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

干扰素诱导的宿主蛋白靶向含有军团菌的液泡

• 批准号: 8284634
• 负责人: Joern Coers
• 金额: $ 19.18万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8284634
• 关键词: Alveolar Macrophages; Bacteria; Binding Proteins; Breathing; Cells; Complement; Development; Drug Delivery Systems; Epitopes; Event; Expression Library; Failure; Genes; Genetic; Genetic Screening; Genomics; Goals; Growth; Guanosine Triphosphate Phosphohydrolases; Helicobacter pylori; Host resistance; Human; Image; Image Analysis; Immune; Immune Targeting; Immune response; Immunity; Immunocompetent; Immunocompromised Host; Individual; Infection; Integration Host Factors; Interferon Type II; Interferons; Knowledge; Legionella; Legionella pneumophila; Legionnaires' Disease; Lung; Mammalian Cell; Mediating; Mediator of activation protein; Microbe; Molecular; Monitor; Mycobacterium tuberculosis; Nature; Nitric Oxide Synthase; Organism; Pathway interactions; Phagocytes; Phagocytosis; Phosphotransferases; Play; Pneumonia; Proteins; RNA Interference; Research Proposals; Resistance; Role; Salmonella enterica; Salmonella typhimurium; Screening procedure; Soil; Structure; Testing; Time; Vacuole; aerosolized; antimicrobial; bactericide; base; cell type; cytokine; gain of function; guanylate; killings; loss of function; macrophage; novel; novel strategies; novel therapeutics; overexpression; pathogen; response; sensor

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.

描述（由申请方提供）：宿主对大量病原体耐药的最重要介质之一是细胞因子干扰素γ（IFN？）。在感染过程中，干扰素？从特化免疫细胞中释放，并在大多数哺乳动物细胞类型中诱导多种抗微生物反应途径。干扰素的重要组成部分？免疫应答是诱导细胞自主抵抗存在于空泡内并在空泡内复制的细菌病原体，包括结核分枝杆菌和嗜肺军团菌。发挥其抗菌活性，许多干扰素？- 诱导的宿主应答蛋白如一氧化氮合酶2（Nos 2）、免疫相关GTP酶（Irg）和鸟苷酸结合蛋白（Gbp）易位至含病原体的空泡（PCV）。宿主细胞识别PCV和靶向IFN的能力的原理？诱导的宿主蛋白对它们的作用目前还不清楚。这项研究的目的是填补这一空白，在我们的知识，通过定义的分子球员，直接定位的干扰素？- 诱导的抗微生物蛋白质对PCV。为了实现这一目标，我们正在采取两个互补的，基因组的方法，目的是确定主机的因素，无论是促进易位的干扰素？- 诱导的蛋白质转移到含军团菌的液泡（LCV）或直接转移到LCV。在具体目标1，我们将使用增益和功能丧失的筛选方法，以确定所需的IFN？- 介导的L.嗜肺菌在巨噬细胞内复制。宿主因子是提供抗L.嗜肺菌在IFN内复制？活化的巨噬细胞将受到机制的研究，以进一步表征其作用，针对干扰素？- 诱导蛋白质转化为PCV。在目标2中，我们将使用表位标记的激酶组表达文库，直接确定在IFN？- 刺激细胞 使用高内涵成像方法。这第二种方法将有可能使我们能够识别功能冗余的主机因素，以及捕捉动态序列的事件参与主机的反应。识别这些靶向通路是理解宿主细胞如何将PCV识别为“非自身”和潜在危险的囊泡结构以及相反地，宿主适应的病原体如何逃避识别的第一步和关键步骤。这些知识将为开发新的治疗方法开辟道路，这些治疗方法旨在提高宿主生物体检测和消除细胞内细菌病原体的固有能力。 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递送到含有病原体（如嗜肺军团菌）的空泡的关键宿主因素。在这些研究中确定的宿主因素可能在宿主对许多病原体（包括结核分枝杆菌和肠道沙门氏菌）的耐药性中发挥重要作用，并可能作为新型基于宿主的抗菌治疗的药物靶点。