Mechanistic Studies of Patatin-like Toxins

描述(申请人提供)：最近发现的Patatin样蛋白家族广泛分布于细菌中。它们由编码磷脂酶A2活性的Patatin结构域的存在来定义。虽然这些蛋白中的绝大多数仍有待鉴定，但到目前为止，大多数被检测的蛋白是由III型、IV型或V型分泌系统分泌的，被转运到宿主细胞中，并具有毒力因子的属性。一旦进入细胞内环境，这些磷脂酶就会破坏正常的宿主细胞生理或导致细胞死亡。铜绿假单胞菌的ExoU是Patatin样蛋白家族最具代表性的代表。这种蛋白在动物模型和人类感染中都与毒力有关，由铜绿假单胞菌III型分泌途径分泌，并通过磷脂酶A2依赖的机制导致广泛的真核细胞快速裂解。这项应用的总体目标是在我们实验室和其他实验室先前工作的基础上，进一步表征ExoU的分子机制，并将这些发现扩展到其他Patatin样蛋白。我们推测，ExoU的膜定位结构域通过与磷脂酰肌醇-4，5-二磷酸结合，将该效应蛋白靶向质膜，这种结合显著增强了ExoU磷脂酶A2的活性。我们假设ExoU在质膜上经历了一次多聚体。最后，我们建议了一种治疗策略，即可以利用ExoU的细胞毒活性来根除铜绿假单胞菌和其他针对中性粒细胞溶解毒素的细菌。在这一应用中，我们提出了旨在检验这些假设的每一个。这些目标的完成将进一步确定类Patatin蛋白的分子机制，并为开发针对感染了中性粒细胞的细菌感染的患者的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.