Project 1: Innate immune responses triggered by Listeria monocytogenes

项目概要/摘要（项目1，Portnoy） 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”。一 我们要解决的中心问题是宿主先天免疫系统如何识别胞内病原体 系统以及如何整合多个信号以诱导适当的响应，以及相反，如何 病原体避免和/或操纵宿主反应以促进它们的发病机制。在项目1中，我们有 选择通过继续详细分析单核细胞增生李斯特菌来解决这个问题， 细胞内，食源性病原体，已被研究了几十年，作为一个模型系统， 剖析感染和免疫的基本方面。以前，我们专注于巨噬细胞转录 对感染的反应，并了解到L。单核细胞增多症从吞噬体中逃脱并分泌 一种称为c-di-AMP的保守且必需的小信号分子，其激活宿主的转录 I型干扰素反应。在与万斯实验室的合作中，我们确定STING为宿主细胞受体 对于c-di-AMP和其他c-di-nucleotides。在目标1中，我们建议扩展这些研究，使用细菌 与小鼠和宿主细胞组合合成或分泌改变水平的c-di-AMP的突变体 具有特定的STING突变，可以改变下游信号。在目标2和3中，我们开始探索 转录后宿主反应的作用，包括细菌和宿主蛋白的泛素化 以及自噬所扮演的角色。在目标2中，我们对初步数据进行了跟踪，这些数据表明L。 单核细胞增多症是受吞噬体自噬，但规避它使用两个决定因素，PlcA 的ActA。我们发现，一个双PlcA/ActA突变体被似乎是LC 3相关的 吞噬作用（phagocytosis，E. coli）。我们将使用细菌和宿主突变体来确定这是否是真的， 细菌避免它，最后，在感染和免疫过程中，细菌避免它的作用是什么。在目标3中， 建议使用基于质谱和生物信息学的工具，在核心C中可用，以确定 泛素化的细菌和宿主蛋白，以及高通量遗传筛选以鉴定宿主 介导泛素化的蛋白质。

Project Summary/Abstract (Project 1, Portnoy) This proposal is Project 1 within a P01 renewal, entitled “Intracellular pathogens and innate immunity.” A central problem that we address is how intracellular pathogens are recognized by the host innate immune system and how multiple signals are integrated to induce an appropriate response, and conversely, how pathogens avoid and/or manipulate host responses to promote their pathogenesis. In Project 1, we have chosen to approach this problem by continuing a detailed analysis of Listeria monocytogenes, a facultative intracellular, food-borne pathogen that has been studied for decades as a model system with which to dissect basic aspects of infection & immunity. Previously, we concentrated on macrophage transcriptional responses to infection and learned that L. monocytogenes escapes from a phagosome and secretes a conserved and essential small signaling molecule called c-di-AMP that activates the transcription of the host type I interferon response. In collaboration with the Vance lab, we identified STING as the host cell receptor for both c-di-AMP and other c-di-nucleotides. In Aim 1, we propose to extend these studies, using bacterial mutants that either synthesize or secrete altered levels of c-di-AMP in combination with mice and host cells that have specific STING mutations that alter downstream signaling. In Aims 2 and 3, we begin to explore the role of post-transcriptional host responses including ubiquitylation of both bacterial and host proteins and the role played by autophagy. In Aim 2, we follow-up on preliminary data showing that L. monocytogenes is subject to autophagy at the phagosome, but circumvents it using two determinants, PlcA and ActA. We show that a double PlcA/ActA mutant is trapped by what appears to be LC3-associated phagocytosis (LAP). We will use bacterial and host mutants to determine if this is in fact LAP, how the bacteria avoid it, and finally, what is the role of LAP avoidance during infection and immunity. In Aim 3, we propose using mass spectrometry-based and bioinformatic tools, available in Core C, to identify the bacterial and host proteins that are ubiquitylated, and high throughput genetic screens to identify the host proteins that mediate ubiquitylation.