Comparative Proteomics and Glycoproteomics Reveal Increased N

Aug 5, 2014 - Campylobacter jejuni is a major cause of bacterial gastroenteritis. C. jejuni encodes a protein glycosylation (Pgl) locus responsible fo...
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Comparative Proteomics and Glycoproteomics Reveal Increased N‑Linked Glycosylation and Relaxed Sequon Specificity in Campylobacter jejuni NCTC11168 O Nichollas E. Scott,†,∇ N. Bishara Marzook,† Joel A. Cain,†,‡ Nestor Solis,†,○ Morten Thaysen-Andersen,§ Steven P. Djordjevic,∥ Nicolle H. Packer,§ Martin R. Larsen,⊥ and Stuart J. Cordwell*,†,‡,# †

School of Molecular Bioscience and ‡Charles Perkins Centre, The University of Sydney, Sydney, New South Wales 2006, Australia § Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia ∥ i3 Institute, University of Technology Sydney, Ultimo, New South Wales 2007, Australia ⊥ Protein Research Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense 5000, Denmark # Discipline of Pathology, School of Medical Sciences, The University of Sydney, Sydney, New South Wales 2006, Australia S Supporting Information *

ABSTRACT: Campylobacter jejuni is a major cause of bacterial gastroenteritis. C. jejuni encodes a protein glycosylation (Pgl) locus responsible for the Nglycosylation 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/EX-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. KEYWORDS: Campylobacter jejuni, Cj0455c, N-linked glycosylation, membrane-associated proteins, bacterial virulence factors

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INTRODUCTION Campylobacter jejuni is a Gram-negative, microaerophilic, spiralshaped motile bacterium that is the most common cause of bacterial gastroenteritis in the developed world,1 infecting ∼1% of the population in the United States and United Kingdom each year.2 C. jejuni infections are predominantly associated with consumption of contaminated poultry, in which the organism is an asymptomatic commensal.3 Symptoms in humans range from mild, noninflammatory diarrhea to severe abdominal cramps, vomiting and inflammation. C. jejuni has also been linked to the development of Guillain−Barré syndrome (GBS), an acute, debilitating immune-mediated disorder of the peripheral nervous system,4 and immunoproliferative small intestine disease, an infection-induced lymphoma originating in the mucosalassociated lymphoid tissue.5 © 2014 American Chemical Society

Studies attempting to understand the human pathogenesis of C. jejuni have typically taken a comparative genomics approach, and multiple strains have been sequenced.6−9 Transcriptomics and transposon mutagenesis have identified factors that correlate with infection;10−12 for example, a transposon screen revealed 195 genes essential for growth at 37 °C, of which 49 have no known function.13 C. jejuni has a highly plastic genome, with ∼21% sequence variation between strains.14 Coupled with an ability to undergo intragenomic recombination in response to Special Issue: Proteomics of Human Diseases: Pathogenesis, Diagnosis, Prognosis, and Treatment Received: June 5, 2014 Published: August 5, 2014 5136

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S/T (X ≠ Pro), where Asn (N) is the attachment site.33 The Nlinked heptasaccharide is encoded by the 16kb pgl (protein glycosylation) gene cluster, and the glycan is attached to proteins exposed to the periplasmic face of the inner membrane by the PglB oligosaccharyltransferase via en bloc transfer from an undecaprenyl phosphate lipid carrier.34−37 Over 80 glycosylation sites have now been identified across several C. jejuni strains.32,38,39 The N-glycan itself can be further modified by the addition of a phosphoethanolamine (pEtN) moiety to the terminal GalNAc of the “canonical” C. jejuni glycan, and this pEtN-modified glycan is attached to at least eight proteins.40 PglB can also liberate the glycan from the lipid carrier to form a periplasmic free oligosaccharide (fOS) that may provide protection against osmotic stress and is thought to be present at 10-fold excess compared to protein-linked glycan.41,42 Removal of genes from the pgl cluster (e.g., pglB) results in C. jejuni that display poor adherence to, and invasion of, epithelial cell lines and reduced chicken colonization;43,44 however, the mechanisms by which this occurs remain to be elucidated. We undertook a quantitative proteomic comparison of C. jejuni NCTC11168 O and GS, which identified proteins involved in adherence, proteolytic activity, cell shape, and capsule biosynthesis associated with the original isolate. We also observed a consistent increase in abundance of proteins encoded by the Pgl pathway. Relative abundance of glycopeptides was determined using label-free approaches alongside a complete glycoproteome analysis of these isolates using CID/HCD tandem mass spectrometry (MS/MS).39 These analyses suggest that the N-linked glycoproteomes of NCTC11168 O and GS are qualitatively similar, and very few glycopeptides from a test set of 30 demonstrated a quantitative difference. For the first time, however, we demonstrate the occurrence of glycopeptides with relaxed N-glycosylation sequon specificity in C. jejuni, one of which was >100-fold increased in abundance in NCTC11168 O.

selective pressures,15 this suggests strains that have been laboratory-adapted may not truly reflect clinical relevance with regard to gene expression associated with human infection or chicken colonization. Comparisons between the widely used genome strain (NCTC11168 GS [“genome sequenced”]) and the original clinical isolate (NCTC11168 O [“original”]) have shown that the infective phenotype (an ability to adhere to, and invade, epithelial cells in culture), as well as typical cell morphology, has been lost during laboratory adaptation.16 Despite this, they are indistinguishable by genotyping. Genome sequencing efforts comparing GS with isolates similar to O have revealed only seven point mutations17 as well as 41 singlenucleotide polymorphisms that involve 20 genes, including those associated with motility and chemotaxis.18 Global transcriptomics revealed marked difference in gene expression related to metabolic processes that suggests adaptation of GS to environments with higher oxygen tension.16 C. jejuni possesses an extensive pan-genome,19 and the comparison of unrelated strains with differing phenotypes can also be problematic due to differences in polymorphic regions. Laboratory-adapted strains, therefore, may contain genes that confer chicken colonization or human infection capability but that may fail to express or posttranslationally modify them correctly in comparison to their progenitor isolates. The C. jejuni NCTC11168 GS genome contains 1643 genes, a major proportion of which (∼10−20%) encodes membraneassociated proteins6,20,21 that are predominantly expressed in vitro but have no functional annotation.22 Human C. jejuni infection is poorly understood, but it involves colonization and adherence followed by internalization, invasion, and finally toxin production, leading to host cell death.3 Outer membrane proteins and processes, including flagellar motility,23,24 as well as surface adhesins, appear to be crucial for both chicken and human epithelial colonization. C. jejuni lacks typical virulenceassociated type III/IV secretion systems employed by enteric bacteria to secrete toxins and proteases that directly interact with host cells. One mechanism by which host interactions may occur is through the packaging of virulence-associated molecules into outer membrane vesicles (OMV) that can induce cytotoxicity to human intestinal epithelial cells.25,26 Colonization is, however, facilitated by the fibronectin (Fn)-binding adhesins CadF27 and FlpA.28 CadF negative mutants poorly colonize the avian host, whereas anti-CadF antibodies block Fn-binding by ∼50%.29 CadF also undergoes proteolytic processing, which results in a loss of recognition by patient sera while retaining Fn binding.30 Proteomic analysis of membrane-associated proteins from a clinical isolate (JHH1) and a laboratory-passaged strain (ATCC700297)22 showed that proteins involved in methylaccepting chemotaxis (MCP)-like signal transduction,31 surface antigens (CjaA and CjaC), and flagellar motility were unique to, or more abundant in, the clinical isolate. These results were similar to those previously observed in transcriptome comparisons of NCTC11168 O and GS.11,16 Several other membraneassociated proteins have also been implicated in C. jejuni adherence and invasion, including JlpA, PEB antigens, and Campylobacter invasion antigens (Cia), many of which are also packaged in OMVs.25 A unique molecular trait of C. jejuni is the ability to posttranslationally modify proteins by the N-linked addition of a seven-residue glycan (GalNAc-α1,4-GalNAc-α1,4-[Glcβ1,3]GalNAc-α1,4-GalNAc-α1,4-GalNAc-α1,3-BacAc2-β1; where BacAc2 is di-N-acetylbacillosamine[2,4-diacetamido-2,4,6 trideoxyglucopyranose])32 at the consensus sequon D/E-X-N-X-

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MATERIAL AND METHODS

Bacterial Strains and Cultivation

C. jejuni NCTC11168 O and GS were kindly supplied by Prof. Victoria Korolik (Griffith University, Australia). O and GS were cultured in parallel, each on 100 Skirrow’s agar plates (Oxoid, Basingstoke UK) in a microaerophilic environment of 5% O2, 5% CO2, and 90% N2 at 37 °C for 48 h. Plates were flooded with 5 mL of sterile phosphate-buffered saline (PBS), and colonies were removed with a cell scraper. Cells were washed three times in PBS, collected by centrifugation at 12 000g, and then snap frozen and lyophilized. Protein Extraction and iTRAQ Labeling

Whole cell lysates and membrane protein-enriched fractions were generated as described.22,39 Proteins from C. jejuni NCTC11168 O and GS whole cell lysates were collected from biological replicates (2 × 100 plate growth experiments), generating a total of four samples. Proteins were suspended in 6 M urea, 2 M thiourea, and 20 mM tetraethylammonium bromide (TEAB) and reduced for 1 h with 10 mM dithiothreitol (DTT) followed by alkylation with 20 mM iodoacetamide for 1 h under exclusion of light. Alkylation was quenched with 10 mM DTT. 1/200 (w/w) of endoproteinase Lys-C (Sigma-Aldrich, St. Louis, MO) was added, and digestion was allowed to proceed for 4 h at 25 °C. Samples were diluted 1:4 with 20 mM TEAB and further digested with 1/50 (w/w) porcine sequencing grade trypsin (Promega, Madison, WI) for 18 h at 25 °C. Peptides were concentrated and desalted by solid-phase extraction using tC18 5137

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within MS/MS scans the signal/noise was sufficient to discern matched peaks from background iTRAQ ratios of peptides containing variable modification, and the corresponding unmodified peptides were consistent across replicates; all major peaks within the MS/MS scan matched the assigned peptide, and only proteins possessing a minimum of two iTRAQ peptides were considered for quantitation (Supporting Information Data S1). Fold-change data were visualized as heat maps using GENE-E software (http://www.broadinstitute.org/ cancer/software/GENE-E/).

Sep-Pak columns (Waters, Milford, MA). Columns were washed with 1 mL of 100% methanol, 1 mL of 100% acetonitrile (MeCN), and 2 mL of 0.1% trifluoroacetic acid (TFA), consecutively. Peptides were acidified to a concentration of 0.1% TFA and passed through a column three times. Peptides were washed with 6 mL of 0.1% TFA and eluted with 1 mL of 70% MeCN, 0.5% formic acid (FA). Samples were dried and stored at −20 °C. Peptides were resuspended in 0.5 M TEAB, and 100 μg of each peptide mix was labeled using 4-plex iTRAQ (ABSciex, Foster City, CA) according to the manufacturer’s instructions. Peptides were labeled for 1 h at 25 °C, and the reaction was terminated by the addition of ultrapure water. Samples were pooled, mixed, and dried by vacuum centrifugation.

Western Blotting

Two milligrams of freeze-dried bacteria was suspended in Laemmli loading buffer (24.8 mM Tris, 10 mM glycerol, 0.5% (w/v) sodium dodecyl sulfate (SDS), 3.6 mM β-mercaptoethanol, and 0.001% (w/v) bromophenol blue (pH6.8)) and heated at 95 °C for 10 min. Insoluble material was removed by centrifugation at 20 000g for 15 min. Supernatants were loaded onto a 16 or 12% polyacrylamide resolving gel (with a 5% stacking gel) for JlpA-specific western and soybean agglutinin (SBA) lectin blotting, respectively. Proteins were separated in a Mini-PROTEAN 3 electrophoresis chamber (Bio-Rad, Hercules, CA) and transferred to poly(vinylidene difluoride) (PVDF) membrane using a Criterion Wet Electrophoretic Transfer Cell (Bio-Rad) for 1 h at 400 mA. Membranes were blocked overnight in 5% bovine serum albumin (Sigma) and probed with either a 1/ 2000 dilution of biotinylated SBA (5U/μL) (Vector Laboratories, Burlingame, CA) or 1/1000 JlpA-specific rabbit antiserum.46 Proteins reactive to biotinylated SBA were detected using a 1/ 4000 dilution of HRP-conjugated streptavidin (Millipore, Billerica, MA); proteins reacting against JlpA-specific rabbit antiserum were detected using a 1/1000 dilution of HRPconjugated goat-anti-rabbit immunoglobulin (Millipore), followed by incubation in Supersignal West Pico Chemiluminescent substrate according to the manufacturer’s instructions (Pierce, Rockford, IL). The resulting blots were visualized using Hyperfilm ECL (GE LifeSciences, Amersham, UK) and a CP100 film processor (AGFA, Mortsel, Belgium). Densitometry was performed using PD-Quest (Bio-Rad).

2D-LC−MS/MS

Peptides were resuspended in buffer A (5 mM phosphate, 25% MeCN, pH 2.7) and loaded onto a PolyLC PolySulfethyl A 200 mm × 2.1 mm 5 μm column (PolyLC Inc., Columbia MD) using an Agilent 1100 (Agilent Technologies, Santa Clara, CA) liquid chromatography (LC) system. Peptide fractionation was achieved by altering the concentration of buffer B (5 mM phosphate, 350 mM KCl, 25% MeCN, pH 2.7). Initially, the column was washed with 10% buffer B for 6 min and then increased from 10 to 45% over 70 min. Buffer B concentration was then adjusted to 100% for 10 min. The system was run with a constant flow rate of 200 μL/min, and fractions were collected every 2 min (43 fractions). A Tempo nanoLC (Eksigent, Dublin, CA) and QStar Elite electrospray ionization mass spectrometer (ABSciex) were used for MS/MS and iTRAQ quantitation. SCX fractions were resuspended in loading solution (0.1% TFA, 2% MeCN), loaded onto a reversed-phase (RP) peptide Captrap column (Michrom Bioresources, Auburn, CA), and desalted at 10 μL/min for 13 min with 98% phase A (0.1% FA)/2% phase B (0.1% FA, 80% MeCN). The trap was switched in line with a 150 μm × 10 cm C18 3 μm 300 Å ProteCol column (SGE, Austin, TX). Phase B concentration was increased from 2 to 90% over 120 min. The column was cleaned with 100% phase B for 15 min and equilibrated with phase A for 30 min before each sample injection. The RP nanoLC elution was infused directly by nanoflow electrospray into the mass spectrometer operated in information-dependent acquisition mode (IDA). A TOF-MS survey scan was acquired (m/z 380−1600, 0.5 s), with the three most intense multiply charged ions (counts >50) subjected to MS/MS. MS/MS spectra were accumulated for 2 s maximum in the mass range m/z 100−1600 with a modified Enhance All Q2 transition setting favoring low mass ions so that iTRAQ reporter ion intensities were enhanced for quantitation.

Preparation of C. jejuni Glycopeptides

Dried membrane protein-enriched fractions were resuspended in 6 M urea, 2 M thiourea, 40 mM NH4HCO3. Proteins were reduced, alkylated, and digested with Lys-C (1/200 w/w) and trypsin (1/50 w/w), as above. Alternatively, proteins were digested with pepsin or thermolysin (Sigma-Aldrich) postreduction/alkylation.47 For pepsin, samples were diluted 1:4 with 0.1% TFA and adjusted to pH 2.5 with 10% TFA. A 1/25 (w/w) ratio of pepsin to protein was added, and proteins were digested for 24 h at 25 °C. For thermolysin, samples were diluted 1:4 with 100 mM NH4HCO3 and 1:25 (w/w) thermolysin to protein added. Samples were incubated for 24 h at 25 °C. All digests were dialyzed against ultrapure water overnight using a Mini Dialysis Kit with a molecular mass cutoff of 1000 Da (Amersham Biosciences, UK) and were then collected and lyophilized. Glycopeptides were enriched from complex mixtures using zwitterionic−hydrophilic interaction liquid chromatography (ZIC-HILIC) as described39 with minor modifications. Briefly, microcolumns composed of 10 μm ZIC-HILIC resin (Sequant, Umeå, Sweden) packed into Proxeon (Odense, Denmark) P10 C8 tips to a bed length of 0.5 cm were washed with ultrapure water. Samples were resuspended in 80% MeCN and 5% FA, and insoluble material was removed by centrifugation at 20 000g for 5 min at 4 °C. Peptides were

Data Processing and Analysis

MS/MS data were processed using ProteinPilot v3.0 (ABSciex). Data were searched against a C. jejuni NCTC11168 FASTA database (NCBI Refseq: NC_002163; 1643 entries) using the Paragon search algorithm with software correction factors provided with the 4-plex iTRAQ labels entered into the isotope correction table. The protein threshold was set at >1.3 (>95% confidence) with false discovery rate (FDR) analysis enabled, giving a final FDR of ∼1%. To further validate the data, all matches with a confidence 2 peptides and with ratios (>1.25 (red scale) and 1.25-fold and 2-fold variation). These were 351ENNDTIAMANHK362 from cytochrome bd oxidase subunit I (CydA; Cj0081) and 21ANTPSDVNQTHTK33 from putative periplasmic protein Cj0168c, which were present at increased abundance in NCTC11168 GS. The final abundance difference was observed in the unique glycopeptide corresponding to residues 55QQVIVLQNQTK65 from Cj0455c, which was present at >100-fold higher abundance in isolate O. Cj0455c was 5145

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Figure 6. Label-free quantitation of identified glycopeptides compared between C. jejuni NCTC11168 O and GS. A total of 30 glycopeptides from 26 glycoproteins were quantified, with only three glycopeptides observed at >2-fold abundance difference between strains: 55QQVIVLQNQTK65 of Cj0455c (>100-fold increase in O), 351ENNDTIAMANHK362 of Cj0081 (2.75-fold increase in GS), and 21ANTPSDVNQTHTK33 of Cj0168c (3.75fold increase in GS). Data used to derive this figure are found in Supporting Information Data S3A and S3B.

tions. Those most associated with isolate O included cell wall and shape (all 13 regulated members present at increased abundance), defense (8/9 proteins elevated in O, including those involved in heat and oxidative stress), and iron-related functions (12/17 proteins elevated in O). Significant virulenceassociated proteins, including the Fn-binding proteins CadF and FlpA, were associated with isolate O, which is consistent with the ability of this isolate to adhere to epithelial cells, whereas elevated CiaB is consistent with the invasive capability of this isolate. The cytolethal-distending toxin CdtA and the Campylobacter adhesion protein A (CapA), however, were significantly more abundant in isolate GS. CapA is a known phase variable protein that, despite evidence for a role in adhesion and invasion of Caco2 cells,55 may be expressed only in GS and not in O.16 Although GS shows a motility defect in comparison to that of O, our data were contradictory for proteins involved in this process, for example, some flagellar proteins (flagellin FlaA and FlgI) were at higher abundance in GS, whereas others (FlgK and FlgS) were elevated significantly in isolate O. Chemotaxis proteins CheV and CheY were also associated with GS. These data are somewhat surprising given that motility and chemotaxis are intimately associated with successful C. jejuni colonization. Several of the observed alterations matched previous transcriptomics analyses, including alterations in gluconeogenesis (Cj1400c-Cj1403 elevated in O compared to that in GS16) and increased abundance of HtrA protease Cj1228c in isolate O.23 Other trends appear unique to our study, such as changes in nutrient and cation transporters and the association between isolate GS and protein translation. We observed elevated NCTC11168 O abundances of proteins within the Pgl N-linked glycosylation pathway. Elevated Pgl proteins in isolate O thus suggested one or more of the following: increased biosynthesis of the N-glycan and attachment to (i) all glycoproteins equally, (ii) specific proteins, and/or (iii) increased periplasmic fOS. Within the Pgl pathway, only PglE

whereas the peptide from Cj0168c was also identified with missed tryptic cleavages. These associated peptides were thus examined to ensure that the observed increases in NCTC11168 GS were consistent. The oxidized peptide 351ENNDTIAMoxANHK362 demonstrated near identical fold change (2.65 compared to 2.75 for the unoxidized form; Supporting Information Data S3B), whereas the missed cleavage forms of 21 ANTPSDVNQTHTK33 showed a range of ratios from −1.4 to 2.9, thus making confident assertions regarding protein abundance and glycosylation site occupancy problematic. Of the 26 proteins containing glycopeptides that were subjected to label-free quantitation, 23 were identified in the original iTRAQ study (Cj0011c, Cj0168c, and Cj1496c were not identified in that data set), and 21/23 showed no change in protein abundance between NCTC11168 O and GS, which is consistent with the glycopeptide data. Cj1032, however, was observed at reduced abundance in isolate O, whereas Cj1126c (the PglB oligosaccharyltransferase) was present at elevated abundance. Despite these changes, no differences were observed in the relative levels of their glycopeptides. This further confirms that specific proteins are the targets of increased glycosylation in isolate O, although apart from Cj0455c, these remain to be identified.

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DISCUSSION

Comparative proteomics of C. jejuni NCTC11168 O and GS was driven by a goal to identify the molecular mechanisms accounting for phenotypic differences between these genetically nearindistinguishable variants.16−18 Such differences include reduced motility, altered cell morphology, changes in glycan binding, poor chicken colonization, and attenuated adherence to, and invasion of, human epithelial cells in the laboratory-adapted GS variant.16,54 Proteins of differential abundance between the two isolates could be clustered into 13 broad functional classifica5146

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forms, whereas in ref 42, MS peak areas for the permethylated fOS (1755.0 m/z) and Asn-bound (1866.1 m/z) glycans were compared using ESI−MS. Permethylation and other sample handling steps may result in a change in LC retention that could influence ESI−MS results, while, additionally, a biological basis for this discrepancy may result from the method of culture: on solid agar plates in our study and in broth in ref 42. Deletion of N-glycosylation reduces the ability of C. jejuni to effectively colonize chickens and to adhere to, and invade, human epithelial cells; however, site-directed mutagenesis of a handful of N-glycosites has been relatively unsuccessful in determining a functional link between N-glycosylation and these phenotypes. Despite this, studies have demonstrated that N-linked glycosylation is required for DNA uptake, as has been seen for the glycoprotein VirB10,62 and for binding of the human macrophage galactose-type lectin.63 As such, the elucidation of modified proteins is critical for providing targets to assess the functional significance of glycosylation on specific proteins; however, the vast majority of identified glycoproteins have no known function (e.g., Table 1). The most significant alteration within the 30 glycopeptides chosen for label-free quantitation was the >100-fold increase in the glycopeptide from Cj0455c 55 QQVIVLQNQTK65. Confirmation of this glycosylation site using ETD fragmentation showed that C. jejuni is able to glycosylate at atypical sequons not consistent with the established D/E-X-N-X-S/T motif. This phenomenon has been documented for other glycosylation systems: the N-linked system of Campylobacter lari64 and in Desulfovibrio desulfuricans.65 In both cases, glycosite occupation, as analyzed with variants of the model bacterial glycosylation substrate AcrA, was only partially dependent on D/E-X-N-X-S/T. In D. desulfuricans, occupation of AcrA N274 was dependent only on T276, whereas in C. lari, examples of AcrA glycosylation at sites containing either N-X-S/T or D/E-X-N could be observed. More recently, the kinetics of C. lari PglB sequon association has been determined and showed strongly reduced affinity for Ala (which mimicked the eukaryotic sequon) in the −2 position.66 Negatively charged amino acids at the −2 position also greatly enhanced the efficiency of glycosylation in test substrates.64 Furthermore, studies of C. jejuni PglB showed Asp compared to Glu at the −2 position leads to a 5-fold increase in glycosylation kinetics on model peptides,67 while a preference for Thr over Ser at the +2 position has also been suggested.66,67 Annotation of the 86 glycosites identified here showed a preference for Asp at the −2 position, consistent with PglB kinetics (54 Asp-containing sequons, 30 Glu-containing sequons; 2 other), but it did not demonstrate any significant bias toward Thr or Ser at the +2 position (with 39 Thr-containing sequons, 46 Ser-containing sequons, and 1 atypical sequon with Ala at the +2 position). Strict assignment of glycosites from MS/MS data without reference to the canonical sequon required detection of glycopeptide forms containing a single Asn residue. This was facilitated by the use of three proteases, two of which are considered to be nonspecific (pepsin and thermolysin). Although these enzymes are beneficial for site localization, they are not suitable for label-free quantitation, as the lack of specificity leads to dilution of the signal across multiple glycopeptides corresponding to the same glycosylation site. The use of multiple proteases enabled the confirmation of 48 glycosylation site assignments (peptides with a single Asn). A further seven sites were localized by the presence of an ion retaining BacAc2 using HCD MS/MS. This approach exploits the stability of N-linked carbohydrates compared to that of internal

deviated from the trend and showed unaltered abundance between O and GS, whereas PglD was not identified. Similar results have been observed in transcriptomic studies where other members of the pgl locus were increased in response to iron limitation, while pglE undergoes a 2-fold decrease in expression.56 Our data support the finding that members of the N-linked glycosylation system can be differentially expressed irrespective of the operon-like nature of the pgl locus.57 Biosynthesis of the initial sugar, 2,4-diacetamido-2,4,6-trideoxyglucopyranose (BacAc2), predominantly depends on the sequential action of PglF, E, and D.58,59 As such, the level of PglE may confer a rate-limiting step in the production of the Nlinked glycan, although our evidence of increased glycosylation of specific proteins does not necessarily support this case. Alternatively, observation of N-linked glycosylation in the absence of pglE, albeit at a significantly lower level,41 suggests other pathways (e.g., pseudaminic acid biosynthesis) could compensate to some degree for the loss of this enzyme. Our assessment of glycosylation at the protein and peptide levels suggests that alterations in the N-linked glycosylation pathway between NCTC11168 O and GS influence glycosylation levels of only a minority of proteins. Because Nglycosylation in C. jejuni is dictated by several factors, including PglB site accessibility on previously folded substrates (occurring mainly at flexible loop regions),36 the kinetics of glycosylation associated with sequon composition, and protein stability in the presence and absence of the glycan, it remains difficult to determine the contribution made by protein abundance alone in any altered glycosylation site occupancy between isolates, as changes to protein structure may also be involved. iTRAQ-based quantitation demonstrated that at the protein level only two glycoproteins differed in their abundances between variants (Cj0143 and Cj1032). Of the altered glycopeptides, we observed no iTRAQ protein abundance differences for either Cj0081 or Cj0168c, which suggests increased site occupancy in GS unrelated to protein abundance; however, Cj0455c glycosylation appeared to be heavily associated with abundance, as the cj0455c gene is expressed at significantly higher levels in O compared with that in GS, consistent with the large increase in glycosylation observed for this peptide. The PglB oligosaccharyltransferase appears to self-regulate utilization of lipid-bound glycan.41 This is thought to allow dynamic changes in the levels of fOS in response to osmotic challenge while maintaining protein glycosylation. We monitored both fOS and Asn-bound glycan between variants using LC−SRM. Variability of quantitation (CVs between 15 and 35%) limited our ability to discern any subtle differences between O and GS; however, CVs were within acceptable limits previously described for analysis of free glycan species.60 It is thus reasonable to conclude that if any differences do exist in free glycan levels then they are 100-fold abundance in the NCTC11168 O variant, and this appears to be predominantly related to genetic expression and protein abundance rather than increased modification of an existing pool of Cj0455c protein.

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ABBREVIATIONS

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REFERENCES

BacAc2, 2,4-di-N-acetylbacillosamine; ETD, electron transfer dissociation; Fn, fibronectin; fOS, free oligosaccharide; GS, genome sequenced isolate of C. jejuni NCTC11168; HCD, higher energy collisional dissociation; O, original clinical isolate of C. jejuni NCTC11168; OMV, outer membrane vesicles; Pgl, protein glycosylation locus; SBA, soybean agglutinin; SRM, selected reaction monitoring; TEAB, tetraethylammonium bromide; ZIC-HILIC, zwitterionic−hydrophilic interaction liquid chromatography

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ASSOCIATED CONTENT

S Supporting Information *

Data S1 contains the data set showing peptide identification and quantitation from iTRAQ analysis of C. jejuni O and GS, and the statistical analysis and false quantitation rate of that data are contained in Data S4. Data S2 contains all glycopeptide MS/MS spectra, and Data S3 contains the label-free glycopeptide quantitation data. Data S5 contains the data used to determine the fOS:Asn-bound N-glycan ratio. Tables S1 and S2 show proteins higher (S1) or lower (S2) in abundance in C. jejuni NCTC11168 O compared with that in GS. Figure S1 shows the additional functional annotations of proteins with altered abundance in C. jejuni NCTC11168 O and GS. Figure S2 shows the ETD MS/MS spectrum of the glycopeptide containing an atypical sequon from Cj0152c. Figure S3 shows glycosylation site localization via fragment ions that retain BacAc2. Figure S4 shows the identification of a previously unrecognized ORF, and Figure S5 shows q-PCR data of cj0455c. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (+61-2) 9351-6050; Fax: (+61-2) 9351-4726; E-mail: [email protected]. Present Addresses ∇

(N.E.S.) Centre for High-Throughput Biology, Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada. ○ (N.S.) Centre for Blood Research, The University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by an Australian Research Council Discovery Project Grant to S.J.C. (ARC DP11053753). N.E.S., N.S., and J.A.C. are recipients of Australian Postgraduate Awards. 5148

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