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Moonlighting and Mimicry: Identification and Functional Characterization of a SAM-dependent Methyltransferase Responsible for Methylation of EF-Tu in Pseudomonas Aeruginosa

Owings, Joshua
Thesis/Dissertation; Online
Owings, Joshua
Goldberg, Joanna
Phosphorylcholine (ChoP) is a charged molecule that, in humans, is the major recognition component of platelet activating factor (PAF) by its cognate receptor, platelet activating factor receptor (PAFR). ChoP-like molecules have been detected on the surfaces of numerous respiratory pathogens where they confer persistence related phenotypes mediated through an increase in adhesion. Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen which is often associated with nosocomial pneumonia. Previously, in P. aeruginosa a ChoP-like modification was detected using ChoP-specific antibodies on surface exposed elongation factor-Tu (EF-Tu), a normally cytoplasmic protein involved in protein chain elongation. Here we identify a gene, eftM, which is required for the modification of EF-Tu. To our surprise, mass spectrometry analysis of modified EF-Tu revealed the modification was not ChoP but rather trimethyl-lysine at amino acid residue 5. This trimethyl-lysine acts as a structural mimic of ChoP and confers similar phenotypes to P. aeruginosa as ChoP does to other respiratory pathogens. Bioinformatic analysis of EftM showed amino acid sequence similarity to S-adenosylmethionine (SAM)-dependent methyltransferases. We confirmed biochemically that EftM is able to bind SAM and used it to directly methylate lysine 5 of EF-Tu both in vivo and in vitro. Mass spectrometry analysis of products from these reactions confirmed that EF-Tu is trimethylated at lysine 5. In vivo, P. aeruginosa methylates EF-Tu only at temperatures closer to ambient, 25°C, and not at body temperature, 37°C. We show that this temperature-dependent methylation phenotype is not due to differences in transcription of eftM. The methyltransferase activity of the laboratory P. aeruginosa strain PAO1 EftM is slightly higher at 25°C compared to 37°C, while the rate of degradation of EftM expressed in P. aeruginosa is significantly decreased at 25°C compared to 37°C allowing for a longer half-life of the protein. The difference in the rate of degradation suggested an increase in protein stability at 25°C and was confirmed by heat pre-treatment of EftM in vitro. Pre-incubation of EftM at 37°C abolished methyltransferase activity while methyltransferase activity was retained when the enzyme was pre-incubated at 25°C. In the absence of evidence for transcriptional regulation of this phenomenon, these results may suggest that the in vivo temperature-dependent phenotype is due to differences in the steady state levels of the EftM protein at different temperatures. In this work, we have discovered a modification that mimics a known bacterial adhesin in respiratory pathogens. It is possible that the ChoP-like modification seen in other respiratory pathogens is actually trimethyl-lysine, the addition of which could be mediated by a methyltransferase similar to EftM. The identification and characterization of EftM could have broad implications for both basic and applied biology. These include the potential identification of a novel export pathway of a normally cytoplasmic protein and insight into the bacterial regulation of protein synthesis. Additionally, the enzyme responsible for the methylation of EF-Tu may be a good target for novel therapeutics to prevent the establishment of respiratory infections.
University of Virginia, Department of Microbiology, Immunology, and Cancer Biology, PHD (Doctor of Philosophy), 2014
Published Date
PHD (Doctor of Philosophy)
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