Genetics of Monocyte Killing by Bacteria

细菌杀死单核细胞的遗传学

• 批准号: 7735942
• 负责人: HOWARD A SHUMAN
• 金额: $ 56.42万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 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/7735942
• 关键词: Actins; Acute Pneumonia; Alleles; Alveolar Macrophages; Bacteria; Binding; Bioinformatics; Biological; Bone Marrow; Breathing; Carrier Proteins; 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; Mutant Strains Mice; 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; 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; public health relevance; tool; trafficking

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: The proposed research will increase understanding about how bacterial pathogens subvert macrophages, a primary defense against infection. Legionella, the agent of Legionnaires' disease is able to grow inside macrophages. During infection, specialized Legionella proteins are delivered to the host macrophages by the bacteria where they wreak havoc with the antimicrobial system of the white cells.

描述（由申请人提供）：该提案的重点是理解肺炎军团菌感染，在内部生存，复制并最终杀死人类巨噬细胞的能力的分子基础。我们建议研究一组新型蛋白质，这些蛋白质是由肺炎乳杆菌制成的，并通过ICM/DOT易位系统转移到宿主细胞上。我们将检验以下假设：易位蛋白与宿主中的细胞器运输途径相互作用并有助于军团菌的细胞内繁殖。发现一种易位的蛋白质VIPA结合肌动蛋白并促进肌动蛋白亚基的聚合。我们还将专注于包含盘绕线圈域的三种蛋白质（LEGC2，LEGC3，LEGC7）。当在模型宿主酿酒酵母中表达时，VIPA和LEGC蛋白都会导致细胞器运输缺陷。该提议的具体目的是：1。确定VIPA活性的分子基础，VIPA是一种肌动蛋白结合，转移的效应子，会干扰内体贩运； 2。检验假设，即VPS/ESCRT复合物的成分与军团菌的细胞内乘法期间的事件有关。 3。效应子基因等位基因的显性阴性和效应子在细胞内乘法中的作用； 4。识别控制宿主细胞酪氨酸激酶，这些激酶控制着效应子易位所需的军团菌与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，我们将利用各种细胞生物学工具，例如通过siRNA耗尽特定细胞器运输组件的细胞，并检查对军团菌感染的影响，将已知的细胞器标记物共定位，具有龙虾菌的液泡和人类含量的人类毛病细胞系。我们还将研究特异性靶向宿主酪氨酸激酶和磷酸酶对军团菌的细胞内繁殖和细胞器运输的影响。我们将使用细菌遗传学来分离编码易位蛋白的基因的显性阴性等位基因，以更好地了解它们在军团菌感染中的作用。所有这些方法都应阐明军团菌用来避免被巨噬细胞杀死并产生成功感染的机制。公共卫生相关性：拟议的研究将增加对细菌病原体如何颠覆巨噬细胞的理解，这是针对感染的主要防御。军团疾病的代理人军团菌能够在巨噬细胞内生长。在感染期间，专门的军团菌蛋白通过细菌将其递送到宿主巨噬细胞中，在那里它们用白细胞的抗菌系统造成破坏。