Wayne Conlan Institute for Biological Sciences, National Research Council, Ottawa, Canada The structure of the lipid A and core region of the lipo-polysaccharide LPS from Francisella tul
Trang 1Structural analysis of Francisella tularensis lipopolysaccharide
Evgeny Vinogradov, Malcolm B Perry and J Wayne Conlan
Institute for Biological Sciences, National Research Council, Ottawa, Canada
The structure of the lipid A and core region of the
lipo-polysaccharide (LPS) from Francisella tularensis (ATCC
29684) was analysed using NMR, mass spectrometry and
chemical methods The LPS contains a
b-GlcN-(1–6)-GlcN lipid A backbone, but has a number of unusual
structural features; it apparently has no substituent at O-1
of the reducing end GlcN residue in the lipid part in the
major part of the population, no substituents at O-3 and
O-4of b-GlcN, and no substituent at O-4of the Kdo
residue The largest oligosaccharide, isolated after strong
alkaline deacylation of NaBH4 reduced LPS had the
fol-lowing structure:
where D-GalNA-(1–3)-b-QuiNAc represents a modified
fragment of the O-chain repeating unit Two shorter
oligo-saccharides lacking the O-chain fragment were also identified
A minor amount of the disaccharide
b-GlcN-(1–6)-a-GlcN-1-P was isolated from the same reaction mixture, indicating the presence of free lipid A, unsubstituted by Kdo and with phosphate at the reducing end.The lipid A, isolated from the products of mild acid hydrolysis, had the structure 2-N-(3-O-acyl4-acyl2)-b-GlcN-(1–6)-2-N-acyl1)3-O-acyl3-GlcN where acyl1, acyl2and acyl3 are 3-hydroxyhexadecanoic or 3-hy-droxyoctadecanoic acids, acyl4is tetradecanoic or (minor) hexadecanoic acids No phosphate substituents were found
in this compound OH-1 of the reducing end glucosamine, and OH-3 and OH-4of the nonreducing end glucosamine residues were not substituted LPS of F tularensis exhibits unusual biological properties, including low endoxicity,
which may be related to its unusual lipid A structure Keywords: Francisella tularensis; lipopolysaccharide; core; lipid A
Francisella tularensisis a Gram-negative bacterium which
causes tularemia, a severe and often fatal disease of humans
and other mammals [1] The bacterium is an intracellular
pathogen and therefore cell-mediated, rather than humoral,
immunity is thought to be required to combat it [1–3]
However, it has also been shown that antibodies directed
against the LPS of F tularensis can ameliorate the course of
tularemia [4,5] Additionally, F tularensis LPS possesses
unusual biological properties that also presumably influence
the disease process For instance, F tularensis LPS lacks
endotoxicity and is a poor inducer of proinflammatory
cytokines [6] On the other hand, it has been shown recently
that subimmunogenic doses of LPS derived from F
tula-rensislive vaccine strain (LVS) can elicit an unusual, and
apparently specific, anti-Francisella resistance that relies on the actions of interferon-gamma and B-cells, but not antibodies, for its expression [7] Knowledge of the fine structure of F tularensis LPS will be needed to explain these biological activities In previous studies [8] we have described the structure of the O-antigen produced by
F tularensisATCC 29684, which proved to be identical to the structure of strain 15 [9] The present study focuses on the structure of the lipid A and core region of the
F tularensisLPS ATCC 29684
E X P E R I M E N T A L P R O C E D U R E S Lipopolysaccharide isolation
F tularensisLVS (ATCC 29684) was grown to D600 1.1
in a 40-L batch in Trypticase soy broth containing 0.1% (w/v) cysteine/HCl and 0.025% (w/v) ferric pyrophosphate Cells were killed by the addition of phenol (final concentra-tion 2%, v/v), and harvested by continuous centrifugaconcentra-tion at
62 000 g (yield 1 g wet wt.ÆL)1) The saline-washed cells (250 g wet wt.) were extracted by stirring with 400 mL 50% (v/v) aqueous phenol at 65C for 15 min [10] The cooled extract was diluted with water (2 vol.) and the cleared extract was dialyzed against tap water until free from phenol The dialyzed retentate was dissolved in 90 mL NaOAc (0.02M,
pH 7.0) and was treated sequentially with RNase, DNase and proteinase K (37C, 2 h each) The ensuing mixture was
Correspondence to Evgeny Vinogradov, Institute for Biological
Sciences, National Research Council, 100 Sussex Dr,
K1A 0R6 Ottawa ON, Canada.
Fax: + 1 613 952 90 92, Tel.: + 1 613 990 0832,
E-mail: evguenii.vinogradov@nrc.ca
Abbreviations: GalA, galacturonic acid; GalNA, galactosaminuronic
acid; D-GalNA, 2,4-dideoxy-2-amino-b- L -threo-hex-4-eno-pyranosyl;
HPAEC, high-performance anion-exchange chromatography;
LPS, lipopolysaccharide; Kdo, 3-deoxy- D -manno-octulosonic acid;
QuiN, 2-amino-2,6-dideoxyglucose.
(Received 3 July 2002, revised 2 October 2002,
accepted 22 October 2002)
Trang 2cleared by low speed centrifugation (3000 g), and subjected
to sequential ultracentrifugation at 27 000 g (precipitate
designated K27), and 60 000 g (precipitate K60), both for
10 h at 4C The precipitated gels were dissolved in distilled
water and lyophilized The K27 fraction (1.72 g) contained
LPS contaminated with 40% (w/w) of an
amylopectin-likeD-glucan The K60 fraction (840 mg) was essentially
pure S-type LPS and was used in subsequent studies
NMR spectroscopy and general methods
1H- and13C-NMR spectra of lipid A were recorded using a
Varian Inova 500 spectrometer in CDCl3–CD3OD (3 : 1,
v/v) or in CDCl3-CD3OH (3 : 1, v/v) Solutions were at
25C and referenced to the residual chloroform signal (1H
7.26 p.p.m.) and MeOH (13C 49.15 p.p.m.); spectra of all
other compounds were recorded at 25C in D2O and
referenced to acetone (dH2.225 p.p.m., dC31.45 p.p.m.)
Varian standard pulse sequences COSY, TOCSY (mixing
time 100 ms), ROESY (mixing time 200 ms), HSQC and
gHMBC (optimized for 5 Hz coupling constant) were used
Electrospray mass spectra were obtained using a Micromass
Quattro spectrometer in 50% (v/v) MeCN with 0.2% (v/v)
HCOOH at a flow rate of 15 lLÆmin)1with direct injection
in negative mode MALDI-TOF mass spectra were
recor-ded in positive mode with a Bruker-Reflex III spectrometer
(Bruker-Franzen Analytik, Bremen, Germany) in both
linear and reflection TOF configurations at an acceleration
voltage of 20 kV and delayed ion extraction The samples
were dissolved in aqueous triethylamine (0.07M) at a
concentration of 2 lgÆlL)1 One lL of the sample was then
mixed with 1 lL of 0.5M matrix solution of
recrystal-lized 2,5-dihydroxybenzoic acid (Aldrich, Deisenhofen,
Germany) in methanol containing 0.1% (v/v) trifluoroacetic
acid Aliquots of 0.5 lL were deposited on a metallic sample
holder and analyzed immediately after drying in a stream of
air The instrument was calibrated externally with similar
compounds of known structure The mass spectra shown
are the sum of at least 50 laser shots GC was performed on
an HP1 column (30 m· 0.25 mm) using an Agilent 6850
chromatograph fitted with a flame ionization detector, or on
a Varian Saturn 2000 ion-trap GC/MS instrument
Lipid A isolation
LPS (200 mg) was hydrolysed with 5% (v/v) AcOH (100C,
4h) and the precipitated product was collected by
centri-fugation at 3000 g and suspended in 2% (v/v) MeOH in
CHCl3 It was applied to a silica gel column (2· 8 cm), then
washed sequentially with 2%, 5%, 10% and 20% (v/v)
MeOH in CHCl3 Clean lipid A (20 mg) was recovered from
the 10% (v/v) MeOH eluate; 5% and 20% (v/v) MeOH
contained minor amounts of lipid-like components
Fatty acid analysis
Lipid (2 mg) was dissolved in CHCl3–MeOH (3 : 1, v/v,
1 mL total volume) and 1MMeONa in MeOH (0.2 mL)
was added The mixture was kept for 24h at 25C, acidified
with trifluoroacetic acid, evaporated and extracted with
hexane Samples of lipid containing hexane extract and
hexane-insoluble material were treated with 1MHCl in MeOH
(1 mL, 100C, 4h), dried, and analysed by GC and GC/MS
Hydrofluoric acid cleavage Lipid A sample (10 mg) was stirred with 48% (v/v) hydrofluoric acid in a volume of 1.0 mL in a poly(ethylene) vial at 20C for 24h, then dried in vacuum dessicator over NaOH The product was analysed by NMR and MS without purification
LPS reduction LPS (150 mg) was dissolved in water (100 mL), then
300 mg NaBH4and a drop of octanol to prevent foaming were added The mixture was kept at room temperature for 24h, then dialysed against water After lyophylization
120 mg of reduced LPS was recovered
Alkaline deacylation Two LPS samples (80 mg each) and NaBH4-reduced LPS (80 mg) were dissolved in 4MKOH (4mL each) To one of the LPS samples 100 mg NaBH4were added, kept overnight
at 100C and neutralized with 2MHCl Precipitated mater-ial was removed by centrifugation at 3000 g and the solutions were passed through SepPak C18 cartridges (washed with MeOH and water before use) and applied to a Sephadex G50 column The fractions were analysed by NMR spectroscopy and ESI/MS, and those containing core oligosaccharides were separated by HPAEC in a gradient of 0.1MNaOH (A)
to 1M AcONa in 0.1M NaOH (B), using 5–50% of B Products were desalted on a Sephadex G15 column Hydrazine treatment
LPS (100 mg) or lipid precipitate from AcOH hydrolysis (30 mg) were dissolved in anhydrous hydrazine (3 mL) and kept at 40C for 1 h Samples were transferred to plastic Petri dishes to provide a large surface for evaporation and hydrazine was removed in a vacuum dessicator over sulfuric acid Products were dissolved in water, precipitates were removed by centrifugation at 5000 g, and the solutions were dried and analysed by NMR spectroscopy Sample obtained from lipid precipitates contained mostly a b-glucan 5, which was purified by ion exchange chromatography on a HiTrap
Q column (Pharmacia) in water (A) to 1MNaCl (B), with a gradient from 0–100% NaCl Sample prepared from LPS was fractionated on Sephadex G50 column to give a-(1–6)-glucan, amylopectin, b-glucan 5, and fragments of the O-chain
R E S U L T S Lipid A was liberated from F tularensis LVS (ATCC 29684) LPS by acetic acid hydrolysis This LPS was more stable to acetic acid hydrolysis than LPSs form most other bacteria, and the lipid A moiety could only be cleaved from the rest of the molecule using hot 5% (v/v) acetic acid rather than the usual 1–2% concentration Lipid A was then purified by conventional silica gel chromatography Com-parison of the 1H-NMR spectra of the unfractionated lipid A and chromatographically fractionated samples indicated that the fraction eluted with 10% (v/v) MeOH
in CHCl3contained the major component It was used in further studies as lipid A
Trang 3Fatty acid analysis of the purified lipid A showed the
presence of C14:0, C16:0, C16:0(3-OH), and C18:0(3-OH)
straight chain acids in the ratio of 1 : 0.2 : 1.6 : 4 In order
to distinguish between ester- and amide-linked acids, the
lipid A was treated with MeONa for O-deacylation,
released acids were extracted into hexane, and both the
hexane extract and the residual material were analyzed by
GC after methanolysis C14:0 and C16:0 acids were found
to be completely released following O-deacylation, whereas
C16:0(3-OH) and C18:0(3-OH) were distributed in both
fractions and were thus present in both ester- and
amide-linked forms
NMR analysis of the lipid using 2D techniques (Table 1,
Fig 1) led to the identification of b-GlcN-(1–6)-GlcN
backbone disaccharide, carrying four acyl residues GlcN
residue A had unsubstituted hydroxyl group at C-1, and was
mostly present in an a-pyranose form Acyl1, acyl2and acyl3
residues had hydroxy or acyloxy groups at C-3 (13C signals
of C-3 at 69.0–72.4p.p.m.), while acyl4had no substituents
The signals of acyl chains could only be identified up to C-4,
H-4, because of the overlap of the remaining signals
Distribution of the acyl residues was deduced from NOE correlations between amide protons and the H-2 of acyl residues, and from HMBC correlations between C-1 of acyl groups and protons at the acylation site (Fig 1) NOE between protons A2 and acyl1-2, and between B2 and acyl2-2 indicated that GlcN A is N-acylated with acyl1, and GlcN B is N-acylated with acyl2 All acyl C-1 signals were identified from H-2:C-1 HMBC correlations C-1 of acyl2 gave HMBC correlation to H-2 of GlcN B; C-1 of acyl4 showed correlation to H-3 of acyl2 C-1 Signals of acyl1and acyl3 overlap, but because acyl1 can be identified as the acylating amino group of GlcN A on the basis of NOE correlation, HMBC correlation at 5.00 (H) to 173.9 (C) p.p.m can only be explained as resulting from the acylation
of A3 with acyl3 O-Acylation of GlcN A at O-3 and of acyl2
at O-3 agreed with the low-field position of the correspond-ing proton signals (Table 1) These results identify the disaccharide backbone structure and acylation pattern except for the length of the carbon chain of the acyl residues Further information on the lipid A structure was obtained from MALDI mass spectra The mass spectrum
Table 1 NMR data for lipid A.
13
13
13
Fig 1 Fragments of HSQCand HMBC spectra of F tularensis (ATCC 29684) lipid A Signals of lipid with the a-anomeric form of GlcN A are labeled.
Trang 4of the lipid A (Fig 2) contained four major clusters of
signals with the first peaks at m/z 1392.6, 1408.6, 1420.7
and 1436.7 These signals correspond to sodium and
potassium adducts of two structural variants with masses
of 1370.0 and 1398.1 Da Since the nonhydroxylated acyl
is known from GC analysis to be mostly C14:0, the
remaining are two C18:0(3-OH) and one C16:0(3-OH)
(1370.0 Da), or three C18:0(3-OH) residues (1398.1 Da),
respectively This was confirmed by the results of
MALDI analysis of a lipid A sample treated with 48%
(v/v) hydrofluoric acid This treatment cleaved the
glycosidic linkage between monosaccharide residues
with-out affecting the acylation The mass spectrum of the
resulting mixture of units A and B contained peaks at
m/z 694.82 (unit B with C14:0 and C18:0(3-OH) +
Na+), 738.9 Da (unit A with C16:0(3-OH) and C18:0
(3-OH) + Na+), and 766.87 Da (unit A with two
C18:0(3-OH) + Na+) Minor peaks of unit A with two
C16:0(3-OH) acyl residues (m/z 710.77), and of unit B
with C16:0 and C18:0(3-OH) acyl residues (m/z
722.83 Da) were also observed These results show that
acyl1 and acyl3 can be C16:0(3-OH) or C18:0(3-OH),
acyl2 is mostly C18:0(3-OH); and acyl4 is C14:0 with
minor amount of C16:0
Combined NMR and MS evidence led to the proposed
structure (Fig 3), where acyl1, acyl2 and acyl3 are
3-hydroxyhexadecanoic or 3-hydroxyoctadecanoic acids,
acyl4is tetradecanoic or (minor) hexadecanoic acids
No core-related products were isolated from the AcOH
hydrolysate of the LPS, probably because of a low content
of rough variants of the LPS, and the presence of
contaminants Deacylation of the LPS with 4MKOH led
to four major products 1a, 2a, 3a and 4, isolated by
HPAEC Compounds 1a, 2a and 3a contained an
unidentified aglycon, derived from GlcN B by alkaline
degradation To avoid this degradation and confirm the
absence of the substituent at O-1 of GlcN A, LPS was
reduced with NaBH4prior to alkaline treatment or treated with KOH in the presence of NaBH4 Both procedures led
to the same products 1b, 2b and 3b, in which Kdo was linked to )6)-b-GlcN-(1–6)-GlcN-ol N-Acetyl group on the QuiN residue J in compounds 3a and 3b was exceptionally stable and was not removed under deacyla-tion condideacyla-tions
NMR spectra of the products 1–4 were assigned (except for aglycon signals in 1a, 2a and 3a) using 2D techniques (Table 2, Fig 4) Monosaccharides were identified on the basis of vicinal proton coupling constants and 13C NMR chemical shifts Anomeric configurations were deduced from the J1,2 coupling constants and chemical shifts of H-1, C-1 and C-5 signals The b-configuration of mannose residue F was confirmed by the observation of strong intraresidual NOE between H-1 and H-3, and between H-1 and H-5 Residue K is a product of an alkaline b-elimination of the O-4substituent from a a-galactos-aminuronamide residue, present in the LPS O-chain [8,9] Connections between monosaccharides were identified on the basis of NOE and HMBC correlations The following NOEs were observed in the product 3b: B1A6, E1C5, E1C7, E1I1, F1E4, F1E6, I1E2, G1F2, G1F3, H1F2, H1G5, J1F4, J1F6 and K1J3 Respective correlations, where applicable, were also observed in the smaller
Fig 2 MALDI mass spectrum of the purified lipid A.
Fig 3 Structure of the lipid A and its fragment, obtained after partial hydrolysis with 48% (v/v) HF Acyl1and acyl3are C16:0(3-OH) or C18:0(3-OH), acyl2 is mostly C18:0(3-OH), and acyl4is C14:0 with minor amount of C16:0.
Trang 5oligosaccharides Structures 1b, 2b and 3b were confirmed
by ESI MS In the structures 1a, 2a and 3a, aglycon R
had mass of 101 Da, the same that was observed for
the products of similar degradation of other LPSs
[E Vinogradov, M B Perry & J W Conlan, unpublished
data] The structure of this fragment was not understood
Together these data led to the structures presented in
Fig 5 Structures 1 and 2 represent LPS lacking O-chain,
and structure 3 contains units K and J, corresponding to a
part of the O-chain repeating unit Thus compound 3 is
derived from smooth LPS form
In an attempt to determine the base labile constituents of the LPS, anhydrous hydrazine treatment (40, 1 h) of the LPS was performed This experiment produced no explain-able results regarding LPS structure O-Chain was depo-lymerized due to the presence of 4-substituted residues of galactosaminuronamide Separation of the products on
Fig 4 1 H NMR spectra of compounds 1a, 2a, 3a and 4 Spectrum of
compound 1a was recorded at 30 C to avoid overlap of water signal
with F1 signal.
Fig 5 Structures of the oligosaccharides, obtained by KOH deacylation
of F tularensis LPS.
Table 2 NMR data for the oligosaccharides 1-3 N-acetyl group signals in 3: H-2 1.89 ppm, C-2 23.2 ppm.
13
13
13
13
13
13
Trang 6Sephadex G50 resulted in the isolation of depolymerized
O-chain and three glucans Lipid A containing compounds
were uniformly spread and mixed with other dominating
components:
[-6)-a-D-Glc-(1-]
[-4)-a-D-Glc-(1-] with - 4,6)-a-D-Glc-(1- branching
(amylopectin)
[-6)-b-D-Glc-(1-]n-3-)-Gro-(1-P-1)-Gro-5 (b-glucan)
The linear a-1-6-glucan had highest molecular mass (eluted
first from Sephadex G-50 gel permeation column) and was
found in minor quantities Amylopectin was present in the
largest amount and constituted 50% of the LPS mass
prior to fractional ultracentrifugation, where it can be
mostly removed at low speed (27 000 g) The b-glucan 5 had
short glucose chains Its mass spectrum corresponded to
8–15 glucose units with a maximum at 12 units The same
b-glucan was isolated from the precipitate (lipid A),
obtained after AcOH hydrolysis of the LPS Treatment of
this precipitate with anhydrous hydrazine led to
solubiliza-tion of compound 5, identical to that obtained directly from
the LPS after hydrazine treatment Probably in its native
form the b-glucan 5 is acylated with fatty acids and thus is a
glycolipid Its structure was determined by NMR (data will
be presented elsewhere)
D I S C U S S I O N
The LPS of F tularensis possesses an unusual lipid A
structure: its major fraction does not contain phosphate
substituents, apparently has reducing glucosamine (residue
A) endgroup, and is not acylated at O-3 of GlcN B Most of
the reported structures of lipid A have phosphate
substit-uents at the reducing end [11] Phosphateless structures with
free reducing end glucosamine have been reported in
Rhodomicrobium vannieliiATCC 17100 and Rhizobium etli
CE3 lipid A [12,13] R etli CE3 lipid A contains
galact-uronic acid at O-4of b-GlcN residue; certain other lipids
from Aquifex pyrophilus [14] and Caulobacter crescentus
(E Vinogradov & M B Perry, unpublished data) contain
galacturonic acid both at O-1 of a-GlcN and at O-4of
b-GlcN residues At the same time F tularensis LPS
preparation contained some of the monophosphorylated
form of the lipid A (its backbone was isolated as compound
4), which is not substituted with Kdo Normally viable
bacteria do not produce lipid A unsubstituted with Kdo; the
inability to transfer Kdo to the lipid moiety is fatal for the
microorganism However, some bacteria, e.g Neisseria, can
be viable without transfer of Kdo to the lipid moiety [15]
Acyl residues at each acylation position in F tularensis
lipid A can be of two different chain lengths (C14/C16, C16/
C18) Such random distribution of fatty acids between
positions of acylation has been found in the lipid A moieties
from other bacterial species [16]
The structure of the LPS core part includes Kdo without
(or with base labile) substituent at O-4, which has not been
observed before This unusual feauture requires, however,
further confirmation since some groups could be lost in
harsh conditions of alkaline deacylation No phosphate
substituents in core region have been found The core part is
small and contains mannose in the inner part instead of the
more common heptose Core structures with Kdo
substi-tuted by mannose residues were reported in several micro-organisms, including Legionella pneumophila, different Rhizobiumspecies, and some other bacteria [17]
It remains to be determined which of the aforementioned structural features of the lipid A and core of F tularensis LPS account for the lack of endotoxic and inflammogenic activity of the intact molecule [6] The presence of longer (compared to the highly endotoxic lipid of E coli) chain fatty acids, the absence of phosphates and of an acyl group
at O-3 of a-GlcN residue could be responsible for the observed weak endotoxic properties of the LPS Weak endotoxicity might account for the fact that F tularensis induces relatively little inflammation at sites of infection compared to other facultative intracellular pathogens [18] Finally, it was noted that the LPS prepared by standard phenol–water extraction was heavily contaminated with an amylopectin-like glucan, a a-(1–6)-linear glucan, and a short chain b-(1–6)-glucan b-(1–6)-Glucans are uncommon in bacterial sources and to our knowledge only one such glucan has been described in Acinetobacter suis [19] The presence of glucans can strongly influence the results of biological activity studies with the LPS, at least distorting quantitative results
A C K N O W L E D G E M E N T S
This work was performed with support from Canadian Bacterial Diseases Network, and by grant AI48474 from the National Institutes
of Health, USA We thank Buko Lindner (Forschungszentrum Borstel, Borstel, FRG) for recording MALDI mass spectra of the lipid A, Bent
O Petersen (Carlsberg Research Center, Denmark) for NMR analysis
of b-glucan, and Don Krajcarsky (National Research Council Canada) for ESI mass spectra.
R E F E R E N C E S
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3 Tarnvik, A., Ericsson, M., Golovliov, I., Sandstrom, G & Sjostedt, A (1996) Orchestration of the protective immune response to intracellular bacteria: Francisella tularensis as a model organism FEMS Immunol Med Microbiol 13, 221–225.
4 Fortier, A.H., Naranayan, R.B., Drabick, J., Williams, J.C & Nacy, C.A (1996) Vaccination with lipopolysaccharide isolated from the live vaccine strain of Francisella tularensis protects against murine tularemia Vaccine Res 5, 193–202.
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6 Sandstrom, G., Sjostedt, A., Johansson, T., Kuoppa, K & Williams, J.C (1992) Immunogenicity and toxicity of lipopolysaccharide from Francisella tularensis LVS FEMS Mi-crobiol Immunol 5, 201–210.
7 Dreisbach, V.C., Cowley, S & Elkins, K.L (1988–96) (2000) Purified lipopolysaccharide from Francisella tularensis live vaccine strain (LVS) induces protective immunity against LVS infection that requires B cells and gamma interferon Infect Immun 68.
8 Conlan, J.W., Vinogradov, E., Monteiro, M.A & Perry, M.B (2002) Mice internally-inoculated with intact lipopolysaccharide, but not lipid A of O-chain from Francisella tularensis LVS rapidly acquire enhanced resistance against systemic and
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15 Steeghs, L., de Cock, H., Evers, E., Zomer, B., Tommassen, J & van der Ley, P (2001) Outer membrane composition of a lipo-polysaccharide-deficient Neisseria meningitidis mutant EMBO J.
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