Microbiology and Infectious Disease

Akkermansia muciniphila identified as key strain to function in pathogen invasion and intestinal stem cell proliferation through Wnt signaling pathway

Xin Ma
Meng Li
Tingting Xu
Xinchen Zhou
Mengqi Qian
Zhiren Yang
Kai Cao
Xinyan Han author has email address

Hainan Institute of Zhejiang University, Yongyou Industry Park, Yazhou Bay Sci-Tech City, Sanya 572000, China
Key Laboratory of Animal Nutrition and Feed Science in East China, Ministry of Agriculture, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China

https://doi.org/10.7554/eLife.92906.1

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Figures and data

Experimental designs of the present study.
(A) Schematic diagram of the FMT administered to the AIMD piglets infected with ETEC K88. (B) Schematic diagram of the alleviatory effects of A. muciniphila and B. fragilis on ETEC K88 infection in mice. (C) Schematic diagram of the alleviatory effects of A. muciniphila on ETEC K88 infection in porcine basal-out and apical-out intestinal organoids.

FMT improved the intestinal morphology and barrier function in AIMD piglets infected with ETEC K88.
(A) SEM images of villi (top) and microvilli (bottom) in the jejunum (scale bars = 300 or 2 μm). (B) The relative mRNA expression of tight junction proteins (ZO-1, claudin and occludin) and adheren junctions (β-catenin and E-cadherin) in the jejunum. (C) The relative protein expression of tight junction proteins (ZO-1, claudin and occludin) and adheren junction proteins (β-catenin and E-cadherin) in the jejunum. (D) Immunofluorescence images of Mucin 2 (MUC2, green) in the jejunum (scale bars = 50 μm). Con: control group, EP: ETEC K88 + PBS group, EF: ETEC K88 + FMT group. Data are expressed as the mean ± SD. *, P < 0.05, **, P < 0.01. Data shown are representative of at least two independent experiments.

FMT changed the composition of gut microbiota and the concentration of SCFAs in AIMD piglets infected with ETEC K88.
(A) Anosim analysis. (B) UPGMA clustering analysis based on Unweighted unifrac distance. (C) LEfSe analysis with LDA score > 4. (D) Quantification of SCFA metabolites (acetic acid, propionic acid, butyric acid, isobutyric acid, isovaleric acid and hexanoic acid). EP: ETEC K88 + PBS group, EF: ETEC K88 + FMT group. Data are expressed as the mean ± SD. *, P < 0.05, **, P < 0.01. Data shown are representative of at least two independent experiments.

A. muciniphila and B. fragilis improved the intestinal morphology of mice infected with ETEC K88.
(A) H&E-stained images of jejunum (scale bars = 50 μm) and the histograms of villus height, crypt depth and the ratio of villus height to crypt depth. (B) TEM images of jejunum and colon (scale bars = 0.5 μm). (C) PAS-stained images of colon (scale bars = 50 μm) and the histogram of crypt depth and number of goblet cell. (D) Immunofluorescence images of Mucin 2 (MUC2, green) in the jejunum and colon (scale bars = 50 μm). C: control group, E: ETEC K88 + PBS group, A: ETEC K88 + A. muciniphila group, B: ETEC K88 + B. fragilis group. Data are expressed as the mean ± SD. *, P < 0.05, **, P < 0.01, ***, P < 0.001. Data shown are representative of at least two independent experiments.

A. muciniphila and B. fragilis regulated the expression of inflammatory cytokines and the balance of Treg and Th17 cells in mice infected with ETEC K88.
(A) The relative mRNA expression of cytokines in the jejunum. (B) Flow cytometric dot plots and proportions of Treg (CD25⁺Foxp3⁺), Th17 (CD4⁺RORγt⁺) and mature dendritic cells (CD86⁺CD11C⁺) in jejunal lamina propria. C: control group, E: ETEC K88 + PBS group, A: ETEC K88 + A. muciniphila group, B: ETEC K88 + B. fragilis group. Data are expressed as the mean ± SD. *, P < 0.05, **, P < 0.01, ***, P < 0.001. Data shown are representative of at least two independent experiments.

A. muciniphila and B. fragilis enhanced the number of Ki67 cells and Lyz Paneth cells in jejunal crypt of mice infected with ETEC K88 by moderately activating the Wnt/β-catenin pathway.
(A) The mRNA expression and immunofluorescence staining images of intestinal epithelial cell proliferation marker Ki67 (red). (B) The mRNA expression and immunofluorescence staining images of Paneth marker Lyz (green). (C) The relative mRNA expression of Lgr5, Wnt3, Notch1 and other genes in jejunum mucosa. (D) The relative protein expression of Wnt/β-catenin pathway and its target genes in jejunum mucosa. C: control group, E: ETEC K88 + PBS group, A: ETEC K88 + A. muciniphila group, B: ETEC K88 + B. fragilis group. Data are expressed as the mean ± SD. *, P < 0.05, **, P < 0.01, ***, P < 0.001. Data shown are representative of at least two independent experiments.

A. muciniphila and B. fragilis promoted the activity of Lgr5⁺ intestinal stem cells as proved by the growth of intestinal organoids.
(A) Morphology of ex vivo culture of crypts isolated from the jejunum of mice in the C, E, A and B groups (Day 5; 4 ×, scale bars = 200 μm; 20 ×, scale bars = 50 μm). (B) The surface area and forming efficiency of intestinal organoids. (C) The relative mRNA expression of genes related to the proliferation and differentiation of the intestinal organoids. C: control group, E: ETEC K88 + PBS group, A: ETEC K88 + A. muciniphila group, B: ETEC K88 + B. fragilis group. Data are expressed as the mean ± SD. *, P < 0.05, **, P < 0.01, ***, P < 0.001. Data shown are representative of at least two independent experiments.

A. muciniphila protected the Lgr5⁺ intestinal stem cell and activated the Wnt/β-catenin signaling pathway of the ETEC K88-induced basal-out intestinal organoids.
(A) The relative mRNA expression of villin, ZO-1, Ki67, Lyz1, MUC2, Lgr5, Wnt3a and β-catenin genes. (B)-(F) Immunofluorescence images of villin (scale bars = 50 μm), Ki67 (scale bars = 50 μm), Lgr5 (scale bars = 20 μm), Wnt3a (scale bars = 50 μm) and β-catenin (scale bars = 50 μm). (G) Fold change of the mean fluorescence intensity. CON: control group, ETEC: ETEC K88 + DMEM/F-12 group, AKK: ETEC K88 + A. muciniphila group, WNT-C59: Wnt-C59 + ETEC K88 + A. muciniphila group. Data are expressed as the mean ± SD. *, P < 0.05, **, P < 0.01, ***, P < 0.001. Data shown are representative of at least two independent experiments.

A. muciniphila alleviated the intestinal epithelial injury of the ETEC K88-induced apical-out intestinal organoids.
(A) Confocal microscope visualization of the apical-out intestinal organoids incubated in FD4 solution (scale bars = 50 μm). (B) The relative mRNA expression of villin, ZO-1, Ki67, Lyz1, MUC2, Lgr5, Wnt3a and β-catenin genes. (C)-(G) Immunofluorescence images of villin, Ki67, Lgr5, Wnt3a and β-catenin (scale bars = 20 μm). (H) Quantification of the apical-out intestinal organoids incubated in FD4 solution that have intact barrier integrity. (I) Fold change of the mean fluorescence intensity. (J) The relative mRNA expression of cytokines in the apical-out intestinal organoids. CON: control group, ETEC: ETEC K88 + DMEM/F-12 group, AKK: ETEC K88 + A. muciniphila group, WNT-C59: Wnt-C59 + ETEC K88 + A. muciniphila group. Data are expressed as the mean ± SD. *, P < 0.05, **, P < 0.01, ***, P < 0.001. Data shown are representative of at least two independent experiments.

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