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

在全国范围内，艰难梭菌感染是抗生素最常见的原因- 相关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 结果显示，残疾退伍军人中艰难梭菌感染的发生率惊人地增加。医院感染 控制程序和谨慎选择抗生素治疗是有益的，但复发和药物治疗- 艰难梭菌的耐药感染持续给退伍军人管理局的医疗系统带来负担。在治疗上，粪便微生物区系 移植已显示出有希望的成功，但缺乏特异性和标准化。寻找新的治疗方法 疾病预防的目标或战略，更好地从根本上了解艰难梭菌毒素如何损害 结肠组织，以及人类宿主的反应，是必不可少的。 艰难梭菌的致病菌株产生毒素A(TcdA)和/或毒素B(TcdB)，直接杀死宿主细胞 并在结肠粘膜中引发炎症反应。艰难梭菌仅编码TcdB的临床分离株 足以在艰难梭菌感染患者中产生完整的病理图谱。贝洛昔单抗是 一种针对TcdB的中和抗体和一种用于预防艰难梭菌复发感染的治疗药物。 因此，Tcdb被认为是人类疾病的主要驱动因素，将是这项提案的具体重点。 最近，在识别宿主细胞表面TcdB受体方面取得了重大进展，特别是Nectin- 3、CSPG4和FrizzledWnt受体蛋白家族。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靶向的亚型以及与这些细胞的相互作用如何在发病过程中起作用 艰难梭菌感染的研究还不够深入。新出现的数据表明，Tcdb专门针对茎和祖先 结肠隐窝底部的细胞可能具有延缓上皮重建的作用。弗里兹莱德夫妇 受体家族是调节结肠干细胞功能的关键。此外，Tcdb可激活 表皮生长因子受体(EGFR)，结肠上皮细胞干细胞行为的中介。这项建议 是基于初步数据表明EGFR、ErbB2和ErbB3受体促进TcdB诱导的细胞死亡。 TcdB对ErbB信号通路的影响尚未在体内进行研究，而这一途径对艰难梭菌的影响尚不清楚。 发病机制尚不清楚。 鉴于ErbB信号与结肠干细胞在稳态和损伤修复中的行为相关，它可能 靶向干细胞或祖细胞是TcdB毒力的关键机制。的主要假说 这一建议是TcdB诱导结肠干/祖细胞中ErbB信号转导的重要途径 艰难梭菌感染发病机制的组成部分。拟议中的实验旨在使用 重组TcdB和同基因艰难梭菌孢子与体内小鼠模型和体外人类结肠癌 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突变型TcdB毒素改变ErbB途径的能力 活体；(2)ErbB抑制TcdB介导的细胞死亡的影响及机制；(3)特定的细胞亚型 在Tcdb介导的结肠损伤过程中受到影响。这项工作的长期目标是揭示 阻断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.