Temporal Requirements for Intracellular Pathogenesis

描述（由申请方提供）：单核细胞增生李斯特菌（Lm）是一种模型细胞内细菌病原体，可导致孕妇、老年人和免疫功能低下个体发生严重食源性疾病。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。在胞内感染期间，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响应环境线索的相互表达是至关重要的细胞外生存和细胞内发病的Lm。ActA是介导基于肌动蛋白的运动性的表面蛋白，而PrfA（DNA结合转录激活因子）和actA转录物的5'非翻译区（5' UTR）用于实现ActA在胞质溶胶中的高水平区室特异性表达。相反，鞭毛运动性在细胞内环境中受到抑制，并在细胞外生长期间通过涉及MogR（DNA结合转录阻遏物）、DegU（反应调节剂）和GmaR（MogR的抗阻遏物）的调节级联反应响应于温度而表达。该建议的重点是阐明鞭毛和肌动蛋白为基础的运动响应于温度和细胞内环境的相互调节的分子机制。在目的I中，将确定PrfA调节的启动子和actA的5' UTR对于胞质溶胶中的区室特异性表达的精确贡献。这将通过在细胞内感染的特定阶段的进展缺陷的野生型和突变株中使用actA-gfpmut 2报告基因融合，并通过分析天然ActA蛋白表达和转录水平来实现。将使用定量荧光和延时视频显微镜来确定单个细菌细胞内的表达水平和动力学。在目的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转录激活的特异性辅助因子。将评价辅助因子突变株的GmaR表达和鞭毛运动性。通过共亲和纯化和凝胶迁移率变动分析确定控制gmaR的温度依赖性活化的蛋白质/DNA相互作用。在低温下细胞内感染期间被抑制的鞭毛表达级联中的精确步骤将通过表征细胞内感染期间DegU、辅助因子和GmaR的产生和功能来确定。在目的III中，我们将定义通过直接GmaR：MogR相互作用介导抗阻遏的精确结构和机制特征。将进行MogR截短蛋白与全长GmaR的共亲和纯化分析，并评价GmaR截短蛋白对鞭毛运动性的互补作用。单核细胞增生李斯特菌（Listeria monocytogenes）是一种生长在人体细胞内的细菌病原体，可导致严重的食源性疾病。在食品加工环境中的持久性和人类细胞的感染是由细菌响应于感测环境信号（例如细胞外温度和细胞内环境）而变得能动的能力促进的。这些研究将有助于确定L.单核细胞增多症用于调节运动性，以响应环境线索，从而提供了有价值的洞察细菌致病的基本机制。

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.