The glucosyltransferase activity of C. difficile Toxin B is required for disease pathogenesis.

Enzymatic inactivation of Rho-family GTPases by the glucosyltransferase domain ofClostridioides difficileToxin B (TcdB) gives rise to various pathogenic effects in cells that are classically thought to be responsible for the disease symptoms associated withC.difficileinfection (CDI). Recentin vitrostudies have shown that TcdB can, under certain circumstances, induce cellular toxicities that are independent of glucosyltransferase (GT) activity, calling into question the precise role of GT activity. Here, to establish the importance of GT activity in CDI disease pathogenesis, we generated the first described mutant strain ofC.difficileproducing glucosyltransferase-defective (GT-defective) toxin. Using allelic exchange (AE) technology, we first deletedtcdAinC.difficile630 Delta ermand subsequently introduced a deactivating D270N substitution in the GT domain of TcdB. To examine the role of GT activityin vivo, we tested each strain in two different animal models of CDI pathogenesis. In the non-lethal murine model of infection, the GT-defective mutant induced minimal pathology in host tissues as compared to the profound caecal inflammation seen in the wild-type and 630 Delta erm Delta tcdA(Delta tcdA) strains. In the more sensitive hamster model of CDI, whereas hamsters in the wild-type or Delta tcdAgroups succumbed to fulminant infection within 4 days, all hamsters infected with the GT-defective mutant survived the 10-day infection period without primary symptoms of CDI or evidence of caecal inflammation. These data demonstrate that GT activity is indispensable for disease pathogenesis and reaffirm its central role in disease and its importance as a therapeutic target for small-molecule inhibition. Author summary Novel non-antibiotic therapies are required for the treatment ofClostridioides difficileinfection (CDI). An emerging class of promising therapeutics for CDI are antivirulence agents that block the actions ofC.difficileToxin B (TcdB), the primary determinant of virulence. In order to develop such treatments, molecular targets and mechanisms must be identified and validated. Historically the glucosyltransferase domain (GTD) represented an ideal target owing to its perceived importance for disease pathogenesis. However, studies capitalizing on recent advances in recombinant TcdB production have unveiled GTD-independent mechanisms of toxicity when applied at high concentrationsin vitro, thus questioning the role of the GTD. Here we generate the first-reported mutant strain ofC.difficileexpressing glucosyltransferase-defective TcdB. Application thereof demonstrates that the GTD is essential for disease in mice and hamsters, thus reoffering the GTD as an ideal candidate for small-molecule inhibitor (SMI) development.