权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Temporal Requirements for Intracellular Pathogenesis

细胞内发病机制的时间要求

基本信息

批准号：
8073203
负责人：
DARREN E HIGGINS
金额：
$ 39.14万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-07-01 至 2013-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8073203
关键词：
5&apos Untranslated Regions Actins Affinity Chromatography Bacteria Biological Assay Cells Cues Cytosol DNA DNA Binding DNA Binding Domain DNA-Protein Interaction Elderly Environment Evaluation Flagellin Fluorescence Fluorescence Microscopy Food Processing Fractionation Gel Gel Chromatography Genetic Transcription Growth High Pressure Liquid Chromatography Human Immunocompromised Host Individual Infection Ion Exchange Kinetics LacZ Genes Length Link Listeria monocytogenes Mediating Membrane Proteins Microbial Biofilms Modeling Molecular Mutagenesis Nature Nutrient Octodon Pathogenesis Phagosomes Pregnant Women Production Promoter Regions Protein Truncation Proteins Regulation Reporter Repression Serum Signal Transduction Staging Swimming Temperature Time Transcript Transcription Coactivator Transcription Repressor/Corepressor Transcriptional Activation Up-Regulation Video Microscopy Western Blotting base cell motility cold temperature extracellular fast protein liquid chromatography foodborne illness genetic regulatory protein glycosyltransferase human NCYM protein insight mutant pathogen prevent promoter protein expression protein protein interaction response

项目摘要

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.

描述(由申请人提供)：单核细胞增生性李斯特菌(Lm)是一种典型的细胞内细菌病原体，可导致孕妇、老年人和免疫功能低下的人患严重的食源性疾病。LM利用鞭毛(游动)运动在细胞外环境中生存，并帮助宿主细胞入侵。在细胞内感染期间，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信号的响应是LM细胞外生存和细胞内发病的关键。ActA是一种表面蛋白，介导基于肌动蛋白的运动，而PrFA(DNA结合转录激活子)和ActA转录本的5‘非翻译区(5’UTR)的功能是实现ActA在细胞质中的高水平隔室特异性表达。相反，鞭毛的运动在细胞内环境中受到抑制，并在细胞外生长过程中对温度做出反应，通过涉及MogR(DNA结合转录抑制因子)、DECU(反应调节因子)和GmaR(MogR的反抑制因子)的调节级联来表达。这一建议的重点是阐明鞭毛和基于肌动蛋白的运动性对温度和细胞内环境的相互调节的分子机制。在目标I中，将确定PRFA调节的启动子和ActA的5‘UTR对细胞质中隔室特异性表达的准确贡献。这将通过在野生型和突变型菌株中融合Acta-gfpmu2报告来实现，这些菌株在细胞内感染的特定阶段存在缺陷，并通过分析本地Acta蛋白表达和转录水平来实现这一点。定量荧光和延时视频显微镜将被用来定义单个细菌细胞内表达的水平和动力学。在AIM II中，将通过DNA亲和纯化和随机转座子突变来鉴定控制DECU介导的gmaR在低温下转录激活的特定辅助因子。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率移动分析来确定。在低温下细胞内感染期间鞭毛表达级联中被抑制的确切步骤将通过表征细胞内感染期间DECU、辅助因子和GmaR的产生和功能来确定。在目标三中，我们将定义通过直接GmaR：MogR相互作用来调节反压制的精确结构和机械特征。将进行mogR截断蛋白与全长GmaR的共亲和纯化分析，并评价用于补充鞭毛运动的gmaR截断蛋白。项目简介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，会导致严重的食源性疾病。细菌对细胞外温度和细胞内环境等环境信号的感知能力有助于食品加工环境中的持久性和人类细胞的感染。这些拟议的研究将确定单核细胞增多性李斯特菌用来调节运动以响应环境提示的分子机制，从而为细菌致病的基本机制提供有价值的见解。