Additional file 1 of Enterocloster clostridioformis protects against Salmonella pathogenesis and modulates epithelial and mucosal immune function

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.

补充材料1：补充示意图
补充图1：肠道菌群对抵抗鼠伤寒沙门氏菌（Salmonella Typhimurium, S. Typhimurium）感染至关重要，但可能干扰小鼠实验研究。(A) 经饮水给药（d.w.）或灌胃（i.g.）给予抗生素后，感染鼠伤寒沙门氏菌的C57BL/6NTac小鼠的体重变化（左）与存活数据（右）。数据汇总自5项独立实验（n=3~18）。(B) 停用抗生素两天后的粪便菌群滴度与鼠伤寒沙门氏菌感染后的存活时长呈显著线性相关。每个数据点代表单只小鼠，本子图整合了饮水给药与灌胃两种给药方案的数据。采用皮尔逊积矩相关系数评估线性相关性，数据汇总自3项独立实验。(C) 饮水给予抗生素后不同天数的粪便共生菌滴度。每个柱状图代表单笼小鼠的平均粪便菌滴度，来自单一项实验。误差棒代表标准误（SEM）。
补充图2：产气荚膜梭菌样杆菌（Eubacterium clostridioformis, E. clostridioformis）不影响鼠伤寒沙门氏菌的毒力基因表达。(A) 在无菌（Germ-free, GF）、单一定植产气荚膜梭菌样杆菌或单一定植大肠杆菌（Escherichia coli, E. coli）的小鼠盲肠内容物中培养18小时后，鼠伤寒沙门氏菌毒力基因的相对表达量。数据汇总自3项实验（n=10~11），采用学生t检验评估统计学显著性，误差棒代表标准误。(B) 在LB肉汤中分别培养大肠杆菌或鼠伤寒沙门氏菌18小时后，鼠伤寒沙门氏菌毒力基因的相对表达量（n=2）。
补充图3：产气荚膜梭菌样杆菌可降低体内病原菌与上皮细胞的接触概率。(A) 感染后1天（days post infection, dpi），鼠伤寒沙门氏菌与盲肠上皮细胞接触量的定量分析（菌落形成单位/毫米上皮组织，CFU/mm）（n=8~12）。(B) 感染后1天的盲肠上皮细胞代表性显微图像：鼠伤寒沙门氏菌经抗O4抗原染色呈红色，宿主细胞核经DAPI染色呈蓝色，细胞骨架经鬼笔环肽染色呈蓝色。(C) 采用体外MODE-K细胞侵袭实验检测胞内鼠伤寒沙门氏菌滴度（n=2）。感染前，MODE-K细胞先用LB培养基或产气荚膜梭菌样杆菌、大肠杆菌的无菌细菌上清液预处理72小时。(A)与(C)的统计学显著性采用威尔科克森检验评估。
补充图4：单一定植产气荚膜梭菌样杆菌可维持上皮组织结构完整。(A) 从无菌、无特定病原体（Specific Pathogen Free, SPF）、单一定植产气荚膜梭菌样杆菌、大肠杆菌或产碱杆菌（Alcaligenes faecalis, A. faecis）的小鼠中分离盲肠上皮细胞，经实时定量聚合酶链式反应（quantitative PCR, qPCR）检测宿主防御基因的相对表达量。数据汇总自6项实验（n=4~14），采用学生t检验结合本雅明-霍赫贝格校正评估统计学显著性，误差棒代表标准误。(B) 无菌、无特定病原体、单一定植产气荚膜梭菌样杆菌或大肠杆菌的小鼠盲肠上皮厚度定量分析。数据汇总自5项实验（n=4~6），采用威尔科克森检验评估统计学显著性。(C) 盲肠上皮组织结构的代表性显微图像。小鼠在采样前经单一定植产气荚膜梭菌样杆菌两周，宿主细胞经Hoescht染色，采用共聚焦显微镜采集图像。
补充图5：结肠与肠系膜淋巴结（mesenteric lymph node）区域的流式细胞术数据。本图展示结肠上皮内室、固有层以及肠系膜淋巴结中额外淋巴细胞群的流式细胞术定量数据。每个数据点代表单只小鼠，数据汇总自3项实验（n=10~12），采用威尔科克森检验结合本雅明-霍赫贝格校正。柱状图与误差棒分别代表中位数±中位数绝对偏差。
补充图6：肠道淋巴细胞流式细胞术分析的设门策略。