title: Role of Glycosyltransferases Modifying Type B Flagellin of Emerging Hypervirulent Clostridium difficile Lineages and Their Impact on Motility and Biofilm Formation.
abstract: Clostridium difficile is the principal cause of nosocomial infectious diarrhea worldwide. The pathogen modifies its flagellin with either a type A or type B O-linked glycosylation system, which has a contributory role in pathogenesis. We study the functional role of glycosyltransferases modifying type B flagellin in the 023 and 027 hypervirulent C. difficile lineages by mutagenesis of five putative glycosyltransferases and biosynthetic genes. We reveal their roles in the biosynthesis of the flagellin glycan chain and demonstrate that flagellar post-translational modification affects motility and adhesion-related bacterial properties of these strains. We show that the glycosyltransferases 1 and 2 (GT1 and GT2) are responsible for the sequential addition of a GlcNAc and two rhamnoses, respectively, and that GT3 is associated with the incorporation of a novel sulfonated peptidyl-amido sugar moiety whose structure is reported in our accompanying paper (Bouché, L., Panico, M., Hitchen, P., Binet, D., Sastre, F., Faulds-Pain, A., Valiente, E., Vinogradov, E., Aubry, A., Fulton, K., Twine, S., Logan, S. M., Wren, B. W., Dell, A., and Morris, H. R. (2016) J. Biol. Chem. 291, 25439-25449). GT2 is also responsible for methylation of the rhamnoses. Whereas type B modification is not required for flagellar assembly, some mutations that result in truncation or abolition of the glycan reduce bacterial motility and promote autoaggregation and biofilm formation. The complete lack of flagellin modification also significantly reduces adhesion of C. difficile to Caco-2 intestinal epithelial cells but does not affect activation of human TLR5. Our study advances our understanding of the genes involved in flagellar glycosylation and their biological roles in emerging hypervirulent C. difficile strains.
authors.affiliation: From 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 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: 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: From 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 Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, United Kingdom.
authors.forename: Mario
authors.initials: M
authors.lastname: Songane
authors.affiliation: From the Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom.
authors.forename: Lisa F
authors.initials: LF
authors.lastname: Dawson
authors.affiliation: From the Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Donahue
authors.affiliation: From the Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Stabler
authors.affiliation: 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: the Department of Life Sciences, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom.|BioPharmaSpec Ltd., Suite 3.1, Lido Medical Centre, St. Saviours Road, Jersey JE2 7LA, United Kingdom.
authors.forename: Howard R
authors.initials: HR
authors.lastname: Morris
authors.affiliation: the Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, United Kingdom.
authors.forename: Mona
authors.initials: M
authors.lastname: Bajaj-Elliott
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 Life Sciences, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom.
authors.forename: Anne
authors.initials: A
authors.lastname: Dell
authors.affiliation: From the Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom, Brendan.wren@lshtm.ac.uk.
authors.forename: Brendan W
authors.initials: BW
authors.lastname: Wren
mesh_terms: D001422:Bacterial Adhesion / Q000502:physiology*; D018441:Biofilms / Q000254:growth & development*; D018938:Caco-2 Cells; D016360:Clostridioides difficile / Q000472:pathogenicity / Q000502:physiology*; D005408:Flagellin / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D016695:Glycosyltransferases / Q000378:metabolism*; D006801:Humans; D051198:Toll-Like Receptor 5 / Q000378:metabolism
chemical_list: C495346:TLR5 protein, human; D051198:Toll-Like Receptor 5; D005408:Flagellin; C416532:flaB flagellin; D016695:Glycosyltransferases
keywords: Clostridium difficile; bacteria; biofilm; flagella; glycosylation; glycosyltransferase; gram positive bacteria; mutant