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.

Fig 1. In vitro characterization of C. difficile mutants.

(A) tcdA and tcdB gene alignments of mutant and wildtype strains to the reference genome of R20291. Red boxes highlight SNMs that deactivate GT catalytic activity. (B) Representative Western blot images of mutant and wildtype strains to confirm secretion (or lack thereof) of TcdA and TcdB. Images directly below each Western blot are stain-free gel images shown as loading controls. The graphs show densitometry analyses from Western blot experiments (n = 3). Dots signify each replicate, bars denote the mean, and error bars represent the standard error of the mean. Significant differences were determined by Tukey’s HSD (* p < 0.05). (C) In vitro growth curves of each strain and mock-inoculated controls (n = 5 per treatment) cultured in BHIS for 24 hours. Dots are the average at each given timepoint, and the error bars depict the 95% confidence interval.

Fig 2. GT-dependent and independent effects on weight loss, diarrhea, and colonization in the mouse model of C. difficile infection.

(A) Visual abstract of the cefoperazone mouse model of CDI used for this study. The figure was created with BioRender (B) Percent weight loss from day 0 for R20291 (n = 22), A_GTX B+ (n = 15), A+ B_GTX (n = 15), A_GTX B_GTX (n = 15), ΔtcdA B_GTX (n = 9), ΔtcdA ΔtcdB (n = 22), and mock (n = 13). Points represent group averages at each day and error bars denote standard error of the mean. (C) Stool scores at 2 dpi. (D) Daily C. difficile burden in stool. Each point is an individual mouse, and the crossbars represent group daily means. Significantly different groups as determined by Dunn’s test are shown in brackets (* p < 0.05; *** p < 0.001).

Fig 3. Glucosyltransferase activity causes edema and inflammation, but epithelial damage is glucosyltransferase independent.

(A) Representative images of ceca and colons from mice inoculated with each treatment at 2 dpi. White bars denote 1 cm. (B) Cecum area and colon length as metrics of organ inflammation. Bars are the mean of each group and points are individuals within the group. Error bars represent standard error of the mean. Differences in cecum area depict that R20291, A_GTX B+, and A+ B_GTX are significantly smaller than the four other groups. Differences in colon length are comparing R20291, A_GTX B+, and A+ B_GTX to mock-inoculated colons. (C) Epithelial damage, inflammation, and edema mean scores as determined by a gastrointestinal pathologist. Points represent each individual animal and error bars are the standard error of the mean (n = 6–9 per treatment). Statistical differences as determined by Tukey’s HSD are shown in brackets (* p < 0.05; *** p < 0.001; **** p < 0.0001) or by letters (p < 0.05). (D) Representative H&E images of mouse ceca inoculated with each strain at 2 dpi. Scale bar, 80 μm.

Fig 4. Epithelial damage is elicited by a glucosyltransferase-independent mechanism during infection.

Representative images of severe epithelial injury in R20291 and A_GTX B_GTX compared to ΔtcdA B_GTX and ΔtcdA ΔtcdB. Scale bar, 80 μm in 20x magnification images. Zoomed in (40x magnification; dashed boxes) images offer a closer view at specific epithelial damage phenotypes. Apoptotic bodies or dying cells are highlighted by blue arrows. Sloughing dead cells and pre-pseudomembrane formation is shown by orange arrowheads. Scale bar, 130 μm.

Fig 5. Glucosyltransferase activity of TcdB is required to elicit acute myeloperoxidase-positive immune cell infiltration.

(A) Number of MPO⁺ cells per single mucosal layer per 20x field of view (FOV). Each point is the average of three 20x FOVs from one animal. Bars depict the group mean and error bars are the standard error of the mean. Statistical differences between R20291 and A_GTX B+ and the other treatments as determined by Tukey’s HSD are shown in a bracket (*** p < 0.001). (B) Representative MPO immunohistochemistry images showing high amounts of immune cell influx into submucosal and mucosal layers in R20291 and A_GTX B+, and lower amounts in A+ B_GTX and A_GTX B_GTX. Scale bar, 80 μm.