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

描述(申请人提供)：恰加斯病是一种热带寄生虫病，在长期感染原虫寄生虫克氏锥虫的个人身上发展多年。作为一种专性的细胞内寄生虫，克氏毛滴虫利用宿主细胞的代谢和细胞过程来促进自身在宿主细胞细胞质中的生长。虽然细胞内的无鞭毛体是克氏锥虫生活史中的关键阶段，但对于支持这种病原体在细胞内生长和存活的宿主细胞需求，人们知之甚少。在这里，我们建议进行全基因组RNA干扰筛选，以确定哺乳动物细胞感染毛滴虫的关键宿主细胞调节因子。这项工作的目标是通过从单基因靶标转向支持细胞内感染所需的宿主细胞生物学途径和细胞网络的综合视图，获得克氏锥虫-宿主细胞界面的关键生物学的全球视角。拟议的siRNA筛选将利用现有的基于细胞的高通量测试来生长克鲁兹毛滴虫，该测试以384孔板的形式测量与稳定表达克鲁兹毛滴虫2-半乳糖苷酶的转基因寄生虫相关的2-半乳糖苷酶活性。我们将筛选一个包含19,470个针对大多数人类基因组的siRNA池的阵列化Dharacon siRNA文库，以确定(在HeLa细胞中)对支持克氏锥虫进入和细胞内生长至关重要的人类基因。我们将独立地验证二次筛选工作中的最高命中结果，并使用现有的生物信息学工具，如独创性路径分析和GeneGo的MetaCore分析，基于生物网络分析对这些命中结果进行优先排序。在研究的第二阶段，将进行基于图像的重点筛选，将siRNA命中结果从影响细胞内复制的事件中大致归类为与复制前事件相关的siRNA命中。我们的筛查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，从而立即提供机会，从整合宿主细胞网络的角度对查格斯病发病机制的关键方面进行机制研究。 公共卫生相关性：恰加斯病是一种热带寄生虫病，在长期感染原虫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，查加斯病在流行国家是一个特别重要的公共卫生问题，在美国也是一个新的移民健康问题。克氏毛滴虫是一种专性的细胞内寄生虫，它利用宿主细胞的代谢和细胞过程来促进其在宿主细胞细胞质中的生长。尽管细胞内的无鞭毛体是克氏锥虫生活史中的关键阶段，可以作为急性和慢性恰加斯病化疗干预的靶标，但对宿主细胞支持这种病原体在细胞内生长和存活的需求知之甚少。这里提出的这项研究试图在全基因组范围内确定宿主细胞途径，这些途径对于支持宿主细胞内的克氏锥虫感染至关重要。这项工作的结果将为了解克氏毛滴虫的发病机制提供直接和重要的见解，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.