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。T. Cruzi利用宿主细胞的代谢和细胞过程来促进其自身在宿主细胞质中的生长。细胞内无鞭毛体是T.尽管克鲁兹生命周期以急性和慢性恰加斯病的化疗干预为目标，但人们对支持该病原体细胞内生长和生存的宿主细胞需求知之甚少。在这里，我们建议进行全基因组RNA干扰筛选，以确定关键的宿主细胞调节T。哺乳动物细胞的Cruzi感染。本研究的目的是在T. cruzi宿主细胞接口的移动远离单基因的目标，对宿主细胞的生物学途径和细胞网络所需的支持细胞内T。克氏感染提出的siRNA筛选将利用预先存在的基于细胞的T. cruzi生长，其测量与稳定的转基因T. cruzi- 2-半乳糖苷酶表达的寄生虫在384孔板格式中的细胞培养。我们将筛选一个包含19，470个靶向人类基因组大部分的siRNA库的阵列Dharmacon siRNA文库，以鉴定对支持T细胞至关重要的人类基因（在HeLa细胞中）。Cruzi进入和细胞内生长。我们将在二级筛选工作中独立验证最高命中率，并使用现有的生物信息学工具（如Inhibitity Pathway Analysis和GeneGo的MetaCore Analysis）基于生物网络分析对这些命中率进行优先排序。在研究的第二阶段，将进行基于图像的集中筛选，以将siRNA命中广泛分类为与影响细胞内复制的那些与复制前事件相关的那些。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恰加斯病社区，因此提供了一个直接的机会，利用我们的发现，从整合的宿主细胞网络的角度对恰加斯病发病机制的关键方面进行机制研究。 公共卫生相关性：查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。作为拉丁美洲心力衰竭的主要原因，恰加斯病在流行国家是一个特别重要的公共卫生问题，在美国也是一个新出现的移民健康问题。T. Cruzi是一种专性细胞内寄生物，其利用宿主细胞代谢和细胞过程来促进其在宿主细胞质中的生长。虽然细胞内无鞭毛体是T.尽管克鲁兹生命周期以急性和慢性恰加斯病的化疗干预为目标，但人们对支持该病原体细胞内生长和生存的宿主细胞需求知之甚少。本文提出的研究旨在在全基因组范围内识别对于支持细胞内T至关重要的宿主细胞途径。宿主细胞中的克氏感染。这项工作的结果将提供直接和重要的见解T。cruzi发病机制，并指导有效预防或控制恰加斯病的努力。

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.