Mechanism of Activation and Membrane Interactions of Pseudomonas toxin ExoU

假单胞菌毒素ExoU的激活机制和膜相互作用

基本信息

批准号：
8860905
负责人：
Jimmy Feix
金额：
$ 30.65万
依托单位：
MEDICAL COLLEGE OF WISCONSIN
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-06-01 至 2019-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8860905
关键词：
Active Sites Adopted Affinity Aspartate Bacterial Infections Bacterial Toxins Binding Biochemical Biophysics Catalysis Cells Characteristics Chemistry Complex Cytosol Development Drug resistance Electron Spin Resonance Spectroscopy Electrons Excision Exhibits Frequencies Genetic Goals Healthcare In Vitro Infection Infectious Diseases Research Laboratories Lipid Bilayers Liposomes Lung Measures Mechanical ventilation Mediating Membrane Methodology Methods Molecular Molecular Chaperones Molecular Conformation Multi-Drug Resistance Mutation Needles Nosocomial Infections Patients Phospholipase Proteins Pseudomonas Pseudomonas aeruginosa Relaxation Role Sepsis Serine Signal Pathway Site Solutions Spin Labels Structure System Therapeutic Agents Tissues Toxin Type III Secretion System Pathway Ubiquitin Universities Virulence Wisconsin Work base biophysical analysis cofactor cystic fibrosis patients cytotoxic design immunosuppressed in vivo inhibitor/antagonist innovation insight intercalation interest medical schools mortality novel pathogen protein protein interaction public health relevance research study scaffold skills structural biology

项目摘要

DESCRIPTION (provided by applicant): Multi-drug resistant (MDR) bacterial infections represent one of the most serious challenges facing health care today. Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is a leading cause of hospital-acquired infections, and is particularly problematic for patients who are immunosuppressed or require mechanical ventilation. P. aeruginosa persists chronically in cystic fibrosis patients, resulting in irreversible lung damage and mortality. Both natural and acquired characteristics, including the expression of several multidrug efflux systems, make P. aeruginosa infections especially difficult to treat. In addition to its intrinsic drug resistance, the infectivity of P. aeruginosa is enhanced by expression of a Type III secretion system (T3SS). This needle-type apparatus directly injects effector proteins into eukaryotic host cells to facilitate bacterial surival and dissemination. The most cytotoxic T3SS effector produced by P. aeruginosa is the phospholipase, ExoU. This application builds upon our previous discovery that ubiquitin and ubiquitinated proteins act as essential cofactors for the activation of ExoU. The interaction of ExoU with ubiquitin is novel in that it does not involve the covalent attachment or removal of ubiquitin subunits, leading us to hypothesize that ubiquitin acts as a scaffold to induce the folding of ExoU into a catalytically-active conformation. We will employ innovative site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) methods in conjunction with genetic and biochemical approaches to characterize the structure of ExoU in solution, elucidate the structure of the ExoU-ubiquitin complex, and investigate ExoU-membrane interactions. These studies will advance the application of new methods to investigate protein-protein interactions, facilitate the development of novel inhibitors of ExoU virulence, and promote our understanding of bacterial toxins that target the membrane interface.

描述（由申请人提供）：多重耐药 (MDR) 细菌感染是当今医疗保健面临的最严峻挑战之一，铜绿假单胞菌是一种革兰氏阴性机会致病菌，是医院获得性感染的主要原因。对于免疫抑制或需要机械通气的患者来说，铜绿假单胞菌在囊性纤维化患者中长期存在，会导致不可逆的肺损伤和自然死亡。铜绿假单胞菌的感染性和获得性特征，包括多种多药外排系统的表达，使得铜绿假单胞菌感染特别难以治疗。这种针型装置直接将效应蛋白注射到真核宿主细胞中，以促进细菌的生存和传播。铜绿假单胞菌产生的最具细胞毒性的 T3SS 效应蛋白是。该应用建立在我们之前的发现之上，即泛素和泛素化蛋白是 ExoU 激活的重要辅助因子。ExoU 与泛素的相互作用是新颖的，因为它不涉及泛素亚基的共价连接或去除，从而导致。我们将利用泛素作为支架来诱导 ExoU 折叠成催化活性构象。定点自旋标记 (SDSL) 电子顺磁共振 (EPR) 方法结合遗传和生化方法来表征溶液中 ExoU 的结构，阐明 ExoU-泛素复合物的结构，并研究 ExoU-膜相互作用。将推进新方法的应用来研究蛋白质-蛋白质相互作用，促进新型 ExoU 毒力抑制剂的开发，并促进我们对针对膜界面的细菌毒素的理解。