Fig. 4: Salmonella type 3 secretion signifies robust live STm mucosal immunogenicity.

a Germ-free mice were enterally conditioned with three successive doses of 10¹⁰ CFU of live STm^Aux (red upright triangles, n = 6 animals examined over two independent experiments), live T3SS-double-deficient STm^Aux (STm^{Aux ΔinvC ΔssaV}, orange squares, n = 6 animals examined over two independent experiments), a live STm^Aux T3SS-double-deficient SPI1/SPI2 double-deletion mutant (STm^{Aux ΔSPI1 ΔSPI2}; purple inverted triangles, n = 6 animals examined over two independent experiments), PAA-killed STm^Aux (blue diamonds, n = 6 animals examined over two independent experiments), or PBS vehicle only (ctrl; gray circles, n = 6 animals examined over two independent experiments). Four weeks after the first treatment (day 0) all mice were challenged with wild-type STm (10³ CFU). Mice were analyzed on day 3 after challenge. Each symbol represents one individual. b Lipocalin-2 concentration in cecal contents at day 3 after challenge. c Cecal histopathology score at day 3 after challenge. Each symbol represents one individual. d Bacterial burden of wild-type STm recoverable from mLNs at day 3 after challenge. e STm-specific titer of intestinal secretory IgA at days 1 and 3 after challenge determined by live bacterial flow cytometry. f Rough STm-specific titer of intestinal secretory IgA at day 3 after challenge determined by live bacterial flow cytometry. Statistics: bars indicate mean (b, d, e) or median values (c). Horizontal, dotted lines indicate the detection limit (b, d, e). Panels b, d, and e were analyzed with ordinary one-way ANOVA and Dunnett’s post hoc test (comparison to STm^Aux group). Panel c was analyzed with a two-sided multicomparisons Kruskal–Wallis test and Dunn’s post hoc test. The data were pooled from three independent experiments. Source data are provided as a Source Data file. Detailed statistical metrics are available in the Supplementary Statistical Analysis file.