Microbial Metabolic Networks at the Mucus Layer Lead to Diet-Independent Butyrate and Vitamin B12 Production by Intestinal Symbionts

Abstract

Akkermansia muciniphila has evolved to specialize in the degradation and utilization of host mucus, which it may use as the sole source of carbon and nitrogen. Mucus degradation and fermentation by A. muciniphila are known to result in the liberation of oligosaccharides and subsequent production of acetate, which becomes directly available to microorganisms in the vicinity of the intestinal mucosa. Coculturing experiments of Amuciniphila with non-mucus-degrading butyrate-producing bacteria Anaerostipes caccae, Eubacterium hallii, and Faecalibacterium prausnitzii resulted in syntrophic growth and production of butyrate. In addition, we demonstrate that the production of pseudovitamin B₁₂ by E. hallii results in production of propionate by A. muciniphila, which suggests that this syntrophy is indeed bidirectional. These data are proof of concept for syntrophic and other symbiotic microbe-microbe interactions at the intestinal mucosal interface. The observed metabolic interactions between Amuciniphila and butyrogenic bacterial taxa support the existence of colonic vitamin and butyrate production pathways that are dependent on host glycan production and independent of dietary carbohydrates. We infer that the intestinal symbiont A. muciniphila can indirectly stimulate intestinal butyrate levels in the vicinity of the intestinal epithelial cells with potential health benefits to the host.IMPORTANCE The intestinal microbiota is said to be a stable ecosystem where many networks between microorganisms are formed. Here we present a proof of principle study of microbial interaction at the intestinal mucus layer. We show that indigestible oligosaccharide chains within mucus become available for a broad range of intestinal microbes after degradation and liberation of sugars by the species Akkermansia muciniphila This leads to the microbial synthesis of vitamin B₁₂, 1,2-propanediol, propionate, and butyrate, which are beneficial to the microbial ecosystem and host epithelial cells.

Keywords: Akkermansia muciniphila; anaerobes; butyrate; cross-feeding; intestine; microbiome; mucus; syntrophy.

FIG 1

Metabolic activity of A. caccae on mucin-derived sugars. A. caccae was grown on monosaccharide present in the glycan chain of mucin. The OD₆₀₀ values and HPLC profiles are shown for the sugars that resulted in positive growth. The sugars that gave positive test results were also used to perform experiments with the addition of 10 mM acetate. The graphs show the mean values for the experiments performed a minimum of three times in duplicate. Values that are significantly different (P < 0.05) in the presence of 10 mM acetate or absence of acetate are indicated by an asterisk. GlucNac, N-acetylglucosamine.

FIG 2

Metabolic activity of E. hallii on mucin-derived sugars. E. hallii was grown on monosaccharide present in the glycan chain of mucin. The OD₆₀₀ value and HPLC profiles are shown for sugars that resulted in positive growth. The sugars that gave positive test results were also used to perform experiments with the addition of 10 mM acetate. The graphs show the mean values for the experiments performed a minimum of three times in duplicate.

FIG 3

A. muciniphila degradation and fermentation of mucus enables cross-feeding by the butyrate-producing gut isolates. (A to C) Cocultures of A. muciniphila with butyrate-producing isolates were performed and measurements of product formation and consumption (A), FISH staining (B), and Q-PCR (C) were performed. (D) Measurement of A. muciniphila metabolites on mucus-containing media without the addition of vitamin B₁₂ or with vitamin B₁₂ from E. hallii or pseudovitamin B₁₂ from E. hallii. The graph shows the mean values for the experiment performed a minimum of three times in duplicate. Asterisks indicate a significant difference (P < 0.05) compared to the condition without vitamin B₁₂ added.

FIG 4

UHPLC-UV chromatogram of E. hallii vitamin B₁₂. (A) Immunoaffinity-purified cell extract of E. hallii (in arbitrary units [AU]) is shown on the y axis, and time (in minutes) is shown on the x axis. Tr, retention time. (B) LC-MS/MS identified a peak at 3.16 min. (C) Chemical structure of pseudovitamin B₁₂ from E. hallii.

FIG 5

Schematic overview of mucus-dependent cross-feeding network. Keystone mucolytic bacteria, such as A. muciniphila, degrade mucin glycans resulting in oligosaccharides (mainly galactose, fucose, mannose, and GlucNac) and SCFAs (acetate, propionate, and 1,2-propanediol) that can be used for growth, as well as propionates, butyrate, and vitamin B₁₂ production by cross-feeding partners. Treg GPR, regulatory T cell G-protein-coupled protein receptor.