Identification and Characterization of Genes Required for Listeria monocytogenes Cytosolic Survival

项目概要： 细胞内病原体是世界范围内发病率和死亡率的重要原因。其中一些病原体， 如单核细胞增生李斯特菌、土拉热弗朗西丝菌、类鼻疽伯克霍尔德菌、立克次氏体属（Rickettsia spp.）甚至 结核分枝杆菌在其宿主细胞的胞质溶胶中度过其感染周期的至少一部分。 对于许多这些生物体来说，进入细胞质并在细胞质中存活对于毒力至关重要。尽管 这些不同的病原体在这个生态位中生存和繁荣的能力，很明显，胞质溶胶是一种 不适合非胞质溶胶适应病原体的环境。然而，人们对这种细胞知之甚少， 细菌在细胞质中遇到的自主防御，以及细胞质中遇到的适应性， 病原体已经进化来克服这些压力。 L.单核细胞增多症是引起李斯特菌病的重要兼性细胞内病原体。L. 单核细胞增多症也是理解复杂的宿主-病原体相互作用的有力工具。在本提案中，我们 将利用一种新的遗传筛选来鉴定L.在宿主细胞中生存所需的单核细胞增生基因 胞质液在该筛选中鉴定的突变体将用于理解细菌逃避宿主的策略。 防御，以确定毒力中胞质存活的重要性，并定义抗菌防御 细胞溶质中的机制。除了基因筛查外，我们还将采取有针对性的方法， 表征已经被鉴定为细胞溶质存活和毒力所必需的基因的子集。具体 我们将确定细菌小分子代谢产物1，4-二羟基-2-萘甲酸酯的未知功能 （DHNA）在细胞溶质存活和毒力中的作用。此外，我们将确定PrkA，a 高度保守的PASTA激酶，作为宿主细胞壁应激反应的主要调节因子发挥作用 胞质液作为一个例子，我们将描述PrkA如何调节新发现的尿苷酰转移酶 高度保守的蛋白YvcK的活性以及这种调节是否在物种间保守。 一旦完成这些目标，我们将确定L所需的基因集合。单核 胞质存活，并将其特征的两个特定的途径（DHNA和PrkA的必要L。 单核细胞增生毒力这些途径可能是治疗干预的新靶点。的确， PASTA激酶已经是我们实验室和其他机构开发新型抗生素的焦点，因此， 了解它们在细菌应激反应和毒力中的作用至关重要。

Project Summary: Intracellular pathogens are a significant cause of morbidity and mortality worldwide. Some of these pathogens, such as Listeria monocytogenes, Francisella tularensis, Burkholderia psuedomallei, Rickettsia spp. and even Mycobacterium tuberculosis, spend at least a portion of their infectious cycle in the cytosol of their host cell. For many of these organisms, access to, and survival in the cytosol, is essential for virulence. Despite the ability of these diverse pathogens to survive and thrive in this niche, it has become clear that the cytosol is an inhospitable environment for non-cytosol adapted pathogens. However, little is known about the cell autonomous defenses that bacteria encounter in the cytosol and in turn the adaptations that cytosolic pathogens have evolved to overcome these stresses. L. monocytogenes is an important facultative intracellular pathogen that causes the disease Listeriosis. L. monocytogenes is also a powerful tool to understand complex host-pathogen interactions. In this proposal we will utilize a novel genetic screen to identify L. monocytogenes genes required for survival in the host cell cytosol. Mutants identified in this screen will be used to understand bacterial strategies for evasion of host defense, to determine the importance of cytosolic survival in virulence, and to define antimicrobial defense mechanisms in the cytosol. In addition to the genetic screen, we will take a targeted approach by characterizing a subset of genes already identified as essential for cytosolic survival and virulence. Specifically we will identify the unknown function of the bacterial small molecule metabolite 1,4-Dihydroxy-2-naphthoate (DHNA) in cytosolic survival and virulence. In addition, we will determine the mechanisms by which PrkA, a highly conserved PASTA kinase, functions as a master regulator of cell wall stress responses in the host cytosol. As one example, we will characterize how PrkA regulates the newly discovered uridyl transferase activity of the highly conserved protein YvcK and whether this regulation is conserved across species. Upon completion of these aims, we will have identified a collection of genes required for L. monocytogenes cytosolic survival and will have characterized two specific pathways (DHNA and PrkA) essential for L. monocytogenes virulence. These pathways may represent novel targets for therapeutic intervention. Indeed, the PASTA kinases are already the focus of novel antibiotic development by our labs and others, and as such, understanding their role in bacterial stress response and virulence is critical.