Tài liệu Báo cáo khoa học: Endotoxic activity and chemical structure of lipopolysaccharides from Chlamydia trachomatis serotypes E and L2 and Chlamydophila psittaci 6BC pdf

Endotoxic activity and chemical structure of lipopolysaccharides
from
Chlamydia trachomatis
serotypes E and L
2
and
Chlamydophila psittaci
6BC
Holger Heine, Sven Mu¨ ller-Loennies, Lore Brade, Buko Lindner and Helmut Brade
Research Center Borstel, Center for Medicine and Biosciences, Borstel, Germany
The lipopolysaccharide (LPS) of Chlamydia trachomatis
serotype E was isolated from tissue culture-grown element-
ary bodies and analyzed structurally by mass spectrometry
and
1
H,
13
Cand
31
P nuclear magnetic resonance. The LPS
is composed of the same pentasaccharide bisphosphate
aKdo-(2–8)-aKdo-(2–4)-aKdo-(2–6)-bGlcN-4P-(1–6)-
aGlcN-1P (Kdo is 3-deoxy-a-
D
-manno-oct-2-ulosonic acid)
as reported for C. trachomatis serotype L
2
[Rund, S., Lind-
ner, B., Brade, H. and Holst, O. (1999) J. Biol. Chem. 274,
16819–16824]. The glucosamine disaccharide backbone is
substituted with a complex mixture of fatty acids with ester

or amide linkage whereby no ester-linked hydroxy fatty
acids were found. The LPS was purified carefully (with
contaminations by protein or nucleic acids below 0.3%) and
tested for its ability to induce proinflammatory cytokines in
several readout systems in comparison to LPS from C. tra-
chomatis serotype L
2
and Chlamydophila psittaci strain 6BC
as well as enterobacterial smooth and rough LPS and syn-
thetic hexaacyl lipid A. The chlamydial LPS were at least 10
times less active than typical endotoxins; specificity of the
activities was confirmed by inhibition with the LPS anta-
gonist, B1233, or with monoclonal antibodies against
chlamydial LPS. Like other LPS, the chlamydial LPS used
toll-like receptor TLR4 for signalling, but unlike other LPS
activation was strictly CD14-dependent.
Keywords: toll-like receptors; innate immunity; MALDI-
TOF MS; ESI-FT-IR MS.
Chlamydia are obligatory intracellular bacteria [1] causing
acute and chronic infections in animals and humans [2,3].
Little is known about the pathogenic mechanisms involved
in chlamydial infections but chronic inflammation is
observed in classical chlamydial diseases such as ocular
trachoma, that is the world’s leading cause of preventable
blindness, or chronic salpingitis, that is the major cause of
secondary female infertility in developed nations. Athero-
sclerosis is a chronic inflammatory disease of the arterial
walls which is presently considered to be associated with
infection by Chlamydophila pneumoniae [4]. Members of the
family Chlamydiaceae are Gram-negative bacteria contain-

ing, in their outer membrane, a lipopolysaccharide (LPS)
harbouring a surface-exposed, family specific epitope that
has been well characterized [5]. It is well known that LPS of
Gram-negative bacteria in general is one of the most potent
stimulators of innate immunity and that the lipid A moiety
of LPS is responsible for this activity [6,7]. Detailed studies
on the structure–function relationships of lipid A have
indicated that the number, type and distribution of fatty
acids in lipid A determine whether it exhibits weak or strong
agonist or antagonist activities [8]. Analytical data have
shown that the fatty acids in LPS of Chlamydiae have acyl
chains with up to 22 carbon atoms and also that 3-hydroxy
fatty acids occur only with amide-linkage [9,10]. Studies on
the biological activity of chlamydial LPS have shown that it
possesses significant lower activity than enterobacterial LPS
in terms of pyrogenicity or Schwartzman-reactivity in
rabbits, lethality in galactosamine-sensitized mice, anti-
complementary activity in guinea-pigs and induction of
proinflammatory cytokines in human peripheral blood
monocytes [10,11]. It was, however, mitogenic for mouse
B-cells and activated mouse peritoneal macrophages, thus,
producing prostaglandin E
2
[10]. These reported data were
obtained on LPS of unknown chemical structure and of ill-
defined purity. Therefore, in this study we have compared
the endotoxic activities of three chlamydial LPS of known
structure and defined purity. Whereas the chemical struc-
tures of the LPS from C. trachomatis serotype L
2

and of
Chl. psittaci 6BC have been reported previously [12,13], that
of the LPS from C. trachomatis serotype E, which is the
Correspondence to H. Brade, Research Center Borstel, Center for
Medicine and Biosciences, D-23845 Borstel, Germany.
Fax: + 49 4537 188 419, Tel.: + 49 4537 188 474,
E-mail:
Abbreviations: CSD, capillary skimmer dissociation; COSY,
correlation spectroscopy; EB, elementary body; ESI-FT-ICR MS,
electrospray ionization Fourier-transform ion cyclotron resonance
mass spectrometry; FBS, fetal bovine serum; HEK, human epithelial
kidney; HMBC, heteronuclear multiple bond correlation; HPAEC,
high-performance anion-exchange chromatography; HSQC, hetero-
nuclear single quantum coherence; Kdo, 3-deoxy-a-
D
-manno-oct-2-
ulopyranosonic acid; LPS, lipopolysaccharide(s); mAb, monoclonal
antibody; MALDI-TOF MS, matrix assisted laser desorption/ion-
ization time-of-flight mass spectrometry; MNC, mononuclear cells;
NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect
spectro-scopy; TLR, Toll-like receptor; TNF, tumor necrosis factor
alpha; ROESY, rotating frame nuclear Overhauser effect spectro-
scopy; TOCSY, total correlation spectroscopy.
Note: H. H. and S. M. L. contributed equally to this study.
(Received 11 July 2002, revised 11 November 2002,
accepted 26 November 2002)
Eur. J. Biochem. 270, 440–450 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03392.x
most frequently occurring serotype in genital tract infec-
tions, is reported here.
Materials and methods

LPS
C. trachomatis serotype E (ATCC) was grown in monolay-
er cultures of mycoplasma-free L929 cells in multilayer trays
(NUNC, 10 · 600 cm
2
) over 2 days. The cultures were
killed by the addition of 0.5% (w/v) of phenol and the
chlamydial elementary bodies (EB) were sedimented to-
gether with the cell debris by centrifugation at 9000 g for 1 h
and lyophilized yielding 8.29 g from 120 000 cm
2
of infec-
ted cells. LPS was obtained by the phenol/water extraction
method, modified as follows. The sediment was suspended
in 200 mL of 45% phenol, containing 2% N-lauroylsarco-
sine sodium salt in water (w/v), heated at 68 °Cfor10min
and cooled on ice. After centrifugation (3000 g, 30 min), the
water phase was removed and the phenol phase was
extracted again with 100 mL of 2% aqueous N-lauroylsar-
cosine sodium. After centrifugation and separation as
before, the extraction of the phenol phase was repeated
again. The water layers were combined, dialyzed against
water and lyophilyzed (yield 1.55 g), then extracted twice
with 35-mL portions of 90% aqueous phenol/chloroform/
light petroleum (boiling point 40–60 °C)2:5:8(v/v/v).
The organic solvents were evaporated and the LPS was
precipitated from the phenol phase by drop-wise addition of
water. The precipitated LPS was washed once with 80%
aqueous phenol and with acetone (yield 54.6 mg), dissolved
in water (10 mgÆmL

)1
) and precipitated with 10 vol. of
ethanol/acetone 9 : 1 (v/v), dried, and dissolved in water
(5 mgÆmL
)1
). Calcium chloride was added to a final
concentration of 0.1
M
, the precipitated LPS was washed,
once with 10 m
M
HCl, once with water, and then neutral-
ized with triethylamine and freeze dried (yield 52 mg).
Quantification of Kdo, glucosamine, phosphorous and fatty
acids was performed as described previously [13]. Contami-
nation with protein or nucleic acid was determined by
amino acid analysis and gas-liquid chromatography mass
spectrometry of alditol acetates, respectively.
LPS of C. trachomatis serotype L
2
[12] and Chl. psittaci
6BC [13] were those described in the reference, LPS from
Salmonella enterica serovar Friedenau, E. coli Re-mutant
strain F515 and synthetic lipid A (compound 506) were
used as controls. De-O-acylation and de-N-acylation were
carried out as described [12]. The deacylated sample was
desalted by gelfiltration on Sephadex G10 in NH
4
HCO
3

and lyophilized three times.
Analytical HPAEC
The deacylated LPS was analysed by analytical high-
performance anion exchange chromatography (HPAEC)
on a column of CarboPak PA1 (4 mm · 250 mm, Dionex)
using the eluents (A) 0.1
M
NaOH and (B) 1
M
NaOAc in
0.1
M
NaOH and a gradient from 30% to 70% of eluent B
in 16 min at a flow-rate of 1 mLÆmin
)1
.Therunwas
monitored by pulsed amperometric detection.
Mass spectrometry
De-O-acylated LPS was analyzed using an Electrospray
Fourier-Transform Ion Cyclotron Resonance (ESI-FT-
ICR) mass spectrometer (APEX II, Bruker Daltonics,
Billerica, MA, USA) equipped with a 7 Tesla actively
shielded magnet and an Apollo ion source. Capillary
skimmer dissociation was induced by increasing the capil-
lary exit voltage from )100 V to )350 V. Samples were
dissolved at a concentration of  20 ngÆlL
)1
in 2-propanol/
water/triethylamine in a ratio of 5 : 5 : 0.01 (v/v/v) and
sprayed at a flow rate of 2 lLÆmin

)1
.
MALDI-TOF MS of LPS and free lipid A were
performed on a Reflex III (Bruker-Daltonik, Bremen,
Germany) in the linear TOF configuration applying an
acceleration voltage of 20 kV. Lipid A samples were
dispersedinmethanol/water/triethylamineinaratioof
5 : 5 : 0.01 (v/v/v) and mixed with a saturated matrix
solution (2,4,6-trihydroxyacetophenon, in 0.1% trifluoro-
acetic acid and acetonitrile in a ratio of 2 : 1 v/v). Aliquots
of sample solution (0.5 lL) were deposited on the metallic
sample holder, dried in a stream of air and analyzed
immediately after evaporation. The spectra are the sum of at
least 50 single laser shot experiments. External mass scale
calibration was performed with E. coli F515 LPS of known
structure.
NMR spectroscopy
The deacylated LPS from C. trachomatis E was investigated
by one-dimensional
1
H-NMR- (600 MHz),
13
C-NMR-
(150 MHz) and
31
P-NMR-spectroscopy (243 MHz) and
two-dimensional
1
H,
1

H-COSY,
1
H,
1
H-ROESY
1
H,
13
C-
HSQC,
1
H,
31
P-HSQC with a Bruker DRX Avance spectro-
meter equipped with a multinuclear z-gradient probehead.
Acetone in D
2
O (2.225 p.p.m.,
1
H) and 1,4-dioxane
(67.4 p.p.m.,
13
C) served as reference. For
31
P-NMR,
85% phosphoric acid was used as reference (set to d
0 p.p.m.) All spectra were recorded at a temperature of
300 K and standard Bruker pulse programs were used in all
experiments. Spectra were recorded from a solution of 1 mg
sample in 0.5 mL D

2
O after addition of 5 lLofa1
M
NaOD
stock solution (1 : 10 dilution in D
2
O of 40% NaOD,
Merck). The final concentration was thus 10 m
M
NaOD. The addition of NaOD was necessary in order to
achieve uniform signals in
31
P-NMR spectra and a better
resolution of deoxy protons belonging to Kdo-residues. The
alkaline conditions led to upfield shifts of signals of H-2
belonging to GlcN-residues. For comparison, the same
spectra and an additional
1
H,
13
C-HMBC NMR spectrum
were recorded under identical conditions on a sample of
aKdo-(2–8)-aKdo-(2–4)-aKdo-(2–6)-bGlcN-4P-(1–6)-
aGlcN-1P (pentasaccharide bisphosphate, PSBP) which
was isolated from Salmonella enterica sv. Minnesota R595-
207 and purified by HPAEC as described previously [14].
An aliquot (10 mg) of this sample was dissolved in 500 lL
D
2
O and 4% NaOD was added until the chemical shifts of

GlcN H-2 signals had the same chemical shift as for the
C. trachomatis E sample. Probably due to residual buffer
from the gel filtration, 30 lL of 4% NaOD had to be added.
Ó FEBS 2003 Structure and function of chlamydial lipopolysaccharide (Eur. J. Biochem. 270) 441
Stimulation of human mononuclear cells (MNC)
Human peripheral blood MNC (Ethical Committee,
University of Luebeck) were isolated from heparinized
blood of healthy adult donors by Ficoll-Isopaque density
gradient centrifugation [15]. Isolated MNC were washed in
NaCl/P
i
and cultured in RPMI 1640 containing 10% human
pooled serum, 100 UÆmL
)1
penicillin and100 lgÆmL
)1
strep-
tomycin (Biochrom, Berlin, Germany). Cells (1 · 10
6
ÆmL
)1
)
were stimulated in duplicates with various amounts of LPS
or the synthetic lipopeptide, Pam
3
Cys-Ser-Lys
4
(P3CSK4;
EMC microcollections, Tu
¨

bingen, Germany). In blocking
experiments, cells were preincubated for 30 min with
10 lgÆmL
)1
of the anti-CD14 Ig MEM-18 [16] or 10 l
M
of
the LPS antagonist, B1233. After 18 h, supernatants were
collected and the content of TNF was quantified by ELISA
(a kind gift of H. Gallati, Intex AG, Muttenz, Switzerland).
CHO/CD14 cells
The CHO/CD14 reporter cell line, clone 3E10, is a stably
transfected CD14-positive CHO cell line that expresses
inducible membrane CD25 (Tac antigen) under transcrip-
tional control of a partial human E-selectin promoter
(pELAM.Tac) which contains an essential nuclear factor,
NF-jB binding site. CHO cells were grown in Ham’s F12
medium containing 10% fetal bovine serum and 1%
penicillin/streptomycin at 37 °C in a humidified 5% CO
2
environment. Medium was supplemented with 400 U of
hygromycin BÆmL
)1
.
Flow cytometry analysis of NF-jB activity
Cells were plated at a density of 2.5 · 10
5
per well in 24-well
plates. The following day, the cells were stimulated for 18 h
in Ham’s F12 medium containing 10% fetal bovine serum

(total volume of 0.3 mL per well). Subsequently, the cells
were harvested with trypsin/EDTA and expression of CD25
with FITC-labelled-antihuman CD25 was analyzed by flow
cytometry (DAKO, Germany). In blocking experiments,
B1233 (10 l
M
) or mAb against chlamydial LPS
(20 lgÆmL
)1
) were added 30 min prior to the stimulation
with LPS or rIL-1b. The preparation of mAb against
chlamydial LPS was described elsewhere [17]. Mab S25-2
and S25-23 recognize, as a minimal structure, the oligosac-
charides aKdo-(2–8)-aKdo and aKdo-(2–8)-aKdo-(2–4)-
aKdo, respectively, representing family specific epitopes of
chlamydial LPS. They were used as immunopurified stock
solutions of 1 mgÆmL
)1
.
Activation of transiently transfected HEK/HEK-CD14 cells
HEK293T cells were stably transfected with an expression
vector for human CD14 (pCDNA3huCD14, a kind gift of
D. Golenbock, Worcester, MA, USA) or with the vector
without insert as control. Stable transfected cell lines (HEK-
vec and HEK-CD14) were isolated and cultured in DMEM
containing 10% fetal bovine serum, 1% penicillin/strepto-
mycin and 400 lgÆmL
)1
, G418 (Invitrogen). For transient
transfection, cells were plated at a density of 2 · 10

5
ÆmL
)1
in 24-well plates in complete medium without G418. The
following day, cells were transiently transfected using
Polyfect (Qiagen) according to the manufacturers’ protocol.
Expression plasmids containing Flag-tagged versions of
human TLR2 and human TLR4 were a kind gift from
P. Nelson, Fred Hutchinson Cancer Research Center,
Seattle, USA and were subcloned into pREP9 (Invitrogen).
The human MD-2 expression plasmid was a kind gift from
K. Miyake, Institute of Medical Science, University of
Tokyo, Tokyo, Japan. All plasmids were used at 400 ng per
transfection, the total DNA content was kept constant at
800 ng per transfection using pCDNA3 (Invitrogen). After
24 h of transfection, cells were washed and stimulated for
another 18 h. Finally, supernatants were collected and the
IL-8 content was quantified using a commercial ELISA
(Biosource).
Results
Isolation and characterization of chlamydial LPS
LPS from C. trachomatis serotype E was obtained from
tissue culture grown bacteria by a modified phenol/water/
detergent extraction, purified by phenol/chloroform/light
petroleum-extraction and ethanol precipitation and conver-
ted into the uniform triethylamine salt form. The total yield
of LPS was approximately 400 ngÆcm
)2
of infected cell
monolayer. Compositional analyses indicated that the

LPS was composed of GlcN (703 nmolÆmg
)1
), Kdo
(981 nmolÆmg
)1
), phosphorus (791 nmol mg
)1
)andfatty
acids (1800 nmolÆmg
)1
) in the molar ratio of 2 : 2.8 :
2.2 : 5.1. Analysis for ribose and amino acids indicated that
the contamination with nucleic acids or protein was below
0.3%. Fatty acid analysis (Table 1) indicated that the
major fatty acids were C14:0, iso-C15:0, C18:0, C20:0 and
3-OH-C20:0.
MALDI-MS and ESI-MS analysis
As a first step in the structural analysis, LPS and de-O-
acylated LPS of C. trachomatis serotype E was analyzed by
MALDI-TOF and ESI-FT-ICR mass spectrometry. The
spectra were compared with corresponding spectra of LPS
samples obtained from C. trachomatis serotype L
2
and
Chl. psittaci strain 6BC, the chemical structures of which
are already known [12,13]. The negative ion MALDI-TOF
mass spectrum of the native LPS (Fig. 1) exhibited a
complex pattern of several groups of ion peaks representing
molecular ions (centered around m/z 2510, 2300 and 2005)
and of laser-induced fragment ions [18] representing the

lipid A moiety of LPS (centered around m/z 1850, 1641, and
1347). The mass difference of D m/z 660 between the
respective groups of ions of the complete LPS and lipid A
indicated that the core oligosaccharide was built up by three
Kdo residues. An enlargement of the spectrum of the
lipid A region is depicted in Fig. 2A in comparison to mass
spectra of lipid A isolated from C. trachomatis L
2
and
Chl. psittaci 6BC (Fig. 2B–C), showing nearly identical
groups of ion peaks for C. trachomatis EandL
2
that could
be assigned to penta-, tetra- and tri-acylated lipid A species,
differing by the mass of C14 : 0 (D m/z 210) and C20 : 0
(D m/z 294) fatty acid. The molecular ion peaks within each
groupdifferedbyD m/z ± 14 and indicated the biological
heterogeneity in the type of fatty acid residues in accordance
442 H. Heine et al. (Eur. J. Biochem. 270) Ó FEBS 2003
to the variety of different fatty acids as detected by chemical
analysis (Table 1). In accordance to published data, the
peak at m/z 1850 corresponded to a bisphosphorylated
GlcN disaccharide carrying five fatty acid residues, two
(3-OH)-C20:0 and one C14:0, C15:0 and C20:0 each, with a
calculated molecular mass of 1850.6 Da. The mass spec-
trum obtained from LPS of Chl. psittaci showed a similar
complex pattern of penta-, tetra-, and tri-acylated lipid A
species, however, shifted by D m/z + 42 (corresponding to
three CH
2

groups) indicating that the lipid A moiety carried
fatty acids with longer chain length. The negative ion CSD
ESI-FT-ICR mass spectrum of de-O-acylated LPS from
C. trachomatis E (Fig. 3A) revealed singly charged molecu-
lar ions in the region around m/z 1779, prominent fragment
ions of the de-O-acylated lipid A moiety around m/z 1119.75,
and ions of minor intensity originating from the consecutive
loss of three Kdo residues. Further ions observed in the
spectrum originated from the loss of phosphate or from
cation adduct ions. Comparison with Fig. 3B and C
demonstrated that the structure of de-O-acylated LPS from
C. trachomatis L
2
and E were nearly identical, whereas the
LPS of Chl. psittaci exhibits a second set of ions representing
molecular species with four Kdo residues (m/z 2000). The
enlargements of the ion region representing ions of lipid A
fragments (right side) showed patterns of isotopic peaks with
mass differences of D m/z ±14and)2 corresponding to
chain length differences and to unsaturated fatty acids,
respectively. The most abundant ion peak of de-O-acylated
lipid A of C. trachomatis L
2
and E at m/z 1119.75 represen-
ted a bisphosphorylated GlcN disaccharide with two amide-
linked (3-OH)-C20:0 fatty acids (calculated monoisotopic
mass of 1120.75). Thus, the intense peaks at m/z 1123.75 and
1137.74 of de-O-acylated lipid A of Chl. psittaci indicated
that in this species (3-OH)-C21 : 0 was the most abundant
amide-linked fatty acid.

Analytical HPAEC and NMR spectroscopy
The LPS of C. trachomatis serotype E was deacylated
and the resulting phosphorylated carbohydrate backbone
Fig. 1. Negative ion MALDI-TOF mass spectrum of LPS from
C. trachomatis serotype E.
Fig. 2. Negative ion MALDI-TOF mass spectra of lipid A isolated from
C. trachomatis serotype E (A) and L
2
(B) andfrom Chl. psittaci 6BC (C).
Table 1. Fatty acid analysis of LPS from C. trachomatis serotype E.
Fatty acid
Amount present
in LPS (nmolÆmg
)1
)
Nonhydroxylated
C14:0 310
iso-C15:0 161
anteiso-C15:0 37
anteiso-C16:0 5
C16:0
a
83
C18:0 141
iso-C19:0 17
anteiso-C19:0 40
C20:0 202
C20:1
b
19

iso-C21:0 16
anteiso-C21:0 17
C22:0 9
Hydroxylated
C20:1
c
83
C21:1
c
14
3-OH-C14:0 3
3-OH-C18:0 33
anteiso-3-OH-C20:0 7
3-OH-C20:0
a
491
iso-3-OH-C21:0 35
anteiso-3-OH-C21:0 55
3-OH-C22:0 22
Total 1800
a
Iso- and anteiso-form not separated.
b
Unsaturated fatty acid with
unknown position of double bond.
c
D2-Unsaturated fatty acid;
assumed derivative of the corresponding 3-hydroxy fatty acids by
a-elimination.
Ó FEBS 2003 Structure and function of chlamydial lipopolysaccharide (Eur. J. Biochem. 270) 443

desalted by gelfiltration. In analytical HPAEC one major
oligosaccharide as well as three minor components were
observed (Fig. 4). Comparison with the HPAEC profile of a
mixture of compounds obtained from deacylation of
S. enterica sv. minnesota R595-207 indicated that these
were aKdo-(2–8)-aKdo-(2–4)-aKdo-(2–6)-bGlcN-4P-(1–6)-
aGlcN-1P (PSBP), pentasaccharide 1-monophosphate (PS
1-MP), tetrasaccharide bisphosphate (TSBP) which lacks
the terminal Kdo, and tetrasaccharide 1-monophosphate
(TS 1-MP). One-dimensional
1
H- and
31
P-, and two-dimen-
sional homo- (
1
H,
1
H-DQF-COSY) and heteronuclear
(
1
H,
13
C-,
1
H,
31
P-HMQC) NMR spectroscopy (Figs 5 and 6)
and assignment of all signals (Table 2) revealed that the
carbohydrate structure of the major component of C. tra-

chomatis E LPS consisted of three Kdo- and two GlcpN-
residues. The
1
H-NMR spectrum was almost identical to a
spectrum of PSBP obtained from S. enterica sv. Minne-
sota R595-207. All
1
H-detected spectra contained three
pairs of deoxy-protons  2 p.p.m. with a characteristic shift
of pyranosidic a-Kdo-residues. The resonance frequencies
and
3
J
1,2
coupling constants of signals belonging to the
anomeric protons of GlcpN-residues revealed that one
was a-andtheotherwasb-configured. These residues
Fig. 3. Negative ion CSD ESI-FT-ICR mass spectra of de-O-acylated LPS from C. trachomatis serotype E (A) and L
2
(B) and from Chl. psittaci 6BC
(C). On the right side the enlargements of the mass region of de-O-acylated lipid A are shown.
Fig. 4. Analytical HPAEC of deacylated LPS obtained from C. tra-
chomatis serotype E on CarboPak PA1 (3 250 mm, Dionex) using the
eluents (A) 0.1
M
NaOH and (B) 1
M
NaOAc in 0.1
M
NaOH and

agradientfrom30%to70%ofeluentBin16minataflow-rateof
1mLÆmin
)1
.
444 H. Heine et al. (Eur. J. Biochem. 270) Ó FEBS 2003
represented the lipid A backbone and an Overhauser
enhancement from B1 to A6a indicated their 1–6-linkage.
Because the signals of deoxy-protons were not well-resolved
and the one-dimensional
31
P-NMR spectrum did not show
any signals indicating several ionic states we have performed
all measurements in 10 m
M
NaOD. Under these conditions,
31
P signals were sharp and proton signals were more
dispersed. However, the signal of the anomeric carbon of
the b-GlcpN was absent from the spectrum under these
conditions and could not be assigned. The severe overlap of
signals belonging to H-4 and H-5 of all Kdo-residues
nevertheless made the unequivocal assignment of spin
systems impossible by
1
H,
1
H-COSY. However, they were
identified by the ROE contact between the equatorial deoxy
proton of residue C and proton 6 of the second Kdo residue
(D). This ROE is characteristic of an a2–4-linked Kdo-

disaccharide [19]. This substitution also leads to a b-shift of
the C-5 carbon whereas carbon 4 resonates at lower field in
comparison to Kdo which is unsubstituted at this position
[19]. Because Kdo lacks an anomeric proton, the substitution
pattern cannot be identified by direct interresidual ROE
across the glycosidic linkage. However, protons H-8a and
H-8b experience a significant shielding upon substitution
with Kdo at the 8-position which then both resonate at
approximately 3.6 p.p.m. vs. 3.7 and 3.9 p.p.m. when
unsubstituted [19]. In accordance with published data, the
substitution of the 8-position of Kdo residue D by the third
Kdo (E) was also indicated by the downfield shifts of protons
H-6 and H-7 of residue D [12]. Two phosphate groups were
identified by
31
P-NMR spectroscopy and the
31
P,
1
H-HSQC
(Fig. 6) spectrum revealed that they were located at positions
A1 (
31
Psignalatd 3.64 p.p.m) and B4 (
31
Psignalatd
4.99 p.p.m) and thus belonged to the lipid A backbone.
In order to verify the assignment, the NMR spectra were
compared with the spectra of PSBP obtained in larger
amounts from S. enterica sv. Minnesota R595-207 [14]

which were measured under the same conditions and
unequivocally assigned by
1
H,
13
C-HSQC,
1
H,
13
C-HMBC,
1
H,
1
H-COSY and
1
H,
1
H-ROESY. The spectra were, apart
from minor differences, identical to the C. trachomatis E
sample.
Taken together, the chemical shift data and ROE
contacts revealed that the carbohydrate structure of the
major component of deacylated LPS from C. trachomatis E
is aKdo-(2–8)-aKdo-(2–4)-aKdo-(2–6)-bGlcN-4P-(1–6)-
aGlcN-1P and thus identical to the one found in C. tra-
chomatis L
2
. HPAEC analysis revealed that small amounts
of PS 1-MP, TSBP and TS 1-MP were present in the
oligosaccharide mixture which gave rise to NMR-signals

with low intensity. Due to the low relative amounts and the
similarity of the compounds (overlapping signals) we have
not attempted to assign their NMR chemical shifts.
Biological activity of chlamydial LPS in human MNC
We then investigated the biological activity of the chlamy-
dial LPS preparations in comparison with a smooth LPS
from Salmonella, a rough E. coli LPS of the Re-type and a
Fig. 5.
1
H,
13
C-HSQC-NMR spectrum of
deac-ylated LPS from C. trachomatis serotype
Ein10m
M
NaOD.
Fig. 6.
1
H,
31
P-HSQC-NMR spectrum of deacylated LPS from
C. trachomatis serotype E in 10 m
M
NaOD.
Ó FEBS 2003 Structure and function of chlamydial lipopolysaccharide (Eur. J. Biochem. 270) 445
synthetic E. coli lipid A. As can be seen in Fig. 7, the LPS of
both C. trachomatis serotypes had almost identical biologi-
cal activity in terms of stimulation of TNF release from
human MNC. In comparison to the other LPS chemotypes,
however, they exhibited substantially less activity with lower

amounts; a minimum of 10 ngÆmL
)1
was required for the
activation of MNC. However, both C. trachomatis prepa-
rations reached the same stimulatory activity as the other
LPS preparations when higher amounts were used
(100 ngÆmL
)1
). In contrast, Chl. psittaci LPS was about
10-fold less active than those from C. trachomatis.
Activation of human MNC by chlamydial LPS can be
inhibited by anti-CD14 Ig and LPS antagonists
The primary binding receptor for LPS is the glycosylphos-
phatidylinositol (GPI)-linked CD14 molecule [20]. At low
LPS concentrations, blocking of CD14 completely abro-
gated stimulation of human MNC. We investigated the
remaining stimulatory activity of all LPS preparations after
preincubation with mAb MEM-18 against CD14 or the
synthetic LPS antagonist, B1233 (Table 3); both com-
pounds blocked completely the activation of cells by all LPS
tested.
Activation of NF-jB in CHO/CD14 cells
In the next set of experiments, a different readout system
was used which was based on the observation that CHO
cells become hypersensitive to LPS upon transfection with
CD14 [21]. Such transfected cells are currently used widely
to analyze LPS signalling pathways. We used CHO/CD14
reporter cells expressing the human CD25 antigen upon
activation of NF-jB [22] to study the activation pathways of
chlamydial LPS. Both C. trachomatis serotypes as well as

Chl. psittaci were able to induce the translocation of NF-jB
in this system (Fig. 8). Again, chlamydial LPS was less
active than the other LPS chemotypes. In comparison to the
Fig. 7. Relative TNF inducing capacity of different LPS chemotypes in
human MNC. Human MNC were stimulated with increasing concen-
trations of LPS chemotypes S. enterica sv. Friedenau (solid circle),
E. coli F515 (solid square), synthetic hexaacylated compound 506
(solid triangle), C. trachomatis L
2
(open square), C. trachomatis E
(open diamond) or Chl. psittaci (open circle) for 18 h. Subsequently,
TNF content of supernatants was determined by ELISA as described
in Materials and methods. The data are calculated from three different
donors and the TNF value obtained by stimulation with 10 ngÆmL
)1
LPS from S. enterica sv. Friedenau was set to 100%. Confidence
values were below 15%.
Table 2.
1
H- and
13
C-NMR chemical shift data of aKdo-(2–8)-aKdo-(2–4)-aKdo-(2–6)-bGlcN-4P-(1–6)-aGlcN-1P (PSBP) obtained from C. tra-
chomatis E (compound 1) and Salmonella enterica sv. Minnesota R595-207 (compound 2) measured in 10 m
M
NaOD. ND, not determined.
Residue Compound
Chemical shift of
1
H (top) and
13

C (bottom) in p.p.m.
1 2 3ax 3eq 4 5 6a 6b 7 8a 8b
Afi6-aGlcN 1P 1 5.399 2.698 3.619 – 3.489 4.074 3.788 4.254 – – –
94.89 55.90 73.93 – 70.28 71.75 69.65 – – – –
2 5.396 2.703 3.618 – 3.464 4.074 3.757 4.243 – – –
94.91 55.84 73.84 – 70.38 71.77 69.77 – – – –
Bfi6-bGlcN 4P 1 4.423 2.689 3.583 – 3.660 3.655 3.478 3.752 – – –
nd 56.66 76.44 – 73.51 74.80 63.35 – – – –
2 4.425 2.670 3.582 – 3.646 3.651 3.446 3.755 – – –
103.40 56.67 76.52 – 73.52 74.83 63.37 – – – –
Cfi4-aKdo 1 – – 1.897 2.062 4.068 4.121 3.744 – 3.904 3.724 3.889
nd nd 33.96 – 70.77 65.59 71.71 – 70.27 63.56 –
2 – – 1.892 2.056 4.047 4.105 3.731 – 3.904 3.702 3.887
175.23 99.9 34.02 – 70.53 65.64 71.64 – 70.41 63.70 –
Dfi8-aKdo 1 – – 1.834 2.139 4.089 4.137 3.877 – 4.247 3.547 3.612
nd nd 34.96 – 66.24 67.50 72.01 – 70.66 63.23 –
2 – – 1.822 2.132 4.089 4.104 3.848 – 4.178 3.535 3.608
175.93 101.1 34.86 – 66.26 67.43 71.70 – 71.05 63.22 –
E aKdo 1 – – 1.788 2.039 4.105 4.100 3.692 – 3.954 3.752 3.933
nd nd 34.48 – 66.20 66.80 71.84 – 69.98 63.35 –
2 – – 1.779 2.032 4.105 4.098 3.675 – 3.934 3.735 3.942
175.57 100.2 34.48 – 66.23 66.81 71.84 – 69.92 63.46 –
446 H. Heine et al. (Eur. J. Biochem. 270) Ó FEBS 2003
stimulation of human MNC, higher LPS concentrations
had to be used under these conditions. Nevertheless, in these
cells the LPS antagonist, B1233, was able to completely
block activation.
Inhibition of cell activation by monoclonal antibodies
against chlamydial LPS
CHO/CD14 reporter cells were also used to analyze the

inhibitory capacity of mAb against chlamydial LPS. Two
different mAbs, S25-23 and S25-2, directed against the
Kdo(2–8)Kdo(2–4)Kdo trisaccharide of chlamydial LPS or
its 2–8-linked disaccharide portion, respectively, were tested,
and both were able to block completely the activation of
CHO reporter cells by LPS (1 lgÆmL
)1
)ofC. trachomatis
serotype E, whereas the activation by LPS of S. enterica sv.
Friedenau was not affected (Fig. 9).
TLR4/MD-2 and CD14 are required for the activation
of cells by chlamydial LPS
Next, we investigated which receptors were involved in the
activation of cells by chlamydial LPS. In the first set of
experiments, HEK293 cells or stable transfected HEK/
CD14 cells were transiently transfected with either Toll-like
receptor (TLR)2 or TLR4/MD-2 and stimulated with
synthetic lipopeptide or LPS from S. enterica sv. Friedenau
or C. trachomatis serotype E. As can be seen in Table 4,
both LPS activated cells exclusively via TLR4/MD-2 only,
Fig. 9. Inhibition of LPS-induced translocation of NF-jB by monoclo-
nal antibodies against chlamydial LPS. CHO/CD14 cells were stably
transfected with a reporter plasmid containing an NF-jB-responsive
promoter driving the expression of human CD25. After preincubation
with 20 lgÆmL
)1
of different mAb against chlamydial-LPS for 30 min,
cells were stimulated with the indicated amounts [lgÆmL
)1
]ofCtra-

chomatis EandS. enterica sv. Friedenau LPS. After 18 h, expression
of CD25 was determined with PE-anti-CD25 mAb by FACS analysis.
Expression of CD25 is given as geometrical fluorescence intensity
(mean ± SD). One representative out of three experiments is shown.
Fig. 8. Inhibition of LPS-induced translocation of NF-jBbytheLPS
antagonist, B1233. CHO/CD14 cells were stably transfected with a
reporterplasmidcontainingaNF-jB-responsive promoter driving the
expression of human CD25. After preincubation with or without 10 l
M
of the lipid A antagonist B1233 for 30 min, cells were stimulated with
indicated amounts [lgÆmL
)1
]ofLPSfromC. trachomatis E(E),
C. trachomatis L
2
(L
2
), Chl. psittaci (6BC), E. coli F515 (Re), synthetic
hexa-acylated compound 506 (506), S. enterica sv. Friedenau (SeF), or
recombinant IL-1b [UÆmL
)1
]. After 18 h, expression of CD25 was
determined with PE-anti-CD25 mAb by FACS analysis. Expression of
CD25 is given as the geometrical fluorescence intensity (mean ± SD).
One representative out of three experiments is shown.
Table 3. Inhibition of LPS-induced TNF release in human MNC by the lipid A antagonist B1233 and the anti-CD14 mAb MEM-18. Human MNC
were preincubated either with the lipid A antagonist, B1233 (10 l
M
) or the anti-CD14-mAb MEM-18 (10 lgÆmL
)1

) and then stimulated with the
indicated amounts of inducers.
Inducer
Concentration
(ngÆmL
)1
)
Amount of TNF (pg/mL) obtained after activation in the presence of:
No inhibitor
Lipid A antagonist
B1233
Anti-CD14-mAb
MEM-18
None – 336 ± 123
a
300 ± 2 159 ± 23
C. trachomatis E 100 1914 ± 25 69 ± 48 194 ± 19
C. trachomatis L2 100 2553 ± 372 235 ± 11 424 ± 48
Chl. psittaci 100 900 ± 166 191 ± 146 176 ± 13
E. coli F515 100 1951 ± 276 98 ± 47 197 ± 21
Compound 506 10 2270 ± 106 114 ± 47 230 ± 25
S. enterica
sv. Friedenau 10 3138 ± 51 149 ± 72 183 ± 24
a
Data are calculated from two independent experiments.
Ó FEBS 2003 Structure and function of chlamydial lipopolysaccharide (Eur. J. Biochem. 270) 447
whereas the activation of cells by the synthetic lipopeptide
Pam
3
CSK

4
required the expression of TLR2. In addition,
the results of these experiments suggested strongly that
chlamydial LPS may be dependent on the expression of
CD14, as C. trachomatis E LPS was unable to stimulate
IL-8 release from HEK control cells transfected with TLR4/
MD-2. As expected, S. enterica serovar Friedenau LPS
activated both cell lines upon transfection with TLR4/
MD-2, with higher IL-8 release in the CD14 expressing
HEK cells (Table 5).
Finally, we tested whether the activity of all investigated
chlamydial LPS was dependent on the presence of CD14.
Surprisingly, both C. trachomatis serotypes as well as
Chl. psittaci LPS showed no stimulatory capacity unless
HEK cells expressing CD14 were used, even up to a
concentration of 1 lgÆmL
)1
(Table 5). In contrast, HEK
cells could be activated by LPS of E. coli F515, synthetic
lipid A and S. enterica LPSataconcentrationaslowas
1–10 ngÆmL
)1
. However, the expression of CD14 enhanced
the sensitivity towards these LPS chemotypes, in particular
at low concentrations.
Discussion
Members of the Gram-negative bacterial family Chlamydi-
aceae cause diseases in man such as ocular trachoma and
infections of the genitourinary tract of men and women. It is
characteristic of all chlamydial infections that they can

persist for many years without clinical symptoms but active
chronic inflammation is also observed where it is unclear
whether living bacteria are essential for the inflammation or
whether bacterial components can sustain the inflammatory
process. It is definitely known from ocular trachoma that no
living bacteria are found in the late stage of the disease that is
characterized by pannus formation and vascularization of
the cornea. There is good evidence that chlamydial heat
shock proteins are involved in the immunopathology of
trachoma [23,24]. The clinical course and the histopathology
observed in occluding salpingitis is very similar to that of
trachoma and there is an ongoing discussion on the putative
role of Chl. pneumoniae in the development of atheroscler-
osis which is also a chronic inflammation of the arterial walls.
One of the most potent bacterial inducers of proinflam-
matory host responses is LPS that is known to be a
constituent of the chlamydial cell wall [25]. It was first
recognized as the major surface antigen of all chlamydiae
and is still an important diagnostic marker. The formerly
called genus-specific epitope (according to taxonomical
changes now called family-specific epitope) is a Kdo
trisaccharide of the sequence Kdo(2–8)Kdo(2–4)Kdo which
represents the saccharide portion of chlamydial LPS [26].
The epitope and the antibodies directed against it have been
characterized; this subject has been reviewed recently [5].
Although we have a detailed understanding of how
endotoxins act on immune cells in general, little is known
about the endotoxic activity of chlamydial LPS. The main
reason for this is the fact that the extensive purification of
LPS and its analytical control require relatively large

quantities. One should keep in mind that even minor
contamination with proteins, lipoproteins or nucleic acids
may be relevant in the sensitive assays available today.
Nevertheless, there is good evidence from our group [10]
and others [11] that chlamydial LPS is less active than
typical enterobacterial LPS in classical endotoxin assays.
When we decided to investigate the biological activity of
chlamydial LPS in more detail we wanted to base this study
on preparations whose structures had been characterized
fully. We have published the structures of C. trachomatis
serotype L
2
[12] and of Chl. psittaci 6BC [13] earlier as these
chlamydial strains are fast growing. As the distribution of
serotype E is worldwide and the most frequently occurring
serotype in genital C. trachomatis infections we decided to
include this strain in our study and, therefore, first had to
determine its structure. We were able to isolate more than
50 mg of LPS from this serotype, a quantity which allowed
extensive purification and structural analyses. Using 500 lg
of LPS each, we determined that contamination by protein
or nucleic acid was below 0.3%. Similarly purified LPS from
E. coli Re-mutants, where up to 20 mg can be used to
measure putative contaminants, have shown that these are
below 0.02%. As seen from the NMR and MS data, the
structure of LPS from serotype E is very similar to that of
serotype L
2
with two GlcN and three Kdo residues, two
phosphates and a heterogeneous acylation pattern. The

Table 4. Comparison of IL-8 release of differently transfected HEK293 cells. HEK-vec or HEK-CD14 cells were transiently transfected with
indicated plasmids as described in ÔMaterials and methodsÕ and stimulated with synthetic lipopeptide Pam
3
CysSK
4
, LPS from S. enterica serovar
Friedenau or C. trachomatis Eor10ngÆmL
)1
recombinant TNF.
Inducer
Concentration
(ngÆmL
)1
)
Relative IL-8 release (%)
a
HEK-vec
+ TLR2
HEK-CD14
+ TLR2
HEK-vec
+ TLR4-MD2
HEK-CD14
+ TLR4-MD2
None – –
b
–– –
Pam
3
CysSK

4
100 78 ± 17 84 ± 21 – –
1000 92 ± 25 104 ± 21 – –
S. enterica 100 – – 44 ± 14 78 ± 8
sv. Friedenau 1000 – – 29 ± 5 72 ± 9
C. trachomatis E 100 – – 3 ± 2 35 ± 15
1000 – – 3 ± 2 32 ± 7
a
Amount of IL-8 release induced by 10 ngÆmL
)1
recombinant TNF was set to 100%. Data are calculated from two independent experi-
ments.
b
Below 2%.
448 H. Heine et al. (Eur. J. Biochem. 270) Ó FEBS 2003
fatty acid composition is also very similar to that reported
for serotype L
2
[9,12] and F [27]. It seems that the LPS
of different serotypes of C. trachomatis show only minor
variations in the acylation pattern which we have observed
also between different batches of LPS from the same
serotype (data not shown). The acylation pattern is,
however, different from that of Chl. psittaci 6BC where
fatty acids with longer chains are found [13].
The biological activity of all three chlamydial LPS was
tested in a variety of readout systems in comparison to
typical endotoxins. In human peripheral MNC, TNF
production could be induced with LPS concentrations
>10 ngÆmL

)1
(Fig. 7). LPS from C. trachomatis sero-
type E and L
2
were of similar activity and both less active
than smooth LPS by a factor of 100 whereas, LPS from
Chl. psittaci was of lower activity than the two other
chlamydial LPS. Nevertheless, we could show that the
observed activities were specific for LPS and not induced by
other chlamydial components. Thus, we could inhibit the
TNF release in human MNC by MEM-18, a monoclonal
antibody against CD14 and we could block the activity with
the LPS antagonist, B1233, that is a synthetic lipid A-like
molecule (Table 3). The inhibition of chlamydial LPS
activity by B1233 was also determined in a second readout
system in which CD14-transfected CHO cells express, in
response to LPS-induced NF-jB translocation, cell surface-
exposed CD25 that can be quantified by flow cytometry
(Fig. 8). In the same readout system, we showed that the
activity of C. trachomatis serotype E LPS could be inhibited
with monoclonal antibodies which recognize carbohydrate
epitopes occurring exclusively in chlamydial LPS (Fig. 9);
the same antibodies had no effect on the activity of LPS
from S. enterica.
Finally, we investigated for the C. trachomatis serotype E
LPS whether TLR2 or TLR4 were involved in the signalling
pathway. As shown in Table 4, signalling occurred only in
cells expressing TLR4 and not in those expressing TLR2.
To our surprise, the chlamydial LPS was only active in cells
coexpressing CD14 and TLR4, unlike LPS from S. enterica

which was able, although to a lesser extent, to activate cells
that did not express CD14. This experiment was then
repeated including the other two chlamydial LPS an E. coli
rough-mutant LPS (Re-LPS) and synthetic lipid A. The
three typical endotoxins smooth and rough LPS and lipid A
were able to stimulate TLR4-expressing HEK cells in the
absence of CD14 whereas none of the chlamydial LPS
showed activity in absence of CD14 (Table 5). This striking
dissimilarity can not be explained simply by the apparent
difference in the biological activity, otherwise the highest
concentration of chlamydial LPS should have shown at
least some activity. We speculate that for chlamydial LPS
CD14 is required for an efficient transfer to the signalling
receptor TLR4.
In summary, we have determined the chemical structure
of the LPS from another serotype of C. trachomatis that
was necessary as our work on serotype L
2
could not be
assumed to be representative for the whole genus Chla-
mydia and the disease caused by the lymphogranuloma
venerum group (serotype L1–L3) is very different from
genital tract infections caused by the serotypes D through
K, among which serotype E is the most abundant. Some
data are available on the serotype F [27], however, in
that study the acylation pattern of one fraction obtained
after extensive degradation was investigated. It would be
worth investigating one of the serotypes A–C which
represent the biovar trachoma group, however, these
strains show – at least in our hands – very poor growth in

tissue culture.
The fact that chlamydial LPS are endotoxically less active
than typical LPS, e.g. enterobacterial LPS does not exclude
its participation in the pathogenesis of chronic inflammation
in chlamydial infections. We know that endotoxins are a
major cause of multiorgan failure and lethal shock in severe
systemic Gram-negative infection but very little is known
about the role of endotoxins in local, particularly chronic
infections like those caused by chlamydiae (e.g. trachoma,
Table 5. Comparison of LPS-induced IL-8 release of HEK-vec and
HEK-CD14 cells. HEK-vec or HEK-CD14 cells were transiently
transfected with indicated plasmids as described in ÔMaterials and
methodsÕ and stimulated with different amounts of indicated LPS
chemotypes or 10 ngÆmL
)1
recombinant TNF.
Inducer
Conc
(ngÆmL
)1
)
Relative IL-8 release (%)
a
HEK-vec HEK-CD14
Control – –
b
–
C. trachomatis E 0.1 – –
1– –
10 – 14 ± 3

100 3 ± 2 41 ± 9
1000 3 ± 2 39 ± 1
C. trachomatis L
2
0.1 – –
1– –
10 – –
100 – 20 ± 3
1000 3 ± 2 36 ± 9
Chl. psittaci 0.1 – –
1– –
10 – 2 ± 1
100 – 19 ± 1
1000 – 35 ± 1
E. coli F515 0.1 – 18 ± 2
1 7±1 43±12
10 32 ± 1 42 ± 4
100 48 ± 17 45 ± 4
1000 58 ± 21 49 ± 6
Compound 506 0.1 – –
1–10±2
10 9 ± 1 41 ± 1
100 26 ± 7 56 ± 6
1000 48 ± 18 56 ± 7
S. enterica 0.1 – 41 ± 1
sv. Friedenau 1 6 ± 2 71 ± 3
10 30 ± 12 62 ± 9
100 56 ± 25 72 ± 6
1000 35 ± 11 68 ± 3
a

Amount of IL-8 release induced by 10 ngÆ mL
)1
recombinant
TNF was set to 100%. Data are calculated from two independent
experiments.
b
Below 2%.
Ó FEBS 2003 Structure and function of chlamydial lipopolysaccharide (Eur. J. Biochem. 270) 449
salpingitis, respiratory tract infections or atherosclerosis).
The microenvironment of an inflammatory focus may
favour the interaction of LPS with its target cells leading to
local concentrations that also make endotoxins of low
activity significant promoters of inflammatory processes.
Acknowledgements
We thank U. Agge, S. Cohrs, R. Engel, I. Goroncy, H. Lu
¨
thje and V.
Susott for technical assistance and S. Kusumoto (Osaka/Japan) for
lipid A (compound 506) and L. Hawkins (North Andover, MA USA)
for compound B1233 and for reading the manuscript. This work was
supported by the Deutsche Forschungsgemeinschaft grant SFB 470 C1
(to L. B) and grant LI-448 (to B. L).
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