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.

 Users may download and print one copy of any publication from the public portal for the purpose of private study or research.  You may not further distribute the material or use it for any profit-making activity or commercial gain  You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

No abstract

Butyrate producing Clostridiales utilize distinct human milk oligosaccharides correlating to early 2 colonization and prevalence in the human gut 3Bacterial growth studies were performed by M.J.P. Proteomic analyses were done by M.J.P and 26 E.S. Protein characterization was done by M.J.P and M.L.L. Enzymatic characterization of 27RiLe a/b 136 was performed by A.G.,To.K., M.S. and Ta.K. Mucin preparation was performed by 28 B.S. and M.J.P. Mucin glycomics were performed by B.S., C.J. and N.G.K. Protein X-ray 29 crystallography was performed by C.Y. and S.F. Metagenome analysis were performed by C.A-30 S and M.A. Experiments were designed by M.J.P and M.A.H. The manuscript was drafted by 31 M.J.P and M.A.H and finalized with contributions of all authors. 32 33 34 35 Abstract 36The early life human gut microbiota exerts life-long health effects on the host, but the mechanisms 37 underpinning its assembly remain elusive. Particularly, the early colonization of Clostridiales from 38the Roseburia-Eubacterium group, associated with protection from colorectal cancer, immune-39and metabolic disorders is enigmatic. Here we unveil the growth of Roseburia and Eubacterium 40 members on human milk oligosaccharides (HMOs) using an unprecedented catabolic apparatus. 41The described HMO pathways and additional glycan utilization loci confer co-growth with 42 Akkermansia muciniphilia via cross-feeding and access to mucin O-glycans. Strikingly, both, HMO 43and xylooligosaccharide pathways, were active simultaneously attesting an adaptation to a mixed 44HMO-solid food diet. Analyses of 4599 Roseburia genomes underscored the preponderance of 45HMO pathways and highlighted different HMO utilization phylotypes. Our revelations provide a 46 possible rationale for the early establishment and resilience of butyrate producing Clostridiales 47and expand the role of specific HMOs in the assembly of the early life microbiota. 48 49 129 R. inulinivorans, respectively. In R. inulinivorans two additional loci encoding sialic acid and 130 fucose catabolism proteins, were also upregulated ( Supplementary Fig. 3). 131 132 133 Fig. 1: Growth of R. hominis, R. inulinivorans and E. ramulus on HMOs and upregulation of core HMOs 134 utilization loci: Growth curves of R. hominis (a) and R. inulinivorans (b) on glucose, LNT, GNB, LNB, and/or purified 135 HMOs from mothers milk compared to a no-carbon source controls over 24 h. c, Growth levels of R. inulinivorans on 136 LNT, LNB, GNB and of E. ramulus on LNT within 24 h including glucose and a non-carbon source controls. d, Growth 137 of R. hominis, R. inulinivorans and E. ramulus on monosaccharides from HMOs and mucin after 24 h. The growth 138 analyses (a-d) on media supplemented with 0.5 % (w/v) carbohydrates (except for R. inulinivorans on 1% (w/v) and 139 4% (w/v) purified HMOs from mothers milk) are means of triplicates with standard deviations. e) HMO and mucin-140 derived oligo-and monosaccharides used for the growth analyses in (a-d). The core HMO utilization loci in R. hominis 141 (f) and R. in...

No abstract

No abstract