Lipid A was isolated by preparative TLC, and its structure determined by MS and NMR to be similar to that of Bacteroides fragilis except for the number of fatty acids.. The structure of
Trang 1Structural study on lipid A and the O-specific polysaccharide
Masahito Hashimoto1,2, Fumiko Kirikae1, Taeko Dohi1, Seizi Adachi3, Shoichi Kusumoto3, Yasuo Suda3,4,*, Tsuyoshi Fujita5, Hideo Naoki5and Teruo Kirikae1
1
Research Institute, International Medical Center of Japan;2Department of Oral Microbiology, Asahi University School of Dentistry, Japan;3Graduate School of Science, Osaka University, Japan;4Department of Bacteriology, Hyogo College of Medicine, Japan;
5 Suntory Institute for Bioorganic Research, Japan
Bacteroides vulgatushas been shown to be involved in the
aggravation of colitis Previously, we separated two potent
virulence factors, capsular polysaccharide (CPS)and
lipo-polysaccharide (LPS), from a clinical isolate of B vulgatus
and characterized the structure of CPS In this study, we
elucidated the structures of O-antigen polysaccharide (OPS)
and lipid A in the LPS LPS was subjected to weak acid
hydrolysis to produce the lipid A fraction and
polysac-charide fraction Lipid A was isolated by preparative TLC,
and its structure determined by MS and NMR to be similar
to that of Bacteroides fragilis except for the number of fatty
acids The polysaccharide fraction was subjected to gel-filtration chromatography to give an OPS-rich fraction The structure of OPS was determined by chemical analysis and NMR spectroscopy to be a polysaccharide composed of the following repeating unit: [fi4)a-L-Rhap(1fi3)b-D -Manp(1fi]
Keywords: Bacteroides vulgatus; fast-atom-bombardment tandem mass spectrometry; lipopolysaccharide; MALDI-TOF-MS; NMR
Commensal flora are thought to be significantly involved in
the pathogenesis of inflammatory bowel diseases, Crohn’s
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-D-Glcp(1fi6)[a-D-GalpNAc(1fi2)b-D-Galp(1fi4)]b-D -GlcpNAc(1fi3)a-D-Galp(1fi4)b-D-Manp(1fi} [7] The structure is completely different from that of the CPS prepared from B fragilis [8] However, the structure of the LPS of B vulgatus has not been fully defined; only SDS/ PAGE profiles [9,10] and the immunochemical character-ization [11] of LPS have been reported In this paper, we describe the structural elucidation of possible virulent factors, O-antigen polysaccharide (OPS)and lipid A moiety, in LPS prepared from B vulgatus
M A T E R I A L S A N D M E T H O D S
Bacteria and LPS
B vulgatus IMCJ 1204 was isolated from the feces of a patient with Crohn’s diseases at the International Medical Center of Japan LPS was separated as described previously [7] Briefly, bacterial cells grown in GAM broth under anaerobic conditions were extracted with phenol/water The
Correspondence to T Kirikae, Research Institute, International
Medical Center of Japan, Shinjuku, Tokyo 162-8655, Japan.
Fax: + 81 3 3202 7364, Tel.: + 81 3 3202 7181 (ext 2838),
E-mail: tkirikae@ri.imcj.go.jp
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)
Trang 2extract 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 0.1M 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
(P)and retained (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 105C for
2.5 h, and the reaction mixture was partitioned with
chloroform/methanol/water (2 : 1 : 3, v/v/v) The
hydro-phobic products were separated by TLC (No 5715; Merck,
Darmstadt, Germany)using the solvent system chloroform/
methanol/water/triethylamine (300 : 120 : 20 : 1, v/v/v/v)
and visualized with anisaldehyde/sulfuric acid reagent
Lipid A was isolated by preparative TLC The hydrophilic
products were subjected to gel-filtration chromatography
on Sephacryl S-200 HR (Amersham Pharmacia Biotech
AB, Uppsala, Sweden) Fractions of 2.5 mL were collected
and monitored by measuring phosphorus and hexose
contents The eluates were combined, dialyzed and
lyo-philized The combined fraction was used as an OPS-rich
fraction and analyzed by the following procedures
Analytical procedures
Phosphorus content was determined by the method of
Bartlett [13] Hexose content was measured by the anthrone/
sulfuric acid method [14]
The sugar constituents of a sample were analysed by the
alditol acetate method [15] Methylation analysis was
carried out using NaOH as described by Ciucanu & Kerek
[16] Absolute configurations of sugars were determined
using R-(+)-butan-2-ol [8] Fatty acids were analyzed by
the method of Ikemoto et al [17] Alditol acetate, partially
methylated alditol acetate, acetylated butyl glycoside and
fatty acid methyl ester were analyzed by GC or GC-MS as
described previously [7]
SDS/PAGE was performed using 15% polyacrylamide
gels by the method of Laemmli [18] The gel was partially
oxidized with periodic acid and then visualized by the silver
staining method [19]
NMR spectroscopy and MS
1H- and13C-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 CDCl3/CD3OD (2 : 1,
v/v) The chemical shifts are expressed as d values using
chloroform (d¼ 7.2 p.p.m.)for1H spectra The spectra of
the OPS-rich fraction were recorded at 303 K at a
concentration of 6 mgÆmL)1in D2O The chemical shifts are expressed as d values using water (d¼ 4.7 p.p.m.)for
1H-NMR spectra and benzene (d¼ 128 p.p.m.)as an external standard for 13C-NMR spectra 1D DANTE, DQF-COSY, TOCSY, ROESY, HMQC and heteronuclear multiple bond connectivity (HMBC)spectra were obtained
as described previously [7]
MALDI-TOF-MS was performed with a Voyager-DE STR (PerSeptive Biosystems, Framingham, MA, USA) instrument Samples were dissolved in dichloromethane/ methanol (2 : 1, v/v), combined with sinapic acid as a matrix, and placed on a sample plate Spectra were obtained using the RDE2000 method
FAB-MS/MS was carried out with a JMS-HX/HX110A tandem mass spectrometer (JEOL)in the negative ion mode Nitrobenzyl alcohol was used as a matrix The sample was ionized with 6 KeV Xe atoms, and the ions were accelerated through 10 KeV Argon was used as the collision gas
R E S U L T S A N D D I S C U S S I O N
Analysis of LPS
As shown in Fig 1, a ladder-like pattern was observed in the SDS/PAGE profiles of LPS from B vulgatus IMCJ
1204, indicating that the LPS contains OPS The repeating unit of the OPS was shorter than that from E coli Breeling
et al [9] analyzed LPS from various strains of B vulgatus using SDS/PAGE and showed that four of eight strains
Fig 1 SDS/PAGE profile of LPS.
Trang 3contained OPS They demonstrated that the LPS with OPS
tended to be associated with immune enhancement of
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 vulgatusATCC 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
LPS was subjected to weak acid hydrolysis to give
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 Dconfiguration 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
the presence of a monophosphoryl lipid A (Fig 3A) The
molecular mass heterogeneity can be explained by the
degree of acylation and the chain length of the fatty acid
The ions at m/z 1688.4, 1674.4, 1660.4, and 1646.3
represented a monophosphoryl lipid A bearing five fatty acids, e.g m/z 1660.4 contains 12 (or 13)-Me-14 : 0, 15 : 0 (3-OH), 16 : 0 (3-OH)and 17 : 0 (3-OH)in the molar proportions 1 : 1 : 2 : 1 The ions at m/z 1432.2, 1420.2, 1406.2 and 1392.2 corresponded to a monophosphoryl lipid A bearing four fatty acids, e.g m/z 1420.2 consists of
12 (or 13)-Me-14 : 0, two 16 : 0 (3-OH) and 17 : 0 (3-OH)
A monophosphoryl lipid A bearing three fatty acids was detected at 1180.0, 1166.0, 1151.9 and 1137.9, e.g m/z 1160.6 includes 12 (or 13)-Me-14 : 0, 16 : 0 (3-OH) and
17 : 0 (3-OH) Diphosphoryl lipid A could not be detected The major components of the hydrophobic products were isolated by preparative TLC and analyzed by MALDI-TOF-MS (data not shown) The negative-ion mode spec-trum of the less hydrophobic component (Rf0.5)revealed monophosphoryl lipid A containing four fatty acids The positive-ion mode spectrum of the more hydrophobic component (Rf0.8)showed a lipid A structure with four fatty acids but no phosphate The latter component may be
a byproduct of the hydrolysis reaction or a natural contaminant These results indicate that the LPS from
B vulgatusmainly contains lipid A carrying four fatty acids and one phosphate Thus, the component with Rf0.5 was further analyzed as the main component of lipid A The structure of the lipid A component was established
by NMR and MS The 1H 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 onlyD-GlcN was observed in the compositional analysis, the sugars were determined as GlcN and designated GlcNIand GlcNIIin order of the1H chemical shift of the anomeric proton (H1) The downfield shift (d¼ 5.34 p.p.m.)and the coupling constant (6.7 Hz for JH,P)of H1-GlcNIshowed a phosphate substitution at the 1-position of GlcNI The coupling constant (3.0 Hz for3J1,2)confirmed the a configuration The coupling constant (8.2 Hz for 3J1,2)for H1-GlcNII showed a b configuration These results indicate that lipid A possesses a common diglucosamine backbone, and GlcNIis located at the reducing end No downfield shift of H4-GlcNII(d¼ 3.16 p.p.m.)revealed a free hydroxy group
at O4-GlcNIIand a monophosphate structure The down-field shift of H3-GlcNI(d¼ 4.94 p.p.m.)indicated an acyl substitution at O3-GlcNI, whereas the signal of H3-GlcNII
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
Trang 4(d¼ 3.28 p.p.m.)did not shift to a lower field, confirming
no acylation at O3-GlcNII The downfield shift of the proton signal for the b-position of fatty acid III (HbIII)at
d¼ 4.99 p.p.m revealed acylation at this position Further characterization was achieved by FAB-MS/MS The frag-mentation patterns of the parent ion at m/z 1420 indicated the fatty acid distribution as shown in Fig 3B, e.g cleavage
A showed a 3-hydroxy fatty acid (17 : 0 or 16 : 0) substitution of N2-GlcNI, while cleavage B–E showed two 3-hydroxy fatty acid (16 : 0 and 17 : 0, or 16 : 0· 2) substitutions of GlcNI and an acyoxyacyl substitution at N2-GlcNII Cleavage F indicated the chain length of the fatty acid on the acyoxyacyl group to be mainly 15, confirming the result of the compositional analysis In the minor triacylated lipid A, the fragmentation patterns of the parent ion at m/z 1166 suggested a lack of fatty acid at O3-GlcNI (data not shown) This result agrees with previous studies [21–23]
The lipid A from B fragilis NCTC 9343 has previously 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-sured at 297 K in CDCl 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 II
3.66 ( 3
J 5,6 2.3) Fatty acids
Fig 3 MALDI-TOF-MS spectrum of hydro-phobic products from the acetic acid hydroly-sate of LPS (A), and FAB-MS/MS spectrum of the parent ion at m/z 1420 (B).
Trang 5an 103-fold higher concentration (data not shown).
Lipid A is an active moiety of LPS in the induction of
cytokines, including tumor necrosis factor, and the
phos-phate residue at the 4¢-position is a critical site for the
activity [24] Therefore, the monophosphoryl lipid A in the
LPS must be responsible for this weak activity
Structure of OPS moiety in LPS
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 aD
configuration and Rha anLconfiguration 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
The1H- and13C-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
and b in order of1H chemical shift The1H signals were
assigned using DQF-COSY, TOCSY and ROESY spectra,
and the13C signals were assigned using HMQC and HMBC
spectra Some of the coupling constants that were not
determined from 1D spectra were estimated using
DQF-COSY spectra; 9–10 Hz for3J3,4of residue a and 1–2 Hz for
3J1,2 of residue b The data are summarized in Table 3
Residue a was assigned as a-L-rhamnopyranose (a-L-Rhap)
The manno-type configuration was clearly revealed by the
characteristic singlet-like signals of H1-a and coupling of
signals H1 to H4 Intraresidual correlation between H1-a
and C5-a in HMBC spectra (Fig 5A)confirmed the
pyranosyl configuration The chemical shift of 1.31 p.p.m
for H6-a and 17.8 p.p.m for C6-a was indicative of a
6-deoxy structure and confirmed this residue to be
rhamno-pyranose The 1JC,H value for the anomeric position of
residue a was determined to be 173 Hz from the
nondecou-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 1JC,Hvalue (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 the1H and13C spectra and assigned asL-Rhap (Table 3) No downfield shift was observed in13C-NMR spectra, indicating a nonsubsti-tuted Rha The signal of H4-a¢ was approximately one third the intensity of that of H1-a or H1-b, indicating its ratio
Fig 4. 1H (A) and13C (B) NMR spectra of the OPS-rich fraction.
Table 3 NMR data for OPS The spectra were measured at 303 K in D 2 O The chemical shifts are expressed as d values (p.p.m.) The coupling constants are in parentheses nd, Not determined; a¢ is estimated to be the nonsubstituted Rha located at the nonreducing terminus of the OPS chain.
Carbohydrate
residues
H1 ( 3 J 1,2 ) C1
H2 ( 3 J 2,3 ) C2
H3 ( 3 J 3,4 ) C3
H4 ( 3 J 4,5 ) C4
H5 C5
H6 ( 3 J 5,6 )
Trang 6The glycosidic linkages were established by the HMBC
experiment (Fig 5A) Long-range coupling from H1-a to
C3-b showed that residue a was linked to O3 of residue b
Coupling from H1-b to C4-a indicated that residue b was
linked to O4 of residue a Interresidual cross-peaks in
ROESY could not be assigned because of the overlapping of
signal, except for the cross-peak between H1-a and H2-b
(Fig 5B) The cross-peak may support the above linkage
These glycosidic linkages are consistent with the
methyla-tion analysis As no other O-substituted sugar was observed
in the methylation analysis, OPS had a linear structure
Thus, the nonsubstituted Rha was estimated to be located at
the nonreducing terminus of the OPS chain Taking these
observations into account, the structure of the OPS moiety
was deduced to be that shown in Fig 5C
In the Bacteroides group, the structure of the
polysac-charide part of LPS from B fragilis NCTC 9343 has been
studied [27] It was shown to lack the OPS moiety but to
contain the Gal-rich core saccharide On the other hand, we
demonstrated that B vulgatus IMCJ 1204 has a short OPS
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 GlcN2backbone with a phosphate and mainly four fatty acids for lipid A, and [fi4)a-L-Rhap (1fi3)a-D-Manp(1fi] for the OPS moiety
A C K N O W L E D G E M E N T S 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
Fig 5 HMBC (A) and ROESY (B) spectra, and proposed chemical structure of the OPS moiety (C).
Trang 7(11A-1)from the Ministry of Health and Welfare of Japan, and
Research for the Future program no 97L00502 from the Japan
Society for the Promotion of Science.
R E F E R E N C E S
1 Sartor, R.B (1997)Pathogenesis and immune mechanisms of
chronic inflammatory bowel diseases Am J Gastroenterol 92,
5S–11S.
2 Elson, C.O., Sartor, R.B., Tennyson, G.S & Riddell, R.H (1995)
Experimental models of inflammatory bowel disease
Gastro-enterology 109, 1344–1367.
3 Onderdonk, A.B., Cisneros, R.L & Bronson, R.T
(1983)En-hancement of experimental ulcerative colitis by immunization with
Bacteroides vulgatus Infect Immun 42, 783–788.
4 Rath, H.C., Herfarth, H.H., Ikeda, J.S., Grenther, W.B., Hamm,
T.E Jr, Balish, E., Taurog, J.D., Hammer, R.E., Wilson, K.H &
Sartor, R.B (1996)Normal luminal bacteria, especially
Bacter-oides species, mediate chronic colitis, gastritis, and arthritis in
HLA-B27/human b2 microglobulin transgenic rats J Clin Invest.
15, 945–953.
5 Rath, H.C., Wilson, K.H & Sartor, R.B (1999)Differential
induction of colitis and gastritis in HLA-B27 transgenic rats
selectively colonized with Bacteroides vulgatus or Escherichia coli.
Infect Immun 67, 2969–2974.
6 Rouyan, G.S., Meisel-Mikolajczyk, F & Rumin, W (1994)The
toxicity of antigens extracted from strains of Bacteroides vulgatus
from different origins to chicken embryos Acta Microbiol Pol 43,
97–101.
7 Hashimoto, M., Kirikae, F., Dohi, T., Kusumoto, S., Suda, Y &
Kirikae, T (2001)Structural elucidation of a capsular
poly-saccharide from a clinical isolate of Bacteroides vulgatus from a
patient with Crohn’s disease Eur J Biochem 268, 3139–3144.
8 Baumann, H., Tzianabos, A.O., Brisson, J.R., Kasper, D.L &
Jennings, H.J (1992)Structural elucidation of two capsular
polysaccharides from one strain of Bacteroides fragilis using
high-resolution NMR spectroscopy Biochemistry 31, 4081–4089.
9 Breeling, J.L., Onderdonk, A.B., Cisneros, R.L & Kasper, D.L.
(1988) Bacteroides vulgatus outer membrane antigens associated
with carrageenan-induced colitis in guinea pigs Infect Immun 56,
1754–1759.
10 Delahooke, D.M., Barclay, G.R & Poxton, I.R (1995)A
re-appraisal of the biological activity of bacteroides LPS J Med.
Microbiol 42, 102–112.
11 Rouyan, G.S., Kaca, W & Meisel-Mikolajczyk, F (1998)
Immunochemical characterization of Bacteroides vulgatus
cell-surface antigens Acta Microbiol Pol 47, 55–63.
12 Fischer, W (1990)Purification and fractionation of
lipopolysac-charide from gram-negative bacteria by hydrophobic interaction
chromatography Eur J Biochem 194, 655–661.
13 Bartlett, G.R (1959)Phosphorus assay in column
chromatogra-phy J Biol Chem 234, 466–468.
14 Ashwell, G (1957)Colorimetric analysis of sugars Methods
Enzymol 3, 73–105.
15 Torello, L.A., Yates, A.J & Thompson, D.K (1980)Critical study of the alditol acetate method for quantitating small quan-tities of hexoses and hexosamines in gangliosides J Chromatogr.
202, 195–209.
16 Ciucanu, I & Kerek, F (1984)A simple and rapid method for the permethylation of carbohydrates Carbohydr Res 131, 209–217.
17 Ikemoto, S., Katoh, K & Komagata, K (1978)Cellular fatty acid composition in methanol-utilizing bacteria J Gen Appl Micro-biol 24, 41–49.
18 Laemmli, U.K (1970)Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680–685.
19 Tsai, C.M & Frasch, C.E (1982)A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels Anal Biochem 119, 115–119.
20 Wollenweber, H.W., Rietschel, E.T., Hofstad, T., Weintraub, A.
& Lindberg, A.A (1980)Nature, type of linkage, quantity, and absolute configuration of (3-hydroxy)fatty acids in lipopoly-saccharides from Bacteroides fragilis NCTC 9343 and related strains J Bacteriol 144, 898–903.
21 Weintraub, A.Z., &hringer, U., Wollenweber, H.W., Seydel, U & Rietschel, E.T (1989)Structural characterization of the lipid A component of Bacteroides fragilis strain NCTC 9343 lipopoly-saccharide Eur J Biochem 183, 425–431.
22 Ogawa, T (1993)Chemical structure of lipid A from Porphyro-monas (Bacteroides) gingivalis lipopolysaccharide FEBSLett 332, 197–201.
23 Kumada, H., Haishima, Y., Umemoto, T & Tanamoto, K (1995) Structural study on the free lipid A isolated from lipopoly-saccharide of Porphyromonas gingivalis J Bacteriol 177, 2098– 2106.
24 Rietschel, E.T., Kirikae, T., Schade, F.U., Mamat, U., Schmidt, G., Loppnow, H., Ulmer, A.J., Za¨hringer, U., Seydel, U & Di Padova, F (1994)Bacterial endotoxin: molecular relationships of structure to activity and function FASEB J 8, 217–225.
25 Tvaroska, I & Taravel, F.R (1995)Carbon–proton coupling constants in the confomational analysis of sugar molecules Adv Carbohydr Chem Biochem 51, 15–61.
26 Bock, K., Pedersen, C & Pedersen, H (1988)Carbon-13 nuclear magnetic resonance data for oligosaccharide Adv Carbohydr Chem Biochem 42, 193–225.
27 Weintraub, A., Za¨hringer, U & Lindberg, A.A (1985)Structural studies of the polysaccharide part of the cell wall lipopoly-saccharide from Bacteroides fragilis NCTC 9343 Eur J Biochem.
151, 657–661.
28 Ogura, Y., Bonen, D.K., Inohara, N., Nicolae, D.L., Chen, F.F., Ramos, R., Britton, H., Moran, T., Karaliuskas, R., Duerr, R.H., Achkar, J.P., Brant, S.R., Bayless, T.M., Kirschner, B.S., Hanauer, S.B., Nunez, G & Cho, J.H (2001)A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease Nature 411, 603–606.
29 Inohara, N., Ogura, Y., Chen, F.F., Muto, A & Nunez, G (2001) Human Nod1 confers responsiveness to bacterial lipopoly-saccharides J Biol Chem 276, 2551–2554.