Mechanism of Phosphorylcholination of EF-Tu on Pseudomonas 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

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.