Periodic Reporting for period 2 - MUC (The microbial degradation and utilization of mucin by Bacteroides in ulcerative colitis)
Reporting period: 2019-09-01 to 2020-08-31
Bacteroides thetaiotaomicron (B. theta), a dominant member of human microbiota, has numerous Polysaccharide Utilization Loci (PULs) encoding dozens of predicted mucin-degradation enzymes (glycoside hydrolases and sulfatases). B. theta was shown to induce ulcerative colitis (UC) in a susceptible animal model. Interestingly, this inflammatory process was dependent on B. theta sulfatases enzymes. Significantly, the enzymatic mechanisms of mucin degradation by this and other gut bacteria remain unclear. This project was designed to investigate the mechanisms of mucin utilization by the human microbiota and its impact on UC development. The main goals are 1) identification of key “early” steps in the depolymerization process, which can be used to block the downstream degradation of mucin glycans, and 2) disclose the mechanism of mucin degradation and utilization by B. theta. These findings will provide insights in the mechanism behind the mucin utilization by gut bacteria and UC development, allowing the development of future therapeutic strategies in IBD. Additionally, the understanding how members of microbiota can alter the mucins compositions present in mucus layer can, potentially, be deployed to ensure that the structure of this ecosystem maximizes human health.
This project disclosed the first model of depolymerization of O-glycans by a single gut bacteria that requires at least 35 enzymes. Additionally, in this study we identified a key sulfatase and two fucosidases that are essential to the utilization of mucin O-glycans by B. theta.
Additionally, the simultaneous deletion of three PULs (previously predicted as mucin PULs) revealed the key role for these enzymes (5 sulfatases and 10 glycoside hydrolases) on utilization of gastric mucin O-glycans (gMO), a substrate enriched in fucose linkages. The deletion of two fucosidases resulted in significant defect in growth on gOS. Indeed, this double fucosidase mutant was unable to utilize fucosylated oligosaccharides, suggesting that these enzymeas are essential to initiate the degradation of fucosylated glycans. The biochemical characterization of the remaining glycoside hydrolases revealed that one enzyme releases long oligosaccharides when incubated with mucins O-glycans. This “endo-mucinase” has a new activity not described in the literature. To understand the specificity determinants, the enzyme was crystallised and the structure was solved. Consistent with the activity, the structure of this enzyme shows an open cleft able to accommodate long O-glycans chains. The characterization of additional glycoside hydrolases present in B. theta mucin PULs revealed that this bacteria encodes exo-active galactosidases, N-acetylglucosaminidases, N-acetylglucosaminidases and fucosidases, that act in the various linkages found in mucins. The characterization of 23 of these enzymes was essential to generate a model of O-glycans utilization by B. theta (Figure 2). Overall, this project demonstrate that mucin utilization by gut bacteria it is initiated by key enzymes that can potential be inhibited blocking this degradative process diseases such as IBD.
This work was presented orally in five international conferences and it will be published in three scientific articles to be submitted in high-impact journals.