title: The Type B Flagellin of Hypervirulent Clostridium difficile Is Modified with Novel Sulfonated Peptidylamido-glycans.
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.
authors.affiliation: From the Department of Life Sciences, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom.
authors.forename: Laura
authors.initials: L
authors.lastname: Bouché
authors.affiliation: From the Department of Life Sciences, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom.
authors.forename: Maria
authors.initials: M
authors.lastname: Panico
authors.affiliation: From the Department of Life Sciences, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom.
authors.forename: Paul
authors.initials: P
authors.lastname: Hitchen
authors.affiliation: BioPharmaSpec, Suite 3.1 Lido Medical Centre, St. Saviours Road, Jersey JE2 7LA, United Kingdom.
authors.forename: Daniel
authors.initials: D
authors.lastname: Binet
authors.affiliation: From the Department of Life Sciences, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom.
authors.forename: Federico
authors.initials: F
authors.lastname: Sastre
authors.affiliation: the Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Faulds-Pain
authors.affiliation: the Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom.
authors.forename: Esmeralda
authors.initials: E
authors.lastname: Valiente
authors.affiliation: the Vaccine Program, Human Health Therapeutics Portfolio, National Research Council, Ottawa, Ontario K1A 0R6, Canada, and.
authors.forename: Evgeny
authors.initials: E
authors.lastname: Vinogradov
authors.affiliation: the Vaccine Program, Human Health Therapeutics Portfolio, National Research Council, Ottawa, Ontario K1A 0R6, Canada, and.
authors.forename: Annie
authors.initials: A
authors.lastname: Aubry
authors.affiliation: the Vaccine Program, Human Health Therapeutics Portfolio, National Research Council, Ottawa, Ontario K1A 0R6, Canada, and.
authors.forename: Kelly
authors.initials: K
authors.lastname: Fulton
authors.affiliation: the Vaccine Program, Human Health Therapeutics Portfolio, National Research Council, Ottawa, Ontario K1A 0R6, Canada, and.
authors.forename: Susan
authors.initials: S
authors.lastname: Twine
authors.affiliation: the Vaccine Program, Human Health Therapeutics Portfolio, National Research Council, Ottawa, Ontario K1A 0R6, Canada, and.
authors.forename: Susan M
authors.initials: SM
authors.lastname: Logan
authors.affiliation: the Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom.
authors.forename: Brendan W
authors.initials: BW
authors.lastname: Wren
authors.affiliation: From the Department of Life Sciences, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom, a.dell@imperial.ac.uk.
authors.forename: Anne
authors.identifier: http://orcid.org/0000-0003-1380-2347
authors.initials: A
authors.lastname: Dell
authors.affiliation: From the Department of Life Sciences, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom.|BioPharmaSpec, Suite 3.1 Lido Medical Centre, St. Saviours Road, Jersey JE2 7LA, United Kingdom.
authors.forename: Howard R
authors.initials: HR
authors.lastname: Morris
mesh_terms: D016360:Clostridioides difficile / Q000737:chemistry* / Q000378:metabolism / Q000472:pathogenicity; D005408:Flagellin / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011135:Polysaccharides, Bacterial / Q000737:chemistry* / Q000378:metabolism
chemical_list: D011135:Polysaccharides, Bacterial; D005408:Flagellin; C416532:flaB flagellin
keywords: Clostridium difficile; Gram-positive bacteria; bacteria; flagellin; glycosylation; mass spectrometry (MS); modification; nuclear magnetic resonance (NMR); sulfonated