C. jejuni Cytolethal Distending Toxin Cell Interactions

描述（由申请方提供）：空肠弯曲菌是一种食源性和水源性病原体，可引起人类严重炎症性腹泻。C.空肠是NIAID B类优先病原体，部分原因是该病原体有可能用作生物战剂，在大的地理区域内污染食物和水供应。了解C.空肠的发病机制，这可能会导致新的战略，规避使用这种细菌作为生物战剂。然而，C.空肠杆菌在宿主体内定殖并引起疾病的机制还知之甚少。这项应用是休斯顿大学和加州大学洛杉矶分校的研究人员合作提出的，目的是研究唯一已知的由C。空肠。我们将探讨C.空肠细胞致死性膨胀毒素（CDT），其引起细胞周期停滞和中毒哺乳动物细胞的最终死亡，并且已经提出帮助重塑体内环境以促进肠道的定殖。在这个R21应用中，我们将开始探索CDT识别并结合敏感细胞表面上的特异性质膜受体作为细胞中毒过程中必不可少的早期步骤的假设。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，休斯顿大学的研究人员将描述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组分以及CDT-受体相互作用的亲和力。此外，我们将建立许多受体是如何存在于每个哺乳动物细胞。此外，由于CDT是包含三个离散亚基（CdtA、CdtB和CdtC）的三方毒素，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。这些实验将是重要的建立框架，为未来的实验，以确定CDT受体的歧视和结合的分子基础。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1阶段，将对细胞系进行诱变并筛选对CDT的敏感性丧失。在第2阶段，将表征灵敏度的丧失，目标是鉴定与CDT结合缺陷的细胞系。这些实验将是至关重要的未来工作，以补充细胞结合缺陷，这将确定假定的CDT受体。本研究的结果将提供有关CDT细胞中毒机制的重要信息，并将为未来开发阻断CDT作用的策略提供基础。空肠发病机制

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.