New approaches to studying host-chlamydia interactions

描述（由申请人提供）：衣原体是专性细胞内寄生虫，是重要的人类病原体。沙眼衣原体是性传播疾病的主要原因，也是西方世界可预防的不孕症的主要原因，也是发展中国家非先天性失明的主要原因。肺炎支原体是人类呼吸道感染的重要原因，并与动脉粥样硬化疾病的发展有关。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。在这个R21探索性拨款中，我们提出了一种新的前向遗传筛选来确定成功感染所需的宿主因子。RNA干扰介导的基因失活提供了一种新的途径，可以很容易地抑制大多数基因的表达，从而导致功能性敲除。它提供了一种进行大规模正向遗传筛选的新方法。黑腹果蝇的基因组相对较小且没有冗余，为研究这些过程提供了理想的“遗传”宿主。果蝇很容易吸收小的干扰RNA，从而使整个果蝇以及果蝇组织培养细胞中的基因有效失活。包括加州大学旧金山分校在内的几个研究小组，在果蝇组织培养细胞中进行全基因组rnai介导的正向遗传筛选，取得了惊人的成功。果蝇幼虫和组织培养细胞最近被用作模型系统来了解几种重要的人类病原体的发病机制。在初步实验中，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。具体目标1：我们将在果蝇S2细胞中使用rna介导的基因失活，系统地失活所有系统发育上保守的基因，以鉴定沙眼衣原体感染所需的宿主基因。我们将定义详细的表型，并剖析每个基因的作用机制，这些基因是沙眼衣原体成功结合、进入和细胞内发育所必需的。特异性目标2：我们将通过在HeLa细胞中灭活人类同源物来验证筛选，并将进一步研究这些宿主基因在沙眼衣原体感染中的作用。总之，这些研究将大大提高我们对传染病发病机制的认识，并为新疗法和新疫苗铺平道路。

DESCRIPTION (provided by applicant): Chlamydia species are obligate intracellular parasites that are important human pathogens. C. trachomatis is the leading cause of sexually transmitted diseases and a key cause of preventable infertility in the western world, and the major cause of non-congenital blindness in developing nations. C. pneumoniae is an important cause of human respiratory infections and has been associated with the development of atherosclerotic disease. 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. In this R21 exploratory grant, we propose a novel forward genetic screen to identify host factors required for successful infection. RNA interference mediated gene inactivation provides a new approach to easily inhibit the expression of most genes, resulting in functional knockouts. It offers a novel approach to carry out large-scale forward genetic screens. Drosophila melanogaster, with its relatively small and non-redundant genome, provides an ideal "genetic" host in which to study these processes. Drosophila readily takes up small interfering RNA, allowing efficient inactivation of genes in whole flies as well as in Drosophila tissue culture cells. Several groups, including at UCSF, have had spectacular success carrying out genome-wide RNAi-mediated forward genetic screens in Drosophila tissue culture cells. Drosophila larvae and tissue culture cells have recently been used as a model system to understand the pathogenesis of several important human pathogens. In preliminary experiments, we have established that C. trachomatis infection of Drosophila tissue culture cells mimics key aspects of initial Chlamydia-mammalian cell interactions. 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. Specific Aim 1: We will use RNA-mediated gene inactivation in Drosophila S2 cells to systematically inactivate all phylogentically conserved genes to identify host genes required for C. trachomatis infection. We will define the detailed phenotype and dissect the mechanism of action of each gene required for successful binding, entry, and intracellular development of C. trachomatis. Specific Aim 2: We will validate the screen by inactivating the human homologs in HeLa cells and will further investigate the role of these host genes in C. trachomatis infection. Together, these studies will significantly advance our knowledge of infectious disease pathogenesis and pave the way for new therapies and vaccines.