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：
9762818
负责人：
Jimmy Feix
金额：
$ 44.77万
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 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9762818
关键词：
ADP Ribose Transferases Acinetobacter baumannii Amino Acids Antibiotic Resistance Bacteria Binding Binding Sites Biochemical Bioinformatics Biological Assay Biological Models Biological Testing Cell Death Cells Cessation of life Chemicals Chronic Complex Cytotoxin Data Disinfectants Electron Spin Resonance Spectroscopy Electrons Electrostatics Elements Endothelium Ensure Enterobacter Enterococcus faecium Environment Enzyme Activation Enzymes Epithelial Eukaryota Family Functional disorder Genetic Genome Goals Human Hydrophobicity Immune Immune Targeting Immunocompromised Host Individual Infection Injections Klebsiella pneumonia bacterium Lead Location Lysophospholipids Mediating Membrane Membrane Proteins Microbial Biofilms Modeling Molecular Conformation Morbidity - disease rate Mutagenesis Needles Normal Cell Nutrient Organism Orthologous Gene Outcome Pathology Phagocytosis Phospholipase Phospholipase A2 Phospholipids Process Property Protein Biosynthesis Protein Family Proteins Pseudomonas aeruginosa Pseudomonas aeruginosa infection Regulatory Element Research Resistance Scanning Series Serine Signal Transduction Signal Transduction Inhibition Spectrum Analysis Staphylococcus aureus Structure Substrate Specificity Surface System Testing Tissues Toxin Type III Secretion System Pathway Ubiquitin base biological systems biophysical analysis biophysical model chronic infection cofactor crosslink cystic fibrosis patients cytotoxic design enzyme activity experimental study health care settings human pathogen inhibitor/antagonist member microbial community molecular modeling monomer mortality novel pathogen rho GTPase-activating protein therapeutic development therapeutic target trafficking

项目摘要

Project Summary Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is problematic for individuals who are immunocompromised. Importantly, P. aeruginosa is responsible for pathology associated with chronic colonization of individuals with cystic fibrosis. The organism is well adapted to health care settings by being able to survive on minimal nutrients, form microbial communities on surfaces and express intrinsic resistance to antibiotics and disinfectants. P. aeruginosa has a large repertoire of destructive enzymes and toxins that aid bacterial replication by neutralizing innate immune cells in human hosts. The mechanisms of action include interference with or inhibition of signal transduction, host protein synthesis, cytoskeletal function or membrane dynamics. Our studies focus on the cytotoxic enzymes or effectors injected by the type III secretion system of P. aeruginosa. These enzymes, ExoS and ExoU, demonstrate broad substrate specificity and can recognize both prokaryotic and eukaryotic targets. The property of broad substrate specificity requires that additional control elements must be in place to keep the effector inactive before delivery to the appropriate host environment. The type III effector, ExoU, a potent phospholipase, is used as a model system to study the dynamic changes that mediate activation of enzyme activity in host cells. We identified ubiquitin (Ub), which is synthesized only by eukaryotes, as the activator required for ExoU- mediated phospholipase activity in host cells. The long-term objectives of this research are to mechanistically describe Ub and substrate induced conformational changes that occur to activate ExoU. We have used molecular modeling, continuous wave and double electron electron resonance spectroscopy to build an initial model of ExoU activation. The model makes specific predictions and provides testable hypotheses that will be challenged in iterative biochemical and biophysical analyses and model refinement with novel mono- and diUb probes. Ub-mediated activation is postulated to serve as a specific, therapeutic target, not limited to ExoU. This property extends to ExoU-orthologs that are encoded in the genomes of a variety of Gram negative human pathogens and opportunists. Overall, these studies aim to contribute a structure-function basis for the rational design of inhibitors that may be applicable for treatment of infections caused by resistant, problematic organisms.

项目概要铜绿假单胞菌是一种革兰氏阴性机会致病菌，对于以下人群来说是有问题的：免疫功能低下。重要的是，铜绿假单胞菌负责与慢性病相关的病理学。囊性纤维化患者的定植。该有机体通过以下方式很好地适应了医疗保健环境：能够依靠最少的营养生存，在表面形成微生物群落并表现出内在的抵抗力抗生素和消毒剂。铜绿假单胞菌具有大量破坏性酶和毒素，有助于通过中和人类宿主的先天免疫细胞来进行细菌复制。作用机制包括干扰或抑制信号转导、宿主蛋白合成、细胞骨架功能或膜动力学。我们的研究重点是III型分泌系统注射的细胞毒性酶或效应物铜绿假单胞菌。这些酶 ExoS 和 ExoU 表现出广泛的底物特异性，并且可以识别原核和真核目标。广泛的底物特异性的特性需要额外的控制元件必须就位，以在将效应器传送到适当的宿主之前保持其不活动状态环境。 III 型效应子 ExoU 是一种有效的磷脂酶，被用作模型系统来研究介导宿主细胞中酶活性激活的动态变化。我们鉴定出仅由真核生物合成的泛素 (Ub) 作为 ExoU- 所需的激活剂介导宿主细胞中的磷脂酶活性。这项研究的长期目标是机械地描述了激活 ExoU 时发生的 Ub 和底物诱导的构象变化。我们已经用过分子建模、连续波和双电子共振光谱建立了初步的 ExoU 激活模型。该模型做出具体预测并提供可检验的假设使用新型单尿素和二尿素进行迭代生化和生物物理分析以及模型细化面临挑战探针。 Ub 介导的激活被认为可作为特定的治疗靶点，而不仅限于 ExoU。这特性延伸至在多种革兰氏阴性人类基因组中编码的 ExoU 直向同源物病原体和机会主义者。总的来说，这些研究旨在为合理的结构功能基础提供基础。设计可能适用于治疗由耐药性、有问题的感染引起的感染的抑制剂有机体。