The structure and biological characteristics of the
Spirochaeta aurantia
outer membrane glycolipid LGL
B
Evgeny Vinogradov
1
, Catherine J. Paul
2
, Jianjun Li
1
, Yuchen Zhou
2
, Elizabeth A. Lyle
3
, Richard I. Tapping
3
,
Andrew M. Kropinski
2
and Malcolm B. Perry
1
1
Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada;
2
Queen’s University, Kingston, ON, Canada;
3
University of Illinois, Urbana, IL, USA
In an attempt t o i solate lipopolysaccharide from Spirocha-
eta aurantia, Darveau-Hancock extraction of the cell mass
was performed. While no lipopolysaccharide was found, two
carbohydrate-containing compounds were detected. They

B
were unable to stimulate any Toll-like receptor (TLR)
examined, including TLR4 a nd TLR2, previously shown
to be sensitive t o lipopolysaccharide and glycolipids from
diverse bacterial origins, including other spirochetes.
Keywords: glycolipid; Spirochaeta aurantia;structure.
Spirochetes are a group of bacteria unified by spiral or
flattened-waveform cell morphology and periplasmic endo-
flagella; Spirochaeta is one of the six genera within this
phylum [1]. This b acterium is a f ree-living nonpathogenic
spirochete, originally isolated from pond mud and able to
fix atmospheric nitrogen [2–4]. Other members of this
phylum include the human pathogens Borrelia burgdorferi
(Lyme disease), the Leptospira (leptospiroses), Treponema
pallidum (syphilis), and T. denticola, T. brennaborense,and
T. maltophilum, which are i mplicated in periodontal disease
[5–7]. Although classified as Gram-negative, controversy
exists over the existence of lipopolysaccharide (LPS) in the
outer membranes o f spirochetes. Clear genetic a nd bio-
chemical evidence exists for the presence of LPS in
Leptospira [8] and for i ts absence i n T. pallidum and Borrelia
[9,10]. Limited structural analysis suggests that several o ral
treponemes (T. brennaborense and T. maltophilium [6],
T. medium [11], and T. denticola [12]) pos sess a surface
glycolipid similar to the lipotechoic acid of Gram-positive
bacteria. Recently, several small surface glycolipids were
identified in B. burgdorferi [13,14].
Toll-like r eceptors (TLR) a re an important component of
the host response to invading bacteria, with TLR4 required
for signal transduction and the inflammatory response

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Abbreviations: EU, endotoxin units; FAME, fatty acid methyl esters;
GalNAcA, N-acetylgalactosaminuronic acid; GSL, glycosphinogo-
lipids; Fuc3N, 3-ami no-3,6-dideoxygalactose; Kd o, 2-keto-3-deoxy-
D
-manno-oct-2-ulosonic acid; LA L, Limulus amebocyte lysate; LBP ,
LPS-binding protein; LPS, lipopolysaccharide; SGM, spirochaete
growth medium; TLR, Toll-like receptor; TNF-a, tumour necrosis
factor-a.
(Received 9 August 2004, revised 30 September 2004,
accepted 13 October 2004)
Eur. J. Biochem. 271, 4685–4695 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04433.x
while superfic ially resembling other spirochetal glycolipids,
LGL
B
is a multisaccharide glycolipid and is unable to
stimulate any TLR examined.
Experimental procedures
Bacterial strain and growth conditions
The S. aurantia strain, M 1, us ed in this study, was obtained
originally from E. P. Greenberg (Ohio State University,
Columbus, OH, USA). It was propagated in spirochete
growth medium (SGM) containing 0.4% (w/v) maltose
(Sigma-Aldrich, St. Louis, MO, USA), 0.2% (w/v) tryptone
and 0.2% (w/v) yeast extract (Difco), at pH 7.5. Cells were
grown at 30 °C with gentle aeration ( 30 r.p.m.; orbital
shaker; Forma Scientific, Marietta, O H, USA) for 24–48 h.
Cell stocks were maintained in SGM in liquid nitrogen.
Glycolipid isolation
Isolation of LGL from S. aurantia. Bacteria were harves-

0.2
M
NaCl; 0.3% (w/v) SDS]. Fractions of  2.1 mL were
collected at an average fl ow rate of 1.5 mLÆmin
)1
.The
fractions containing the l ow m olecular mass m aterial
(LGL
B
), as determined by standard SDS/PAGE with silver
stain [23], were pooled, precipitated with cold 0.375
M
MgCl
2
in 95% (w/v) ethanol, suspended in distilled water
and subjected to a second chromatography to ensure
homogeneity. Material was then reprecipitated, suspended
in distilled water, dialyzed, lyophilized and weighed in
preparation for further analysis.
Tricine–SDS/PAGE. Tricine–SDS/PAGE [15% (w/v)
resolving gel; 1 0% (w/v) s pacer g el; 4.5% ( w/v) stacking
gel) was u sed to e xamine the low molecular mass portions of
LPS and LGL [24]. LPS from Salmonella enterica sv.
typhimurium wild type , Sal. enterica sv. t yphimurium TV 119
(Ra mutant) a nd Sal. enterica sv. m innesota R5 (Rc mutant)
were p urchased f rom S igma-Aldrich. Products in acryl-
amide gels we re visualized by silve r staining [23].
NMR spectroscopy and general methods
NMR spectra were recorded at 25 °CinD
2

formed by collision activation of selected precursor ions
with nitro gen in the RF-only q uadrupole c ollision ce ll, were
recorded by a t ime-of-flight mass analyzer. Collision
energies were typically 120 eV (laboratory f rame of refer-
ence).
Hydrolysis. Hydrolysis was p erformed with 4
M
CF
3
CO
2
H
(110 °C, 3 h), monosaccharides were conventionally con-
verted into the alditol acetates and analysed by GLC on an
Agilent 6850 chromatograp h equipped with a DB-17
(30 m · 0.25 mm) fused-silica column using a temperature
gradient of 180 °C(2 min) fi 240 °C, at 2 °CÆmin
)1
.GC-
MS was performed on the Varian Saturn 2000 system with
an ion-trap mass spectral detector using the same column.
Gel chromatography. Gel chromatography was carried out
on Sephadex G-50 (2.5 · 95 cm) and Sephadex G -15
columns (1.6 · 80 cm) in pyridinium-acetate buffer,
pH 4.5 (4 mL of pyridine and 10 mL of AcOH in 1 L of
water), and the eluate was monitored by a refractive index
detector.
Configuration experiments
For determining the absolute configuration of t he mono-
saccharides, product 2 (1 mg) was treated with (S)-2-

The absolute configuration of
L
-aspartic acid was deter-
mined by chiral HPLC of the oligosaccharide hydrolysate
on a Chirex D p enicillamine column (250 · 4.6 mm;
Phenomenex) in 15% (v/v) methanol containing 2 m
M
CuSO
4
, with UV detection at 2 54 nm.
Fatty acid methyl esters (FAMEs) were generated from
1 mg s amples of LGL
B
by the addition of 1 mL o f 3
M
HCl
in methanol (Alltech Associates, Inc., Deerfield, I L, USA)
and incubation at 100 °C for 18 h. Following liberation of
the FAMEs, the hydrolysates were neutralized with 0.46 g
of silver carbonate and doped with 204.5 lg of tridecanoic
acid (in n-pentanol) as an internal standard. The samples
were centrifuged and the FAMEs were resolved by
PerkinElmer Sigma 3 gas chromatography, equipped with
a glass column [3.05 m · 2 mm internal diameter, packed
with 3% (w/v) SP-2100 DOH, 100/120 Supelcoport w ith
carrier gas (N
2
)], at a flow rate of 50 mlÆmin
)1
. The oven was

Flow (Amersham Pharmacia Biotech) in a gradient of
water/1
M
NaCl over 1 h with UV detection at 220 nm. The
products were desalted by gel chromatography on a
Sephadex G-15 column.
Biological assays
LAL a ssays were conducted by the Associates of Cape Cod,
Inc. (Cape Cod, MA, USA), by using the gel-clot method,
and the number of endotoxin units (EU) was compared
with control standard endotoxin from Escherichia coli
O113. The activation o f TLRs w as measur ed by quantifying
the production of tumour necrosis factor-a (TNF-a)by
whole blood cells, in response to a panel of TLR agonists, a s
described by Tapping et al. [26]. Briefly, whole blood from
healthy donors was collected into tubes containing heparin
and d iluted 1 : 4 in RPMI 1640. Sa mples were a liquoted
into 96-well plates, agonist was added, and incubatio n was
carried out at 37 °C in an atmosphere of 5% carbon dioxide
for 6 h. Cell supernatants were removed a nd assayed for
cytokine production by standard sandwich ELISA in
96-well Immunlon plates (Dynatech L aboratories, Chant-
illy, VA, USA). The TNF-a ELISA was p erformed by using
mAbs 68B6A3 or 68B2B3 for capture and t he biotinylated
mAb 68B3C5 (Biosource International, Camarillo, CA,
USA), followed by streptavidin-conjugated horseradish
peroxidase (HRP), for d etection. ELISAs were d eveloped
by using o-phenylenediamine as a substrate, and the
absorbance was measured at 490 nm by using a Spectramax
plate reader and software (Molecular Devices, Sunnyvale,

based upon the cell dry wieght. T his h igh y ield is not
unexpected as the surface to volume ratio of this bacterium
is 13.6Ælm
)1
, approximately 3 .5 times higher t han that of
E. coli or Sal. enterica sv. typhimurium (3.9Ælm
)1
). The
Darveau-Hancock procedure does not discriminate between
high (ÔsmoothÕ)orlow(ÔroughÕ)molecularmassLPS,
provides a high yield of product and should apply equally to
polysaccharides or glycolipids [22]. Potential complex
glycolipids were separated from previously characterized
glycogen storage granules by size exclusion chromatography
with examination of the fractions for carbohyd rates and
hexosamines [27]. A low molecular mass carbohydrate-
containing material (LGL
B
) was isolated, and when
examined by Tricine–SDS/PAGE [24] , demonstrated mobil-
ity s imilar t o the rough LPS of a Sal. enterica sv. typhimurium
TV 119 Ra mutant (Fig. 1). Another material, LGL
A
,was
identified as a larger glycolipid and is thought to contain
O-antigen like r epeats, contributing to the b anding pattern
observed in crude S. aurantia extract ( data not shown).
Preliminary colorimetric analysis [28] indicated that
LGL
B

NMR spectra of both oligosaccharides were completely
assigned by using 2 D N MR techniques ( Figs 2–4, Table 2).
Monosaccharides were identified o n the basis o f vicinal
proton coupling constants and
13
C NMR 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 position of C-6 signals of uronic acids was
found from HMBC correlations to H-5 protons. Connec-
tions between monosaccharides were identified on the basis
of NOESY ( Fig. 3) and HMBC correlations. The following
inter-residual NOEs were observed in oligosaccharides 1
and2:P1G4(in1),C1G4(in2),andG1A2,G1A1,A1G5,
A1L4, A1L3, L1E4, E 1F4, F1I4, I1D4, D1N3, D1N4,
N1Q1, B1K4, K1E3, O1I3, and M1D3. These correlations
include several contacts to nontransglycosidic protons next
to the linkage position, and between H-1 o f a monosac-
charide a nd H-5 of a glycosylating residue in the e vent of an
a-(1–2)-linkage. R espective H MBC c orrelations between
H-1 and a carbon at the transglycosidic position were
identified for all linkages. Amide linkage between C-6 of
residue E and an amino group of the aspartic acid was
identified on t he basis of the HMBC correlation b etween
H-2 of aspartic acid and C-6 of the GalA E, thu s showing
that aspartic acid is amide linked through i ts amino group to
C-6 of galacturonic acid E (Fig. 4).
Absolute configuration o f the monosaccharides was
determined by GC analysis of acety lated 2-butyl glycosides.

lane 5, Sal. enterica sv. minnesota R5 (Rc m u tant), 2 lg.
Table 1. Fatty acid methyl e ster (FAME) analysis, GLC and GLC-MS
indicated that the majority of fatty acids contained in Spirochaeta
aurantia LGL
B
are either branched or unsaturated. Values stated a re the
average n molÆmg
)1
with standard deviations (±) obtained from
quantifying and averaging areas under specific peaks from GLC
analysis of four separate samples of LGL
B
.
Identity of fatty acid LGL
B
(nmolÆmg
)1
)
Tetradecanoic acid (C14:0) 34.9 ± 1.3
13-Methyltetradecanoic acid (iC15:0) 224.1 ± 9.0
15-Methylpentadecanoic acid (iC16:0) 117.6 ± 12.4
9-Hexadecenoic acid (C16:1
9
) 155.3 ± 10.5
9-Octadecenoic acid (C18:1
9
) 58.3 ± 5.6
4688 E. Vinogradov et al. (Eur. J. Biochem. 271) Ó FEBS 2004
methanol, a nd the product w as peracetylated in order to
reacetylate free amino groups; it was checked by GC-MS

O
72
N
4
: 2358.6633 Da). The
MS for oligosaccharide 2 showed a m olecular mass of
2375.56 Da (calculated exact mass for C
85
H
129
O
74
N
3
:
2375.6422). In addition, an ammonium adduct of com-
pound 2 with m/z 1197.25 was observed as the most
abundant ions (observed molecular mass: 2392.50 Da). The
composition details, as well as some sequence information
of those two major c omponents w ith m/z 1180.24 and
1197.25, were further characterized by tandem mass
spectrometry (MS/MS). The fragmentation of cationic
oligosaccharides typically proceeds by cleavage at the
glycosidic bonds, which provides sequence and branching
information [31]. The charge state of a fragment ion is then
identified by u sing the isotope profile, owing to the high
resolution provided by the TOF mass analyser. The
product-ion s pectrum ( MS/MS s pectrum), obtained from
a doubly charged ion a t m/z 1197.25, is illustrated in Fig. 6.
This spectrum revealed two major doubly charged ions at

E12
E13
F12
F13
G15
F14
I12
I13
I14
E45
G12
G14
G13
K12
K13
K14,15
L12
L13,15
L14
F45
I45
D45
I24
E34
E24
I34
D34
N14
I23
F34

E5:F2
E1:F4
F1:I4
E45
I1:D4
I12
K1:E3
K15
K12
F45
O1:I3
M1:D3
I34
I35
F34
N15
N13
N1:Q1
M15
O15
O13
Fig. 3. Fragments of TOCSY (left) an d NOESY (right) s pectra o f oligosaccharide 2. Intraresidual correlations are labeled with a letter designation o f
the m o nosaccharide residue and numbers o f the c orrelating protons. Inte r-residual correlations a re labeled withlettersforbothmonosaccharides.
Ó FEBS 2004 Analysis of Spirochaeta aurantia glycolipid LGL
B
(Eur. J. Biochem. 271) 4689
fragment ions at m/z 292.06, 468.09, 613.1 6, 789.21, 1006.27,
1182.24, and 1344.37, respectively. The fragment ion at m/z
556.15 corresponds to the unit I-D-N, which might result
from the loss of Q from I-D-N-Q (m/z 648.20) o r from the

The gelation of LAL is a standard assay based on the
nonspecific immune response of the horseshoe crab, and is
used to assess the endotoxic potential of various substances
[32]. LGL
B
displayed a 100- fold less endot oxic potential,
registering 2.5 · 10
5
EUÆmg
)1
when compared to an E. coli
O113 LPS control (1 · 10
7
EUÆmg
)1
) in a LAL gel clot
assay.
LGL
B
was also examined for its ability to act as a TLR
agonist. Attempts to measure a reaction f rom cells trans-
fected specifically with human TLR2 or TLR4 were
unsuccessful, regardless of the concentration of LGL
B
examined (data not shown). T he whole blood assay u ses
fresh human blood (which contains a variety of Toll
receptors) and measures the total r elease of TNF-a by
ELISA [26]. Cells were stimulated with defined TLR
agonists (zymosan a nd heat-killed Staph. aureus for
TLR2; PolyIC for TLR3; E. coli Re595 LPS for TLR4;

B
were added, no production of
TNF-a was detected, showing t hat this large glycolipid
cannot stimulate TLR2, -3, -4, -7 or -9.
Discussion
Although s ome s tructural i nformation has been obtained
from other spirochetes, the complete elucidation o f t he
LGL
B
from S. aurantia represents the first complete
structure o f a large glycolipid from these bacteria. The
dodecasaccharide LGL
B
is anchored by a diacyl glycerol.
A glycolipid containing a single sugar, BbGL-II, and also
anchored on a g lycerol, has been identified in B. burg-
dorferi [13]. I t i s s urface localized, and antibodies to this
molecule were detected in patients with Lyme disease. A
diacyl glycerol anchor has also been purposed for the
glycolipids of T. denticola, T. maltophilum,andT. brenn-
aborense [6,12]. A glycolipid identified in T. pectinovorum
contained glycerol, and the majority of fatty acids were
branched, although on the basis of detection of Kdo in
this material, the authors designated it LPS. A diacyl
glycerol anchor may substitute for lipid A, an observa-
tion supported by the absence of any homologs to genes
involved in lipid A biosynthesis in the completed ge-
nomes of B. burgdorferi, T. pallidum or T. denticola
[9,10,33].
All o f t he treponemal g lycolipids i dentified have either

C 96.5 68.7 79.3 79.4 72.4 175.6
E, a-GalA6Asp
1
H 5.20 4.12 4.20 4.70 5.05
13
C 100.8 68.8 79.4 79.4 72.2 170.6
F, a-GalA
1
H 5.18 3.89 4.21 4.50 4.79
13
C 99.3 68.9 69.6 80.3 72.8 176.0
G, a-GlcA (1)
1
H 5.19 3.69 4.06 3.83 4.30
13
C 101.6 73.1 75.0 77.3 73.7 177.2
G, a-GlcA (2)
1
H 5.19 3.69 4.06 3.82 4.30
13
C 101.6 73.1 75.0 77.8 73.7 177.2
I, a-GalNAcA
1
H 5.11 4.46 4.10 4.69 4.76
13
C 99.3 49.8 77.3 77.6 72.9 176.0
K, b-GlcA
1
H 4.80 3.50 3.86 3.91 3.90
13

13
C 178.8 52.3 39.1 178.8
Q, Gro, 1 and 2
1
H 3.85 4.03 3.68
4.00 3.76
13
C 72.1 71.7 63.7
Q, Gro, LGL
B
1
H 3.84 5.32 4.15
3.84 4.50
13
C 69.8 72.0 64.8
Ó FEBS 2004 Analysis of Spirochaeta aurantia glycolipid LGL
B
(Eur. J. Biochem. 271) 4691
1000 1100 1200 1300 1400
m/z
1197.25
1189.28
1180.30
1116.27
1131.28
1000 1 100 1200 1300 1400
1180.25
1114.29
1
2

I
1612.48
D NQL OM
C-G-A,NH
3
Fig. 6. MS/MS spectrum obtained from a doubly charged ion at m/z 1197.25 of o ligosaccharide 2.
4692 E. Vinogradov et al. (Eur. J. Biochem. 271) Ó FEBS 2004
unsaturated acyl group of BbGL-II. Schultz et al.
indicated that the presence of fatty acid branching in
T. denticola is analogous to adap tations in Gram-positive
bacteria to alter membrane fluidity [12]. Gram-negative
bacteria are known to m odify the d egree of saturation in
their fatty acids to modulate membrane fluidity [34,35].
LGL
B
contained both unsaturated and branched fatty
acids (i.e. C14:0, iC15:0, C16:1), t he only spirochete
glycolipid identified, to date, with both of these modi-
fications, suggesting LGL
B
may form highly fluid mem-
branes.
S. aurant ia LGL
B
comprises 15% lipid by mass, corres-
ponding well with the p roportion of fatty acids in the
glycolipid OML521 (10.7%) from T. denticola, a glycolipid
that is also estima ted t o be similar in size to Ra LPS [12]. Ra
LPS is t he minimum L PS unit required for efficient and
proper folding, and functioning, of porin [36]. T. denticola

Toll-like r eceptors [12,20], suggesting t hat a t a functional
level they possess some similarity. LGL
B
was able to gel
LAL, but did not stimulate any TLR examined: this is
an unusual situation, paralleled in the spirochete litera-
ture only by the inability of the Borrelia glycolipids to
activate TLR2 o r -4 [13]. TNF-a release was measured
following the e xposure o f h uman mononuclear cells to
two diffe rent GSLs from S. paucimobilis: t he mono-
glycosylated GSL-1, and the tetraglycosylated GSL-4A.
GSL-1 was unable to activate the release of monokines,
in contrast to the larger GSL-4A, although induction was
still 10 000-fold below that of the LPS standard [44].
While this appears to be s imilar to the situation with the
monoglycosylated BbGL-II, the inability of LGL
B
to
stimulate TNF-a release precludes size as the only
explanation for the difference in biological activity
observed with GSLs.
Another oral spirochete implicated in periodontal dis-
ease, T. medium, contains the glycolipid, Tm-Gp, which
abrogates TLR activation through interactions with
LPS-binding protein (LBP) and CD14, two important
components of TLR-mediated innate immunity [45]. The
blocking by Tm-Gp was dependent on the lipid portion of
the molecule, but whether S. aurantia LGL
B
would block a

could abrogate the
interaction with LBP and prevent any release of TNF-a in
the whole blood assay for TLR activation. Specific struc-
tural e ntities of LPS, producing certain biological effects,
have been extensively studied given the central role of this
molecule in pathogenesis and vaccine development. Char-
acterization of any biological activity o f spirochete glyco-
lipids is important for similar reasons, especially in the case
of B. burgdorferi BbGL-II, given the difficulties in develop-
ing an effective proteinaceous vaccine targeting this organ-
ism [13,47].
0
5
10
15
20
25
30
35
40
TNF-α (ng/mL)
No Zymosan HKSA PolyIC Re LPS R848 CpG LGL
B
Fig. 7. Tumour necrosis factor-a (TNF-a) production through activation
of Toll-like receptors (TLR) in the presence of different agonists. Con-
trols for different TLR were as follows: zymosan and h eat-killed
Staphylococcus aureus (HK SA) for TLR2; PolyIC fo r TLR3;
Escherichia coli Re595 lipopolysaccharide (LPS) for TLR4; R848 for
TLR7; and CpG Oligo for TLR9. Error bars represent the standard
deviation of cellular a ctivation e xperiments pe rforme d in t riplicate.

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