Diet-driven differential response of Akkermansia muciniphila modulates pathogen susceptibility

The erosion of the colonic mucus layer by a dietary fiber-deprived gut microbiota results in heightened susceptibility to an attaching and effacing pathogen, Citrobacter rodentium. Nevertheless, the questions of whether and how specific mucolytic bacteria aid in the increased pathogen susceptibility remain unexplored. Here, we leverage a functionally characterized, 14-member synthetic human microbiota in gnotobiotic mice to deduce which bacteria and functions are responsible for the pathogen susceptibility. Using strain dropouts of mucolytic bacteria from the community, we show that Akkermansia muciniphila renders the host more vulnerable to the mucosal pathogen during fiber deprivation. However, the presence of A. muciniphila reduces pathogen load on a fiber-sufficient diet, highlighting the context-dependent beneficial effects of this mucin specialist. The enhanced pathogen susceptibility is not owing to altered host immune or pathogen responses, but is driven by a combination of increased mucus penetrability and altered activities of A. muciniphila and other community members. Our study provides novel insights into the mechanisms of how discrete functional responses of the same mucolytic bacterium either resist or enhance enteric pathogen susceptibility.


Figure EV2
. Acetylated galactoglucomannan supplementation is insufficient to rescue the resistant phenotype of the FR-fed mice, while short-term feeding of the FF diet is sufficient to develop a susceptible phenotype.
(A) Relative abundances of 14SM bacteria among mice fed a fiber-free (FF) diet supplemented with 7.5% acetylated galactoglucomannan (AcGGM).Abundances were calculated from 16S rRNA gene sequencing results after initial colonization (day 8), prior to diet change (day 13), during the feeding period (day 15-54), or during C. rodentium infection.Ten sampling points, n = 1-5 per sampling point.(B) Fecal C. rodentium load of 14SM FR-, FF-and AcGGM-fed Swiss Webster mice during the 10 days of infection (n = 5-11 per group following the exclusion of three outliers).Error bars show SEM and the curves the arithmetic mean; one-way ANOVA with adjustment for multiple comparisons between FF and AcGGM (top significance labels, yellow text) or between FF and FR (bottom significance labels, green text) group using the Benjamini-Hochberg method.ns, non-significant; *, adjusted p < 0.1; **, adjusted p < 0.01; ***, adjusted p < 0.001.(C) Fecal LCN-2 concentration of infected 14SM FR-, FF-, and AcGGM-fed mice on the final day of their infection (n = 4-7 per group, no outliers).Error bars show SD and the center of the arithmetic mean; one-way ANOVA with adjustment for multiple comparisons using the Benjamini-Hochberg method.ns non-significant.(D) Colon length of infected 14SM FR-, FF-, and AcGGM-fed mice on the final day of their infection (n = 10-16 per group, no outliers).Error bars show SD and the center of the arithmetic mean; one-way ANOVA with adjustment for multiple comparisons using the Benjamini-Hochberg method.ns non-significant.(E) Experimental timeline for short-term feeding experiment.Age-matched germ-free Swiss Webster mice were gavaged with one of the different synthetic gut microbiota (SM) on 3 consecutive days.These mice were maintained for at least 14 days on the fiberrich (FR) diet, after which mice were switched to a fiber-free (FF) for either 20 days (FF, n = 3) or 5 days (FF, n = 4) before they were infected with Citrobacter rodentium.After infection, mice were closely observed for up to 10 days.(F) Relative abundances of 14SM bacteria among mice fed a fiber-free diet for 20 (left) and 5 (right) days prior to infection.Abundances were calculated from 16S rRNA gene sequencing results, and the number of sampling points varies by feeding regimen, with 16 for the FF diet with a 20-day feeding period and 10 for the FF diet with a 5-day feeding period, n = 1-4 mice per sampling point.(G) Fecal C. rodentium load of 14SM mice fed the FF diet for 5, 20, or 40 days before infection.5-day feeding n = 4, 20-day feeding n = 3, 40-day feeding n = 12.Some days it was impossible to recover fecal pellets from certain mice.Error bars show SEM and the curves the arithmetic mean; one-way ANOVA with adjustment for multiple comparisons between 5-day feeding and 40-day feeding (top significance labels, blue text) groups or between 20-day feeding and 40-day feeding (bottom significance labels, olive green text) groups using the Benjamini-Hochberg method.N/A, no statistic available as n = 1 for one group; ns non-significant; *, adjusted p < 0.1; **, adjusted p < 0.01; ***, adjusted p < 0.001.

Figure EV3. Host immune cell populations remain largely unchanged between dietary groups after infection with C. rodentium.
(A) Non-significantly altered immune cell population as a percent of the parent population among mice at 3 DPI, as determined by fluorescence-activated cell sorting (FACS).Population percentages were analyzed by two-way ANOVA (main effects only) with adjustment for multiple comparisons between SMs of the same diet (shown) and between diets of the same SM (see Source Data) using the Benjamini-Hochberg method (n = 4-6 per group, following exclusion of one outlier in 13SM FF CD + GATA3+ group).Error bars show SD and the center of the arithmetic mean; ns non-significant (adjusted p > 0.1).(B) Gating strategy for FACS analysis.

Molecular Systems Biology
Mathis Wolter et al

Figure EV4 .
Figure EV4.Distinct effects of diet and community composition on bacterial co-abundance correlations.
Correlation matrices and networks of relative abundances in Dataset EV1 across all timepoints (14 FR, n = 177; 14SM FF, n = 200; 13SM FR, n = 20; 13SM FF, n = 19).In the matrix (lower left), all correlations are shown.Correlations were calculated using the Pearson method.In the network plot (upper right), only correlations with |r| > 0.2 are shown.Figure EV5.Bacterial transcriptomes altered by A. muciniphila presence in fiber-deprived mice.Volcano plots of synthetic microbiota members with >10 DEGs in the 13SM FF vs 14SM FF contrast (B.caccae, B. ovatus, B. thetaiotaomicron, B. uniformis, C. symbiosum, C. aerofaciens, and E. coli).RNA was extracted from cecal contents at 3 DPI in 13SM FF (C.rodentium resistant) and 14SM FF (C.rodentium susceptible) mice and individually mapped to the genome specified in Salmon.Features were filtered out if <2.5 cpm in four samples.Significance based on adjusted p value <0.05 based on Wald test adjusted using the Benjamini-Hochberg method in DESeq2.GH glycoside hydrolase, OMP outer membrane protein, RT reverse transcriptase.