Mechanistic Studies of Patatin-like Toxins

描述（由申请人提供）：最近发现的马铃薯糖蛋白样蛋白家族广泛分布于细菌中。它们通过存在马铃薯糖蛋白结构域来定义，该结构域编码磷脂酶A2活性。虽然这些蛋白质中的绝大多数仍有待表征，但迄今为止所研究的大多数蛋白质是由III型、IV型或V型分泌系统分泌的，被易位到宿主细胞中，并且具有毒力因子的属性。一旦在细胞内环境中，这些磷脂酶引起正常宿主细胞生理学的破坏或细胞死亡。马铃薯糖蛋白样蛋白家族的最佳表征代表是铜绿假单胞菌的ExoU。这种蛋白质与动物模型和人类感染中的毒力相关，由铜绿假单胞菌III型分泌途径分泌，并通过磷脂酶A2依赖性机制引起广谱真核细胞的快速裂解。本申请的总体目标是建立在我们实验室和其他人先前工作的基础上，进一步表征ExoU的分子机制，并将这些发现扩展到其他马铃薯糖蛋白样蛋白。我们假设ExoU的膜定位结构域通过结合磷脂酰肌醇-4，5-二磷酸将该效应蛋白靶向质膜，并且这种结合显著增强ExoU磷脂酶A2活性。我们假设ExoU在质膜处经历一次多聚化。最后，我们提出了一种治疗策略，其中可以利用ExoU的细胞毒性活性来根除铜绿假单胞菌和其他将裂解毒素靶向中性粒细胞的细菌。在这个应用程序中，我们提出的目的是测试这些假设中的每一个。这些目标的完成将进一步确定马铃薯糖蛋白样蛋白的分子机制，并为开发新型治疗干预措施奠定基础。

DESCRIPTION (provided by applicant): The recently recognized family of patatin-like proteins is widely distributed among bacteria. They are defined by the presence of a patatin domain, which encodes phospholipase A2 activity. Although the vast majority of these proteins remain to be characterized, most of those examined to date are secreted by type III, type IV, or type V secretion systems, are translocated into host cells, and have attributes of virulence factors. Once in the intracellular environment, these phospholipases cause disruption of normal host cell physiology or cell death. The best characterized representative of the patatin-like family of proteins is ExoU of Pseudomonas aeruginosa. This protein, which is associated with virulence in both animal models and human infections, is secreted by the P. aeruginosa type III secretion pathway and causes rapid lysis of a broad spectrum of eukaryotic cells by a phospholipase A2 dependent mechanism. The overall goal of this application is to build upon prior work by our laboratory and others to further characterize the molecular mechanisms of ExoU and to extend these findings to other patatin-like proteins. We hypothesize that the membrane localization domain of ExoU targets this effector protein to the plasma membrane by binding phosphatidylinositol-4,5-bisphosphate and that this binding markedly enhances ExoU phospholipase A2 activity. We hypothesize that ExoU undergoes multimerization once at the plasma membrane. Finally, we suggest a therapeutic strategy whereby the cytotoxic activity of ExoU can be exploited to eradicate P. aeruginosa and other bacteria that target lytic toxins to neutrophils. In this application, we propose aims to test each of these hypotheses. The completion of these aims will further define the molecular mechanisms of patatin-like proteins and lay the foundation for the development of novel therapeutic interventions for patients infected by bacteria that lyse neutrophils.