Novel approaches to identify host genes required for Chlamydia pathogenesis

鉴定衣原体发病机制所需宿主基因的新方法

• 批准号: 7790778
• 负责人: Joanne N. Engel
• 金额: $ 37.51万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-15 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7790778
• 关键词: Actins; Affect; Alleles; Anabolism; Animal Model; Binding; Biochemical; Bioinformatics; Biological; Candidate Disease Gene; Cell Communication; Cell Line; Cell physiology; Cells; Cellular biology; Chlamydia; Chlamydia Infections; Data; Development; Diagnostic; Disease; Dissection; Dominant-Negative Mutation; Drosophila genus; Drosophila melanogaster; Enzymes; Fruit; Gene Silencing; Genes; Genetic; Genetic Screening; Genome; Goals; Grant; Harvest; Hela Cells; Heparan Sulfate Proteoglycan; Human; Infection; Infection prevention; Integration Host Factors; Knock-out; Knowledge; Lead; Libraries; Life Cycle Stages; Mammalian Cell; Mediating; Methods; Modeling; Organism; PDGFRA gene; PDGFRB gene; Pathogenesis; Pharmaceutical Preparations; Phenotype; Phosphotransferases; Platelet-Derived Growth Factor Receptor; Prevention; Process; Protein Tyrosine Kinase; Proteins; RNA Interference; Research Personnel; Role; Signal Pathway; Site; Small Interfering RNA; Staging; Technology; Testing; Transfection; Vaccines; Vacuole; Virulence Factors; Work; bacterial genetics; base; fly; gene function; genome-wide; human disease; insight; interest; macrophage; microbial; novel; novel strategies; novel therapeutics; obligate intracellular parasite; p21 activated kinase; pathogen; polymerization; research study; success; tissue culture; tissue/cell culture; tool

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.

描述(申请人提供)：衣原体是一种专性的细胞内寄生虫，是导致多种人类疾病的重要原因。衣原体物种有独特的细胞内生命周期，但由于生物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导致人类疾病的。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R01中，我们使用了一种新的正向遗传筛查的结果，该筛查使用了果蝇S2细胞中全基因组RNAi来确定成功感染所需的宿主因素。在以前的工作中，我们已经证实，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。我们已经完成了第一次和第二次筛选，鉴定了大约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过程中的作用。在第一种方法中，我们将验证并进一步研究宿主基因，这些基因的功能由同源性推断，导致基于先前存在的数据(“收获低洼的果实”)的明显的可检验预测。在第二种方法中，我们将研究宿主基因，这些基因的功能尚不清楚，但有可能产生新的见解(“走向未知”)。在每种情况下，我们都将通过在哺乳动物(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确认有希望的候选基因，当有成分活性或显性负性等位基因时，将其导入，或适当的敲除细胞。我们将通过量化宿主基因耗尽对结合、进入和细胞内发育的影响，来确定每个感兴趣的宿主分子所需的步骤。最后，我们将使用新的基于RNAi的技术来确定宿主基因是否在动物感染模型中是必需的。总之，这些方法将最大限度地发挥这一新筛查的潜力，系统和全面地识别在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知识。此外，它们还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一个重要原因。这笔赠款将发现感染所需的宿主基因。这可能会使新药和疫苗靶点的开发成为可能。