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（由应用提供）：在机会性病原体中，铜绿假单胞菌，我们发现通常的细胞质翻译起始因子伸长因子TU（EF-TU）表面暴露并通过对磷酸胆碱（PC）的抗体进行检测而被检测到。这种修饰在25°C下生长的铜绿假单胞菌菌株中更为突出，而在初步研究中，我们测试了与野生型菌株相比，我们在已知与胆碱合成，代谢或运输的基因中具有突变的菌株，对PC表达没有影响。然后，我们筛选了整个全面的铜绿假单胞菌菌株PA14转座子突变库文库，并找到了一个负责EF-TU翻译后修饰的单个基因。与野生型菌株相比，该基因已删除的菌株（称为EFTM）粘附不太粘附于气道上皮细胞和 在急性肺炎的小鼠模型中，殖民地的殖民地不太好。通过方法的组合，包括纯化的修饰或未修饰的EF-TU的质谱，关键保留的位置定向诱变以及功能遗传丧失/功能研究的遗传丧失/功能研究的丧失，我们证明了铜绿假单胞菌模拟了裂解层与EF-TU结构相似的质子5甲基5的存在在裂纹5中的存在，该甲基属于三个甲基5。但是，这种翻译后修改如何影响EF-TU的出口和/或正常功能。这可能代表了这种丰富的细菌蛋白的新型控制机制，并且在细菌蛋白合成的调节中可能暗示。我们将采用多收益的方法来解决EF-TU如何导出以及这种修饰如何影响蛋白质合成。我们将在细胞中定位这种修饰的位置，以及使用亚细胞分馏，电子显微镜和质谱法进行了表面暴露的EF-TU区域。我们将进一步确定改良的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，抑制可能定义了中断细菌 - 宿主相互作用的新目标，从而定义了铜绿假单胞菌的发病机理。