The role of toxins in Clostridium difficile infection pathogenesis

毒素在艰难梭菌感染发病机制中的作用

• 批准号: 10412917
• 负责人: Dana Borden Lacy
• 金额: --
• 依托单位: VETERANS HEALTH ADMINISTRATION
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-10-01 至 2023-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10412917
• 关键词: ADP Ribose Transferases; ADP ribosylation; Actins; Adherence; Affinity; Alpaca; Anaerobic Bacteria; Antibiotic Therapy; Antibiotics; Antigens; Binding; Biological Assay; Bone Marrow; Caring; Cell physiology; Cells; Clinical; Clostridium difficile; Colon; Complex; Cryoelectron Microscopy; Dendritic Cells; Diarrhea; Disease; Elements; Endocytosis; Endosomes; Enzymes; Epithelial; Epithelial Cells; Epitopes; Family; Flow Cytometry; Foundations; G Actin; Genetic Transcription; Goals; Immunize; Incidence; Infection; Inflammasome; Intoxication; Knowledge; Left; Libraries; Life; Lipolysis; Lipoprotein Receptor; Mediating; Microtubules; Modeling; Morbidity - disease rate; Mus; Mutagenesis; Mutate; Mutation; Nosocomial Infections; Pathogenesis; Pathology; Perforation; Phage Display; Play; Polysaccharides; Prevalence; Process; Proteins; Pseudomembranous Colitis; Reagent; Recurrence; Relapse; Reproduction spores; Role; Sepsis; Severities; Source; Structure; Symptoms; TLR2 gene; Testing; Toxic Megacolon; Toxin; Transferase; United States; VDAC1 gene; Vaccine Antigen; Veterans; Veterans Health Administration; Virulence Factors; alpha Toxin; eosinophil; host microbiome; military veteran; mortality; mutant; nanobodies; receptor; response; tool; treatment strategy; vaccine development; vaccine trial

Project Summary Clostridioides difficile is a gram-positive, spore-forming anaerobe, a leading cause of nosocomial infection in the United States, and a significant problem for Veterans receiving care in Veterans Health Administration (VHA) facilities. The disease state is most often preceded by disruption of the host microbiome in response to antibiotic treatment and is characterized by mild to severe (and often recurrent) diarrhea. Left untreated, C. difficile infection (CDI) can be life threatening with sequelae that include antibiotic-associated pseudomembranous colitis, toxic megacolon, and sepsis. CDI is dependent on the secretion of one or more AB-type toxins: toxin A (TcdA), toxin B (TcdB), and the C. difficile transferase toxin (CDT, or binary toxin). While TcdA and TcdB are considered the primary virulence factors, multiple studies suggest that CDT increases the severity of CDI. The increasing prevalence of CDT containing clinical isolates and the potential for developing CDT as an effective vaccine antigen necessitate a deeper understanding of the CDT mechanism of action. CDT belongs to the Iota family of binary toxins and consists of two proteins: an ADP-ribosyltransferase (CdtA) and a cell binding and pore-forming protein (CdtB). CdtB engages host cells by binding the lipolysis stimulated lipoprotein receptor (LSR). Proteolytic cleavage promotes CdtB oligomerization into a prepore which allows for CdtA binding. The CdtA-CdtB prepore complex is internalized by endocytosis, and endosome acidification leads to CdtB pore formation and CdtA translocation into the cell. CdtA-mediated disruption of actin cytoskeletal structure has been associated with the formation of microtubule protrusions that could play a role in C. difficile adherence to the epithelium. CDT can also synergize with TcdA/TcdB to promote inflammasome activation and suppression of an otherwise protective eosinophil response. We have obtained structures of the CdtB prepore and CdtB pore by cryo-electron microscopy. We propose to define the structures of CdtB bound to CdtA and the LSR receptor (Aim 1), and to dissect the structural features involved in epithelial cell intoxication (Aim 1) and inflammasome priming (Aim 2) using structure-guided mutagenesis and functional studies. In parallel, we will generate a panel of CdtB specific nanobodies that can be used to define the important epitopes needed for high affinity binding and neutralization (Aim 3). The over-arching goal is to provide the mechanistic foundation for understanding the role of CDT function in pathogenesis and the tools to advance this knowledge into effective vaccine development strategies.

项目概要 艰难梭菌是一种革兰氏阳性、产芽孢厌氧菌，是医院内感染的主要原因 美国的感染，以及在退伍军人健康中心接受护理的退伍军人面临的一个重大问题 管理 (VHA) 设施。疾病状态通常先于宿主微生物组的破坏 对抗生素治疗的反应，其特征是轻度至重度（且经常复发）腹泻。左边 如果不进行治疗，艰难梭菌感染 (CDI) 可能会危及生命并产生后遗症，包括抗生素相关的后遗症 伪膜性结肠炎、中毒性巨结肠和败血症。 CDI 依赖于一种或多种 AB 型毒素的分泌：毒素 A (TcdA)、毒素 B (TcdB) 和 艰难梭菌转移酶毒素（CDT，或二元毒素）。虽然 TcdA 和 TcdB 被认为是主要毒力 多项研究表明 CDT 会增加 CDI 的严重程度。 CDT 的患病率不断增加 包含临床分离株和开发 CDT 作为有效疫苗抗原的潜力需要 加深对CDT作用机制的了解。 CDT 属于二元毒素 Iota 家族，由两种蛋白质组成：ADP-核糖基转移酶 (CdtA) 和细胞结合和成孔蛋白 (CdtB)。 CdtB 通过结合脂肪分解作用与宿主细胞结合 刺激脂蛋白受体（LSR）。蛋白水解促进 CdtB 寡聚化成前孔， 允许 CdtA 结合。 CdtA-CdtB 前孔复合物通过内吞作用内化，内体 酸化导致 CdtB 孔形成和 CdtA 易位到细胞中。 CdtA 介导的肌动蛋白破坏 细胞骨架结构与微管突起的形成有关，微管突起可能在 艰难梭菌粘附于上皮。 CDT还可以与TcdA/TcdB协同促进炎症小体 激活和抑制其他保护性嗜酸性粒细胞反应。 我们通过冷冻电子显微镜获得了 CdtB 预孔和 CdtB 孔的结构。我们 提议定义与 CdtA 和 LSR 受体结合的 CdtB 的结构（目标 1），并剖析 涉及上皮细胞中毒（目标 1）和炎性体启动（目标 2）的结构特征 结构引导诱变和功能研究。同时，我们将生成一个 CdtB 特定面板 可用于定义高亲和力结合和中和所需的重要表位的纳米抗体 （目标 3）。总体目标是为理解 CDT 的作用提供机制基础 发病机制中的功能以及将这些知识推进有效疫苗开发策略的工具。