Identification of receptor-binding domains of Bacteroidales antibacterial pore-forming toxins

Bacteroidales are abundant Gram-negative bacteria present in the gut microbiota of most animals, including humans, where they carry out vital functions for host health. To thrive in this competitive environment, Bacteroidales use sophisticated weapons to outmatch competitors. Among these, Bacteroidales secreted antimicrobial proteins (BSAPs) represent a novel class of bactericidal pore-forming toxins that are highly specific to their receptor, typically targeting only a single membrane protein or lipopolysaccharide. The molecular determinants conferring this high selectivity remain unknown. In this study, we therefore investigated the model protein BSAP-1 and determined which of its domains is involved in providing receptor specificity. We demonstrate that receptor recognition is entirely driven by the C-terminal domain (CTD) of BSAP-1 using a combination of in vivo competition assays, in vitro protein binding studies, and mutational analysis. Specifically, we show that deletion of the CTD abrogates BSAP-1 bactericidal activity by preventing receptor binding, whereas grafting the CTD to unrelated carrier proteins enables CTD-driven interaction with the BSAP-1 receptor. Combining structural investigation of a BSAP-1-receptor complex with mutational analysis, we unveil that this interaction is driven by electrostatic interactions. Building upon this discovery, we show that BSAPs can be categorized according to the structure of their CTD, suggesting a strong CTD structure-receptor type correlation. In summary, our research demonstrates that BSAP receptor recognition is driven by their CTD and paves the way for future applications.