Intracellular niche-specific profiling reveals transcriptional adaptations required for the cytosolic lifestyle of Salmonella enterica
Fig 3
Iron-associated genes are induced in the epithelial cytosol.
(A) Epithelial cells seeded on coverslips were infected wild-type mCherry-S. Typhimurium harboring gfpmut3 transcriptional reporters. At 8 h p.i., cells were fixed and stained with Hoechst 33342 to detect DNA. Representative confocal microscopy images show induction of iroB, nrdH, sitA, sufA, yjjZ and STnc3080 promoters in cytosolic bacteria. Green = transcriptional reporter, red = S. Typhimurium, blue = DNA. Scale bars are 10 μm. (B) Quantification of the mean fluorescence intensity (MFI) of GFP signal by fluorescence microscopy and ImageJ. Bacteria were designated as being cytosolic (Cyto) or vacuolar (Vac) if residing within cells with ≥100 bacteria or 2–40 bacteria, respectively. t0 represents the infection inoculum i.e. bacteria grown to late log phase in LB-Miller broth. 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 PsitA-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).