TY - JOUR
T1 - Comparative proteomics and glycoproteomics reveal increased n-linked glycosylation and relaxed sequon specificity in Campylobacter jejuni NCTC11168 O
AU - Scott, Nichollas E.
AU - Marzook, N. Bishara
AU - Cain, Joel A.
AU - Solis, Nestor
AU - Thaysen-Andersen, Morten
AU - Djordjevic, Steven P.
AU - Packer, Nicolle H.
AU - Larsen, Martin R.
AU - Cordwell, Stuart J.
PY - 2014/11/7
Y1 - 2014/11/7
N2 - 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.
AB - 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.
KW - Campylobacter jejuni
KW - Cj0455c
KW - N-linked glycosylation
KW - bacterial virulence factors
KW - membrane-associated proteins
UR - http://www.scopus.com/inward/record.url?scp=84908879032&partnerID=8YFLogxK
U2 - 10.1021/pr5005554
DO - 10.1021/pr5005554
M3 - Article
C2 - 25093254
AN - SCOPUS:84908879032
SN - 1535-3893
VL - 13
SP - 5136
EP - 5150
JO - Journal of Proteome Research
JF - Journal of Proteome Research
IS - 11
ER -