Temporal Requirements for Intracellular Pathogenesis

项目摘要/摘要 单核细胞增多性李斯特菌是一种典型的细胞内致病菌，可引起严重的食源性疾病 孕妇、老年人和免疫功能低下者的疾病。LM使用鞭毛(游泳) 在细胞外环境中生存的能动性，并帮助宿主细胞入侵。在.期间 在细胞内感染时，LM使用基于肌动蛋白的运动性在胞浆内移动，并在细胞间传播。 鞭毛和基于肌动蛋白的运动性对环境提示的反应是关键的 肺泡巨噬细胞的细胞外存活和细胞内发病机制。ActA是一种表面蛋白，介导肌动蛋白- 基于运动性，而PrFA(一种DNA结合转录激活子)和5‘非翻译区(5’UTR) ActA转录本的功能是实现ActA在细胞质中的高水平隔室特异性表达。在……里面 相反，鞭毛的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)的调节级联反应来响应温度的生长 抑制因子)、DECU(反应调节因子)和GmaR(MogR的反抑制因子)。这项提案的重点是 旨在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性。在Aim I中， PrFA调控的启动子和ActA的5‘UTR将在细胞质中进行隔室特异性表达 下定决心。这将通过在野生型和突变株中进行Acta-GFP报告融合来实现 在细胞内感染的特定阶段有进展的缺陷。定量荧光和时间- 失效视频显微镜将被用来确定单个细菌内表达的水平和动力学 细胞。在AIM II中，控制DECU介导的gmaR转录激活的特定辅助因子在 利用DNA亲和纯化和随机转座子鉴定对低温的响应 诱变。将对辅助因子突变株进行GmaR表达和鞭毛运动的评估。 控制gmaR依赖于温度的激活的蛋白质/DNA相互作用将由 共亲和纯化及凝胶迁移率漂移分析。在目标III中，我们将定义精确的结构和 通过直接GmaR：MogR相互作用调节反压抑的机械性特征。共亲和纯化 含全长GmaR的MogR截断蛋白的分析及GmaR截断蛋白的评价 鞭毛运动的补充性将被执行。在目标IV中，深入了解宿主派生的信号 Acta的表达调控将通过血清来源的分离和纯化来确定 激活Acta-GFP融合结构的组件。鞭毛表达级联中的精确步骤 在低温下细胞内感染期间被抑制的将通过以下特征来确定 细胞内感染过程中DECU、辅助因子和GmaR的产生和功能。项目叙事 单核细胞增多性李斯特菌是一种生长在人类细胞内的细菌病原体，它会导致严重的 食源性疾病。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 细菌对感知环境信号，如胞外信号作出反应而变得能动的能力 温度和细胞内环境。拟议中的研究将确定分子 单核细胞增多性李斯特氏菌用来调节运动以响应环境提示的机制，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.