Targeting of Interferon-induced host proteins to Legionella-containing vacuoles

描述（由申请人提供）：宿主抵抗大量病原体的最重要介质之一是细胞因子干扰素γ (IFN?)。感染期间，IFN？从专门的免疫细胞中释放，并在大多数哺乳动物细胞类型中诱导多种抗菌反应途径。IFN的重要组成部分？反应是诱导细胞自主抵抗在液泡内居住和复制的细菌病原体，包括结核分枝杆菌和嗜肺军团菌。为了发挥抗菌作用，大量IFN？诱导的宿主反应蛋白如一氧化氮合酶2 （Nos2）、免疫相关gtp酶（Irg）和鸟苷酸结合蛋白（Gbp）转运到含病原体液泡（PCVs）上。宿主细胞识别pcv和靶IFN的基本原理？-诱导的宿主蛋白对它们的作用目前还不清楚。本研究计划的目标是通过定义指导IFN定位的分子参与者来填补我们知识中的空白。诱导的抗菌蛋白。为了实现这一目标，我们正在采取两种互补的基因组方法，旨在确定促进IFN易位的宿主因素？诱导蛋白转移到含军团菌液泡（LCV）或直接转移到LCV。在具体目标1中，我们将使用功能获得和功能丧失筛选方法来确定IFN？-介导的巨噬细胞内嗜肺乳杆菌复制的限制。宿主因子对IFN内嗜肺乳杆菌复制提供抗性至关重要？-活化的巨噬细胞将进行机制研究，以进一步表征其靶向IFN的作用。诱导蛋白对pcv的影响。在Aim 2中，我们将使用表位标记的激酶表达文库直接识别IFN？刺激细胞

DESCRIPTION (provided by applicant): One of the most important mediators of host resistance to a vast number of pathogens is the cytokine Interferon gamma (IFN?). During infection, IFN? is released from specialized immune cells and induces multiple antimicrobial response pathways in most mammalian cell types. An important component of the IFN? response is the induction of cell-autonomous resistance towards bacterial pathogens residing and replicating within vacuoles, including Mycobacterium tuberculosis and Legionella pneumophila. To exert their antimicrobial activities, numerous IFN? -induced host response proteins like Nitric oxide synthase 2 (Nos2), Immunity Related GTPases (Irg) and Guanylate binding proteins (Gbp) translocate to pathogen-containing vacuoles (PCVs). The principles that underlie the ability of the host cell to recognize PCVs and target IFN? -induced host proteins to them are currently not well understood. The goal of this research proposal is to fill this gap in our knowledge by defining the molecular players that direct the localization of IFN? -induced antimicrobial proteins to PCVs. Towards this goal, we are taking two complementary, genomic approaches that aim to identify host factors that either facilitate the translocation of IFN? -induced proteins to a Legionella-containing vacuole (LCV) or translocate to LCVs directly. In Specific Aim 1, we will use gain- and loss-of-function screening approaches to identify host factors required for IFN? -mediated restriction of L. pneumophila replication inside macrophages. Host factors critical for providing resistance to L. pneumophila replication inside IFN? -activated macrophages will be subjected to mechanistic studies to further characterize their role in targeting IFN? - induced proteins to PCVs. In Aim 2, we will use an epitope-tagged kinome expression library to directly identify kinases localizing to PCVs in IFN? -stimulated cells using a high-content imaging approach. This second approach will potentially allow us to identify functionally redundant host factors, as well as capture the dynamic sequence of events involved in the host response. Identification of these targeting pathways is a first and critical step towars understanding how host cells recognize PCVs as 'non-self' and potentially dangerous vesicular structures and, conversely, how host-adapted pathogens can evade recognition. This knowledge will open up avenues for the development of novel therapeutics that aim to boost the inherent ability of the host organism to detect and eliminate intracellular bacterial pathogens.