title: Comprehensive analysis of flagellin glycosylation in Campylobacter jejuni NCTC 11168 reveals incorporation of legionaminic acid and its importance for host colonization.
abstract: Campylobacter jejuni is the leading cause of bacterial gastroenteritis. It relies on several virulence factors for host colonization, including glycosylated flagella. C. jejuni NCTC 11168 modifies its flagellins with pseudaminic acid derivatives. It is also presumed to modify these proteins with legionaminic acid, although no glycopeptide evidence was available at the onset of this study. The enzyme encoded by cj1319 can be used to make legionaminic acid in vitro, but the pathway for legionaminic acid synthesis partially inferred by knockout mutagenesis in Campylobacter coli VC167 excludes Cj1319. To address this contradiction, we examined the presence of legionaminic acid in flagellin glycopeptides of wild-type (WT) C. jejuni NCTC 11168 and of a cj1319 knockout mutant. We used high-energy collision-induced dissociation to obtain amino acid sequences while also visualizing signature sugar oxonium ions. Data analysis was performed with PEAKS software, and spectra were manually inspected for glycopeptide determination and verification. We showed that legionaminic acid is present on the flagellins of C. jejuni NCTC 11168 and that flagellin glycosylation is highly heterogeneous, with up to six different sugars singly present at a given site. We found that the cj1319 mutant produces more legionaminic acid than WT, thus excluding the requirement for Cj1319 for legionaminic acid synthesis. We also showed that this mutant has enhanced chicken colonization compared with WT, which may in part be attributed to the high content of legionaminic acid on its flagella.
authors.affiliation: Department of Microbiology and Immunology and.
authors.forename: Najwa
authors.initials: N
authors.lastname: Zebian
authors.affiliation: Department of Microbiology and Immunology and.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Merkx-Jacques
authors.affiliation: Department of Biochemistry, Western University, London, ON N6A 5C1, Canada.
authors.forename: Paula P
authors.initials: PP
authors.lastname: Pittock
authors.affiliation: INRS-Institut Armand Frappier, Laval, QC H7V 1B7, Canada.
authors.forename: Sebastien
authors.initials: S
authors.lastname: Houle
authors.affiliation: INRS-Institut Armand Frappier, Laval, QC H7V 1B7, Canada.
authors.forename: Charles M
authors.initials: CM
authors.lastname: Dozois
authors.affiliation: Department of Biochemistry, Western University, London, ON N6A 5C1, Canada.
authors.forename: Gilles A
authors.initials: GA
authors.lastname: Lajoie
authors.affiliation: Department of Microbiology and Immunology and ccreuzen@uwo.ca.
authors.forename: Carole
authors.initials: C
authors.lastname: Creuzenet
mesh_terms: D016123:Campylobacter jejuni / Q000235:genetics / Q000472:pathogenicity; D005408:Flagellin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006020:Glycopeptides / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D054884:Host-Pathogen Interactions / Q000235:genetics; D006801:Humans; D016296:Mutagenesis; D012794:Sialic Acids / Q000737:chemistry / Q000378:metabolism*; D013400:Sugar Acids / Q000737:chemistry / Q000378:metabolism*
chemical_list: C513444:5,7-diacetamido-3,5,7,9-tetradeoxynonulosonic acid; C110570:5,7-diacetamido-8-O-acetyl-3,5,7,9-tetradeoxy-glycero-talo-nonulosonic acid; D006020:Glycopeptides; D012794:Sialic Acids; D013400:Sugar Acids; D005408:Flagellin
keywords: Campylobacter jejuni; flagellins; glycopeptide; legionaminic acid; pseudaminic acid