The gut commensal Blautia maintains colonic mucus function under low-fiber consumption through secretion of short-chain fatty acids

Abstract

Beneficial gut bacteria are indispensable for developing colonic mucus and fully establishing its protective function against intestinal microorganisms. Low-fiber diet consumption alters the gut bacterial configuration and disturbs this microbe-mucus interaction, but the specific bacteria and microbial metabolites responsible for maintaining mucus function remain poorly understood. By using human-to-mouse microbiota transplantation and ex vivo analysis of colonic mucus function, we here show as a proof-of-concept that individuals who increase their daily dietary fiber intake can improve the capacity of their gut microbiota to prevent diet-mediated mucus defects. Mucus growth, a critical feature of intact colonic mucus, correlated with the abundance of the gut commensal Blautia, and supplementation of Blautia coccoides to mice confirmed its mucus-stimulating capacity. Mechanistically, B. coccoides stimulated mucus growth through the production of the short-chain fatty acids propionate and acetate via activation of the short-chain fatty acid receptor Ffar2, which could serve as a new target to restore mucus growth during mucus-associated lifestyle diseases.

Fig. 1. Human-derived microbiota prevent mucus defects in mice fed a Western-style diet (WSD) in a fiber-dependent manner.

HD Habitual diet, HF High-fiber diet, m male, f female. A Selection of participants from a previously published intervention study, in which participants increased their dietary fiber intake for 12 weeks. Participants (n = 67) were ranked based on their improvement in waist-to-hip ratio, body mass index, free fat mass, cholesterol, triglyceride, and glucose levels (heatmap: rows correspond to individual participants, columns correspond to each metabolic parameter). From the top ten responders, shifts in bacterial composition, based on Bray‒Curtis and weighted UniFrac distance metric, were calculated, and after combination with the metabolic score, 5 participants within the top 6 responders were selected. Statistical significance was determined by the Wilcoxon matched-pairs signed-rank test. B Shift in gut bacteria community structure of the 5 human participants selected as donors for microbiota transplantation before and after HF intervention. C Schematic representation of the human-to-mouse FMT experiment using antibiotic-treated mice. Following the first FMT, mice were fed a standard chow diet or a WSD (n = 8 mice/group). D Mucus growth rate and E mucus thickness of the inner colonic mucus layer. F Colon length. G Absolute quantification of host defense protein/peptide transcripts in distal colon; Statistical significance in D-G was determined by 2-way ANOVA and Tukey’s multiple comparison test within microbiota transplant groups. Data in A and D–G are presented as mean ± SD with p_(adj) < 0.05 (*), p_(adj) < 0.01 (**), p_(adj) < 0.001 (***) and p_(adj) < 0.0001(****) considered statistically significant. All P values are two-sided. Parts of A and C were created with BioRender.com. Source data are provided as a Source Data file.

Fig. 2. Relative abundance of the gut commensal Blautia correlates with mucus function.

A Weighted UniFrac PCoA and (B) relative abundance of bacterial genera before (baseline) and after (termination) human-to-mouse FMT. C Abundance of bacterial taxa in fresh stool samples from mice transplanted with habitual diet (HD)- or high-fiber diet (HF)-derived human microbiota and fed a chow or Western-style diet (WSD) at phylum and (D) genus level (n = 8 mice/group). Relative abundance taxa plots (left) display the top 30 bacterial taxa. Boxplots (right) display Centered log-ratio (CLR) transformed abundance counts of selected taxa, with data presented as median with upper and lower quartiles. Statistical significance was determined by 2-way ANOVA and Tukey’s multiple comparison test. E Spearman correlation analysis between mucus growth rate in the distal colon and centered log ratio (Clr)-transformed relative abundance of selected genera. Data was tested for normal distribution using the D’Agostino & Pearson test and P values were adjusted for multiple comparisons using the Benjamini‒Hochberg procedure. For adjusted P values in (D) and (E), p_(adj) < 0.05 (*), p_(adj) < 0.01 (**), p_(adj) < 0.001 (***) and p_(adj) < 0.0001 (****) were considered statistically significant. F Relative abundance of Blautia in human participants (n = 67) before and after 12 weeks of high-fiber intervention. Data is presented as mean ± SD and statistical significance was determined by the Wilcoxon matched-pairs signed-rank test, with p < 0.05 (*) considered statistically significant. All P values are two-sided. Source data are provided as a Source Data file.

Fig. 3. Human-derived high-fiber microbiota ameliorate intestinal infection.

A Enzymatic activity of carbohydrate-active enzymes (CAZy) in the cecal content of human microbiota-transplanted mice (n = 8 mice/group) against various carbohydrate structures. Statistical significance was determined with 2-way ANOVA and Tukey’s multiple comparison test. B Normalized CAZy activity of enzymes primarily targeting dietary fiber (red/orange) and mucus glycans (green). C Schematic representation of the human FMT and Citrobacter rodentium infection experiment (n = 9 mice/group). D Change in CFUs of C. rodentium in stool samples and (E) change in body weight after C. rodentium infection. F Number of goblet cells per crypt in the distal colon and (G) CFUs of C. rodentium in the cecum 7 days post infection (n = 9 mice/group). (H) Weighted UniFrac PCoA and (I) relative abundance of bacterial genera before (baseline) (n = 9 mice/group) and after human-to-mouse FMT (HD: n = 6 mice; HF: n = 8 mice). J Relative genus abundance in cecal content of the transplanted and infected mice on day 1 (D1) and day 5 (D5) post infection; K Relative abundance of Blautia at D1 post infection; Statistical significance was determined with the Mann‒Whitney U test. L Spearman correlation between the relative abundance of Blautia and C. rodentium CFUs in stool at D1 post infection. Statistical significance was determined using the Mann‒Whitney U test for (D)–(G). For (D–H) and (K), data are presented as mean ± SD, and normal distribution was tested with the D’Agostino & Pearson test. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) and p < 0.0001 (****) were considered statistically significant. All P values are two-sided. HD habitual diet, HF high-fiber diet. Parts of (C) were created with BioRender.com. Source data are provided as a Source Data file.

Fig. 4. Blautia coccoides improves mucus function in WSD-fed mice.

A Schematic representation of B. coccoides supplementation through drinking water to WSD-fed mice. Mice were supplemented with media control (n = 4 mice) or B. coccoides (n = 6 mice) for a period of 33 days, whereupon mucus function was investigated. B Mucus growth rate of inner colonic mucus layer. C Representative confocal images (top) and processed iso-surface images (bottom) from distal colon explants stained for DNA (green), fucosylated mucin glycans (UEA1; purple), sialylated mucin glycans (WGA; orange) overlaid with 1 µm fluorescent microspheres (blue). D Position-based microsphere (bead) distribution within mucus layer per group (left) and individual mouse (right), obtained from processed images (2–3 images/mouse). Red shading indicates zone within 10 µm from colonic epithelium. E Fraction of microspheres (beads) penetrating red zone (within 10 µm from colonic epithelium) of the mucus layer as indicated in D. Values are an average of 2–3 analyzed images/mouse. F Schematic representation of B. coccoides supplementation through oral gavage of WSD-fed mice. Mice were supplemented with viable or heat-killed B. coccoides (n = 6 mice/group) through repeated oral gavage over a period of 35 days, whereupon mucus function was investigated. G Mucus growth rate of inner colonic mucus. H Representative confocal images (top) and processed iso-surface images (bottom) from distal colon explants stained as described for (C). I Position-based microsphere (bead) distribution within mucus layer per group (left) and individual mouse (right), obtained from processed images (2–4 images/mouse). Red shading indicates zone within 10 µm from colonic epithelium. J Fraction of microspheres (beads) that penetrate the red zone (within 10 µm from colonic epithelium) of the mucus layer as indicated in I. Values are the average of 2–4 analyzed images per mouse. Data in (B), (G), (E), and (J) are presented as mean ± SD. Data in (D) and (I) are presented as median and quartiles. Normal distribution was tested with the D’Agostino & Pearson test. Statistical significance was determined using the Mann–Whitney U test, with p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) considered statistically significant. All P values are two-sided. Parts of (A) and (F) were created with BioRender.com. Source data are provided as a Source Data file.

Fig. 5. Blautia coccoides partly ameliorates Citrobacter rodentium infection.

A Schematic representation of the B. coccoides supplementation experiment in C. rodentium-infected mice. Mice fed a WSD and supplemented with B. coccoides or media control through drinking water were infected with C. rodentium 8 days after the start of treatment (n = 8 mice/group). B Monitoring of C. rodentium CFUs from stool, LOD = limit of detection. C Mucus growth rate and thickness measured 7 days post infection. D Absolute quantification of host defense protein/peptide transcripts in the distal colon. E Weighted UniFrac PCoA and relative abundance of bacterial genera before (baseline) and after switching to WSD feeding, infecting with C. rodentium and supplementing with B. coccoides or media control (termination); Data in (B–D) are presented as mean ± SD and normal distribution was tested with the D’Agostino & Pearson test. Statistical significance was determined by the Mann‒Whitney U test with p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) considered statistically significant. All P values are two-sided. Parts of (A) were created with BioRender.com. Source data are provided as a Source Data file.

Fig. 6. Blautia coccoides stimulates mucus growth through short-chain fatty acid production.

A PLS-DA of mucus metabolites. WSD-fed mice (n = 10) were supplemented with B. coccoides (red, n = 6) or control media (blue, n = 4) through drinking water. B Unsupervised hierarchical cluster analysis using Euclidian distance measurements of the 25 most altered metabolites. Color scale indicates fold change in normalized peak intensity. C Peak area of altered SCFAs, hexanoate and heptanoate. D Spearman correlation between colonic mucus growth rate and SCFAs, hexanoate and heptanoate peak intensity in mucus of individual mice. E Spearman correlation between colonic mucus growth rate and SCFAs, hexanoate and heptanoate peak intensity in the cecum of WSD-fed mice transplanted with human microbiota. F Ex vivo mucus growth rate in distal colon tissue explants with supplementation of propionate, G supernatant of a 24 h B. coccoides culture (n = 5) and (H) supernatant of a 24 h cecal community supplemented with B. coccoides (n = 6), with respective controls. I Quantification of acetate and propionate in supernatant of a 24 h B. coccoides culture (n = 3) and (J) supernatant of a 24 h cecal community supplemented with B. coccoides (n = 3). K Ex vivo stimulation of distal colon tissue explants with acetate as described above (n = 6 mice/group). L Fold change in acetate and propionate peak area in supernatants of a 48 h B. coccoides culture incubated in GAM media supplemented with fucose (control n = 2; supernatant n = 3). M Ex vivo stimulation and mucus growth measurement of colonic tissue stimulated with acetate or propionate, with and without a Ffar2 chemical antagonist (n = 5), N with a chemical agonist of Ffar2 (n = 5) and (O) with supernatant of a 24–48 h B. coccoides culture with and without a Ffar2 chemical antagonist (n = 5). Statistical significance was determined by Kruskal‒Wallis test and Dunn’s multiple comparisons test (F, K) or Mann‒Whitney U test (C, G, H, L, M, N, O). Normal distribution of the data was tested with the D’Agostino & Pearson test, and data are presented as mean ± SD with p < 0.05 (*) and p < 0.01 (**) considered statistically significant. All P values are two-sided. Source data are provided as a Source Data file.