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The microbial degradation and utilization of mucin by Bacteroides in ulcerative colitis

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

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 “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.
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. Recent work using a model of spontaneously colitis provided 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. To understand the role of sulfatase in O-glycan degradation, 24 sulfatases were cloned and the proteins were recombinant expressed. The biochemical characterization of these proteins revealed the specificity for twelve sulfatases targeting all the different linkages present in O-glycans. Consistent with an essential role of sulfatases in mucin utilization, a mutant lacking sulfatase activity was unable to grow on porcine colonic mucin O-glycans (cMO), a highly sulfated substrate. This phenotype was also observed with a mutant lacking a single sulfatase, suggesting that this enzyme is required to initiate the depolymerization of sulfated cMO. Additionally, the in vivo competition of this single sulfatase mutant against the WT strain revealed that this enzyme is also an important fitness factors during gut colonization. Together this data suggests that a single sulfatase enzyme is critical to the utilization of sulfated mucin oligosaccharides.
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.
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 characterization of these endo-mucinases and additional glycoside hydrolases resulted in a proposed model for the degradation of mucin O-glycans by a single gut bacterium. Additionally, the glycomics analysis also contributed to understand the role of these bacterial enzymes in O-glycosylation alterations described in UC. Overall, 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.