Báo cáo Y học: Structural analysis of Francisella tularensis lipopolysaccharide potx

Structural 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-4 of b-GlcN, and no substituent at O-4 of the Kdo
residue. The largest oligosaccharide, isolated after strong
alkaline deacylation of NaBH
4
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-
acyl
4
-acyl
2

)-b-GlcN-(1–6)-2-N-acyl
1
)3-O-acyl
3
-GlcN where
acyl
1
,acyl
2
and acyl
3
are 3-hydroxyhexadecanoic or 3-hy-
droxyoctadecanoic acids, acyl
4
is 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-4 of 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 tularensis is 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-
rensis live 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. tularensis ATCC 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. tularensis LPS ATCC 29684.
EXPERIMENTAL PROCEDURES
Lipopolysaccharide isolation
F. tularensis LVS (ATCC 29684) was grown to D
600
 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 centrifugation at
62 000 g (yield  1gwetwt.ÆL
)1
). The saline-washed cells
(250 g wet wt.) were extracted by stirring with 400 mL 50%

(v/v) aqueous phenol at 65 °C 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 (0.02
M
,
pH 7.0) and was treated sequentially with RNase, DNase
and proteinase K (37 °C, 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:
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)
Eur. J. Biochem. 269, 6112–6118 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03321.x
cleared 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 4 °C. 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-
like
D
-glucan. The K60 fraction (840 mg) was essentially
pure S-type LPS and was used in subsequent studies.
NMR spectroscopy and general methods
1
H- and
13
C-NMR spectra of lipid A were recorded using a
Varian Inova 500 spectrometer in CDCl
3
–CD
3
OD (3 : 1,
v/v) or in CDCl
3
-CD
3
OH (3 : 1, v/v). Solutions were at
25 °C and referenced to the residual chloroform signal (
1
H
7.26 p.p.m.) and MeOH (
13
C 49.15 p.p.m.); spectra of all
other compounds were recorded at 25 °CinD
2
Oand
referenced to acetone (d

H
2.225 p.p.m., d
C
31.45 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
)1
with 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 (0.07
M
)ata
concentration of 2 lgÆlL
)1
.OnelL of the sample was then
mixedwith1lLof0.5
M
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 (100 °C,
4 h) and the precipitated product was collected by centri-
fugation at 3000 g and suspended in 2% (v/v) MeOH in
CHCl
3
. It was applied to a silica gel column (2 · 8cm),then
washed sequentially with 2%, 5%, 10% and 20% (v/v)
MeOH in CHCl
3
. 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 CHCl
3
–MeOH (3 : 1, v/v,
1 mL total volume) and 1
M
MeONa in MeOH (0.2 mL)
was added. The mixture was kept for 24 h at 25 °C, acidified
with trifluoroacetic acid, evaporated and extracted with
hexane. Samples of lipid containing hexane extract and
hexane-insoluble material were treated with 1

M
HCl in MeOH
(1 mL, 100 °C, 4 h), 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 20 °C for 24 h, 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 NaBH
4
and a drop of octanol to prevent foaming
were added. The mixture was kept at room temperature for
24 h, then dialysed against water. After lyophylization
120 mg of reduced LPS was recovered.
Alkaline deacylation
Two LPS samples (80 mg each) and NaBH
4
-reduced LPS
(80 mg) were dissolved in 4
M
KOH (4 mL each). To one of
the LPS samples 100 mg NaBH
4
were added, kept overnight
at 100 °C and neutralized with 2
M
HCl. 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 0.1
M
NaOH (A)
to 1
M
AcONa in 0.1
M
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 40 °C 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 1
M
NaCl (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.
RESULTS
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
1
H-NMR spectra of the unfractionated
lipid A and chromatographically fractionated samples
indicated that the fraction eluted with 10% (v/v) MeOH
in CHCl
3
contained the major component. It was used in
further studies as Ôlipid AÕ.
Ó FEBS 2002 Francisella tularensis lipopolysaccharide (Eur. J. Biochem. 269) 6113
Fatty 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. Acyl
1
,acyl
2
and acyl
3
residues had hydroxy or acyloxy groups at C-3 (
13
C signals
of C-3 at 69.0–72.4 p.p.m.), while acyl
4
had 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 acyl
1
-2, and between B2and
acyl
2
-2 indicated that GlcN A is N-acylated with acyl

1
,and
GlcN B is N-acylated with acyl
2
. All acyl C-1 signals were
identified from H-2:C-1 HMBC correlations. C-1 of acyl
2
gave HMBC correlation to H-2 of GlcN B; C-1 of acyl
4
showed correlation to H-3 of acyl
2
. C-1 Signals of acyl
1
and
acyl
3
overlap, but because acyl
1
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 A3withacyl
3
. O-Acylation of GlcN A at O-3 and of acyl
2
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.
Unit Nucleus 1 2(/2b or /NH) 3 4 5 6a 6b HMBC from C-1
A
1
H 4.92 3.94/7.09 5.00 3.34 3.88 3.56 3.89
13
C 92.0 52.8 75.0 69.6 71.2 69.7
B
1
H 4.32 3.41/7.62 3.34 3.20 3.13 3.56 3.69
13
C 102.5 56.9 75.6 71.5 76.7 62.4 A6
acyl
11
H 2.06/2.15 3.70 1.30
13
C 173.9 43.9 69.3 37.9 A2
acyl
21
H 2.28/2.33 5.01 1.46 1.14
13
C 172.9 42.2 72.4 34.9 26.0 B2
acyl
31
H 2.22/2.32 3.81 1.25
13
C 173.9 43.3 69.0 37.9 A3
acyl

41
H 2.14 1.43 1.14
13
C 175.4 35.2 25.7 30.1 acyl
2
H-3
Fig. 1. Fragments of HSQC and HMBC
spectra of F. tularensis (ATCC 29684) lipid A.
Signals of lipid with the a-anomeric form of
GlcN A are labeled.
6114 E. Vinogradov et al.(Eur. J. Biochem. 269) Ó FEBS 2002
of 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
acyl
1
and acyl
3
can be C16:0(3-OH) or C18:0(3-OH),
acyl
2
is mostly C18:0(3-OH); and acyl
4
is C14:0 with
minor amount of C16:0.
Combined NMR and MS evidence led to the proposed
structure (Fig. 3), where acyl
1
,acyl
2
and acyl
3
are
3-hydroxyhexadecanoic or 3-hydroxyoctadecanoic acids,
acyl
4

is 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 4
M
KOH led
to four major products 1a, 2a, 3a and 4,isolatedby
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,LPSwas
reduced with NaBH
4
prior to alkaline treatment or treated
with KOH in the presence of NaBH
4
. Both procedures led
to the same products 1b, 2b and 3b,inwhichKdowas
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 conditions.
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
13
CNMR
chemical shifts. Anomeric configurations were deduced

from the J
1,2
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-4 substituent 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, J1F6andK1J3. 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. Acyl
1
and acyl
3
are C16:0(3-OH) or
C18:0(3-OH), acyl
2
is mostly C18:0(3-OH), and acyl
4
is C14:0 with
minor amount of C16:0.
Ó FEBS 2002 Francisella tularensis lipopolysaccharide (Eur. J. Biochem. 269) 6115
oligosaccharides. 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.
Residue Nucleus 1 2 (3ax) 3 (3eq) 4 5 6 (6a) 7 (6b) 8a 8b
C, 1,2,3
1
H 1.70 2.02 4.17 4.10 3.65 3.80 3.67 3.90
13
C 36.2 66.3 77.3 72.3 70.4 64.1

E, 1,2,3
1
H 5.13 4.26 4.01 3.93 4.00 3.74 3.74
13
C 100.3 78.0 69.5 76.5 72.5 60.5
F, 1
1
H 4.79 4.29 3.83 3.68 3.45 3.79 3.95
13
C 100.4 75.4 74.3 67.4 77.3 61.5
F, 2
1
H 4.86 4.40 4.01 3.93 3.48 3.80 3.95
13
C 100.1 73.1 79.1 67.7 76.9 61.0
F, 3
1
H 4.82 4.37 4.03 4.23 3.44 3.72 3.94
13
C 100.1 74.0 78.2 72.9 76.3 60.5
G, 2
1
H 5.36 3.62 3.59 3.37 3.80 3.71 3.91
13
C 100.1 71.8 73.8 70.3 73.5 61.5
G, 3
1
H 5.43 3.59 3.58 3.37 3.78 3.70 3.88
13
C 100.2 72.2 74.4 70.4 73.7 61.5

H, 1
1
H 5.57 3.53 4.15 3.99 4.30 3.66 3.66
13
C 96.7 51.9 67.2 68.7 71.6 61.2
H, 2
1
H 5.71 3.57 4.11 3.96 4.23 3.65 3.70
13
C 95.2 51.7 67.1 68.7 72.2 61.4
H, 3
1
H 5.65 3.56 4.13 3.97 4.24 3.64 3.68
13
C 95.5 51.8 67.1 68.7 72.4 61.5
I, 1,2,3
1
H 4.50 3.32 3.51 3.39 3.48 3.72 3.90
13
C 102.5 73.6 76.2 70.4 76.7 61.6
J
1
H 4.60 3.77 3.88 3.43 3.54 1.31
13
C 100.8 55.1 78.9 76.2 72.5 17.1
K
1
H 5.76 3.44 4.46 5.85
13
C 96.9 53.6 62.8 109.1

6116 E. Vinogradov et al.(Eur. J. Biochem. 269) Ó FEBS 2002
Sephadex 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 (Ôlipid AÕ),
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).
DISCUSSION
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.Mostof
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 vannielii ATCC 17100 and Rhizobium etli
CE3 lipid A [12,13]. R. etli CE3 lipid A contains galact-
uronic acid at O-4 of 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-4 of
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
Rhizobium species, 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.
ACKNOWLEDGEMENTS
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.
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