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

• 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
• 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

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类生物恐怖制剂，因此了解调节其毒性的分子和细胞机制具有更高的优先级。两个可能在致病性中起作用的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靶点。我们提出了一种正向遗传学方法，利用蛋白质的过表达来鉴定调节吞噬和PI 3-激酶信号传导的基因。在Aim 1中，我们将在一个可诱导的溶组织芽孢杆菌表达系统中生成一个溶组织芽孢杆菌cDNA文库，并将该文库转染到溶组织芽孢杆菌滋养体中。然后，我们将使用基于人类红细胞内化（装载代谢毒素，结核菌素）或基于第二种代谢毒素，亚甲基二膦酸盐内化的筛选，从转化者群体中选择吞噬突变体和胞饮突变体。从幸存者中重新分离表达质粒并对cDNA插入物进行测序，将允许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耐渥曼宁突变体。Wortmannin是pi3激酶的抑制剂，表达Wortmannin靶点或其下游效应物水平升高的细胞应耐受更高水平的药物。鉴定幸存者中过表达的cdna将揭示wortmannin的靶标和pi3激酶的下游效应物。这将提供一个全局视图的PI 3-激酶信号网络的溶组织芽孢杆菌。这些研究的完成将对吞噬和PI 3-激酶信号传导的调控基因提供重要的见解，这些基因是溶组织芽胞杆菌的重要毒力功能。这些研究的完成也将导致一个新的研究工具的发展，一个诱导cDNA表达文库。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类生物恐怖制剂，因此了解调节其毒性的分子和细胞机制具有更高的优先级。拟议的研究将确定在这种生物体中调节吞噬和信号转导的基因。由于这些细胞过程对溶组织芽胞杆菌的毒力很重要，这项工作将大大提高我们对致病性的理解。