Bifidobacterium bifidum Suppresses Antibiotic-Induced InflammationThese results indicate that probiotic supplementation is effective, but only when antibiotics cause proinflammatory species-induced gut inflammation, suggesting that the necessity of probiotic supplementation is strongly influenced by the type of disturbance introduced to the community.

Mucinolytic bacteria modulate host-microbiota symbiosis and dysbiosis through their ability to degrade mucin O-glycans. However, how and to what extent bacterial enzymes are involved in the breakdown process remains poorly understood. Here, we focus on a glycoside hydrolase family 20 sulfoglycosidase (BbhII) from Bifidobacterium bifidum, which releases N-acetylglucosamine-6sulfate from sulfated mucins. Glycomic analysis showed that, in addition to sulfatases, sulfoglycosidases are involved in mucin O-glycan breakdown in vivo and that the released Nacetylglucosamine-6-sulfate potentially affects gut microbial metabolism, both of which were also supported by a metagenomic data mining analysis. Enzymatic and structural analysis of BbhII reveals the architecture underlying its specificity and the presence of a GlcNAc-6S-specific carbohydrate binding module (CBM) 32 with a distinct sugar recognition mode that B. bifidum takes advantage of to degrade mucin O-glycans. Comparative analysis of the genomes of prominent mucinolytic bacteria also highlights a CBM-dependent O-glycan breakdown strategy utilised by B. bifidum. Main textacetylglucosamine-6-sulfate (GlcNAc-6S) from porcine gastric mucin (PGM) in vitro. This activity does not seem to be the result of N-acetylglucosaminidase promiscuity, as BbhII showed 400-fold higher activity toward GlcNAc-6S over GlcNAc residues 15 . B. bifidum is a Gram-positive anaerobe capable of assimilating host glycans such as human milk oligosaccharides and mucin O-glycans but is incapable of plant polysaccharide degradation 16,17 . This bacterium possesses cell surface-anchored GHs acting on almost all glycosidic linkages in O-glycans, with the known exception of α-linked N-acetylgalactosaminides 16 . As a possible reflection of this repertoire of GHs, B. bifidum colonises the intestines of a wide range of mammals 18 . However, its in vivo mucin O-glycan degradative capability has not been addressed and is controversial 19 . Here, through structural, glycomic, and informatics studies on BbhII combined with animal and human sample analyses, we not only demonstrate the in vivo relevance of sulfoglycosidase to intestinal mucin O-glycan breakdown but also reveal the mechanistic basis of how B. bifidum takes advantage of a novel GlcNAc-6S-specific carbohydrate-binding module (CBM) 32 13 , found within BbhII to degrade O-glycans. Comparative genomic analysis of mucinolytic microbes highlights a CBM-dependent mucin O-glycan strategy employed by B. bifidum. Results B. bifidum affects intestinal O-glycan metabolism in miceUsing conventional mice we examined how B. bifidum administration affects intestinal mucin Oglycan breakdown (Fig. 1a). Quantitative PCR analysis indicated, at day 5, B. bifidum-colonization of the caecum contents and faeces, but not of the intestinal surface, at an abundance of 0.062% and 3.7% per total 16S rRNA genes, respectively (Supplementary Table 1). The data were comparable with faecal microbiota composition analysis (Extended Data Fig. 1a). Using faecal extracts contain...

Ovomucin, a hen egg white protein, is characterized by its hydrogel-forming properties, high molecular weight, and extensive O-glycosylation with a high degree of sialylation. As a commonly used food ingredient, we explored whether ovomucin has an effect on the gut microbiota. O-Glycan analysis revealed that ovomucin contained core-1 and core-2 structures with heavy modification by N-acetylneuraminic acid and/or sulfate groups. Of the two mucin-degrading gut microbes we tested, Akkermansia muciniphila grew in medium containing ovomucin as a sole carbon source during a 24 h culture period, whereas Bifidobacterium bifidum did not. Both gut microbes, however, degraded ovomucin O-glycans and released monosaccharides into the culture supernatants in a speciesdependent manner, as revealed by semi-quantified mass spectrometric analysis and anion exchange chromatography analysis. Our data suggest that ovomucin potentially affects the gut microbiota through O-glycan decomposition by gut microbes and degradant sugar sharing within the community. Akkermansia muciniphila, Bifidobacterium bifidum, gut microbiota, mucin degradation, O-glycan, ovomucin.

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