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Clostridioides difficile causes antibiotic-associated diseases in humans, ranging from mild diarrhea to severe pseudomembranous colitis and death. A major clinical challenge is the prevention of disease recurrence, which affects nearly ~20 to 30% of the patients with a primary C. difficile infection (CDI). During CDI, C. difficile forms metabolically dormant spores that are essential for recurrence of CDI (R-CDI). In prior studies, we have shown that C. difficile spores interact with intestinal epithelial cells (IECs), which contribute to R-CDI. However, this interaction remains poorly understood. Here, we provide evidence that C. difficile spores interact with E-cadherin, contributing to spore adherence and internalization into IECs. C. difficile toxins TcdA and TcdB lead to adherens junctions opening and increase spore adherence to IECs. Confocal micrographs demonstrate that C. difficile spores associate with accessible E-cadherin; spore-E-cadherin association increases upon TcdA and TcdB intoxication. The presence of anti-E-cadherin antibodies decreased spore adherence and entry into IECs. By enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and immunogold labeling, we observed that E-cadherin binds to C. difficile spores, specifically to the hairlike projections of the spore, reducing spore adherence to IECs. Overall, these results expand our knowledge of how C. difficile spores bind to IECs by providing evidence that E-cadherin acts as a spore adherence receptor to IECs and by revealing how toxin-mediated damage affects spore interactions with IECs.

Indexación Scopus Background: Fidaxomicin has novel pharmacologic effects on C. difficile spore formation including outgrowth inhibition and persistent spore attachment. However, the mechanism of fidaxomicin attachment on spores has not undergone rigorous microscopic studies. Materials & methods: Fidaxomicin attachment to C. difficile spores of three distinct ribotypes and C. difficile mutant spores with inactivation of exosporium or spore-coat protein-coding genes were visualized using confocal microscopy with a fidaxomicin-bodipy compound (green fluorescence). The pharmacologic effect of the fidaxomicin-bodipy compound was determined. Confocal microscopy experiments included direct effect on C. difficile wild-type and mutant spores, effect of exosporium removal, and direct attachment to a comparator spore forming organism, Bacillus subtilis. Results: The fidaxomicin-bodipy compound MIC was 1 mg/L compared to 0.06 mg/L for unlabeled fidaxomicin, a 16-fold increase. Using confocal microscopy, the intracellular localization of fidaxomicin into vegetative C. difficile cells was observed consistent with its RNA polymerase mechanism of action and inhibited spore outgrowth. The fidaxomicin-bodipy compound was visualized outside of the core of C. difficile spores with no co-localization with the membrane staining dye FM4-64. Exosporium removal reduced fidaxomicin-bodipy association with C. difficile spores. Reduced fidaxomicin-bodipy was observed in C. difficile mutant spores for the spore surface proteins CdeC and CotE. Conclusion: This study visualized a direct attachment of fidaxomicin to C. difficile spores that was diminished with mutants of specific exosporium and spore coat proteins. These data provide advanced insight regarding the anti-spore properties of fidaxomicin. © 2021 The Authors https://www-sciencedirect-com.recursosbiblioteca.unab.cl/science/article/pii/S1075996421000354?via%3Dihub