title: Comparative proteomics and glycoproteomics reveal increased N-linked glycosylation and relaxed sequon specificity in Campylobacter jejuni NCTC11168 O.
abstract: Campylobacter jejuni is a major cause of bacterial gastroenteritis. C. jejuni encodes a protein glycosylation (Pgl) locus responsible for the N-glycosylation of membrane-associated proteins. We examined two variants of the genome sequenced strain NCTC11168: O, a representative of the original clinical isolate, and GS, a laboratory-adapted relative of O. Comparative proteomics by iTRAQ and two-dimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC-MS/MS) allowed the confident identification of 1214 proteins (73.9% of the predicted C. jejuni proteome), of which 187 were present at statistically significant altered levels of abundance between variants. Proteins associated with the O variant included adhesins (CadF and FlpA), proteases, capsule biosynthesis, and cell shape determinants as well as six proteins encoded by the Pgl system, including the PglK flippase and PglB oligosaccharyltransferase. Lectin blotting highlighted specific glycoproteins more abundant in NCTC11168 O, whereas others remained unaltered. Hydrophilic interaction liquid chromatography (HILIC) and LC-MS/MS identified 30 completely novel glycosites from 15 proteins. A novel glycopeptide from a 14 kDa membrane protein (Cj0455c) was identified that did not contain the C. jejuni N-linked sequon D/E-X-N-X-S/T (X ≠ Pro) but that instead contained a sequon with leucine at the -2 position. Occupied atypical sequons were also observed in Cj0958c (OxaA; Gln at the -2 position) and Cj0152c (Ala at the +2 position). The relative O and GS abundances of 30 glycopeptides were determined by label-free quantitation, which revealed a >100-fold increase in the atypical glycopeptide from Cj0455c in isolate O. Our data provide further evidence for the importance of the Pgl system in C. jejuni.
authors.affiliation: School of Molecular Bioscience and ‡Charles Perkins Centre, The University of Sydney , Sydney, New South Wales 2006, Australia.
authors.forename: Nichollas E
authors.initials: NE
authors.lastname: Scott
authors.forename: N Bishara
authors.initials: NB
authors.lastname: Marzook
authors.forename: Joel A
authors.initials: JA
authors.lastname: Cain
authors.forename: Nestor
authors.initials: N
authors.lastname: Solis
authors.forename: Morten
authors.initials: M
authors.lastname: Thaysen-Andersen
authors.forename: Steven P
authors.initials: SP
authors.lastname: Djordjevic
authors.forename: Nicolle H
authors.initials: NH
authors.lastname: Packer
authors.forename: Martin R
authors.initials: MR
authors.lastname: Larsen
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Cordwell
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D016123:Campylobacter jejuni / Q000235:genetics / Q000378:metabolism*; D002236:Carbohydrate Conformation; D002352:Carrier Proteins / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D006023:Glycoproteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D040901:Proteomics / Q000379:methods*; D013045:Species Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C107872:CadF protein, Campylobacter jejuni; D002352:Carrier Proteins; C119770:FlpA protein, bacteria; D006023:Glycoproteins; D014157:Transcription Factors
keywords: Campylobacter jejuni; Cj0455c; N-linked glycosylation; bacterial virulence factors; membrane-associated proteins