Abstract
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,-structure; see text- 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, -structure; see text- (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 alpha1-->2-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-GlcpN4Pbeta1-->6D-GlcpNalpha1P. 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.
Highlights
From the Division of Medical and Biochemical Microbiology, Research Center Borstel, Center for Medicine and Biosciences, D-23845 Borstel, Germany
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)
In the core region of LPS from K. pneumoniae ssp. pneumoniae R20, we determined as a novel structure an ␣132linked D,D-heptoglycan and found that phosphate residues present in all core regions of LPS from other enterobacterial genera [30] are absent
Summary
Bacteria and Bacterial LPS—K. pneumoniae ssp. pneumoniae rough strain R20 (O1Ϫ:K20Ϫ) was obtained from W. 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 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. 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]
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