Identification of a Novel Heptoglycan of alpha 1right-arrow 2-Linked D-glycero-D-manno-Heptopyranose
CHEMICAL AND ANTIGENIC STRUCTURE OF LIPOPOLYSACCHARIDES FROM KLEBSIELLA PNEUMONIAE SSP. PNEUMONIAE ROUGH STRAIN R20 (O1-:K20-)*

Miriam Süsskind, Lore Brade, Helmut Brade, and Otto HolstDagger

From the Division of Medical and Biochemical Microbiology, Research Center Borstel, Center for Medicine and Biosciences, D-23845 Borstel, Germany

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  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,
<AR><R><C><UP><B>Gal</B></UP><B><IT>p</IT><UP>A&bgr;1→6</UP></B></C></R><R><C></C></R><R><C></C></R><R><C><B><IT>threo</IT><UP>-hex-4-enurono</UP><IT>p</IT></B></C></R><R><C></C></R><R><C></C></R><R><C></C></R></AR><AR><R><C><B><IT>Glcp</IT><UP>&bgr;1→4He</UP></B></C></R><R><C><B><UP>3</UP></B></C></R><R><C><B><UP>↑</UP></B></C></R><R><C><B><UP>1 → 3Hep</UP><IT>p</IT><UP>&agr;1</UP></B></C></R><R><C><B><UP>7    </UP></B></C></R><R><C><B><UP>↑    </UP></B></C></R><R><C><B><UP>Hep</UP><IT>p</IT><UP>&agr;1   </UP></B></C></R></AR><AR><R><C><B><UP>p</UP><IT>p</IT><UP>&agr;1→5Kd</UP></B></C></R><R><C><B><UP>4</UP></B></C></R><R><C><B><UP>↑</UP></B></C></R><R><C><B><UP>Kdo&agr;2</UP></B></C></R><R><C></C></R><R><C></C></R><R><C></C></R></AR><AR><R><C><B><UP>o&agr;2→6Glc</UP></B></C></R><R><C><B><UP>4</UP></B></C></R><R><C><B><UP>↑</UP></B></C></R><R><C><B><UP>P</UP></B></C></R><R><C></C></R><R><C></C></R><R><C></C></R></AR><AR><R><C><B><IT>p</IT><UP>N&bgr;1→6Glc</UP><IT>p</IT><UP>N&agr;1→P</UP></B></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R></AR>
<SC><UP><B>Structure</B></UP></SC><UP><B> 1</B></UP>
where Kdo was 3-deoxy-D-manno-oct-2-ulopyranosonic acid and Hepp was manno-heptopyranose. The presence of the threo-hex-4-enuronopyranosyl residue indicated a substituent at O-4 of the second GalA residue linked to O-3 of the second L,D-Hep residue, which had been eliminated by treatment with hot alkali. We now report the complete structure of lipopolysaccharide, which was elucidated by additional characterization of isolated core oligosaccharides and analysis of the lipid A. The substituent at O-4 of the second GalpA is D-GlcpN, which in a fraction of the LPS is substituted at O-6 by three or four residues of D-glycero-D-manno-heptopyranose (D,D-Hepp). The complete carbohydrate backbone of the LPS is as follows,
<AR><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C><SC>d</SC>,</C></R><R><C></C></R><R><C></C></R><R><C><SC><UP><B>d</B></UP></SC><UP><B>,</B></UP><SC><UP><B>d</B></UP></SC><UP><B>-He</B></UP></C></R><R><C><UP><B>2</B></UP></C></R><R><C><UP><B>↑</B></UP></C></R><R><C><SC><UP><B>d</B></UP></SC><UP><B>,</B></UP><SC><UP><B>d</B></UP></SC><UP><B>-Hep</B></UP><B><IT>p</IT><UP>*&agr;1 </UP></B></C></R></AR><AR><R><C></C></R><R><C></C></R><R><C></C></R><R><C><SC><B><UP>d</UP></B></SC><B><UP>,</UP></B></C></R><R><C></C></R><R><C></C></R><R><C><SC><B><UP>d</UP></B></SC><B><UP>-He</UP></B></C></R><R><C><B><UP>2</UP></B></C></R><R><C><B><UP>↑</UP></B></C></R><R><C><B><UP>p</UP><IT>p</IT><UP>&agr;1</UP></B></C></R><R><C></C></R><R><C></C></R><R><C></C></R></AR><AR><R><C></C></R><R><C></C></R><R><C></C></R><R><C><SC><B><UP>d</UP></B></SC><B><UP>-He</UP></B></C></R><R><C><B><UP>2</UP></B></C></R><R><C><B><UP>↑</UP></B></C></R><R><C><B><UP>p</UP><IT>p</IT><UP>&agr;1</UP></B></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R></AR><AR><R><C><B><UP>Gal</UP><IT>p</IT><UP>A*&bgr;1→6Glc</UP><IT>p</IT><UP>&bgr;1→4</UP></B><SC><B><UP>l</UP></B></SC><B><UP>,</UP></B><SC><B><UP>d</UP></B></SC><B><UP>-Hep</UP></B></C></R><R><C><B><UP>3</UP></B></C></R><R><C><B><UP>↑</UP></B></C></R><R><C><B><UP>p</UP><IT>p</IT><UP>*&agr;1→6Glc</UP><IT>p</IT><UP>N&agr;1→4Gal</UP><IT>p</IT><UP>A&agr;1→3</UP></B><SC><B><UP>l</UP></B></SC><B><UP>,</UP></B><SC><B><UP>d</UP></B></SC><B><UP>-Hep</UP><IT>p</IT><UP>&agr;1</UP></B></C></R><R><C><B><UP>7    </UP></B></C></R><R><C><B><UP>↑    </UP></B></C></R><R><C><SC><B><UP>l,d</UP></B></SC><B><UP>-Hepp&agr;1   K</UP></B></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R></AR><AR><R><C><B><IT>p</IT><UP>&agr;1→5Kdo</UP></B></C></R><R><C><B><UP>4</UP></B></C></R><R><C><B><UP>↑</UP></B></C></R><R><C><B><UP>Kdo&agr;2</UP></B></C></R><R><C><B><UP>4    </UP></B></C></R><R><C><B><UP>↑    </UP></B></C></R><R><C><B><UP>do&agr;2*    </UP></B></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R></AR><AR><R><C><B><UP>&agr;2→6Glc</UP></B></C></R><R><C><B><UP>4</UP></B></C></R><R><C><B><UP>↑</UP></B></C></R><R><C><B><UP>P</UP></B></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R></AR><AR><R><C><B><IT>p</IT><UP>N&bgr;1→6Glc</UP><IT>p</IT><UP>N&agr;1</UP><IT>→</IT><UP>P</UP></B></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R></AR>
<SC><UP><B>Structure</B></UP></SC><UP><B> 2</B></UP>
(L-glycero-D-manno-heptopyranose; L,D-Hepp), where all hexoses possess the D-configuration. Sugars marked with an asterisk are present in nonstoichiometric amounts. The structure is unique with regard to the presence of an alpha 1right-arrow2-linked D-glycero-D-manno-heptoglycan (oligosaccharide), which has not been described to date, and does not contain phosphate substituents in the core region. Fatty acid analysis of lipid A identified (R)-3-hydroxytetradecanoic acid as sole amide-linked fatty acid and (R)-3-hydroxytetradecanoic acid, tetradecanoic acid, small amounts of 2-hydroxytetradecanoic acid, hexadecanoic acid, and traces of dodecanoic acid as ester-linked fatty acids, substituting the carbohydrate backbone D-GlcpN4Pbeta 1right-arrow6D-GlcpNalpha 1P. The nonreducing GlcN carries four fatty acids, present as two 3-O-tetradecanoyltetradecanoic acid residues, one of which is amide-linked and the other ester-linked to O-3'. The reducing GlcN is substituted in a nature fraction of lipid A by two residues of (R)-3-hydroxytetradecanoic acid, one in amide and the other in ester linkage at O-3. Two minor fractions of lipid A were identified; in one, the amide-linked (R)-3-hydroxytetradecanoic acid at the reducing GlcN is esterified with hexadecanoic acid, resulting in 3-O-hexadecanoyltetradecanoic acid, and in the second, one of the 3-O-tetradecanoyltetradecanoic acid residues at the nonreducing GlcN is replaced by 3-O-dodecanoyltetradecanoic acid. Thus, the complete structure of LPS is as shown in Fig. 1.

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
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 beta -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 alpha -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
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Abstract
Introduction
Procedures
Results
Discussion
References

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 delta H 2.225; delta 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, delta H 1.95 ppm; delta 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
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Abstract
Introduction
Procedures
Results
Discussion
References

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: beta -D-GalA, residue J; Kdo, residue P; D,D-Hep residues L, M, N, O, and 16:0.

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 beta -threo-hex-4-enuronopyranosyl residue, indicating the loss of at least one substituent at O-4 of a GalA residue by beta -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, beta -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.

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 alpha - (residues H and K) and one beta -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 alpha -linked Kdo as the 10th residue. Residue K was established as alpha -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.

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 alpha - and beta -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.

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 alpha -GalA (H, 5.303 ppm), alpha -GlcN (K, 4.940 ppm), and beta -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)), alpha -D-GlcN (K, 5.143 ppm), alpha -D-GalA (H, 5.262 ppm), beta -D-Glc (I, 4.550 ppm), and beta -D-GalA (J, 4.473 ppm). The signals of H-3ax (at 1.802 ppm) and H-3eq (at 2.114 ppm) identified one alpha -linked Kdo residue (C). In general, the obtained NMR spectra showed broad similarity to those of decasaccharide 3; however, the additional beta -D-GalA resulted in a shift to lower field of C-6, H-6a, and H-6b of beta 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.

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 alpha -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 beta D-GalA (J). The chemical shift values of C-6, H-6a, and H-6b of beta 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 alpha -D-GlcN (K), beta D-Glc (I), alpha D-GalA (H), and beta 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.

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 alpha 1right-arrow2-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-GlcpNbeta 1right-arrow6alpha -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."

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).

                              
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Table IV
Reactivity of selected monoclonal antibodies against K. pneumoniae R20 in various serological assays

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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Table V
Inhibition of mAbs S47-7 and S47-19 in EIA
The solid phase antigen was LPS (250 ng/well).


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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).

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).

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha 1right-arrow2-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 alpha -D-Glc residue located in the position of residue K (alpha -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-Hepalpha 1right-arrow7L,D-Hepalpha 1right-arrow3L,D-Hepalpha 1right-arrow5alpha -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-Hepalpha 1right-arrow6D-GlcNAcbeta 1right-arrow4alpha D-GalA in P. mirabilis strain R110/1959 (34) and D,D-Hepalpha 1right-arrow6D-GlcNalpha 1right-arrow4alpha 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 alpha 1right-arrow3-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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Research Center Borstel, Parkallee 22, D-23845 Borstel, Germany. Tel.: 49-4537-188472; Fax: 49-4537-188419.

  

1 The abbreviations used are: LPS, lipopolysaccharide; Kdo, 3-deoxy-D-manno-oct-2-ulopyranosonic acid; D,D-Hep, D-glycero-D-manno-heptose; L,D-Hep, L-glycero-D-manno-heptose; HPAE, high performance anion exchange chromatography; GLC, gas-liquid chromatography; PAGE, polyacrylamide gel electrophoresis; LD, laser desorption; FAB, fast atom bombardment; MS, mass spectrometry; COSY, correlated spectroscopy; NOE, nuclear Overhauser enhancement; NOESY, nuclear Overhauser enhancement spectroscopy; TOCSY, total correlation spectroscopy; HMQC, heteronuclear multiple quantum coherence; EIA, enzyme immunoassay.

2 Süsskind, M., Brade, H., and Holst, O., unpublished observations.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Montgomerie, J. Z. (1979) Rev. Infect. Dis. 1, 736-753[Medline] [Order article via Infotrieve]
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