艰难梭菌感染(CDI)是抗生素相关肠道疾病的主要原因,导致严重的腹泻和致命的伪膜性结肠炎。TcdB,这种细菌分泌的必需毒力因子之一,诱导宿主细胞凋亡通过一个知之甚少的机制。这里,我们进行了针对Caco-2细胞定制的RNA干扰(RNAi)筛选,肠上皮的细胞系模型,发现参与TcdB诱导细胞凋亡的宿主因子。我们鉴定了血红蛋白,也称为结斑血红蛋白(JUP)或γ-catenin,catenin家族的一员,作为一种新的宿主因子和先前已知的细胞死亡相关染色质因子,高迁移率组盒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从粘附连接重新分布到线粒体,并与抗凋亡因子Bcl-XL共定位。JUP蛋白可以渗透线粒体膜,导致细胞色素c的释放。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。用甘草酸治疗,HMGB1抑制剂,在培养的细胞和小鼠结扎的结肠环模型中,对TcdB诱导的上皮损伤的抵抗力显着增加。这些发现证明了JUP和HMGB1在TcdB诱导的上皮细胞凋亡中的关键作用。重要性艰难梭菌感染(CDI)是医院获得性腹泻的主要原因。毒素,尤其是TcdB,引起上皮细胞凋亡,但是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制造的小干扰(siRNA)文库进行聚焦凋亡的RNAi筛选,我们发现了一个新的宿主因素,血红蛋白(γ-连环蛋白),作为TcdB诱导细胞凋亡所需的关键因素。在TcdB刺激后,斑红蛋白靶向并渗透线粒体,证明了该连环蛋白家族成员在肠上皮细胞凋亡中的作用。我们还发现了一个以前已知的细胞死亡相关染色质因子,探讨了HMGB1对CDI体内治疗的抑制作用。
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-XL.
JUP proteins could permeabilize the mitochondrial membrane, resulting in the release of cytochrome c. 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. IMPORTANCE 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 in vivo.