C. jejuni Cytolethal Distending Toxin Cell Interactions

描述（由申请人提供）：空肠弯曲杆菌是一种食物和水源性病原体，可导致人类严重炎症性腹泻。空肠弯曲菌被列为 NIAID B 类优先病原体，部分原因是该病原体有可能被用作生物战剂，污染大片地理区域的食品和水供应。了解空肠弯曲菌发病机制的基本生物学非常重要，这可能会产生新的策略来规避这种细菌作为生物战剂的使用。然而，人们对空肠弯曲菌在宿主体内定殖并引起疾病的致病机制知之甚少。该应用程序是休斯顿大学和加州大学洛杉矶分校研究人员之间的合作提案，旨在研究空肠弯曲菌分泌的唯一已知外毒素。我们将研究空肠弯曲菌细胞致死膨胀毒素（CDT）的细胞机制，该毒素会导致细胞周期停滞并最终导致中毒哺乳动物细胞死亡，并已被提议帮助重塑体内环境以促进肠道定植。在此 R21 应用中，我们将开始探索这样的假设：CDT 区分并结合敏感细胞表面上的特定质膜受体，作为细胞中毒过程中重要的早期步骤。在此应用中，我们提出了在分子水平上研究 CDT 与靶细胞相互作用的实验。在具体目标 1 中，休斯顿大学研究人员将描述 CDT 与敏感哺乳动物细胞系的相互作用。我们将确定特异性和非特异性成分以及 CDT 受体相互作用的亲和力。此外，我们将确定每个哺乳动物细胞中受体的存在方式。此外，由于 CDT 是一种三部分毒素，由三个离散的亚基（CdtA、CdtB 和 CdtC）组成，因此我们将确定每个亚基对毒素结合的贡献。最后，我们将开始描述 CDT 受体的性质。这些实验对于建立未来实验的框架非常重要，以确定 CDT 受体区分和结合的分子基础。在具体目标 2 中，我们将鉴定和表征对 CDT 具有抗性的突变细胞系。加州大学洛杉矶分校的研究人员将使用涉及两个基本阶段的遗传方法。在第一阶段，细胞系将被诱变并筛选是否丧失对 CDT 的敏感性。在第 2 阶段，将表征敏感性丧失，目标是识别与 CDT 结合缺陷的细胞系。这些实验对于未来补充细胞结合缺陷的工作至关重要，这将鉴定假定的 CDT 受体。这项研究的结果将提供有关 CDT 细胞中毒机制的重要信息，并将为未来制定阻断 CDT 作用的策略作为减轻空肠弯曲菌发病机制的方法奠定基础。

DESCRIPTION (provided by applicant): Campylobacter jejuni is a food and water borne pathogen responsible for severe inflammatory diarrhea in humans. C. jejuni is a NIAID Category B Priority Pathogen because, in part, of the potential of this pathogen to be used as a biowarfare agent to contaminate food- and water supplies over large geographical areas. It is important to understand the basic biology of C. jejuni pathogenesis, which may lead to novel strategies for circumventing the use of this bacterium as a biowarfare agent. However, the pathogenic mechanisms used by C. jejuni to colonize and cause disease in the host are poorly understood. This application is a collaborative proposal between investigators at the University of Houston and UCLA to study the only known exotoxin secreted by C. jejuni. We will investigate the cellular mechanisms of the C. jejuni cytolethal distending toxin (CDT), which causes cell cycle arrest and eventual death of intoxicated mammalian cells, and has been proposed to assist in remodeling the in vivo environment to facilitate colonization of the intestinal tract. In this R21 application, we will begin to explore the hypothesis that CDT discriminates for and binds to a specific plasma membrane receptor on the surface of sensitive cells as an essential early step during cellular intoxication. In this application, we propose experiments for investigating at the molecular level the interactions of CDT with target cells. In Specific Aim 1, University of Houston researchers will characterize the interactions of CDT with sensitive mammalian cell lines. We will determine the specific and non-specific components as well as the affinity of CDT-receptor interactions. In addition, we will establish many how receptors are present per mammalian cell. Moreover, because CDT is a tri-partite toxin comprising three discrete subunits (CdtA, CdtB, and CdtC), we will establish the contribution of each subunit to binding of the toxin. Finally, we will begin to characterize the nature of the CDT receptor. These experiments will be important for establishing the framework for future experiments to identify the molecular basis for CDT receptor discrimination and binding. In Specific Aim 2, we will identify and characterize mutant cell lines that are resistant to CDT. UCLA researchers will use a genetic approach involving two fundamental phases. In phase 1, cell lines will be mutagenized and screened for a loss of sensitivity to CDT. In phase 2, the loss of sensitivity will be characterized, with the goal being the identification of a cell line that is deficient in binding to CDT. These experiments will be crucial for future work to complement the cell-binding defect, which will identify the putative CDT receptor. Results from this research will provide important information about the mechanism of CDT cellular intoxication, and will provide the basis for future work to develop strategies for blocking the action of CDT as an approach for attenuating C. jejuni pathogenesis.