Mechanism of Phosphorylcholination of EF-Tu on Pseudomonas aeruginosa

EF-Tu对铜绿假单胞菌的磷酸胆酸化机制

• 批准号: 8638629
• 负责人: Joanna B Goldberg
• 金额: $ 23.19万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-15 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8638629
• 关键词: Acute Pneumonia; Address; Adherence; Adhesions; Affect; Affinity; Antibiotic Resistance; Antibodies; Bacterial Proteins; Binding; Biological Assay; Biotin; Cell Fractionation; Cell surface; Cells; Chemical Structure; Choline; Chromatin; Chronic; Communities; EEF1A1 gene; Electron Microscopy; Environment; Epithelial Cells; Epitopes; Escherichia coli; Genes; Genetic; Gram-Negative Bacteria; Grant; Guanosine Triphosphate; Histidine; Histones; Hospitals; Housekeeping; Human; Hydrolysis; Immune response; Immunocompromised Host; Immunoelectron Microscopy; Individual; Infection; Knowledge; Label; Libraries; Life; Lung; Lysine; Mass Spectrum Analysis; Mediating; Membrane; Membrane Proteins; Metabolism; Microbe; Modification; Mono-S; Mutation; Pathogenesis; Patients; Peptide Elongation Factor Tu; Peptide Initiation Factors; Peptides; Phosphorylcholine; Platelet Activating Factor; Pneumonia; Post-Translational Protein Processing; Protein Biosynthesis; Proteins; Pseudomonas aeruginosa; Recombinants; Regulation; Respiratory System; Respiratory tract structure; Role; Site-Directed Mutagenesis; Surface; Testing; Therapeutic Intervention; Transfer RNA; Ventilator; Vesicle; Virulence; cystic fibrosis patients; gain of function; in vitro Assay; inhibitor/antagonist; insight; loss of function; methyl group; mouse model; mutant; novel; pathogen; periplasm; platelet activating factor receptor; prevent; public health relevance; streptavidin-agarose; tool

DESCRIPTION (provided by applicant): In the opportunistic pathogen, Pseudomonas aeruginosa, we have found that the normally cytoplasmic translation initiation factor, elongation factor-Tu (EF-Tu), is surface exposed and modified as detected with antibodies to phosphorylcholine (PC). This modification is more prominent on P. aeruginosa strains grown at 25¿C compared to 37¿C. In Preliminary Studies, we tested strains with mutations in genes known to be involved in choline synthesis, metabolism, or transport and found no effect on PC expression, compared to the wild-type strain. We then screened the entire comprehensive P. aeruginosa strain PA14 transposon mutant library and have found a single gene that is responsible for the post-translational modification of EF-Tu. Compared to the wild-type strain, a strain deleted for this gene, referred to as eftM, adheres less well to airway epithelial cells and colonizes less well in a mouse model of acute pneumonia. Through a combination of approaches, including mass spectrometry of purified modified or unmodified EF-Tu, site-directed mutagenesis of key residues, and genetic loss of function/gain of function studies, we demonstrate that P. aeruginosa mimics platelet-activating factor (PAF) by the presence of three methyl groups on lysine 5 of EF-Tu resulting in a chemical structure similar to PC. However how this post-translational modification affects the export and/or normal function of EF-Tu is not known. This may represent a novel mechanism of control for this abundant bacterial protein and may be implicated in the regulation of bacterial protein synthesis. We will take a multipronged approach to address how EF-Tu is exported and how this modification affects protein synthesis. We will localize where in the cell this modification occurs and which regions of EF-Tu are surface exposed and PC modified, using subcellular fractionation, electron microscopy, and mass spectrometry. We will further determine how modified EF-Tu affects protein synthesis. The knowledge and the tools generated from these studies will provide insights into this novel post-translational modification, inhibition of which may define new targets for interrupting bacterial-host interactions and thus the pathogenesis of P. aeruginosa.

描述（由申请方提供）：在条件致病菌铜绿假单胞菌中，我们发现正常细胞质翻译起始因子-延伸因子-Tu（EF-Tu）表面暴露并被修饰，如磷酸胆碱（PC）抗体所检测。与37 ℃相比，这种修饰在25 ℃下生长的铜绿假单胞菌菌株上更为突出。在初步研究中，我们测试了已知参与胆碱合成、代谢或转运的基因突变的菌株，发现与野生型菌株相比，对PC表达没有影响。然后，我们筛选了整个综合性铜绿假单胞菌菌株PA 14转座子突变体文库，并发现了负责EF-Tu翻译后修饰的单个基因。与野生型菌株相比，缺失该基因的菌株（称为eftM）与气道上皮细胞的粘附较差， 在急性肺炎的小鼠模型中的定殖效果较差。通过结合的方法，包括纯化的修饰或未修饰的EF-Tu的质谱分析，关键残基的定点诱变，和遗传功能丧失/功能获得的研究，我们证明，铜绿假单胞菌模拟血小板活化因子（PAF）的EF-Tu的赖氨酸5上的三个甲基的存在下，导致类似于PC的化学结构。然而，这种翻译后修饰如何影响EF-Tu的输出和/或正常功能尚不清楚。这可能代表了这种丰富的细菌蛋白质的一种新的控制机制，并可能涉及细菌蛋白质合成的调节。我们将采取多管齐下的方法来解决EF-Tu如何输出以及这种修饰如何影响蛋白质合成。我们将本地化在细胞中发生这种修改，EF-Tu的哪些区域是表面暴露和PC修改，使用亚细胞分级，电子显微镜和质谱。我们将进一步确定修饰的EF-Tu如何影响蛋白质合成。从这些研究中产生的知识和工具将提供对这种新型翻译后修饰的见解，抑制这种修饰可能会定义新的靶点，用于中断细菌-宿主相互作用，从而中断铜绿假单胞菌的发病机制。