Type III effector-cofactor dynamics within the cellular environment

描述（由申请人提供）：铜绿假单胞菌是一种机会性革兰氏阴性病原体，对抗菌药物具有内在耐药性，难以治疗。该微生物具有III型分泌系统，对于逃避先天免疫和在受损患者中建立急性感染至关重要。由铜绿假单胞菌产生的最具毒性和破坏性的III型效应子是ExoU，其表达与人类感染的不良临床结果相关。ExoU具有磷脂酶A2活性，只有当真核辅因子与膜底物一起提供时，才能从重组酶制剂中检测到该活性。在这个新的应用中，ExoU的动态特征将作为模型系统进行分析，以了解分泌的T3S效应子如何与真核辅因子相互作用以影响细胞生理学。随着泛素作为ExoU介导的磷脂酶活性的主要辅因子的发现，已经取得了重大进展。与真核细胞骨架沿着，泛素系统是病原体操纵以促进细菌复制和传播到新宿主的重要靶标。泛素介导的ExoU活化代表了操纵宿主的新机制。本研究的目的是功能性地确定参与膜底物和泛素识别的ExoU结构域。响应于同源结合伴侣和脂质体的ExoU和泛素的区域和全局变化将利用最先进的生物物理方法来测量，以表征溶液中的天然蛋白质结构。这些目标的完成将为新型抑制剂的设计提供必要的信息，并有助于生物学研究。 研究与ExoU同源的重要真核过程和其他毒力因子的工具。

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that is intrinsically resistant to antimicrobials and difficult to treat. The organism possesses a type III secretion system that is critical for evading innate immunity and establishing acute infections in compromised patients. The most toxic and destructive type III effector produced by P. aeruginosa is ExoU, whose expression is correlated with poor clinical outcomes in human infections. ExoU possesses phospholipase A2 activity, which is detectable from recombinant enzyme preparations only when a eukaryotic cofactor is provided with membrane substrates. In this new application the dynamic features of ExoU will be analyzed as a model system for understanding how the secreted T3S effectors interact with eukaryotic cofactors to affect cellular physiology. A major advance has been achieved with the discovery of ubiquitin as the main cofactor for ExoU-mediated phospholipase activity. Along with the eukaryotic cytoskeleton, the ubiquitin system is a significant target that pathogens manipulate to facilitate bacterial replication and transmission to new hosts. Ubiquitin mediated activation of ExoU represents a novel mechanism of manipulation of the host. This study is designed to functionally define the domains of ExoU involved in recognition of membrane substrates and ubiquitin. Regional and global changes in both ExoU and ubiquitin in response to cognate binding partners and liposomes will be measured utilizing state-of-the art biophysical approaches for characterizing native protein structure in solution. The completion of these aims will provide essential information towards the design of novel inhibitors and contribute biological tools to study essential eukaryotic processes and other virulence factors that are homologous to ExoU.