The early life human gut microbiota exerts lifelong health effects on the host, but the mechanisms underpinning its assembly remain elusive. Particularly, the early colonization of Clostridiales from the Roseburia-Eubacterium group, associated with protection from colorectal cancer, immune-and metabolic disorders is enigmatic. Here, we describe catabolic pathways that support the growth of Roseburia and Eubacterium members on distinct human milk oligosaccharides (HMOs). The HMO pathways, which include enzymes with a previously unknown structural fold and specificity, were upregulated together with additional glycanutilization loci during growth on selected HMOs and in co-cultures with Akkermansia muciniphila on mucin, suggesting an additional role in enabling cross-feeding and access to mucin O-glycans. Analyses of 4599 Roseburia genomes underscored the preponderance and diversity of the HMO utilization loci within the genus. The catabolism of HMOs by butyrateproducing Clostridiales may contribute to the competitiveness of this group during the weaning-triggered maturation of the microbiota.

The mucolytic human gut microbiota specialist Akkermansia muciniphila is proposed to boost mucin-secretion by the host, thereby being a key player in mucus turnover. Mucin glycan utilization requires the removal of protective caps, notably fucose and sialic acid, but the enzymatic details of this process remain largely unknown. Here, we describe the specificities of ten A. muciniphila glycoside hydrolases, which collectively remove all known sialyl and fucosyl mucin caps including those on double-sulfated epitopes. Structural analyses revealed an unprecedented fucosidase modular arrangement and explained the sialyl T-antigen specificity of a sialidase of a previously unknown family. Cell-attached sialidases and fucosidases displayed mucin-binding and their inhibition abolished growth of A. muciniphila on mucin. Remarkably, neither the sialic acid nor fucose contributed to A. muciniphila growth, but instead promoted butyrate production by co-cultured Clostridia. This study brings unprecedented mechanistic insight into the initiation of mucin O-glycan degradation by A. muciniphila and nutrient sharing between mucus-associated bacteria.

The gut mucolytic specialist Akkermansia muciniphila is strongly associated with the integrity of the mucus layer. Mucin glycan utilization requires the removal of diverse protective caps, notably, fucose and sialic acid, but the enzymatic details of this process remain largely unknown. Here, we describe the specificities of ten A. muciniphila glycoside hydrolases, which collectively remove all known sialyl and fucosyl mucin caps including those with double sulphated epitopes. Structural analyses revealed an unprecedented fucosidase modular arrangement and explained the exclusive sialyl T-antigen specificity of a sialidase of a previously unknown family and catalytic apparatus. Key cell attached sialidases and fucosidases conferred mucin-binding and their inhibition abolished growth of A. muciniphila on mucin. Remarkably, the sialic acid fucose did not contribute to A. muciniphila growth, but instead promoted butyrate production by co-cultured Clostridia. This study brings unique mechanistic insight into the initiation of mucin O-glycan degradation by A. muciniphila and the nutrient sharing between key mucus-associated bacteria.