Temporal Requirements for Intracellular Pathogenesis

项目总结/摘要 单核细胞增生李斯特菌（Listeria monocytogenes，Lm）是一种典型的胞内致病菌， 孕妇、老年人和免疫功能低下者的疾病。LM使用鞭毛（游泳） 在细胞外环境小生境中存活的运动性并有助于宿主细胞的侵入。期间 在胞内感染中，Lm使用基于肌动蛋白的运动性在胞质溶胶内移动并从细胞扩散到细胞。 鞭毛和肌动蛋白为基础的运动性对环境线索的反应的相互表达是至关重要的 细胞外存活和细胞内发病机制的Lm。ActA是一种介导肌动蛋白的表面蛋白， 基于运动性，而PrfA（DNA结合转录激活因子）和5 '非翻译区（5' UTR）的 actA转录物的功能是实现ActA在胞质溶胶中的高水平区室特异性表达。在 相反，鞭毛运动在细胞内环境中受到抑制，在细胞外环境中表达。 通过涉及MogR（DNA结合转录因子）的调节级联反应响应温度的生长 阻遏物）、DegU（反应调节物）和GmaR（MogR的抗阻遏物）。本提案的重点 阐明鞭毛和肌动蛋白相互调节的分子机制， 对温度和细胞内环境作出反应的运动。在目标I中， 用于在胞质溶胶中区室特异性表达的actA的PrfA调节的启动子和5 'UTR将被克隆。 测定这将在野生型和突变株中使用actA-gfp报告基因融合物来实现 在细胞内感染的特定阶段有缺陷的。定量荧光和时间- 延时视频显微镜将用于确定单个细菌内表达的水平和动力学， 细胞在目的II中，控制DegU介导的gmaR转录激活的特异性辅助因子， 将使用DNA亲和纯化和随机转座子鉴定对低温的响应 诱变将评价辅助因子突变株的GmaR表达和鞭毛运动性。 控制gmaR的温度依赖性活化的蛋白质/DNA相互作用将通过以下方法确定： 共亲和纯化和凝胶迁移率变动分析。在目标III中，我们将定义精确的结构和 通过直接GmaR：MogR相互作用介导抗阻遏的机制特征。共亲和纯化 分析具有全长GmaR的MogR截短蛋白并评估GmaR截短蛋白用于 将进行鞭毛运动性的互补。在Aim IV中，深入了解宿主来源的信号， 将通过分级分离和纯化血清来源的蛋白质来确定被感测以调节ActA的表达。 激活actA-gfp融合构建体的组分。鞭毛表达级联的精确步骤 在低温下胞内感染期间被抑制的蛋白质将通过表征 DegU、辅助因子和GmaR在细胞内感染期间的产生和功能。项目叙述 单核细胞增生李斯特菌是一种细菌病原体，生长在人体细胞内， 食源性疾病在食品加工环境中的持久性和人类细胞的感染是由以下因素促进的： 细菌响应于感知环境信号（例如细胞外信号）而变得能动的能力 温度和细胞内环境。拟议的研究将确定分子 机制，L。单核细胞增多症用于调节运动性以响应环境线索，因此 为细菌致病的基本机制提供了有价值的见解。

Project Summary/Abstract Listeria monocytogenes (Lm) is a model intracellular bacterial pathogen that causes serious foodborne illness in pregnant women, the elderly, and immunocompromised individuals. Lm uses flagellar (swimming) motility to survive in extracellular environmental niches and aid in the invasion of host cells. During intracellular infection, Lm uses actin-based motility to move within the cytosol and spread from cell-to-cell. Reciprocal expression of flagellar and actin-based motility in response to environmental cues is critical to extracellular survival and intracellular pathogenesis of Lm. ActA is a surface protein that mediates actin- based motility, while PrfA (a DNA binding transcriptional activator) and the 5' untranslated region (5' UTR) of actA transcripts function to achieve high-level compartment-specific expression of ActA in the cytosol. In contrast, flagellar motility is repressed in the intracellular environment and expressed during extracellular growth in response to temperature via a regulatory cascade involving MogR (a DNA binding transcriptional repressor), DegU (a response regulator), and GmaR (an anti-repressor for MogR). The focus of this proposal is to elucidate the molecular mechanisms governing the reciprocal regulation of flagellar and actin-based motility in response to temperature and the intracellular environment. In Aim I, the precise contribution of the PrfA-regulated promoter and 5' UTR of actA for compartment-specific expression in the cytosol will be determined. This will be accomplished using actA-gfp reporter fusions in wild-type and mutant strains defective for progression through specific stages of intracellular infection. Quantitative fluorescence and time- lapse video microscopy will be used to define the level and kinetics of expression within individual bacterial cells. In Aim II, specific accessory factors that govern DegU-mediated transcriptional activation of gmaR in response to low temperature will be identified using DNA affinity purification and random transposon mutagenesis. Accessory factor mutant strains will be evaluated for GmaR expression and flagellar motility. The protein/DNA interactions controlling temperature-dependent activation of gmaR will be determined by co-affinity purification and gel mobility shift analysis. In Aim III, we will define the precise structural and mechanistic features that mediate anti-repression via direct GmaR:MogR interaction. Co-affinity purification analysis of MogR truncation proteins with full-length GmaR and evaluation of GmaR truncation proteins for complementation of flagellar motility will be performed. In Aim IV, insight into host-derived signals that are sensed to regulate expression of ActA will be determined by fractionation and purification of serum-derived components that activate actA-gfp fusion constructs. The precise step in the flagellar expression cascade that is inhibited during intracellular infection at low temperature will be determined by characterizing production and function of DegU, accessory factors, and GmaR during intracellular infection. Project Narrative Listeria monocytogenes is a bacterial pathogen that grows inside of human cells and causes serious foodborne illness. Persistence in food processing environments and infection of human cells is facilitated by the ability of bacteria to become motile in response to sensing environmental signals such as extracellular temperature and the intracellular environment. The proposed studies will determine the molecular mechanisms that L. monocytogenes uses to regulate motility in response to environmental cues, thus providing valuable insight into fundamental mechanisms of bacterial pathogenesis.