Genetics of Monocyte Killing by Bacteria

描述(由申请人提供)：这项提案的重点是了解嗜肺军团菌感染、存活、复制并最终杀死人类巨噬细胞的分子基础。我们打算研究一组由嗜肺乳杆菌制造并通过ICM/Dot转位系统转位到宿主细胞的新蛋白。我们将检验这一假设，即转位的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增殖。其中一种转位蛋白VIPA被发现与肌动蛋白结合并促进肌动蛋白亚基的聚合。我们还将重点介绍三种含有卷曲结构域的蛋白质(LegC2、LegC3、LegC7)。当VIPA和LegC蛋白在模式宿主酿酒酵母中表达时，都会导致细胞器运输缺陷。这项建议的具体目的是：1.确定VIPA活性的分子基础，VIPA是一种肌动蛋白结合的易位效应器，干扰内体运输；2.检验VPS/ESCRT复合体的成分与军团菌细胞内增殖事件相关的假设；3.效应基因的显性-负性干扰等位基因以及效应器在细胞内增殖中的作用；4.识别控制军团菌与宿主细胞之间最初相互作用的宿主细胞酪氨酸激酶；5.确定LegC2、LegC3和LegC7的相互作用伙伴。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耗尽特定细胞器运输成分的细胞并检测其对军团菌感染的影响，将已知的细胞器标记与含军团菌的液泡共定位，以及在人巨噬细胞系中异位表达军团菌基因。我们还将研究特定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澄清军团菌用来避免被巨噬细胞杀死并成功感染的机制。与公共卫生相关：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，巨噬细胞是抵御感染的主要防御措施。军团菌是军团病的病原体，能够在巨噬细胞内生长。在感染期间，特殊的军团菌蛋白被细菌传递到宿主巨噬细胞，在那里它们破坏了白细胞的抗菌系统。

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.