Mechanism of Phosphorylcholination of EF-Tu on Pseudomonas aeruginosa

描述(申请人提供)：在条件致病菌铜绿假单胞菌中，我们发现正常的细胞质翻译起始因子Ef-Tu(EF-Tu)是表面暴露的，并被磷胆碱(PC)抗体检测到。与37℃相比，这种修饰在25℃下生长的铜绿假单胞菌上更加显著。在初步研究中，我们测试了已知参与胆碱合成、代谢或转运基因突变的菌株，与野生型菌株相比，我们没有发现对PC表达的影响。然后，我们对完整的铜绿假单胞菌PA14转座子突变文库进行了筛选，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，该基因缺失的菌株被称为EFTM，它与呼吸道上皮细胞的粘附性较差 在急性肺炎的小鼠模型中定植效果较差。通过对纯化的修饰或未修饰的EF-Tu进行质谱分析、关键残基的定点突变和功能遗传丧失/功能获得的研究，我们证明了铜绿假单胞菌通过在EF-Tu的赖氨酸5上存在三个甲基来模拟血小板激活因子(PAF)，从而产生与PC相似的化学结构。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。这可能代表了对这种丰富的细菌蛋白的一种新的控制机制，并可能与细菌蛋白质合成的调节有关。我们将采取多管齐下的方法来解决EF-Tu是如何输出的，以及这种修饰如何影响蛋白质合成。我们将使用亚细胞分离、电子显微镜和质谱学来定位这种修饰发生在细胞中的哪里，以及EF-Tu的哪些区域被表面暴露和PC修饰。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一新颖的翻译后修饰提供见解，对其的抑制可能为阻断细菌与宿主的相互作用从而阻止铜绿假单胞菌的发病机制定义新的靶点。

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.