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

这项提案是P01更新中的项目1，题为“细胞内病原体和先天免疫”。 单核细胞增多性李斯特菌是一种高度顺应性的细胞内致病菌，它能诱导强健和长寿。 细胞免疫。先前提案的目标是确定单核细胞增多性李斯特菌的决定因素。 激活先天免疫的宿主胞浆监视途径(CSP)，从而导致 IFNB和共同调控基因。我们假设，这一途径对有效和 单核细胞增多性李斯特菌诱导的长寿细胞免疫。使用基因筛查和随后的 生物化学，我们确定c-diamp是通过多药外排泵分泌的李斯特配体。 并激活CSP。因此，c-di-AMP是一种新发现的PAMP。C-di-AMP也是一种新颖的 保守的小细菌信号分子，似乎是细菌生长所必需的。我们已经确定了 二腺苷环化酶(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，并产生一组表征良好的 表达c-diamp水平增强或降低的菌株。我们还将解决 以下问题：调节c-di-AMP生产的细菌成分是什么？是不是c-di- AMP从细菌的内部和/或外部起作用？C-diAMP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-diamp在巨噬细胞感染过程中的作用。 以及体外培养的树突状细胞。我们还将评估纯化的c-diAMP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突变来识别新的宿主因子 它们控制着CSP。在初步数据中，我们发现Sting(干扰素基因刺激物)是一个关键的 对单核细胞增多性乳杆菌、结核分枝杆菌和c-二-AMP反应所必需的宿主成分。在决赛中 目的验证c-diAMP的产生对L. 单核细胞增多症的发病机制，以及重要的是，有助于诱导细胞免疫。我们 将研究c-二-AMP作为佐剂的作用。

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.