Listeria Virulence Gene Expression Within Host Cells

性状（由申请方提供）：单核细胞增生李斯特菌是一种兼性胞质内细菌病原体，是人类严重食源性感染的重要病原体。作为一种常见于环境中的细菌，L.单核细胞增多症能够介导从土壤中的生命到其生存和复制的人类细胞中的生命的复杂过渡。该提案旨在确定环境细菌介导从土壤中的外部生命到哺乳动物细胞内生命的转换所需的条件。正在进行的工作集中在一个关键的转录激活因子的毒力基因表达的L。称为PrfA的单核细胞增多症。PrfA是迄今发现的几乎所有单核细胞增多性李斯特菌毒力因子表达所必需的，然而，李斯特菌使用的机制并不清楚。单核细胞增多症协调宿主细胞内基因产物表达的机制尚不清楚。PrfA以高活性和低活性状态存在，该提议的工作假设是PrfA活化的调节对于L.单核细胞增多症成功地从外部环境过渡到哺乳动物细胞内的生活。本研究将结合遗传学和生物化学的方法来阐明L。单核细胞增多症调节宿主细胞内毒力基因的表达。它将检查PrfA激活的致病后果，并将定义在宿主细胞内诱导的细菌基因产物，这些产物直接或间接依赖于PrfA的表达。目的1将集中在突变激活形式的PrfA的生化分析和评估的影响PrfA激活L。单核细胞增生生理学和发病机制。目的2阐明新发现的PrfA依赖基因产物对L.单核细胞增多症发病机制目的3进行功能特性的突变位于以外的prfA基因座，改变单核细胞增生李斯特菌毒力基因表达的调节。拟议工作的最终目标是阐明使细菌在宿主细胞内存活的调控途径，以及支持宿主内复制的其他细菌因子的功能鉴定。

DESCRIPTION (provided by applicant): Listeria monocytogenes is a facultative intra-cytoplasmic bacterial pathogen that is an important agent of serious human food-borne infections. As a bacterium commonly found in the environment, L. monocytogenes is able to mediate the complex transition from life in the soil to life in human cells where it survives and replicates. This proposal seeks to determine what is required for an environmental bacterium to mediate the switch from outside life in the soil to life inside of mammalian cells. On-going work has focused on a key transcriptional activator of virulence gene expression in L. monocytogenes known as PrfA. PrfA is required for the expression of nearly every L monocytogenes virulence factor identified to date, however the mechanisms used by L. monocytogenes to coordinate the expression of gene products within host cells are unknown. PrfA exists in high activity and low activity states, and the working hypothesis of this proposal is that the regulation of PrfA activation is critical for L. monocytogenes to successfully transition from the outside environment to life within mammalian cells. This proposal will use a combination of genetic and biochemical approaches to elucidate the mechanisms by which L. monocytogenes regulates the expression of virulence genes within host cells. It will examine the pathogenic consequences of PrfA activation, and will define bacterial gene products that are induced within host cells and that are directly or indirectly dependent upon PrfA for expression. Aim 1 will focus on the biochemical analysis of mutationally activated forms of PrfA and assessment of the impact of PrfA activation on L. monocytogenes physiology and pathogenesis. Aim 2 will elucidate the contributions of newly identified PrfA-dependent gene products to L. monocytogenes pathogenesis. Aim 3 undertakes functional characterization of mutations located outside of the prfA locus that alter the regulation of L monocytogenes virulence gene expression. The ultimate goal of the proposed work is to elucidate the regulatory pathways that enable bacterial survival within host cells, and the functional identification of additional bacterial factors that support replication within the host.