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.
