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

Thomas W Cullen,James A Madsen,Petko L Ivanov,Jennifer S Brodbelt,M Stephen Trent

doi:10.1074/jbc.m111.321737

Abstract

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 (Thr(75)) 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.

Highlights

C. jejuni modify the flagellar protein FlgG and lipid A with phosphoethanolamine
Considering recent work in Neisseria sp. showing that modification of the Type IV pili with phosphoryl substituents was important for antigenic diversity and involved in dissemination of N. meningitidis upon cell contact [18], we felt it was important to map the actual site of pEtN modification on FlgG
The bacterial cell surface and appendages protruding from it interface with the surrounding environment bringing them into contact with host cells and immunological defenses, making bacterial surface structures important for evasion of host defenses

Summary

Background

Expected massa effffllllpggggtFFFGCGGGh, ,,,ifslfff6glllgggϩGFFFhGGGishhh6iiiϩsss666(((TTT777555ASY)))ϩϩϩ a Average masses. Complementation of flgFG-deficient C. jejuni strains with flgFGhis demonstrated wild type motility, whereas those complemented with site-directed mutations of flgFGhis showed motility defects similar to that of eptC-deficient strains (Fig. 7), with T75Y SDM strains showing the greatest defect These results suggest that the observed motility phenotypes are not the result of changes in the outer membrane but rather directly related to modification of FlgG with pEtN. The eptC-deficient C. jejuni strain showed a dramatic decrease in resistance to PMB (MIC of 0.8 Ϯ 0.2 ␮g/ml) when compared with wild type (MIC of 17.3 Ϯ 3.3 ␮g/ml), indicating a loss of pEtN modification of the lipid A backbone (Table 4). These data suggest that in C. jejuni, pEtN modification of lipid A provides resistance to CAMPs, whereas modification of FlgG with pEtN plays no role in CAMP resistance

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION