Mucin O-glycan degradation by GH101 underpins mucosal persistence of Bifidobacterium bifidum PRL2010

Abstract Host-derived mucin O-glycans constitute a key chemical component of the human intestinal niche and are continuously encountered by gut-adapted bifidobacterial cells, thereby playing a pivotal role in host–microbe interactions. Among these microbes, Bifidobacterium bifidum is one of the most persistent members of the human gut microbiota. Comparative genomic analyses of mucosa-associated strains of this species have revealed a gene conserved across its pangenome, predicted to encode a GH101 glycoside hydrolase involved in mucin degradation. In this study, we performed a molecular characterization of the GH101 enzyme encoded by B. bifidum PRL2010. Insertional mutagenesis of the GH101 gene from the PRL2010 genome resulted in a marked reduction in bacterial adhesion to mucin-secreting epithelial cells, along with a significant growth difference when N-acetyl-galactosamine was provided as the sole carbon source. These findings indicate that GH101 mediates the initial cleavage of O-GalNAc core 1 structures, representing a crucial step for the adhesion to human mucosa. However, this enzyme represents only one component of the broader set of glycosidases required for the complete degradation of host mucins. Overall, our results establish a mechanistic link between metabolic specialization and ecological fitness, providing insights into how mucin-adapted commensals may contribute to interactions with the human gut mucosa. Key points • The GH101 glycoside hydrolase of Bifidobacterium bifidum PRL2010 plays a pivotal role in host mucin utilization by initiating the cleavage of O-GalNAc core 1 structures. • Insertional mutant of the GH101 gene impairs both mucosal adhesion and growth on N-acetyl-galactosamine. • These findings reveal a mechanistic connection between mucin glycan metabolism and ecological fitness by mucin-adapted bifidobacterial commensals. Graphical Abstract Within the intestinal ecosystem, Bifidobacterium bifidum PRL2010 utilizes epithelial mucin through the activity of glycoside hydrolase (GH) enzymes, releasing sugars that support microbial cross-feeding. GH101-deficient mutants lose mucin-degrading capacity and show impaired growth on mucin, demonstrating the essential role of GH101 in mucin utilization and gut microbe interactions. Created in BioRender. Turroni, F. (2026) https://BioRender.com/jhll48f