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：
7876802
负责人：
LESLY A TEMESVARI
金额：
$ 18.56万
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 2012-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7876802
关键词：
1-Phosphatidylinositol 3-Kinase Amebic colitis Biology Bioterrorism Candidate Disease Gene Categories Cell physiology Cells Chemicals Complementary DNA 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）：Entamoeba Histolictica是Amoebic痢疾和肝脓肿的病因。由于该病原体被归类为B类生物恐怖剂，因此优先级升高以了解调节其毒力的分子和细胞机制。可能在致病性中起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，对这些过程的更好理解可能会揭示针对反amoebic策略的新目标。我们提出了一种正向遗传学方法，该方法使用蛋白质过表达来鉴定调节吞噬作用和PI 3-激酶信号传导的基因。在AIM 1中，我们将在诱导的E. histoltica表达系统中生成一个溶组织溶性cDNA库，并将该文库转染到Histolodictica滋养体中。然后，我们将根据人类红细胞的内在化（含有代谢毒素，结核素）的内在化或第二次代谢毒素，甲基二磷酸甲基二磷酸甲基甲酸盐的内在化，从而选择转化体群的吞噬作用突变体和吞噬突变体。从幸存者中重新分离表达质粒和cDNA插入物的测序将允许鉴定调节这些内吞过程的基因。在AIM 2中，我们将从转化子种群中选择抗浆素的耐磨牙突变体。 Wortmannin是PI 3-激酶和细胞的抑制剂，表达麦芽汁靶标的水平增加，或其下游效应子应耐受较高的药物水平。在幸存者中过表达的CDNA的鉴定将揭示毛摩尔蛋白酶和PI 3-激酶的下游效应子的靶标。这将提供E. issolytica中PI 3-激酶信号网络的全局视图。这些研究的完成将为调节吞噬作用和PI 3-激酶信号传导的基因提供重要的见解，这是溶作息肉大肠杆菌中重要的毒力功能。这些研究的完成还将导致开发一种新的研究工具，即诱导的cDNA表达式文库。公共卫生相关性：Entamoeba Histolictica是半oe虫和肝脓肿的病因。由于该病原体也被归类为B级生物恐怖剂，因此优先级升高以了解调节其毒力的分子和细胞机制。拟议的研究将确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溶组织性的毒力很重要，因此这项工作将显着增强我们对致病性的理解。