Characterization of Unique Modification of Flagellar Rod Protein FlgG by Campylobacter jejuni Lipid A Phosphoethanolamine Transferase, Linking Bacterial Locomotion and Antimicrobial Peptide Resistance

• Published in: The Journal of biological chemistry Vol. 287; no. 5; pp. 3326 - 3336
• Main Authors: Cullen, Thomas W., Madsen, James A., Ivanov, Petko L., Brodbelt, Jennifer S., Trent, M. Stephen
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 27.01.2012; American Society for Biochemistry and Molecular Biology
• Subjects: Antimicrobial Cationic Peptides; Antimicrobial Peptide; Antimicrobial Peptides; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Campylobacter jejuni - genetics; Campylobacter jejuni - metabolism; Campylobacter jejuni - pathogenicity; Drug Resistance, Bacterial - drug effects; Drug Resistance, Bacterial - physiology; Ethanolaminephosphotransferase - genetics; Ethanolaminephosphotransferase - metabolism; Flagella; Flagella - genetics; Flagella - metabolism; Glycerophospholipid; Lipid A; Lipid A - genetics; Lipid A - metabolism; Lipid Metabolism; Lipopolysaccharide; Lipoproteins - genetics; Lipoproteins - metabolism; Membrane; Microbiology; Outer Membrane; Post-translational Modification; Protein Processing, Post-Translational - physiology; Outer Membrane; Glycerophospholipid; Post-translational Modification; Antimicrobial Peptide; Lipopolysaccharide; Lipid Metabolism; Membrane; Flagella; Antimicrobial Peptides; Lipid A
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.321737

Gram-negative bacteria assemble complex surface structures that interface with the surrounding environment and are involved in pathogenesis. Recent work in Campylobacter jejuni identified a gene encoding a novel phosphoethanolamine (pEtN) transferase Cj0256, renamed EptC, that serves a dual role in modifying the flagellar rod protein, FlgG, and the lipid A domain of C. jejuni lipooligosaccharide with a pEtN residue. In this work, we characterize the unique post-translational pEtN modification of FlgG using collision-induced and electron transfer dissociation mass spectrometry, as well as a genetic approach using site-directed mutagenesis to determine the site of modification. Specifically, we show that FlgG is modified with pEtN at a single site (Thr75) by EptC and demonstrate enzyme specificity by showing that EptC is unable to modify other amino acids (e.g. serine and tyrosine). Using Campylobacter strains expressing site-directed FlgG mutants, we also show that defects in motility arise directly from the loss of pEtN modification of FlgG. Interestingly, alignments of FlgG from most epsilon proteobacteria reveal a conserved site of modification. Characterization of EptC and its enzymatic targets expands on the increasingly important field of prokaryotic post-translational modification of bacterial surface structures and the unidentified role they may play in pathogenesis. Background:C. jejuni modify the flagellar protein FlgG and lipid A with phosphoethanolamine. Results: The C. jejuni phosphoethanolamine transferase EptC modifies FlgG at position Thr75. Conclusion: Phosphoethanolamine modification of FlgG is required for motility, whereas modification of lipid A with phosphoethanolamine results in resistance to antimicrobial peptides. Significance: Understanding how bacterial surface modifications influence and regulate virulence is key for the development of novel therapeutics.