Age-Dependent Enterocyte Invasion and Microcolony Formation by Salmonella
Figure 2
Comparative analysis of WT and invasion-deficient Salmonella.
(A–C) Organ counts in liver (A), spleen (B) and MLN (C) 1, 2 and 4 days after oral infection of 1-day-old mice with 102 CFU WT (open circles), ΔinvC SPI1 mutant (filled circles) or complemented ΔinvC pinvC (open squares) S. Typhimurium. The results represent the median values from 3–4 independent experiments (n = 9–15 per group). (D) Immunostaining of S. Typhimurium (red) in small intestinal tissue sections of 1-day-old mice 4 days p.i. with 102 CFU WT or SPI1 mutant (ΔinvC) S. Typhimurium. Counterstaining with E-cadherin (green) and Dapi (blue). Bar, upper panel = 20 µm; lower panel = 5 µm. (E) Viable bacteria cultured from gentamicin treated primary enterocytes isolated at day 4 p.i. with WT or SPI1 mutant (ΔinvC) S. Typhimurium. The results represent the median values from one out of two independent experiments (n = 4–6 per group). (F) Median of the number of intraepithelial gentamicin-protected bacteria as shown in (E) divided by the total number of bacteria cultured from untreated isolated IECs (in %). (G) Flow cytometric analysis of infected enterocytes (Fl-1) isolated at day 4 after infection of 1-day-old mice with 102 CFU WT or SPI1 mutant (ΔinvC) (GFP+) S. Typhimurium. Cells were gated for the epithelial cell marker EpCAM (APC). The number of GFP-positive enterocytes of all EpCAM+ cells is indicated (%). One representative data set of three independent experiments is shown. Note that due to the relatively high autofluorescence of enterocytes, cell infected by low numbers of bacteria may remain undetected; the indicated gating might therefore underestimate the number of Salmonella infected cells. (H) Organ counts in liver, spleen and MLN after oral infection of 6-week-old streptomycin (20 mg) pretreated mice infected with 2×108 CFU WT (open circles, n = 21) or isogenic ΔinvC SPI1 mutant (filled circles, n = 18) S. Typhimurium (n = 6). The results represent the median values from three experiments.