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

胃肠细胞类型特异性信号传导和艰难梭菌毒素发病机制

• 批准号: 10513320
• 负责人: Nicholas O Markham
• 金额: --
• 依托单位: VETERANS HEALTH ADMINISTRATION
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-10-01 至 2026-09-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10513320
• 关键词: Address; Affect; Antibiotic Therapy; Antibodies; Bacterial Infections; CSPG4 gene; Cell Death; Cell Death Induction; Cell Surface Receptors; Cells; Cessation of life; Clinical; Clostridium; Clostridium difficile; Colon; Colon Injury; Communities; Data; Disabled Persons; Disease; Drug resistance; ERBB2 gene; Epidermal Growth Factor Receptor; Epithelial Cells; Epithelium; Exposure to; Family; Genes; Genetic Transcription; Goals; Growth Factor Receptors; Healthcare Systems; Homeostasis; Hospitals; Human; Human Cell Line; Immunofluorescence Immunologic; In Situ; In Vitro; Incidence; Infection; Infection Control; Inflammatory Response; Knowledge; Ligands; Link; Measures; Mediating; Mediator; Microbiology; Modeling; Molecular; Mucous Membrane; Mus; Nosocomial Infections; Oncogenes; Oral; Pathogenesis; Pathogenicity; Pathology; Pathway interactions; Patients; Pharmaceutical Preparations; Phosphorylation; Preventive; Receptor Signaling; Recombinants; Recurrence; Reproduction spores; Resolution; Role; Science; Signal Pathway; Signal Transduction; Small Interfering RNA; Specificity; Standardization; Targeted Toxins; Techniques; Testing; Therapeutic; Tissues; Toxin; Transgenic Mice; Translating; Tyrosine Kinase Inhibitor; Tyrosine Phosphorylation; United States; Veterans; Virulence; Work; antibiotic-associated diarrhea; base; beta catenin; cell behavior; cell injury; cell killing; cell type; chronic infection; colonic crypt; disorder prevention; epidemiologic data; epidemiology study; epithelial stem cell; experimental study; fecal transplantation; fluorophore; gastrointestinal; gastrointestinal epithelium; human disease; in vivo; injury and repair; innovation; knock-down; lentivirally transduced; member; mouse model; mutant; nectin-3; neutralizing antibody; new therapeutic target; novel; novel therapeutics; pharmacologic; prevent; programs; protein expression; receptor; receptor binding; receptor expression; reconstitution; response; self-renewal; small hairpin RNA; small molecule inhibitor; stem; stem cell function; stem cells; success; targeted treatment; therapeutic target; three dimensional cell culture; trend

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.

在全国范围 相关的腹泻和最常见的医院获得感染。最近的流行病学研究已经 表明残疾退伍军人的艰难梭菌感染发生率令人震惊。基于医院的感染 控制程序和仔细选择抗生素治疗是有益的，但是经常性和药物 - 抗艰难梭菌感染持续负担VA医疗保健系统。在治疗上，粪便菌群 移植表现出了有希望的成功，但缺乏特异性和标准化。识别新的治疗 预防疾病的目标或策略，对艰难梭菌毒素的损害如何更好地理解 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），它们直接杀死宿主细胞 并在结肠粘膜中诱发炎症反应。单独编码TCDB的艰难梭菌的临床分离株 足以在艰难梭菌感染患者中产生全部病理。 Bezlotoxumab是 针对TCDB的中和抗体和一种用于预防艰难梭菌感染的治疗药物。 因此，TCDB被认为是人类疾病的杰出驱动力，将是该提案的特定重点。 最近，已经取得了重大进展，以鉴定TCDB的宿主细胞表面受体，特别是nectin-- 3，CSPG4和Wnt受体蛋白的毛躁家族。这些受体在人类结肠中的表达 组织及其对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促进病程中的发病机理 艰难梭菌感染的研究被研究了。新兴数据表明TCDB专门针对茎和祖细胞 结肠隐窝底部的细胞可能会延迟上皮重建的作用。卷曲 受体家族对于调节结肠干细胞功能至关重要。另外，TCDB反式激活 表皮生长因子受体（EGFR），是结肠上皮细胞干细胞行为的介体。这个建议 基于表明EGFR，ERBB2和ERBB3受体的初步数据促进了TCDB诱导的细胞死亡。 TCDB对ERBB信号的影响尚未在体内研究，该途径对艰难梭菌的影响 发病机理尚不清楚。 鉴于ERBB信号传导与稳态和损伤修复中的结肠干细胞行为的相关性，它可能 靶向茎或祖细胞是TCDB毒力的关键机制。主要假设 该建议是TCDB在结肠茎/祖细胞中诱导ERBB信号传导是重要的 艰难梭菌感染发病机理的成分。提出的实验旨在使用 重组TCDB和ISEGENIC C.艰难梭菌具有体内小鼠模型和离体人类结构群至 了解TCDB如何与胃肠道上皮细胞中的ERBB受体相互作用。结果将确定： （1）野生型和特异性突变体TCDB毒素缺乏受体结合以改变ERBB途径的能力 体内（2）ERBB抑制对TCDB介导的细胞死亡的影响和机制； （3）特定的细胞亚型 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目标的目标 缠绕艰难梭菌感染的发病机理的治疗策略。