Tài liệu Báo cáo Y học: The structures of the lipooligosaccharide and capsule polysaccharide of Campylobacter jejuni genome sequenced strain NCTC 11168 pdf

The structures of the lipooligosaccharide and capsule polysaccharide
of
Campylobacter jejuni
genome sequenced strain NCTC 11168
Frank St. Michael, Christine M. Szymanski, Jianjun Li, Kenneth H. Chan, Nam Huan Khieu,
Suzon Larocque, Warren W. Wakarchuk, Jean-Robert Brisson and Mario A. Monteiro
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Canada
Campylobacter jejuni infections are one of the leading causes
of human gastroenteritis and are suspected of being a pre-
cursor to Guillain–Barre
´
and Miller–Fisher syndromes.
Recently, the complete genome sequence of C. jejuni NCTC
11168 was described. In this study, the molecular structure of
the lipooligosaccharide and capsular polysaccharide of
C. jejuni NCTC 11168 was investigated. The lipooligosac-
charide was shown to exhibit carbohydrate structures anal-
ogous to the GM1a and GM2 carbohydrate epitopes of
human gangliosides (shown below):
The high M
r
capsule polysaccharide was composed of
b-
D
-Ribp, b-
D
-GalfNAc, a-
D
-GlcpA6(NGro), a uronic
acid amidated with 2-amino-2-deoxyglycerol at C-6, and
6-O-methyl-

D
-glycero-a-
L
-gluco-heptopyranose as a side-
branch (shown below):
The structural information presented here will aid in the
identification and characterization of specific enzymes that
are involved in the biosynthesis of these structures and may
lead to the discovery of potential therapeutic targets. In
addition, the correlation of carbohydrate structure with gene
complement will aid in the elucidation of the role of these
surface carbohydrates in C. jejuni pathogenesis.
Keywords: lipooligosaccharide; capsule; electron spray
ionization mass spectrometry; high-resolution magic angle
spinning NMR; heptose.
In humans, Campylobacter jejuni infection often gives rise to
enteritis and, in some regions, this Gram-negative bacterium
surpasses Salmonella, Shigella and Escherichia as the
primary cause of gastrointestinal disease [1,2]. Moreover,
C. jejuni infections have been linked to the more severe
clinical outcomes caused by Guillain–Barre
´
[3,4] and
Miller–Fisher syndromes [5]. The subsequent paralysis
observed in Guillain-Barre
´
and Miller–Fisher syndromes
is thought to be an autoimmune reaction due to molecular
mimicry of gangliosides by C. jejuni lipooligosaccharides
(LOS) [6,7].

In the pioneering studies carried out by Aspinall and
coworkers on the cell-surface carbohydrates from
Campylobacter species, it was observed that insoluble gels
from phenol-water extractions of bacterial cells yielded
mainly low M
r
LOS, with core oligosaccharide linked to
lipid A, and the aqueous phases from such extractions
furnished high M
r
glycans with extended polymers with
no attachment to lipid A as seen in the teichoic acid-like
P/PEtn
GM1a GM2 fl
6
b-Gal-(1fi3)- b-
D
-GalNAc-(1fi4)-b-
D
-Gal-(1 fi3)-b-
D
-Gal-(1fi3)-
L
-a-
D
-Hep-(1fi3)-
L
-a-
D
-Hep-(1fi5)Kdo

32 2 4
›› › ›
21 1 1
a-Neu5Ac a-
D
-Gal b-
D
-Glc b-
D
-Glc
6-O-Me-
D
-a-
L
-glcHepp
1
fl
3
fi2)-b-
D
-Ribf-(1-5)-b-
D
-Galf NAc-(1-4)-a-
D
-GlcpA6(NGro)-(1fi
0
B
B
B
B

@
0
B
B
B
B
@
1
C
C
C
C
A
Correspondence to J R. Brisson, Institute for Biological Sciences,
National Research Council of Canada, Ottawa, Canada, K1A 0R6.
Fax: + 1 613 952 9092, Tel.: + 1 613 990 3244,
E-mail: ; M. Monteiro, Wyeth Vaccines
Research, 211 Bailey Road, West Henrietta, NY, 14586, USA.
Fax: + 1 585 273 751, Tel.: + 1 585 273 7667,
E-mail:
Abbreviations: CE, capillary electrophoresis; ESI-MS, electron spray
ionization mass spectrometry; FAB, fast-atom bombardment;
HMBC, heteronuclear multiple bond coherence; HMQC, heteronu-
clear multiple quantum coherence; HR-MAS, high-resolution magic
angle spinning; HSQC, heteronuclear single quantum coherence;
KmR, kanamycin resistance; LOS, lipooligosaccharide; LPS, lipo-
polysaccharide; OS, oligosaccharide.
Dedication: The authors would like to dedicate this manuscript to
Professor Gerald Aspinall.
(Received 3 July 2002, revised 16 August 2002,

accepted 21 August 2002)
Eur. J. Biochem. 269, 5119–5136 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03201.x
polymer from C. coli serotype HS:30, the repeating disac-
charide from C. jejuni serotype HS:3, and the poly(tetra-
glycosylphosphates) from C. lari [6].
Analysis of the genome sequence of C. jejuni NCTC
11168 revealed that the strain possessed a type II/III capsule
locus found in other organisms such as E. coli K1 and
Neisseria meningitidis group B [8,9]. This led to the
realization that what was once believed to be high molecular
weight lipopolysaccharides (LPS) was actually capsule and
that capsule was the main serodeterminant of the Penner
typing scheme [10,11].
Campylobacter LOS and capsule are important in
adherence and invasion in vitro [10,12], colonization and
disease in vivo [10], molecular mimicry of gangliosides [6,7],
possible autoimmunity leading to Guillain–Barre
´
and
Miller–Fisher syndromes [5,13], maintenance of cell surface
charge [10], antigenic and phase variation [10,12,14–17], and
serum resistance [10,15]. Also, C. jejuni NCTC 11168 was
recently sequenced [8] and serves as a reference strain for the
understanding of the genetics of this significant food-borne
pathogen. We therefore elucidated the structures of these
major cell surface carbohydrates to make functional
analysis of the genes in the respective genetic loci possible.
EXPERIMENTAL PROCEDURES
Bacterial strains and plasmids
C. jejuni NCTC 11168 (HS:2) was originally isolated from a

case of human enteritis [18] and later sequenced by Parkhill
et al.[8].E. coli DH10B (Invitrogen) was used as the host
for the cloning experiments. Plasmid pPCR-Script Amp
(Stratagene) was used as the cloning vector.
Media and growth conditions
C. jejuni NCTC 11168 was routinely grown on Mueller
Hinton agar (Difco) under microaerophilic conditions at
37 °C. E. coli clones were grown on Luria S-gal agar
(Sigma) at 37 °C. When appropriate, antibiotics were added
to the following final concentrations: kanamycin,
30 lgÆmL
)1
and ampicillin, 150 lgÆmL
)1
.
Generation of LOS and capsule
Campylobacter biomass was harvested from overnight
liquid cultures by centrifugation. Carbohydrates were
isolated by the hot water/phenol extraction of bacterial
cells [19] as a gel-like pellet upon ultracentrifugation of the
aqueous phase. The LOS pellet was lyophilized, then
purified on a column of Bio-Gel P-2 (1 cm · 100 cm) with
water as eluent. Some of the LOS preparation was then
treated with 1% acetic acid at 100 °C for 1 h with
subsequent removal of the insoluble lipid A by centrifuga-
tion (5000 g) to yield the core oligosaccharide (OS).
The supernatant from ultracentrifugation was purified on
Bio-Gel P-2 with water as eluent and lyophilized to obtain
the capsule polysaccharide (PS-1).
Sugar composition and methylation linkage analysis

Sugar composition analysis was performed by the alditol
acetate method [20]. The hydrolysis was performed in 4
M
trifluoroacetic acid for 4 h at 100 °C followed by reduction
with NaBD
4
in H
2
O overnight, then acetylated with acetic
anhydride at 100 °C for 2 h using residual sodium acetate as
catalyst. The alditol acetate derivatives were characterized
by GLC-MS using a Hewlett-Packard chromatograph
equipped with a 30-
M
DB-17 capillary column (180 °C to
260 °C at 3.5 °CÆmin
)1
). MS was performed in the electron
impact mode and recorded on a Varian Saturn II mass
spectrometer. Absolute configuration was assigned by
characterization of the but-2-yl glycosides in GLC-MS
[21]. Methylation analysis was carried out by the NaOH/
dimethylsulfoxide/CH
3
I procedure [22] using GLC-MS in
the electron impact mode to characterize the sugars as
above. A portion (1/4) of the methylated sample was used
for fast-atom bombardment-mass spectrometry (FAB-MS)
in positive ion mode. The Jeol JMS-AX505H mass
spectrometer was used with a matrix of glycerol/thioglycerol

1 : 3 and 3 kV as the tip voltage.
Smith degradation
The polysaccharide sample ( 5 mg) was oxidized with
40 m
M
sodium metaperiodate in 0.1
M
sodium acetate at
4 °C for 72 h [23]. The product was isolated on a Bio-Gel
P-2 as per above then reduced with NaBD
4
and acidified
with cation exchange resin (J. T. Baker). The product was
then hydrolyzed with 1
M
trifluoroacetic acid at 45 °C for
1 h, reduced with NaBD
4
and re-acidified. Then, finally the
sample was fractionated on a Bio-Gel P-2 column and
fractions analyzed.
CE-ESI-MS and CE-ESI-MS/MS
A crystal Model 310 capillary electrophoresis (CE)
instrument (AYI Unicam) was coupled to an API 3000
mass spectrometer (Perkin-Elmer/Sciex) via a microIon-
spray interface. A sheath solution (isopropanol/methanol
2 : 1) was delivered at a flow rate of 1 lLÆmin
)1
to a low
dead volume tee (250 lm internal diameter, Chromato-

graphic Specialties). All aqueous solutions were filtered
through a 0.45-lm filter (Millipore) before use. An
electrospray stainless steel needle (27 gauge) was butted
against the low dead volume tee and enabled the delivery
of the sheath solution to the end of the capillary column.
The separations were obtained on about 90-cm length
bare fused-silica capillary using 10 m
M
ammonium acetate/
ammonium hydroxide in deionized water, pH 9.0, con-
taining 5% methanol. A voltage of 20 kV was typically
applied at the injection. The outlet of the capillary was
tapered to c.15lm internal diameter using a laser puller
(Sutter Instruments). Mass spectra were acquired with
dwell times of 3.0 ms per step of 1 m/z
)1
unit in full-mass
scan mode. The MS/MS data were acquired with dwell
times of 1.0 ms per step of 1 m/z
)1
unit. 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.
Construction and characterization
of insertional mutants
For construction of the Cj1428c mutant, genes Cj1427c to
Cj1428c were PCR amplified from C. jejuni NCTC 11168
5120 F. St. Michael et al. (Eur. J. Biochem. 269) Ó FEBS 2002
with the following primer pair: Cj1427cF918 (5¢-AA

CTTTCATCATTTTAAACGCTCTT-3¢)andfclR51
(5¢-TACAGCATTGGTAGAAAACTTACAA-3¢). For
construction of the kpsM mutant, gene Cj1448c was PCR
amplified with: kpsMF771 (5¢- TACCGCCGTTAAAGCT
TGTCTATTA-3¢) and kpsMR73B (5¢- TATATATGGGT
AGTTGGGGAGCCTA-3¢). For construction of the
Cj1439c mutant, gene Cj1439c was PCR amplified with:
glfF1081 (5¢-TTTTACAAAATAATAATGCCGATCT-3¢)
and glfR6 (5¢-TGATTATTTAATTGTTGGTTCTGG
A-3¢). The PCR products were ligated to pPCR-Script
Amp according to the manufacturer’s instructions. A blunt-
ended kanamycin resistance (KmR) cassette from pILL600
[24] was inserted into the filled-in BglII restriction site of
Cj1428c to create pCSc28, into the NheI restriction site
of kpsM to create pCSc48, and into the BsaBI restriction
site of Cj1439c to create pCSc39. The orientation of the
KmR cassette was determined by sequencing with the
ckanB primer (5¢-CCTGGGTTTCAAGCATTAG-3¢)
using terminator chemistry and AmpliTaq DNA polym-
erase FS cycle sequencing kits (Perkin Elmer-Applied
Biosystems) and analyzed on an Applied Biosystems 373
DNA sequencer. The mutated plasmid DNA was used for
electroporation into C. jejuni NCTC 11168 [25] and KmR
transformants were characterized by PCR to confirm that
the incoming plasmid DNA had integrated by a double
cross-over event.
Reverse transcriptase-polymerase chain reaction
It has previously been shown that gene insertion of the
Campylobacter KmR cassette in a nonpolar orientation has
no effect on transcription of downstream genes [26].

Sequencing results confirmed that the KmR insertion in
both kpsM and Cj1439c was in the nonpolar orientation.
However, the KmR cassette inserted in a polar orientation
into Cj1428c so, we wanted to determine whether Cj1428c
disruption had a polar affect on downstream genes. RNA
was isolated from C. jejuni wild type and the Cj1428c
mutant using the RNeasy mini kit (Qiagen). RT-PCR
reactions, along with controls, were performed using the
OneStep RT-PCR kit (Qiagen). First-strand synthesis was
carried out as described by the manufacturer at 50 °C for
30 min. PCR conditions were 95 °C for 15 min followed by
30 cycles at 94 °C for 30 s, 59 °C for 4 min, and 72 °C for
30 s followed by a final annealing at 72 °C for 10 min using
primers: Cj1427cF918 (5¢-AACTTTCATCATTTTAAAC
GCTCTT-3¢) and Cj1427cR10 (5¢-AAAGTTTTAATTAC
AGGTGGTGCAG-3¢).
Deoxycholate-PAGE analysis and silver staining
of polysaccharides
Proteinase K-treated whole cells of C. jejuni were
prepared according to the method of Salloway et al.
[27] based on the original method of Hitchcock and
Brown [28]. The samples were separated on 16.5%
deoxycholate-PAGE [29]. After electrophoresis, the gels
were silver stained according to the method of Tsai and
Frasch [30]. However, gels were fixed for 2–5 h rather
than overnight to prevent elution of the high molecular
weight polysaccharides [31]. In addition, we have recently
improved the silver staining procedure by visualizing the
carbohydrates with commercially available developer
(Bio-Rad [32]).

Nuclear magnetic resonance
NMR experiments were acquired on Varian Inova 600,
500 and 400 MHz spectrometers using a 5-mm triple
resonance probe with the
1
H coil nearest to the sample
and with a Z gradient coil. All measurements were made
at 25 °C on 2–5 mg of sample dissolved in 0.6 mL of
D
2
O, pH 6–7. Experiments in 90% H
2
O were carried
out at pH 4–5. The methyl resonance of acetone was
used as an internal reference at 2.225 p.p.m. for
1
H
spectra and 31.07 p.p.m. for
13
C spectra. Standard
homo and heteronuclear correlated 2D techniques were
used for general assignments: COSY, TOCSY, NOESY,
HMQC or HSQC, HMQC-TOCSY and HMBC.
Selective 1D experiments were performed for the
determination of accurate coupling constants and NOEs
andtoperformthe1Danalogofa3D-TOCSY-
NOESY experiment [33]. High resolution magic angle
spinning (HR-MAS) experiments were performed using
a gradient 4 mm indirect detection nano-NMR probe
(Varian) with a broadband decoupling coil. Proton

spectra of killed cell pellets were acquired as described
previously [34].
Molecular modeling
The conformational analysis for trisaccharide BCD of PS-1
was performed using the Metropolis Monte-Carlo method
as previously described [35]. The PFOS potential was used
[36]. Residue B was modeled as a glucuronic acid. Minim-
ized coordinates for the monosaccharides were obtained
using MM3(92) available from the Quantum Chemistry
Program Exchange. The minimum energy conformation for
each disaccharide was used as the starting conformation for
the trisaccharide. The (O5–C5–C6–O6) torsion angle was
restricted to the )60° conformer for the
DL
form of residue
D
andto+60° for the
LD
form. For the trisaccharide, 5 · 10
3
macro moves were used with a step length of 10° for the
glycosidic linkage and pendant groups and a temperature of
10
3
K resulting in an acceptance ratio of 0.5. Distances were
extracted from the saved coordinates at each macro move.
The molecular model for the trisaccharides were generated
using the minimum energy conformer. Molecular drawings
were performed using Schakal97 from E. Keeler, University
of Freiburg, Germany.

Vacuum MD simulation were performed with the
DISCOVER
-3 program from Accelrys Inc. (MSI) using the
AMBER FORCEFIELD
version 1.0–1.6, with Homans’ param-
eters applicable to saccharides [37], on a SGI Indigo 2 Solid
Impact R10000 195 MHz. As before, the (O5–C5–C6–O6)
torsion angle for residue D was restrained. The initial
structure was subjected to a 300-step energy minimization
(BFGS method), followed by 50 ps dynamics simulation at
298 K. Initial velocities were generated from a Maxwell–
Boltzmann distribution, and the temperature was controlled
by the direct velocity scaling method. The Verlet algorithm
of integrating the Newton’s equations of motion was
applied with 1 fs timestep for the simulation where a
distance-dependent dielectric of the form er (r ¼ the
distance between atoms) was used. Distances were extracted
Ó FEBS 2002 LOS and capsule structures of C. jejuni NCTC 11168 (Eur. J. Biochem. 269) 5121
from a trajectory file of 5000 frames stored after each MD
run.
RESULTS
Structural determination of the LOS
The alditol acetate derivatives [20] of
D
-glucose (
D
-Glc),
D
-galactose (
D

-Gal), N-acetyl-
D
-galactosamine (
D
-GalNAc)
and
L
-glycero-
D
-manno-heptose (
LD
-Hep), in the respective
ratios of 3 : 2.3 : 1 : 1.8, were detected in both the lipid
A-free core OS and intact LOS, by GLC-MS. The absolute
configurations of the sugars mentioned above were deduced
by the identification of but-2-yl chiral glycosides in GLC-
MS [21]. Sugar linkage analysis (Table 1), by the methyla-
tion procedure [22], on the LOS revealed the following sugar
linkage types: two units of terminal
D
-Glc, one unit of each
terminal
D
-Gal, 2,3-substituted
D
-Gal and 3,4-disubstituted
D
-Gal, traces of 4-substituted
D
-Gal, one unit of terminal

D
-GalNAc, trace amounts of 3-substituted
D
-GalNAc,
one unit of 2,3-disubstituted
LD
-Hep, and trace amounts
of 3,4-disubstituted
LD
-Hep. A parallel linkage analysis on
the liberated core OS, after removal of lipid A with 1% acetic
acid, afforded the same sugar linkage types, but in addition it
showed a significant decrease of 3,4-disubstituted
D
-Gal and
a greater amount of 4-substituted
D
-Gal (Table 1).
To gain a quick insight into the overall composition of the
LOS, a series of ESI-MS experiments were performed on
the core OS. The ESI-MS spectrum (Fig. 1a) of the core OS
showed a heterogeneous mixture (Table 2) with the pres-
ence at the reducing-end of the anhydro form of 3-deoxy-
manno-octolusonic acid (Kdo) as a distinct marker. The
primary molecular ion at m/z 1759 [)18 (H
2
O)] correspon-
dedtoacompositionofHex
5
,Hep

2
, GalNAc, Kdo and
2-amino ethyl phosphate (PEtn), and ion m/z 1716 ()18)
belonged to a composition of Hex
5
,Hep
2
, GalNAc, Kdo
and phosphate (P). Trace amounts of the phosphate-free
core OS (Hex
5
,Hep
2
, GalNAc, Kdo) at m/z 1636 ()18)
was also observed, along with small amounts of m/z of 1877
and 1921, which corresponded to the addition of an extra
hexose to both phosphorylated core OSs. For both
molecular ions, a significant ion at m/z 2007 ()18) (Hex
5
,
Hep
2
, GalNAc, Kdo, Neu5Ac, P) and m/z 2050 ()18)
(Hex
5
,Hep
2
, GalNAc, Kdo, Neu5Ac, PEtn) pointed
towards the presence of sialic acid (Neu5Ac) in the core
OS. Indeed, ESI-MS on core OS preparations that were

obtained by a harsher treatment with 5% acetic acid, to
intentionally remove any acid labile Neu5Ac, yielded the
same primary ions as discussed above, but no ions
containing sialic acid. Taking into account the previous
observed variation between 3,4-disubstituted Gal and
3-substituted Gal in the core OS, before and after mild acid
treatment, and the detection of the acid sensitive sialic acid
in ESI-MS, suggested that Neu5Ac may be attached at O-3
of the 3,4-disubstituted Gal.
The CE-MS/MS spectra for the components having a
total mass of m/z 2050 or precursor ions at m/z 1026 (doubly
protonated) are presented in Fig. 1b. The fragment ions
observed at m/z 1848 and 1760 clearly indicated that one
HexNAc residue and one Neu5Ac residue were present as
terminal units. As shown in the Fig. 1b, the fragment ion of
m/z 1848, arising from the loss of HexNAc, subsequently
loses one hexose (m/z 1686), one Neu5Ac (m/z 1394.5), three
hexoses (m/z 1232, 1070, 908) and finally one heptose
residue (m/z 716). The fragment ion at m/z 366 suggested
that the HexNAc was attached to a Hex unit, and the
fragment ion at m/z 454 was indicative a Hex-Neu5Ac
disaccharide. Moreover, fragment ion m/z 554 suggested the
existence of PEtn-Hep-Kdo moiety. It was also observed
that a minor glycoform, with an extra Hex (composition of
Hex
6
HexNAc
1
PEtn
1

Hep
2
Kdo
1
) was present in the LOS
of C. jejuni NCTC 11168. The tandem mass spectrum of ion
m/z 1005, which corresponded to the morpholine adduct of
the LOS, is presented in Fig. 1c. The doubly charged ion
yielded a fragment ion at m/z 1922 when the morpholine ion
was cleaved off. The dissociation of the core oligosaccharide
gave rise to consecutive losses of Hex, HexNAc, and five
hexoses. In contrast with Fig. 1(b), in which the ion m/z 292
corresponded to a protonated Neu5Ac, no sialic acid
fragment ion was found in this glycoform.
For the core structural features of oligosaccharide from
C. jejuni NCTC 11168, we have shown evidence for the
proposed structure as indicated in the inset of Fig. 1b.
Solid information regarding the sequences of sugar units
was obtained by FAB-MS of the methylated core OS
derivative. Figure 2 and Table 3 shows a series of A-type
primary glycosyl oxonium ions, and secondary ions, of
defined compositions at m/z 260fi228 (GlcNAc)
+
, m/z
376fi344 (Neu5Ac)
+
, m/z 464 (Gal, GalNAc), m/z 826
(GalNAc, Neu5Ac, Gal)
+
, m/z 872 (GalNAc,Gal

3
)
+
, m/z
1029 (Hex
2
, GalNAc, Neu5Ac)
+
, m/z 1120 (GalNAc, Hex
3
,
Hep)
+
, m/z 1233 (GalNAc, Hex
3
,Neu5Ac)
+
, m/z 1324
(GalNAc, Hex
4
,Hep)
+
, m/z 1407 (Hex
4
,Hep
2
,P)
+
, m/z
1477 (Hex

4
,Hep,Hep,PEtn)
+
, m/z 1685 (GalNAc, Hex
4
,
Neu5Ac, Hep
2
)
+
, m/z 1857 (GalNAc, Hex
5
,Hep
2
,P)
+
and
m/z 1928 (GalNAc, Hex
5
,Hep
2
, PEtn]
+
. A double cleavage
ion containing a phosphate moiety was also see at m/z 547
(Hep,Glc,P)
+
.
Combining the FAB-MS sequence data (Fig. 2, Table 3)
with the information obtained from the linkage analysis

(Table 1) and from the selective ESI-MS experiments
(Fig. 1, Table 2). The following provisional structural
arrangement for the core OS region can be proposed
(Hex ¼ Glc or Gal):
Table 1. Methylation linkage analysis of C. jejuni NCTC 11168 intact
LOS, core OS and Smith degradation products.
Linkage type LOS OS
Smith degradation
product
Glc-(1fi 22
Gal-(1fi 113
fi3)-Gal-(1fi 1
fi4)-Gal-(1fi Traces < 1 Traces
fi2,3)-Gal-(1fi 11
fi3,4)-Gal-(1fi 1 < 1 Traces
GalNAc-(1fi 1 1 Traces
fi3)-GalNAc-(1fi Traces Traces
fi2,3)-Hep-(1fi 11
– fi3,4)-Hep (1fi Traces Traces
fi3)-Man-(1fi 3
fi5)-3d-Hexitol
a
0.5
fi5)-3d-Hexitol
b
0.5
a,b
Two isomeric forms of 3-deoxy-1,1,2,6-tetra-
2
H-5-O-acetyl-

1,2,4,6-tetra-O-methyl-hexitol (from 5-substituted Kdo).
5122 F. St. Michael et al. (Eur. J. Biochem. 269) Ó FEBS 2002
A Smith degradation [23] was strategically performed on
the core OS to disentangled the linkages at the branch
points. Prior to periodate oxidation, the core OS was
reduced with NaBD
4
for the incorporation of deuterium at
the Kdo terminus so that this unit could be detected in the
final product. Periodate oxidation of the reduced core OS
was followed by reduction with NaBD
4
, mild acid hydro-
lysis, and a final reduction with NaBD
4
. Sugar linkage
analysis of the final product (Table 1) showed the presence
of terminal Gal (from 3,4-substituted Gal), 3-substituted
Gal (from 2,3-substituted Gal), 3-substituted Man-O-6-
2
H
(from 2,3- and 3,4-substituted
LD
-Hep units) and two
isomeric units of 3-deoxy-1,1,2,6-tetra-
2
H-5-O-acetyl-
1,2,4,6-tetra-O-methyl-hexitol (from 5-substituted Kdo), in
the approximate ratios of 3 : 1: 3 : 0.5 : 0.5. There were also
traces of 4- and 3,4-substituted Gal and terminal GalNAc;

these two derivatives originated from an extended molecule
that contained a hexose at the nonreducing terminus of
the core {Hex-(1fi4)-GalNAc-(1fi4)[Neu5Ac-(1fi3)]-
Gal… inner core}.Theisomerichexitolderivativeswere
recognized as originating from a modified 5-linked Kdo
termini as seen in all C. jejuni strains. The backbone Gal and
LD
-Hep units are thus joined by 1fi3 linkages and the inner
most
LD
-Hep is linked to O-5 of Kdo. The FAB-MS
Fig. 1. Electron spray ionization-mass spectr-
ometry. C. jejuni NCTC 11168 core OS
showing a heterogeneous mixture (a). CE-MS/
MS (+ ion mode, produces ions of m/z 1026)
analysis of C. jejuni NCTC 11168 core OS (b).
CE-MS/MS (+ ion mode, produces ions of
m/z 1005) analysis of LOS from C. jejuni
NCTC 11168 core OS (c).
P/PEtn
fl
[
D
-Hex-(1fi3)]±-
D
-GalNAc-(1fi3 or 4)-
D
-Gal-(1fi2 or 3)-
D
-Gal-(1fi2 or 3)-

LD
-Hep-(1fi3 or 4)-
LD
-Hep-(1fiKdo
3or4 2or3 2or3 3or4
›› › ›
21 1 1
Neu5Ac
D
-Hex
D
-Hex
D
-Hex
Ó FEBS 2002 LOS and capsule structures of C. jejuni NCTC 11168 (Eur. J. Biochem. 269) 5123
spectrum (Table 3) of the methylated final product from the
Smith degradation yielded some limited, but corroborating,
sequence data (* ¼ deuterium) stemming from the terminus
showing m/z 219 (Gal)
+
, m/z 424 [Gal-(1fi3)-Man*]
+
,and
m/z 289 (Neu5Ac*)
+
, m/z 493 [Neu5Ac*(1fi3)-Gal]*, and
m/z 902 [Neu5Ac*(2fi3)-Gal-(1fi3)-Gal-(1fi3)-Man*]
+
.
Two final products were thus recovered from the Smith

degradation (shown below), one was terminated by a GM2
structure, and the major product was a linear Gal-Man-
Man-3dhex (shown below) backbone:
Two unresolved anomeric resonances, characteristic of
sugars with the mannose configuration, were observed at d
5.207 and d 5.07 in the
1
H nuclear magnetic resonance
spectrum of the Smith degradation product. The same
spectrum also showed one b anomeric resonance at 4.55
(J
1,2
7.2 Hz). The
1
H NMR data just described indicated
that the Man (
LD
-Hep) units possessed an a anomeric
configuration, whereas the 2,3-disubstituted
D
-Gal had a b
anomeric configuration. Therefore, at this time, the follow-
ing structure for the core OS region, where Hex represents
Glc or Gal, was proposed:
The sugar linkage analysis (Table 1) performed on the
core OS suggested the presence of slightly more than one
unit of terminal Gal and two units of terminal Glc. Given
the fact that the hexose at the nonreducing terminus was
only present in trace amounts, as observed by linkage
analysis (traces of 3-substituted GalNAc), ESI-MS and

FAB-MS, the three side-branches hexoses could be
assigned to two units of Glc and one unit of Gal. The
nonstoichiometric hexose present in trace amounts that is
connected to the O-3 position of GalNAc was a Gal
residue.
The
1
H NMR spectrum, in combination with a
1
H–
1
H
TOCSY experiment, of the core OS yielded three a
anomeric protons, at d 5.67 (J
1,2
3Hz)thatwasassignedto
an a-
D
-Gal residue [H-2 d 3.79 (J
2,3
9.5 Hz) and H-3 d 3.93
(J
3,4
3Hz)],andatd 5.40 (unresolved doublet) [H-2 d 4.39
(J
2,3
2Hz) and H-3 d 4.28 (J
3,4
3 Hz)], and at 5.10
(unresolved doublet) [H-2 d 4.17 (J

2,3
2 Hz)], typical of
a-
D
-Man configurations and were thus assigned to the
L
-a-
D
-Hep residues, as were observed in the Smith
degradation
1
H NMR product described above. All other
anomeric resonances detected possessed b anomeric con-
figuration and could be seen at d 4.99 (J
1,2
)7Hz),d 4.88
(J
1,2
)7Hz),d 4.69 (J
1,2
)7Hz),d 4.66 (J
1,2
)7Hz)andd
4.62 (J
1,2
)7 Hz). To situate the side-branch hexoses, a 2D
1
H–
1
H NOESY experiment was performed and conclusive

evidence, an inter-NOE between H-1 (d 5.67) of the a-Gal
and H-2 (d 3.98) of the residue with the b anomeric H-1
(d 4.99), was obtained that placed the sole a-
D
-Gal side-
branch at O-2 of the unit with the anomeric at d 4.99,
which has to belong to the sole 2-substituted unit, that
being the 2,3-disubstituted Gal in the backbone. The
other side-branch hexoses, two Glc units, must then
have the b anomeric configuration and be attached to
the
L
-a-
D
-Hep residues (b-
D
-Glc-(1fi2)-
L
-a-
D
-Hep) and
(b-
D
-Glc-(1fi4)-
L
-a-
D
-Hep).
The GM2 and GM1a ganglioside mimics in C. jejuni
NCTC 11168 LOSs were covalently attached to the inner

core region (Fig. 3) composed of basal core OS units, Gal,
LD
-Hep and Glc. The GM2 and GM1a epitopes completed
a core OS similar to that present in C. jejuni serogroup
HS:1 [6]. In addition, the innermost
LD
-Hep was phos-
phorylated by a monoester phosphate or by a 2-amino
ethyl phosphate.
Structure of the capsule polysaccharide
The polysaccharide, obtained from the aqueous phase after
ultracentrifugation, was purified on a Bio-Gel P-2 (PS-1).
Alditol acetate analysis revealed the presence of
D
-Glc,
Table 2. Negative ion ESI-MS data and proposed compositions for
C. jejuni NCTC 11168 core OS and de-O-acylated LOS (masses include
the addition of water)
a
.
Core OS
Observed molecular
mass (Da)
Proposed
structure
1635 ()18) HexNAcÆHex
5
ÆHep
2
ÆKdo

1714 ()18) HexNAcÆHex
5
ÆHep
2
ÆPÆKdo
1758 ()18) HexNAcÆHex
5
ÆHep
2
ÆPEtnÆKdo
1876 HexNAcÆHex
6
ÆHep
2
ÆPÆKdo
1920 HexNAcÆHex
6
ÆHep
2
ÆPEtnÆKdo
2005 ()18) Neu5AcÆHexNAcÆHex
5
ÆHep
2
ÆPÆKdo
2049 ()18) Neu5AcÆHexNAcÆHex
5
ÆHep
2
ÆPEtnÆKdo

a
Residues used and their molecular mass: Neu5Ac, 291; HexNAc,
203; Hex, 162; Hep, 192; PEtn, 123; P, 79; Kdo, 220.
P/PEtn
fl
[
D
-Hex-(1fi3)]±-
D
-GalNAc-(1fi4)-
D
-Gal-(1fi3)-b-
D
-Gal-(1fi3)-
L
-a-
D
-Hep-(1fi3)-L-a-
D
-Hep-(1fi5)-Kdo
32 2 4
›› › ›
21 1 1
Neu5Ac
D
-Hex
D
-Hex
D
-Hex

Major Smith degradation product
[
D
-GalNAc-(1fi4)-
D
-Gal-(1fi3)]±-
D
-Gal-(1fi3)-
D
-Man*-(1fi3)-
D
-Man*-(1fi)-3-d-hexitol****
3
›
2
[Neu5Ac*]±
5124 F. St. Michael et al. (Eur. J. Biochem. 269) Ó FEBS 2002
D
-Gal,
LD
-Hep (from the core region) and
D
-GlcNAc as
minor components, and as major units
D
-ribose (
D
-Rib),
6-O-methyl-heptose (6-O-Me-Hep), and N-acetyl-
D

-gal-
actosamine (
D
-GalNAc). From the alditol acetate analysis
it was observed that PS-1 was slightly contaminated with
LOS, additional efforts at purification were not successful.
The methylation linkage analysis revealed the presence
of 2-substituted
D
-Rib, 4-substituted
D
-GalNAc, and a
terminal heptose unit. No substituted heptose was detected,
and thus the 6-O-Me-Hep was present as a side chain
residue.
For further characterization of the structure of PS-1, the
sample was acid hydrolyzed under mild conditions (1
M
HCl, 100 °C for 5 min) and the resultant hydrolysate was
purified on a Bio-Gel P-2 column. The sample was then
analyzed by CE-ESI-MS (Fig. 4a) and gave rise to two
components having a mass of 791 as the major product and
a minor mass of 762. The MS/MS spectra of m/z 791
(Fig. 4b) revealed fragments m/z 588 and 585. This showed
the loss of either 6-O-Me-Hep or GalNAc, respectively; thus
illustrating that they were terminal units in this OS from
acid hydrolysis of PS-1. This finding was consistent with
the previous observation in the linkage analysis that the
6-O-Me-Hep was a terminal unit. The fragment ion at 382
arose from the loss of both the 6-O-Me-Hep and GalNAc,

whichthenleadtothelossofRibwithafinalmassof
250 Da. The MS/MS spectra of m/z 762 (Fig. 4c), as
with m/z 791, showed the loss GalNAc (m/z 558) and
6-O-Me-Hep (m/z 555) as terminal residues. It can also be
observed, as in the previous example, that after the loss of
the 6-O-Me-Hep and the GalNAc leading to fragment ion
m/z 352, Rib is also lost, which furnished a final mass of
220 Da. Therefore, from this CE-MS/MS analysis it was
suspected that there might be two polysaccharide chains
present or that one of the components in the capsule can be
modified.
The
1
H NMR spectrum of PS-1 (Fig. 5a,b) revealed the
presence of four anomerics. COSY, NOESY, and TOCSY
NMR experiments were performed on PS-1, but the
heterogeneity lead to broad lines, making interpretation
difficult. This lead to the use of HR-MAS NMR to examine
capsular polysaccharide resonances on intact Campylobacter
cells without the need for extensive growth and purification
[34]. HR-MAS spectra of wild-type and capsule mutants
were used for the initial screening and selection of a mutant
that lacked the 6-O-Me-Hep, as this sugar was suspected to
be a side chain. The disappearance of one anomeric and the
loss of the OMe resonance at 3.56 p.p.m. (Fig. 5c,d) were
used to ascertain this. Once an appropriate mutant was
generated (Cj1428c mutant; see below), its polysaccharide
was purified (the absence of the 6-O-Me-Hep was also
verified by alditol acetate analysis, results not shown). The
polysaccharide (isolated as described above) of the Cj1428c

mutant was denoted as PS-2 (Fig. 6). The proton spectrum
of PS-2 was more homogeneous and had sharper lines
than the proton spectrum of PS-1. As the spectrum of PS-2
was less complex than the spectrum of the native PS-1,
its structural determination was first undertaken.
NMR methods, as outlined before [33,38,39], were used
for the structural determination of polysaccharides 1 and
Fig. 2. FAB-MS spectra of the methylated
C. jejuni NCTC 11168 core OS.
Ó FEBS 2002 LOS and capsule structures of C. jejuni NCTC 11168 (Eur. J. Biochem. 269) 5125
Table 3. Interpretation of m/z ions in the FAB-MS spectrum of the methylated core OS from C. j ejuni NCTC 11168.
Primary m/z ion Secondary m/z ion Double cleavage m/z ion Proposed structure
260 228 (260–32) GlcNAc
+
376 344 (376–32) Neu5Ac
+
464 432 (464–32) Gal-(1fi3)-GalNAc
+
547 Glc-(1fi4)-Hep
+
›
P
668 GalNAc-(1fi4)-Gal-(1fi3)-Gal
+
826 GalNAc-(1fi4)-Gal
+
3
›
2
Neu5Ac

872 GalNAc-(1fi4)-Gal-(1fi3)-Gal
+
3
›
2
Neu5Ac
1029 Gal-(1fi3)-GalNAc-(1fi4)-Gal
+
3
›
2
Neu5Ac
1120 GalNAc-(1fi4)-Gal-(1fi3)-Gal-(1fi3)-Hep
+
2
›
1
Gal
1233 GalNAc-(1fi4)-Gal-(1fi3)-Gal
+
32
››
21
Neu5Ac Gal
1324 GalNAc-(1fi4)-Gal-(1fi3)-Gal-(1fi3)-Hep
+
22
››
11
Gal Glc

1407 P
fl
Gal-(1fi3)-Hep-(1fi3)-Hep
+
22 4
›› ›
11 1
Gal Glc Glc
1477 PEtn
fl
Gal-(1fi3)-Hep-(1fi3)-Hep
+
224
›› ›
111
GalGlc Glc
1685 GalNAc-(1fi4)-Gal-(1fi3)-Gal-(1fi3)-Hep
+
322
›››
211
Neu5Ac Gal Glc
5126 F. St. Michael et al. (Eur. J. Biochem. 269) Ó FEBS 2002
2 obtained from C. jejuni. 1D selective NMR methods
were also used to characterize individual components [33].
1D-TOCSY experiments were used to detect the coupled
spin systems and spin simulation was used to obtain
accurate coupling constants (Figs 7a–d). HMQC was used
to assign CH, CH
2

and CH
3
carbon resonances (Fig. 7f).
The assignments were verified by means of an HMQC-
TOCSY experiment. HMBC was used to locate the C¼O
resonances (Fig. 7g). A correlation was also observed
between C-7C and H-8C for the NAc group of residue C
(results not shown). Location of nitrogen-bearing groups
was carried out by performing experiments in 90% H
2
Oin
order to detect the NH resonances (Fig. 7i). A COSY
experiment (results not shown) was used to detect the NH-
C-H correlation and assign the NH resonances. The
NOESY experiment was used to detect NOEs between
the pendant groups and the ring protons. Finally the
HMBC experiment was used to detect the multiple bond
correlation between the C¼O and NH resonances (Fig. 7i).
Table 3. (Continued).
1857 P
fl
GalNAc-(1fi4)-Gal-(1fi3)-Gal-(1fi3)-Hep-(1fi3)-Hep
+
322 4
››› ›
211 1
Neu5Ac Gal Glc Glc
1928 PEtn
fl
GalNAc-(1fi4)-Gal-(1fi3)-Gal-(1fi3)-Hep-(1fi3)-Hep

+
3224
››› ›
211 1
Neu5Ac Gal Glc Glc
Atomic mass units of the residues discussed above: Glc/Gal GalNAc Hep Neu5Ac P PEtn
Terminal 219 260 263 376 94 166
Monosubstituted 204 245 248
Disubstituted 189 233
Fig. 3. The complete structure of C. jejuni
NCTC 11168 LOS.
Fig. 4. CE-ESI-MS and CE-MS/MS analysis. CE-ESI-MS of PS-1
after acid hydrolysis (1
M
HCl, 100 °C for 5 min) and Bio-Gel P-2
purification(a),CE-MS/MSofm/z 791 (b), CE-MS/MS of m/z 762 (c).
Fig. 5. Proton spectra of C. jejuni whole cells and isolated polysaccha-
rides. HR-MAS spectrum of C. jejuni whole cells (a) and its isolated
polysaccharide (b). HR-MAS spectrum of C. jejuni Cj1428c mutant
whole cells (c) and its isolated polysaccharide (d). The anomeric
resonances are labeled according to the structures shown in Fig. 6.
Ó FEBS 2002 LOS and capsule structures of C. jejuni NCTC 11168 (Eur. J. Biochem. 269) 5127
As the absolute configuration of the sugars was known from
the chemical analysis, from a comparison of chemical shifts
and coupling constants with those of monosaccharides,
residue A was assigned as b-
D
-Ribf, residue B as the amide
of a-
D

-GlcpA with -NH-CH
2
-CH
2
OH at C-6, and residue C
as b-
D
-GalfNAc. The sequence of sugars was established by
an HMBC experiment (Fig. 7h). The (H-1 A, C-5C) (H-1C,
C-4B) and (H-1B, C-2 A) HMBC correlations established
the (-A-C-B-)
n
polymeric sequence (Fig. 6). The NMR data
areshowninTable4.
The linkage analysis of the Cj1428c mutant, PS-2, by the
methylation method [22] revealed the same 2-substituted
Rib and 4-substituted GalpNAc,whichinthiscase,as
observed by NMR, is a 5-substituted GalfNAc. As expected,
this PS-2 lacked the terminal 6-O-Me-Hep. Confirmation of
the repeat established by NMR was observed in the FAB-
MS and MALDI-MS (Fig. 8) of the methylated PS-2. The
MALDI-MS spectra shows a series of A-type primary
glycosyl oxonium ions and secondary ions of defined
compositions at m/z 260fi228 (GalNAc)
+
, m/z 420 (Gal-
NAc, Rib)
+
, m/z 695 (GalNAc, Rib, GlcA), m/z 941
(GalNAc

2
,Rib,GlcA)
+
, m/z 1101 (GalNAc
2
,Rib
2
,
GlcA)
+
, m/z 1620 (GalNAc
3
,Rib
2
,GlcA
2
)
+
, m/z 1781
Fig. 6. Structure of polysaccharides PS-1 and PS-2 from C. jejuni,and
labeling of the residues and atoms. Residue A is b-
D
-Ribf,residueBis
the amide of a-
D
-GlcpA with ethanolamine at C-6 for PS-2 and with
2-amino-2-deoxyglycerol at C-6 for PS-1, residue C is b-
D
-GalfNAc,
and residue D is

D
-glycero-a-
L
-gluco-heptopyranose.
Fig. 7. NMR spectrum of PS-2. Spin simula-
ted spectra for residue A (a), residue B (b),
residue C (c) and the substituent at C-6 of
residue B (d), along with the resolution
enhanced proton spectrum (e). In (f) is the
HMQC spectrum of the ring protons. In (g)
is the HMBC spectrum showing the C¼O
region for assignment of C-6B and C-7C. The
HMBC spectrum in (h) shows the intergly-
cosidic
3
J(C,H) correlations. In (i) the proton
spectrum, NOESY and HMBC correlations
for the NH resonances obtained in 90% H
2
O
are shown.
5128 F. St. Michael et al. (Eur. J. Biochem. 269) Ó FEBS 2002
(GalNAc
3
,Rib
3
,GlcA
2
)
+

and m/z 2301 (GalNAc
4
,Rib
3
,
GlcA
3
)
+
.
Similarly to PS-2, the 1D-TOCSY experiments were used
to identify the spin systems for each residue and pendant
groups of PS-1. The 1D-TOCSY for anomeric resonance of
residue D is shown in Fig. 9a. The HMQC spectrum was
used to assign the
13
C resonances (Fig. 9g). Residues A, B
and C were found to be the same as in PS-2 except for the
pendant group at C-6B. The sequence of the sugars for PS-1
was established from detection of the inter-residue NOE
between the anomeric resonance and aglycon resonance.
Because the (H-1A, H-5C) (H-1C, H-4B) (H-1B, H-2A)
NOEs were present, the polymeric sequence (-A-C-B-)
n
was
thus found to be the same as in PS-2. Residue D was found
to be linked at C-3 of residue B from the presence of the
(H-1D, H-3B) NOE. From the (H-7B, C-8,9B) HMBC
correlations (Fig. 9h), the 1D-TOCSY for H-9B, and the
NOEs for the NH resonances at C-6B (results not shown),

the pendant group was identified as -NH-CH-(CH
2
OH)
2
.
Its chemical shifts were identical to those of a similar
pendant group observed at C-6 of GlcA [40]. Small signals
(< 10%) from the -NH-CH
2
-CH
2
OH group were still
observed (Fig. 5), due to heterogeneity at C-6 of the GlcA
residue in PS-1.
For residue D in PS-1, a 1D-TOCSY on the H-1D
resonance could detect resonances up to H-5 (Fig. 9a). The
location of the OMe group at C-6 was determined from the
(H-8D, C-6D) and (H-8D, C-6D) HMBC correlations
(Fig. 9h), and from the (H-8D, H-6D) NOE (Fig. 9b).
Finally, the H-7 resonances were located by means of a
1D-NOESY-TOCSY with selective excitation of the H-8D
resonance followed by selective excitation of the H-6D
Table 4. NMR data for PS-1 and PS-2. The CH
3
signal of acetone was set at 2.225 p.p.m. for
1
H and 31.07 p.p.m. for
13
C.Averageerrorof
± 0.2 p.p.m. for d

C
, ± 0.01 p.p.m. for d
H
,and±0.5HzforJ.J-values were obtained from the spin-simulated spectra. Vicinal
3
J values are
positive and geminal
2
J values are negative.
Atom
PS-2 PS-1
Type d
C
d
H
J Type d
C
d
H
1 A CH 105.9 5.37 1.0 CH 106.0 5.36
2 A CH 81.1 4.18 5.0 CH 81.2 4.22
3 A CH 71.0 4.26 7.2 CH 70.8 4.32
4 A CH 84.0 4.12 2.6 CH 84.0 4.13
5A CH
2
63.2 3.86
3.67
6.5
)12.3
CH

2
63.2 3.88
3.69
1B CH 98.9 5.20 3.9 CH 98.9 5.14
2B CH 71.9 3.70 9.5 CH 73.2 3.95
3B CH 72.0 3.88 9.2 CH 73.6 4.08
4B CH 79.1 3.65 9.8 CH 75.9 3.92
5B CH 72.0 4.36 CH 72.6 4.36
6B C¼O 171.7 C¼O 171.8
7B CH
2
42.7 3.47
3.21
5.8
)14.0
CH 54.0 4.05
8B CH
2
60.8 3.67
3.65
5.8
)14.0
CH
2
61.3 3.73
3.66
9B CH
2
61.3 3.73
3.66

6B NH 8.51 5.7 NH 8.33
1C CH 106.8 4.87 2.0 CH 105.2 5.05
2C CH 64.0 4.14 4.5 CH 63.0 4.08
3C CH 76.5 4.17 7.0 CH 74.7 4.24
4C CH 82.9 4.22 3.2 CH 82.1 4.24
5C CH 77.6 3.96 4.0 CH 77.9 3.89
6C CH
2
62.2 3.80
3.75
6.7
)12.3
CH
2
62.0 3.81
3.77
7C C¼O 174.9 C¼O 175.2
8C CH
3
23.0 2.02 CH
3
23.0 2.05
2C NH 8.29 NH 8.21
1D CH 98.2 5.60
2D CH 72.3 3.52
3D CH 73.8 3.74
4D CH 70.4 3.57
5D CH 72.1 4.15
6D CH 79.6 3.80
7D CH

2
63.0 3.87
8D CH
3
60.6 3.56
Ó FEBS 2002 LOS and capsule structures of C. jejuni NCTC 11168 (Eur. J. Biochem. 269) 5129
resonance in the final TOCSY step (Fig. 9c). From the
1D-TOCSY of H-1D (Fig. 9a), vicinal J(Hn,Hn +1)
values were found to be 3.6, 9.5, 9.5, 9.7 Hz ± 0.5 Hz for
the H-1, H-2, H-3 and H-4 resonances, respectively,
indicative of the gluco-pyranose conformation for the ring.
J(H5, H6) was found to be < 2 Hz. Due to overlap of
resonances, the coupling constant between the H-6 and H-7
resonances could not be determined. Hence, residue D was
Fig. 8. MALDI-MS of the methylated PS-2
from the Cj1428c mutant showing the repeat of
the polysaccharide.
Fig. 9. NMR spectra for PS-1. (a) 1D-
TOCSY of H-1D resonance at 5.60 p.p.m.
with 150 ms mixing time. (b) 1D-NOESY
with 200 mixing time of the OMe (H-8D)
resonance at 3.56 p.p.m. (c) Subsequent
1D-NOESY-TOCSY (H-8D, 200 ms; H-6D,
60 ms) for detection of the H-7D and H-7¢D
resonances. (d) 1D-TOCSY-NOESY (H-4D
H-8D, 30 ms; H-5D, 200 ms) for the detection
of NOEs for H-5D. (e) 1D-TOCSY-NOESY
(H-5D, 40 ms; H-4D, 400 ms) for detection of
the NOEs for H-4D. (f) 1D-TOCSY-NOESY
(H-2D, 40 ms; H-3D, 400 ms) for detection of

NOEs for H-3D. (g) HMQC spectrum and
assignment of
13
C resonances. (h) HMBC
spectra for location of -NH-CH-(CH
2
OH)
2
substituent on residue B and OMe group on
residue D. (i) NOESY spectrum and deter-
mination of the sequence from inter-residue
NOE correlations.
5130 F. St. Michael et al. (Eur. J. Biochem. 269) Ó FEBS 2002
determined to be glycero-a-gluco-heptopyranose. The
NMR data for PS-1 are given in Table 4.
The absolute configuration of residues A, B and C could
be determined from MS based methods [21,41]. The
absolute configuration of residue D was determined by
NOEs (Fig. 9) in conjunction with molecular modeling.
This method has been previously used to establish the
absolute configuration of sugars in cases where conven-
tional methods are not amenable [42]. The use of selective
experiments, especially the 1D-TOCSY-NOESY, permitted
the detection of NOEs from proton resonances within the
ring. This allowed NOE constraints to be obtained which
cannot be otherwise determined from 2D experiments [33].
NOEs are highly dependent of interproton distances
(r · 10
)6
) and thus on the conformation of the molecule.

In our case, molecular modeling was performed for the
trisaccharide BCD, with different absolute configurations
for residue D and the
D
-configuration for residues B and C.
It was found that some of the interproton distances between
residues C and D were highly dependent on the relative
absolute configuration of the monosaccharides. NOEs
between the glycerol side chains and the ring protons of
residue D could also be used to determine the relative
configuration of residue D.
The strong NOEs between 5D-77¢D (Fig. 9d) and the
absence of the 4D-77¢D NOE (Fig. 9e) indicated either the
D
-glycero-
L
-gluco-heptopyranose or
L
-glycero-
D
-gluco-
heptopyranose configurations. Strong inter-residue NOEs
5D-2C, 5D-4C (Fig. 9d), 3D-2C and 3D-4C were observed.
These inter-residue NOEs were similar in intensity to the
intraresidue NOE 3D-5D, indicating that interproton pairs
were in the 2–3 A
˚
range. The average distances obtained
from molecular dynamics for interproton pairs having
strong NOEs are shown in Table 5. The standard deviation

is due to flexibility, mainly about the glycosidic bonds. Only
for residue D having the
D
-glycero-
L
-gluco-heptopyranose
absolute configuration are short inter-residue interproton
distances possible, consistent with the observed NOEs.
Average distances obtained from a Metropolis Monte-
Carlo calculation gave similar results. Minimum energy
conformers from the Metropolis Monte-Carlo calculations
are shown in Fig. 10. When residue D has the
L
-glycero-
D
-
gluco-heptopyranose absolute configuration, the range of
inter-residue interproton distances being sampled are not
consistent with the observed NOEs, especially for the 5D-
2C NOE.
Genetic analysis of the capsule polysaccharide
Using gene mutation to manipulate the capsular structure in
combination with the use of HR-MAS NMR, we were able
to compare the complex capsular carbohydrate structure of
wild-type NCTC 11168 with the simpler capsule of the
Cj1428c mutant (Figs 5c and 11a). Cj1428c is homologous
to fcl of E. coli whose product is involved in the conversion
of GDP-4-keto-6-deoxy-
D
-mannose to GDP-4-keto-6-

L
-
deoxygalactose to GDP-
L
-fucose in the formation of fucose
in the colanic acid extracellular polysaccharide [43]. The role
of Cj1428c in Campylobacter polysaccharide biosynthesis is
currently unknown. There is no evidence for the presence of
fucose in any carbohydrate structures of NCTC 11168
identified to date, but we are currently investigating this
possibility. Alternatively, this enzyme may be involved in
the epimerization reaction necessary for the biosynthesis of
the capsular heptopyranose as alditol acetate analysis
(results not shown) and HR-MAS NMR (Fig. 5c) demon-
strated the lack of 6-O-Me-Hep in the Cj1428c mutant.
RT-PCR demonstrated that the mutation was nonpolar
(results not shown). Interestingly, deoxycholate-PAGE
demonstrated that the repeat was polymerized even in the
absence of 6-O-Me-Hep (Fig. 11b).
Capsular Cj1439c exhibits significant homology to E. coli
UDP-galactopyranose mutase, glf, responsible for the
biosynthesis of galactofuranose [44]. Interestingly,
HR-MAS NMR (results not shown) and deoxycholate-
PAGE (Fig. 11b) data demonstrate that mutation of NCTC
11168 Cj1439c results in an acapsular phenotype similar to
the acapsular transport mutant, kpsM (Fig. 11b) [10,11].
GalpNAc may be converted to GalfNAc by the mutase
and is consistent with the observation that the capsular
Fig. 10. Molecular model of residues BCD for PS 2 with residue D being
a

D
-glycero-
L
-gluco-heptopyranose in (a) and an
L
-glycer o-
D
-gluco-
heptopyranose in (b). Residue B was modeled as a glucuronic acid. The
hydroxyl protons are not shown and the exocyclic chain of residue C is
not shown in (a). The absolute configuration of residue D was estab-
lished to be
D
-glycero-
L
-gluco-heptopyranose, consistent with the
5D-77¢D NOEs and the inter-residue 3D-2C, 3D-4C, 5D-2C, 5D-4C
NOEs.
Table 5. Experimental NOEs and interproton distances (average
± standard deviation) obtained from molecular dynamics for the BCD
trisaccharide with different absolute configurations for the heptose
residue D.
H–H NOE
a
DL
-Hep
LD
-Hep
r (A
˚

) r (A
˚
)
5D)3D 1 2.6 ± 0.2 2.6 ± 0.3
5D)6D 1 2.4 ± 0.1 2.5 ± 0.1
5D)77¢D 1 3.2 ± 0.6 3.1 ± 0.6
5D)2C 1 3.3 ± 0.8 6.4 ± 1
5D)4C 0.4 4.1 ± 1.3 6.2 ± 1.5
3D)5D 1 2.6 ± 0.3 2.6 ± 0.3
3D)2C 0.3 4.3 ± 1.1 5.8 ± 1.3
3D)4C 0.9 3.6 ± 1.2 5.2 ± 1.8
a
Normalized integrals to the intraresidue 5D)3D and 3D)5D
NOE. Estimated error of ± 0.2.
Ó FEBS 2002 LOS and capsule structures of C. jejuni NCTC 11168 (Eur. J. Biochem. 269) 5131
N-acetylhexosamine is in the furanose configuration. It is
interesting to note that loss of GalfNAc, one of the three
main sugars in the polymer repeat, results in loss of capsule
polymerization in contrast to loss of the 6-O-MeHep which
does not affect polymerization of the capsule sugars.
Cj1441c in the capsular gene cluster is homologous to
E. coli kfiD which encodes an UDP-glucose dehydrogenase
involved in the biosynthesis of glucuronic acid [45]. There-
fore, it is not surprising that the capsular polysaccharide of
NCTC 11168 contains glucuronic acid. We are currently
making a mutant in Cj1441c to determine whether loss of
this gene also results in an acapsular phenotype. The
capsular polysaccharide of serostrain HS:19 has also been
reported to exhibit a repeating glucuronosyl residue,
modified with N-linked glycerol, and b-1,3 linked to

GlcNAc [40]. Comparison of NCTC 11168 and HS:19
capsular loci sequences demonstrates that HS:19 also
contains a Cj1441c homolog (unpublished results).
Analysis of the genome sequence of NCTC 11168
demonstrates that the strain contains two gene clusters
involved in heptose biosynthesis [8]. One cluster, located in
the LOS gene cluster, is similar to that found in other Gram-
negative bacteria and is necessary for the biosynthesis of the
L
-glycero-
D
-manno-heptopyranoses commonly found in
LPS of many Gram-negative bacteria. The second cluster,
found in the capsule gene cluster, is homologous to
genes recently identified in the Gram-positive, A. thermo-
aerophilus, involved in the biosynthesis of
D
-glycero-
D
-manno-heptopyranose in the S-layer glycoprotein [46].
Campylobacter species have been shown to produce both
LD
-heptoses in their LOS cores and other heptose isomers in
their capsular polysaccharides (for examples see [6]).
Therefore, it was not surprising to find
L
-glycero-
D
-manno-
heptopyranose in the LOS core and 6-O-Me-Hep in the

capsule polysaccharide in this study. We are currently
characterizing the heptose biosynthetic pathways in NCTC
11168.
The sugar composition of the NCTC 11168 polysaccha-
rides was examined in earlier studies by Naess and Hofstad
[47]. The authors actually noted the presence of ribose in
their preparations but attributed the finding to incomplete
digestion with ribonuclease [47]. Here, we demonstrate that
ribose is indeed present in stoichiometric amounts in the
capsular polysaccharide. The genes necessary for ribose
biosynthesis are not obvious in the capsule gene cluster, thus
we speculate that the sugar is synthesized during normal cell
metabolism and that the genes required for biosynthesis are
located elsewhere on the chromosome.
As already mentioned, both N-linked glycerol and
ethanol modifications were found on the glucuronosyl
residue of wild-type capsule. Further NMR analysis of the
Cj1428c mutant demonstrated that the glycerol modifica-
tion was absent suggesting that we had selected a possible
phase variant, which only produced the ethanol modifica-
tion. In addition, we observed variation in the 6-O-methyl
modification on
D
-glycero-
L
-gluco-heptopyranose. This was
first noted when different preparations of NCTC 11168
whole cells were examined by HR-MAS NMR and showed
variations in the size of the methyl peak as well as what
appeared to be splitting of the heptose anomeric peak

(results not shown). We have isolated a natural phase
variant that produces the capsular polysaccharide without
the methyl modification and this variant is currently under
investigation.
DISCUSSION
The LOS outer core region of C. jejuni NCTC 11168 has
been found to consist predominately of a GM2 ganglioside
mimic, but also in smaller amounts, of a GM1a ganglioside
mimic lacking the terminal b-1,3-linked galactose (Fig. 3).
In the strain investigated here, wlaN encodes the b-1,3-
galactosytransferase which adds the terminal Gal to
GalNAc. Linton et al. showed that the homopolymeric
C-tract within wlaN was subject to frequent on/off switching
through a mechanism of slipped-strand mispairing during
DNA replication [17]. The authors predominantly observed
thein-frameversionofwlaN while our laboratory observes
Fig. 11. Mutagenesis and analysis of capsule mutants of C. jejuni NCTC 11168. (a) Schematic of the capsule gene cluster with gray arrows
representing genes mentioned in the text. The kps genes involved in capsule transport and assembly are shown along with possible hypervariable
regions within the cluster (due to homopolymeric C tracts or a 21-bp repeat, R) that may be responsible for the structural variability described. The
constructs used in this study along with the direction of the KmR cassette insertion are shown. (b) Silver-stained deoxycholate-PAGE of proteinase
K whole cell digests of wild-type NCTC 11168 and isogenic mutants. Lane 1: wild-type NCTC 11168; lane 2: kpsM mutant; lane 3: 1428c mutant;
lane 4: 1439c mutant. The capsular repeat is indicated by the arrow.
5132 F. St. Michael et al. (Eur. J. Biochem. 269) Ó FEBS 2002
mostly the truncated version of the gene (results not shown).
Examination of LOS cores isolated from growth of single
colonies of our NCTC 11168 by deoxycholate-PAGE and
MS yielded structures with and without the terminal
galactose (results not shown). These results demonstrate
that the population analyzed contained a mixture of GM1a
and GM2 core types with the GM2 core type mimic

predominating.
Comparison of LOS core structures of NCTC 11168
(HS:2) with the HS:2 serostrain indicated that both the
terminal galactose and GalNAc are missing in the latter [6].
As already mentioned, the missing galactose can be
attributed to the phase variability of wlaN. The lack of
terminal GalNAc in this strain has recently been shown by
Gilbert et al. to be due to the inactivation of the N-acetyl-
galactosaminyltransferase, CgtA, by one amino acid sub-
stitution [48]. Recently, the structure of an aerotolerant
HS:2 serostrain was also described [49] which was demon-
strated to be similar to that described for the HS:2 parent
with the exception of a missing sialic acid. Again, this
discrepancy in core structure may be due to an inactive or
truncated version of the sialyltransferase encoded by cstIII
[15].
The most noticeable feature of the NCTC 11168 LOS,
whichhasalsobeenobservedinotherC. jejuni LOSs, was
the presence of sialic acid [6]. Sialic acids are an important
surface component of many human and bacteria cell-
surface molecules, especially human ganglioside epitopes.
This type of molecular mimicry in C. jejuni has been given
substantial attention due to the fact that it may be involved
in the onset of Guillain–Barre
´
and Miller–Fisher syndromes
[5,50].
A single capsular gene locus of approximately 42 kb with
35 putative genes was recently identified in C. jejuni NCTC
11168 [8]. The overall genetic organization of this locus is

similar to group II/III capsule loci with genes involved in
capsule biosynthesis being flanked by the kps genes involved
in the transport of the sugar polymer (Fig. 11a) [9–11]. Class
II/III capsules have been shown to be attached to the
membrane by glycerophosphate anchors [9]. However, we
were unable to conclusively demonstrate the linkage of the
capsule polysaccharide to its membrane anchor and are
currently trying to determine the nature of the membrane
linkage.
It is currently unknown whether the polysaccharide that
we describe is polymerized by a processive or blockwise-
dependent method, although the ability to visualize a ladder
by silver staining (Fig. 11b) would suggest block transfer of
sugars differing by increments of one repeat unit [51]. Also,
mutation of Cj1439c, which we believe is involved in
GalfNAc biosynthesis, resulted in an acapsular phenotype
again suggesting a blockwise polymerization mechanism, in
contrast to the processive polymerization described for
group II/III capsular polysaccharides [9]. Deoxycholate-
PAGE (Fig. 11b) and HR-MAS NMR (Fig. 5c) analysis of
the Cj1428c mutant demonstrated that the capsule repeats
were still produced in the absence of the 6-O-Me-Hep
branch. These results suggest that NCTC 11168 possesses a
promiscuous polymerase which assembles the carbohydrate
repeats even in the absence of the branch sugar. This
observation is similar to that described for the E. coli K4
chondroitin polymerase which assembles the capsular
polysaccharide backbone of GlcAb(1–3)-GalNAc b(1–4)
even in the absence of the b-fructose branch at position C-3
of the glucuronosyl residue [52].

There have been increasing reports in the literature
describing the decoration of carbohydrates and capping of
sugar polymers with variable functional groups and/or
other sugars. For example, the acetamido groups of the
pseudaminic acid moieties on C. jejuni 81–176 flagellin are
substituted with variable acetamidino and hydroxypropri-
onyl groups [53]. Soil bacteria, collectively called rhizobia,
produce host-specific nodulation factors that decorate
backbone LOS with varying modifications (methylation,
carbamoylation, acetylation, and fucosylation) [54]. Phos-
phocholine functions are present on pilin proteins of
pathogenic Neisseria and on LPS sugars of commensal
Neisseria [55]. The NMe moiety on polylegionaminic acid is
a phase-variable epitope of Legionella pneumophila LPS
[56]. LPS of Rhizobium etli exhibits structural heterogeneity
with O-acetyl and O-methyl substitutions and with a
portion of the LPS containing 2,3,4-tri-O-methylfucosyl
caps [57]. Methyl capping of terminal perosamines of LPS
has also been described in Klebsiella, E. coli, Rhizobium and
Vibrio as well as on the S-layer glycoprotein of Geobacillus
stearothermophilus [51,57–59].
In this report, we describe variable methyl, glycerol, and
ethanol modifications on the capsule polysaccharides of
C. jejuni NCTC 11168. As mentioned above, methyl
addition is one of the more common carbohydrate modi-
fications described. In contrast, aminoglycerols have only
been described in Vibrio cholerae LPS and in the C. jejuni
HS:19 serostrain capsule polysaccharide [40,41]. Interest-
ingly, glycerophosphate modification has been described on
the pilin of Neisseria meningitidis [60]. Ethanol modification

on sugars has not been described except for its presence in
phosphoethanolamine, a common variable modification on
bacterial lipid As, including that of C. jejuni.
In summary, we have presented the complete structures
of the LOS and capsule polysaccharide of the genome
sequenced strain C. jejuni NCTC 11168. The outer core
LOS of NCTC 11168 has structural homology with the
human gangliosides, GM2 and GM1a. As demonstrated
previously in NCTC 11168 [16,17,48] and in 81–176 [12],
C. jejuni can exhibit variable ganglioside mimics due to
variation of the LOS core. The mimicry of human cell-
surface glycolipids and glycoproteins appears to be a general
trend of mucosal pathogens such as Haemophilus, Neisse-
ria, Helicobacter and Campylobacter (for reviews see
[7,61]). The peculiar property of producing structures
analogous to cell-surface molecules of the host and having
the ability to vary these structures plays an important role in
pathogenesis and survival within the environment.
In the capsular polysaccharide of NCTC 11168, we have
identified two new sugars that have not been described
before. As far as we are aware, this is the first report of
GalfNAc in the literature. Molecular modeling also dem-
onstrated that the absolute configuration of the heptopyr-
anose is
D
-glycero-
L
-gluco-heptopyranose. This is the first
demonstration of the
L

-gluco conformer in nature. Further-
more, we have observed variable glycerol, ethanol, and
methyl modifications on the sugar repeats suggesting that a
single capsular polysaccharide in C. jejuni can exhibit
different modifications. This observation was also made in
the C. jejuni capsular polysaccharides of the HS:23 and
HS:36 serostrains [6]. In addition, it was previously shown
Ó FEBS 2002 LOS and capsule structures of C. jejuni NCTC 11168 (Eur. J. Biochem. 269) 5133
that C. jejuni 81–176 can exhibit two different high
molecular weight polysaccharides, one of which is phase
variable [10].
Carbohydrate modifications have been shown to be
involved in important cell processes such as resistance to
antimicrobial peptides [62], possible alteration of surface
charge [57], susceptibility to bactericidal killing [55], poten-
tial termination signals for polymerization [51,58], persist-
ence in microbial niches [55], protection against host
immune responses by variation and molecular mimicry
[56,62], and cellular signaling with host cells through signal
transduction pathways [63]. We are currently investigating
the importance of C. jejuni carbohydrate modification and
variability in the disease process.
ACKNOWLEDGMENTS
The authors would like to thank Michel Gilbert and Susan Logan for
cell growth, John Nash and Simon Foote for primer design, Anna
Cunningham for sequencing, and Nicolas Cadotte for help with figures
and references. We would also like to thank Bernd Kneidinger for
helpful discussions. Funding for this work has been provided through
the National Research Council Genomics and Health Initiative.
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