Temporal Requirements for Intracellular Pathogenesis

描述（由申请人提供）：单核增生李斯特菌（Lm）是一种典型的细胞内细菌病原体，在孕妇、老年人和免疫功能低下的个体中引起严重的食源性疾病。Lm利用鞭毛（游泳）运动在细胞外环境中生存，并帮助入侵宿主细胞。在细胞内感染期间，Lm利用基于肌动蛋白的运动在细胞质内移动并在细胞间传播。鞭毛和肌动蛋白运动响应环境信号的相互表达对Lm的细胞外存活和细胞内发病至关重要。ActA是一种介导基于肌动蛋白的运动的表面蛋白，而PrfA（一种DNA结合转录激活因子）和ActA转录本的5‘未翻译区（5’ UTR）的功能是实现ActA在细胞质中高水平的区室特异性表达。相比之下，鞭毛运动在细胞内环境中受到抑制，并在细胞外生长过程中响应温度，通过涉及MogR （DNA结合转录抑制因子）、DegU（反应调节因子）和GmaR （MogR的抗抑制因子）的调节级联表达。本研究的重点是阐明鞭毛和肌动蛋白运动在响应温度和细胞内环境时相互调节的分子机制。在Aim I中，将确定prfa调控的启动子和actA的5' UTR在细胞质中对室特异性表达的精确贡献。这将通过分析天然ActA蛋白的表达和转录水平，在细胞内感染的特定阶段有进展缺陷的野生型和突变株中使用ActA -gfpmut2报告基因融合来完成。定量荧光和延时视频显微镜将用于确定单个细菌细胞内表达的水平和动力学。在Aim II中，将使用DNA亲和纯化和随机转座子诱变来鉴定控制degu介导的gmaR转录激活以响应低温的特定辅助因子。辅助因子突变株将评估GmaR表达和鞭毛运动性。控制gmaR温度依赖性激活的蛋白质/DNA相互作用将通过共亲和纯化和凝胶迁移分析来确定。低温下细胞内感染时鞭毛表达级联被抑制的精确步骤将通过细胞内感染时DegU、辅助因子和GmaR的产生和功能来确定。在Aim III中，我们将定义通过直接GmaR:MogR相互作用介导抗抑制的精确结构和机制特征。将进行MogR截断蛋白与全长GmaR共亲和纯化分析，并评估GmaR截断蛋白对鞭毛运动的互补作用。单核增生李斯特菌是一种生长在人体细胞内的细菌病原体，可引起严重的食源性疾病。细菌能够对感知环境信号（如细胞外温度和细胞内环境）做出反应，从而变得能动，从而促进了食物加工环境中的持久性和人类细胞的感染。所提出的研究将确定单核增生乳杆菌在响应环境信号时调节运动的分子机制，从而为细菌发病的基本机制提供有价值的见解。

DESCRIPTION (provided by applicant): 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-gfpmut2 reporter fusions in wild-type and mutant strains defective for progression through specific stages of intracellular infection and by analysis of native ActA protein expression and transcript levels. 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. 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. 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. 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.