Fig 4. Analysis of sopBE₂ and sopAE₂ double mutant S. Typhimurium.

(A-C) 1-day-old C57BL/6 mice were orally infected with 100 CFU wild type (WT) (filled circles), isogenic ΔsopBE₂ (inverted open triangles), or ΔsopAE₂ (open triangles) S. Typhimurium. Viable counts in (A) isolated gentamicin-treated enterocytes and (B) total liver tissue homogenate at 4 days p.i.. (C) Quantitative RT-PCR for Cxcl2 mRNA in total RNA prepared from enterocytes isolated at 4 days p.i.. Values were normalized to uninfected age-matched control animals (crosses). Individual values and the mean from at least two independent experiments are shown (n = 3–6 animals per group). The data for uninfected control animals and Salmonella WT infected mice are identical to Fig 1A–1C. (D) Immunostaining for Salmonella (red) in small intestinal tissue sections at 4 days p.i. with 100 CFU ΔsopE₂, ΔsopBE₂, or ΔsopAE₂ S. Typhimurium. Counterstaining with E-cadherin (green), WGA (white) and DAPI (blue). Bar, 5 μm. (E) Percentage of epithelial cells positive for single bacteria or microcolonies (>1 intraepithelial bacterium) at 4 days p.i. with ΔsopBE₂ or ΔsopAE₂ S. Typhimurium. 30 Salmonella-positive epithelial cells per infected neonate (n = 8–13) were analyzed. Results represent the mean ± SD. (F) Co-immunostaining for Salmonella ΔsopBE₂ and ΔsopAE₂ (green) and LAMP1 (red) in small intestinal tissue sections at day 4 p.i.. Counterstaining with E-cadherin (white) and DAPI (blue). Bar, 5 μm. (G) Quantitative evaluation of the percentage of intraepithelial ΔsopBE₂ and ΔsopAE₂ S. Typhimurium associated with LAMP1 staining. All microcolonies from three tissue sections per infected neonate were analyzed (n = 3–4) at day 4 p.i.. Results represent the mean ± SD. (H) Co-immunostaining for ΔsopBE₂ and ΔsopAE₂ Salmonella (red) and the GFP-expressing SPI2 reporter (pM973; green) in small intestinal tissue sections at day 4 p.i.. Counterstaining with E-cadherin (white) and DAPI (blue). Bar, 5 μm. (I) Quantitative analysis of the percentage of intraepithelial S. Typhimurium expressing the SPI2 reporter. All microcolonies from three tissue sections per infected neonate were analyzed (n = 3–4) at day 4 p.i.. Results represent the mean ± SD.