A forward genetics screen for genes regulating phagocytosis and signaling in Enta

Enta 中调节吞噬作用和信号传导基因的正向遗传学筛选

基本信息

批准号：
7573223
负责人：
LESLY A TEMESVARI
金额：
$ 21.25万
依托单位：
CLEMSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-06-19 至 2011-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7573223
关键词：
1-Phosphatidylinositol 3-Kinase Amebic colitis Biology Bioterrorism Candidate Disease Gene Categories Cell physiology Cells Chemicals Complementary DNA Developed Countries Developing Countries Development Dose Endocytosis Entamoeba histolytica Episome Erythrocytes Expression Library Gene Expression Profile Genes Genetic Genetic Screening Genomics Human Infection Kinetics Libraries Liquid substance Liver Abscess Mammalian Cell Metabolic Molecular Oral Organelles Organism Parasites Pathogenicity Phagocytosis Phagosomes Pharmaceutical Preparations Phase Pinocytosis Plasmids Play Population Process Protein Overexpression Proteins Research Resistance Role Signal Transduction Survivors System Tetracyclines Time Toxin Tubercidin Vesicle Virulence Work base cDNA Expression cDNA Library gene discovery high throughput screening inhibitor/antagonist innovation insight knock-down methylene diphosphonate mutant overexpression pathogen phosphatidylinositol 3-phosphate prevent protein expression public health relevance small molecule tool wortmannin

项目摘要

DESCRIPTION (provided by applicant): Entamoeba histolytica is the causative agent of amoebic dysentery and liver abscess. Since this pathogen is categorized as a Class B bioterrorism agent, there is elevated priority to understand the molecular and cellular mechanisms that regulate its virulence. Two cellular processes that may play a role in pathogenicity are phagocytosis and PI 3-kinase-based signal transduction. Therefore, a better understanding of these processes may reveal new targets for anti-amoebic strategies. We propose a forward genetics approach that uses overexpression of proteins to identify genes that regulate phagocytosis and PI 3-kinase signaling. In Aim 1, we will generate an E. histolytica cDNA library in an inducible E. histolytica expression system and transfect this library into E. histolytica trophozoites. We will then select for phagocytosis mutants and pinocytosis mutants from the population of transformants using screens based on the internalization of human red blood cells (loaded with a metabolic toxin, tubercidin) or on the internalization of a second metabolic toxin, methylenediphosphonate. Re-isolation of the expression plasmids from survivors and sequencing of the cDNA inserts will allow for the identification of genes that regulate these endocytic processes. In Aim 2, we will select for wortmannin-resistant mutants from the population of transformants. Wortmannin is an inhibitor of PI 3-kinases and cells that express increased levels of wortmannin targets or their downstream effectors should tolerate higher levels of the drug. Identification of cDNAs overexpressed in survivors will reveal targets of wortmannin and downstream effectors of PI 3-kinases. This will provide a global view of PI 3-kinase signaling networks in E. histolytica. Completion of these studies will provide significant insight into the genes that regulate phagocytosis and PI 3-kinase signaling, important virulence functions in E. histolytica. Completion of these studies will also result in the development of a new research tool, an inducible cDNA expression library. PUBLIC HEALTH RELEVANCE: Entamoeba histolytica is the causative agent of amoebic dysentery and liver abscess. Since this pathogen is also categorized as a Class B bioterrorism agent, there is elevated priority to understand the molecular and cellular mechanisms that regulate its virulence. The proposed studies will identify genes that regulate phagocytosis and signal transduction in this organism. Since these cellular processes are important to E. histolytica virulence, this work will significantly enhance our understanding of pathogenicity.

描述（由申请人提供）：溶组织内阿米巴是阿米巴痢疾和肝脓肿的病原体。由于这种病原体被归类为 B 类生物恐怖主义制剂，因此需要优先了解调节其毒力的分子和细胞机制。可能在致病性中发挥作用的两个细胞过程是吞噬作用和基于 PI 3 激酶的信号转导。因此，更好地了解这些过程可能会揭示抗阿米巴策略的新目标。我们提出了一种正向遗传学方法，利用蛋白质的过度表达来识别调节吞噬作用和 PI 3 激酶信号传导的基因。在目标 1 中，我们将在诱导型溶组织内阿米巴表达系统中生成溶组织内阿米巴 cDNA 文库，并将该文库转染到溶组织内阿米巴滋养体中。然后，我们将使用基于人红细胞（装载有代谢毒素，结核菌素）的内化或基于第二种代谢毒素，亚甲基二磷酸盐的内化的筛选，从转化体群体中选择吞噬作用突变体和胞饮作用突变体。从幸存者中重新分离表达质粒并对 cDNA 插入片段进行测序将有助于鉴定调节这些内吞过程的基因。在目标 2 中，我们将从转化体群体中选择渥曼青霉素抗性突变体。渥曼青霉素是 PI 3 激酶的抑制剂，表达增加的渥曼青霉素靶标或其下游效应子水平的细胞应该耐受更高水平的药物。对幸存者中过度表达的 cDNA 进行鉴定将揭示渥曼青霉素的靶标和 PI 3 激酶的下游效应子。这将提供溶组织内阿米巴中 PI 3 激酶信号网络的全局视图。这些研究的完成将有助于深入了解调节吞噬作用和 PI 3 激酶信号传导的基因以及溶组织内阿米巴的重要毒力功能。这些研究的完成还将导致新研究工具——诱导型 cDNA 表达文库的开发。公共卫生相关性：溶组织内阿米巴是阿米巴痢疾和肝脓肿的病原体。由于这种病原体也被归类为 B 类生物恐怖主义制剂，因此优先了解调节其毒力的分子和细胞机制。拟议的研究将鉴定调节该生物体吞噬作用和信号转导的基因。由于这些细胞过程对于溶组织内阿米巴毒力很重要，因此这项工作将显着增强我们对致病性的理解。