Intracellular niche-specific profiling reveals transcriptional adaptations required for the cytosolic lifestyle of Salmonella enterica
Fig 6
SPI1-associated genes are up-regulated in the epithelial cytosol.
(A) Epithelial cells seeded on coverslips were infected with 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 sicA, sopE2, sopF, siiA and lpxR promoters in cytosolic bacteria. PipB is a type III effector translocated by T3SS2 and the PpipB-gfpmut3 reporter served as a control for vacuole-specific gene induction. Green = transcriptional reporter, red = S. Typhimurium, blue = DNA. Scale bars are 10 μm. (B) Quantification of the 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 2–3 experiments. Acquisition parameters (exposure time and gain) were set up using PsicA-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).