The role of toxins in Clostridium difficile infection pathogenesis

项目摘要 艰难梭状芽胞杆菌是一种革兰氏阳性、芽胞形成的厌氧菌，是医院感染的主要原因 美国的感染，以及退伍军人接受退伍军人健康护理的一个重大问题 行政(VHA)设施。在疾病状态之前，最常见的是宿主微生物群的破坏。 对抗生素治疗有反应，以轻度至重度(经常是反复发作)腹泻为特征。左边 如果不治疗，艰难梭菌感染(CDI)可能会危及生命，并产生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：A毒素(TcdA)、B毒素(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 多项研究表明，CDT增加了CDI的严重程度。CDT的患病率越来越高 包含临床分离株和开发CDT作为有效疫苗抗原的潜力需要一种 对CDT的作用机制有更深入的了解。 CDT属于IOTA二元毒素家族，由两种蛋白质组成：ADP-核糖基转移酶 (CDtA)和一种细胞结合和孔道形成蛋白(CDtB)。CDtB通过结合脂解作用与宿主细胞接触 刺激的脂蛋白受体(LSR)。蛋白水解性切割促进CdtB齐聚成前孔， 允许CDtA绑定。CdtA-cdtB前孔复合体通过内吞作用和内吞作用内化 酸化导致镉B孔的形成和镉A向细胞内的移位。CDtA对肌动蛋白的干扰作用 细胞骨架结构与微管突起的形成有关，微管突起可能在 艰难梭菌与上皮细胞的粘附性。CDT还可与TcdA/TcdB协同促炎症作用 激活和抑制本来具有保护性的嗜酸性粒细胞反应。 用低温电子显微镜观察到了CdtB的前孔和孔的结构。我们 建议定义与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)和炎性小体启动(目标2)的结构特征 结构导向诱变和功能研究。同时，我们将生成一个特定于CdtB的面板 可用于定义高亲和力结合和中和所需的重要表位的纳米体 (目标3)。总体目标是为理解CDT的作用提供机制基础 在发病机制中的作用以及将这一知识推进到有效的疫苗开发战略中的工具。

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.