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Abstract Infections by Clostridioides difficile , a bacterium that targets the large intestine (colon), impact a large number of people worldwide. Bacterial colonization is mediated by two exotoxins: toxins A and B. Short peptides that can be delivered to the gut and inhibit the biocatalytic activity of these toxins represent a promising therapeutic strategy to prevent and treat C. diff . infection. We describe an approach that combines a Pep tide B inding D esign (PepBD) algorithm, molecular-level simulations, a rapid screening assay to evaluate peptide:toxin binding, a primary human cell-based assay, and surface plasmon resonance (SPR) measurements to develop peptide inhibitors that block Toxin A in colon epithelial cells. One peptide, SA1, is found to block TcdA toxicity in primary-derived human colon (large intestinal) epithelial cells. SA1 binds TcdA with a K D of 56.1 ± 29.8 nM as measured by surface plasmon resonance (SPR). 

Novel human colonocyte monolayer cultures, benchmarked by transcriptomics for physiological relevance, detect early cytopathic impacts of Clostridioides difficile toxins TcdA and TcdB. A fluorescent ZO-1 reporter in primary human colonocytes is used to track epithelial barrier disruption in response to TcdA. Basal TcdA/B exposure generally caused more rapid onset and cytotoxicity than apical exposure. Transcriptomics demonstrate changes in tight junction, chemokine, and cytokine receptor gene expression post-TcdA exposure. Diclofenac-induced leaky epithelium enhanced apical exposure toxicity.