Listeria Virulence Gene Expression Within Host Cells

描述（由申请人提供）：单核细胞增生李斯特菌是一种兼性细胞质内细菌病原体，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，单核增生乳杆菌能够介导从土壤中的生命到人类细胞中的生命的复杂过渡，并在那里生存和复制。这个提议试图确定环境细菌需要什么来调节从土壤中的外部生命到哺乳动物细胞内的生命的转换。正在进行的工作主要集中在单核增生乳杆菌毒力基因表达的关键转录激活因子PrfA上。迄今为止，几乎所有单核增生乳杆菌毒力因子的表达都需要PrfA，然而单核增生乳杆菌在宿主细胞内协调基因产物表达的机制尚不清楚。PrfA存在高活性和低活性两种状态，本研究的工作假设是，PrfA激活的调控是单核增生乳杆菌成功从外部环境过渡到哺乳动物细胞内生活的关键。本研究将采用遗传和生化相结合的方法来阐明单核增生乳杆菌调控宿主细胞内毒力基因表达的机制。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，这些基因产物直接或间接依赖于PrfA的表达。目的1将侧重于突变激活形式的PrfA的生化分析和评估PrfA激活对单核增生乳杆菌生理和发病机制的影响。目的2将阐明新发现的prfa依赖基因产物在单核增生乳杆菌发病机制中的作用。Aim 3对位于prfA位点外的突变进行功能表征，这些突变改变了L单核细胞增生基因毒力基因表达的调控。这项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.