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: 8912974
• 负责人: Joanna B Goldberg
• 金额: $ 19.29万
• 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 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8912974
• 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; Health; 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; 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; 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; streptavidin-agarose; tool; ventilator-associated pneumonia

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)，在磷酸化胆碱（PC）抗体检测中被表面暴露和修饰。与37℃相比，这种修饰在25℃下生长的铜绿假单胞菌菌株中更为突出。在初步研究中，我们测试了已知参与胆碱合成、代谢或运输的基因突变的菌株，与野生型菌株相比，没有发现对PC表达的影响。然后，我们筛选了铜绿假单胞菌PA14转座子突变体文库，发现了一个负责EF-Tu翻译后修饰的基因。与野生型菌株相比，该基因缺失的菌株（称为eftM）与气道上皮细胞的粘附性较差

项目成果

Lysine trimethylation of EF-Tu mimics platelet-activating factor to initiate Pseudomonas aeruginosa pneumonia.

• DOI: 10.1128/mbio.00207-13
• 发表时间: 2013-05-07
• 期刊: mBio
• 影响因子: 6.4
• 作者: Barbier M;Owings JP;Martínez-Ramos I;Damron FH;Gomila R;Blázquez J;Goldberg JB;Albertí S
• 通讯作者: Albertí S