 4.

Invasive capacity for Cos-1 fibroblast cells of the ΔTCS-RR mutants. Invasion assays were performed with all the L. monocytogenes ΔTCS-RR mutants, and the results for a subset of the mutants are shown. For invasion assays, the cells were inoculated with bacteria at a multiplicity of infection of about 10 bacteria per cell in RPMI 1640 medium without fetal calf serum. After centrifuging the bacteria onto the Cos-1 cells for 10 min, the cells were further incubated together with the bacteria for 50 min at 37°C to allow entry. The cells were then washed three times with phosphate buffered saline (PBS) and inoculated with fresh RPMI 1640 medium containing gentamicin (50 μg ml⁻¹) for 1 h at 37°C and then lysed by adding a PBS/Triton X-100 solution (0.1%) and the titer of intracellular bacteria was determined by spreading onto BHI plates. The numbers of bacteria recovered (expressed as percentages of the number recovered for the control, taken as 100%) are shown. Values are means and standard deviations (error bars) of the results of one representative experiment run in triplicate. Open bars indicate that differences from the control are statistically significant.