Division of Natural and Applied Sciences, Duke Kunshan Universitygrid.448631.c, Kunshan, Jiangsu, China.
Department of Biomedical Sciences, Jockey Club College of Veterinary Medicine and Life Sciences, City University of Hong Konggrid.35030.35, Hong Kong SAR, China.
mBio. 2022 Oct 26;13(5):e0184922. doi: 10.1128/mbio.01849-22. Epub 2022 Aug 31.
Clostridioides difficile infection (CDI) is the leading cause of antibiotic-associated intestinal disease, resulting in severe diarrhea and fatal pseudomembranous colitis. TcdB, one of the essential virulence factors secreted by this bacterium, induces host cell apoptosis through a poorly understood mechanism. Here, we performed an RNA interference (RNAi) screen customized to Caco-2 cells, a cell line model of the intestinal epithelium, to discover host factors involved in TcdB-induced apoptosis. We identified plakoglobin, also known as junction plakoglobin (JUP) or γ-catenin, a member of the catenin family, as a novel host factor and a previously known cell death-related chromatin factor, high-mobility group box 1 (HMGB1). Disruption of those host factors by RNAi and CRISPR resulted in resistance of cells to TcdB-mediated and mitochondrion-dependent apoptosis. JUP was redistributed from adherens junctions to the mitochondria and colocalized with the antiapoptotic factor Bcl-X. JUP proteins could permeabilize the mitochondrial membrane, resulting in the release of cytochrome . Our results reveal a novel role of JUP in targeting the mitochondria to promote the mitochondrial apoptotic pathway. Treatment with glycyrrhizin, an HMGB1 inhibitor, resulted in significantly increased resistance to TcdB-induced epithelial damage in cultured cells and a mouse ligated colon loop model. These findings demonstrate the critical roles of JUP and HMGB1 in TcdB-induced epithelial cell apoptosis. Clostridioides difficile infection (CDI) is the leading cause of hospital-acquired diarrhea. Toxins, especially TcdB, cause epithelial cell apoptosis, but the underlying cell death mechanism is less clear. Through an apoptosis-focused RNAi screen using a bacterium-made small interfering (siRNA) library customized to a human colonic epithelial cell model, we found a novel host factor, plakoglobin (γ-catenin), as a key factor required for cell apoptosis induced by TcdB. Plakoglobin targets and permeabilizes mitochondria after stimulation by TcdB, demonstrating a hitherto underappreciated role of this catenin family member in the apoptosis of intestinal epithelial cells. We also found a previously known cell death-related chromatin factor, HMGB1, and explored the inhibition of HMGB1 for CDI therapy .
艰难梭菌感染（CDI）是抗生素相关性肠道疾病的主要原因，可导致严重腹泻和致命的伪膜性结肠炎。TcdB 是该细菌分泌的一种重要毒力因子，通过一种尚未完全了解的机制诱导宿主细胞凋亡。在这里，我们针对肠道上皮细胞模型 Caco-2 细胞进行了 RNA 干扰（RNAi）筛选，以发现参与 TcdB 诱导凋亡的宿主因子。我们鉴定出桥粒斑蛋白（也称为连接斑蛋白（JUP）或γ-连环蛋白）作为一种新型宿主因子和先前已知的细胞死亡相关染色质因子高迁移率族蛋白 1（HMGB1）。RNAi 和 CRISPR 对这些宿主因子的破坏导致细胞对 TcdB 介导的和线粒体依赖性凋亡的抗性。JUP 从黏附连接重新分布到线粒体，并与抗凋亡因子 Bcl-X 共定位。JUP 蛋白可以使线粒体膜通透，导致细胞色素 c 释放。我们的结果揭示了 JUP 在靶向线粒体以促进线粒体凋亡途径中的新作用。用 HMGB1 抑制剂甘草酸处理可显著增加培养细胞和结扎结肠环模型中 TcdB 诱导的上皮损伤的抗性。这些发现表明 JUP 和 HMGB1 在 TcdB 诱导的上皮细胞凋亡中起关键作用。艰难梭菌感染（CDI）是医院获得性腹泻的主要原因。毒素，特别是 TcdB，导致上皮细胞凋亡，但潜在的细胞死亡机制尚不清楚。通过使用针对人类结肠上皮细胞模型定制的细菌小干扰（siRNA）文库进行以凋亡为重点的 RNAi 筛选，我们发现了一种新型宿主因子桥粒斑蛋白（γ-连环蛋白），它是 TcdB 诱导细胞凋亡所必需的关键因子。TcdB 刺激后，桥粒斑蛋白靶向并使线粒体通透，证明了这种连接蛋白家族成员在肠上皮细胞凋亡中的作用尚未得到充分认识。我们还发现了先前已知的细胞死亡相关染色质因子 HMGB1，并探讨了抑制 HMGB1 治疗 CDI。
