Efficient mucin O-glycan degradation by specific mucin degrading intestinal bacteria: towards understanding enzyme-glycan interactions

Intestinal mucin glycan-degrading bacteria are important for mucus turnover, stimulating mucus production, and producing beneficial metabolites. The mucin-degrading bacteria require various enzymes to break down mucin O-glycans. In this study, mucin glycan-degrading bacteria Akkermansia muciniphila, Ruminococcus torques, and Bacteroides thetaiotaomicron, were grown on porcine gastric mucin in monocultures, co-cultures, and a synthetic bacterial community. Enzyme extracts from these cultures were incubated with a selection of glycans, varying in sugar and linkage composition, to investigate enzyme specificities. Proteomics identified β-galactosidases, α-N-acetylgalactosaminidases, β-N-acetylglucosaminidases, α-fucosidases, α-sialidases, sulphatases, carbohydrate esterases, and polysaccharide lyases involved in O-glycan degradation. Enzymes produced by A. muciniphila and R. torques efficiently cleaved β-linked galactose and N-acetylgalactosamine. B. thetaiotaomicron enzymes minimally cleaved mucin glycans although multiple β-galactosidases and β-N-acetylglucosaminidases were produced. A. muciniphila favoured removal of fucose linked to non-terminal sugars whereas R. torques and B. thetaiotaomicron favoured removal of fucose linked to terminal sugars. A. muciniphila enzymes favoured cleavage of fucose α1-2 linked over α1-3 linked and cleavage of N-acetylglucosamine β1-3 linked over β1-4 linked. Both A. muciniphila and B. thetaiotaomicron favoured cleavage of galactose β1-4 linked over β1-3 linked and sialic acid α2-3 linked over α2-6 linked. Removal of sulphate from mucin structures was only observed by B. thetaiotaomicron. Bacterial co-cultures and the synthetic community produced all enzymes identified in the monocultures resulting in efficient mucin O-glycan degradation. Combining proteomics and glycan linkage cleavage by bacterial enzymes, showed differences in glycan degradation by the bacteria. This highlighted the importance of intestinal bacterial composition in mucin glycan degradation.