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传播的病原体，可导致人类严重的炎症性腹泻。空肠梭菌是NIAID的B类优先病原体，部分原因是这种病原体有可能被用作生物战剂，污染大地理区域的食物和水供应。了解空肠梭菌的基本生物学机制是非常重要的，这可能会导致新的策略来避免将这种细菌用作生物战剂。然而，空肠梭菌在宿主中定植和致病的致病机制尚不清楚。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建议，用于研究空肠梭菌分泌的唯一已知外毒素。我们将研究空肠梭菌细胞致死膨胀毒素（CDT）的细胞机制，CDT可导致哺乳动物细胞周期阻滞和最终死亡，并被认为有助于重塑体内环境，促进肠道定植。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的特定质膜受体的假设，这是细胞中毒过程中必不可少的早期步骤。在这个应用中，我们提出在分子水平上研究CDT与靶细胞的相互作用的实验。在Specific Aim 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成分以及cdt受体相互作用的亲和力。此外，我们将建立许多受体如何存在于每个哺乳动物细胞。此外，由于CDT是由三个离散亚基（CdtA， CdtB和CdtC）组成的三方毒素，我们将建立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。这些实验将为今后确定CDT受体识别和结合的分子基础的实验建立框架。在特异性目标2中，我们将鉴定和表征抗CDT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对CDT敏感性的丧失。在第二阶段，敏感性的丧失将是特征，目标是鉴定缺乏与CDT结合的细胞系。这些实验将对未来的工作至关重要，以补充细胞结合缺陷，这将确定假定的CDT受体。本研究结果将为CDT细胞中毒的机制提供重要信息，并为今后开发阻断CDT作用的策略作为减轻空肠梭菌发病机制的方法提供基础。

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.