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 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 demonstrate that both TcdA and TcdB contribute to disease pathogenesis 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.

Toxin-mediated downregulation of absorptive ion transporters NHE3, DRA, and SGLT1 in the colon contributes to diarrhea associated withClostridioides difficileinfection

“…Strains were stored at -80 o C in 20% glycerol stocks for long-term use. Spore stocks were prepared as previously described and stored at 4 o C until use 28,46 .…”

“…Stool samples were scored using a modified Bristol Stool Chart 1-4 scale by color and consistency. In this scoring scheme 1 = normal stool, 2 = well-formed but discolored stool, 3 = moist, soft, and discolored stool, and 4 = wet tail, watery diarrhea, and empty bowels 28 . Stool was then weighed, homogenized in 500 µl sterile PBS pH 7.4, and serially diluted on TCCFA media (TA 10% w/v; D-cycloserine 10 mg/ml; cefoxitin 10 mg/ml; fructose) to quantify C. difficile shed during infection.…”

“…Additionally, TcdB concentrations at and above 0.1 nM induce glucosyltransferase-independent epithelial injury through the production of reactive oxygen species that results in a necrotic cell death [25][26][27][28] . Despite the understanding that TcdA and TcdB are the main determinants of disease, the role of each toxin during infection is not fully defined [28][29][30][31][32] .…”

“…Additionally, TcdB concentrations at and above 0.1 nM induce glucosyltransferase-independent epithelial injury through the production of reactive oxygen species that results in a necrotic cell death [25][26][27][28] . Despite the understanding that TcdA and TcdB are the main determinants of disease, the role of each toxin during infection is not fully defined [28][29][30][31][32] . A recent study from our lab reaffirmed that TcdA and TcdB both cause symptoms in the murine model of infection and suggested that they synergize to worsen disease outcomes, including the severity of diarrhea 28 .…”

“…Despite the understanding that TcdA and TcdB are the main determinants of disease, the role of each toxin during infection is not fully defined [28][29][30][31][32] . A recent study from our lab reaffirmed that TcdA and TcdB both cause symptoms in the murine model of infection and suggested that they synergize to worsen disease outcomes, including the severity of diarrhea 28 .…”