Gastrointestinal cell type-specific signaling and C. difficile toxin pathogenesis

在全国范围内，艰难梭菌（Clostridium difficile）感染是抗生素引起的最常见原因。 相关的腹泻和最常见的医院获得性感染。最近的流行病学研究表明 残疾退伍军人中艰难梭菌感染的发病率惊人增加。医院感染 控制计划和仔细选择抗生素治疗是有益的，但反复发作和药物治疗 耐药性艰难梭菌感染持续给退伍军人管理局医疗系统带来负担。治疗上，粪便微生物群 移植已显示出可喜的成功，但缺乏特异性和标准化。确定新的治疗方法 疾病预防的目标或策略，更好地了解艰难梭菌毒素如何造成损害 结肠组织以及人类宿主的反应至关重要。 艰难梭菌致病菌株产生毒素 A (TcdA) 和/或毒素 B (TcdB)，直接杀死宿主细胞 并诱发结肠粘膜炎症反应。单独编码 TcdB 的艰难梭菌临床分离株 足以产生艰难梭菌感染患者的全谱病理学。贝兹妥单抗是 针对 TcdB 的中和抗体和用于预防复发性艰难梭菌感染的治疗药物。 因此，TcdB 被认为是人类疾病的重要驱动因素，并将成为该提案的具体重点。 最近，在鉴定 TcdB 宿主细胞表面受体（特别是 Nectin）方面取得了重大进展。 3、CSPG4 和 Wnt 受体蛋白 Frizzled 家族。这些受体在人结肠中的表达 组织及其各自对 TcdB 功能的贡献尚不清楚。此外，上皮细胞 TcdB 靶向的亚型以及在病程中与这些细胞的相互作用如何促进发病机制 艰难梭菌感染的机制尚未得到充分研究。新数据表明 TcdB 专门针对茎和祖细胞 结肠隐窝底部的细胞可能具有延迟上皮重建的作用。卷曲的 受体家族对于调节结肠干细胞功能至关重要。此外，TcdB 反式激活 表皮生长因子受体（EGFR），结肠上皮细胞干细胞行为的介质。这个提议 基于初步数据表明 EGFR、ErbB2 和 ErbB3 受体促进 TcdB 诱导的细胞死亡。 TcdB 对 ErbB 信号传导的影响尚未在体内研究，该途径对艰难梭菌的影响 发病机制尚不清楚。 鉴于 ErbB 信号传导与结肠干细胞稳态和损伤修复行为的相关性，它可能 靶向干细胞或祖细胞是 TcdB 毒力的关键机制。主要假设为 该提议是 TcdB 在结肠干/祖细胞中诱导 ErbB 信号传导作为重要的 艰难梭菌感染的发病机制的组成部分。所提出的实验旨在使用 重组 TcdB 和同基因艰难梭菌孢子与体内小鼠模型和离体人类结肠 了解 TcdB 如何与胃肠道上皮细胞中的 ErbB 受体相互作用。结果将决定： (1) 受体结合缺陷的野生型和特定突变型 TcdB 毒素改变 ErbB 途径的能力 体内； (2) ErbB抑制对TcdB介导的细胞死亡的影响及机制； (3)特定细胞亚型 在 TcdB 介导的结肠损伤期间受到影响。这项工作的长期目标是揭示 阻止艰难梭菌感染发病机制的治疗策略。

Nationwide, Clostridioides (formerly Clostridium) difficile infection is the most common cause of antibiotic- associated diarrhea and the most frequent hospital-acquired infection. Recent epidemiological studies have shown an alarming increase in incidence of C. difficile infection in disabled Veterans. Hospital-based infection control programs and careful choice of antibiotic treatments have been beneficial, but recurrent and drug- resistant C. difficile infections persistently burden the VA healthcare system. Therapeutically, fecal microbiota transplant has shown promising success but lacks specificity and standardization. To identify new therapeutic targets or strategies for disease prevention, a better fundamental understanding of how C. difficile toxins damage colonic tissue, and how the human host responds, is essential. Pathogenic strains of C. difficile produce toxin A (TcdA) and/or toxin B (TcdB), which directly kill host cells and induce an inflammatory response in the colonic mucosa. Clinical isolates of C. difficile encoding TcdB alone are sufficient to produce the whole spectrum of pathology in patients with C. difficile infection. Bezlotoxumab is a neutralizing antibody against TcdB and a therapeutic drug for preventing recurrent C. difficile infection. Therefore, TcdB is considered a prominent driver of human disease and will be the specific focus of this proposal. Recently, significant progress has been made to identify host cell surface receptors for TcdB, specifically Nectin- 3, CSPG4, and the Frizzled family of Wnt receptor proteins. The expression of these receptors in human colonic tissue and their respective contribution to TcdB function is not clearly understood. Furthermore, the epithelial cell subtypes targeted by TcdB and how interactions with these cells contribute to pathogenesis during the course of C. difficile infection is understudied. Emerging data suggest TcdB specifically targets the stem and progenitor cells in the base of colonic crypts potentially with the effect of delaying epithelial reconstitution. The Frizzled family of receptors are critical for regulating colonic stem cell function. Additionally, TcdB transactivates the epidermal growth factor receptor (EGFR), a mediator of colonic epithelial cell stem cell behavior. This proposal is based on preliminary data suggesting EGFR, ErbB2, and ErbB3 receptors facilitate TcdB-induced cell death. The effect of TcdB on ErbB signaling has not been studied in vivo, and the impact of this pathway on C. difficile pathogenesis is not known. Given the relevance of ErbB signaling to colonic stem cell behavior in homeostasis and injury repair, it may be that targeting stem or progenitor cells is a critical mechanism of TcdB virulence. The main hypothesis of this proposal is TcdB induces ErbB signaling in colonic stem/progenitor cells as an important component of the pathogenesis of C. difficile infection. The proposed experiments are aimed at using recombinant TcdB and isogenic C. difficile spores with in vivo mouse models and ex vivo human colonoids to understand how TcdB interacts with ErbB receptors in gastrointestinal epithelial cells. The results will determine: (1) the ability of wild type and specific mutant TcdB toxins deficient in receptor binding to alter ErbB pathways in vivo; (2) the impact and mechanism of ErbB inhibition on TcdB-mediated cell death; (3) the specific cell subtypes that are affected during TcdB-mediated colonic injury. The long-term goal of this work is to reveal targets for therapeutic strategies that interdict the pathogenesis of C. difficile infection.