Fig. 5.

Strains containing prfA P219S are hyperinvasive for tissue culture cells. (a) Bacterial invasion and intracellular growth in monolayers of PtK2 epithelial cells. PtK2 epithelial cells grown on glass coverslips were infected with the indicated strains at an m.o.i. of 100 : 1. Monolayers were washed and gentamicin was added 1 h post-infection (p.i.); coverslips were removed at the indicated time points for lysis of host cells and enumeration of intracellular bacteria. The number of bacteria recovered from the prfA P219S mutant was significantly different from the number recovered from the wild-type strain at 3, 5 and 7 h p.i., while the differences between the prfA G145S mutant and wild-type were significant at 3 and 7 h p.i. **P≤0.005, ***P≤0.0005 using an unpaired two-tailed Student’s t-test (GraphPad Prism v.5.0A). In addition, the P219S mutant was significantly different from the G145S mutant at 3 and 5 h p.i. (**P≤0.005). Data shown represent the mean±sem of three independent experiments done in triplicate. (b) Bacterial infection of L2 fibroblast cells. The ability of the prfA P219S mutant and wild-type L. monocytogenes to invade, multiply and spread cell-to-cell within fibroblast tissue culture cell monolayers was determined by assessing plaque formation following infection with an m.o.i. of 10 : 1 or 1 : 1. At 1 h p.i. the cells were washed and gentamicin was added. Plaques were visualized 3 days p.i. Data shown are representative of three independent experiments done in duplicate.