Type III effector-cofactor dynamics within the cellular environment

细胞环境中的 III 型效应子-辅因子动力学

基本信息

批准号：
8828548
负责人：
Dara W. Frank
金额：
$ 38.25万
依托单位：
MEDICAL COLLEGE OF WISCONSIN
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-05-23 至 2016-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8828548
关键词：
ATP phosphohydrolase Acute Affect Amino Acids Activation Antimicrobial Resistance Bacteria Binding Biochemical Biological Biological Assay Biological Models C-terminal Cell membrane Cell physiology Cells Cellular biology Characteristics Clinical Complex Cytoskeleton Cytotoxin Data Development Electron Spin Resonance Spectroscopy Electrons Ensure Environment Enzymes Equilibrium Escherichia coli Eukaryotic Cell Evolution Family Future Genetic Global Change Glycine Goals Health Human In Vitro Infection Injection of therapeutic agent Kinetics Liposomes Mammalian Cell Measurement Measures Mediating Membrane Modeling Molecular Chaperones Molecular Conformation Natural Immunity Needles Nuclear Magnetic Resonance Organism Outcome Pathway interactions Patients Phospholipase Phospholipase A2 Preparation Process Prokaryotic Cells Property Protein Isoforms Proteins Pseudomonas aeruginosa Recombinants Research Resolution Series Site-Directed Mutagenesis Solutions Structure System Testing Toxin Type III Secretion System Pathway Ubiquitin Virulence Factors Yeasts base biophysical techniques cofactor cytotoxic cytotoxicity design enzyme activity inhibitor/antagonist insight novel novel therapeutics pathogen protein complex protein folding protein structure research study response tool trafficking transmission process

项目摘要

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.

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，其本质上对抗菌药物具有耐药性且难以治疗。该生物体拥有 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。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，只有当膜底物提供真核辅因子时，才能从重组酶制剂中检测到该活性。在这个新应用中，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助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助因子的发现，我们取得了重大进展。与真核细胞骨架一样，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目标。泛素介导的 ExoU 激活代表了一种新的宿主操纵机制。本研究旨在从功能上定义参与膜底物和泛素识别的 ExoU 结构域。将利用最先进的生物物理方法来测量 ExoU 和泛素响应同源结合伴侣和脂质体的区域和全局变化，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重要信息，并有助于生物学研究基本真核过程和与 ExoU 同源的其他毒力因子的工具。