The role of Listeria cyclic-di-AMP during infection and immunity

李斯特菌环二腺苷在感染和免疫过程中的作用

• 批准号: 8234225
• 负责人: DANIEL A PORTNOY
• 金额: $ 43.31万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-15 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8234225
• 关键词: Address; Adenylate Cyclase; Adjuvant; Bacteria; Biochemistry; Cellular Immunity; Collaborations; Communicable Diseases; Data; Dendritic Cells; Dendritic cell activation; Ethylnitrosourea; Genes; Genetic Screening; Goals; Growth; Immune response; Immunity; In Vitro; Infection; Integration Host Factors; Interferons; Lead; Life; Ligands; Listeria; Listeria monocytogenes; Modeling; Molecular; Mutagenesis; Mycobacterium tuberculosis; Natural Immunity; Pathogenesis; Pathway interactions; Physiological; Production; Role; Signaling Molecule; Testing; efflux pump; macrophage; novel; pathogen; phosphoric diester hydrolase; response; therapeutic vaccine; vaccine development

This proposal is Project 1 within a P01 renewal, entitled "Intracellular pathogens and innate immunity." Listeria monocytogenes is a highly amenable model intracellular pathogen that induces robust and long-lived cell mediated immunity. The goal of the previous proposal was to identify L. monocytogenes determinants that activate a host cytosolic surveillance pathway (CSP) of innate immunity that leads to the expression of IFNB and co-regulated genes. We hypothesize that this pathway contributes significantly to the potent and long-lived cell-mediated immunity induced by L. monocytogenes. Using a genetic screen and subsequent biochemistry, we identified c-di-AMP as the listerial ligand that is secreted through a multidrug efflux pump and activates the CSP. Thus, c-di-AMP is a newly discovered PAMP. C-di-AMP is also a novel and conserved small bacterial signaling molecule that appears essential for bacterial growth. We have identified the di-adenylate cyclase (DacA) and c-di-AMP phosphodiesterase (Pde). In Aim 1, we propose to further characterize the molecular determinants that lead to expression of c-di-AMP and generate a panel of well-characterized strains that express either enhanced or diminished levels of c-di-AMP. We will also address the following questions: What are the bacterial components that regulate c-di-AMP production? Does c-di- AMP act from the inside and/or the outside of bacteria? What is the physiologic role of c-di-AMP? In Aim 2, we will use the strains constructed above to determine the role of c-di-AMP during infection of macrophages and dendritic cells in vitro. We will also evaluate the effects of purified c-di-AMP on dendritic cell activation. In collaboration with the Vance Lab (Project 3), we will use ENU mutagenesis to identify novel host factors that control the CSP. In preliminary data, we identified Sting (Stimulator of interferon genes) as a critical host component necessary for response to L. monocytogenes, M. tuberculosis, and c-di-AMP. In the final Aim, we will test the hypothesis that c-di-AMP production is essential for the full expression of L. monocytogenes pathogenesis, and importantly, contributes to the induction of cell-mediated immunity. We will examine the role of c-di-AMP as an adjuvant.

该提案是 P01 更新中的项目 1，题为“细胞内病原体和先天免疫”。 单核细胞增生李斯特氏菌是一种高度顺应的细胞内病原体模型，可诱导强健且长寿的细菌 细胞介导的免疫。先前提案的目标是确定单核细胞增生李斯特菌决定因素 激活先天免疫的宿主细胞溶质监视途径（CSP），从而导致表达 IFNB 和共同调控的基因。我们假设该途径对有效和有效的作用有显着贡献 由单核细胞增生李斯特菌诱导的长寿命细胞介导的免疫。使用基因筛选和后续 生物化学中，我们确定 c-di-AMP 是通过多药外排泵分泌的李斯特菌配体 并激活CSP。因此，c-di-AMP是新发现的PAMP。 C-di-AMP 也是一种新颖且 保守的小细菌信号分子，似乎对细菌生长至关重要。我们已经确定 二腺苷酸环化酶 (DacA) 和 c-二-AMP 磷酸二酯酶 (Pde)。在目标 1 中，我们建议进一步 表征导致 c-di-AMP 表达的分子决定因素并生成一组充分表征的 表达 c-di-AMP 水平增强或降低的菌株。我们还将针对 以下问题：调节 c-di-AMP 产生的细菌成分是什么？ c-di- 吗？ AMP 从细菌内部和/或外部起作用？ c-di-AMP 的生理作用是什么？在目标 2 中， 我们将使用上面构建的菌株来确定 c-di-AMP 在巨噬细胞感染过程中的作用 和体外树突状细胞。我们还将评估纯化的 c-di-AMP 对树突状细胞激活的影响。 与万斯实验室（项目 3）合作，我们将使用 ENU 诱变来识别新的宿主因子 控制 CSP。在初步数据中，我们将 Sting（干扰素基因刺激剂）确定为关键的 对单核细胞增生李斯特菌、结核分枝杆菌和 c-di-AMP 作出反应所必需的宿主成分。在决赛中 目标是，我们将检验 c-di-AMP 的产生对于 L 的完整表达至关重要的假设。 单核细胞增生李斯特菌的发病机制，重要的是，有助于诱导细胞介导的免疫。我们 将检查 c-di-AMP 作为佐剂的作用。