Comparative Genomic and Functional Profiling of ECM-Targeting Enzymes in Bacteroides, a Key Genus of the Human Gut Microbiome.

PURPOSE: The human extracellular matrix (ECM) provides essential cues for intestinal homeostasis. While most studies focus on ECM degradation by host cells, our prior work suggests that commensal gut microbes may also contribute to these remodeling processes. Here, we continue exploring this novel dimension of host-microbe interactions by profiling the proteolytic diversity and substrate-specific activity of ECM-targeting enzymes across species of Bacteroides, a dominant and metabolically versatile gut genus. METHODS: We curated a custom ECM-specific enzyme database from the BRENDA repository and used it to perform comparative genomic analyses across 11 Bacteroides species, mapping the diversity and abundance of candidate ECM-degrading proteases and carbohydrate active enzymes (CAZymes). Functional activity was evaluated via in vitro degradation assays using purified substrates. Family-specific protease inhibitors were used to confirm the major catalytic classes involved. RESULTS: ECM-targeting CAZymes and proteases were broadly encoded across all 11 genomes, with gene counts positively correlated with genome size and GAG-associated genes comprising the largest substrate category. Experimental degradation assays revealed species- and substrate-specific activity patterns, including elastin degradation restricted to a subset of species, a capacity previously undocumented in intestinal Bacteroides. Genomic predictions showed limited concordance with measured enzymatic activity, suggesting context-dependent regulation of ECM-degrading enzymes. Inhibitor experiments confirmed that collagen degradation is driven primarily by metalloproteases and secondarily by serine proteases across representative species. CONCLUSIONS: Our findings position commensal Bacteroides as a rich, yet underappreciated, source of ECMdegrading enzymes. This work underscores the need to consider microbiota as key modulators of host tissue homeostasis and potential targets for therapeutic modulation.