Purification and Characterization of His-Tagged Recombinant Bacteroides fragilis Toxin-2 Variants In Vitro and In Vivo.

Bacteroides fragilis is a major commensal bacterium of the human colon. However, enterotoxigenic B. fragilis (ETBF) secretes B. fragilis toxin (BFT), a zinc-dependent metalloprotease that cleaves E-cadherin and promotes chronic inflammation and colorectal tumorigenesis. Despite extensive research, the cellular receptor for BFT remains unidentified. In this study, we developed His-tagged recombinant BFT variants including both catalytically active and inactive forms to facilitate biochemical and functional analyses. Functional assays confirmed that the active variant retained proteolytic activity and induced characteristic cellular responses, while the inactive variant served as an effective negative control. These results establish a robust experimental platform for BFT receptor identification and mechanistic studies of BFT-host interactions. The active and inactive BFT variants provide essential molecular tools for investigating ETBF pathogenicity and developing therapeutic interventions.