FIG. 1.

Identification of new effectors required for Golgi localization of SCVs in HeLa cells. (A and B) HeLa cells were infected with GFP-expressing wild-type (wt) Salmonella or mutants lacking different effectors and fixed at 8 h postinfection. (A) Cells were immunostained for Lamp1 and the Golgi marker Giantin and scored by immunofluorescence for the intracellular positioning of bacteria. Only bacteria enclosed in a Lamp1-positive compartment that were either completely or partially surrounded by the Golgi marker were counted as being Golgi associated. Bacterial clusters that were found adjacent to the Golgi but did not fulfill the above criteria were counted as nonassociated. At least 50 host cells, corresponding to more than 100 bacteria, were scored blind in each experiment. Statistical analysis for comparison of wild-type Salmonella and mutant strains indicated a significant difference for the ssaV, sseF, and sifA strains (P < 0.001), whereas no significant difference was observed in comparison of the wt to other mutants or the complemented sseF and sifA strains (P > 0.05). (B) Confocal immunofluorescence images of the dispersed distribution of the sseF mutant in contrast with the Golgi localization of the wt strain. The cell shape is marked. Bars, 10 μm. (C) Intracellular replication of Salmonella strains. Values indicate the fold increase, calculated as the ratio of intracellular bacteria between 2 and 16 h after invasion. Statistical analysis indicated a significant difference between mutant strains and wild-type Salmonella (P < 0.001), whereas no significant difference was observed between mutant strains. (D) Complementation of the intracellular positioning of the sseF and sifA mutants to the Golgi region by ectopic expression of myc-SseF and myc-SifA, respectively. (A, C, and D) Standard deviations of the means are shown and correspond to three independent experiments.