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.
Highlights
Glycosylation of Campylobacter flagellin is required for the biogenesis of a functional flagella filament
Metabolomic Analysis of C. coli VC167 by CE-ESMS—Using CE-ESMS and precursor ion scanning, cell lysates from wildtype C. coli VC167 were found to contain an intracellular pool of CMP-linked sugars at m/z 638 and 637 that were indicative of CMP-nonulosonic acids as observed before [18]
Oxonium ions corresponding to CMP-329 were observed at m/z 653, and tandem mass spectrometry experiments gave rise to a product ion corresponding to the Da carbohydrate moiety at m/z (Fig. 1B)
Summary
Pse5Ac7Ac, 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid ( known as pseudaminic acid); Pse5Ac7Am, 5-acetamido-7-acetamidino-3,5,7,9-tetradeoxyL-glycero-L-manno-nonulosonic acid; CE, capillary electrophoresis; CE-ESMS, capillary electrophoresis electrospray ionization-mass spectrometry; HILIC-MS, hydrophilic interaction liquid chromatographymass spectrometry; HMBC, heteronuclear multiple bond correlation; HSQC, heteronuclear single quantum coherence; Leg5Ac7Ac, 5,7diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-nonulosonic acid; Leg5Am7Ac, 5-acetamidino-7-acetamido-3,5,7,9,-tetradeoxy-D-glycero-Dgalacto-nonulosonic acid; Leg5AmNMe7Ac, 5-E/Z-N-(N-methylacetimidoyl)-7-acetamidino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-nonulosonic acid; Neu5Ac, N-acetylneuraminic acid; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; TDP, thymidine diphos-. In addition to Pse, C. coli VC167 appears to synthesize a structurally and serologically distinct acetamidino sugar having the same mass (315 Da) as the Pse5Ac7Am modification found on flagellin from C. And coworkers [18] were able to exploit this focused metabolomics approach and use C. jejuni 81–176 and the isogenic mutant strain pseC as a source for large-scale purifications of biosynthetic sugar-nucleotides relevant to the flagellin glycosylation process for precise structural analysis by NMR. The success of using targeted metabolomics strategies to elucidate unknown gene functions and identify novel biosynthetic substrates prompted us to employ this strategy to investigate the ptm flagellin glycosylation locus using C. coli VC167 as a model strain
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