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， 显示出C.残疾退伍军人的艰难感染。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- 抗性C.艰难感染持续加重VA医疗保健系统的负担。从治疗学上讲， 移植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 疾病预防的目标或策略，更好地了解C。艰难梭菌毒素损伤 结肠组织以及人类宿主的反应是至关重要的。 病原菌株C.艰难梭菌产生毒素A（TcdA）和/或毒素B（TcdB），其直接杀死宿主细胞 并在结肠粘膜中诱导炎症反应。临床分离的C.单独编码TcdB的艰难梭菌 足以在C.艰难感染Bezlotoxumab是 抗TcdB的中和抗体和用于预防复发性C.艰难感染 因此，TcdB被认为是人类疾病的主要驱动因素，并将成为本提案的具体重点。 最近，在鉴定TcdB的宿主细胞表面受体，特别是Nectin-1方面取得了重大进展。 3、CSPG 4和Wnt受体蛋白的Frizzled家族。这些受体在人结肠组织中的表达 组织和它们各自对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病程中促成发病机制 梭艰难梭菌感染研究不足。新出现的数据表明TcdB特异性靶向干细胞和祖细胞 结肠隐窝底部的细胞，可能具有延迟上皮重建的作用。的Frizzled 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导者。这项建议 基于初步数据，表明EGFR、ErbB 2和ErbB 3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C.艰难 发病机制尚不清楚。 考虑到ErbB信号与结肠干细胞稳态和损伤修复行为的相关性， 靶向干细胞或祖细胞是TcdB毒力的关键机制。的主要假设 该建议是TcdB诱导结肠干/祖细胞中的ErbB信号传导作为重要的 C.艰难感染拟议的实验旨在使用 重组TcdB和同基因C.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异性突变TcdB毒素改变ErbB途径的能力 ErbB抑制剂对TcdB介导的细胞死亡的影响及其机制; 在TcdB介导的结肠损伤中受到影响。这项工作的长期目标是揭示目标， 阻断C.艰难感染

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.