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

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

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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（应用程序提供）：多药耐药（MDR）细菌感染是当今医疗保健面临的最严重挑战之一。铜绿假单胞菌是一种革兰氏阴性的机会性病原体，是医院获得感染的主要原因，对于免疫抑制或需要机械通气的患者而言，尤其是问题。铜绿假单胞菌在囊性纤维化患者中长期存在，导致不可逆的肺损伤和死亡率。天然和获得的特征，包括几种多饮用外系统的表达，使铜绿假单胞菌感染特别困难。除了其内在的耐药性外，铜绿假单胞菌的感染还通过表达III型分泌系统（T3SS）增强。该针型设备将效应蛋白直接注入真核生物宿主细胞中，以促进细菌的外观和传播。铜绿假单胞菌产生的最多的细胞毒性T3SS效应子是磷脂酶Exou。该应用是基于我们以前的发现，即泛素和泛素蛋白是激活Exou的必需辅助因子。外来与泛素的相互作用是新颖的，因为它不涉及共价附着或去除泛素亚基，从而使我们假设泛素是将海洋折叠成催化活性构象的折叠。我们将采用创新的位置定向的自旋标记（SDSL）电子顺磁共振（EPR）方法，并结合遗传和生化方法来表征溶液中的Exoun结构，阐明了Expiquitin络合物的结构，并研究了Exou-Membrane相互作用。这些研究将推动新方法研究蛋白质蛋白质相互作用，促进新型蛋白质抑制剂的发展，并促进我们对靶向膜界面的细菌毒素的理解。