Mechanism of Phosphorylcholination of EF-Tu on Pseudomonas aeruginosa

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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.