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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - MUC (The microbial degradation and utilization of mucin by Bacteroides in ulcerative colitis)

Teaser

Summary

The human gastrointestinal track is colonized by a diverse microbial community (microbiota) which as a significant impact in human health and disease. In the colon, the mucus layer provides a physical barrier that separates the microbiota and the intestinal epithelium preventing the close contact and inflammation (Figure 1A and B). This barrier is organized in a dense inner mucus layer that is almost devoid of bacteria and an outer mucus layer heavily colonized by the microbiota (Figure 1A). The major component of the mucus layer is MUC2, a mucin glycoprotein that is heavily O-glycosylated (Figure 1C). The combination of increased mucus degrading bacteria and the corresponding disruption of the mucus barrier have been proposed to promote inflammatory bowel diseases (IBD) (Figure 1B), a disease for which the incidence is rising in the Western society.
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 â€œearlyâ€ steps in the depolymerization process, which could block the downstream degradation of mucin glycans, 2) disclose the mechanism of mucin degradation and utilization by B. theta and, 3) understand the role of bacterial enzymes in O-glycosylation alterations described in UC. 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.

Work performed

Recent work using a model of spontaneously colitis provide evidences that B. theta sulfatases are required to trigger the inflammatory process that results in colitis in a susceptible animal model. However, the key sulfatases implicated in this process remain unclear. B. theta genome encodes 31 sulfatases however, only 4 of these, which are involved in glycosaminoglycan degradation, have been characterized. Additionally, three of B. theta sulfatases are predicted to be inactive. To understand the role of these enzymes in O-glycan degradation, 24 sulfatases were cloned and the proteins were recombinant expressed and tested against defined sulfated-oligosaccharides. This biochemical characterization revealed the specificity for twelve sulfatases targeting galactose-3-sulfate (Gal-3S, three enzymes), Gal-6S (four enzymes), Gal-4S (two enzymes), N-acetylglucosamine-6S (GlcNAc-6S, two enzymes), and GlcNAc-3S (one enzyme). Consistent with an essential role of sulfatases, a mutant lacking sulfatase activity was unable to grow on O-glycan from pig colonic mucin (cOS), a highly sulfated substrate. This phenotype was also observed with a mutant lacking a single Gal-3S sulfatase, suggesting that this enzyme is required to initiate the depolymerization of sulfated cOS. Additionally, the in vivo competition of a mutant lacking al the Gal-3S sulfatases against the WT strain revealed that these enzymes are also important fitness factors. Together this data suggests that Gal-3S sulfatases are critical enzymes to the utilization of sulfated mucin oligosaccharides.
Additionally, the simultaneous deletion of three PULs (previously predicted as mucin PULs) revealed a critical role for these enzymes (5 sulfatases and 10 glycoside hydrolases) on gastric O-glycans (gOS), a substrate enriched in 1,2-fucose linkages. The deletion of two fucosidases revelead to have a 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 O-glycans. The biochemical characterization of the remain 8 glycoside hydrolases revealed that one enzyme releases long oligosaccharides when incubated with mucins O-glycans. This is the first report of an â€œendo-mucinaseâ€ enzyme 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 B1,4-galactosidases, B1,3-N-acetylglucosaminidases, B1,3-N-acetylglucosaminidases, a-galactosidases, a1,3/1,4-fucosidases and a1,2-fucosidases. The characterization of these enzymes was essential to generate a model of O-glycans utilization by B. theta (Figure 2).
In this project, the combination of biochemical and genetic approaches in in vitro and in vivo models allowed the identification of key enzymes (sulfatases and fucosidases) required to O-glycans degradation that have the potential to be explored as drug targets in IBD.

Final results

The microbiota is one of the key factors implicated in IBD, a disease with a rising incidence in the Western society. The mucus layer, which is heavily colonized by the microbiota, has a barrier effect preventing the close contact between the bacteria and the intestinal epithelium and subsequent inflammation. Some members of the microbiota can degrade this mucus layer and, if this degradation overcomes the mucus production, the bacteria can reach the epithelium leading to inflammation. Significantly, the enzymatic mechanisms of mucin degradation by gut bacteria remain unclear. This project revealed that sulfatases and fucosidases are key enzymes in the initiation of mucin depolymerization. Additional biochemical characterization of glycoside hydrolases allowed the characterization of 12 sulfatases and the identification of enzymes displaying a novel activity (endo-mucinase). The ongoing characterization of these endo-mucinases and additional glycoside hydrolases will allow validate the proposed model for the degradation of mucin O-glycans by a single gut bacterium. Additionally, further glycomics analysis will also contribute to understand the role of these bacterial enzymes in O-glycosylation alterations described in UC. This project provides novel insights into the mechanism of mucin utilization by the human microbiota and the identification of key enzymes (sulfatases and fucosidases) contributes for the development of future therapeutic strategies to improve human health.