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，标题为“细胞内病原体和先天免疫”。 单核细胞增生李斯特菌是一种高度可及的模型细胞内病原体，可诱导健壮且长期寿命 细胞介导的免疫力。先前建议的目的是识别单核细胞增生李斯特菌的决定因素 激活先天免疫的宿主胞质监视途径（CSP），导致表达 IFNB和共同调节的基因。我们假设该途径对有效和 长乳杆菌诱导的长寿命细胞介导的免疫。使用遗传屏幕和随后的 生物化学，我们将C-Di-Amp确定为通过多药外排泵分泌的层次配体 并激活CSP。因此，C-Di-Amp是新发现的弹药。 C-Di-Amp也是一部小说， 保守的小细菌信号传导分子似乎对细菌生长至关重要。我们已经确定了 二苯基酸环化酶（DACA）和C-DI-AMP磷酸二酯酶（PDE）。在AIM 1中，我们建议进一步 表征导致C-DI-AMP表达并产生一个良好特征的分子决定因素 表达增强或减少C-DI-AMP的菌株。我们还将解决 以下问题：调节C-DIAMP生产的细菌成分是什么？ c-di- 放大器从内部和/或细菌的外部起作用？ C-DI-AMP的生理作用是什么？在AIM 2中， 我们将使用上面构造的菌株来确定C-DI-AMP在巨噬细胞感染中的作用 和树突状细胞体外。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诱变来识别新型宿主因素 控制CSP。在初步数据中，我们将刺刺（干扰素基因的刺激剂）确定为关键 响应单核细胞增生李斯特菌，结核分枝杆菌和C-DI-AMP所需的宿主成分。在决赛中 目的，我们将检验以下假设：C-DI-AMP生产对于L的全部表达至关重要。 单核细胞增生的发病机理，重要的是，有助于诱导细胞介导的免疫。我们 将检查C-DI-AMP作为辅助的作用。