Bacteroides fragilis toxin (BFT) detection and neutralization using phage display-derived synthetic hagfish VLRBs

Enterotoxigenic Bacteroides fragilis (ETBF) weakens the intestinal epithelial barrier and promotes inflammatory signaling through secretion of B. fragilis toxin (BFT), a metalloprotease associated with chronic intestinal inflammation. By triggering the breakdown of the E-cadherin glycoprotein in cellular junctions, BFT compromises tissue integrity and affects pathways involved in tumor suppression and signal transduction. Despite growing evidence implicating BFT in colorectal carcinogenesis, its role remains ambiguous, in part due to limited availability of suitable tools for its detection and neutralization. Selective binding molecules targeting BFT are, therefore, critical for understanding the role of ETBF in these clinical pathologies. Here, we used phage display screening to develop hagfish VLRB-derived consensus scaffold, termed ccombodies, as alternative non-immunoglobulin molecules targeting BFT. Three candidate binders (B8-1, C2-6, and G8-5) were expressed and purified for characterization. Specificity analyses against a range of bacterial virulence factors confirmed the selective recognition of BFT. Binding affinity was determined by ELISA titration and biolayer interferometry (BLI) yielding apparent K _D values in the nanomolar range. The ccombodies detected BFT at low nanogram levels by Western blot and exhibited high specificity and sensitivity in toxin recognition. Furthermore, treatment with ccombody attenuated BFT-induced cytotoxicity in Caco-2 and HT-29 epithelial cells. These findings establish ccombodies as a novel class of BFT-targeting molecules with potential applications in selective toxin detection, passive immunoprophylaxis, and antivirulence intervention.