Elucidation of Akkermansia muciniphila Probiotic Traits Driven by Mucin Depletion

Abstract

Akkermansia muciniphila is widely considered a next-generation beneficial microbe. This bacterium resides in the mucus layer of its host and regulates intestinal homeostasis and intestinal barrier integrity by affecting host signaling pathways. However, it remains unknown how the expression of genes encoding extracellular proteins is regulated in response to dynamic mucosal environments. In this study, we elucidated the effect of mucin on the gene expression and probiotic traits of A. muciniphila. Transcriptome analysis showed that the genes encoding most mucin-degrading enzymes were significantly upregulated in the presence of mucin. By contrast, most genes involved in glycolysis and energy metabolic pathways were upregulated under mucin-depleted conditions. Interestingly, the absence of mucin resulted in the upregulation of 79 genes encoding secreted protein candidates, including Amuc-1100 as well as members of major protein secretion systems. These transcript level changes were consistent with the fact that administration of A. muciniphila grown under mucin-depleted conditions to high-fat diet-induced diabetic mice reduced obesity and improved intestinal barrier integrity more efficiently than administration of A. muciniphila grown under mucin-containing conditions. In conclusion, mucin content in the growth medium plays a critical role in the improvement by A. muciniphila of high-fat diet-induced obesity, intestinal inflammation, and compromised intestinal barrier integrity related to a decrease in goblet cell density. Our findings suggest the depletion of animal-derived mucin in growth medium as a novel principle for the development of A. muciniphila for human therapeutics.

Keywords: Akkermansia muciniphila; extracellular protein; metabolic disorder; microbiome analysis; mucus layer.

FIGURE 1

Transcriptomic dynamics of A. muciniphila under mucin-rich and mucin-depleted conditions. (A) Schematic of experimental design employed in this study. (B) Principal component analysis (PCA) of whole-transcriptome RNA data. (C) Sample-to-sample Pearson correlation coefficients between the data sets. (D) Volcano plot of genes sequenced under mucin-rich and mucin-depleted conditions. Differentially expressed genes (DEGs) are in blue. (E) Significantly enriched Gene Ontology (GO) molecular function terms among upregulated (red) and downregulated (blue) differentially expressed genes between mucin-rich and mucin-depleted conditions (Benjamini-Hochberg corrected P < 0.05).

FIGURE 2

Differentially expressed genes involved in mucin-degradation pathway and glycolytic/gluconeogenesis in A. muciniphila. Enzymes are represented according to their corresponding mucin-degrading reactions, as follows: A, N-acetylgalactosaminidase; B, L-fucosidase; C, sulfatase; D, galactosidase; E, neuraminidase. The heat map indicates RNA expression levels and log₂ fold changes. Gene list and expression values are also shown in Supplementary Table S4.

FIGURE 3

Analysis of extracellular proteins and protein secretion systems in A. muciniphila. (A) Workflow to identify the extracellular proteins encoded in A. muciniphila genome. (B) Significantly enriched Gene Ontology (GO) terms among the predicted extracellular proteins. GO terms belonging to the biological process and molecular function categories are shown in gray and blue, respectively. (C) Volcano plots summarizing the differentially expressed genes encoding extracellular proteins. The negative log₁₀ of the Padj is plotted on the Y-axis, and the log₂ of the fold change is plotted on the X-axis. (D) Differentially expressed genes involved in protein secretion pathways in A. muciniphila. ^∗Padj < 0.01.

FIGURE 4

Effects of A. muciniphila on HFD-induced obesity. (A) Body weight and (B) blood glucose change (%) after 4 weeks of treatment. (C) Representative H&E-stained images and size distribution of epididymal adipose tissue deposits. Scale bars, 100 μm. (D) Blood glucose change and the mean area under the curve (AUC) measured during the IP-GTT. (E) Blood glucose change and the mean area under the curve (AUC) measured during the IP-ITT. (F) Fasting plasma insulin and (G) HOMA-IR index measured after 4 weeks of treatment. Data are means ± SEM (n = 4–5 for each group). ^#P < 0.05, High fat diet (HFD) vs. Normal chow diet (ND) group, ^∗P < 0.05, HFD vs. A. muciniphila-treated group (two-tailed Student’s t-test). ^abcMeans not sharing a common letter are significantly different at P < 0.05 (Tukey-Kramer HSD test).

FIGURE 5

Effects of A. muciniphila on intestinal barrier function and inflammation. (A) Circulating plasma LPS measured after 4 weeks of treatment. (B) Expression of genes encoding barrier- or pore-forming tight junction proteins, (C) proinflammatory cytokines, and (D) mucin in the ileum. (E) Representative PAS-stained images and (F) mucus-containing goblet cell density in the ileum. Scale bars, 100 μm. Data are means ± SEM (n = 4–5 for each group). ^#P < 0.05, High fat diet (HFD) vs. Normal chow diet (ND) group, ^∗P < 0.05, HFD vs. A. muciniphila-treated group (two-tailed Student’s t-test). ^abcMeans not sharing a common letter are significantly different at P < 0.05 (Tukey-Kramer HSD test).