Revisiting Hyaluronan Catabolism in Bacteroides: Pathway Conservation, Overlooked Proteins, and Predictive Accuracy

ABSTRACT The ability of gut microbes to degrade host‐ and diet‐derived glycans is central to microbiome ecology and host interactions, yet predicting these functions in silico remains challenging. Hyaluronan (HA), a glycosaminoglycan (GAG) abundant in host tissues and dietary supplements, is depolymerized by specialized polysaccharide utilization loci (PULs) in Bacteroides. Here, we combined comparative protein analysis, functional assays, and quantitative proteomics to evaluate the reliability of sequence‐based predictions of HA utilization. Clustering of more than 3900 PL8 and GH88 protein sequences from 54 Bacteroides species did not distinguish known HA degraders from nondegraders, underscoring the limited predictive power of these enzymes alone. Experimental validation in Bacteroides acidifaciens DSM 111135 and Bacteroides thetaiotaomicron DSM 2079 confirmed HA degradation, as HA‐derived fragments were identified by liquid chromatography–mass spectrometry. Proteomic profiling revealed coordinated induction of both canonical GAG‐specific PULs‐encoded proteins and noncanonical accessory proteins (BT4410/BT4411) in response to HA in both species. Incorporating such noncanonical components into comparative frameworks may improve prediction of glycan utilization potential and help link microbial genomic content to ecological function in the gut.