FIG. 7.

Reduced PC-PLC activity in ΔprsA2::erm strains impairs bacterial vacuolar escape, resulting in reduced intracellular replication. (A) Henle human epithelial cells were infected with the WT or ΔprsA2::erm, Δhly, ΔprsA2::erm Δhly, or ΔplcB Δhly mutant as described in Materials and Methods. After 30 min, infected cells were washed with PBS followed by the addition of gentamicin (50 μg/ml) at 1 h postinfection. The data shown are representative of three independent experiments done in duplicate. □, WT; ▴, ΔprsA2::erm; •, Δhly; ▵, ΔprsA2::erm Δhly double mutant; ▪, ΔplcB Δhly double mutant. (B) Coverslips were also processed for microscopy at 10.5 h postinfection. Cell monolayers were fixed, permeabilized, and stained for L. monocytogenes using an anti-Listeria antibody and tetramethylrhodamine-conjugated goat anti-rabbit secondary antibody to detect bacteria (red) and NBD (7-nitrobanz-2-oxa-1,3-diazole)-phallacidin to detect F-actin (green) at a ×1,000 magnification.