The role of toxins in Clostridium difficile infection pathogenesis

项目摘要 艰难梭菌是一种革兰氏阳性，孢子形成厌氧菌，是导致医院感染的主要原因。 感染在美国，和一个重大问题，退伍军人接受照顾退伍军人健康 管理（VHA）设施。疾病状态最常见的是宿主微生物组的破坏， 对抗生素治疗有反应，其特征为轻度至重度（且经常复发）腹泻。左 未处理的C.艰难梭菌感染（CDI）可能危及生命，并伴有后遗症，包括与糖尿病相关的 假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）和毒素A（TcdA）。 C.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的发病率不断增加 含有临床分离株和开发CDT作为有效疫苗抗原的潜力， 更深入地了解CDT的作用机制。 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）和细胞结合和孔形成蛋白（CdtB）。CdtB通过结合脂解作用与宿主细胞结合， 刺激脂蛋白受体（LSR）。蛋白水解切割促进CdtB寡聚化进入前孔， 允许CdtA结合。CdtA-CdtB前孔复合物通过内吞作用内化， 酸化导致CdtB孔形成和CdtA易位进入细胞。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， C.难以粘附于上皮。CDT还可协同TcdA/TcdB促进炎性小体 激活和抑制其它保护性嗜酸性粒细胞反应。 我们已经获得了CdtB前孔和CdtB孔的结构，通过冷冻电子显微镜。我们 提出定义与CdtA和LSR受体结合的CdtB的结构（Aim 1），并剖析 上皮细胞中毒（Aim 1）和炎性小体引发（Aim 2）的结构特征， 结构导向诱变和功能研究。同时，我们将生成一组CdtB特异性 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 (Aim 3）。本文的总体目标是为理解CDT的作用提供机制基础 在发病机制中的作用以及将这些知识转化为有效疫苗开发策略的工具。

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.