Figure 3. SarA is translocated through both T3SSs of S. Typhimurium.

a, SarA-TEM fusions are translocated into host cells. HeLa cells infected with wild-type S. Typhimurium showed blue fluorescence, indicating that the SarA-TEM fusion was translocated into the host cell cytoplasm. b, SarA translocation occurs primarily through the SPI-2 T3SS. HeLa cells infected with ΔssaT S. Typhimurium showed a reduced number of cells with SarA-TEM activity in the cytosol. c, Quantification of SarA-TEM translocation by flow cytometry. 11 replicates over 4 experiments. d, Wild-type and ΔssaT invasion are equal. HeLa cells were infected with wild-type and ΔssaT containing pSarA-TEM on a kanamycin resistant plasmid as described, lysed 2 hours post infection and plated to determine invasion. P-values calculated using unpaired student’s t test with Welch’s correction; error bars represent SEM. e, Mutation of both SPI-1 and SPI-2 is required to completely block SarA-induced IL-10 production in LCLs (GM19154). Disruption of the SPI-1 T3SS (ΔprgH) resulted in a modest decrease in the amount of IL-10 induced by S. Typhimurium that did not reach statistical significance when entry is facilitated by S. Typhi. Disruption of the SPI-2 secretion system (ΔssaT) results in a significant decrease in the amount of IL-10 induced by S. Typhimurium. Disruption of both the SPI-1 and SPI-2 secretion systems (ΔprgHΔssaT) results in no induction of IL-10 by S. Typhimurium. IL-10 represented as femtograms per infected cell; infection measured by GFP+ S. Typhimurium in LCLs by flow cytometry. Data are presented as IL-10 per infected cell. IL-10 concentration and invasion percentages are depicted separately in Fig. S5. 6 biological replicates over 3 experiments. P-values calculated using ANOVA with Sidak’s multiple comparison post-hoc test, error bars represent SEM.