Structural study on lipid A and the O-specific polysaccharide
of the lipopolysaccharide from a clinical isolate of
Bacteroides
vulgatus
from a patient with Crohn’s disease
Masahito Hashimoto
1,2
, Fumiko Kirikae
1
, Taeko Dohi
1
, Seizi Adachi
3
, Shoichi Kusumoto
3
, Yasuo Suda
3,4,
*,
Tsuyoshi Fujita
5
, Hideo Naoki
5
and Teruo Kirikae
1
1
Research Institute, International Medical Center of Japan;
2
Department of Oral Microbiology, Asahi University School of Dentistry,
Japan;
3
Graduate School of Science, Osaka University, Japan;

disease and ulcerative colitis (reviewed in [1]). As chronic
intestinal inflammation in several rodent models is prevent-
ed in a germ-free environment [2], efforts have been made to
identify the organisms responsible for the induction or
perpetuation of enterocolitis. Bacteroides are Gram-nega-
tive rods and the predominant anaerobes in endogenous
intestinal flora. Among these species, Bacteroides vulgatus
has been shown to be involved in the aggravation of colitis.
For example, immunization of guinea pigs with B. vulgatus
before administration of carrageenan and feeding with
viable B. vulgatus resulted in more rapid ulceration, whereas
a phenotypically similar organism, Bacteroides fragilis,had
no such effect [3]. HLA-B27 transgenic rats colonized with a
mixture of six different obligate and facultative anaerobic
bacteria including B. vulgatus developed a much more
active colitis and gastritis than littermates colonized with the
same mixture without B. vulgatus [4]. B27 transgenic rats
monoassociated with B. vulgatus developed colitis compa-
rable to that in rats colonized with the above bacterial
mixture, but Escherichia coli-monoassociated rats showed
no evidence of colitis [5].
Surface components of many enteric bacteria are impor-
tant for their virulence. Capsular polysaccharide (CPS) and
lipopolysaccharide (LPS) are two well-described virulence
factors. The CPS and LPS of B. vulgatus have been
suggested to play key roles in its virulence [6]. Previously,
we separated CPS from a clinical isolate of B. vulgatus and
characterized its structure as a novel polysaccharide
composed of the following repeating unit: {fi3)
b-

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Abbreviations: CPS, capsular polysaccharide; FAB-MS/MS, fast atom
bombardment-tandem mass spectrometry; HMBC, heteronuclear
multiple bond connectivity; LPS, lipopolysaccharide; OPS, O-antigen
polysaccharide.
*Present address: Department of Nanostructure and Advanced
Materials, Graduate School of Science and Engineering,
Kagoshima University, Japan.
(Received 25 March 2002, revised 5 June 2002,
accepted 20 June 2002)
Eur. J. Biochem. 269, 3715–3721 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03062.x
extract was subjected to enzymatic digestion with DNase
and RNase followed by proteinase K, and then phenol/
water extraction again to yield the crude LPS preparation.
LPS was separated by hydrophobic interaction chroma-
tography [12]. The preparation was subjected to stepwise
separation on octyl-Sepharose using 0.1
M
acetate buffer
(pH 4.5) containing 15% propan-1-ol and the same acetate
buffer containing 60% propan-1-ol to give the pass-through
(OS-P) and retained (OS-R) fractions, respectively. The OS-
R fraction contained LPS from the SDS/PAGE analysis as
described in Results and discussion. LPS from B. fragilis
NCTC 10581 was prepared by a procedure similar to that
described above. LPS from E. coli O111:B4 was purchased
from Sigma (St Louis, MO, USA).
Chemical degradation and separation
LPS was hydrolyzed with 0.6% acetic acid at 105 °Cfor

staining method [19].
NMR spectroscopy and MS
1
H- and
13
C-NMR spectra were recorded on a JMN-LA500
spectrometer (JEOL, Tokyo, Japan) equipped with an
indirect detection gradient probe, IDG500-5VJ (Nanorac
Cryogenics, Martinez, CA, USA) at 500 and 126 MHz,
respectively. Spectra of lipid A were obtained at 327 K at a
concentration of 0.6 mgÆmL
)1
in CDCl
3
/CD
3
OD (2 : 1,
v/v). The chemical shifts are expressed as d values using
chloroform (d ¼ 7.2 p.p.m.) for
1
H spectra. The spectra of
the OPS-rich fraction were recorded at 303 K at a
concentration of 6 mgÆmL
)1
in D
2
O. The chemical shifts
are expressed as d values using water (d ¼ 4.7 p.p.m.) for
1
H-NMR spectra and benzene (d ¼ 128 p.p.m.) as an

colitis. On the other hand, a closely related LPS from
B. fragilis did not possess OPS (Fig. 1) as described
previously [10]. B. fragilis has been reported to have less
ability for immune enhancement of ulcerative colitis than
B. vulgatus [3]. The OPS portion of B. vulgatus LPS is
therefore probably important in colitis.
The chemical composition of the LPS is summarized in
Table 1. It contains sugars, amino sugar, fatty acids and
phosphate. The fatty acid components were similar to those
of B. fragilis [20,21], suggesting structural similarity in the
lipid A moiety. The sugar components were different from
those from B. fragilis, which lacks OPS [21], and those from
B. vulgatus ATCC 8482 [11]. We previously reported that
the sugar components of CPS from B. vulgatus IMCJ 1204
were also different from those of B. vulgatus ATCC 8482
[7]. These results indicate that the structural variation in
surface glycoconjugates among the strains of B. vulgatus is
great, and structural differences may affect virulence [6].
Structure of lipid A moiety in LPS
LPSwassubjectedtoweakacidhydrolysistogive
hydrophilic and hydrophobic products. The chemical
compositions of the hydrophobic products are summarized
in Table 1. GlcN, fatty acids and phosphate were present in
the molar proportions 2 : 3.3 : 1.4. Absolute configuration
analysis confirmed that GlcN has a
D
configuration. On
TLC analysis, two major and several minor spots were
detected among the hydrophobic products (Fig. 2). The
negative-ion mode MALDI-TOF mass spectrum revealed

and one phosphate. Thus, the component with R
f
0.5 was
further analyzed as the main component of lipid A.
The structure of the lipid A component was established
by NMR and MS. The
1
H NMR signals of the isolated
lipid A were assigned using DQF-COSY and TOCSY, and
the data are summarized in Table 2. Two sets of sugar
signals were observed. The coupling constants of the signals
revealed a glucopyranosyl configuration. As only
D
-GlcN
was observed in the compositional analysis, the sugars were
determined as GlcN and designated GlcN
I
and GlcN
II
in
order of the
1
H chemical shift of the anomeric proton (H1).
The downfield shift (d ¼ 5.34 p.p.m.) and the coupling
constant (6.7 Hz for J
H,P
) of H1-GlcN
I
showed a phosphate
substitution at the 1-position of GlcN

II
Fig. 2. TLC profile of the hydrophobic products from the acetic acid
hydrolysate of LPS.
Table 1. Chemical composition of the LPS from B. vulgatus
IMCJ1204. nd, Not detected.
Component
Amount (lmolÆmg
)1
)
LPS
Hydrophobic
products
OPS-rich
fraction
Sugars 2.22 0.81 3.28
Rha 0.71 ND 1.32
Fuc 0.19 ND ND
Man 0.43 ND 1.38
Gal 0.41 ND 0.37
Glc 0.37 ND 0.21
GlcN 0.11 0.81 ND
Fatty acids 0.70 1.33 ND
12-Me-13 : 0 0.03 0.04
14 : 0 0.01 0.02
13-Me-14 : 0 0.07 0.11
12-Me-14 : 0 0.06 0.13
15 : 0 0.01 0.02
15 : 0 (3-OH) 0.10 0.15
16 : 0 (3-OH) 0.29 0.57
15-Me-16 : 0 (3-OH) 0.05 0.15

been isolated and characterized as having a penta-acyl and
monophosphoryl structure [21]. The lipid A from a closely
related bacterium, Porphyromonas gingivalis, has been
reported to mainly contain one phosphate and three
(P. gingivalis 381) [22] or four (P. gingivalis SU63) [23] fatty
acids. These observations indicate that the fundamental
structure of lipid A from Bacteroidaceae is similar but the
number of acyl substituents is variable. The LPS showed
significantly less activity than E. coli LPS in inducing
production of tumor necrosis factor in human peripheral
whole blood cells, with a dose–response curve that shifted to
Table 2.
1
H-NMR data for isolated lipid A. The spectra were mea-
suredat297KinCDCl
3
/CD
3
OD (2 : 1, v/v). The chemical shifts are
expressed as d values (p.p.m.). The coupling constants are shown in
parentheses.
Proton
Chemical shift
(coupling constant)
GlcN
I
H1 5.34 (
3
J
1,2

GlcN
II
H1¢ 4.36 (
3
J
1,2
8.2)
H2¢ 3.37 (
3
J
2,3
9.9)
H3¢ 3.28 (
3
J
3,4
8.9)
H4¢ 3.16 (
3
J
4,5
9.4)
H5¢ 3.07
H6¢ 3.52 (
3
J
5,6
6.0,
2
J

To analyze the structure of the OPS moiety, the hydrophilic
products from the acetic acid hydrolysate of LPS were
separated by gel-filtration chromatography to give the high-
molecular-mass OPS-rich fraction (30%). Mainly two
sugars, Rha and Man, were detected in the OPS-rich
fraction on analysis of the sugar constituents (Table 1). The
approximate molar ratio of Rha to Man was 1 : 1. Abso-
lute configuration analysis demonstrated that Man has a
D
configuration and Rha an
L
configuration. On methylation
analysis, 2,3,4-tri-O-methyl-6-deoxyhexose, 2,3-di-O-
methyl-6-deoxyhexose and 2,4,6-tri-O-methyl-hexose were
mainly observed.
The
1
H- and
13
C-NMR spectra of the OPS-rich fraction
are shown in Fig. 4. Two anomeric signals were mainly
observed, and the corresponding sugars were designated as a
andbinorderof
1
H chemical shift. The
1
H signals were
assigned using DQF-COSY, TOCSY and ROESY spectra,
and the
13

pling DEPT spectrum indicating the a configuration [25].
The downfield shift of C4-a showed that a glycoside is
attached at O4 of residue a [26]. Residue b was assigned as
b-
D
-mannopyranose (b-
D
-Manp). The mannopyranosyl
configuration was clearly revealed by the characteristic
singlet-like signals of H1-b and H2-b, coupling of signals H2
to H4, and intraresidual correlation between H1-b and C5-b
in HMBC spectra (Fig. 5A). Intraresidual correlations
between H1 and H5 in the ROESY spectrum revealed the
b configuration (Fig. 5B). The
1
J
C,H
value (164 Hz) con-
firmed the anomeric configuration. The downfield shift of
C3-b indicated a 3-O-substituted structure. Some minor
signals (designated as a¢) were observed in the
1
Hand
13
C
spectra and assigned as
L
-Rhap (Table 3). No downfield shift
was observed in
13

(
3
J
3,4
)
C3
H4
(
3
J
4,5
)
C4
H5
C5
H6
(
3
J
5,6
)
C6 (
3
J
5,6
,
2
J
6,6
)

consisting of Rha and Man. Although we have not studied
the structure of the core saccharide, it would be made up of
Gal and Glc. The results of this study showed that the
polysaccharide region of LPS from Bacteroides has wide
structural variation. As the structure of the lipid A moiety is
similar to that of B. fragilis but the polysaccharide part is
completely different, the difference in structure of the
polysaccharide region may reflect the virulence of LPS in
inflammatory bowel diseases. Recently, Ogura et al.[28]
demonstrated that a frameshift mutation in NOD2 was
associated with susceptibility to Crohn’s disease. NOD2
seems to function as a receptor for LPS with the leucine-rich
repeat motif [29]. The structure of LPS responsible for the
recognition of NOD2 is so far unknown, but it may
recognize the polysaccharide region of LPS.
In summary, we found the structure of lipid A and the
OPS moiety in LPS from a clinical isolate of B. vulgatus,
IMCJ 1204, to be a GlcN
2
backbone with a phosphate and
mainly four fatty acids for lipid A, and [fi4)a-
L
-Rhap
(1fi3)a-
D
-Manp(1fi]fortheOPSmoiety.
ACKNOWLEDGEMENTS
This study was supported in part by a grant from the Ministry of
Education, Science and Culture of Japan (13670289 to T. K.), grants
and contracts from International Health Cooperation Research

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Ó FEBS 2002 Lipopolysaccharide of B. vulgatus (Eur. J. Biochem. 269) 3721