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Báo cáo khoa học: The HS:19 serostrain of Campylobacter jejuni has a hyaluronic acid-type capsular polysaccharide with a nonstoichiometric sorbose branch and O-methyl phosphoramidate group docx

The HS:19 serostrain of Campylobacter jejuni has a
hyaluronic acid-type capsular polysaccharide with a
nonstoichiometric sorbose branch and O-methyl
phosphoramidate group
David J. McNally, Harold C. Jarrell, Nam H. Khieu, Jianjun Li, Evgeny Vinogradov,
Dennis M. Whitfield, Christine M. Szymanski and Jean-Robert Brisson
Institute for Biological Sciences, National Research Council of Canada, Ottawa Ontario, Canada
Campylobacter jejuni is one of the leading causes of
human gastroenteritis and surpasses Salmonella, Shig-
ella and Escherichia in some regions as the primary
cause of gastrointestinal disease [1–3]. There is also a
convincing body of evidence linking C. jejuni infections
to the onset of Guillain–Barre
syndrome [4–11].
Although of relatively rare occurrence, this syndrome
is the most common cause of acute neuromuscular
paralysis since the eradication of polio. It is character-
ized by weakness in the limbs and respiratory muscles,
with paralysis generally occurring 1–3 weeks after
infection [12,13]. Penner’s passive haemagglutination

Campylobacter jejuni; capsular
polysaccharide; high-resolution magic angle
spinning (HR-MAS) NMR; phosphoramidate;
J R. Brisson, Institute for Biological
Sciences, National Research Council of
Canada, 100 Sussex Drive, Ottawa Ontario,
Canada, K1A 0R6
Fax: +1 613 9529092
Tel: +1 613 9903244
E-mail: jean-robert.brisson@nrc-cnrc.gc.ca
(Received 3 April 2006, revised 2 June
2006, accepted 29 June 2006)
A recent study that examined multiple strains of Campylobacter jejuni
reported that HS:19, a serostrain that has been associated with the onset of
syndrome, had unidentified labile, capsular polysaccharide
(CPS) structures. In this study, we expand on this observation by using
current glyco-analytical technologies to characterize these unknown groups.
Capillary electrophoresis electrospray ionization MS and NMR analysis
with a cryogenically cooled probe (cold probe) of CPS purified using a gen-
tle enzymatic method revealed a hyaluronic acid-type [-4)-b-d-GlcA6NGro-
repeating unit, where NGro is 2-aminoglycerol. A
labile a-sorbofuranose branch located at C2 of GlcA was determined to
have the l configuration using a novel pyranose oxidase assay and is the
first report of this sugar in a bacterial glycan. A labile O-methyl phosphor-
amidate group, CH
)(OR) (MeOPN), was found at C4 of Glc-
NAc. Structural heterogeneity of the CPS was due to nonstoichiometric
glycosylation with sorbose at C2 of GlcA and the nonstoichiometric, vari-
ably methylated phosphoramidate group. Examination of whole bacterial
cells using high-resolution magic angle spinning NMR revealed that the
MeOPN group is a prominent feature on the cell surface for this sero-
strain. These results are reminiscent of those in the 11168 and HS:1 strains
and suggest that decoration of CPS with nonstoichiometric elements such
as keto sugars and the phosphoramidate is a common mechanism used by
this bacterium to produce a structurally complex surface glycan from a lim-
ited number of genes. The findings of this work with the HS:19 serostrain
now present a means to explore the role of CPS as a virulence factor in
C. jejuni.
CPS, capsular polysaccharide; CE-ESI-MS, capillary electrophoresis electrospray ionization mass spectrometry; HMBC, heteronuclear
multiple-bond correlation; HR-MAS NMR, high-resolution magic angle spinning nuclear magnetic resonance spectroscopy; HSQC,
heteronuclear single-quantum coherence; MeOPN, O-methyl phosphoramidate CH
FEBS Journal 273 (2006) 3975–3989 ª 2006 The Authors Journal compilation ª 2006 FEBS 3975
assay [14] was used to show that 81% of C. jejuni iso-
lates from patients with Guillain–Barre
syndrome in
Japan belonged to Penner’s HS:19 serotype. In the
United States, 33% of C. jejuni isolates from such
patients were classified as HS:19 [11,15,16].
It is now widely accepted that the major antigenic
component of Penner’s serotyping system for C. jejuni
is capsular polysaccharide (CPS) [17]. This was not
always the case, as lipopolysaccharide was for many
years thought to be the basis for Penner’s classification
system [8]. Because CPS is the outermost structure on
the bacterial cell, it plays a key role in the interaction
between the pathogen, host, and environment [18] and
is generally thought to be important for bacterial survi-
val and persistence in the environment [19]. By mimick-
ing host cell antigens and through structural variation,
CPSs also convey evasion from host immune responses
and are therefore considered important virulence fac-
tors. CPS production in C. jejuni remained unnoticed
until the genome sequencing of C. jejuni NCTC11168
in 2000 and the identification of genes implicated in
CPS biosynthesis [20]. Since this discovery, genetic, bio-
chemical and microscopy studies have demonstrated
CPS production in different strains of C. jejuni
[17,21,22]. Our laboratory has shown that it is possible
to study CPS directly on the surface of intact C. jejuni
cells using high-resolution magic angle spinning NMR
spectroscopy (HR-MAS NMR) [18,23,24]. On the basis
of its role in epithelial cell invasion, diarrhoeal disease,
serum resistance and maintenance of bacterial cell sur-
face hydrophilicity [17,22], CPS is thought to play a
critical role in pathogenesis for C. jejuni.
The precise mechanisms by which CPS conveys viru-
lence to C. jejuni are poorly understood; however,
structural variation of this surface glycan is emerging
as a possible mechanism [23,24]. The CPSs produced
by C. jejuni are structurally diverse and there are more
than 60 serostrains described for this bacterium, exclu-
ding nontypeable strains, each having a different CPS
structure [25]. For each serostrain, there are often
phase-variable structural modifications such as the
incorporation of methyl, ethanolamine and amino-
glycerol groups on CPS sugars [18,23,24]. The most
unusual of these modifications is the O-methyl phos-
phoramidate CH
)(OR) (MeOPN), which
is a highly labile phosphorylated structure that was
first described on the GalfNAc CPS sugar for the
11168 strain [18]. By using mild extraction conditions
to purify CPS and HR-MAS NMR to study CPS
in vivo, it was recently shown that the HS:1 serostrain
also expresses this phosphorylated modification on two
labile fructofuranose branches. By placing variable
MeOPN groups on labile branches, the HS:1 strain
was shown to produce a structurally variable and
therefore highly complex CPS despite having a small
CPS biosynthetic locus containing 11 genes [23,25].
Sequencing the CPS biosynthetic regions for several
strains of C. jejuni uncovered evidence for multiple
mechanisms of CPS variation, including exchange of
capsular genes and entire clusters by horizontal trans-
fer, gene duplication, deletion, fusion and contingency
gene variation [25]. Of particular interest, the CPS
locus for the HS:19 serostrain was shown to contain
only 13 genes, including a udg homologue responsible
for producing b-d-GlcA6NGro [25]. This finding corre-
lated well with the CPS structure reported for the
HS:19 serostrain, which was shown to consist of a [-4)-
repeating unit
[5,6,12]. The CPS locus of the HS:19 strain was also
shown to contain HS19.07, a homologue of cj1421 in
the 11168 strain, which is speculated to be a MeOP N
transferase responsible for adding MeOPN to CPS
sugars [24]. By examining a partially purified CPS sam-
ple prepared from HS:19 cells, the latter study
observed unidentified labile groups and speculated that
one of these was a MeOPN modification similar to the
one reported for other strains of C. jejuni [18,23–25].
In this study, we thoroughly investigate the complete
CPS structure for the HS:19 serostrain of C. jejuni by
using the latest glyco-analytical technologies to charac-
terize these unknown labile groups. Initially, HR-MAS
NMR was used to examine CPS directly on the surface
of whole HS:19 cells. To study the structure of the
CPS in greater detail, we isolated it using a mild enzy-
matic extraction method that preserves labile groups
[23]. In previous studies [5,6,12], the classic hot
water ⁄ phenol extraction method [26] was used, as this
structure was originally thought to be a high-molecu-
lar-mass lipopolysaccharide. High-resolution NMR at
600 MHz (
H) with an ultra-sensitive cryogenically
cooled probe (cold probe), and capillary electro-
phoresis electrospray ionization mass spectrometry
(CE-ESI-MS) with in-source collision-induced dissoci-
ation [27] were then used to determine the structure of
purified CPS. Herein we report the complete structure
for the CPS of the HS:19 serostrain of C. jejuni and
discuss the biological significance of these new struc-
tural findings for this organism.
The results generated by HR-MAS NMR and high-
resolution NMR, CE-ESI-MS and chemical ⁄ enzymatic
analyses revealed a hyaluronic acid-type CPS with a
unit (Fig. 1). This finding is in agreement with
Campylobacter jejuni HS: 19 CPS D. J. McNally et al.
3976 FEBS Journal 273 (2006) 3975–3989 ª 2006 The Authors Journal compilation ª 2006 FEBS
previous studies that examined CPS in the HS:19 sero-
strain of C. jejuni [5,6,12]. However, the complete
structure of the CPS was found to be more complex
because of a nonstoichiometric a-l-sorbose branch
located at C2 of b-d-GlcA and a variably methylated
nonstoichiometric MeOPN group at C4 of b-d-Glc-
HR-MAS NMR spectroscopy of whole cells
Examination of CPS on the surface of whole HS:19
cells with HR-MAS NMR revealed multiple signals
originating from the cell surface (Fig. 2). The
HR-MAS NMR spectrum exhibited broad lines, how-
ever; a doublet characteristic of a MeOPN group at d
3.77 p.p.m. was observed to protrude above the broad
CPS signals (Fig. 2A). A scalar coupling of 12.0 Hz
was measured for this doublet, which is in good agree-
ment with
couplings determined for the MeOPN
in the 11168 and HS:1 serostrains [18,23,24]. A 1D
heteronuclear single-quantum coherence (HSQC)
experiment that specifically selects for the MeOPN
group (
P decoupled, MeOPN-filtered
P HSQC) con-
firmed that this doublet originated from a MeOPN
group (Fig. 2B). The chemical shift of the MeOPN
signal at d
14.7 p.p.m., determined using a 2D
HSQC experiment (Fig. 2C), is highly unique to a
phosphoramidate bond and is consistent with MeOPN
signals observed in other strains of C. jejuni, which
range from d
13.1 p.p.m. to 14.7 p.p.m. [18,23,24]. For
HR-MAS NMR of whole C. jejuni cells, we typically
observe only the
P correlation between the phos-
phorus and the sharp methyl group resonance of the
MeOPN. The correlation between phosphorus of the
MeOPN and the ring protons of pyranose sugars is not
observed [24], probably because of short T
- -Sor
- -GlcA NGro
- -GlcNAc
Fig. 1. The complete structure of the repeating unit for the CPS
of the HS:19 serostrain of C. jejuni. The repeating unit of the
CPS consists of [-4)-b-
with an
L-sorbofuranose branch located at C2 of GlcA and an MeOPN
group at C4 of GlcNAc. Structural heterogeneity is due to the non-
stoichiometric sorbofuranose branch and the variably methylated
nonstoichiometric MeOPN group. Residue A represents b-
ronic acid-6-N-glycerol, B is 2-acetamido-2-deoxy-b-
D-glucose, C is
L-sorbofuranose, and D is MeOPN.
Fig. 2. HR-MAS NMR spectroscopy of intact
C. jejuni HS:19 cells. (A)
spectrum (256 transients) showing a doublet
originating from a MeOPN group. (B) 1D
P-HSQC ‘MeOPN-filtered’ HR-MAS NMR
spectrum (
P-decoupled, 256 transients,
¼ 10 Hz) showing a broad signal ori-
ginating from a MeOPN group. (C) 2D
P-HSQC HR-MAS NMR spectrum showing
two MeOPNs (256 transients, 128 incre-
¼ 10 Hz).
D. J. McNally et al. Campylobacter jejuni HS: 19 CPS
FEBS Journal 273 (2006) 3975–3989 ª 2006 The Authors Journal compilation ª 2006 FEBS 3977
relaxation times and the lower intensity of the broad
GlcNAc H4 resonance due to homonuclear couplings
and structural heterogeneity of the CPS. A second
minor MeOPN signal observed at d
14.3 p.p.m. indi-
cated structural heterogeneity for CPS on the cell sur-
face. Spectral lines originating from CPS on the surface
of HS:19 cells were too broad to draw additional con-
clusions regarding its structure, necessitating its isola-
tion for further study.
Isolation of CPS
Previous studies that have examined CPS in the HS:19
serostrain used Westphal’s classic hot water ⁄ phenol
extraction method to isolate the polysaccharide
[5,6,12,26]. Our results using this method closely
agreed with these studies in terms of the quantity and
purity of CPS obtained. However, CE-ESI-MS and
NMR analyses of hot water ⁄ phenol-purified CPS
revealed that labile groups such as the MeOPN and
sorbose branch were mostly absent (not shown). On
the basis of these observations, we concluded that the
hot water ⁄ phenol method was responsible for remov-
ing labile CPS groups, and that removal of these labile
modifications by this method resulted in them being
overlooked by previous studies. These findings support
a recent study that found that the hot water⁄ phenol
method hydrolyzed labile groups that are present on
the CPS of the HS:1 serostrain of C. jejuni [23]. With a
less harsh enzymatic purification method,  5 mg CPS
was obtained from a 6-L culture of HS:19 cells (7 g
cells, wet pellet mass). CPS isolated with this gentler
technique contained a moderately higher concentration
of nucleic acid and protein impurities. However, this
enzymatic extraction method preserved the sought
after labile groups, facilitating the characterization of
the complete CPS structure.
High-resolution NMR spectroscopy
of purified CPS
Examination of purified CPS with NMR at 600 MHz
H) with a cold probe revealed a [-4)-b- d-GlcA6N-
backbone and showed
the unknown labile CPS structures to be a MeOPN
group and an a-sorbofuranose branch (Table 1,
Fig. 3). The proton spectrum of purified CPS closely
resembled CPS on the cell surface in that a doublet
originating from a MeOPN group (d
3.77 p.p.m.,
¼ 12.0 Hz) was observed to protrude above
broad CPS signals (Fig. 3A). Selective 1D TOCSY and
1D NOESY experiments were used to assign the pro-
ton resonances for GlcA, GlcNAc and sorbose. 1D
TOCSY of the sorbose H3 resonance revealed overlap-
ping H4 and H5 signals (Fig. 3B), and simultaneous
excitation of the H4 and H5 resonances showed signals
corresponding to H6 ⁄ H6¢ (Fig. 3C). As C2 of sorbose
does not have a proton, a 1D NOESY experiment of
the sorbose H3 resonance was used to assign H1 ⁄ H1¢
resonances (Fig. 3D).
P correlation observed between the MeOPN
group and H4 of GlcNAc at d
14.7 p.p.m.
indicated the location of the MeOPN at C4 of Glc-
NAc (Fig. 3E). Carbon assignments were determined
H correlations observed using a
experiment (Fig. 3F). With the exception of sorbose
resonances, signals within the
C-HMQC spectrum
for purified HS:19 CPS were generally broad and
therefore weak. In particular,
H correlations for
C3 and C4 of GlcNAc and C1 of GlcA and GlcNAc
were visible only at higher temperature (40 °C) and
at 600 MHz (
H) with a cryogenically cooled probe.
Proton and carbon resonances determined from
C-HMQC and heteronuclear multiple-bond correla-
tion (HMBC) experiments were consistent with those
reported for b-GlcA6NGro and b-GlcNAc [5,6,12], a
MeOPN group [18,23,24] and a-sorbofuranose [28–30].
Structural heterogeneity generated by the sorbose
branch and MeOPN group was indicated by two sets
H correlations for C2 and C3 of GlcA, as well
Table 1. NMR proton and carbon chemical shifts d (p.p.m.) for CPS
purified from HS:19 serostrain of C. jejuni.
Residue Position d
b-D-GlcA6NGro (A) A1 4.65 101.0
A2 3.71 73.8
A3 3.71 73.8
A4 3.90 79.0
A5 3.94 75.1
A6 170.7
A7 4.07 53.9
A8 ⁄ A8¢ 3.65 ⁄ 3.75 61.5
D-GlcNAc (B) B1 4.62 100.7
B2 3.97 56.1
B3 4.24 75.8
B4 4.26 74.2
B5 3.61 75.5
B6 ⁄ B6¢ 3.74 ⁄ 3.93 61.3
B7 175.0
B8 2.11 23.5
L-Sorf (C) C1 ⁄ 1¢ 3.64 ⁄ 3.73 61.5
C2 104.3
C3 4.17 79.2
C4 4.41 76.1
C5 4.39 79.2
C6 ⁄ C6¢ 3.69 ⁄ 3.79 62.9
MeOPN (D) D1 3.77 54.8
Campylobacter jejuni HS: 19 CPS D. J. McNally et al.
3978 FEBS Journal 273 (2006) 3975–3989 ª 2006 The Authors Journal compilation ª 2006 FEBS
as for C4 and C5 of GlcNAc (Fig. 3F, indicated with
asterisks). The chemical shifts of these extra resonances
were in excellent agreement with those reported for
nonphosphoramidated, nonsorbosylated CPS [5,6,12].
Compared with nonphosphoramidated CPS, our
results indicated that the MeOPN caused a downfield
shift in the H4 resonance of GlcNAc (0.71 p.p.m.).
This observation is consistent with the effects of phos-
phoramidation reported for the 11168 and HS:1
strains, where the presence of the MeOPN group
caused the signals for neighbouring protons to shift by
0.6–0.8 p.p.m. [18,23,24].
MS analysis of purified CPS
Because of the large molecular mass of the HS:19
CPS, a high orifice voltage (+ 200 V) was used to
promote in-source collision-induced dissociation [27]
to facilitate its analysis with CE-ESI-MS (Fig. 4).
CE-ESI-MS analysis of purified CPS corroborated the
hyaluronic acid-like structure deduced from chemical
analyses and NMR spectroscopy (Fig. 4A, Table 2).
Interestingly, ions observed at m⁄ z 514.4, 532.4, 694.5
and 984.8 indicated the presence of an O-phosphor-
amidate group OP(O)(NH
)(OR) without O-methyla-
tion, suggesting that the nonstoichiometric MeOPN
group is also variably methylated for the HS:19 sero-
strain. CE-ESI-MS ⁄ MS analysis of m ⁄ z 708.5, corres-
ponding to one complete repeat of the CPS, revealed
fragment ions at m ⁄ z 297.0 and m ⁄ z 412.3, confirming
the location of the MeOPN group on GlcNAc and
the presence of a single sorbose branch on GlcA
(Fig. 4B).
Determination of absolute configuration
for CPS sugars
By comparing the GC retention times of the R- and
S-butyl glycosides of authentic standards with the
R-butyl glycosides prepared from a purified CPS sam-
ple, b-GlcA and b-GlcNAc were shown to have the
d configuration (not shown). As the chiral alcohol
Fig. 3. High-resolution NMR spectroscopy
of CPS purified from the HS:19 serostrain of
C. jejuni. (A)
H NMR spectrum (1024 tran-
sients) showing a doublet originating from
the MeOPN group. (B) 1D TOCSY (30 ms)
of Sor H3. (C) 1D TOCSY (30 ms) of Sor H4
and H5. (D) 1D NOESY (400 ms) of Sor H3.
P-HSQC spectrum (64 transients, 32
¼ 7 Hz). (F)
spectrum (128 transients, 128 increments,
¼ 150 Hz). For selective 1D experi-
ments, excited resonances are underlined.
Residues with asterisks correspond to struc-
tural heterogeneity generated by the non-
stoichiometric MeOPN group and
L-sorbofuranose branch. Annotations for
residues are the same as Fig. 1 and sm
represents sorbose monosaccharide.
D. J. McNally et al. Campylobacter jejuni HS: 19 CPS
FEBS Journal 273 (2006) 3975–3989 ª 2006 The Authors Journal compilation ª 2006 FEBS 3979
method cannot be used to determine the absolute con-
figuration of keto sugars [31], we report for the first
time the use of pyranose oxidase for this purpose. Pyr-
anose oxidase from the white rot fungus Trametes
multicolor is reported to oxidize l-sorbose at the C5
position forming 5-keto-d-fructose and hydrogen per-
oxide [32–34]. However, it was not known if this
enzyme was specific to the l isoform. By incubating
pyranose oxidase with d-sorbose and l-sorbose
standards, we established that this enzyme oxidizes
l-sorbose but not d-sorbose (Figs 5A–D). Incubating
hydrolyzed purified HS:19 CPS with pyranose oxidase
resulted in the disappearance of signals associated with
the sorbose monosaccharide and the production of the
same oxidation product as observed for the l-sorbose
standard (Figs 5E,F). On the basis of these results,
sorbose was concluded to have the l absolute
Branching pattern for sorbose
The results of an HMBC experiment confirmed the
glycosidic linkages within the [-4)-b-d-GlcA6NGro-(1–
repeating unit of the purified CPS
and showed that the a-l-Sor branch was located at C2
or C3 of GlcA (not shown). However, we were unable
to determine the precise location of the a-l-Sor branch
using the HMBC experiment because of spectral over-
lap for the resonances originating from positions 2 and
3 of GlcA which have identical
C and
H chemical
shifts (Fig. 3F, Table 1). The results of methylation
analysis were inconclusive because of the poor solubil-
Fig. 4. MS analysis of CPS that was purified
from the HS:19 serostrain of C. jejuni. (A)
CE-ESI-MS total ion spectrum (positive ion
mode, orifice voltage +200 V). (B) CE-ESI-
MS ⁄ MS analysis of m ⁄ z 708.5, which cor-
responds to one complete repeat of the
Campylobacter jejuni HS: 19 CPS D. J. McNally et al.
3980 FEBS Journal 273 (2006) 3975–3989 ª 2006 The Authors Journal compilation ª 2006 FEBS
ity of the CPS in organic solvent, the labile Sor glycos-
idic bond, and the heterogeneous substitution of Sor
within the CPS. Furthermore, because signals for CPS
protons were broad and several positions for Sor,
GlcA and GlcNAc have overlapping proton signals,
NOEs could not be used to resolve the location of the
Sor branch. Similar problems were reported by a study
that discovered a nonstoichiometric fructose branch
in the lipopolysaccharide of Vibrio cholerae 0139
Bengal [35].
Nevertheless, two lines of evidence point to the
location of the Sor branch at C2 of GlcA. Compar-
ison of
C chemical shifts for intact and acid-hydro-
lyzed CPS revealed that substitution with Sor causes
an upfield shift in the C3 signal for GlcA
(0.35 p.p.m.) and a downfield shift in the C2 signal of
GlcA (0.73 p.p.m.). Although we were unable to
locate data for sorbose in the literature, substitution
with fructose was shown to cause a small downfield
shift in the signals originating from the carbon atoms
Table 2. Positive ion CE-ESI-MS data for CPS isolated from the HS:19 serostrain of C. jejuni. Isotope-averaged masses of residues were
used for calculation of total molecular masses based on the following proposed compositions: Hex (a-
L-Sor), 162.1; HexNAc (b-D-GlcNAc),
203.2; HexANGro (b-
D-GlcA6NGro), 249.2; MeOPN[O-methyl phosphoramidate, CH
)], 93.2; NGro (N-glycerol), 73.1; OPN
(O-phosphoramidate), 79.0; H
O, 18.0. For these gas-phase (IS-CID) degradation products, no H
O molecule is added to the residue unless
specifically indicated.
Molecular mass (m ⁄ z)
Observed Calculated Difference
74.0 74.1 0.1 NGro – H
149.8 150.1 0.3 HexNAc – (H
168.2 168.2 0.0 HexNAc – (H
186.0 186.2 0.2 HexNAc – (H
204.3 204.2 0.1 HexNAc – H
221.5 221.2 0.3 HexNAc
231.5 231.2 0.3 HexANGro – (H
243.3 243.2 0.1 HexNAcMeOPN–(H
250.0 250.2 0.2 HexANGro – H
279.3 279.2 0.1 HexNAcMeOPN–(H
297.0 297.2 0.2 HexNAcMeOPN–H
412.3 412.4 0.1 HexANGro + Hex – H
453.3 453.4 0.1 HexANGro + HexNAc – H
514.4 514.4 0.0 HexANGro + HexNAcOPN–(H
528.3 528.4 0.1 HexANGro + HexNAcMeOPN–(H
532.3 532.4 0.1 HexANGro + HexNAcOPN–H
546.3 546.4 0.1 HexANGro + HexNAcMeOPN–H
615.4 615.6 0.2 HexANGro + HexNAc + Hex – H
656.5 656.6 0.1 HexANGro + (HexNAc)
690.5 690.8 0.3 HexANGro + HexNAcMeOPN + Hex – (H
694.2 694.5 0.3 HexANGro + HexNAcOPN + Hex – H
708.5 708.6 0.1 HexANGro + HexNAcMeOPN + Hex – H
749.4 749.6 0.2 HexANGro + HexNAcMeOPN + HexNAc – H
795.4 795.7 0.3 (HexANGro)
842.4 842.7 0.3 HexANGro + (HexNAcMeOPN)
887.5 887.8 0.3 (HexANGro)
+ (HexNAc)
905.8 905.8 0.0 (HexANGro)
+ (HexNAc)
957.8 957.8 0.0 (HexANGro)
+ HexNAcMeOPN + Hex – H
984.5 984.8 0.3 (HexANGro)
+ HexNAcOPN + HexNAc – H
998.7 998.8 0.1 (HexANGro)
+ HexNAcMeOPN + HexNAc – H
1059.8 1059.8 0.0 (HexANGro)
+ HexNAcMeOPN + HexNAcOPN–(H
1078.0 1077.8 0.2 (HexANGro)
1091.8 1091.9 0.1 (HexANGro)
+ (HexNAcMeOPN)
1108.5 1108.8 0.3 (HexANGro)
+ (HexNAcMeOPN)
1253.0 1253.0 0.0 (HexANGro)
+ (HexNAcMeOPN)
+ Hex – H
1357.5 1357.2 0.3 (HexANGro)
+ (HexNAc)
1681.5 1681.5 0.0 (HexANGro)
+ (HexNAc)
+ (Hex)
1844.0 1843.7 0.3 (HexANGro)
+ (HexNAc)
+ (Hex)
D. J. McNally et al. Campylobacter jejuni HS: 19 CPS
FEBS Journal 273 (2006) 3975–3989 ª 2006 The Authors Journal compilation ª 2006 FEBS 3981
of pyranose rings [36–38]. Furthermore, compared
with acid-hydrolyzed CPS, we observed substantial up-
field shifts for the C1 resonance of GlcA (2.8 p.p.m.)
and for the C3 resonance of GlcNAc (7.5 p.p.m.). As
substitution with a keto sugar or a phosphoramidate
group causes a relatively small change (< 1 p.p.m.)
[3,18,23], the magnitude of these differences for the C1
signal of GlcA (A) and C3 signal of GlcNAc (B) indi-
cated a change about the GlcA-(1–3)-GlcNAc (A–B)
glycosidic bond.
To understand the effects of various substitutions
on the conformation of the glycosidic bonds in the
CPS, we modelled the B–A–B unit [GlcNAc-(1–4)-
GlcA6NGro-(1–3)-GlcNAc] with and without residues
C(a-l-Sor) and D (MeOPN). We would expect major
changes in the distribution of u (O5–C1–O1–Cx) and
w (C1–O1–Cx–Cx)1), with x being the aglyconic link-
age site, to be accompanied by a change in
C chem-
ical shifts for C1 and Cx [39–41]. Compared with
acid-hydrolyzed CPS (B–A–B), we observed large
changes in the
C chemical shifts for C1 of A and
C3 of B (A–B) on substitution with C and D, but not
for C1 of B or C4 of A (B–A). Modelling of the
B–A–B unit with residue D on both B residues was
performed to ascertain whether the presence of the
MeOPN group by itself caused significant changes
about the glycosidic bonds in the B–A–B unit. As can
be observed by comparing (uw) maps in Fig. 6A–D,
no significant changes were observed. Then, by model-
ling the DB–A–BD unit with residue C at C3 of
residue A (C-3 A) or at C2 of residue A (C-2A), it
was observed that a major change in the distribution
of (uw) occurred for the B–A linkage for C-3A
(Fig. 6F) and for the A–B linkage for C-2A (Fig. 6G).
Hence, the NMR and molecular modelling results
indicate the location of the a-l-Sor branch to be at
C2 of GlcA.
A model of a minimum energy conformer for the
B–A–B unit representing the [-4)-b-d-GlcA6NGro-(1–
repeating unit with MeOPN
groups positioned at C4 of two GlcNAc residues and
an a-l-sorbofuranose branch at C2 of GlcA is shown
in Fig. 7. As can be observed, the sorbose branch at
C2 of GlcA influences the conformation about the
GlcA-(1–3)-b-d-GlcNAc glycosidic linkage because of
steric hindrance between sorbose and the N-acetyl
group of the GlcNAc residue. Based on the positions
of the sorbose branch and MeOPN group that extend
away from the repeating unit, it is expected that they
are prominent structural features on the surface of
HS:19 cells.
The HS:19 serostrain of C. jejuni is an important Pen-
ner type, as it is commonly associated with gastrointes-
tinal infections and has been linked to the onset of
syndrome in the United States and
Japan [11,15,16,42]. A recent study [25] that examined
a partially purified CPS sample for this serostrain
H (p.p.m.)
3.95 3.85 3.75 3.65 3.55 3.45
Fig. 5. Determination of absolute configur-
ation for sorbose in the HS:19 CPS with pyr-
anose oxidase. (A)
H-NMR spectrum of
D-sorbose. (B)
H-NMR spectrum of the
D-sorbose sample incubated with
pyranose oxidase. (C)
H-NMR spectrum of
L-sorbose. (D)
H-NMR spectrum of
L-sorbose incubated with pyranose oxidase.
H-NMR spectrum of CPS that was
purified from the HS:19 serostrain of
C. jejuni and treated with mild acid to
liberate the sorbose monosaccharide.
H-NMR spectrum of the same CPS
sample incubated with pyranose oxidase.
Campylobacter jejuni HS: 19 CPS D. J. McNally et al.
3982 FEBS Journal 273 (2006) 3975–3989 ª 2006 The Authors Journal compilation ª 2006 FEBS
observed labile unidentified CPS structures that were
not reported in previous studies [5,6,8,12]. In this
study, we have characterized the complete CPS struc-
ture for the HS:19 serostrain and have shown that
these labile groups are an a-l-sorbofuranose branch
attached at C2 of b-d-GlcA and a MeOPN modifica-
tion located at C4 of b-d-GlcNAc.
There are very few reports for sorbose in the litera-
ture and it is an unusual sugar to find in a bacterial
polysaccharide. Although this sugar is found in the
plant kingdom [30], this is the first report of sorbose
in a bacterial glycan. Multiple
H correlations
observed for the C2 position of the b-d-GlcA residue
indicated that the sorbose branch is nonstoichiometric
and is therefore a source of structural heterogeneity
for the CPS. The complete CPS structure for HS:19
resembles that produced by the HS:1 serostrain, which
was shown to have two nonstoichiometric fructose
branches [23]. Although the biological role of these
modifications is not clear, these similarities suggest
that decoration of CPS with nonstoichiometric keto
sugar branches is a common structural feature in
C. jejuni. Genetic determinants for the biosynthesis of
these keto sugars were not readily detected within the
CPS biosynthetic locus of either strain [25]. This dis-
crepancy points to the novelty of these modifications
and the lack of other bacterial homologues in the
current databases. Alternatively, the biosynthesis genes
may reside elsewhere in the genome. Studies that have
examined CPS biosynthesis in Escherichia coli K4,
which has chondroitin-type [-4)-b-d-GlcA-(1–3)-b-d-
CPS with a fructose branch at C3 of
GlcA, showed that the Fru residue is added after CPS
chain elongation is complete [43,44]. Because the Fru
and Sor branches in HS:1 and HS:19 are nonstoichio-
metric, addition of these keto sugars to CPS might be
achieved using the mechanism described for
E. coli K4. Our results indicate that the sorbose
branch is a prominent feature on the cell surface and
that sorbose induces a conformational change in the
structure of the CPS. One might speculate that CPS,
with and without sorbose, presents different epitopes,
which has implications for how the HS:19 serostrain is
perceived by its hosts and also in the success of CPS-
based vaccine development.
The MeOPN modification is found on the CPSs of
several strains of C. jejuni, including 11168, HS:1,
81-176, HS:36 [18,23,24] and now HS:19, suggesting
that the MeOPN is a common feature in this bacter-
ium. On the basis of the location of the MeOPNon
both pyranose and furanose sugars, it was speculated
Fig. 6. Molecular modelling of glycosidic
torsional angles u and w for the GlcA6NGro-
(1–3)-GlcNAc (A–B) and GlcNAc-(1–4)-
GlcA6NGro (B–A) glycosidic linkages. For
each linkage map (A–B or B–A), the unit
modelled is indicated above. For (c–h),
residue D was modelled at C4 of residue B.
Glycosidic torsional angles are defined as
u ¼ O5–C1–O1–Cx and w ¼ C1–O1–Cx–
Cx)1, with x being the aglyconic linkage
site. Annotations for residues are the same
as Fig. 1.
Fig. 7. Molecular model for the repeating unit of the CPS of the
HS:19 serostrain of C. jejuni. The CPS consists of a [-4)-b-
repeating unit with an a-L-sorbofuranose
branch located at C2 of GlcA and a MeOPN group at C4 of GlcNAc.
OH groups have been removed to simplify the appearance of the
D. J. McNally et al. Campylobacter jejuni HS: 19 CPS
FEBS Journal 273 (2006) 3975–3989 ª 2006 The Authors Journal compilation ª 2006 FEBS 3983
that transfer of the MeOPN to CPS sugars requires
more than one gene product [24]. cj1422c, a putative
glycosyl transferase in 11168, was singled out as one of
the genes [25], and cj1421c was speculated to be the
other. Our results support cj1421c as a possible
MeOPN transferase, as the CPS biosynthetic locus in
HS:19 contains only the cj1421 homologue HS19.07
[25]. Multiple
H correlations observed for C4 of
GlcNAc indicated that the MeOPN group is nonstoi-
chiometric and is therefore an additional source of
structural variation. Moreover, our MS results showed
that the MeOPN group in HS:19 is variably methyla-
ted. Because fragment ions corresponding to nonmeth-
ylated MeOPN were not observed for the CPS of
11168 or HS:1 [23,24], those observed for HS:19 were
concluded to be real and not an artefact of the MS
analyses. These observations suggest that the MeOPN
is synthesized in a nonmethylated form and is methyla-
ted at a later point in time. Despite having a small
CPS locus with only 13 genes [25], the HS:19 sero-
strain produces a structurally heterogeneous CPS
through the addition of nonstoichiometric elements
such as a sorbose branch and MeOPN modification.
These findings support the incorporation of these mod-
ifications as a mechanism to produce a structurally
complex surface glycan from a limited number of
As hyaluronic acid is a common constituent of con-
nective tissue and biological fluids in mammals, it is
believed that several pathogenic bacteria produce a
hyaluronic acid-type CPS to evade the host immune
response. Among these bacteria, Streptococcus
pyogenes (group A streptococcus) has been studied the
most [45]. By examining CPS-deficient mutants, several
studies have shown that the hyaluronic acid CPS pro-
duced by S. pyogenes is an important virulence factor
and a prerequisite for infection. For instance, the CPS
was shown to offer protection from phagocytes and
epithelial cells [46–50], induce apoptosis in host cells
[51], increase invasiveness and adherence [52], be
important in mucoidy and biofilm formation [48,53],
and was found to be responsible for subcutaneous
spread of the bacterium [54]. The CPS in S. pyogenes
was also shown to act as a universal adhesin by bind-
ing to CD44, a hyaluronic acid-binding glycoprotein
found on pharyngeal and epidermal keratinocytes in
humans [47]. On the basis of these virulence roles that
have been demonstrated for the hyaluronic acid-type
CPS produced by S. pyogenes, the importance of the
structurally similar CPS produced by the HS:19 sero-
strain of C. jejuni merits investigation.
The HS:19 serostrain of C. jejuni is known to pro-
duce oligosaccharide structures in the outer core of its
lipo-oligosaccharide that are identical with peripheral
nerve gangliosides. As a result, patients with gastroen-
teritis resulting from infection with C. jejuni occasion-
ally develop antiganglioside antibodies. This molecular
mimicry was proposed to result in antibody-mediated
nerve damage to Schwann cells expressing these gan-
gliosides thereby explaining paralysis in patients with
syndrome [11,42]. Interestingly, there
are also an increasing number of population studies
that indicate a link between C. jejuni infections and
acute reactive arthritis [55–66]. In the light of the pre-
valence of the HS:19 serostrain and the similarities
shared by its CPS structure and the connective tissues
of mammals, a link between HS:19 infections and
reactive arthritis cannot be ruled out.
The discovery of labile groups such as the nonstoichio-
metric a-l-sorbose branch and a variably methylated
MeOPN modification in the HS:19 serostrain reinfor-
ces the importance of using mild analytical methods
for examining the CPSs produced by C. jejuni.We
believe that the analytical methods described here will
be useful for solving the structures of complex CPSs
in other bacteria that contain labile components.
Although CPS is recognized as a major virulence fac-
tor in C. jejuni, the precise mechanisms explaining how
CPS conveys virulence and the importance of the
extensive phase-variable modifications decorating the
CPSs are not known. Because production of a hyalu-
ronic acid-type CPS is a prerequisite for infection in
other pathogens, most notably group A streptococcus,
the role of the CPS in the infection process of HS:19
merits investigation. Based on the wealth of knowledge
that is available for the genetics, regulation and bio-
synthesis of hyaluronic acid-type CPSs in other organ-
isms and the findings of the current study, the HS:19
serostrain now presents an interesting model to explore
CPS as a virulence factor in C. jejuni.
Experimental procedures
Solvents and reagents
Unless otherwise stated, solvents and reagents were pur-
chased from Sigma Biochemicals and Reagents (Oakville,
ON, Canada).
Media and growth conditions
The HS:19 strain (ATCC 43446, designation MK104 [14])
of C. jejuni was routinely maintained on Mueller Hinton
Campylobacter jejuni HS: 19 CPS D. J. McNally et al.
3984 FEBS Journal 273 (2006) 3975–3989 ª 2006 The Authors Journal compilation ª 2006 FEBS
(MH) agar (Difco, Kansas City, MO, USA) plates under
microaerophilic conditions (10% CO
, 85% N
37 °C. For large-scale extraction of CPS, 6 L C. jejuni
HS:19 was grown in brain heart infusion (BHI) broth
(Difco) under microaerophilic conditions at 37 °C for 24 h
with agitation at 100 r.p.m. Bacterial cells were then har-
vested by centrifugation (9000 g, 20 min) and placed in
70% ethanol. Cells were removed from the ethanol solution
by centrifugation (9000 g, 20 min), and the bacterial pellet
was refrigerated until extraction.
Isolation of CPS
A gentle enzymatic method was used to isolate CPS from
bacterial cells in order to preserve labile groups [23].
Briefly, cells harvested from 6 L BHI broth were suspended
in NaCl ⁄ P
buffer (pH 7.4). Lysozyme was then added to a
final concentration of 1 mgÆmL
before the addition of
mutanolysin to a final concentration of 67 UÆmL
. The
bacterial cell suspension was then incubated for 24 h at
37 °C with agitation at 100 r.p.m. The mixture was then
emulsiflexed twice (144 789.75 kPa; 21 000 p.s.i.) to lyse
cells, and DNAse I and RNAse (130 lgÆmL
and RNAse) was added before being incubated for 4 h at
37 °C with agitation at 100 r.p.m. Pronase E (type XIV
from Streptomyces griseus, EC, 5.2 UÆmg
) and
protease (type XXIV from Bacillus licheniformis,EC, 8.8 UÆmg
) were both added to a final concen-
tration of 200 lgÆmL
followed by incubation at 37 °C
overnight with agitation at 100 r.p.m. The crude CPS
extract was then dialyzed against running water for 72 h
(molecular mass cut-off 12 kDa), ultracentrifuged for 2 h
(140 000 g ,15°C), and the supernatant was lyophilized.
Crude CPS was resuspended in water and purified using a
SephadexÒ superfine G-50 column equipped with a Waters
differential refractometer (model R403; Waters, Mississ-
auga, ON, Canada). Fractions containing CPS were com-
bined and lyophilized. Semi-purified CPS was then
resuspended in water and purified using a Gilson liquid
chromatograph (model 306 and 302 pumps, 811 dynamic
mixer, 802B manometric module; Gilson, Middleton, WI,
USA) with a Gilson UV detector (220 nm; model UV ⁄
Vis-151 detector; Gilson) equipped with a tandem QHP
HiTrapÔ ion-exchange column (Amersham Biosciences,
Piscataway, NJ, USA). Fractions containing CPS were
combined and lyophilized. Purified bacterial CPS was then
desalted using a SephadexÒ superfine G-15 column, lyophi-
lized, and stored at )20 °C until further analysis.
HR-MAS NMR and high-resolution NMR
HR-MAS NMR analysis of intact bacterial cells was per-
formed as described by McNally et al. [23]. For high-reso-
lution NMR spectroscopy, 3 mg CPS isolated from the
HS:19 serostrain of C. jejuni was suspended in 150 lL
ammonium bicarbonate-buffered 99% D
O (50 mm
, pD 8.2; Cambridge Isotopes Laboratories Inc,
Andover, MA, USA) and placed in a 3 mm NMR tube
(Wilmad, Buena, NJ, USA). NMR experiments were per-
formed at 600 MHz (
H) using a Varian 5 mm, Z-gradient,
triple-resonance, cryogenically cooled probe, and at
500 MHz (
H) with a Varian Inova spectrometer equipped
with a Varian 3 mm, Z-gradient, triple-resonance (
P) probe (Varian, Palo Alto, CA, USA). 1D
P spectra
were acquired using a Varian Mercury 200 MHz (
H) spec-
trometer and a Nalorac 5 mm four nuclei probe. Standard
homonuclear and heteronuclear correlated 1D and 2D pulse
sequences from Varian were used for general assignments,
and selective 1D TOCSY and NOESY experiments with a
Z-filter were used for complete residue assignment and
characterization of individual spin systems [67,68]. NMR
experiments were typically performed at 25 °C, with sup-
pression of the HOD resonance at 4.78 p.p.m. The methyl
resonance of acetone was used as an internal reference (d
2.225 p.p.m. and d
31.07 p.p.m.), and
P NMR spectra
were referenced to an external 85% phosphoric acid stand-
ard (d
0 p.p.m.).
MS analysis of purified CPS
CPS isolated from the HS:19 serostrain of C. jejuni was
mass-analyzed using in-source collision-induced dissociation
CE-ESI ⁄ MS [27] with a Crystal model 310 capillary
electrophoresis instrument (ATI Unicam, Boston, MA,
USA) coupled to an API 3000 mass spectrometer (Applied
Biosystems ⁄ Sciex, Concord, ON, Canada) via a microIon
spray interface. A sheath solution (propan-2-ol ⁄ methanol,
2 : 1, v ⁄ v) was delivered at a flow rate of 1 lLÆmin
. Sepa-
rations were achieved on  90 cm of bare fused-silica capil-
lary (190 lm outside diameter · 50 lm internal diameter;
Polymicro Technologies, Phoenix, AZ, USA) using 15 mm
ammonium acetate ⁄ ammonium hydroxide in deionized
water (pH 9.0) containing 5% methanol as separation buf-
fer. A voltage of 20 kV was typically applied during CE
separation, and +5 kV was used as electrospray voltage.
Mass spectra were acquired with dwell times of 3.0 ms per
step of 0.1 m ⁄ z unit in full-mass scan mode. Fragment ions
formed by collision activation of selected precursor ions
with nitrogen in the RF-only quadrupole collision cell were
mass analyzed by scanning the third quadrupole. All sam-
ples were analyzed in positive ion mode using an orifice
voltage of +200 V.
Determination of absolute configuration
for CPS sugars
The absolute configuration (d or l) of GlcA and GlcNAc
CPS sugars isolated from the HS:19 serostrain of C. jejuni
was assigned by characterization of their R-butyl glyco-
D. J. McNally et al. Campylobacter jejuni HS: 19 CPS
FEBS Journal 273 (2006) 3975–3989 ª 2006 The Authors Journal compilation ª 2006 FEBS 3985
sides using GC as described by McNally et al. [23]. The
absolute configuration of sorbofuranose was assigned en-
zymatically using pyranose oxidase (EC; oxygen
2-oxidoreductase) from Coriolus sp. Although l-sorbose
has been reported to be a preferred substrate of this
enzyme [32–34], its specificity for the l-isoform was not
known. To investigate the specificity of this enzyme, 1 mg
l-sorbose or d-sorbose was incubated with 2 mg pyranose
oxidase (27 UÆmL
) overnight at 37 °C in 200 lLD
phosphate buffer (100 mm KH
, 100 mm NaCl,
pD 5.6), and the resulting oxidation products were ana-
lyzed with NMR. To determine the absolute configuration
of sorbose within the HS:19 CPS, 1 mg pure CPS was
first hydrolyzed overnight by mild acid treatment (addition
of dilute HCl, pH 3.0) to liberate sorbose monosaccharide.
The hydrolyzed CPS sample was then neutralized, lyophi-
lized, and resuspended in 200 lLD
O phosphate buffer
(100 mm KH
, 100 mm NaCl, pD 5.6). The hydro-
lyzed CPS sample containing the sorbose monosaccharide
was then incubated overnight at 37 °C with 2 mg pyranose
oxidase (27 UÆmL
), and the reaction products were ana-
lyzed by NMR.
Molecular modelling and molecular dynamics
Molecular dynamics simulations were performed for one
repeat unit of the CPS [b-d-GlcNAc-(1–4)- b-d-GlcA6N-
Gro-(1–3)-b-d-GlcNAc] without and with the MeOPNat
C4 of GlcNAc, and without a-l-sorbose and with a-l-sor-
bose at C2 or C3 of GlcA. Molecular structures were
constructed using the Biopolymer Module running on the
Insight II environment (Accelrys Inc., San Diego, CA,
USA). Glycosidic torsional angles, defined as u ¼ O5–C1–
O1–Cx and w ¼ C1–O1–Cx–Cx)1, with x being the agly-
conic linkage site, were chosen from potential energy maps
generated for CPS constituent disaccharides. All molecular
structures were subjected to 1000-step energy minimization
using a conjugate gradient method. Potential energy calcu-
lations, energy minimizations and dynamics simulations
were performed using the Discover-3 program (Accelrys
Inc.). Atomic potentials and charges were assigned using
an Amber force field supported by Discover-3. A group-
based nonbond method with a cut-off distance of 9.5 A
and a distance-dependent dielectric value of 4 were used
for all calculations. Energy-minimized structures were then
subjected to a 500-ps dynamics simulation in vacuum with
the first 100 ps being discarded. A Verlet algorithm with
a 1-fs time step was used for simulations, with trajectory
frames being saved every 0.25 ps. During simulations, the
initial chair conformations for six-membered sugar rings
were conserved using a periodic cosine function with a
force constant of 100 kcalÆmol
to restrain three of
the possible six ring torsions. The conformations of six-
member rings, such as the pyran rings of sugars, are well
studied from a conformational perspective [69]. If torsion
angles are used to describe ring puckering and if ring clo-
sure and redundancy conditions are taken into considera-
tion, then it is possible to define the conformation of any
six-membered ring using a minimum of three torsion
angles [70]. For this reason, it was possible to conserve
the initial chair conformations of the six-membered sugar
rings during molecular dynamics simulations by restraining
three torsion angles. Scatter plots for the glycosidic torsion
angles u and w shown in Fig. 6 were generated using the
Analysis Module running on Insight II. The molecular
model for the CPS presented in Fig. 7 was drawn using
Visual Molecular Dynamics [71].
We are grateful to Dr Michel Gilbert for the HS:19
serostrain, Dr Malcolm Perry for insightful discus-
sions, Dr Dietmar Haltrich for helpful discussions per-
taining to pyranose oxidase and to Marc Lamoureux
for assistance with bacterial growths.
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