Genetics of Monocyte Killing by Bacteria

描述（由申请方提供）：本提案的重点是了解嗜肺军团菌感染人巨噬细胞、在其中存活、在其中复制并最终杀死人巨噬细胞的能力的分子基础。我们打算研究一组由L. pneumophila中的蛋白质通过Icm/Dot易位系统易位到宿主细胞中。我们将测试的假设，易位的蛋白质相互作用的细胞器运输途径在主机和军团菌细胞内增殖。其中一种转位蛋白VipA被发现结合肌动蛋白并促进肌动蛋白亚基的聚合。我们还将关注三种含有卷曲螺旋结构域的蛋白质（LegC 2，LegC 3，LegC 7）。VipA和LegC蛋白在模型宿主酿酒酵母中表达时均引起细胞器运输缺陷。这项建议的具体目标是：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，可干扰内体运输; 2.检验Vps/ESCRT复合物的组分与军团菌细胞内增殖过程中的事件相关的假设; 3.效应基因的显性负性干扰等位基因及效应子在细胞内增殖中的作用; 4.确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，我们将利用各种细胞生物学工具，例如通过siRNA消耗细胞的特定细胞器运输组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的空泡的共定位以及军团菌基因在人巨噬细胞系中的异位表达。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负等位基因，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用于避免巨噬细胞杀死并产生成功感染的机制。公共卫生相关性：这项拟议中的研究将增加对细菌病原体如何破坏巨噬细胞的理解，巨噬细胞是对抗感染的主要防御。军团菌是军团菌病的病原体，能够在巨噬细胞内生长。在感染期间，特化的军团菌蛋白质被细菌递送到宿主巨噬细胞，在那里它们对白色细胞的抗微生物系统造成严重破坏。

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.