Genetics of Monocyte Killing by Bacteria

细菌杀死单核细胞的遗传学

• 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
• 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' 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

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.

项目摘要 这项建议的重点是了解嗜肺军团菌能力的分子基础， 感染、在其中存活、在其中复制并最终杀死人类巨噬细胞。我们建议研究一组 由L. pneumophila，并通过Icm/Dot易位转移到宿主细胞 系统我们将测试这一假设，即易位蛋白质与细胞器运输途径相互作用， 宿主并有助于军团菌细胞内增殖。其中一种转运蛋白VipA， 发现其结合肌动蛋白并促进肌动蛋白亚基的聚合。我们还将关注三种蛋白质（LegC 2， LegC3、LegC7）。VipA和LegC蛋白都能引起细胞器的运输 缺陷时，在模型宿主，酿酒酵母中表达。这项建议的具体目标是 至：1.确定VipA活性的分子基础，VipA是一种肌动蛋白结合的易位效应物， 干扰内体运输; 2.检验Vps/ESCRT复合物的组分是 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 效应子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 控制军团菌和宿主细胞之间的初始相互作用所需的效应易位; 5. 确定LegC 2、LegC 3和LegC 7的互动伙伴。为了实现这些目标，我们将 各种细胞生物学工具的优点，例如消耗特定细胞器运输的细胞 通过siRNA和检查对军团菌感染的影响，已知细胞器的共定位， 标记与军团菌含有空泡和异位表达军团菌基因是人类 巨噬细胞系。我们还将研究特异性靶向宿主酪氨酸激酶的作用， 磷酸酶对军团菌细胞内增殖和细胞器运输的影响。我们将利用细菌遗传学 分离编码易位蛋白质的基因的显性负等位基因，以更好地了解其 在军团菌感染中的作用。所有这些方法都应该阐明军团菌用于 避免被巨噬细胞杀死并产生成功的感染。