Listeria virulence gene expression within host cells

描述(申请人提供)：这项研究的长期目标是了解环境细菌如何获得从土壤有机体过渡到哺乳动物细胞内生命的能力。单核细胞增多性李斯特菌(Lm)是一种兼性胞内细菌，是严重食源性感染的日益重要的病原体，并导致了美国历史上一些最大的食品召回事件。这项提案的重点是 基本毒力调节蛋白，称为PRFA，协调复杂和多方面的LM从外部环境中的生命过渡到受感染宿主内的生命。PRFA功能的关键是从在宿主细胞外复制的细菌中发现的低PRFA活性状态转变为细菌进入宿主细胞后的高活性状态。PrFA的胞内激活不仅对LM毒力基因产物的表达和分泌产生深刻影响，而且对LM的代谢状态和细菌对胁迫条件的响应能力也有深远的影响。这一建议的中心假设是，PRFA激活的调节对于LM成功地平衡细菌在外部环境中的生存和哺乳动物细胞内的生命至关重要。将结合遗传、生化和体内方法来阐明调节PRFA激活状态的机制，并从功能上破译PRFA激活如何协调LM应激抗性、细菌细胞信号和细菌生理的不同方面，以促进细菌在细胞质中的生长和存活。目的1将阐明PRFA激活、LM应激敏感性的不同方面与抗性之间的机制联系。实验将阐明PRFA的激活如何选择性地影响应激抗性，以增强宿主细胞内的细菌生存，并将确定由于宿主炎症反应而发生的自由基损害的靶点。目标2的重点是在PRFA激活的条件下促进胞浆中细菌活力的机制。实验将从功能上确定两种分泌伴侣和一种名为Lmo2637的信息素编码脂蛋白在发病机制中的作用；这些基因产物的分泌是由PRFA激活诱导的，所有这些都在维持细胞质中的LM活性方面具有明显的作用。目标3将阐明导致宿主内PRFA激活的分子途径，并将寻求确定触发PRFA激活的辅因子。这项提议的最终目标将是阐明促进LM在宿主细胞内存活的分子途径，以及土壤中的居民转变为细胞入侵者。

DESCRIPTION (provided by applicant): The long-term objective of this research is to understand how an environmental bacterium gains the ability to transition from soil organism to life within mammalian cells. Listeria monocytogenes (Lm) is a facultative intra- cytosolic bacterium that is an increasingly important agent of serious food-borne infections and has been responsible for some of the largest food recalls in US history. The focus of this proposal is on an essential virulence regulatory protein known as PrfA that coordinates the complex and multifaceted Lm transition from life in the outside environment to life within an infected host. Critical to PrfA function is the shift from the low PrfA activity state found in bacteria replicatig outside of host cells to a high activity state following bacterial entry into the host cell. Cytosoic activation of PrfA has profound effects not only on the expression and secretion of Lm virulence gene products, but also on the metabolic state of Lm and on the ability of the bacterium to respond to conditions of stress. The central hypothesis of this proposal is that the regulation of PrfA activation is critical for Lm to successfully balance bacterial survival in the outside environment with life within mammalian cells. A combination of genetic, biochemical, and in vivo approaches will be used to elucidate the mechanisms that regulate PrfA activation status as well as to functionally decipher how PrfA activation coordinates different aspects of Lm stress resistance, bacterial cell signaling, and bacterial physiology to enhance bacterial growth and viability within the cytosol. Aim 1 will elucidate the mechanistic links between PrfA activation an different facets of Lm stress susceptibility and resistance. Experiments will clarify how PrfA activation selectively influences stress resistance to enhance bacterial survival within host cells and will identify targets of free radical damage that occur as a result of host inflammatory responses. The focus of Aim 2 is on mechanisms that promote bacterial viability in the cytosol under conditions of PrfA activation. Experiments will functionally define the contributions of two secretion chaperones and a pheromone-encoding lipoprotein known as Lmo2637 to pathogenesis; the secretion of these gene products is induced by PrfA activation and all have apparent roles in maintaining Lm viability within the cytosol. Aim 3 will clarify the molecular pathways leading to PrfA activation within the host and will seek to identify the cofactor that triggers PrfA activation. The ultimate goal of this proposal will be to elucidate the molecular pathways that promote Lm survival within host cells and the transformation of a soil dweller into a cell invader.