Glycobiology, 2001, Vol. 11, No. 8 703-709
© 2001 Oxford University Press
The structure of the lipooligosaccharide (LOS) from the 
-1,2-N-acetyl glucosamine transferase (rfaKNMB) mutant strain CMK1 of Neisseria meningitidis: implications for LOS inner core assembly and LOS-based vaccines
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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The inner core structures of the lipooligosaccharides (LOS) of Neisseria meningitidis are potential vaccine candidates because both bactericidal and opsonic antibodies can be generated against these epitopes. In an effort to better understand LOS biosynthesis and the potential immunogenicity of the LOS inner core, we have determined the LOS structure from a meningococcal rfaK mutant CMK1. The rfaK gene encodes the transferase that adds an
-N-acetylglucosaminosyl residue to O-2 of the inner core heptose (Hep) II of the LOS. The LOS oligosaccharide from this mutant was previously shown to contain only Hep, 3-deoxy-D-manno-2-octulosonic acid (Kdo), and multiple phosphoethanolamine (PEA) substituents (Kahler et al., 1996a
, J. Bacteriol., 178, 1265–1273). The complete structure of the oligosaccharide (OS) component of the LOS from mutant CMK1 was determined using glycosyl composition and linkage analyses, and 1H, 13C, and 31P nuclear magnetic resonance spectroscopy. The CMK1 OS structure contains a PEA group at O-3 of Hep II in place of the usual glucosyl residue found at this position in the completed L2 LOS glycoform from the parent NMB strain. The PEA group at O-6 of Hep II, however, is present in both the CMK1 mutant LOS and parental NMB L2 LOS structures. The structure of the OS from CMK1 suggests that PEA substituents are transferred to both the O-3 and O-6 positions of Hep II prior to: (1) the incorporation of the -GlcNAc on Hep II; (2) the synthesis of the -chain on Hep I; and (3) the substitution of the glycosyl residue at the O-3 Hep II, which distinguishes L2 and L3 immunotypes. The LOS structure of the CMK1 mutant makes it a candidate immunogen that could generate broadly cross-reactive inner-core LOS antibodies. Key words: lipooligosaccharide/lipopolysaccharide/Neisseria meningitidis
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Introduction |
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Neisseria meningitidis serogroup B is an important obligate human pathogen and is the leading cause of bacterial sepsis and meningitis in many countries (Brandtzaeg et al., 1989
; Virji, 1996; Kahler and Stephens, 1998). Antibodies to meningococcal lipooligosaccharides (LOSs) have been detected in convalescent sera (Estabrook et al., 1990, 1993; Plested et al., 2000) and have been shown to have both bactericidal and opsonic functions (Plested et al., 1999, 2000; Verheul et al., 1991; Kahler and Stephens, 1998). N. meningitidis can express at least 12 immunologically distinct LOS structures (Kahler and Stephens, 1998). The different structures, classified as immunotypes, are generated by varying the composition of the -chain oligosaccharide (OS) attached to heptose (Hep) I, and the attachment of alternative glucose (Glc), N-acetyl glucosamine (GlcNAc), and phosphoethanolamine (PEA) substituents to Hep II. Previous studies have shown that the expression of different LOS immunotypes (Moran et al., 1994) and truncated LOS structures effect the level of resistance of meningococci to killing by normal human serum (Kahler et al., 1998), thereby substantiating the importance of these structures for the immunogenicity and pathogenicity of N. meningitidis.
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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Considerable interest has recently focused on elucidating the sequence of steps required for the variable addition of glucose or PEA to Hep II of the meningococcal LOS inner core. In the L2 immunotype LOS, Glc and PEA are attached to O-3 and O-6 of Hep II, respectively, whereas the classic L3 immunotype has PEA attached to O-3 of Hep II with no PEA at O-6 (Gamain et al., 1992
; Verheul et al., 1993). Potentially, the L2 immunotype substitution pattern could be explained by the competitive addition of Glc or PEA to O-3 of Hep II by the corresponding transferases. Therefore, in those isolates expressing L3 LOS structures only, the putative -1,3-glucosyltransferase encoded by lgtG would be inactive either by phase variation or mutation. A recent report supports the view that there is phase variable expression of lgtG, the gene that encodes the putative -1,3-glucosyltransferase (Banerjee et al., 1998).
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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Composition analysis
The neutral glycosyl and fatty acyl residues were determined for the LOS, hydrogen fluoride (HF)–treated LOS (LOS-HF), OS, OS-HF, and lipid A. The results, Table I, verified those of an earlier report (Kahler et al., 1996a
), and showed that the intact CMK1 mutant LOS contains Hep, GlcNAc, and 3-deoxy-D-manno-2-octulosonic acid (Kdo). Also, as noted in the earlier report, the level of Hep increased after HF treatment due to the removal of phosphate and/or PEA substituents that allowed the detection of previously phosphorylated Hep residues. The OS preparation, obtained by mild acid hydrolysis of the LOS, contained only Hep and Kdo, indicating that the GlcNAc residue observed in the intact LOS was derived from the lipid A. Fatty acid analysis of the lipid A preparation showed that it contained dodecanoic, 3-hydroxydecanoic, and 3-hydroxytetradecanoic acids in a ratio consistent with that previously reported for the lipid A from N. meningitidis strain M986-NCV1 (Kulshin et al., 1992). Determination of the absolute configurations of the heptosyl residues present in OS revealed that they all have the L-glycero-D-manno-configuration.
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Glycosyl linkage analysis
The glycosyl linkages for the LOS, LOS-HF, OS, and OS-HF are shown in Table II. The LOS contained both 6-linked GlcNAc and 3-linked Hep (I), and the OS contained only 3-linked Hep. The 6-linked GlcNAc in the intact LOS is due to the presence of lipid A and is not present in the OS because the lipid A was removed by mild acid hydrolysis. Glycosyl linkage analysis after aqueous 48% HF treatment of LOS (LOS-HF) and OS (OS-HF), which removes phosphate and PEA substituents, resulted in the appearance of a terminally linked Hep (II) residue in addition to 3-linked Hep in both the LOS-HF and OS-HF samples (Table II). This result is consistent with an earlier report for the polar rfaK mutant 559 (Kahler et al., 1996a
). The appearance of terminally linked Hep after HF treatment indicates that this residue contained phosphate or PEA substituent prior to HF treatment.
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The locations of PEA or phosphate substituents were determined by methylation of OS, followed by HF treatment and ethylation. In this method the location of an ethyl group indicates the location of the phosphate or PEA substituent in the original LOS or OS sample. Analysis of the partially methylated/ethylated alditol acetates showed the presence of 1,5-di-O-acetyl-3,6-di-O-ethyl-2,4,7-tri-O-methylheptitol and small quantities of 1,5-di-O-acetyl-3-O-ethyl-2,4,6,7-tetra-O-methylheptitol and 1,5-di-O-acetyl-6-O-ethyl-2,3,4,7-tetra-O-methylheptitol. These results show that the major form of LOS contains PEA or phosphate groups at both the 3 and 6 positions of Hep II with very minor amounts of LOS in which Hep II is substituted at either the 3 or 6 positions.
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 (<2–3 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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The sequence of glycosyl residues was determined from a rotating-frame overhauser spectroscopy (ROSEY) experiment (Figure 3B and Table IV). In addition to intraresidue nuclear overhauser effect (NOE) contacts of AH-1 to AH-2 and AH-4, there is an interresidue contact to BH-3. Because residue B is the 3-linked Hep residue, the following glycosyl sequence was established as
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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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Previous work on another rfaK mutant, strain 559, showed that the truncated LOS contained only heptose and Kdo and that it contained multiple PEA substituents at unknown locations (Kahler et al., 1996a
). The detailed structural analysis of the rfaK mutant CMK1, presented here, shows that these PEA groups are located at both O-3 and O-6 of Hep II. These results have several implications regarding the biosynthesis of meningococcal LOS as suggested in Figure 5. The structure of the CMK1 LOS indicates that PEA is transferred to both O-3 and O-6 of Hep II prior to the incorporation of the -GlcNAc residue at O-2, and, also, that without the GlcNAc residue, synthesis of the -chain cannot be initiated. Thus, both O-2 and O-3 PEA substituents appear necessary for GlcNAc addition to Hep II and for the subsequent action of the LgtE, LgtA, and LgtB glycosyl transferases. Because the LOS from the wild-type NMB parent consists of a mixture of structures 5 (L3), 6 (L2), and 7 (Figure 5), this would indicate (a) that the PEA substituent on Hep II O-3 would have to be removed to allow for the addition of the -Glc residue at O-3 of Hep II, and (b) that the -GlcNAc residue on O-2 of Hep II would have to be removed to form structure 7. Thus, the fact that NMB LOS consists of structures 5, 6, and 7 implies that there are as yet unidentified PEA and glycosyl hydrolases that remove the PEA and -GlcNAc substituents, respectively, from Hep II.
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In the biosynthetic scheme shown in Figure 5, it is suggested that LgtG (the putative Hep II
-1,3-glucosyl transferase), and the proposed PEA, and N-acetylglucosaminosyl hydrolases act on the mature L3 LOS (structure 5) and, thus, should be regarded as enzymes that process the L3 LOS into structure 6 (L2) and then into structure 7. An alternative possibility is that these L3 processing enzymes could act on any of several intermediate products formed prior to the mature L3 structure. For example, the PEA hydrolase activity could act on structures 3, 4, or on the products of LgtE, LgtA, or LgtB. Similarly the LgtG glycosyl transferase could act on any of the PEA hydrolysis products, and the -GlcNAc hydrolase could act on structure 4, or on the products of LgtE, LgtA, or LgtB. However, these alternatives would probably result in the formation of a much greater variety of LOS structures than is observed for strain NMB. The fact that NMB produces only structures 5, 6, and 7 suggests that this processing is more likely to occur on the mature L3 structure, not prior to its formation. However, the point in LOS biosynthesis at which the O-3 PEA group is removed and replaced by -Glc will require determining the structures, including the location of PEA substituents, of the LOS OSs from LgtF, LgE, LgtA, LgtB, and LgtG mutants.
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 70–90% 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-Kdo2–lipid 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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Bacterial strains and condition of growth
The conditions for the bacterial growth requirements for mutant CMK1 (NMBrfaK::
) have been previously described (Kahler et al., 1996a). The following antibiotic concentrations were used to maintain the bacteria in GC (Difco) and brain heart infusion media supplemented with 2.5% fetal calf serum:5 µg of tetracycline (Sigma Chemical Co., St. Louis, MO) per ml and 80 µg of spectinomycin (chloride salt) per ml. Kanamycin selection was performed in brain heart infusion medium at 60 µg/ml (sulfate salt).
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-chloroform–petroleum 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 phenol–sulfuric 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, 4–5 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 GLC–mass 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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The authors thank Dr. John Gluska for assistance in obtaining the two-dimensional NMR spectra. This work was supported by Public Health Service grant AI-33517 from the National Institutes of Health to D.S.S. and by a grant from the Department of Energy (DE-FG05-93ER20097) to the Complex Carbohydrate Research Center.
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Abbreviations |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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COSY, correlation spectroscopy; GLC, gas-liquid chromatography; Glc, glucose; GlcNAc, N-acetyl glucosamine; Hep, heptose; HF, hydrogen fluoride; HSQC, heteronuclear single quantum correlation; Kdo, 3-deoxy-D-manno-2-octulosonic acid; LOS, lipooligosaccharide; LPS, lipopolysaccharide; MS, mass spectrometry; NMR, nuclear magnetic resonance; OS, oligosaccharide; PEA, phosphoethanolamine; PL, phospholipid; TOCSY, total correlation spectroscopy.
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Footnotes |
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1 To whom correspondence should be addressed
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References |
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