Novel approaches to identify host genes required for Chlamydia pathogenesis

描述（由申请人提供）：衣原体物种是专性细胞内寄生虫，是广泛的人类疾病的重要原因。衣原体物种具有独特的细胞内生命周期，但由于生长困难和缺乏遗传学，对其细节和疾病发病机制的了解一直受到阻碍。我们的长期目标是了解衣原体是如何引起人类疾病的。我们的短期目标是确定衣原体发病所需的宿主基因。在这个新的R01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细胞中使用全基因组RNAi来鉴定成功感染所需的宿主因子。在之前的工作中，我们已经确定了沙眼衣原体感染果蝇组织培养细胞模拟了最初衣原体与哺乳动物细胞相互作用的关键方面。我们已经完成了一次和二次筛选，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。我们提出综合和互补的方法来验证这些宿主基因在沙眼衣原体感染哺乳动物细胞中的作用。在第一种方法中，我们将验证并进一步研究宿主基因，其功能通过同源性推断，导致基于预先存在的数据（“收获低洼的果实”）的明显可测试的预测。在第二种方法中，我们将研究功能未知但有可能产生新见解的宿主基因（“走向未知”）。在每种情况下，我们将通过在哺乳动物（HeLa）细胞中执行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、转染构成型活性或显性负等位基因（当可用时）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时如何影响结合、进入和细胞内发育来确定每个感兴趣的宿主分子在哪个步骤是必需的。最后，我们将使用基于rnai的新技术，确定宿主基因在动物感染模型中是否必需。总之，这些方法将最大限度地发挥这种新型筛选的潜力，系统地、全面地鉴定衣原体感染发病机制中重要的新宿主基因。这些发现将增加我们对细胞内感染发病机制的基本认识。此外，它们有潜力为开发新的治疗、诊断和预防疗法确定新的靶点。

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.