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: 10516088
• 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/10516088
• 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; Clostridium difficile; Colon; Complex; Cryoelectron Microscopy; Cytoskeleton; Dendritic Cells; Diarrhea; Disease; Elements; Endocytosis; Endosomes; Enzymes; Epithelial Cells; Epithelium; 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; NF-kappa B; Nosocomial Infections; Pathogenesis; Pathology; Perforation; Phage Display; 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; eosinophil; host microbiome; military veteran; mortality; mutant; nanobodies; receptor; response; synergism; 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.

项目摘要 梭状芽胞杆菌艰难梭菌是一种革兰氏阳性，形成孢子的厌氧菌，是医院的主要原因 美国的感染，这是退伍军人卫生护理的重大问题 管理（VHA）设施。疾病状态通常是在宿主微生物组中断之前 对抗生素治疗的反应，其特征是轻度至重度（并且经常复发）腹泻。左边 未经治疗的艰难梭菌感染（CDI）可能会因续集威胁，包括抗生素相关 假膜性结肠炎，有毒的巨型巨麦和败血症。 CDI取决于一种或多种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）。而TCDA和TCDB被认为是主要的毒力 因素，多项研究表明CDT增加了CDI的严重程度。 CDT的患病率增加 含有临床分离株和开发CDT作为有效疫苗抗原的潜力是 对CDT作用机理的更深入了解。 CDT属于二进制毒素的IOTA家族，由两种蛋白质组成：ADP-核糖基转移酶 （CDTA）和细胞结合和孔形成蛋白（CDTB）。 CDTB通过结合脂解会使宿主细胞接合 刺激的脂蛋白受体（LSR）。蛋白水解裂解将CDTB的寡聚化促进到一个预矿中 允许CDTA结合。 CDTA-CDTB预倍体复合物通过内吞作用和内体内在化。 酸化导致CDTB孔的形成，CDTA转移到细胞中。 CDTA介导的肌动蛋白的破坏 细胞骨架结构与可能在 艰难梭菌遵守上皮。 CDT还可以与TCDA/TCDB协同促进炎症体 激活和抑制原本保护性的嗜酸性粒细胞反应。 我们通过冷冻电子显微镜获得了CDTB预球和CDTB孔的结构。我们 建议定义与CDTA和LSR受体结合的CDTB的结构（AIM 1），并剖析 使用上皮细胞中毒（AIM 1）和炎性体启动（AIM 2）的结构特征使用 结构引导的诱变和功能研究。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化 （目标3）。总结的目标是为理解CDT的作用提供机械基础 在发病机理和将这些知识推向有效疫苗开发策略的工具中起作用。