Abstract
Glycosylation of flagellins is a well recognized property of many bacterial species. In this study, we describe the structural characterization of novel flagellar glycans from a number of hypervirulent strains of C. difficile. We used mass spectrometry (nano-LC-MS and MS/MS analysis) to identify a number of putative glycopeptides that carried a variety of glycoform substitutions, each of which was linked through an initial N-acetylhexosamine residue to Ser or Thr. Detailed analysis of a LLDGSSTEIR glycopeptide released by tryptic digestion, which carried two variant structures, revealed that the glycopeptide contained, in addition to carbohydrate moieties, a novel structural entity. A variety of electrospray-MS strategies using Q-TOF technology were used to define this entity, including positive and negative ion collisionally activated decomposition MS/MS, which produced unique fragmentation patterns, and high resolution accurate mass measurement to allow derivation of atomic compositions, leading to the suggestion of a taurine-containing peptidylamido-glycan structure. Finally, NMR analysis of flagellin glycopeptides provided complementary information. The glycan portion of the modification was assigned as α-Fuc3N-(1→3)-α-Rha-(1→2)-α-Rha3OMe-(1→3)-β-GlcNAc-(1→)Ser, and the novel capping moiety was shown to be comprised of taurine, alanine, and glycine. This is the first report of a novel O-linked sulfonated peptidylamido-glycan moiety decorating a flagellin protein.
Highlights
The reasons for the increase in severity of C. difficile infection caused by hypervirulent strains are still not well understood
Its flagellin is substituted with the legionaminic acid derivative 7-acetamido-5-(N-methyl-glutam-4-yl)-amino-3,5,7,9-tetradeoxyD-glycero-␣-D-galacto-nonulosonic acid (␣Leg5GluNMe7Ac) [12] In contrast, C. difficile post-translational modifications appear to be quite different in structural composition from those that have been found in Gram-negative organisms
In the study presented here, following nano-LC-MS and MS/MS analysis, a number of putative glycopeptides derived from RT0207 and other flagellins were identified via a search for oxonium ions (e.g. m/z 204) in the data sets
Summary
We describe the structural characterization of novel flagellar glycans from a number of hypervirulent strains of C. difficile. A variety of electrospray-MS strategies using Q-TOF technology were used to define this entity, including positive and negative ion collisionally activated decomposition MS/MS, which produced unique fragmentation patterns, and high resolution accurate mass measurement to allow derivation of atomic compositions, leading to the suggestion of a taurinecontaining peptidylamido-glycan structure. We describe the chemical characterization of this newly discovered structure using Q-TOF technology [15, 16] applied in a variety of structural elucidation strategies [17, 18], including positive and negative ion collisionally activated decomposition (CAD) MS/MS to produce unique and interpretable fragmentation patterns and high resolution accurate mass measurement to allow derivation of detailed atomic compositions.
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