title: The post-translational modification of the Clostridium difficile flagellin affects motility, cell surface properties and virulence.
abstract: Clostridium difficile is a prominent nosocomial pathogen, proliferating and causing enteric disease in individuals with a compromised gut microflora. We characterized the post-translational modification of flagellin in C. difficile 630. The structure of the modification was solved by nuclear magnetic resonance and shown to contain an N-acetylglucosamine substituted with a phosphorylated N-methyl-l-threonine. A reverse genetics approach investigated the function of the putative four-gene modification locus. All mutants were found to have truncated glycan structures by LC-MS/MS, taking into account bioinformatic analysis, we propose that the open reading frame CD0241 encodes a kinase involved in the transfer of the phosphate to the threonine, the CD0242 protein catalyses the addition of the phosphothreonine to the N-acetylglucosamine moiety and CD0243 transfers the methyl group to the threonine. Some mutations affected motility and caused cells to aggregate to each other and abiotic surfaces. Altering the structure of the flagellin modification impacted on colonization and disease recurrence in a murine model of infection, showing that alterations in the surface architecture of C. difficile vegetative cells can play a significant role in disease. We show that motility is not a requirement for colonization, but that colonization was compromised when the glycan structure was incomplete.
authors.affiliation: London School of Hygiene and Tropical Medicine, Keppel Street, London, WC1E 7HT, UK.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Faulds-Pain
authors.forename: Susan M
authors.initials: SM
authors.lastname: Twine
authors.forename: Evgeny
authors.initials: E
authors.lastname: Vinogradov
authors.forename: Philippa C R
authors.initials: PC
authors.lastname: Strong
authors.forename: Anne
authors.initials: A
authors.lastname: Dell
authors.forename: Anthony M
authors.initials: AM
authors.lastname: Buckley
authors.forename: Gillian R
authors.initials: GR
authors.lastname: Douce
authors.forename: Esmeralda
authors.initials: E
authors.lastname: Valiente
authors.forename: Susan M
authors.initials: SM
authors.lastname: Logan
authors.forename: Brendan W
authors.initials: BW
authors.lastname: Wren
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid; D016360:Clostridioides difficile / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D003015:Clostridium Infections / Q000382:microbiology / Q000473:pathology; D004195:Disease Models, Animal; D005408:Flagellin / Q000737:chemistry* / Q000378:metabolism*; D008124:Locomotion; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D011499:Protein Processing, Post-Translational; D012008:Recurrence; D013499:Surface Properties; D053719:Tandem Mass Spectrometry; D014774:Virulence
chemical_list: D005408:Flagellin