泄漏的人类结肠模型揭示了在早期梭状芽孢杆菌毒素暴露的早期梭状芽胞杆菌毒素暴露中未偶联的顶端/基底细胞毒性

梭状芽胞杆菌艰难梭菌（艰难梭菌）毒素A（TCDA）和B（TCDB）部分通过破坏上皮屏障功能而导致抗生素相关的结肠炎。准确的体外模型对于检测早期毒性动力学，研究病因学和开发新疗法的临床前模型是必需的。癌细胞系和类器官的特性固有地限制了这些努力。我们开发了具有屏障功能的分化人类结肠上皮（HCE）的成年干细胞衍生的单层，研究了毒素对单层顶端/基础方面的影响，并评估了上皮屏障是否增强毒性。单细胞RNA-sequencing（SCRNASEQ）将与艰难梭菌相关的基因映射到人类谱系。转录组学比较HCE与CACO-2，知情的体外干细胞分化时机，并揭示了对TCDA的转录反应。旋转电阻（TEER）和荧光通透性测定测量的细胞毒性。 TCDB毒性的贡献在双氯芬酸诱导的泄漏肠模型中进行了评估。 Scrnaseq表现出广泛而可变的毒素受体表达。体内的吸收性结肠细胞显示出增加的毒素受体，RHO GTPase和细胞连接基因表达。晚期TCDA毒性通常会降低细胞因子/趋化因子，并增加紧密连接和死亡受体基因。分化的CACO-2细胞保持不成熟，而HCE单层与体内成熟的结肠细胞相似。与顶型暴露相比，TCDA/B的基础暴露会导致更大的毒性和凋亡。双氯芬酸增强了对毒素的顶端暴露。根尖/基底毒性与更快的发作和幅度后毒素暴露量增加。泄漏的连接可以增强顶端TCDB暴露的毒性。 HCE单层代表了一种与生理相关和敏感的系统，以评估微生物毒素对肠道上皮的影响。

Clostridioides difficile (C. difficile) toxins A (TcdA) and B (TcdB) cause antibiotic-associated colitis in part by disrupting epithelial barrier function. Accurate in vitro models are necessary to detect early toxicity kinetics, investigate disease etiology, and develop preclinical models for new therapies. Properties of cancer cell lines and organoids inherently limit these efforts. We developed adult stem cell-derived monolayers of differentiated human colonic epithelium (hCE) with barrier function, investigated the impact of toxins on apical/basal aspects of monolayers, and evaluated whether a leaky epithelial barrier enhances toxicity. Single-cell RNA-sequencing (scRNAseq) mapped C. difficile-relevant genes to human lineages. Transcriptomics compared hCE to Caco-2, informed timing of in vitro stem cell differentiation, and revealed transcriptional responses to TcdA. Transepithelial electrical resistance (TEER) and fluorescent permeability assays measured cytotoxicity. Contribution of TcdB toxicity was evaluated in a diclofenac-induced leaky gut model. scRNAseq demonstrated broad and variable toxin receptor expression. Absorptive colonocytes in vivo displayed increased toxin receptor, Rho GTPase, and cell junction gene expression. Advanced TcdA toxicity generally decreased cytokine/chemokine and increased tight junction and death receptor genes. Differentiated Caco-2 cells remained immature whereas hCE monolayers were similar to mature colonocytes in vivo. Basal exposure of TcdA/B caused greater toxicity and apoptosis than apical exposure. Apical exposure to toxins was enhanced by diclofenac. Apical/basal toxicities are uncoupled with more rapid onset and increased magnitude postbasal toxin exposure. Leaky junctions enhance toxicity of apical TcdB exposure. hCE monolayers represent a physiologically relevant and sensitive system to evaluate the impact of microbial toxins on gut epithelium.NEW & NOTEWORTHY 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.