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

鉴定克氏锥虫宿主细胞调节因子的功能基因组筛选

基本信息

批准号：
7978709
负责人：
BARBARA A BURLEIGH
金额：
$ 24.53万
依托单位：
HARVARD SCHOOL OF PUBLIC HEALTH
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-06-01 至 2012-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7978709
关键词：
Acute Bioinformatics Biological Biological Assay Biology Cell Communication Cell physiology Cells Cellular Metabolic Process Cellular biology Chagas Disease Cherry - dietary Chronic Communities Computer software Country Cytoplasm Databases Development Disease Outcome Disease Progression Environment Event Funding Galactosidase Gene Proteins Gene Targeting Genes Goals Growth Health Heart failure Hela Cells Host-Parasite Relations Human Human Genome Image Immigrant Immunofluorescence Immunologic Individual Infection Inflammation Intervention Latin America Libraries Life Cycle Stages Link Maintenance Mammalian Cell Measures Mediating Metabolic Nature Parasites Parasitic Diseases Pathogenesis Pathway Analysis Pathway interactions Phase Phenotype Prevention Proteins Public Health RNA Interference Reporting Research Resources Reverse Transcriptase Polymerase Chain Reaction Role Screening Result Screening procedure Small Interfering RNA Staging Study models Therapeutic Tissues Transgenic Organisms Trypanosoma cruzi United States Validation Western Blotting Work base functional genomics genome wide association study genome-wide high throughput screening insight novel obligate intracellular parasite pathogen prevent public health relevance tool

项目摘要

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.

描述（由申请人提供）：Chagas的疾病是一种热带寄生虫病，在长期感染原生动物寄生虫Cruzi Cruzi的个体中，多年来发展了多年。作为强制性细胞内寄生虫，Cruzi利用宿主细胞代谢和细胞过程来促进其自身在宿主细胞质中的生长。虽然细胞内杂物代表了克鲁齐（T.在这里，我们建议进行全基因组的RNA干扰筛查，以鉴定哺乳动物细胞T. cruzi感染的关键宿主细胞调节剂。这项工作的目的是通过从单个基因靶标转移到宿主细胞生物学途径和支持细胞内T. cruzi感染所需的宿主细胞生物学途径和细胞网络的综合视图，从而获得在T. Cruzi-Host细胞界面上对关键生物学的全局视图。所提出的siRNA屏幕将利用基于细胞的基于细胞的高通量测定法对Cruzi的生长进行，该测定法测量了与稳定的转基因T. cruzi-2-galactosidase寄生虫相关的2-半乳糖苷酶活性，在384孔板格式中。我们将筛选一个阵列的佛法siRNA库，其中包含针对大多数人类基因组的19,470个siRNA池，以鉴定人类基因（在HELA细胞中），这对于支持Cruzi的进入和细胞内生长至关重要。我们将使用可用的生物信息学工具（例如Ingenuity途径分析）和Genego的Metacore分析来独立验证二级筛查工作中的最高点击，并根据生物网络分析进行优先级。功能分析将提供给更广泛的Chagas疾病界，因此提供了一个即时的机会，从综合宿主细胞网络的角度来看，我们的发现来利用我们的发现，用于对Chagas疾病发病机理的关键方面进行机械研究。公共卫生相关性：Chagas疾病是一种热带寄生虫病，在长期感染原生动物寄生虫Crypanosoma Cruzi的个体中，多年来发展了多年。作为拉丁美洲心力衰竭的主要原因，查加斯疾病代表了在流行国家以及美国新兴的移民健康问题中非常重要的公共卫生问题。克鲁兹（T. cruzi）是一种强制性细胞内寄生虫，可利用宿主细胞代谢和细胞过程，以促进其在宿主细胞质中的生长。尽管细胞内杂物代表了克鲁齐（T.这里提出的研究旨在以全基因组量表鉴定宿主细胞途径，这对于支持宿主细胞中的细胞内T. cruzi感染至关重要。这项工作的结果将提供对克鲁兹氏菌发病机理的立即和重要见解，并指导有效预防或控制查加斯疾病的努力。