Novel approaches to identify host genes required for Chlamydia pathogenesis

描述（由申请人提供）：衣原体属是专性细胞内寄生虫，是多种人类疾病的重要原因。衣原体物种有一个独特的细胞内生命周期，但了解其细节和疾病发病机制一直受到生物体生长困难和缺乏遗传学的阻碍。我们的长期目标是了解衣原体如何导致人类疾病。我们的短期目标是确定衣原体致病所需的宿主基因。在这个新的R 01中，我们使用了一种新的正向遗传筛选的结果，该筛选在果蝇S2细胞中采用全基因组RNAi来识别成功感染所需的宿主因子。在以前的工作中，我们已经建立了C。沙眼衣原体感染果蝇组织培养细胞模拟初始衣原体-哺乳动物细胞相互作用的关键方面。我们已经完成了初步和二次筛选，确定了约125个影响结合，进入和/或早期液泡形成的宿主基因。我们提出了全面和互补的方法来验证这些宿主基因在C。沙眼感染哺乳动物细胞。在第一种方法中，我们将验证和进一步研究宿主基因的功能，推断同源性，导致明显的可测试的预测的基础上预先存在的数据（“收获低洼水果”）。在第二种方法中，我们将研究宿主基因，其功能仍然未知，但有可能产生新的见解（“走向未知”）。在每种情况下，我们将通过在哺乳动物（HeLa）细胞中进行RNAi介导的基因失活来评估候选基因在哺乳动物细胞感染中的作用。我们将使用药理学方法，转染组成型活性或显性负等位基因（如果可用）或适当的敲除细胞来确认有希望的候选基因。我们将通过量化宿主基因耗尽后如何影响结合、进入和细胞内发育来确定在哪个步骤需要每个感兴趣的宿主分子。最后，我们将使用新的基于RNAi的技术来确定宿主基因是否是感染动物模型所必需的。总之，这些方法将最大限度地发挥这种新的屏幕的潜力，系统和全面地确定新的宿主基因重要的衣原体感染的发病机制。这些发现将增加我们对细胞内感染发病机制的基础知识。此外，它们有可能为开发新的治疗、诊断和预防疗法确定新的靶点。 衣原体是世界范围内人类疾病的重要原因。这项资助将发现感染所需的宿主基因。这可能有助于开发新的药物和疫苗靶点。

DESCRIPTION (provided by applicant): Chlamydia species are obligate intracellular parasites that are important causes of a wide range of human diseases. Chlamydia species have a unique intracellular life cycle but understanding its details and the mechanisms of disease pathogenesis has been hampered by the difficulty in growing the organism and the lack of genetics. Our long term goal is to understand how Chlamydia causes disease in humans. Our short term goals are to identify host genes required for Chlamydial pathogenesis. In this new R01, we use the results of a novel forward genetic screen that employed genome-wide RNAi in Drosophila S2 cells to identify host factors required for successful infection. In previous work, we have established that C. trachomatis infection of Drosophila tissue culture cells mimics key aspects of initial Chlamydia-mammalian cell interactions. We have completed a primary and secondary screen which has identified ~125 host genes that affect binding, entry, and/or early vacuole formation. We propose comprehensive and complementary approaches to validate the role of these host genes during C. trachomatis infection of mammalian cells. In the first approach, we will validate and further study host genes whose function, inferred by homology, leads to obvious testable predictions based on pre-existing data ("harvesting the low lying fruit"). In the second approach, we will investigate host genes whose functions remain unknown but which have the potential to yield novel insights ("going for the unknown"). In each case, we will assess the role of the candidate gene in mammalian cell infections by performing RNAi-mediated gene inactivation in mammalian (HeLa) cells. We will confirm promising candidate genes using pharmacologic approaches, transfection of constitutively active or dominant negative alleles when available, or appropriate knock-out cells. We will determine at which, step each host molecule of interest is required, by quantifying how binding, entry, and intracellular development is affected upon depletion of the host genes. Finally, we will determine whether the host gene is required in an animal model of infection, using novel RNAi-based technologies. Together, these approaches will maximize the potential of this novel screen to systematically and comprehensively identify new host genes important in the pathogenesis of chlamydial infections. These findings will increase our basic knowledge of the pathogenesis of intracellular infections. In addition, they have the potential to identify new targets for the development of new therapeutic, diagnostic, and preventative therapies. Chlamydia species are an important cause of human diseases world-wide. This grant will discover what host genes are required for infection. This may allow the development of new drug and vaccine targets.