C. jejuni Cytolethal Distending Toxin Cell Interactions

空肠弯曲菌细胞致死膨胀毒素细胞相互作用

• 批准号: 7133491
• 负责人: Steven R. Blanke
• 金额: $ 25.88万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7133491
• 关键词: CHO cells; Campylobacter; bacterial cytopathogenic effect; bacterial genetics; cell line; cell surface receptors; chemical association; exotoxins; flow cytometry; genetic screening; host organism interaction; iodination; mutant; polymerase chain reaction; radiotracer; receptor binding; receptor expression

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.

描述（由申请人提供）：弯曲杆菌空肠是一种食物和水传播的病原体，负责人类严重的炎症性腹泻。空肠梭菌是B类优先病原体，因为该病原体的潜力部分是用作生物果料剂，以污染大型地理区域上的食品和供水。重要的是要了解空肠梭菌发病机理的基本生物学，这可能会导致新的策略来规避这种细菌作为生物乳化剂的使用。然而，对宿主在宿主中定殖并引起疾病的病原体机制知之甚少。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一项协作建议，研究了C. Jejuni唯一已知的Exotoxin。我们将研究Jejuni C. c. jejuni细胞降低毒素（CDT）的细胞机制，该毒素（CDT）导致细胞周期停滞和最终死亡醉酒的哺乳动物细胞，并已提议协助重塑体内环境以促进肠道的结肠化。在此R21应用中，我们将开始探讨CDT区分并结合敏感细胞表面上特定的质膜受体并结合的假设，这是细胞中毒过程中必不可少的早期步骤。在此应用中，我们提出了在分子水平研究CDT与靶细胞的相互作用的实验。在特定的目标1中，休斯敦大学的研究人员将表征CDT与敏感哺乳动物细胞系的相互作用。我们将确定特定和非特异性组件以及CDT受体相互作用的亲和力。此外，我们将建立许多受体如何每个哺乳动物细胞存在。此外，由于CDT是一种三方毒素，其中包括三个离散亚基（CDTA，CDTB和CDTC），因此我们将建立每个亚基对毒素结合的贡献。最后，我们将开始表征CDT受体的性质。这些实验对于建立未来实验的框架以确定CDT受体歧视和结合的分子基础将很重要。在特定目标2中，我们将识别并表征对CDT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，从而丧失对CDT的敏感性。在第2阶段，将表征灵敏度的丧失，目的是识别与CDT结合不足的细胞系。这些实验对于将来的工作至关重要，以补充细胞结合缺陷，这将确定推定的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，并将为未来的工作提供基础，以制定阻止CDT作为减弱Jejuni发病机理的方法的策略。