DOI: 10.6084/m9.figshare.28517140.v1
title: Additional file 1 of Enterocloster clostridioformis protects against Salmonella pathogenesis and modulates epithelial and mucosal immune function

description: Supplementary Material 1: Supplementary figures: Supplementary Fig. 1. The gut microbiota is essential for resistance against S. Typhimurium infection but can confound mouse studies. (A) Weight loss (left) and survival (right) data for C57BL/6NTac mice infected with S.Tm following antibiotic administration in drinking water (d.w.) or by intragastric gavage (i.g.). Data shown are pooled from five experiments (n = 3–18). (B) Faecal microbiota titres at two days post cessation of antibiotics correlate with duration of survival following S.Tm infection. Points represent individual mice. This subfigure combines data from both d.w. and i.g. protocols. Linear correlation was assessed using the Pearson product-moment correlation coefficient. Data are pooled from three independent experiments. (C) Titres of faecal commensal bacteria in the days following antibiotic treatment in the drinking water. Each bar represents the mean faecal titres from a different cage of mice from one experiment. Error bars indicate SEM. Supplementary Fig. 2. S.Tm virulence gene expression is unaffected by E. clostridioformis. (A) Relative expression of S.Tm virulence genes following 18 h culture in caecal contents from GF, E. clostridioformis-, or E. coli-monocolonised mice. Data shown are pooled from three experiments (n = 10–11). Significance was assessed using student’s t-tests. Error bars indicate SEM. (B) Relative expression of S.Tm virulence genes following 18 h culture of E. coli or S.Tm in LB broth (n = 2). Supplementary Fig. 3. E. clostridioformis is associated with less pathogen–epithelium contact in vivo. (A) Quantification of S.Tm contact with the caecal epithelium (CFU/mm epithelium) at 1 dpi (n = 8–12). (B) Representative micrographs of caecal epithelium 1dpi stained for S.Tm (anti-O4 antigen, red), and host nuclei (DAPI, blue) and cytoskeleton (phalloidin, blue). (C) Intracellular S.Tm titres using an in vitro MODE-K invasion assay (n = 2). MODE-K cells were pretreated for 72 h with LB or sterile bacterial spent media from E. clostridioformis or E. coli prior to infection. Wilcoxon tests were used to assess statistical significance in (A) and (C). Supplementary Fig. 4. Epithelial architecture is maintained after E. clostridioformis monocolonisation. (A) Relative expression by qPCR of host defence genes in caecal epithelial cells isolated from GF, SPF, or E. clostridioformis-, E. coli-, or A. faecis-monocolonised mice. Data shown are pooled from six experiments (n = 4–14). Significance was assessed using student’s t-tests with Benjamini–Hochberg correction. Error bars indicate SEM. (B) Quantification of caecal epithelium thickness from GF, SPF, and E. clostridioformis- and E. coli-moncolonised mice. Data are pooled from 5 experiments (n = 4–6). Significance was assessed using Wilcoxon tests. (C) Representative micrographs showing the caecal epithelium architecture. Mice were monocolonised with E. clostridioformis for two weeks prior to sampling. Host cells were stained with Hoescht and micrographs taken using confocal microscopy. Supplementary Fig. 5. Flow cytometric data for colon and mesenteric lymph node compartments. Flow cytometric quantification data for additional lymphocyte populations from the colon intraepithelial compartment and lamina propria as well as mesenteric lymph nodes. Each data point represents a single mouse. Pooled data are shown from three experiments (n = 10–12). Wilcoxon tests with Benjamini–Hochberg correction. Bars and error bars represent median ± median absolute deviation, respectively. Supplementary Fig. 6. Gating strategy for flow cytometric analysis of intestinal lymphocytes.