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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 is limited. Moreover, no model is available for examining C. difficile-induced enteritis, an understudied health problem. We hypothesized that 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 quantitative PCR, we paradoxically found that HIEs expressed greater quantities of toxin receptor mRNA and yet exhibited decreased sensitivity to toxins when compared with 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 mucin 2 (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 that HIEs can provide important insights into the intricate interactions between toxins and the human intestinal epithelium.<\/jats:p><jats:p>NEW &amp; NOTEWORTHY In this article, we developed a novel model of Clostridioides difficile-induced enteritis using jejunal-derived human intestinal enteroids (HIEs) transduced with fluorescently tagged F-actin. Using live-imaging, we identified that jejunal HIEs express high levels of TcdA and CDT receptors, are more sensitive to TcdA than TcdB, and secrete mucus, which delays toxin-epithelial interactions. This work also optimizes optically clear C. difficile-conditioned media suitable for live-cell imaging.<\/jats:p>","DOI":"10.1152\/ajpgi.00045.2020","type":"journal-article","created":{"date-parts":[[2020,3,30]],"date-time":"2020-03-30T07:03:43Z","timestamp":1585551823000},"page":"G870-G888","source":"Crossref","is-referenced-by-count":37,"title":["Human intestinal enteroids as a model of<i>Clostridioides difficile<\/i>-induced enteritis"],"prefix":"10.1152","volume":"318","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9742-9932","authenticated-orcid":false,"given":"Melinda A.","family":"Engevik","sequence":"first","affiliation":[{"name":"Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas"},{"name":"Department of Pathology, Texas Children\u2019s Hospital, Houston, Texas"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Heather A.","family":"Danhof","sequence":"additional","affiliation":[{"name":"Alkek Center for Metagenomic and 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