The Type B Flagellin of Hypervirulent Clostridium difficile Is Modified with Novel Sulfonated Peptidylamido-glycans

• Published in: The Journal of biological chemistry 2016-12, Vol.291 (49), p.25439-25449
• Main Authors: Bouché, Laura, Panico, Maria, Hitchen, Paul, Binet, Daniel, Sastre, Federico, Faulds-Pain, Alexandra, Valiente, Esmeralda, Vinogradov, Evgeny, Aubry, Annie, Fulton, Kelly, Twine, Susan, Logan, Susan M., Wren, Brendan W., Dell, Anne, Morris, Howard R.
• Format: Article
• Language: English
• Subjects: bacteria; Clostridioides difficile - chemistry; Clostridioides difficile - metabolism; Clostridioides difficile - pathogenicity; Clostridium difficile; flagellin; Flagellin - chemistry; Flagellin - metabolism; Glycobiology and Extracellular Matrices; Glycosylation; Gram-positive bacteria; mass spectrometry (MS); modification; nuclear magnetic resonance (NMR); Nuclear Magnetic Resonance, Biomolecular; Polysaccharides, Bacterial - chemistry; Polysaccharides, Bacterial - metabolism; sulfonated
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M116.749481

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.