A functional genomic screen to identify host cell regulators of Trypanosoma cruzi

描述（由申请人提供）：恰加斯病是一种热带寄生虫病，在慢性感染原生动物寄生虫克氏锥虫的个体中发展多年。作为一种专性细胞内寄生虫，克氏锥虫利用宿主细胞的代谢和细胞过程来促进其在宿主细胞质中的生长。虽然在急性和慢性恰加斯病中，细胞内无尾线虫是克氏锥虫生命周期中以化疗干预为目标的关键阶段，但人们对支持这种病原体细胞内生长和存活的宿主细胞需求知之甚少。在此，我们建议进行全基因组RNA干扰筛选，以确定哺乳动物细胞感染T. cruzi的关键宿主细胞调节因子。这项工作的目标是通过从单一基因靶点转向支持细胞内克氏t感染所需的宿主细胞生物学途径和细胞网络的综合视图，获得克氏t -宿主细胞界面关键生物学的全局视图。拟议的siRNA筛选将利用预先存在的基于细胞的高通量克氏绦虫生长试验，该试验在384孔板格式中测量与稳定的转基因克氏绦虫- 2-半乳糖苷酶表达寄生虫相关的2-半乳糖苷酶活性。我们将筛选一个包含19,470个siRNA库的排列的Dharmacon siRNA文库，该文库针对大多数人类基因组，以鉴定（在HeLa细胞中）支持T. cruzi进入和细胞内生长的关键人类基因。我们将在二次筛选工作中独立验证最受欢迎的药物，并使用现有的生物信息学工具（如Ingenuity Pathway analysis和GeneGo的MetaCore analysis）根据生物网络分析对这些药物进行优先排序。在研究的第二阶段，将进行集中的基于图像的筛选，将siRNA撞击分为与复制前事件相关的撞击和影响细胞内复制的撞击。我们的筛选工作和初步功能分析的结果将提供给更广泛的南美锥虫病社区，因此提供了一个直接的机会，利用我们的发现从宿主细胞网络的角度对南美锥虫病发病机制的关键方面进行机制研究。

DESCRIPTION (provided by applicant): Chagas' disease is a tropical parasitic disease that develops over a number of years in individuals that are chronically infected with the protozoan parasite, Trypanosoma cruzi. As an obligate intracellular parasite, T. cruzi exploits host cell metabolic and cellular processes to fuel its own growth in the host cell cytoplasm. While intracellular amastigotes represent the critical stage in the T. cruzi life cycle to target for chemotherapeutic intervention in both acute and chronic Chagas' disease, very little is known regarding the host cell requirements to support intracellular growth and survival of this pathogen. Here we propose to conduct a genome-wide RNA interference screen to identify critical host cell regulators of T. cruzi infection of mammalian cells. The goal of this work is to obtain a global view of the critical biology at the T. cruzi-host cell interface by moving away from single gene targets, toward an integrated view of the host cell biological pathways and cellular networks required to support intracellular T. cruzi infection. The proposed siRNA screen will exploit a pre-existing cell-based high-throughput assay for T. cruzi growth, which measures 2-galactosidase activity associated with stable transgenic T. cruzi- 2-galactosidase-expressing parasites in a 384-well plate format. We will screen an arrayed Dharmacon siRNA library containing 19,470 siRNA pools targeting the majority of the human genome, to identify human genes (in HeLa cells) that are critical for supporting T. cruzi entry and intracellular growth. We will independently validate the top hits in secondary screening efforts and prioritize these based on biological network analysis using available bioinformatics tools such as Ingenuity Pathway Analysis" and GeneGo's MetaCore Analysis. In the second phase of the study, a focused image-based screen will be conducted to broadly categorize siRNA hits into those associated with pre-replication events from those that impact intracellular replication. Results from our screening efforts and the preliminary functional analysis will be made available to the broader Chagas' disease community and therefore provide an immediate opportunity to exploit our findings for mechanistic studies of critical aspects of Chagas' disease pathogenesis from the standpoint of integrated host cell networks. PUBLIC HEALTH RELEVANCE: Chagas' disease is a tropical parasitic disease that develops over a number of years in individuals that are chronically infected with the protozoan parasite, Trypanosoma cruzi. As a leading cause of heart failure in Latin America, Chagas' disease represents a public health problem of exceptional importance in endemic countries as well as an emerging immigrant health issue in the United States. T. cruzi is an obligate intracellular parasite that exploits host cell metabolic and cellular processes to fuel its growth in the host cell cytoplasm. Although intracellular amastigotes represent the critical stage in the T. cruzi life cycle to target for chemotherapeutic intervention in both acute and chronic Chagas' disease, very little is known regarding the host cell requirements to support intracellular growth and survival of this pathogen. The study proposed here seeks to identify host cell pathways, on a genome-wide scale, that are critical for supporting intracellular T. cruzi infection in host cells. Results from this work will provide immediate and important insights into T. cruzi pathogenesis and guide efforts toward effective prevention or control of Chagas' disease.