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

描述(申请人提供)：宿主对大量病原体的抗性最重要的媒介之一是细胞因子干扰素？在感染过程中，干扰素？从专门的免疫细胞中释放出来，并在大多数哺乳动物细胞类型中诱导多种抗菌素反应途径。干扰素的重要成分？应答是诱导细胞对居住和复制在空泡中的细菌病原体产生抵抗力，包括结核分枝杆菌和嗜肺军团菌。为了发挥其抗菌活性，许多干扰素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)和鸟苷结合蛋白(GBP)被转移到含有病原体的空泡(PCV)。宿主细胞识别PCV并针对干扰素诱导的宿主蛋白的能力的原理目前还不是很清楚。这项研究提案的目标是通过定义将干扰素诱导的抗菌蛋白定位到PCV的分子角色来填补我们知识中的这一空白。为了实现这一目标，我们采取了两种互补的基因组方法，旨在确定促进干扰素诱导的蛋白转位到军团菌含液泡(LCV)或直接转位到LCV的宿主因子。在具体目标1中，我们将使用获得和功能丧失的筛选方法来确定干扰素？介导的限制嗜肺乳杆菌在巨噬细胞内复制所需的宿主因素。在干扰素激活的巨噬细胞内提供对肺炎链球菌复制的抵抗力的宿主因素将受到机制研究，以进一步表征它们在将干扰素诱导的蛋白靶向PCV中的作用。在目标2中，我们将使用一个表位标记的Kinome表达文库来直接在干扰素刺激的细胞中鉴定定位于PCV的激酶。 使用高含量成像方法。第二种方法可能使我们能够识别功能上的冗余主机因素，以及捕获主机响应中涉及的事件的动态序列。识别这些靶向途径是了解宿主细胞如何识别PCV为“非我”和潜在危险的囊泡结构的第一步，也是关键的一步，反过来，宿主适应的病原体如何逃避识别。这一知识将为开发旨在提高宿主机体检测和消除细胞内细菌病原体的固有能力的新疗法开辟道路。 公共卫生相关性：该提案旨在部署新的(基于基因组学的)方法，以确定负责将抗菌蛋白输送到含有病原体(如嗜肺军团菌)的空泡的关键宿主因素。这些研究中确定的宿主因子可能在宿主对包括结核分枝杆菌和肠道沙门氏菌在内的许多病原体的耐药性中发挥重要作用，并可能成为基于宿主的新型抗菌疗法的药物靶点。

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. PUBLIC HEALTH RELEVANCE: This proposal aims to deploy new (genomics-based) approaches to identify the key host factors responsible for the delivery of antimicrobial proteins to vacuoles that contain pathogens such as the bacterium Legionella pneumophila. Host factors identified in these studies are likely to play an important role in host resistance to many pathogens, including Mycobacterium tuberculosis and Salmonella enterica, and may serve as drug targets for novel host-based antimicrobial therapies.