Genetics of Monocyte Killing by Bacteria

项目总结 这项建议的重点是了解嗜肺军团菌能力的分子基础 感染、存活、复制并最终杀死人类巨噬细胞。我们建议研究一组 嗜肺乳杆菌制造的通过ICM/Dot易位转移到宿主细胞的新蛋白 系统。我们将检验这一假设，即转位的蛋白质与细胞器运输途径相互作用。 并有助于军团菌胞内增殖。其中一种易位蛋白VIPA是 发现能结合肌动蛋白并促进肌动蛋白亚基的聚合。我们还将重点研究三种蛋白质(LegC2， LegC3、LegC7)，其包含盘绕的线圈结构域。VIPA和LegC蛋白都能引起细胞器运输 在模式宿主酿酒酵母中表达的缺陷。这项提议的具体目的是 目的：1.确定VIPA活性的分子基础，VIPA是一种肌动蛋白结合、易位的效应器， 干扰内体转运；2.检验VPS/ESCRT复合体的成分是 与军团菌胞内增殖相关事件；3.军团菌显性-负性干扰等位基因 效应基因及其在细胞内增殖中的作用；4.宿主细胞酪氨酸激酶的鉴定 控制效应器转位所需的军团菌与宿主细胞之间的初始相互作用； 确定LegC2、LegC3和LegC7的交互合作伙伴。为了实现这些目标，我们将采取 各种细胞生物学工具的优势，例如特定细胞器的耗尽细胞运输 通过siRNA和检测对军团菌感染的影响，共定位已知的细胞器 含有军团菌空泡和军团菌基因异位表达的标记是人类的 巨噬细胞系。我们还将研究特定靶向宿主酪氨酸激酶和一个 磷酸酶对军团菌胞内增殖和细胞器转运的影响。我们将使用细菌遗传学 分离编码易位蛋白的基因的显性负等位基因，以更好地了解它们的 在军团菌感染过程中的作用。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。

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.