Identification of novel carbohydrate modifications
on Campylobacter jejuni 11168 flagellin using
metabolomics-based approaches
Susan M. Logan
1
, Joseph P. M. Hui
2
, Evgeny Vinogradov
1
, Annie J. Aubry
1
, Jeremy E. Melanson
2
,
John F. Kelly
1
, Harald Nothaft
1
and Evelyn C. Soo
2
1 NRC-Institute for Biological Sciences, Ottawa, Canada
2 NRC-Institute for Marine Biosciences, Halifax, Canada
Campylobacter is an important human pathogen and
the most prevalent causative agent of bacterial gastro-
enteritis worldwide, with Campylobacter jejuni repre-
senting over 90% of all Campylobacter infections [1].
Most cases of Campylobacter infections are sporadic
and can be traced to the consumption of undercooked
(or the handling of) contaminated chicken, but out-
breaks, although rare, do occur mainly as a result of
the consumption of contaminated sources of water or

11168 remain elusive due to lability and respective levels of individual gly-
can modifications. We report the characterization of the carbohydrate
modifications on C. jejuni 11168 flagellin using metabolomics-based
approaches. Detected as their corresponding CMP-linked precursors, struc-
tural information on the flagellin modifications was obtained using a com-
bination of MS and NMR spectroscopy. In addition to the pseudaminic
acid and legionaminic acid sugars known to be present on Campylobacter
flagellin, two unusual 2,3-di-O-methylglyceric acid modifications of a nonu-
losonate sugar were identified. By performing a metabolomic analysis of
selected isogenic mutants of genes from the flagellin glycosylation locus of
this pathogen, these novel CMP-linked precursors were confirmed to be
di-O-methylglyceric acid derivatives of pseudaminic acid and the related
acetamidino sugar. This is the first comprehensive analysis of the flagellar
modifications in C. jejuni 11168 and structural elucidation of di-O-methyl-
glyceric acid derivatives of pseudaminic acid on Campylobacter flagellin.
Abbreviations
HILIC, hydrophilic interaction liquid chromatography; HMBC, heteronuclear multiple bond correlation; Pse, pseudaminic acid.
1014 FEBS Journal 276 (2009) 1014–1023 ª 2009 The Authors Journal compilation ª 2009 FEBS
post-infection neuropathy Guillain–Barre
´
Syndrome [3]
and rare malignant lymphomas of the small intestine
known as immunoproliferative small intestine disease
[4].
Flagellae comprise an important virulence factor of
many bacterial pathogens that confers motility and
allows colonization of host cells. In Campylobacter, the
major structural protein FlaA must be glycosylated for
flagellar filament assembly [5] and, given the impor-
tance of motility in infectivity, there is tremendous

lished proteomics-based approaches were unsuccessful.
In addition, although the identification of biosynthetic
genes had been made via mutagenesis studies [5], the
functional characterization of the flagellar glycan bio-
synthetic enzymes and nonulosonate sugar pathways
was poorly described.
Metabolomics has recently emerged as an invalu-
able tool for the study of poorly characterized meta-
bolic pathways. In the first metabolomic study of
Campylobacter, a targeted metabolomic screen of
C. jejuni 81-176 revealed the tremendous potential for
using metabolomics to identify unknown substrates
and elucidate the role of genes in the biosynthesis of
the novel flagellin glycan structures [11]. This work
led to expanded studies of the flagellin glycosylation
locus in Campylobacter [12,13] and highlighted the
innovative use of metabolomics as an alternative to
proteomics-based approaches [8–10,14] to gain precise
structural information on novel carbohydrate moieties
that glycosylate the flagellar protein. The use of
hydrophilic interaction liquid chromatography (HI-
LIC)-MS in these recent studies was critical because
it allowed discrimination of metabolites with the same
mass-to-charge (m ⁄ z) ratios, which would otherwise
be indistinguishable using MS alone. The HILIC-MS
method allowed the separation of complex mixtures
of CMP-linked nonulosonic acids and the large-scale
purification of these novel metabolites for NMR
analysis. The resolution afforded by HILIC for
sugar-nucleotides ultimately led to the unexpected

the wild-type strain (Fig. 1). Upon closer examination
of these CMP-linked sugars, it was observed that the
retention times of six of the CMP-linked sugars and
S. M. Logan et al. Flagellin glycosylation in C. jejuni
FEBS Journal 276 (2009) 1014–1023 ª 2009 The Authors Journal compilation ª 2009 FEBS 1015
their corresponding m ⁄ z values were consistent with
those obtained in earlier metabolomic studies of
C. jejuni 81-176 and C. coli VC167 [12,13], identify-
ing these metabolites as the CMP-linked precur-
sors of Pse5Ac7Ac, Pse5Ac7Am, Leg5Ac7Ac,
Leg5Am7Ac, Leg5MeAm7Ac and Neu5Ac (Leg is
5,7-diamino-3,5,7,9-tetradeoxy-d-glycero-d-galacto-non
ulosonic acid). The presence of these CMP-linked pre-
cursors in C. jejuni 11168 is not surprising because
the genes known to be involved in their biosynthesis
in C. jejuni 81-176 (Pse5Ac7Ac, Pse5Ac7Am and
Neu5Ac) or C. coli VC167 (Leg5Ac7Ac, Leg5Am7Ac,
Leg5MeAm7Ac) are also present in the 11168 strain.
However, in addition to these well-characterized
CMP-linked intermediates, two unknown CMP-linked
precursors were also observed in the metabolome of
C. jejuni 11168. As shown in Fig. 1, one of these
novel CMP-sugars was detected at 13.6 min as
[M ) H]
)
ions at m ⁄ z 712, whereas the second novel
CMP-sugar was observed at 17.3 min at m ⁄ z 711. In
the positive mode, oxonium ions corresponding to
these novel CMP-linked sugars are produced and
these were observed as precursor ions at m ⁄ z 714 (I)

ions at m ⁄ z 390.2 and 391.2 (underlined) and related degradation
products. Loss of water molecules from the m ⁄ z 391.2 ion yields
the strong fragment ions at m ⁄ z 373.2 and 355.2, respectively,
whereas the ion at m ⁄ z 346.2 arises from the loss of CO
2
from the
oxonium ion at m ⁄ z 390.2. The ion at m ⁄ z 1943.4 corresponds to
the intact peptide ion (singly charged) having lost the glycan modifi-
cation. (B) MS ⁄ MS spectrum of the doubly charged ion at m ⁄ z
1064.5 corresponding to the glycopeptide, T
463–479
, also modified
with a single glycan moiety. In this instance, the peptide appears to
be modified predominantly with the 389 Da glycan as its oxonium
ion and the related degradation products are dominant. Regions of
both spectra have been expanded to highlight some of the less
abundant but informative fragment ions.
Flagellin glycosylation in C. jejuni S. M. Logan et al.
1016 FEBS Journal 276 (2009) 1014–1023 ª 2009 The Authors Journal compilation ª 2009 FEBS
nucleotides within the metabolome. However, to derive
meaningful structural information on the novel CMP-
sugars and reduce ambiguity, it was necessary to
employ high resolution MS for subsequent experi-
ments. Accurate mass measurements of I and II were
performed on a Waters Q-ToF Premier mass spec-
trometer (Waters Corp., Milford, MA, USA) and
accurate masses of the protonated [M + H]
+
ions of I
and II were revealed as 714.2222 and 713.2403 Da,

, sug-
gesting that these novel metabolites are CMP-linked
(data not shown). In the positive mode, MS ⁄ MS of I
and II revealed major fragment ions at m ⁄ z 391.1723
and 390.1887, which correspond to the masses of the
two novel carbohydrate moieties (Fig. 3D,E). To gener-
ate structural information on these carbohydrate moie-
ties, further tandem MS experiments were carried out
to fragment the novel carbohydrates. As shown in
Fig. 4A,B, the second generation product ion spectra
for I and II revealed fragmentation patterns that are
typically observed for nonulosonic acids [9,10,12–14].
For example, characteristic and consecutive neutral
losses of water, ammonia and formic acid were
observed in the second generation product ion spectra
of both I and II. It is noteworthy that a prominent loss
of the acetamidino functionality, CH
3
C(=NH)NH
Fig. 3. Accurate mass measurements of unknown CMP-sugars detected in C. jejuni 11168. (A) Extracted ion chromatogram of m ⁄ z 714 (I)
and 713 (II). (B) MS at 14.6 min showing the accurate mass of I. (C) MS at 17.9 min showing accurate mass of II. (D) Corresponding
MS ⁄ MS spectrum of I. (E) Corresponding MS ⁄ MS spectrum of II.
S. M. Logan et al. Flagellin glycosylation in C. jejuni
FEBS Journal 276 (2009) 1014–1023 ª 2009 The Authors Journal compilation ª 2009 FEBS 1017
(neutral loss of 58 Da), was also observed in the second
generation product ion spectrum of II (Fig. 4B). Based
on our existing knowledge of Campylobacter flagellar
glycans, it is highly plausible that II is the related ace-
tamidino derivative of I because such a feature appears
to be prevalent among the nonulosonic sugars in Cam-

H
6
O
2
(theoretical mass = 74.0367 Da), thus hav-
ing the overall formula C
5
H
9
O
3
. To unequivocally
assign the structures of I and II, large-scale purifica-
tions of the metabolites were achieved, as described
previously [12,13] for NMR structural elucidation.
Structural analysis of II by NMR spectroscopy
By contrast to the earlier work on C. jejuni 81-176 and
C. coli VC167, the novel CMP-linked metabolites I
and II detected in C. jejuni 11168 were rather unstable
compounds. MS analysis of the purified substrates
revealed that approximately 20 lg of each metabolite
was isolated but, upon their analysis by NMR, it was
observed that degradation of the metabolites had
occurred. This was particularly true for I where com-
plete degradation of the metabolite appeared to have
taken place, and therefore it was only possible to pur-
sue the structural analysis of II by NMR spectroscopy.
In addition to the problem of instability, it was also
observed that the sample contained appreciable
amounts of impurities, including glycerol, lactic acid

13
C signals of these groups (see Table S2) cor-
responded to 2,3-di-O -methyl-glycerate. The COOH
group signal was not observed in HMBC due to its
presence in concentrations below the limit of detection;
thus, the linkage of dimethylglycerate to the nonulo-
sonic acid could not be directly confirmed. However,
Fig. 4. Second generation product ion spectrum of (A) I and (B) II.
Broken arrows indicate the possible neutral loss of the 2,3-di-O-
methyl-glyceramide (C
5
H
11
NO
3
).
Flagellin glycosylation in C. jejuni S. M. Logan et al.
1018 FEBS Journal 276 (2009) 1014–1023 ª 2009 The Authors Journal compilation ª 2009 FEBS
given that the exact theoretical mass of CMP-Pse5acy-
l7Ac (where acyl is 2,3-di-O-methylglycerate) (i.e.
C
25
H
42
N
6
O
16
P
1

C chemical shifts of II with model
compounds bearing either two acetyl groups (Pse5A-
c7Ac) or one acetyl group and one amidino group at
N-7 (Pse5Ac7Am) showed good agreement of the
7-amidino derivative with the data for the analyzed
compound (see Table S2). The 7-N-acetyl derivative
had C-7 signal shifted more than 4 p.p.m. upfield com-
pared to that of the 7-amidino derivative. This differ-
ence is characteristic for the amidine substitution and
has been reported also with other sugars [16]. Thus, II
is likely to be substituted with the dimethylglycerate
group at N-5 and with amidine at N-7.
Metabolomic analysis of pseB and pseC Pse
biosynthetic genes
Earlier studies on Pse biosynthesis in Campylobacter
identified key roles for pseB and pseC in the biosyn-
thetic process [12,17,18] and the insertional inactiva-
tion of these two genes had led to the disappearance
of CMP-Pse5Ac7Ac and CMP-Pse5Ac7Am from the
metabolome of C. jejuni 81-176 [12,17,18]. The analysis
of I and II by MS and NMR analyses in the present
study strongly suggests that these two CMP-sugars are
novel modifications of Pse. However, considering the
complexity of the flagellin glycosylation locus of
C. jejuni 11168 and the ability of this strain of Cam-
pylobacter to synthesize both Pse and legionaminic
acid sugars, it would be invaluable to obtain support-
ing biological data to confirm these CMP-sugars as
novel derivatives of Pse. Given that pseB and pseC are
exclusively involved in Pse5Ac7Ac biosynthesis, there

l-manno-nonulosonic acid (Fig. 5).
Fig. 5. Proposed structures of I and II.
S. M. Logan et al. Flagellin glycosylation in C. jejuni
FEBS Journal 276 (2009) 1014–1023 ª 2009 The Authors Journal compilation ª 2009 FEBS 1019
Discussion
Metabolomics has provided a unique opportunity to
highlight subtle differences in the nature of the glyco-
syl moieties that decorate the flagellin of different
Campylobacter strains through structural elucidation
of their corresponding biosynthetic intermediates. Ear-
lier studies of C. coli VC167 had revealed the potential
for Campylobacter to synthesize a variety of legionami-
nic acid sugars, and this capability was again utilized
in the present study to investigate C. jejuni 11168. In
addition to previously well characterized glycosyl mod-
ifications, two novel carbohydrate modifications were
also detected in the metabolome of C. jejuni 11168 as
their corresponding CMP-linked intermediates. Exten-
sive structural analysis of these CMP-sugars using a
combination of high resolution MS and NMR spec-
troscopy identified these metabolites as the dimethyl-
glyceric acid derivatives of Pse and the related
acetamidino derivative. It is noteworthy that the use of
high resolution MS in the present study was instru-
mental in elucidating the structures of the carbohy-
drate moieties. This was particularly true of I, where
complete degradation of the metabolite had occurred
during NMR analysis and structural information could
only be gained using high resolution MS. Although the
absolute configuration of the novel glycans could not

present study can now also be explored.
Campylobacter is not unique in attaching novel
nonulosonate sugar derivatives to its flagellin. Flagel-
lins from a strain of Campylobacter botulinum,a
Gram-positive spore forming anaerobe, have also been
shown to be glycosylated with a novel legionaminic
acid derivative, 7-acetamido-5-(N-methyl-glutam-4-yl)-
amino-3,5,7,9-tetradeoxy-d-glycero-a-d-galacto-nonulo-
sonic acid (Leg5GluMe7Ac) [25], whereas other strains
appear to produce related structures. It is not yet
known whether these modifications also contribute to
the colonization ability of C. botulinum isolates in
distinct animal hosts.
Experimental procedures
Bacterial strains and growth conditions
C. jejuni 11168 and isogenic mutants pseB and pseC were
grown using the procedure as described previously [12].
Purification of flagellin
Flagellin was purified as previously described [26], although
the solubilization step in 1% SDS was eliminated and the
crude pellet after ultracentrifugation was characterized
directly by MS.
LC-MS/MS analysis of flagellin
Flagellin protein was digested overnight with trypsin (50–
200 lg; Promega, Madison, WI, USA) at a ratio of 30 : 1
(protein : enzyme, v ⁄ v) in 50 mm ammonium bicarbonate
at 37 °C. Protein digests were analyzed by MS as previ-
ously described [10].
Construction of C. jejuni 11168 pseB and pseC
mutant strains

mutants pseB and pseC were prepared and extraction of
intracellular sugar-nucleotides from the cell lysates was
achieved using ENVI-Carb (Supelco, Bellefonte, PA, USA)
solid phase extraction cartridges, as described previously
[12].
MS
Cell lysates of parent strain C. jejuni 11168 and isogenic
mutants pseB and pseC were probed for intracellular sugar-
nucleotides using HILIC-MS and a precursor ion scanning
method, as described previously [12]. The intracellular
sugar-nucleotides were separated by HILIC on a TSKgel
Amide80 column (inner diameter 250 · 4.6 mm; Tosoh Bio-
science, Montgomeryville, PA, USA) using an Agilent 1100
Series LC system (Santa Clara, CA, USA) and detected by
precursor ion scanning on a 4000 QTRAP hybrid triple
quadrupole linear ion trap mass spectrometer (AB ⁄ MDS
Sciex, Concord, ON, Canada). For large-scale purifications
of the unknown intermediates, the flow from the LC system
was split 2 : 8 v ⁄ v to the mass spectrometer and the
fractions collected and pooled for subsequent structural
analyses.
For high resolution MS experiments, an Agilent 1100
Series LC system was coupled to a Q-ToF Premier hybrid
quadrupole TOF mass spectrometer equipped with an Elec-
trospray Ionization (ESI) LockSprayÔ modular source
(Waters Corp.). Calibration was performed using the
MS ⁄ MS fragment ions of [Glu
1
]-Fibrinopeptide B (1 pmo-
lÆlL

Inova 600 spectrometer (Varian, Palo Alto, CA, USA) with
a cold probe in D
2
O (Cambridge Isotopes Laboratories
Inc., Andover, MA, USA) solutions at 25 °C with acetone
standard (2.23 p.p.m. for
1
H and 31.5 p.p.m. for
13
C) using
standard COSY, TOCSY (mixing time 120 ms), ROESY
(mixing time 200 ms), HSQC and HMBC (100 ms long-
range transfer delay).
Acknowledgements
We would like to thank Dr C. Szymanski, University
of Alberta, for providing pseB and pseC isogenic
mutants of 11168 and Tom Devecseri, NRC-IBS, for
his assistance in preparing the figures.
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