Intracellular niche-specific profiling reveals transcriptional adaptations required for the cytosolic lifestyle of Salmonella enterica
Fig 7
A subset of “up cytosol” genes is required for optimal proliferation in the epithelial cell cytosol.
(A) Epithelial cells were infected with wild-type (WT) bacteria or the indicated gene deletion mutants and the proportion of cytosolic bacteria at 7 h p.i. was quantified using the CHQ resistance assay. n≥4 independent experiments for each strain. Asterisks indicate data significantly different from WT (ANOVA with Dunnett’s post-hoc test). (B) Genetic complementation. Epithelial cells were infected with WT bacteria or deletion mutants harboring an empty vector (-), pWSK29 or pWKS30, or the respective vector encoding the indicated gene(s), except for the ΔfepB mutant, which was complemented with a chromosomal copy of fepB at the glmS site. The proportion of cytosolic bacteria at 7 h p.i. was quantified using the CHQ resistance assay. Large dots indicate the mean of each experiment (n≥3). Asterisks indicate data significantly different from WT (ANOVA with Dunnett’s post-hoc test). (C) Epithelial cells were infected with WT bacteria or the indicated gene deletion mutants and the proportion of cytosolic bacteria at 90 min p.i. was quantified using the CHQ resistance assay. Large dots indicate the mean of each experiment (n≥3). (D) Epithelial cells seeded on coverslips were infected with WT bacteria or gene deletion mutants harboring pCHAR-Duo(ASV), a plasmid-borne dual reporter–constitutive mCherry expression is driven by the synthetic ProB promoter and gfpmut3.1(ASV) (encoding for destabilized GFP) is under the control of the glucose-6-phosphate responsive uhpT promoter from S. Typhimurium. The number of cytosolic (GFP+, mCherry+) and vacuolar (GFP-, mCherry+) bacteria in each infected cell was blindly scored by fluorescence microscopy. Small dots represent individual bacteria; large dots indicate the mean of each experiment; horizontal bars indicate the average of 3 experiments. Asterisk indicates data significantly different from WT (Kruskal-Wallis test). (E) Data from (D) was reanalyzed to determine the percentage of infected cells containing >50 cytosolic bacteria. Large dots indicate the mean of each experiment. Asterisks indicate data significantly different from WT (ANOVA with Dunnett’s post-hoc test).