LISTERIA HEMOLYSIN AND ESCAPE FROM A VACUOLE

描述(改编自申请人的摘要)：单核细胞增多性李斯特菌 是一种典型的兼性细胞内病原体，主要感染 孕妇和免疫功能受损的个人。一个主要的决定因素 单核细胞增多性乳杆菌致病机制是一种分泌性的孔道形成蛋白，指 为李斯特溶素O(LLO)。LLO在很大程度上是导致心脏破裂的原因 宿主空泡是吞噬作用的结果。Perfringolysin O(PFO)是一种 相关的孔形成蛋白，参与了血管内皮细胞瘤的发病机制 由细胞外病原体引起的感染。当用L. 单核细胞增多症，PFO介导从吞噬空泡中逃逸，但有毒性 送到牢房里。正常情况下，LLO在感染期间持续表达，但 与PFO不同的是，它在胞浆中被蛋白质降解，并且 与MHC I类分子结合出现在细胞表面。它 假设最适pH和胞浆处理是必不可少的 LIO编码的决定因素，将其与PFO区分开来。 当前提案的重点是定义准确的结构和 LLO的机械特征使其有别于 硫醇激活的溶细胞素家族，促进其空泡内活动， 并在细胞质中决定它的命运。在AIM I中，负责的蛋白质序列 对于LLO的酸性，最适pH和在寄主胞浆中的处理将是 已确认身份。这将通过域和子域交换来实现 LLO和PFO之间，以及改良的荷电到丙氨酸扫描突变。 LLO/PFO嵌合体将从大肠杆菌中纯化并鉴定 生化方面的。接下来，嵌合体将被引入单核细胞增多性乳杆菌。 并以感染的组织培养模型为特征。 在AIM II中，LLO在寄主细胞质中的加工途径将完全 已评估。将通过单核细胞增多性乳杆菌的代谢标记鉴定L10 在受感染的宿主细胞内，随后进行免疫沉淀。 前体/产品关系将通过脉冲追逐来确定 实验。将使用特定的抑制剂来评估该蛋白的作用 蛋白质小体和其他降解中的蛋白酶。LLO的角色 磷酸化将通过生物化学和遗传方法进行评估。 在Aim III中，单核细胞增生性李斯特菌吞噬小体的确切性质将是 其特征是pH、穿孔时间和 内切体/溶酶体成熟途径。最适pH的作用和L. 单核细胞增多性磷脂酶将用突变体和嵌合体进行评估 菌株。 在目标四中，研究人员将评估使用大肠杆菌的可行性 表达LLO的K12及其重组蛋白系统的研究 外来蛋白质进入哺乳动物的胞浆中，用于抗原呈递。

DESCRIPTION (Adapted from the applicant's abstract): Listeria monocytogenes is a model facultative intracellular pathogen which primarily infects pregnant women and immunocompromised individuals. A primary determinant of L. monocytogenes pathogenesis is a secreted pore-forming protein referred to as listeriolysin O (LLO). LLO is largely responsible for rupture of the host vacuole which results from phagocytosis. Perfringolysin O (PFO) is a related pore-forming protein which is involved in the pathogenesis of infections by an extracellular pathogen. When expressed by L. monocytogenes, PFO mediates escape from the phagocytic vacuole, but is toxic to the cell. Normally, LLO is continually expressed during infection, but in contrast to PFO, it is proteolytically degraded in the cytosol and presented on the cell surface in association with MHC class I molecules. It is hypothesized that pH optimum and cytosolic processing are essential LLO-encoded determinants which distinguish it from PFO. The focus of the current proposal is to define the precise structural and mechanistic features of LLO which differentiate it from other members of the family of thiol-activated cytolysins, facilitate its intravacuolar activity, and direct its fate in the cytosol. In Aim I, protein sequences responsible for LLO's acidic pH optimum and processing in the host cytosol will be identified. This will be accomplished by domain and sub-domain swapping between LLO and PFO, and modified charged-to-alanine scanning mutagenesis. The LLO/PFO chimeras will be purified from E. coli and characterized biochemically. Next, the chimeras will be introduced into L. monocytogenes and characterized in tissue culture models of infection., In Aim II, the pathway of LLO processing in the host cytosol will be fully evaluated. LLO will be identified by metabolic labeling of L. monocytogenes within infected host cells, followed by immunoprecipitation. Precursor/product relationships will be determined by pulse-chase experiments. Specific inhibitors will be used to evaluate the role of the proteosome and other proteases in degradation. The role of LLO phosphorylation will be evaluated biochemically and genetically. In Aim III, the precise nature of the L. monocytogenes phagosome will be characterized with regard to pH, time of perforation, and markers of the endosome/lysosome pathway of maturation. The role of pH optimum and the L. monocytogenes phospholipases will be evaluated by using mutant and chimeric strains. In Aim IV, the investigators will evaluate the feasibility of using E. coli K12 expressing LLO and a recombinant protein as a novel system to introduce foreign proteins into the mammalian cytosol for antigen presentation.