Functional specialization of bacterial cellular and extracellular fractions contributes to polysaccharide degradation

Bacteroides thetaiotaomicron (Bt), a key commensal of the human gut microbiota, exhibits robust capacity to degrade dietary polysaccharides via carbohydrate-active enzymes (CAZymes). However, the exact spatial distribution and the functional coordination of these enzymes remain poorly understood. In this study, we characterized the “labor division” during inulin degradation across four cellular fractions of Bt: cytoplasm, outer membrane, extracellular vesicles (EVs), and supernatant. In vitro degradation confirmed that all four fractions exhibited potent hydrolytic activity. Proteomic analysis showed that all four fractions contained inulin-hydrolyzing CAZymes-primarily glycoside hydrolases and polysaccharide lyases-albeit with marked differences in hydrolase composition across fractions. Furthermore, untargeted metabolomic analysis of the hydrolysates identified 45 shared differential metabolites in pairwise comparisons, including carbohydrates and short-chain fatty acids. These results indicate that there are significant differences in the hydrolytic capacity among the various fractions. Notably, cross-feeding experiments revealed that the hydrolytic products generated by EVs can support the growth of bacteria with limited utilization capabilities. Taken together, our results highlight a synergistic multi-fraction strategy for inulin degradation in Bacteroides, offering novel insights into the polysaccharide utilization and a foundation for developing of postbiotics and functional microbial products.