Glucosyltransferase-dependent and independent effects of Clostridioides difficile toxins during infection

Abstract

Clostridioides difficile infection (CDI) is the leading cause of nosocomial diarrhea and pseudomembranous colitis in the USA. In addition to these symptoms, patients with CDI can develop severe inflammation and tissue damage, resulting in life-threatening toxic megacolon. CDI is mediated by two large homologous protein toxins, TcdA and TcdB, that bind and hijack receptors to enter host cells where they use glucosyltransferase (GT) enzymes to inactivate Rho family GTPases. GT-dependent intoxication elicits cytopathic changes, cytokine production, and apoptosis. At higher concentrations TcdB induces GT-independent necrosis in cells and tissue by stimulating production of reactive oxygen species via recruitment of the NADPH oxidase complex. Although GT-independent necrosis has been observed in vitro, the relevance of this mechanism during CDI has remained an outstanding question in the field. In this study we generated novel C. difficile toxin mutants in the hypervirulent BI/NAP1/PCR-ribotype 027 R20291 strain to test the hypothesis that GT-independent epithelial damage occurs during CDI. Using the mouse model of CDI, we observed that epithelial damage occurs through a GT-independent process that is does not involve immune cell influx. The GT-activity of either toxin was sufficient to cause severe edema and inflammation, yet GT activity of both toxins was necessary to produce severe watery diarrhea. These results indicate that both TcdA and TcdB contribute to infection when present. Further, while inactivating GT activity of C. difficile toxins may suppress diarrhea and deleterious GT-dependent immune responses, the potential of severe GT-independent epithelial damage merits consideration when developing toxin-based therapeutics against CDI. SIGNIFICANCEClostridioides difficile is the leading cause of antibiotic-associated diarrhea in hospitals worldwide. This bacterium produces two virulence factors, TcdA and TcdB, which are large protein toxins that enter host colon cells to cause inflammation, fluid secretion, and cell death. The enzymatic domain of TcdB is a target for novel C. difficile infection (CDI) therapeutics since it is considered the major factor in causing severe CDI. However, necrotic cell death due to non-enzymatic TcdB-host interactions have been reported in cell culture and intoxicated tissue. Here, we generated C. difficile strains with enzyme-inactive toxins to evaluate the role of each toxin in an animal model of CDI. We observe an additive role for TcdA and TcdB in disease and both glucosyltransferase-dependent and independent phenotypes. These findings are expected to inform the development of toxin-based CDI therapeutics.