Human intestinal enteroids as a model of Clostridioides difficile-induced enteritis.

Clostridioides difficile is an important nosocomial pathogen that produces toxins to cause life-threatening diarrhea and colitis. Toxins bind to epithelial receptors and promote the collapse of the actin cytoskeleton. C. difficile toxin activity is commonly studied in cancer-derived and immortalized cell lines. However, the biological relevance of these models are limited. Moreover, no model is available for examining C. difficile-induced enteritis, an understudied health problem. We hypothesized human intestinal enteroids (HIEs) express toxin receptors and provide a new model to dissect C. difficile cytotoxicity in the small intestine. We generated biopsy-derived jejunal HIE and Vero cells which stably express LifeAct-Ruby, a fluorescent label of F-actin, to monitor actin cytoskeleton rearrangement by live-cell microscopy. Imaging analysis revealed that toxins from pathogenic C. difficile strains elicited cell rounding in a strain-dependent manner and HIEs were tenfold more sensitive to toxin A (TcdA) than toxin B (TcdB). By qPCR we paradoxically found that HIEs expressed greater quantities of toxin receptor mRNA and yet exhibited decreased sensitivity to toxins when compared to traditionally used cell lines. We reasoned that these differences may be explained by components, such as mucins, that are present in HIEs cultures that are absent in immortalized cell lines. Addition of human-derived MUC2 to Vero cells delayed cell rounding, indicating that mucus serves as a barrier to toxin-receptor binding. This work highlights that investigation of C. difficile infection in HIEs can provide important insights into the intricate interactions between toxins and the human intestinal epithelium.