Mass spectrometric analysis of intact lipooligosaccharide: direct evidence for O-acetylated sialic acids and discovery of O-linked glycine expressed by Campylobacter jejuni.
The lipooligosaccharides (LOS) of Campylobacter jejuni is an important virulence factor. Its core oligosaccharide component is frequently sialylated and bears a close resemblance with host gangliosides. The display of ganglioside mimics by this bacterium is believed to trigger the onset of the autoimmune condition Guillain-Barré syndrome (GBS) in some individuals. Considerable effort has been directed toward the structural characterization of the glycan component of the LOS of C. jejuni strains isolated from GBS patients. Capillary electrophoresis-mass spectrometry (CE-MS) has been a particularly useful analytical technique applied toward this task. Conventional analysis of bacterial LOS by CE-MS has generally involved the prior removal of O-acyl lipid chains, which is necessary for the effective solubilization and separation of the heterogeneous ensemble of LOS species. Unfortunately, O-deacylation causes the undesired removal of important glycan-associated O-linked modifications, such as O-acetate and O-linked amino acids. In this report, we describe a CE-MS technique developed for the rapid analysis of fully intact LOS from C. jejuni. Using this method, we report the structural characterization of the glycan from 10 GBS-associated strains and two enteritis strains, using material isolated from as little as one colony. The application of this technique has enabled us to unambiguously identify LOS-bound O-acetylated sialic acid in a number of strains and has revealed for the first time that C. jejuni frequently modifies its core with O-linked glycine. Our studies demonstrate that MS-based structural analysis of bacterial LOS can be optimized to the level where only a single-colony quantity of material is required and time-consuming chemical treatments can be avoided.
Dzieciatkowska
Monika
M
Institute for Biological Sciences, National Research Council Canada, Ottawa, Ontario, Canada.
Brochu
Denis
D
van Belkum
Alex
A
Heikema
Astrid P
AP
Yuki
Nobuhiro
N
Houliston
R Scott
RS
Richards
James C
JC
Gilbert
Michel
M
Li
Jianjun
J
D000107:Acetylation; D016123:Campylobacter jejuni / Q000378:metabolism*; D002240:Carbohydrate Sequence; D019075:Electrophoresis, Capillary; D005998:Glycine / Q000737:chemistry* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000737:chemistry* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
D008070:Lipopolysaccharides; C023023:lipid-linked oligosaccharides; D019158:N-Acetylneuraminic Acid; D005998:Glycine