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

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%

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

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

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)-

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

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

+
), 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

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

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

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-

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

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
Ó FEBS 2002 Francisella tularensis lipopolysaccharide (Eur. J. Biochem. 269) 6117
aerogenic challenge with the pathogen. Microbial Pathogenesis in
press.
9. Vinogradov, E.V., Shashkov, A.S., Knirel, Y.A., Kochetkov,
N.K., Tochtamysheva, N.V., Averin, S.P., Goncharova, O.V. &
Khlebnikov, V.S. (1991) Structure of the O-antigen of Francisella
tularensis strain 15. Carbohydr. Res. 214, 289–297.
10. Westphal, O. & Jann, K. (1965) Bacterial lipopolysaccharides.
Extraction with phenol-water and further applications of the
procedure. Methods Carbohydr. Chem. 5, 83–91.
11. Za
¨
hringer, U., Lindner, B. & Rietschel, E.T. (1999) Chemical
structure of lipid A: recent advances in structural analysis of
biologically active molecules. In Endotoxin in Health and Disease.
(Brade, H., Opal, S.M., Vogel, S.N. & Morrison, D.C, eds.),
pp. 93–114. Marcel Dekker, NY-Basel.
12. Holst, O., Borowiak, D., Weckesser, J. & Mayer, H. (1983)
Structural studies on the phosphate-free lipid A of Rhodomicro-

monocytogenes, Francisella tularensis,andSalmonella typhimurium
involves lysis of infected hepatocytes by leukocytes. Infect. Immun.
60, 5164–5171.
19. Monteiro, M.A., Slavic, D., St Michael, F., Brisson, J.R.,
MacInnes, J.I. & Perry, M.B. (2000) The first description of
a(1fi 6)-b-
D
-glucan in prokaryotes: (1 fi 6)-b-
D
-glucan is a
common component of Actinobacillus suis and is the basis for
aserotypingsystem.Carbohydr. Res. 329, 121–130.
6118 E. Vinogradov et al.(Eur. J. Biochem. 269) Ó FEBS 2002