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.

项目摘要铜绿假单胞菌是一种革兰氏阴性的机会性病原体，对于那些个体来说是有问题的被免疫受损。重要的是，铜绿假单胞菌负责与慢性有关的病理具有囊性纤维化个体的定殖。该有机体很好地适应了医疗保健环境能够在最小养分上生存，在表面上形成微生物群落并表达内在的抗性抗生素和消毒剂。铜绿假单胞菌具有大量的破坏性酶和毒素的曲目细菌复制通过中和人类宿主中的先天免疫细胞。行动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。我们的研究集中于由III型分泌系统注入的细胞毒性酶或效应子 P.铜绿。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靶标。广泛的底物特异性的属性要求额外控制元素必须到位才能使效应器无效，然后再传递到适当的主机环境。 III型效应子Exou，一种有效的磷脂酶，被用作研究的模型系统动态变化，介导宿主细胞中酶活性的激活。我们确定了仅由真核生物合成的泛素（UB），为外来所需的激活剂宿主细胞中介导的磷脂酶活性。这项研究的长期目标是机械描述UB和底物引起的构象变化，这些变化是激活Eseo的。我们已经使用过分子建模，连续波和双电子谐振光谱，以构建初始 EXOU激活的模型。该模型做出特定的预测，并提供可检验的假设在迭代的生化和生物物理分析和模型改进中受到挑战探针。 UB介导的激活被假定是一种特定的治疗靶标，而不限于欧洲。这属性扩展到编码在各种革兰氏阴性人基因组中的外来传播物病原体和机会主义者。总体而言，这些研究旨在为理性贡献结构功能基础设计可能适用于耐药性，有问题的感染的抑制剂的设计有机体。