The complete catalog of antimicrobial resistance secondary active transporters in Clostridioides difficile: evolution...

Secondary active transporters shuttle substrates across eukaryotic and prokaryotic membranes, utilizing different electrochemical gradients. They are recognized as one of the antimicrobial efflux pumps among pathogens. While primary active transporters within the genome of C. difficile 630 have been completely cataloged, the systematical study of secondary active transporters remains incomplete. Here, we not only identify secondary active transporters but also disclose their evolution and role in drug resistance in C. difficile 630. Our analysis reveals that C. difficile 630 carries 147 secondary active transporters belonging to 27 (super)families. Notably, 50 (34%) of them potentially contribute to antimicrobial resistance (AMR). AMR-secondary active transporters are structurally classified into five (super)families: the p-aminobenzoyl-glutamate transporter (AbgT), drug/metabolite transporter (DMT) superfamily, major facilitator (MFS) superfamily, multidrug and toxic compound extrusion (MATE) family, and resistance-nodulation-division (RND) family. Surprisingly, complete RND genes found in C. difficile 630 are likely an evolutionary leftover from the common ancestor with the diderm. Through protein structure comparisons, we have potentially identified six novel AMR-secondary active transporters from DMT, MATE, and MFS (super)families. Pangenome analysis revealed that half of the AMR-secondary transporters are accessory genes, which indicates an important role in adaptive AMR function rather than innate physiological homeostasis. Gene expression profile firmly supports their ability to respond to a wide spectrum of antibiotics. Our findings highlight the evolution of AMR-secondary active transporters and their integral role in antibiotic responses. This marks AMR-secondary active transporters as interesting therapeutic targets to synergize with other antibiotic activity.