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INTRODUCTION |
Chlamydia are obligatory intracellular bacteria (1)
causing acute and chronic infections in animals and humans (2, 3). Chlamydia trachomatis is the world's leading cause of
preventable blindness in developing countries in North Africa and Asia.
It is also a predominant pathogen in sexually transmitted urogenital infections in developed countries and is considered as the major cause
of secondary infertility in women resulting from tubal damage and
occlusion. Reactive arthritis is another syndrome resulting from
primary genital C. trachomatis infection.
Chlamydia pneumoniae is a pathogen of the respiratory tract
with which more than half of the adult population is infected in
different geographic areas all over the world (4). It is well
established that the infection is usually mild in immunocompetent hosts, but severe pneumonias are observed in immunocompromised patients. There is an ongoing discussion on the putative association of
C. pneumoniae infections with atheroscleriosis in general
and myocardial infarction in particular (5). If this hypothesis would
be verified it is necessary to identify the mechanisms by which
chlamydiae can provide a continuous stimulus of inflammation, which at
the same time would help to understand the chronic inflammatory processes in trachoma and pelvic inflammatory disease.
Chlamydiae possess a lipopolysaccharide
(LPS)1 of low endotoxic
activity (6, 7), which is attributed to the higher hydrophobicity of
its lipid A moiety with fatty acids of longer chain length and the
presence of nonhydroxylated fatty acids ester-linked to the sugar
backbone. Chlamydial LPS harbors also a genus-specific epitope composed
of a 3-deoxy-D-manno-oct-2-ulopyranosonic acid (Kdo) trisaccharide with the sequence
Kdo
2
8Kdo
2
4Kdo
2
,which is surface-exposed and highly
immunogenic (8). It is used in clinical microbiology laboratories as a
marker for the whole genus and can be detected with monoclonal
antibodies against it. This genus-specific Kdo trisaccharide is
biosynthetically assembled by a single Kdo transferase and can be
synthesized by recombinant Escherichia coli bacteria
expressing chlamydial Kdo transferase genes (9). The availability of
LPS of such recombinant bacteria actually allowed us to determine the
chemical and antigenic structure of the Kdo region (10), because
chlamydial LPS was, due to its obligate intracellular growth, so far
only available in quantities not allowing a structural analysis with
the methods used at that time. With the improvements in analytical
chemistry, particularly in NMR spectroscopy and mass spectrometry, the
required amounts of LPS were reduced to levels that can now be prepared
even from tissue culture-grown chlamydiae. Recently, Qureshi et
al. (11) have investigated by mass spectrometry the acylation
pattern of the lipid A moiety of the LPS of C. trachomatis
serotype F. Here, we report the structure of the phosphorylated
carbohydrate backbone of chlamydial LPS as determined by NMR
spectroscopy, which, together with compositional analysis, mass
spectrometric data on the de-O-acylated LPS, and the data of
Qureshi et al. (11), show the first complete structure of a
major LPS molecular species in C. trachomatis.
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EXPERIMENTAL PROCEDURES |
General--
The following experiments were performed as
described: the determination of the absolute configuration of GlcN
(12), analytical procedures for the quantification of Kdo, GlcN, and
phosphate (12), and high-performance anion-exchange chromatography
(13), with the modification that in semi-preparative high-performance anion-exchange chromatography, the column was eluted with a linear gradient of 30-70% 1 M sodium acetate in 0.1 M NaOH for 16 min, and then eluted isocratically with 70%
1 M sodium acetate in 0.1 M NaOH, and that
fractions were desalted by gel-permeation chromatography (P5-Econo-Pac
cartridges, Bio-Rad, Munich, Germany). The fatty acid analyses were
performed according to Wollenweber and Rietschel (14).
Cultivation of Bacteria--
C. trachomatis serotype
L2 was grown in monolayer cultures of mycoplasma-free L929 cells in
multilayer trays (NUNC, 10 × 600-cm2) for 2 days.
The cultures were killed by the addition of 0.5% (w/v) phenol, and the
chlamydial elementary bodies were sedimented with the cell debris by
centrifugation (Beckman J2-21 centrifuge, JA10 rotor, 9,000 × g) yielding 2.88 g from 50,000 cm2 of
infected cells. LPS was obtained by using the phenol/water method (15),
modified as follows. The sediment was suspended in 75 ml of 45%
phenol, containing 2% N-lauroylsarcosine sodium salt in
water (w/v), heated at 68 °C for 10 min, and cooled on ice. After
centrifugation (3,000 × g, 30 min), the water phase was removed and the phenol phase was extracted again with 35 ml of 2%
aqueous N-lauroylsarcosine sodium. After centrifugation and
separation as before, the extraction was repeated again. The water
layers were combined, dialyzed against water, and lyophilyzed (yield
261 mg), then extracted twice with 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 dropwise addition of water. The precipitated LPS was washed
with acetone (yield 25.6 mg) dissolved in water (5 mg/ml) and
precipitated with 10 volumes of ethanol/acetone 9:1 (v/v), dried,
dissolved in water (5 mg/ml), and freeze-dried (yield 22.3 mg).
Deacylation Procedure--
Deacylation of LPS (2 mg) was
according to the method of Holst et al. (16) with the
modifications that reactions were performed in the same reaction tube
and that, after extraction of the fatty acids, the water phase was
dialyzed using a dialyzer system (volume, 1 or 1.5 ml, Sialomed, CA)
with cellulose acetate membranes (molecular weight cut off, 500).
Matrix-assisted Laser Desorption Ionization Mass Spectrometry
(MALDI-MS)--
MALDI-MS of LPS and de-O-acylated LPS was
performed with a Bruker-ReflexII (Bruker-Franzen Analytik, Bremen,
Germany) in the linear time-of-flight configuration, with continuous
ion extraction in the negative ion mode at an acceleration voltage of
30 kV. Samples (<10 nmol) were dispersed in 15 µl of aqueous
triethylamine solution (0.18 M) and treated with small
amounts of Amberlite IR-120 (H+) cation-exchanger (Merck,
Darmstadt, Germany) to remove excess sodium and potassium ions. The
solutions were mixed with an equal volume of matrix solution (0.5 M 2,4,6-trihydroxyacetophenone in methanol; Aldrich,
Steinheim, Germany) and aliquots of 0.5 µl were deposited on a
metallic sample holder and analyzed immediately after drying in a
stream of air. The mass spectra shown are the sum of at least 50 laser shots.
NMR Spectroscopy--
One-dimensional 1H-NMR and
two-dimensional 1H,1H and
1H,13C NMR (pD 5.0) spectra were recorded with
a Bruker AMX-600 spectrometer, using a microprobe head (Bruker PHTXI
600SB H-C/N-D-02.5) and Bruker standard software. The 1H
resonances were measured relative to internal acetone (2.225 ppm), and
coupling constants were determined on the first-order basis (±0.5 Hz).
The assignment of the proton chemical shifts was achieved by
correlation spectroscopy, total correlation spectroscopy, and
double-quantum-filtered correlation spectroscopy experiments. The
assignment of carbon chemical shifts was achieved by
1H,13C heteronuclear multiple quantum coherence
experiments and by comparison to published 13C NMR data
(13, 17, 18). Nuclear Overhauser effect contacts were identified using
rotating frame nuclear Overhauser effect spectroscopy and nuclear
Overhauser effect spectroscopy experiments. 13C resonances
were determined relative to internal dioxane (67.4 ppm).
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RESULTS |
Isolation and Characterization of the LPS--
Preliminary
experiments had shown that the extraction of chlamydial elementary
bodies together with the cell debris gave higher yields of LPS than
applying the phenol/chloroform/light petroleum method to purified
elementary bodies. However, the LPS was equally distributed between the
water and phenol phase. When 2% of N-lauroyl sarcosyl
sodium were added to the phenol/water mixture, the large majority
(>90%) of the LPS was found in the water phase (data not shown).
Because phenol/water-extracted LPS contains a considerable amount of
protein and nucleic acid, the extracted material was further purified
by the phenol/chloroform/light petroleum method and by precipitation
with ethanol/acetone from an aqueous solution. Sugar and phosphate
analyses of the LPS identified Kdo, GlcN, and phosphate in the molar
ratio of approximately 2.8:2.0:2.1, respectively. Fatty acid analysis
revealed the presence of tetradecanoic, iso- and anteiso-branched
pentadecanoic, hexadecanoic, octadecanoic, and eicosanoic acids as
major fatty acids. Hexadecanoic, heptadecanoic, nonadecanoic, and
heneicosanoic acids (all present as iso- and anteiso-branched fatty
acids) are minor ester-linked fatty acids. It should be noted that no
ester-linked 3-hydroxy fatty acids were identified.
(R)-3-Hydroxyeicosanoic acid (158 nmol/mg LPS) was found to
be the major amide-linked fatty acid, and
(R)-3-hydroxyoctadecanoic (47 nmol/mg),
(R)-3-hydroxynonadecanoic (traces), iso- and
anteiso-branched (R)-3-hydroxyeicosanoic (traces), iso- (43 nmol/mg) and anteiso- (71 nmol/mg) branched
(R)-3-hydroxyheneicosanoic, and
(R)-3-hydroxydodecosanoic (16 nmol/mg) acids are present as
minor amide-linked fatty acids.
MALDI-MS Analysis--
Molecular mass analysis of the purified LPS
by MALDI-MS revealed the presence of molecular species consisting of
lipid A (two GlcN and two phosphate residues, and 4-5 fatty acid
residues) alone or substituted by 1, 2, or 3 Kdo residues (data not
shown). No higher masses were detected. Because the mass resolution was low due to a considerable heterogeneity in fatty acid substitution and
cation attachment, de-O-acylated LPS was prepared and
analyzed using MALDI-MS (Fig. 1). The
mass spectrum exhibited one major ion at m/z
1781.1 corresponding to an [M-H]
ion consisting of 3 Kdo, 2 GlcN, 2 phosphate, and 2 amide-bound (R)-3-hydroxyeicosanoic acid residues. Less intense signals
with mass differences of ±14 (CH2) were additionally
identified, indicating also a heterogeneity of amide-bound fatty acids.
This was confirmed by analysis of amide-bound fatty acids after HCl
hydrolysis of de-O-acylated LPS. Minor ions were observed
that represent molecular species representing de-O-acylated
lipid A alone or substituted by 1 or 2 Kdo residues. However, it could
not be decided to which extent these ions reflect intrinsic biological
heterogeneity or are fragment ions produced in the ion source during
laser desorption. In addition, an ion of low intensity at
m/z 1701.1 corresponding to monophosphorylated
de-O-acylated LPS was found.
Deacylation of LPS and Characterization of the Products--
To
analyze the carbohydrate backbone of the LPS from C. trachomatis L2, we applied the deacylation procedure of LPS
published in Ref. 18 that comprises successive hydrazinolysis and hot KOH treatment. Fig. 2 shows the
high-performance anion-exchange chromatography analyses of aliquots of
the obtained products from LPS of C. trachomatis L2
(Fig. 2A) and recombinant E. coli F515-207 (Fig.
2B) (12). The major peak (peak II) in Fig.
2A possesses a retention time identical to that of
Kdo
2
8Kdo-
2
4Kdo
2
6D-GlcpN
1
6D-GlcpN
1,4'-bisphosphate (pentasaccharide bisphosphate), which has been isolated previously from LPS of E. coli F515-207 (12). In
addition, a small peak (Fig. 2A, peak I) was
detected in the chromatogram with a retention time identical to that of
Kdo
2
4Kdo
2
6D-GlcpN
1
6D-GlcpN
1,4'-bisphosphate (tetrasaccharide bisphosphate) of LPS from E. coli F515-207 (12). From deacylated LPS, oligosaccharide II (Fig.
2A, 110 µg, 5.5% LPS) was isolated using high-performance anion-exchange chromatography and investigated by NMR spectroscopy. The
1H and 13C chemical shift assignments (Table
I) are based on two-dimensional correlation spectroscopy, nuclear Overhauser effect spectroscopy, rotating frame nuclear Overhauser effect spectroscopy, total
correlation spectroscopy (Fig. 3), and a
heteronuclear multiple quantum coherence experiment. Chemical shifts
were similar to those of pentasaccharide bisphosphate (13). Larger
differences were observed in the chemical shifts of H-1 and C-1 of
residue A (see Fig. 4 for labeling of residues), caused by a different pD of the sample in Ref. 13. In the
1H NMR spectrum, two signals were found in the anomeric
region (at 5.64 and 4.65 ppm) that could be assigned to one
-linked [3JH-1,H-2 4.0 Hz] and one
-linked [3JH-1,H-2 7.0 Hz] GlcN
residue (A and B), respectively. The H, P-coupling constant
[3JH-1,P 7.0 Hz] indicated the
substitution of O-1 of the reducing GlcN by a phosphate residue. The
-pyranosidic configuration of Kdo residues C, D, and E was deduced
from the proton signals of H-3ax and H-3eq, which were
in the region 1.7-2.2 ppm, characteristic for deoxyprotons (16, 19).
This was further confirmed by the chemical shifts of the H-4 protons of
residues C, D, and E, which were identified between 4.05-4.15 ppm.
Comparison of the proton signals with published data (13, 18)
identified E as a terminal Kdo residue. The chemical shifts of protons
H-8a,b of residue D were shifted upfield proving (13, 17) the
substitution of Kdo D at O-8 by another Kdo residue. Also, the chemical
shifts of H-6a,b of GlcN residues A and B were shifted downfield,
indicating the substitution of both sugars at O-6. In the
13C NMR spectrum, the signal of C-4 of Kdo C was shifted
downfield (about 4 ppm), whereas a slight
-shift of about 1 ppm was
found for C-5 indicating the substitution of residue C at position O-4. The second phosphate residue was linked to O-4 of residue B, as shown
by the downfield chemical shift of C-4 (73.4 ppm). The downfield shifts
of the C-6 signals of residues A and B confirmed their substitution at
O-6; that of residue A was also proven by a nuclear Overhauser effect
contact between H-1 of residue B and H-6 of residue A. The observed
contacts between protons H-3eq of residue C and
H-6 of residue D proved the sequence Kdo
2
4Kdo
2
(C-D) (13).
No other nuclear Overhauser effect contacts were identified.

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Fig. 2.
Analytical high-performance anion exchange
chromatogram of deacylated LPS from C. trachomatis L2
(A) and E. coli F515-207
(B). TSBP, tetrasaccharide bisphosphate; PSBP,
pentasaccharide bisphosphate.
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Table I
1H and 13C chemical shifts of oligosaccharide II
isolated from LPS of C. trachomatis L2
Spectra were recorded at 600 MHz (1H) and 125.7 MHz
(13C) in 2H2O relative to acetone (1H,
2.225 ppm) or dioxane (13C, 67.4 ppm). For residues A-E see
Fig. 4.
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Fig. 3.
Total correlation spectroscopy spectrum of
Kdo 2 8Kdo 2 4Kdo 2 6D-GlcpN 1 6D-GlcpN
1,4'-bisphosphate isolated from LPS of C. trachomatis
L2. The spectrum was recorded at 600 MHz and 27 °C.
The letters refer to the carbohydrate residues as shown in
Fig. 4, and the Arabic numerals to the protons in the
respective residue.
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Fig. 4.
Structure of de-O-acylated
LPS from C. trachomatis L2. This structure
corresponds to the major peak at m/z = 1781 in Fig. 1 and was determined by chemical analysis, MALDI-MS, and NMR
spectroscopy.
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Taken together, our data establish (i) the structure of
oligosaccharide II as
Kdo
2
8Kdo
2
4Kdo
2
6D-GlcpN
1
6D-GlcpN
1,4'-bisphosphate, which is identical to pentasaccharide bisphosphate
isolated from LPS of recombinant E. coli F515-207 (12) and
(ii) the structure of de-O-acylated LPS as shown
in Fig. 4.
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DISCUSSION |
The increasing biomedical interest in Chlamydia has
inititated investigations more than a decade ago on the molecular
organization of this obligatory intracellular bacterium. Because of its
intracellular growth, these studies were hampered by the availability
of only minute quantities of the microorganism and isolated compounds. When it was found that chlamydial genes can be expressed in E. coli (20), the knowledge on a number of immunogenic proteins grew
rapidly. A complex glycolipid antigen was also recognized at that time
as a major surface antigen with genus specificity but nothing was known
on its chemical or antigenic structure. In the 1980s, independent
reports were published on the relatedness of this antigen to
enterobacterial rough LPS of the Re-chemotype (containing Kdo as the
only core sugar), which was based on compositional analysis,
polyacrylamide gel electrophoresis profiles and serology (7, 21, 23).
Finally, the preparation of monoclonal antibodies against the
genus-specific epitope allowed the identification of a cloned
chlamydial gene as a Kdo transferase, which was able to synthesize the
chlamydial epitope in an E. coli background (9). These
recombinant bacteria gave us access to a structural analysis of the
genus-specific epitope that turned out to be a Kdo trisaccharide of the
sequence Kdo
2
8Kdo
2
4Kdo
2
(10). The
assumption that the same structure occurs also in chlamydial LPS was
based only on the reactivity of monoclonal antibodies and on a mass
spectrometric "finger-print" of a chlamydial LPS fragment (10).
There was never the slightest doubt from our or other groups on the
correctness of this assumption, but when we and others became
interested in the chlamydial LPS as an endotoxin and in its
biosynthesis, it was necessary to investigate again chlamydial LPS to
determine the following: (i) the structure of the lipid
A moiety (which in the LPS of the recombinant bacteria was of the
E. coli type); (ii) the linkage point of the Kdo
to the lipid A region; and (iii) whether components other
than the Kdo trisaccharide were present in the core region. The latter point was of particular interest because meanwhile it became known that
the Kdo transferase of Chlamydia psittaci was able to
assemble a branched Kdo tetrasaccharide in the E. coli
background (13).
Because the endotoxic activity of LPS depends mainly on the structure
of the lipid A moiety (22), we first discuss this part of the molecule.
It is known from earlier reports that chlamydial lipid A is of less
endotoxic potency than enterobacterial lipid A, which was attributed to
the lower number of acyl chains and to their longer chain length (6,
7). Compositional analyses had indicated that there were on the average
two hydroxy and 3.2 nonhydroxy fatty acids in amide- and ester-linkage,
respectively, and two phosphate groups/2 mol of glucosamine suggesting,
in analogy to many lipid A structures, the presence of a
bisphosphorylated glucosamine backbone but the lack of ester-linked
hydroxy fatty acids (7, 23). Recently, Qureshi et al. (11)
showed in a detailed mass spectrometric analysis of a major molecular
monophosphoryl lipid A species that indeed only two hydroxy fatty acids
are present in chlamydial lipid A and that these are exclusively
amide-linked. After the analysis of hundreds of lipid A structures,
this is the first example that positions 3 and 3' of the lipid A
backbone are not substituted with 3-hydroxy fatty acids. These authors also determined the number and distribution of fatty acids showing that
a tetra- and pentaacyl species predominate, the latter containing a
single acyloxyacyl residue in amide-linkage on the distal sugar unit.
This work also confirmed the enormous heterogeneity of the acyl chains
described earlier (7, 23). The structural analysis of the lipid A
backbone and its substitution with phosphate was not determined.
Therefore, we isolated the complete carbohydrate backbone after
successive deacylation with hydrazine and potassium hydroxide and
investigated the purified product by NMR spectroscopy. The data clearly
identified the lipid A backbone as
GlcpN
1
6GlcpN
1,4'
P2. Because glycosidic linkages are not cleaved
under the conditions used for deacylation, the sugar composition of the core region could be analyzed on the same compound. The Kdo
trisaccharide of the sequence Kdo
2
8Kdo
2
4Kdo
2
was
identified by various one- and two-dimensional NMR experiments in
comparison to reference compounds (12), and the linkage of this
trisaccharide to the lipid A moiety was determined as 2
6. MALDI-MS
analysis of de-O-acylated chlamydial LPS identified as a
major fraction a molecular species composed of Kdo, GlcN, phosphate,
and 3-hydroxyeicosanoic acid in a ratio of 3:2:2:2, respectively. This
indicates that the major portion of chlamydial LPS does not contain
components other than those described here, unless these would be
present in hydrazine-labile linkage.
In summary, our data are in agreement with those published by us and
others and add important structural details not known before. Taken
together, the structure of LPS of C. trachomatis serotype L2
is as shown in Fig. 5. It is noted that
the data on fatty acid composition and distribution determined here for
serotype L2 are similar to those described by Qureshi et al.
(11) for serotype F. Based on this knowledge, we have recently started to synthesize the chlamydial lipid A to study its endotoxic activity and its function as an acceptor for chlamydial Kdo transferases, studies which so far could not be done on homogenous compounds.

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Fig. 5.
Chemical structure of a major molecular
species of LPS from C. trachomatis L2. The
structure of the phosphorylated carbohydrate backbone was determined by
NMR spectroscopy; the acylation pattern was determined by fatty acid
analysis and MALDI-MS; the position of the acyloxyacyl residue is drawn
in analogy to the data obtained for lipid A of serovar F (11).
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