From the Division of Medical and Biochemical Microbiology, Research
Center Borstel, Center for Medicine and Biosciences,
D-23845 Borstel, Germany
In a preliminary
investigation (Süsskind, M., Müller-Loennies, S., Nimmich,
W., Brade, H., and Holst, O. (1995) Carbohydr. Res. 269, C1-C7), we identified after deacylation of lipopolysaccharides (LPS)
from Klebsiella pneumoniae ssp. pneumoniae
rough strain R20 (O1
:K20
) as a major
fraction the
oligosaccharide,
After immunization of BALB/c mice, two monoclonal antibodies
were obtained that were shown to be specific for the core of LPS from
K. pneumoniae ssp. pneumoniae, since they did
not react with LPS or whole-cell lysates of a variety of other
Gram-negative species. Both monoclonal antibodies could be inhibited by
LPS but not by isolated oligosaccharides and are thus considered to recognize a conformational epitope in the core
region.
 |
INTRODUCTION |
Klebsiella pneumoniae is an important
Gram-negative pathogenic bacterium associated with nosocomial
infections (1). It represents a major cause of mortality in
hospital-acquired infections (2) and is (following Escherichia
coli) the second most frequent microorganism isolated from
patients with Gram-negative septicemia. Capsular polysaccharides and
LPS1 are important virulence
factors of K. pneumoniae (1, 2), and structures of the
former (3) and of O-antigens (see Ref. 4 for references) from LPS have
been investigated extensively. A structural investigation of the LPS
core region began only recently (4, 5). In a preliminary investigation
(4) of LPS from the rough mutant K. pneumoniae ssp.
pneumoniae R20 (O1
:K20
), we
isolated from deacylated LPS the major fraction of the carbohydrate backbone and characterized its structure. It possessed a terminal threo-hex-4-enuronic acid residue, which resulted from
-elimination under the alkaline conditions used, indicating a
further substitution at position O-4 of a second GalA residue. In
another investigation (5), the structure of the core region of LPS from
serotype O8 was found to be similar to that of LPS of serotype O1. In
this case, the eliminated substituent was
-D-Glcp linked at O-4 of the second GalA
residue. Both core regions lack phosphate residues, which is so far
unique in enterobacterial LPS.
Several attempts have been made to develop anti-LPS antibodies as
therapeutic agents in septic patients. All three regions of LPS,
i.e. the O-specific polysaccharide, the core
region, and the lipid A, can act as immunogen; however, the O-antigen
expresses high structural variability, even within one species, and
lipid A antigenicity is cryptic in LPS and exposed as a neoantigen only after removal of the lipid A-distal saccharide moiety (6). The core
region has been identified as a suitable target for the induction of
antibodies with broad cross-reactivity among all E. coli
strains. One antibody, named WN1 222-5 (7, 8), recognizes an epitope
present in all core types of E. coli, Salmonella
enterica, and Shigella, which is primarily constituted
by sugars of the inner core region. The outer core oligosaccharide and
phosphate substituents seem to stabilize a given conformation also
required for binding. Since our preliminary investigation of the
Klebsiella core had indicated a similar structure of the
heptose-Kdo region as in E. coli, except for the absence of
phosphate groups, we investigated details of the chemical structure and
its relation to immunoreactivity in Klebsiella.
 |
EXPERIMENTAL PROCEDURES |
Bacteria and Bacterial LPS--
K. pneumoniae ssp.
pneumoniae rough strain R20
(O1
:K20
) was obtained from W. Nimmich
(Institute of Medical Microbiology, University of Rostock, Germany).
Its cultivation and the isolation of LPS from dried bacteria have been
reported earlier (4). LPS from E. coli with core types R1,
R2, R3, R4, and K-12 strain W3100, and S. enterica sv.
Minnesota R60 were obtained from our LPS collection. Clinical isolates
of Enterobacter, Serratia, Hafnia,
Citrobacter, Proteus vulgaris, Morganella
morganii, Neisseria sicca, S. enterica sv.
Panama, Providencia alcaligenes, Pseudomonas
aeruginosa, Shigella sonnei, Hemophilus
influenzae, and Bacteroides fragilis were kindly provided by R. Podschun (Department of Medical Microbiology and Virology, University of Kiel, Germany).
Isolation of Oligosaccharides--
The LPS (208 mg) was
hydrolyzed in 1% acetic acid (100 °C, 45 min), and the precipitate
was removed by centrifugation (2,500 × g, 1 h)
and lyophilized to give lipid A (94 mg, 45% of LPS). The supernatant
was evaporated to dryness, dissolved in water, centrifuged
(100,000 × g, 4 °C, 2 h) and separated by gel
permeation chromatography on TSK HW40 (S). The main fraction (52 mg,
25% of LPS) was separated by high performance anion exchange
chromatography (HPAE), from which, after desalting by gel permeation
chromatography, oligosaccharides 1-5 could be isolated in pure form:
heptasaccharide 1 (3.7 mg, 1.8% of the LPS), octasaccharide 2 (13.4 mg, 6.4% of the LPS), decasaccharide 3 (4 mg, 1.9% of the LPS),
undecasaccharide 4 (4.5 mg, 2.2% of the LPS), and dodecasaccharide 5 (1.8 mg, 0.9% of the LPS).
Preparation of Dephosphorylated Lipid A--
The LPS was
dephosphorylated (48% HF, 4 °C, 48 h; yield was 76% of the
LPS) and hydrolyzed (1% acetic acid, 100 °C, 90 min), and the
dephosphorylated lipid A was extracted with
CH2Cl2, dried, and subjected to laser
desorption mass spectrometry (LD-MS).
General and Analytical Methods--
Compositional analysis, gel
permeation chromatography on TSK HW40 (S), and the determination of the
absolute configuration of Glc and GlcN were performed as described (9).
For the determination of the absolute configuration of GalA, LPS was
methanolyzed (0.5 M methanolic HCl, 85 °C, 40 min) and
centrifuged, and the supernatant was carboxyl-reduced
(NaBH4, 4 °C, 24 h). The content of the resulting D-Gal was measured using D-galactose oxidase
(Sigma) and a peroxidase assay (Boehringer Mannheim), according to the
supplier's instructions. The absolute configuration of the hydroxy
fatty acids was determined by GLC of the phenylethylamide derivatives
(10). Analyses for ester- and amide-linked fatty acids and ester-linked
acyloxyacyl groups were performed as described (11), as was SDS-PAGE
(10 and 18% acrylamide) (12), gels of which were stained with silver nitrate for the detection of LPS (13) or with Coomassie Brilliant Blue
for the detection of proteins. Analytical and semipreparative HPAE were
performed as described (14), with the modification that in
semipreparative HPAE the CarboPac PA1 column was eluted at 3 ml
min
1, using a gradient program of isocratically 1% B
(0-10 min), then linearly to 2% B over 20 min, linearly to 3% B over
10 min, linearly to 5% B over 10 min, and linearly to 7% over
10 min (A, deionized water; B, 1 M sodium acetate, pH
6.0).
Methylation Analyses--
Methylation of carboxyl-reduced
oligosaccharide 1 (2 mg) was carried out according to Ciucanu and Kerek
(15). The methylated sample was hydrolyzed (2 M
trifluoroacetic acid, 100 °C, 2 h), reduced
(NaB2H4), acetylated, and analyzed using
GLC-MS. Methylation analysis of the Kdo region was performed on
dephosphorylated LPS (6 mg) as published (16).
Gas-Liquid Chromatography and Mass Spectrometry--
GLC and
GLC/MS were carried out as described (9). The temperature program in
GLC was as follows: 110 °C for 3 min and then 3 °C
min
1 to 270 °C. LD-MS was performed on a laser
microprobe mass analyzer 500 (Leibold-Heraeus, Cologne, Germany),
equipped with a microprobe and a time-of-flight mass analyzer. The
intensity of the laser beam was 1012 watts
cm
1. Between 5 and 20 pg of the sample were analyzed per
laser pulse. For fast atom bombardment (FAB)-MS, LPS preparations were
dissolved in water at a final concentration of 10 mg ml
1.
Positive and negative ion mode FAB-MS were carried out using the first
two sectors of a Jeol JMS-SX/SX 102A tandem mass spectrometer (Department of Mass Spectrometry, Utrecht University, The Netherlands) operating at 10 kV (mass range, m/z 20-2400) accelerating
voltage. The FAB gun was operated at an emission current of 10 mA with xenon as the bombarding gas. Spectra were scanned at a speed of 30 s for the full mass range specified by the accelerating voltage used
and were recorded and processed on a Hewlett-Packard HP 9000 data
system. In all experiments, the matrix used was thioglycerol (2-3
µl), and the probe was loaded with 1 µl of sample solution.
NMR Spectroscopy--
For structural assignments, NMR spectra
were recorded on solutions (0.5 ml) of oligosaccharides 1, 3, and 4 (2-3 mg each), 2 (13 mg), and 5 (1.5 mg) in
2H2O with a Bruker AMX 600 spectrometer
(1H NMR, 600.13 MHz, 13C NMR 125.77 MHz) at
27 °C. The resonances were measured relative to internal acetone
((CH3)2CO
H 2.225;
C 31.07). One-dimensional 1H,
13C, and 31P NMR spectra were recorded with a
Bruker AM 360 L spectrometer (1H, 360 MHz; 13C,
90.6 MHz; 31P, 145.8 MHz) at 23 °C (CH3CN,
H 1.95 ppm;
C 1.70 ppm) to confirm purity. Coupling constants (±0.5 Hz) were determined on a first order
basis. The phase-sensitive COSY experiments were performed using double
quantum filtering (17, 18) with the Bruker COSYPHDQ microprogram, using
1,024 t1 increments and a sweep width of 2.5 KHz
and collecting 4,096 data points in the F2
dimension. The data matrix was zero-filled in the
F1 dimension to give a matrix of 4 × 2,048 points and was resolution-enhanced in both dimensions by a shifted
sine-bell function before Fourier transformation. The total correlation
spectroscopy (TOCSY) (19) and the nuclear Overhauser enhancement
spectroscopy (NOESY) (20) experiments were performed according to the
method of States et al. (21) in the phase-sensitive mode. A
decoupling in the presence of scalar interactions (DIPSI-2) (22) spin
lock pulse sequence was used in the TOCSY experiment with a mixing time
of 125 ms and a spin lock power of 7,300 Hz. The NOESY experiments were
performed with a mixing time of 200 ms. The intensities of NOESY
cross-peaks were classified as strong, medium, or weak, using
cross-peaks from intraring proton-proton contacts for calibration. The
13C,1H COSY spectra were measured in the
1H detected mode via multiple quantum coherence (HMQC)
(23), using a GARP sequence (24) to decouple 13C couplings
during acquisition. The experiments were carried out in the
phase-sensitive mode by the States time-proportional phase incrementation phase-cycling method (25) acquiring a total of 4,096 points over a sweep width of 4,545 Hz in F2 and
1,024 in F1 over 13,500 Hz. Processing was
performed using standard Bruker software after zero-filling to 2,048 in
F1. The COSY, TOCSY, NOESY, and HMQC spectra
were assigned using the computer program PRONTO (26), which allows the
simultaneous display of different two-dimensional spectra and the
individual labeling of cross-peaks.
Monoclonal Antibodies--
Monoclonal antibodies were prepared
by conventional protocols after immunization of BALB/c mice with
heat-killed bacteria (K. pneumoniae ssp.
pneumoniae rough strain R20) by intravenous injection on
days 0, 7, 14, and 21 with 20, 20, 60, and 120 µg, respectively. On
days 90, 91, and 92, mice received a booster injection of 200 µg
each; the first injection was intravenous, and the last two were
intraperitoneal. Fusion was performed on day 94. Cell culture, media,
and the fusion protocol were described previously (27). Primary
hybridomas were screened by Western blot with bacterial whole-cell
lysates as antigen, and relevant hybridomas were cloned at least three
times by limiting dilution, isotyped using an isotyper kit (Bio-Rad),
and adapted to serum-free medium supplement with Ultroser (Life
Technologies, Inc.). Culture supernatants were prepared in at least
100-ml quantities, and antibodies were purified on protein G-Sepharose
(Pharmacia Biotech Inc.) according to the supplier's instructions.
Purification was ascertained by SDS-PAGE, and protein concentrations
were determined by using the bicinchoninic acid assay (Pierce). mAb WN1
222-5 (IgG2a) binding to the core region of all E. coli strains has been described earlier (7, 8).
Serology--
All serological methods such as enzyme immunoassay
(EIA), using LPS as solid phase antigen, EIA inhibition,
hemagglutination and inhibition of hemagglutination, and the Western
blot technique were described previously (27, 28).
 |
RESULTS |
Structural Analysis of the Carbohydrate Backbone of
LPS--
Compositional analysis of the LPS (structure shown in Fig.
1) revealed the presence of
D-Glc; D-GlcN; D-GalA;
L,D-Hep; D,D-Hep; Kdo;
(R)-3-hydroxytetradecanoic acid; tetradecanoic acid; small amounts of 2-hydroxytetradecanoic acid, hexadecanoic acid, and dodecanoic acid; and phosphate.

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Fig. 1.
The structure of LPS from K. pneumoniae ssp. pneumoniae strain R20.
Nonstoichiometric substitutions: -D-GalA, residue J;
Kdo, residue P; D,D-Hep residues L, M, N, O,
and 16:0.
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In an earlier report (4) on the structure of the core region from LPS
of K. pneumoniae ssp. pneumoniae strain R20, we
presented as a major product obtained from deacylated LPS one
oligosaccharide representing the carbohydrate backbone of lipid A and
part of the core region (Structure 1). This oligosaccharide possessed a
terminal
-threo-hex-4-enuronopyranosyl residue,
indicating the loss of at least one substituent at O-4 of a GalA
residue by
-elimination due to treatment with hot KOH. To elucidate
the complete structure of the core region, we treated the LPS with 1%
acetic acid to cleave the ketosidic Kdo-lipid A linkage and isolated
the resulting core oligosaccharides. In a first step, two fractions
were obtained from gel permeation chromatography of the hydrolysate,
the second of which contained mainly Kdo and was thus not further
investigated. The major fraction was separated by HPAE at pH 6.0 (Fig.
2), yielding oligosaccharides 1-5 (Fig. 3). Monosaccharide analysis of the
oligosaccharides revealed that they all contained D-Glc,
L,D-Hep, Kdo, and D-GlcN. In
oligosaccharides 3-5, D,D-Hep was additionally
identified.

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Fig. 2.
HPAE chromatogram of core oligosaccharides
1-5 obtained after hydrolysis of LPS with 1% acetic acid. The
analysis was performed using a CarboPac PA 100 column (4 × 250 mm, Dionex Corp.) at 1 ml min 1, using a gradient program
of 1-10% 1 M aqueous sodium acetate (pH 6.0) over 18 min.
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Fig. 3.
Structures of oligosaccharides M3, M4, and
1-5. R1, -GalA J in M3, M4, 2, 4, and 5;
R2, D,D-Hep residues L,
M, N, and O (beginning from the reducing terminus) in 3-5. Except
where indicated, hexoses are D-configured.
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The structures of the isolated oligosaccharides were characterized by
NMR spectroscopy and are presented in Fig. 3. Chemical shifts of
1H and 13C NMR spectra were assigned using
COSY, TOCSY, NOESY, and HMQC experiments and the computer program
PRONTO (26). The data are given in Tables I-III. Nine signals were
identified in the anomeric region of the 1H NMR spectrum of
decasaccharide 3 (Figs. 4 and
5, Table
I), six of which (residues E, F, G, L, M,
N, O; compare Fig. 1) were assigned to manno-configured
heptose residues as established by small
JH-1,H-2 coupling constants (
2 Hz, except in
the case of residue E, which possessed JH-1,H-2
2.5 Hz) and by the coupling constants of the other ring protons. Three
other anomeric signals were attributed to two
- (residues H and K)
and one
-linked (I) hexoses possessing an axial H-2, as
characterized by the chemical shifts and
JH-1, H-2 coupling constants (5.322 ppm (3.8 Hz), residue H; 5.127 ppm (4.0 Hz), residue K; 4.551 ppm (7.8 Hz),
residue I). Characteristic high field signals of deoxy protons at 1.810 ppm (H-3ax) and 2.119 ppm (H-3eq) identified
-linked Kdo as the 10th residue. Residue K was established as
-linked GlcN by characteristic high field shifts of H-2 (3.233 ppm)
and C-2 (54.5 ppm). The 13C NMR spectrum was assigned by an
HMQC experiment (Fig. 6, Table II). It contained eight signals in the
anomeric region, one of which (at 100.7 ppm) consisted of three
nonresolved resonances. Low field shifted signals indicated the
substitution at O-2 of residues L and M (C-2 at 78.8 ppm, nonresolved),
at O-4 of H (C-4 at 79.6 ppm), at O-5 of C (C-5 at 74.6 ppm), at O-6 of
K (C-6 at 65.2 ppm), at O-3 (C-3 at 75.8 ppm) and O-4 (C-4 at 74.1 ppm) of E, and at O-3 (C-3 at 79.6 ppm) and O-7 (C-7 at 69.9 ppm) of F. Residues G, I, and N are terminal residues.

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Fig. 4.
Anomeric regions of the 1H NMR
spectra of oligosaccharides 1-5. Spectra were recorded at 600 MHz
and 27 °C. The letters refer to the carbohydrate residues
as shown in Fig. 1, and the Arabic numerals refer to the
proton in the respective residue.
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Fig. 5.
TOCSY spectrum (anomeric region) of
oligosaccharide 3. The spectrum was recorded at 600 MHz and
27 °C. The cross-peaks are labeled as explained in the legend to
Fig. 4.
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Table I
1H NMR chemical shift data for oligosaccharides derived
from K. pneumoniae R20 lipopolysaccharide
Chemical shifts are expressed relative to acetone (2.225 ppm at
27 °C): monosaccharides C-O are as shown in Fig. 1.
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Fig. 6.
Part of the 1H,13C
HMQC spectrum of oligosaccharide 3. The letters refer
to the carbohydrate residues as shown in Fig. 1, and the Arabic
numerals refer to the proton/carbon in the respective residue.
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Table II
13C NMR chemical shift data for oligosaccharides derived from
K. pneumoniae R20 lipopolysaccharide
Spectra were recorded at 150.9 MHz in 2H2O relative to
acetone (31.07 ppm at 27 °C): monosaccharides C-O are as shown in
Fig. 1.
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The sequence of the monosaccharides in decasaccharide 3 was established
by NOESY experiments. Interresidual NOE contacts were identified
between protons H-1 of the substituting residues and the respective
protons at the linkage sites of the glycosylated sugars (Table
III), thus allowing the unambiguous
characterization of the monosaccharide sequence in decasaccharide 3 as
depicted in Fig. 3. In particular, strong NOE signals were identified
between proton N1 (terminal heptose) and protons M1 and M2, M1 and L1 and L2, and L1 and K6. In addition, intraresidual contacts confirmed the
- and
-configuration of residues I and H, respectively.
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Table III
NOE signals of the oligosaccharides 1-5, observed in the NOESY
spectra
Shown are signals that were important for the structural determination;
monosaccharides C-O are as shown in Fig. 1.
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Methylation analysis of decasaccharide 3 employed the
carboxyl-reduction of methyl esters with
NaB2H4, converting galacturonic acid to
galactose. Analysis using GLC/mass spectrometry revealed terminal
glucose and heptose; 6-substituted glucosamine; 2-substituted heptose;
4-substituted galactose
(1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl-6,6'-[2H]galactitol);
and one 3,4- and one 3,7-disubstituted heptose.
Structural analysis of heptasaccharide 1 was performed using COSY and
TOCSY experiments. The anomeric region of the 1H NMR
spectrum (Fig. 4, Table I) possessed six signals, three of which
corresponded to H-1 of heptose residues (residues E (5.078 ppm), F
(5.212 ppm), and G (4.904 ppm)). The
Jn, n+1 coupling constants of the ring
protons of these residues confirmed the manno-configuration.
However, residue E possessed an unusual big
JH-1, H-2 coupling constant of 3 Hz, similar to
that of decasaccharide 3. The other three signals in the anomeric
region were assigned to
-GalA (H, 5.303 ppm),
-GlcN (K, 4.940 ppm), and
-Glc (I, 4.563 ppm). The seventh residue was Kdo. The
assignment of the 13C NMR spectrum (Table II) using HMQC
experiments confirmed these results. Low field shifted signals
indicated the substitution at O-4 of residue H (C-4 at 79.9 ppm), at
O-5 of C (C-5 at 74.6 ppm), at O-3 (C-3 at 76.1 ppm) and O-4 (C-4 at
74.3 ppm) of E, and at O-3 (C-3 at 79.6 ppm) and O-7 (C-7 at 69.9 ppm)
of F. Residues G, I, and K are terminal residues, in the last case
indicated by an upfield shift of about 5 ppm of the signal for C-6
compared with the chemical shifts of decasaccharide 3.
In a NOESY spectrum, interresidual NOE signals (Table III) were
observed between H-1 of residue K and H-4 of H, H-1 of H and H-3 of F,
H-1 of G and H-7a,b of F, H-1 of F and H-3 of E, H-1 of I and H-4 of E,
and H-1 of E and H-5 of C, identifying the structure labeled as
1 in Fig. 3. Thus, heptasaccharide 1 represents a partial
structure of decasaccharide 3.
Eight signals were identified in the anomeric region of the
1H NMR spectrum (Fig. 4, Table I) of undecasaccharide 4, two of which possessed double intensity. They were assigned by
1H,1H (Fig. 7),
and 1H,13C COSY and TOCSY experiments to six
heptose residues (E (5.073 ppm), F (5.105 ppm), G (4.880 ppm), L (5.098 ppm), M (5.262 ppm), and N (4.964 ppm)),
-D-GlcN (K,
5.143 ppm),
-D-GalA (H, 5.262 ppm),
-D-Glc (I, 4.550 ppm), and
-D-GalA (J,
4.473 ppm). The signals of H-3ax (at 1.802 ppm) and
H-3eq (at 2.114 ppm) identified one
-linked Kdo residue
(C). In general, the obtained NMR spectra showed broad similarity to
those of decasaccharide 3; however, the additional
-D-GalA resulted in a shift to lower field of C-6, H-6a,
and H-6b of
D-Glc I. Low field shifted signals in the
13C NMR spectrum (assigned by HMQC and HMQC-TOCSY
experiments) indicated the substitution at O-4 of residue H (C-4 at
79.2 ppm), at O-5 of C (C-5 at 74.4 ppm), at O-6 of I (C-6 at 68.6 ppm)
and K (C-6 at 65.0 ppm), at O-3 (C-3 at 77.4 ppm, the signal for C-4
could not be attributed) of E, at O-3 (C-3 at 79.4 ppm) and O-7 (C-7 at
69.8 ppm) of F, and at O-2 of residues L and M (C-2 at 78.7 ppm,
respectively). Residues G, J, and N are terminal residues.

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Fig. 7.
Part of the 1H,1H
COSY spectrum of oligosaccharide 4. The spectrum was recorded at
600 MHz and 27 °C, and the cross-peaks are labeled as explained in
the legend to Fig. 4.
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The chemical shift values of H-5 of heptose residues M and N and the
primary structure of undecasaccharide 4 were determined by NOESY
experiments. All heptose and hexose residues possessed strong NOE
signals between their H-1 and the corresponding proton at the
attachment site of the substituted sugar (Table III). The NOE signals
from M1 to N5 and from L1 to M5 confirmed the substitution of residues
L and M at O-2. Thus, undecasaccharide 4 contains a heptoglycan of
three residues (L, M, and N) that substitutes O-6 of
-D-GlcN (unit K) as in decasaccharide 3.
The 1H NMR spectrum of octasaccharide 2 possessed seven
signals in the anomeric region (Fig. 4, Table I), which were similar to
the corresponding signals of heptasaccharide 1. An additional doublet
at 4.482 ppm could be assigned by a 1H,1H-COSY
experiment to
D-GalA (J). The chemical shift values of C-6, H-6a,
and H-6b of
D-Glc (I) were upfield in comparison with those of
heptasaccharide 1. The 13C NMR spectrum (Table II) showed
two signals in the region 175-176 ppm, one of which possessed double
intensity, belonging to the three carboxylic carbons of GalA residues H
and J and Kdo C. The monosaccharide sequence was unambiguously
established by NOESY experiments (Table III); thus, octasaccharide 2 represents a partial structure of undecasaccharide 4.
The 1H NMR spectrum of dodecasaccharide 5 contained 10 signals in the anomeric region (Fig. 4, Table I), one of which with double intensity was assigned to the anomeric protons of heptose residues M and N, as determined by the
Jn, n+1 coupling constants. Five other
anomeric protons (at 5.081, 5.125, 4.889, 5.103, and 4.972 ppm)
possessed also small JH-1, H-2 (2 Hz) and were
attributed to the heptose residues E, F, G, L, and O, respectively. The
other four signals with coupling constants JH-1, H-2 3.3-8.1 Hz corresponded to H-1 of
-D-GlcN (K),
D-Glc (I),
D-GalA (H),
and
D-GalA (J), as assigned by 1H,1H-COSY,
TOCSY, and 13C,1H-COSY experiments. NOE
contacts from H-1 of all substituting sugars to their respective
linkage sites were detected (Fig. 8, Table III). The observed NOE contact between protons O1 and N2 proved
the attachment of heptose O to O-2 of heptose N. The shifted 13C resonances of C-2 of N (+9 ppm) and the NOE
signals between N1 and O5 confirmed this substitution. Thus,
dodecasaccharide 5 is composed of undecasaccharide 4 plus one heptose
residue extending the heptoglycan.

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Fig. 8.
NOESY spectrum (anomeric region) of
oligosaccharide 5. The spectrum was recorded at 600 MHz and
27 °C, and the cross-peaks are labeled as explained in the legend to
Fig. 4.
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The distribution of L,D- and
D,D-Hep in the core region was investigated as
follows. The coupling constants in the 1H NMR spectra of
oligosaccharides 1-5 identified the manno-configuration of
all heptose residues. Neutral sugar analyses of heptasaccharide 1 and
octasaccharide 2 identified exclusively
L,D-Hep, proving that no
D,D-Hep is present in the inner core region of
the LPS. Neutral sugar analysis of decasaccharide 3 revealed the
presence of L,D-Hep and
D,D-Hep in a molar ratio of approximately 2:3, indicating that the
1
2-linked heptoglycan consists exclusively of
D,D-Hep. In summary, the complete carbohydrate
backbone of the core region obtained after acetic acid hydrolysis of
LPS from K. pneumoniae ssp. pneumoniae R20 is
represented by the structure of dodecasaccharide 5 (Fig. 3).
Identification of oligosaccharides of various length (Fig. 3,
1-5) illustrates the heterogeneity of the LPS.
A FAB mass spectrum (obtained in the positive mode) of the main
oligosaccharide fraction after gel permeation chromatography gave six
pseudomolecular ions [M + H]+. The ions at m/z
1314, 1490, 1890, 2066, and 2258 were consistent with the molecular
masses of oligosaccharides 1-5. An additional pseudomolecular ion at
m/z 1874 of minor intensity corresponded to the molecular
mass of oligosaccharide 2 plus two heptose residues that could not be
isolated, due to its low abundance.
Analysis of Fatty Acid Substitution--
The nature and
distribution of fatty acids in lipid A were determined by chemical
analysis and LD-MS of dephosphorylated lipid A. The absolute
configuration of 14:0(3-OH) was R. After
de-O-acylation of LPS with 0.25 M methanolic
sodium methoxide followed by esterification of the released fatty acids
with diazomethane, quantification revealed that 14:0, 16:0, trace
amounts of 12:0, and about half of the amount of 14:0(3-OH) had been
released, indicating their ester linkage. Investigation of the fatty
acid content before and after esterification with diazomethane (8)
identified 3-O-tetradecanoyltetradecanoic acid as the sole
ester-linked acyloxyacyl structure. Fatty acid analysis of
de-O-acylated LPS identified 14:0(3-OH) as the sole amide-linked fatty acid. Thus, 14:0 and 16:0 were ester-linked to
amide-bound 14:0(3-OH).
The carbohydrate backbone of the lipid A consists of
D-GlcpN
1
6
-D-GlcpN
1,4'-bisphosphate, which is substituted at O-6' by the core
oligosaccharide (4). Thus, positions O-3, O-4, and O-3' may be
acylated. The location of acyl residues in lipid A was studied by LD-MS
analysis of dephosphorylated lipid A. The LD-mass spectrum obtained in
the positive mode in the presence of CsI is shown in Fig.
9a. Four ion clusters
representing molecular masses were identified, i.e. at
m/z 2053, 2037, and 2009 (heptaacyl lipid A species, minor
portion); at m/z 1814, 1798, and 1770 (hexaacyl lipid A
species, major), at m/z 1588, 1572, and 1544 (pentaacyl lipid A species, minor), and another minor portion of tetraacylated lipid A is indicated by the ion at m/z 1362. The mass
differences of 239, 226, and 210 Da between the hepta-, hexa-, penta-,
and tetraacylated species are due to the absence of one 16:0,
14:0(3OH), and 14:0, respectively. Further heterogeneity was shown by
mass differences of 16 and 28 Da in the ion clusters, indicating the presence of minor portions possessing in addition either 14:0(OH) alone
or both 14:0(OH) and 12:0, replacing 14:0.

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Fig. 9.
Laser desorption mass spectra of
dephosphorylated lipid A in the presence of CsI (a) or NaI
(b). Mtetra,
Mpenta, etc. indicate the ion clusters that
represent the molecular masses of tetraacylated, pentaacylated, etc.
lipid A moieties. A represents the fragment of the reducing
terminus, and B represents that of the the nonreducing
terminus. E and F are diagnostic ions for the
identification of fatty acid distribution as explained under "Results."
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Another LD-MS spectrum in the presence of NaI led to a higher degree of
fragmentation (Fig. 9b). Three ions could be assigned to the
nonreducing GlcN residue substituted by four (two 14:0(3OH), two 14:0,
m/z 1075), three (two 14:0(3OH), one 14:0, m/z
865), or two (one 14:0(3OH), one 14:0, m/z 639) acyl
residues. The ions marked E are of diagnostic value. They
originated from cleavages between C-4/5 and C-5/ring oxygen of the
reducing GlcN during the laser desorption process (29), resulting in
ions that represent the molecular masses of variously acylated
nonreducing GlcN residues plus 42 Da. The ion at m/z 1117 represents such a tetraacylated (two
3-O-tetradecanoyltetradecanoic acid residues) fragment. The difference of 226 Da between E ions at m/z 907 and 681 identified the above mentioned replacement of 14:0 by 14:0(OH) at the
nonreducing GlcN residue. The ions at m/z 893 and 655 originated from the reducing GlcN residue carrying either two
(14:0(3OH)) or three (one additional 16:0) fatty acids,
respectively.
In conclusion, we have characterized the complete structure of LPS from
K. pneumoniae ssp. pneumoniae strain R20 as shown in Fig. 1.
Immunization of Mice and Preparation of Monoclonal
Antibodies--
BALB/c mice were immunized with heat-killed bacteria
and tested on day 28 for serum antibodies against LPS as solid-phase antigen in EIA, and the animal with the highest titer was used for
fusion on day 94. Spleen cells (2.5 × 108) were
fused, 1.1 × 108 cells of which were seeded into 720 primary wells; 575 primary hybridomas were obtained, 73 of which
produced specific antibody, screened by the Western blot technique. For
further selection, the hybridoma supernatants were tested in EIA and
hemagglutination. We selected those two hybridomas (named S47-7 and
S47-19) that showed strong positive reaction in Western blot and EIA
or hemagglutination. After cloning by limiting dilution and isotyping
(Table IV), monoclonal antibodies were
purified by affinity chromatography on protein G and checked for purity
by SDS-PAGE (data not shown).
Specificity of Monoclonal Antibodies--
The two antibodies
S47-7 and S47-19 were tested by hemagglutination using sheep
erythrocytes coated with LPS and by EIA using LPS as antigen. The
results are shown in Table IV. S47-19 reacted at 4 times lower
concentration in hemagglutination than in EIA. Taking into account that
hemagglutination is at least 10 times less sensitive than EIA, this is
a significant difference. The opposite result was obtained with the
other clone S47-7, which reacted at 80 times lower concentration in
EIA than in hemagglutination. Further binding characteristics of the
monoclonal antibodies were determined in EIA by checkerboard titrations
using antigen coating concentrations in a range of 10-0.08 µg/ml and
antibody concentrations of 2.5 ng/ml to 5 µg/ml and 25 ng/ml to 50 µg/ml for S47-7 and S47-19, respectively (see Fig.
10). The data confirmed that S47-7 and
S47-19 reacted in EIA over a broad range of antigen concentrations, whereby mAb S47-7 bound at approximately 40 times lower
concentrations. Next, the oligosaccharides (see Fig. 3) that were
isolated from the LPS after acetic acid hydrolysis or after KOH
treatment were used as inhibitors in inhibition assays. Since clone
S47-19 exhibited better reactivity in hemagglutination, inhibition
tests were done first in this test system; however, strong spontaneous
agglutination of sheep erythrocytes by the oligosaccharides alone did
not allow interpretation of this assay. Therefore, both clones were
tested in EIA inhibition. The results are shown in Table
V. Both clones were efficiently inhibited
by LPS, whereas none of the oligosaccharides was active in
concentrations of up to 1 mg/ml. The specificity of the antibodies was
confirmed in Western blots of different LPS and whole-cell lysates of
clinical isolates separated by SDS-PAGE. Fig.
11a shows the silver-stained
gel after SDS-PAGE of LPS from K. pneumoniae ssp.
pneumoniae strain R20 (lanes 1 and
21), from E. coli with different core types
(lanes 2-6), from S. enterica sv. Minnesota R60
(lane 7), and of whole-cell lysates of various clinical
isolates of different Gram-negative species. Three parallel gels were
run and blotted onto nitrocellulose, two of which were developed in
Western blot with mAb S47-7 or S47-19. Both clones gave the same
pattern and were highly specific for K. pneumoniae; the
result obtained with S47-19 is shown in Fig. 11c.

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Fig. 10.
Binding curves of monoclonal antibodies
S47-7 (a) and S47-19 (b) in EIA using
K. pneumoniae R20 LPS as solid-phase antigen. Plates
were coated with graded concentrations of antigen corresponding to 10 (filled circles), 5 (filled squares), 2.5 (filled triangles), 1.25 (filled diamonds), 0.63 (open circles), 0.32 (open squares), 0.16 (open triangles), and 0.08 (open diamonds)
µg/ml using 50 µl/well and reacted with mAb concentrations
indicated on the abscissa. Values are the means of
quadruplicates (confidence values do not exceed 10%).
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Fig. 11.
Reactivity of monoclonal antibodies with LPS
in Western blot. LPS was separated by SDS-PAGE using a 15%
separating gel and stained with alkaline silver nitrate (a)
or blotted onto nitrocellulose and stained with mAb WN1 222-5
(b) or mAb S47-19 (c). Lanes contain the LPS (2 µg/lane) of K. pneumoniae R20 (lanes 1 and
21), E. coli R1 (lane 2), R2
(lane 3), R3 (lane 4), R4 (lane 5),
K-12 W3100 (lane 6), S. enterica sv. Minnesota
R60 (lane 7), and whole-cell lysates (clinical isolates) of
Enterobacter (lane 8), Serratia
(lane 9), Hafnia (lane 10),
Citrobacter (lane 11), P. vulgaris
(lane 12), M. morganii (lane 13),
N. sicca (lane 14), S. enterica sv.
Panama (lane 15), P. alcaligenes (lane
16), P. aeruginosa (lane 17), S. sonnei (lane 18), H. influenzae (lane 19), and B. fragilis (lane 20).
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In Fig. 11b, the broad cross-reactivity of mAb WN1 222-5 is
shown. This mAb reacted with the LPS of all E. coli core
types and with the S. enterica core. Positive reactions can
be also seen with different clinical isolates such as Hafnia
(lane 10), Salmonella (lane 15), and
Shigella (line 18).
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DISCUSSION |
We present the first complete structure of LPS from K. pneumoniae. In the core region of LPS from K. pneumoniae ssp. pneumoniae R20, we determined as a
novel structure an
1
2-linked
D,D-heptoglycan and found that phosphate
residues present in all core regions of LPS from other enterobacterial
genera (30) are absent. A core structure identified in LPS of a strain
from serotype O8 of K. pneumoniae ssp. ozaenae
(5) displays similarities to those described in this paper; however, it
is terminated by an
-D-Glc residue located in the
position of residue K (
-D-GlcN) of the R20 core and
possesses no D,D-Hep.
The core region of strain R20 shares with all other characterized
enterobacterial core regions (30) the structural element L,D-Hep
1
7L,D-Hep
1
3L,D-Hep
1
5
-Kdo.
In core regions of the S. enterica type (e.g. in
LPS of S. enterica, E. coli, and Hafnia alvei), this structure is not substituted at O-4 of the first L,D-Hep residue by D-Glc or a
disaccharide, but it carries instead phosphoryl substituents. In the
core regions of three other enterobacterial species, i.e.
Yersinia enterocolitica (serotypes O:3 and O:9) (31-33),
Proteus mirabilis, and K. pneumoniae, O-4 of the
first L,D-Hep residue is substituted by
D-Glc or a disaccharide. Of these, the core regions of LPS
from P. mirabilis strain R110/1959 and K. pneumoniae ssp. pneumoniae R20 are structurally most
related, since the first L,D-Hep residue is
substituted in both cases at O-4 by a disaccharide, and the second is
substituted at O-3 by similar trisaccharide moieties
(D,D-Hep
1
6D-GlcNAc
1
4
D-GalA in P. mirabilis strain R110/1959 (34) and
D,D-Hep
1
6D-GlcN
1
4
D-GalA in K. pneumoniae ssp. pneumoniae R20). However,
only one D,D-Hep residue was identified in the
P. mirabilis strain. We are presently investigating this LPS
for the presence of heptoglycan structures.
The herein reported heptoglycan is the first one identified in
enterobacterial LPS; however, similar
1
3-linked
D,D-heptoglycans were identified in LPS of some
strains of Helicobacter pylori (35-37), where they build up
an intervening region between the core region and the O-antigen. Also,
2- and 2/7-substituted D,D-Hep residues were
found in LPS of Helicobacter felis (38), but no structural
data have been reported. One may speculate that
D,D-heptoglycans represent an optional fourth
LPS region (in addition to lipid A, the core region, and the
O-antigen); however, their genetics and biosynthesis are presently not
known. D,D-Hep was identified as a biosynthetic
precursor of L,D-Hep (39-41). It was shown
that a mutation in the gene encoding for the
ADP-D,D-Hep-6-epimerase converting
D,D-Hep to L,D-Hep
(rfaD) resulted in (a) the loss of enzyme
activity, (b) an incomplete LPS terminated by
D,D-Hep, and (c) the accumulation of
ADP-D,D-Hep (40). However, there exist several
LPS that contain both heptoses (42), but it is always
L,D-Hep that is introduced in the early steps
of LPS biosynthesis. D,D-Hep residues are
transferred in later steps, and they may decorate the inner core region
(e.g. in Y. enterocolitica O:3 and O:9) or be
found in more remote parts of the carbohydrate chain (e.g.
in P. mirabilis strain R110/1959, K. pneumoniae
ssp. pneumoniae R20, and strains of H. pylori).
The regulation of this distribution of heptoses between early and later
steps in core biosynthesis is not understood; down-regulation of the
ADP-D,D-Hep-6-epimerase, which converts
D,D-Hep to L,D-Hep in
late core biosynthesis could be one plausible mechanism. However,
heptosyl transferases may use both L,D-Hep and
D,D-Hep as donor substrates in early and late
core biosynthesis, as indicated by the following findings: (a) the terminal D,D-Hep present in
the core region of P. mirabilis strain R110/1959 is replaced
by L,D-Hep in a strain of serotype O28 (43);
(b) an L,D-Hep residue is present at
the nonreducing terminus of the core region of E. coli K-12
(44, 45); and (c) a mutant of S. enterica sv.
Typhimurium in which the gene encoding for
ADP-D,D-Hep-6-epimerase (rfaD) was
defective used D,D-Hep for biosynthesis of the
inner core region (46).
The structure of the lipid A of K. pneumoniae ssp.
pneumoniae R20 possesses all characteristic features of
enterobacterial lipid A, with an asymmetrical acylation pattern. As in
lipid A of S. enterica sv. Minnesota strain R595 and
P. mirabilis strain R45 (47), the lipid A moiety from
K. pneumoniae ssp. pneumoniae R20 contains a
heptaacyl component in varying amounts, depending on the growth
conditions (data not shown). The structure resembles that of lipid A
from K. pneumoniae O3 (48); however, as proven by
31P NMR spectroscopy of de-O-acylated LPS and by
the bisphosphorylated products obtained after alkaline treatment of
de-O-acylated LPS (4), it lacks the
4-amino-4-deoxy-L-arabinose that substitutes the phosphate
residue at O-4' in lipid A from K. pneumoniae O3. The
structure of lipid A from K. pneumoniae ssp.
pneumoniae R20 is also similar to that of P. mirabilis (47).
Due to the presence of structurally conserved elements in
enterobacterial core regions, anti-core antibodies with broad
cross-reactivity and cross-protectivity are of tremendous biomedical
interest particularly for the immunotherapy of Gram-negative sepsis. We
have reported on a monoclonal antibody named WN1 222-5 recognizing the
core region of S. enterica, E. coli, and
Shigella but not that of P. aeruginosa, P. mirabilis, and K. pneumoniae LPS (7, 8). The lack of
reactivity of K. pneumoniae LPS with WN1 222-5 can be
attributed to the structural features of this LPS, the presence of
uronic acids and the heptoglycan, substitution at O-4 of the second
L,D-Hep residue by saccharide structures
instead of a phosphate residue, and the complete lack of phosphate
groups in the core region. We have obtained two monoclonal antibodies
with specificity for the distinct structural features of the core
region of K. pneumoniae ssp. pneumoniae R20 LPS.
Both antibodies do not react with LPS from a broad variety of other
Gram-negative bacteria, including clinical isolates (see Fig. 11). The
antibodies bind to LPS from all O-serotypes of K. pneumoniae
(data not shown). Therefore, we are presently investigating the
potential use of these mAbs to differentiate rapidly
Klebsiella strains from other Gram-negative bacteria in a
clinical setting. The epitope specificity of the mAbs could not be
fully determined, since the preparation of the complete carbohydrate
backbone by KOH treatment is not possible due to the elimination
reaction described. Nevertheless, the following discussion may help to
add to our knowledge of LPS immunoreactivity. mAb WN1 222-5 requires
for binding the sugar residues of the inner core region and the lipid A
backbone but no acyl residues. Removal of phosphate groups from LPS or
lack of the outer core region did not abolish binding of the antibody, but LPS lacking both the phosphates and the outer core region did not
bind anymore (7). These data suggested that mAb WN1 222-5 binds to
sugars of the inner core region in a specific conformation that is
stabilized by the outer core region and phosphate groups. The data
reported here further support this hypothesis. Although the
oligosaccharides obtained after acid hydrolysis and KOH treatment represent the outer and inner core region, respectively, none of them
was able to inhibit the mAbs. Considering that only three or four
monosaccharide residues fit into the binding pocket of immunoglobulins
(49), the epitope seen by our antibodies must be present in the
isolated oligosaccharides, since they represent broadly overlapping
part structures of the carbohydrate backbone. The lack of fatty acids
also cannot be the reason for the negative outcome of the inhibition
assays, since the LPS of K. oxitoca mutant strain R29, the
core structure of which comprises residues C, D, E, F, and I (see Fig.
1),2 was also not active. In
addition, we have shown for many other antibodies against LPS and even
against lipid A that none of them required acyl chains for binding (6).
In summary, we consider the postulation of a conformational epitope is
the most plausible interpretation.
One should also note the fact that the terminal heptoglycan did not
induce specific antibodies, which could be expected considering the
rare occurrence of this structure in nature. We have observed that
rough LPS from S. enterica sv. Minnesota chemotypes
Rd1 and Rd2, which both terminate with
L,D-Hep, were very poor immunogens for mice
(50). Obviously, the manno-configurated heptoses resemble mannose that is present in many glycoconjugates of procaryotes and
eucaryotes, thus not allowing an immune response.
We thank K. Bock and B. Petersen (Carlsberg
Laboratory, Valby, Denmark) for 600-MHz NMR spectroscopy; the
Carlsberg Laboratory for providing temporary accommodation and
facilities to M. Süsskind; B. Lindner for LD-MS; M. M. A. Olsthoorn and J. E. Thomas-Oates (Bijvoet Center for
Biomolecular Research, Utrecht University, The Netherlands) for FAB-MS;
M. Willen and S. Ruttkowski for expert technical assistance; H.-P.
Cordes for help with 360-MHz NMR spectroscopy; H. Moll for help
with GLC-MS; W. Nimmich (Institute of Medical Microbiology,
University of Rostock, Germany) for the strain; R. Podschun (Department
of Medical Microbiology and Virology, University of Kiel,
Germany) for various clinical isolates; and M. Richter for cultivation
of bacteria.