Butyrate producing Clostridiales utilize distinct human milk oligosaccharides correlating to early colonization and prevalence in the human gut

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...