Discovery of a secreted <i>Bacteroides fragilis</i> mucinase that cleaves mucins with bis-T O-glycans through a carbohydrate binding module-dependent mechanism

Degradation of mucins at the host-microbial mucus interphase involves glycosidases that release monosaccharides from O-glycans and mucinases that cleave the mucin protein backbone. Mucinases recognize and cleave peptide bonds at specific sequence motifs with varying O-glycan structures required and/or permissible. Mucinases that digest mucins with intact O-glycans can potentially destroy the protective mucus, while mucinases that only digest mucins with partially degraded O-glycans may serve at a later stage of nutrient sourcing from mucins. Here, we discovered nine CBM-bearing M60-like mucinases across gut commensals and opportunists, including a conserved <i>Bacteroides fragilis</i> mucinase denoted HC11. We also investigated the previously described <i>Bacteroides thetaiotaomicron</i> mucinase BT4244, which together delineates two functional classes with distinct preferences: BT4244 for bis-Tn (GalNAcα1-O-Ser/Thr) and HC11 for bis-T (Galβ1-3GalNAcα1-O-Ser/Thr) O-glycans. Both mucinases harbor carbohydrate-binding modules (CBM32) that bind their cognate O-glycan motifs and are required - together with the catalytic domains - for efficient cleavage of extended mucin domains, which is consistent with cooperative engagement, but are not required for the cleavage of short glycopeptides. We show <i>B. fragilis</i> strains secrete HC11 and degrade mucins only after the removal of sialic acids. Together, these findings expand the mucinase repertoire by nine enzymes spanning commensals and opportunists, demonstrate that CBM32 domains are essential for efficient cleavage of extended mucin substrates likely by promoting multivalent engagement and substrate positioning, and nominateidentify CBM-catalytic cooperation as a mechanism and intervention point for controlling mucus turnover and barrier integrity.