FIGURE 5.

Analysis of point mutants of SifA: stability of the vacuole, replication in macrophages, and virulence in mice. A, HeLa cells were infected for 16 h with GFP-Salmonella strains expressing or not the 2HA-tagged version of wild-type or point-mutation variants of SifA, fixed, and immunostained for the SCV marker LAMP1. Percentages of bacteria in SCVs were scored. B, infected RAW 264.7 mouse macrophage cells were infected for 16 h, fixed, and examined by epifluorescence microscopy for enumeration of intracellular bacteria. Percentages of infected cells in which bacteria replicated (cells with >10 bacteria) were determined. A and B, results are the average of three independent experiments in which >50 cells were scored. p values are indicated and were obtained by comparing indicated strains. C, mice were inoculated intraperitoneally with a mixture of two strains. At day 2 after injection, spleens were harvested for bacterial counts. Competitive indexes of ΔsifA against wild-type Salmonella or ΔsifA strains expressing wild-type or point-mutation variants of SifA were determined. Each symbol represents the competitive index from one mouse, and horizontal bars correspond to the mean ± S.D. of all of the mice, as indicated. ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; and ***, p ≤ 0.001.