Genetics of Monocyte Killing by Bacteria

细菌杀死单核细胞的遗传学

基本信息

批准号：
8278661
负责人：
HOWARD A SHUMAN
金额：
$ 54.97万
依托单位：
UNIVERSITY OF CHICAGO
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：
2009-06-15 至 2014-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8278661
关键词：
Actins Acute Pneumonia Alleles Alveolar Macrophages Bacteria Binding Biochemical Bioinformatics Biological Breathing Cell Line Cells Coiled-Coil Domain Complex Defect Disease Dominant-Negative Mutation Ectopic Expression Endoplasmic Reticulum Ensure Environment Event Exhibits Funding Genes Genetic Golgi Apparatus Growth Human Individual Infection Legionella Legionella pneumophila Legionnaires&apos Disease Leukocytes Lung Lysosomes Mammalian Cell Measures Membrane Microbial Biofilms Modeling Molecular Organelles PTPRC gene Pathway interactions Phagosomes Phenotype Phosphoric Monoester Hydrolases Phosphotransferases Plumbing Property Protein Family Protein Tyrosine Kinase Protein Tyrosine Phosphatase Protein translocation Proteins Research Role SNAP receptor Saccharomyces cerevisiae Small Interfering RNA Sorting - Cell Movement Source System Testing Vacuolar Protein Sorting Vacuole Vesicle Water Work Yeasts aerosolized antimicrobial bacterial genetics base inhibitor/antagonist killings macrophage monocyte mutant novel null mutation pathogen polymerization tool trafficking

项目摘要

PROJECT SUMMARY This proposal is focused on understanding the molecular basis for the ability of Legionella pneumophila to infect, survive within, replicate within, and eventually kill human macrophages. We propose to study a group of novel proteins that are made by L. pneumophila and translocated to host cells by the Icm/Dot translocation system. We will test the hypothesis that the translocated proteins interact with organelle trafficking pathways in the host and contribute to Legionella intracellular multiplication. One of the translocated proteins, VipA, was found to bind actin and promote polymerization of actin subunits. We will also focus on three proteins (LegC2, LegC3, LegC7) that contain coiled coil domains. VipA and the LegC proteins all cause organelle trafficking defects when expressed in the model host, Saccharomyces cerevisiae. The specific aims of this proposal are to : 1. Determine the molecular basis for the activity of VipA, an actin-binding, translocated effector that interferes with endosomal trafficking; 2. Test the hypothesis that components of the Vps/ESCRT complex are related to events during intracellular multiplication of Legionella; 3. Dominant-negative interfering alleles of effector genes and the role of effectors during intracellular multiplication; 4. Identify host cell tyrosine kinases that control the initial interactions between Legionella and host cells required for effector translocation; 5. Identify interaction partners of LegC2, LegC3 and LegC7. In order to carry out these Aims we will take advantage of a variety of cell biological tools such as depleting cells of specific organelle trafficking components by siRNA and examining the effect on Legionella infection, co-localization of known organelle markers with the Legionella -containing vacuole and ectopic expression of Legionella genes is human macrophage cell lines. We will also examine the effects of specifically targeting host tyrosine kinases and a phosphatase on Legionella intracellular multiplication and organelle trafficking. We will use bacterial genetics to isolate dominant-negative alleles of the genes encoding the translocated proteins to better understand their role during Legionella infection. All of these approaches should clarify the mechanisms that Legionella uses to avoid killing by macrophages and produce a successful infection.

项目概要该提案的重点是了解嗜肺军团菌能力的分子基础感染人类巨噬细胞，在其中生存、复制并最终杀死人类巨噬细胞。我们建议研究一组由嗜肺军团菌产生并通过 Icm/Dot 易位易位至宿主细胞的新型蛋白质系统。我们将测试易位蛋白与细胞器运输途径相互作用的假设宿主并有助于军团菌细胞内增殖。其中一种易位蛋白 VipA 是发现结合肌动蛋白并促进肌动蛋白亚基的聚合。我们还将重点关注三种蛋白质（LegC2、 LegC3、LegC7) 包含卷曲线圈域。 VipA 和 LegC 蛋白都会导致细胞器贩运在模型宿主酿酒酵母中表达时存在缺陷。该提案的具体目标是目的： 1. 确定 VipA 活性的分子基础，VipA 是一种肌动蛋白结合易位效应子，干扰内体运输； 2. 检验 Vps/ESCRT 复合物的组成部分的假设与军团菌细胞内增殖过程中的事件有关； 3. 显性负干扰等位基因效应基因和效应子在细胞内增殖过程中的作用； 4. 鉴定宿主细胞酪氨酸激酶控制效应器易位所需的军团菌和宿主细胞之间的初始相互作用； 5. 确定 LegC2、LegC3 和 LegC7 的互动伙伴。为了实现这些目标，我们将采取各种细胞生物学工具的优势，例如消耗特定细胞器运输的细胞 siRNA 成分并检查对军团菌感染的影响，已知细胞器的共定位带有含有军团菌的液泡的标记和军团菌基因的异位表达是人类的巨噬细胞系。我们还将研究专门针对宿主酪氨酸激酶和磷酸酶对军团菌细胞内增殖和细胞器运输的影响。我们将使用细菌遗传学分离编码易位蛋白的基因的显性失活等位基因，以更好地了解它们在军团菌感染期间的作用。所有这些方法都应该阐明军团菌用于治疗的机制。避免被巨噬细胞杀死并产生成功的感染。