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（由申请人提供）：在机会性病原体铜绿假单胞菌中，我们发现正常的细胞质翻译起始因子、延伸因子-Tu (EF-Tu) 是表面暴露的，并且用磷酸胆碱 (PC) 抗体检测到被修饰。这种修饰在 25° 生长的铜绿假单胞菌菌株上更为明显。 C 与 37¿ C. 在初步研究中，我们测试了已知参与胆碱合成、代谢或运输的基因突变的菌株，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铜绿假单胞菌。菌株 PA14 转座子突变体文库，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，删除该基因的菌株（称为 eftM）粘附较少。对气道上皮细胞有很好的作用 通过结合多种方法，包括纯化的修饰或未修饰的 EF-Tu 的质谱分析、关键残基的定点突变以及遗传功能丧失/功能获得研究，我们证明了其在急性肺炎小鼠模型中的定植效果较差。铜绿假单胞菌通过 EF-Tu 赖氨酸 5 上存在三个甲基来模仿血小板激活因子 (PAF)，从而产生与 PC 相似的化学结构。翻译后修饰是否影响 EF-Tu 的输出和/或正常功能尚不清楚，这可能代表了一种控制这种丰富细菌蛋白质的新机制，并且可能与细菌蛋白质合成的调节有关。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，我们将使用亚细胞分级、电子显微镜和质量来定位细胞中这种修饰发生的位置以及 EF-Tu 的哪些区域是表面暴露和 PC 修饰的。我们将进一步确定修饰的 EF-Tu 如何影响蛋白质合成，这些研究产生的知识和工具将为这种新颖的翻译后修饰提供见解，对其的抑制可能会定义中断细菌与宿主相互作用的新靶标。铜绿假单胞菌的发病机制。