Targeted Metabolomics Analysis of Campylobacter coli VC167 Reveals Legionaminic Acid Derivatives as Novel Flagellar Glycans

Abstract

Glycosylation of Campylobacter flagellin is required for the biogenesis of a functional flagella filament. Recently, we used a targeted metabolomics approach using mass spectrometry and NMR to identify changes in the metabolic profile of wild type and mutants in the flagellar glycosylation locus, characterize novel metabolites, and assign function to genes to define the pseudaminic acid biosynthetic pathway in Campylobacter jejuni 81-176 (McNally, D. J., Hui, J. P., Aubry, A. J., Mui, K. K., Guerry, P., Brisson, J. R., Logan, S. M., and Soo, E. C. (2006) J. Biol. Chem. 281, 18489 -18498). In this study, we use a similar approach to further define the glycome and metabolomic complement of nucleotide-activated sugars in Campylobacter coli VC167. Herein we demonstrate that, in addition to CMP-pseudaminic acid, C. coli VC167 also produces two structurally distinct nucleotide-activated nonulosonate sugars that were observed as negative ions at m/z 637 and m/z 651 (CMP-315 and CMP-329). Hydrophilic interaction liquid chromatography-mass spectrometry yielded suitable amounts of the pure sugar nucleotides for NMR spectroscopy using a cold probe. Structural analysis in conjunction with molecular modeling identified the sugar moieties as acetamidino and N-methylacetimidoyl derivatives of legionaminic acid (Leg5Am7Ac and Leg5AmNMe7Ac). Targeted metabolomic analyses of isogenic mutants established a role for the ptmA-F genes and defined two new ptm genes in this locus as legionaminic acid biosynthetic enzymes. This is the first report of legionaminic acid in Campylobacter sp. and the first report of legionaminic acid derivatives as modifications on a protein.Campylobacter sp. are among the most frequent cause of bacterial diarrhea worldwide and the leading cause of foodborne illness in North America (1, 2). Motility is critical to intestinal colonization by Campylobacter and is required for invasion of epithelial cells in vitro. Campylobacter flagella also function as secretory organelles in the absence of specialized type III secretion systems in this pathogen (3, 4). As such, flagella are recognized as a major virulence factor for Campylobacter sp. (5). The flagellar filament structural protein, FlaA, is the immunodominant protein recognized during infection and has been shown to be an immunoprotective antigen (6 -9). Flagellins from numerous strains of Campylobacter jejuni and the related organism Campylobacter coli have been shown to be among the most heavily glycosylated prokaryotic proteins described (10 -12), and the glycosyl modifications appear to be surface-exposed in the assembled filament and highly immunogenic (13,14). In addition, it appears that unique forms of these glycosyl modifications contribute to the serospecificity of the flagellar filament (11) and the glycans are responsible for autoagglutination and microcolony formation (15).All genes known to be involved in glycosylation of Campylobacter flagellins map near the flaA and flaB structural genes in a region that is one of the most hypervariable in th...

Diversity Within the O-linked Protein Glycosylation Systems of Acinetobacter Species

“…S1B and S4C). This finding demonstrates A. baumannii is one of the many bacterial species now recognized to utilize legionaminic/pseudaminic acid or their de-rivatives within protein attached glycans (45,53,54). Within the best characterized of these systems, the O-linked glycosylation system of C. jejuni, these sialic acid analogs are required for ideal protein function where they are essential for autoagglutination, modulation of the hydrophobicity of the flagellin and dampening of the inflammation response by SigLec-10 binding (55)(56)(57).…”

Preparation of legionaminic acid analogs of sialo-glycoconjugates by means of mammalian sialyltransferases

“…These are all considerably larger substrates than the ones we examined previously [1,2] so their successful modification extends the scope of these reactions to more biologically interesting substances, which was not possible with the bacterial sialyltransferases. However the recent finding of natural antibodies to legionaminic acid in human sera [21] rules out their use as therapeutics, though its known derivatives such as the 5-acetamidino form [22] may not be as reactive. (upper panel) and the GM1a starting material (lower panel).…”

Flagellar Glycosylation: Current Advances

“…In strain 81-176, the probable flagellin glycosylation related genes are largely involved in pseudaminic acid and acetamidino pseudaminic acid biosynthesis (Pse family), and lie downstream of the flagellin gene (about 27 kb) (Guerry et al, 2006). Similarly, Pse family glycosylation islands were identified in both C. coli VC167 and C. jejuni NCTC 11168, and in addition, in C. coli VC167 other flagellin modification genes which are involved in the synthesis of legionaminic acid and its derivatives (ptm family) were identified (McNally et al, 2007). However, the glycosyltransferase which catalyzes the addition of sugar residues to the protein backbone has not been identified.…”

“…This glycosylation island could be divided into two regions, a variable region which was located immediately downstream of the flagellin gene, and a subsequent conserved region. The carbohydrate biosynthesis genes, which are significantly related to the legionaminic acid biosynthesis genes (ptm family) in Campylobacter coli, were encoded in the variable region, whereas the conserved region also encoded the carbohydrate biosynthesis genes (McNally et al, 2007). The C. botulinum strain Langeland was found to have homologous proteins to the capsular biosynthetic proteins from Streptococcus agalactia, including those derived from a second set of the sialic acid biosynthetic genes, neuA and neuB.…”