Intracellular niche-specific profiling reveals transcriptional adaptations required for the cytosolic lifestyle of Salmonella enterica
Fig 4
Manipulation of cellular iron levels primarily affects cytosolic bacteria.
(A) Epithelial cells seeded on coverslips were left untreated (control) or treated overnight in growth media supplemented with 10 μM, 30 μM, 100 μM or 300 μM ferric ammonium citrate (FAC). Cells were infected with wild-type mCherry-S. Typhimurium harboring PiroN-gfpmut3, PsitA-gfpmut3 or PyjjZ-gfpmut3 reporters, fixed at 8 h p.i. and the MFI of bacterial GFP signal was quantified by fluorescence microscopy and Image J. Bacteria were designated as being cytosolic (Cyto) or vacuolar (Vac) if residing within cells with ≥100 bacteria or 2–40 bacteria, respectively. Small dots represent individual bacteria; large dots indicate the mean of each experiment; horizontal bars indicate the average of two experiments. Acquisition parameters (exposure time and gain) were set-up using PiroN-gfpmut3 (the highest GFP intensity) and these same parameters were applied throughout. Dashed lines indicate the range of background fluorescence in the GFP channel measured for mCherry-S. Typhimurium (no reporter plasmid). Asterisks indicate data significantly different from control (Kruskal-Wallis test). (B) Epithelial cells seeded on coverslips were left untreated (control) or treated overnight with growth media containing 200 μM 2,2’-dipyridyl (DPI), a metal-chelating compound, 200 μM DPI and 200 μM ferric ammonium citrate (DPI+FAC), or 200 μM FAC alone (FAC). Infection strains and quantification of GFP signal were as described for (A). Asterisks indicate data significantly different from control (Kruskal-Wallis test). (C) Cells were treated as in (B) and infected with wild-type bacteria. The number of intracellular bacteria was quantified by gentamicin protection assay at 1 h and 8 h p.i. Fold-replication is CFUs at 8 h/1 h. The mean from each experiment is represented as a large dot (n = 3). Asterisks indicate data significantly different from control (ANOVA with Dunnett’s post-hoc test). (D) Cells were treated as in (B) and infected with wild-type bacteria. The proportion of cytosolic bacteria was quantified by CHQ resistance assay at 7 h p.i. The mean from each experiment is represented as a large dot (n≥3). Asterisk indicates data significantly different from control (ANOVA with Dunnett’s post-hoc test). (E) Cells were treated as in (B) and infected with mCherry-S. Typhimurium harboring a plasmid-borne reporter of cytosolic access, pNF101, and fixed at 8 h p.i. Left panel: the proportion of infected cells containing only cytosolic (all bacteria are GFP+, mCherry+), only vacuolar (all bacteria are GFP-, mCherry+) or a mixed population (Cyto&Vac) of bacteria was blindly scored by fluorescence microscopy. n≥5 independent experiments. Right panel: the number of cytosolic or vacuolar bacteria in each cell was blindly scored. Small dots represent individual bacteria; large dots indicate the mean of each experiment; horizontal bars indicate the average of two experiments.