Genetics of Monocyte Killing by Bacteria

描述（由申请人提供）：本提案的重点是了解嗜肺军团菌感染、在人巨噬细胞内存活、复制并最终杀死人巨噬细胞的能力的分子基础。我们建议研究一组由嗜肺乳杆菌产生并通过Icm/Dot易位系统转运到宿主细胞的新蛋白。我们将验证易位蛋白与宿主细胞器运输途径相互作用并促进军团菌细胞内增殖的假设。其中一种易位蛋白VipA被发现与肌动蛋白结合并促进肌动蛋白亚基的聚合。我们还将重点介绍包含卷曲线圈结构域的三种蛋白质（LegC2, LegC3, LegC7）。VipA和LegC蛋白在模型宿主酿酒酵母中表达时都会引起细胞器运输缺陷。本建议的具体目的是：1。确定VipA活性的分子基础，VipA是一种干扰内体运输的肌动蛋白结合易位效应物；2. 验证Vps/ESCRT复合物组分与军团菌细胞内增殖有关的假设；3. 效应基因的显性-负干扰等位基因及效应基因在细胞内增殖中的作用4. 鉴定控制军团菌与宿主细胞之间初始相互作用的宿主细胞酪氨酸激酶，这些相互作用是效应易位所必需的；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，如通过siRNA消耗细胞中的特定细胞器运输成分，并检查对军团菌感染的影响，已知细胞器标记物与含军团菌液泡共定位以及军团菌基因的异位表达是人类巨噬细胞系。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。我们将利用细菌遗传学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。所有这些方法都应该阐明军团菌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破坏巨噬细胞的理解，巨噬细胞是抵抗感染的主要防御。军团菌，军团病的病原体能够在巨噬细胞内生长。在感染过程中，特殊的军团菌蛋白由细菌传递到宿主巨噬细胞，在那里它们对白细胞的抗菌系统造成严重破坏。

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.