2Complex Carbohydrate Research Center, The University of Georgia, 220 River Bend Road, Athens, GA 30602, USA, and 3Departments of Medicine and Microbiology, Emory University School of Medicine, Atlanta, GA 30303, USA and Department of Veterans Affairs Medical Center, Atlanta, GA, USA
Received on February 8, 2001; revised on March 27, 2001; accepted on April 2, 2001.
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Abstract |
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Key words: lipooligosaccharide/lipopolysaccharide/Neisseria meningitidis
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Introduction |
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The structures of the LOS from serogroup B N. meningitidis strain NMB have been determined (Rahman et al., 1998) and are shown in Figure 1. The LOSs of strain NMB are structurally heterogeneous due to several factors. First, the OS that is present in the largest amount (about 75% of the LOSs) is of the L2 immunotype (Figure 1A) which contains the lacto-N-neotetraose
-chain attached to the Hep I residue, and is substituted at O-2 and O-3 of the Hep II residue by
-GlcNAc and
-Glc, respectively. Second, the OS present in about 15% of the LOSs is of the L3 immunotype (Figure 1B), consisting of the lacto-N-neotetraose
-chain, with
-GlcNAc and PEA at O-2 and O-3, respectively, of Hep II. A third novel OS structure (about 9%; Figure 1C) is also present which contains the
-chain and the terminal
-Glc at O-3 of Hep II, but lacks the terminal
-GlcNAc residue that is attached to O-2 of Hep II. Last, a proportion of both the L2 and L3 immunotype LOSs from NMB also contain a terminal sialic acid residue linked to O-3 of the
-chain terminal Gal residue. These findings support the notion that the heterogeneity of meningococcal LOS structures may be generated by postconstruction processing via a variety of enzymes acting on the LOS inner core and terminal
-chain.
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However, an alternative model for the sequence of events that results in LOS inner core modification is possible. In this scenario, the L2 LOS structure is processed from a basal L3 structure by the removal of PEA from the O-3 of Hep II and its subsequent replacement with Glc by LgtG. This model is suggested based on the structure of the truncated LOS produced by CMK1, which is the subject of this report. The CMK1 mutant is a derivative of strain NMB in which rfaK encoding the -1,2-N-acetylglucosaminosyl transferase has been insertionally inactivated. Previous glycosyl composition and linkage analysis showed that the LOS from CMK1 lacked both the GlcNAc that is normally attached to O-2 of Hep II and the complete
-chain region that is normally attached to Hep I (Kahler et al., 1996a
,b). The results described herein show that the CMK1 OS has PEA substituents at both O-3 and O-6 of Hep II. Because the O-3 PEA is characteristic of an L3 LOS structure and O-6 PEA is typical of the predominant L2 LOS structure expressed by strain NMB, the alternative model is proposed in which the L3 structure is processed into the L2 structure. The implications of this model for meningococcal LOS biosynthesis and the use of this structure as a vaccine candidate are discussed.
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Results |
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Methylation analysis using the modified procedure designated to determine Kdo and sialic acid linkages (Edebrink et al., 1994) showed that the intact LOS contained terminally and 4,5-linked Kdo and did not contain detectable levels of sialic acid.
Nuclear magnetic resonance (NMR) analysis
The glycosyl sequence of the OS and the location of the PEA substituents were verified by NMR analysis. The 1H-NMR spectrum of the OS (Figure 2) shows two anomeric proton signals, labeled A and B. Both signals were assigned to the two -linked L-glycero-D-manno-heptosyl residues due to their small J1,2 coupling (<23 Hz) and their chemical shifts (
> 5.00 p.p.m.). With the aid of correlation spectroscopy (COSY) (spectrum not shown), total correlation spectroscopy (TOCSY) (Figure 3A), and heterogeneous single quantum correlation (HSQC) (spectrum not shown) analyses, the 1H and 13C NMR signals could be assigned as shown in Table III. Relative to residue A, the H-3 and C-3 chemical shifts of residue B are slightly upfield and downfield, respectively. This is consistent with residue B being the 3-linked Hep residue (Hep I), and residue A the terminally linked, PEA-substituted Hep residue (Hep II). The additional signals between
1.7 and 2.7 were characteristic for Kdo H-3 protons. The multiple Kdo signals are due to the fact that mild acid hydrolysis can lead to OSs in which the reducing end Kdo is present as pyranosyl, furanosyl, and anhydro-Kdo residues. The Kdo residue was designated as C. The proton resonances at
3.29 and 4.17 are due to the PEA substituents (O-CH2CH2-NH2 and O-CH2CH2-NH2, respectively).
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Residue B has a strong interresidue NOE contact from BH-1 to CH-5 (in addition to intraresidue NOE contact to BH-2 and BH-3), indicating that residue B is linked to the 5-position of residue C. Thus the trisaccharide element A-B-C was established as
Methylation analysis (described above) indicated that the terminal Hep II residue contained phosphate or PEA substituents at positions 3 and 6. The location of the PEA groups was further established by 31P NMR spectroscopy. The one-dimentional 31P NMR spectrum (Figure 4, top) of the OS showed that it contained three different phosphate NMR signals at 0.30, 0.30, and 1.30. One of these three phosphorus NMR signals, the signal at
1.30, was due to contaminating phosphoglycerol because its chemical shifts were identical to standard phosphoglycerol. The phosphoglycerol probably originates from phospholipid contamination of the original LOS preparation. The locations of PEA groups in this OS were determined by a two-dimensional coupled 1H-31P heterogeneous multiple quantum correlation (HMQC) experiment (Figure 4, bottom). In this experiment the phosphate signal at
0.30 was coupled to an H-6 proton (
4.60) of residue A (Hep II) and to the O-CH2CH2NH2 protons of ethanolamine (
4.17). The other phosphorus signal at
0.30 was coupled to the H-3 proton (
4.41) of residue A (Hep II) and to the O-CH2CH2NH2 protons of ethanolamine (
4.17). These NMR data, together with the methylation results, show that there are PEA groups located at both O-3 and O-6 of residue A (Hep II). Thus, the complete OS structure is:
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Discussion |
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Plested et al. (1999) have recently shown that a monoclonal antibody designated MAB B5 that recognizes the O-3 PEA linked to HepII, reacted with 7090% of all major N. meningitidis serogroups. Although this antibody did not have significant bactericidal activity, it was shown to improve opsonophagocytosis of meningococci. Those isolates that were not recognized by MAB B5 expressed LOS inner cores with O-6 PEA (L2, L4, and L6 immunotypes) or had no PEA (L5 immunotype) substitution of the inner core. Although antibodies have been raised against O-6 PEA LOS inner cores, these antibodies were poorly immunogenic (Verheul et al., 1991
). The model proposed in Figure 5 for the biosynthesis of the LOS inner core suggests that, although successful antibodies may be raised against the LOS inner core with both O-3 and O-6 PEA groups, meningococci have evolved at least two strategies to overcome this potential problem. Thus, the CMK1 OS structure may be a common precursor to many of the LOS immunotypes and, therefore, a potential inner core glycoform for N. meningitidis vaccine development. However, the ability of N. meningitidis to express different inner core structures suggests that multiple glycoforms or glycoforms that are broadly cross-reactive may need to be included in such vaccines.
In summary, the biosynthetic pathway for LOS assembly in N. meningitidis appears to require a Hep2-Kdo2lipid A intermediate with PEA substituents at both the O-3 and O-6 positions of Hep II. Antibodies directed at this inner core structure could be broadly cross-reactive, and, thus, the CMK1 LOS structure may prove useful in meningococcal vaccine development.
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Materials and methods |
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Isolation of LOS and preparation of LOS OSs
The LOS was prepared from 5 g (dry weight) of cells harvested from an overnight aerobic culture, by a modified version of the phenol-chloroformpetroleum ether extraction procedure as previously described (Kahler et al., 1996a). The LOS sample (20 mg) was hydrolyzed in aqueous 1% acetic acid (10 ml) for 2 h at 100°C. The hydrolysate was centrifuged at 10,000 x g for 20 min, and the supernatant was collected. The pellet was washed once with 5 ml of water and centrifuged again. The water wash was added to the supernatant, and any remaining lipid A was extracted with diethyl ether (three times, 5 ml volumes each time). The aqueous phase, containing the OSs, was lyophilized. The lyophilized OSs were dissolved in 0.5 ml of water, filtered with Microfilterfuge tubes containing 0.45-µm pore size Nylon-66 membrane filters, applied to a Bio-Gel P-4 column (70 x 1.6 cm), and eluted with water containing 1% 1-butanol. Fractions were assayed for carbohydrate using the phenolsulfuric acid assay. The OS eluted as a single peak just after the void volume and was lyophilized. The yield of OS was 13 mg.
Preparation of LOS-HF
The LOS and OS samples (10 mg) were placed in 1.5-ml polypropylene tubes. The samples were treated with cold aqueous 48% HF (100 µl) and kept for 24 h at 4°C (Kenne et al., 1993). The HF was removed by flushing under a stream of air, followed by addition of diethyl ether (600 µl) and drying with a stream of air. This step was repeated three times. The dry pellet was dissolved in water and lyophilized. The lyophilized, dephosphorylated OSs were further purified as described in the previous paragraph.
NMR spectroscopy
Samples, 45 mg, were prepared for NMR analysis by a twofold lyophilization from D2O, dissolved in D2O, and analyzed. Spectra were recorded at 60°C. Chemical shifts are reported in p.p.m., using sodium 3-trimethylsilylpropanoate-d4 (H 0.00) and acetone (
C 31.00) as internal references. All NMR spectra were recorded on Varian 300 or 500 MHz spectrometers. The Varian software was used to collect the two-dimensional DQF-COSY, TOCSY, HSQC, and NOESY datasets. The TOCSY experiments contained MLEV17 (Bax and Davis, 1985
) mixing sequences ranging from 60 ms to 320 ms, and the NOESY mixing delay was 200 ms.
Glycosyl composition analyses
The glycosyl composition of the LOS and OS (0.5 mg each) were performed by hydrolysis in 2 M trifluoroacetic acid (0.5 ml) in a closed vial at 120°C for 3 h. The glycoses in the hydrolysate were reduced with NaBH4, acetylated, and analyzed by gas-liquid chromatography (GLC) and combined GLCmass spectrometry (MS) (York et al., 1985). For the determination of Kdo, the LOS sample (0.5 mg) was dried in vacuum and methanolyzed in 1 ml of MeOH-2 N HCl at 80°C for 16 h. The resulting methyl glycosides were trimethylsilylated, and the mixture was analyzed by GLC-MS (York et al., 1985
). The absolute configurations of the glycoses were determined by GLC-MS analysis of the trimethylsilylated (S)-(+)-2-butyl and (S)-(-)-2-butyl glycosides (Gerwig et al., 1979
).
Glycosyl linkage analyses
Glycosyl linkage analysis was carried out using a modified NaOH method (Ciucanu and Kerek, 1984; McConville et al., 1990
). The sample (1 mg) was dissolved in dimethyl sulfoxide (100 µl), powdered NaOH (100 mg) was added, and the reaction mixture was stirred rapidly at room temperature for 30 min. Methylation was performed by the sequential additions of methyl iodide (10, 10, and 20 µl) at 10-min intervals. After an additional 20 min stirring, 1 ml of 1 M sodium thiosulfate was added and the methylated glycans were recovered in the organic phase by extraction with chloroform (0.5 ml x 3). The permethylated product was further purified by reverse-phase chromatography using a Sep-Pak C18 cartridge (Waeghe et al., 1983
). The methylated glycan was hydrolyzed with 2 M trifluoroacetic acid (120°C, 3 h), reduced with NaB2H4, acetylated, and analyzed by GLC and GLC-MS (York et al., 1985
). For determining the Kdo linkage, the permethylated LOS sample (0.5 mg) was dried in vacuum and methanolyzed in 1 ml of methanolic 2 N HCl at 80°C for 4 h. The released partially methylated methyl glycosides were acetylated with a addition of 100 µl pyridine and 100 µl acetic anhydride (120°C, 1 h), and the mixture was analyzed by GLC-MS (Edebrink et al., 1994
).
Lipid A purification
Lipid A was released from the LOS (10 mg) by hydrolysis in aqueous 1% acetic acid (5 ml) at 100°C for 2 h. The precipitated lipid A was collected by centrifugation (10,000 x g). The precipitate was resuspended with water (2 ml) and partitioned with chloroform (2 ml). The chloroform layer, containing purified lipid A, was concentrated to dryness.
Fatty acid analysis
Total fatty acids were released by methanolysis of lipid A with methanolic 1 M HCl at 80°C for 16 h and were trimethylsilylated. The resulting fatty acid methyl esters were analyzed by GLC-MS (York et al., 1985). Ester- and amide-linked fatty acids were distinguished by preferential release of the ester-linked fatty acids using anhydrous sodium methoxide (Wollenweber and Rietschel, 1990
), and the products were analyzed by GLC-MS.
Chromatographic and spectrometric techniques
GLC and GLC-MS analyses were performed using capillary columns (length, 30 m; inner diameter, 0.32 mm) with helium as the carrier. A DB-5 column (J&W Scientific) was used for aminoglycosyl derivatives, and an SP2330 column (Supelco, Bellefonte, PA) was used for the neutral glycosyl derivatives. GLC equipment consisted of HP5890 gas chromatograph equipped with a flame ionization detector (Hewlett-Packard). GLC-MS (electron ionization) was performed using a Hewlett-Packard 5970 MSD.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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Bax, A., and Davis, D.G. (1985) MLEV-17-based two-dimensional homonuclear magnetization transfer spectroscopy. J. Magn. Reson., 65, 355360.[ISI]
Brandtzaeg, P., Kierulf, P., Gaustad, P., Skulberg, A., Bruun, J.N., Halvorsen, S., and Sorensen, E. (1989) Plasma endotoxin as a predictor of multiple organ failure and death in systemic meningococcal disease. J. Infect. Dis., 159, 195203.[ISI][Medline]
Ciucanu, I., and Kerek, F. (1984) A simple and rapid method for the permethylation of carbohydrates. Carbohydr. Res., 131, 209217.[ISI]
Edebrink, P., Jansson, P.-E., Rahman, M.M., Widmalm, G., Holme, T., Rahman, M., and Weintraub, A. (1994) Structural studies of the O-polysaccharide from the lipopolysaccharide of Moraxella (Branhamella) catarrhalis serotype A (strain ATCC 25238). Carbohydr. Res., 257, 269284.[ISI][Medline]
Estabrook, M.M., Mandrell, R.E., Apicella, M.A., and Griffiss, J.M. (1990) Measurement of the human immune response to meningococcal lipooligosaccharide antigens by using serum to inhibit monoclonal antibody binding to purified lipooligosaccharide. Infect. Immun., 58, 22042213.[ISI][Medline]
Estabrook, M.M., Baker. C.J., and Griffiss, J.M. (1993) The immune response of children to meningococcal lipooligosacchrides during disseminated disease is directed primarily against two monoclonal antibody-defined epitopes. J. Infect. Dis., 167, 966970.[ISI][Medline]
Gamain, A., Beurret, M., Michon, F., Brisson, J.-R., and Jennings, H.J. (1992) Structure of the L2 lipopolysaccharide core oligosaccharides of Neisseria meningitidis. J. Biol. Chem., 267, 922925.
Gerwig, G.J., Kamerling, J.P., and Vliegenthart, J.F.G. (1979) Determination of the absolute configuration of monosaccharides in complex carbohydrates by capillary G.L.C. Carbohydr. Res., 77, 17.[ISI]
Kahler, C.M., and Stephens, D.S. (1998) Genetic basis for biosynthesis, structure, and function of meningococcal lipooligosaccharide (endotoxin). Crit. Rev. Microbiol., 24, 281334.[ISI][Medline]
Kahler, C.M., Carlson, R.W., Rahman, M.M., Martin, L.E., and Stephens, D.S. (1996a) Inner core biosynthesis of lipooligosaccharide (LOS) in Neisseria meningitidis serogroup B: Identification and role in LOS assembly of the 1, 2 N-acetylglucosamine transferase (rfaK). J. Bacteriol., 178, 12651273.[Abstract]
Kahler, C.M., Carlson, R.W., Rahman, M.M., Martin, L.E., and Stephens, D.S. (1996b) Two glycosyltransferase genes, lgtF and rfaK, constitute the lipooligosaccharide ice (inner core extension) biosynthesis operon of Neisseria meningitidis. J. Bacteriol., 178, 66776684.[Abstract]
Kahler, C.M., Martin, L.E., Shih, G.C., Rahman, M.M., Carlson, R.W., and Stephens, D.S. (1998) The (2-8)-linked polysialic acid capsule and lipooligosaccharide structure both contribute to the ability of serogroup B Neisseria meningitidis to resist the bactericidal activity of normal human serum. Infect. Immun., 66, 59395947.
Kenne, L., Lindberg, B., Rahman, M.M., and Mosihuzzaman, M. (1993) Structural studies of Vibrio fluvialis M-940 O-antigen polysaccharide. Carbohydr. Res., 242, 181189.[ISI][Medline]
Kulshin, V.A., Zahringer, U., Lindner, B., Frasch, C.E., Tsai, C., Dmitriev, B.A., and Rietschel, E.T. (1992) Structural characterization of the lipid A component of pathogenic Neisseria meningitidis. J. Bacteriol., 174, 17931800.[Abstract]
McConville, M.J., Homans, S.W., Thomas-Oates, J.E., Dell, A., and Bacic, A. (1990) Structure of the glycoinositol phospholipids from Leishmania major, a family of novel galactofuranose containing glycolipids. J. Biol. Chem., 265, 73857390.
Moran, E.E., Brandt, B.L., and Zollinger, W.D. (1994) Expression of the L8 lipopolysaccharide determinant increases the sensitivity of Neisseria meningitidis to serum bactericidal activity. Infect. Immun., 62, 52905295.[Abstract]
Plested, J.S., Gidney, M.A.J., Coull, P.A., Griffiths, H.G., Herbert, M.A., Bird, A.G., Richards, J.C., and Moxon, E.R. (2000) Enzyme linked immunosorbent assay (ELISA) for the detection of serum antibodies to the inner core lipopolysaccharide of Neisseria meningitidis group B. J. Immunol. Meth., 237, 7384.[ISI][Medline]
Plested, J.S., Makepeace, K., Jennings, M.P., Gidney, M.A.J., Lacelle, S., Brisson, J.-R., Cox, A.D., Martin, A., Bird, A.G., Tang, C.M., and others (1999) Conservation and accessibility of an inner core lipopolysaccharide epitope of Neisseria meningitidis. Infect. Immun., 67, 54175426.
Rahman, M.M., Stephens, D.S., Kahler, C.M., Glushka, J., and Carlson, R.W. (1998) The lipooligosaccharide (LOS) of Neisseria meningitidis serogroup B strain NMB contains L2, L3, and novel oligosaccharides, and lacks the lipid-A 4'-phosphate substituent. Carbohydr. Res., 307, 311324.[ISI][Medline]
Verheul, A.F., Braat, A.K., Leenhouts, J.M., Hoogerhout, P., Poolman, J.T., Snippe, H., and Verhoef, J. (1991) Preparation, characterization, and immunogenicity of meningococcal immunotype L2 and L3, 7, 9 phophoethanolamine group-containing oligosaccharide-protein conjugates. Infect. Immun., 59, 843851.[ISI][Medline]
Verheul, A.F.M., Snippe, H., and Poolman, J.T. (1993) Meningococcal lipopolysaccharides: virulence factor and potential vaccine component. Microbiol. Rev., 57, 3449.[Abstract]
Virji, M. (1996) Meningococcal disease: epidemiology and pathogenesis. Trends Microbiol., 4, 466470.[ISI][Medline]
Waeghe, T.J., Darvill, A.G., McNeil, M., and Albersheim, P. (1983) Determination, by methylation analysis, of the glycosyl-linkage compositions of microgram quantities of complex carbohydrates. Carbohydr. Res., 123, 281304.[ISI]
Wollenweber, H.-W., and Rietschel, E.T. (1990) Analysis of lipopolysaccharide (lipid A) fatty acids. J. Microbiol. Meth., 11, 195211.[ISI]
York, W.S., Darvill, A.G., McNeil, M., Stevenson, T.T., and Albersheim, P. (1985) Isolation and characterization of plant cell walls and cell wall components. Meth. Enzymol., 118, 340.[ISI]