Báo cáo khoa học: Identification of a novel inner-core oligosaccharide structure in Neisseria meningitidis lipopolysaccharide docx

Identification of a novel inner-core oligosaccharide structure in Neisseria meningitidis lipopolysaccharide Andrew D. Cox 1 , J. Claire Wright 2 , Margaret A. J. Gidney 1 , Suzanne Lacelle 1 , Joyce S. Plested 2 , Adele Martin 1 , E. Richard Moxon 2 and James C. Richards 1 1 Institute for Biological Sciences, National Research Council, Ottawa, Canada; 2 Institute for Molecular Medicine, John Radcliffe Hospital, University of Oxford, UK The structure of the lipopolysaccharide (LPS) from three Neisseria meningitidis strains was elucidated. These strains were nonreactive with mAbs that recognize common inner- core epitopes from meningococcal LPS. It is well established that the inner core of meningococcal LPS consists of a diheptosyl-N-acetylglucosamine unit, in which the distal heptose unit (Hep II) can carry PEtn at the 3 or 6 position or not at all, and the proximal heptose residue (Hep I) is sub- stituted at the 4 position by a glucose residue. Additional substitution at the 3 position of Hep II with a glucose residue is also a common structural feature in some strains. The structures of the O-deacylated LPSs and core oligosaccha- rides of the three chosen strains were deduced by a combi- nation of monosaccharide analysis, NMR spectroscopy and MS. These analyses revealed the presence of a structure not previously identified in meningococcal LPS, in which an additional b-configured glucose residue was found to sub- stitute Hep I at the 2 position. This provided the structural basis for the nonreactivity of LPS with these mAbs. The determination of this novel structural feature identified a further degree of variability within the inner-core oligosac- charide of meningococcal LPS which may contribute to the interaction of meningococcal strains with their host. Keywords: lipopolysaccharide; mass spectrometry; Neisse- ria meningitidis; NMR; oligosaccharide. The lipopolysaccharide (LPS) of Neisseria meningitidis contains a core oligosaccharide unit with a conserved inner-core diheptose-N-acetylglucosamine backbone, in which the two L -glycero- D -manno-heptose (Hep) residues can provide a point of attachment for the outer-core oligosaccharide residues [1]. Meningococcal LPS has been classified into 12 distinct immunotypes (L1–L12), originally defined by mAb reactivities [2], but further defined by structural analyses. The structures of LPS from immuno- types L1/6 [3,4], L2 [5], L3 [6], L4/7 [7], L5 [8] and L9 [9] have been elucidated. The structural basis of the immuno- typing scheme is governed by the location of a phospho- ethanolamine (PEtn) moiety on the distal heptose residue (Hep II) at either the 3 or 6 position, or absent, or at both positions simultaneously [10]. The length and nature of oligosaccharide extension from the proximal heptose residue (Hep I) and the presence or absence of a glu- cose sugar at Hep II also dictates the immunotype. The enzyme UDP glucose 4-epimerase (GalE) is essential for N. meningitidis to synthesize UDP-Gal for incorporation of galactose into its LPS and is encoded by the gene galE [11]. The absence of galactose residues from the conserved inner-core structure of meningococcal LPS has led to the utilization of mutants defective in the enzyme, resulting in truncation of the oligosaccharide chains of the LPS at the glucose residue at Hep I, and galE mutants have been used by our group to derive mAbs to inner-core LPS epitopes [12] (unpublished data). Identified in this way, mAbs with an absolute requirement for PEtn at the 3 position [12], or the 6 position (unpublished data) of Hep II were developed. mAbs were also produced that had specificities for PEtn at the 6 position of Hep II coupled with the presence of a glucose residue at the 3 position of Hep II, glucose at the 3 position of Hep II coupled with the absence of PEtn at the 6 position of Hep II and specific for an epitope where there was no substitution with PEtn or glucose at Hep II (unpublished data). During the course of these studies, we specifically selected LPS from clinical isolates that were not reactive with these mAbs. Structural analysis of meningococcal clinical isolates revealed an additional and uniquely located glucose residue in the core oligosaccharide that had not previously been identified in meningococcal LPS. Materials and methods Growth of organism and isolation of LPS N. meningitidis strains 1000 (NRCC No. 6156) and 1000 galE (NRCC No. 6109) were grown in a 28-L fermenter as described previously [12], yielding  100gwetweightfor each growth. Strains 425/93 and NM115 are from the Correspondence to A. D. Cox, Institute for Biological Sciences, 100 Sussex Drive, National Research Council, Ottawa, ON K1A 0R6, Canada. Fax: + 1 613 952 9092, Tel.: + 1 613 991 6172, E-mail: Andrew.Cox@nrc-cnrc.gc.ca Abbreviations: LPS, lipopolysaccharide; PEtn, phosphoethanolamine; ESI, electrospray ionization; HSQC, heteronuclear single quantum coherence. (Received 21 January 2003, revised 18 February 2003, accepted 24 February 2003) Eur. J. Biochem. 270, 1759–1766 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03535.x culture collection of E. R. Moxon (University of Oxford, UK) and were grown on BHI agar plates as described [12]. Strain 1000 originates from a global collection of 34 representative serogroup B strains [13]. Strain 425/93 is a serogroup B carriage strain from the collection of P. Kriz (National Institute of Public Health, Prague, Czech Repub- lic) isolated from the Czech Republic in 1993 [14]. Strain NM115 is a serogroup B strain from R. Heyderman’s group (Imperial College, London, UK) [15]. LPS was extracted from the fermenter-grown strains by the hot phenol/water method as described previously and purified from the dia- lysed aqueous phase by ultracentrifugation (45 000 r.p.m., 4 °C, 5 h) after treatment with DNase, RNase and pro- teinase K [12], yielding  200 mg LPS for each strain. LPS was extracted from the plate-grown strains by the hot phenol/water method and ethanol precipitation of the aqueous phase as described [12], yielding  12 mg LPS for each strain. O-Deacylated LPS (LPS-OH) was prepared as described previously [16], with yields of  50%. Core oligosaccharide was prepared by the following procedure. LPS was hydrolysed at 100 °C for 2 h in 2% acetic acid. Insoluble material was removed by centrifugation (8000 r.p.m., 20 min), and the supernatant was lyophilized, resulting in  50% yield of core oligosaccharide. Analytical methods Sugars were determined as their alditol acetate derivatives by GLC-MS as described previously [16]. Mass spectrometry All electrospray ionization (ESI)-MS and capillary electro- phoresis (CE)-ESI-MS analyses were carried out as described previously [17]. NMR spectroscopy NMRexperimentswereperformedonVarianINOVA500 and 400 NMR spectrometers as described previously [10]. Results LPS was isolated from plate-grown (425/93 and NM115) or fermenter-grown (1000 and its galE mutant) strains by standard methods. Sugar analysis of the LPS-derived alditol acetates from the parent strains revealed glucitol, galactitol, glucosaminitol and L -glycero- D -manno-heptitol in approximately equimolar ratios. Sugar analysis of the LPS-derived alditol acetates from the galE mutant of strain 1000 revealed glucitol, glucosaminitol and L -gly- cero- D -manno-heptitol in approximately equimolar ratios. O-Deacylated LPS (LPS-OH) from all strains was prepared by hydrazinolysis, and initial analyses were carried out by negative-ion ESI-MS and CE-ESI-MS (Table 1). MS-MS enabled the size of the core oligosac- charide and lipid A moiety to be deduced. A consistent variation in the size of the lipid A moiety was again observed; this is due to a difference in the phosphory- lation pattern of the lipid A region (unpublished data). MS analysis indicated that there was no PEtn in the core oligosaccharide, and this was consistent with these strains not reacting with mAbs requiring the presence of PEtn in the core oligosaccharide (unpublished data). Sialylated glycoforms were only observed in strains NM115 and 425/93. 4 Hex and sialylated 4 Hex were the major glycoforms observed for all three strains but not for the galE mutant strain, and indeed were the only glycoforms observed for strain NM115, whereas 3 Hex glycoforms were also observed for strains 425/93 and 1000 (Table 1). Core oligosaccharide from the 1000 galE mutant strain was also prepared and examined by MS. A range of molecular masses was found for the core oligosaccharide consistent with a composition of Kdo, 2Hep, GlcNAc, 2Glc with nonstoichiometric substitution with glycine and acetyl groups as observed previously for meningococcal LPS (Table 1) [18]. To elucidate the exact locations and linkage patterns of the oligosaccharide chains in the LPS, NMR studies were performed on the three LPS-OHs from the parent strains. LPS-OH from strain 425/93 (Fig. 1) and strain NM115 gave good 1 H-NMR spectra simply by dissolving in D 2 O. However, LPS-OH from strain 1000 initially gave a poor 1 H-NMR spectrum, which was better resolved on the addition of deuterated SDS (5 mg) and EDTA (0.5 mg). The 1 H-NMR spectra of the three LPS-OH samples were very similar. However, the anomeric signals of the lipid A amino sugars were only weakly visible for the LPS-OH from strain 1000, presumably because of extensive aggre- gation of this region of the molecule to suppress their signals. 1 H resonances of the LPS-OH from the three strains were assigned by COSY and TOCSY experiments. Figure 2 shows a region of the TOCSY spectrum for the O-deacylated LPS from strain 425/93. Assignments were made by comparison with reported data for other meningococcal oligosaccharides [5–8,10], and are summar- ized in Table 2. In addition to the assignments tabulated, peaks corresponding to the axial ( 1.80 p.p.m.) and equatorial ( 2.75 p.p.m.) H-3 protons of the sialic acid residue were observed in the 1 H-NMR spectra of strains 425/93 and NM115. Peaks corresponding to the acetyl groups of the N-acetylglucosamine residues and the equatorial and the axial H-3 protons of the Kdo residues were unresolved because of overlap with the N-linked fatty acid residues and the axial H-3 resonance of the sialic acid, respectively. In the representative spectrum of the LPS-OH from strain 425/93, spin systems arising from heptose residues (Hep I and Hep II) were readily identified from their anomeric 1 H resonances at 5.45 and 5.42 p.p.m. (Hep I) and 5.54 p.p.m. (Hep II) and from the appearance of their spin systems, which pointed to manno-pyranosyl ring systems. The heterogeneity observed for the ano- meric proton of Hep I was thought to be due to variation in phosphate substitution in the lipid A region of the molecule (unpublished data). The a-configurations were evident for the heptosyl residues from the occur- rence of intraresidue NOEs between the H-1 and H-2 resonances only. The remaining resolved residues in the a-anomeric region at 5.07 and 5.39 p.p.m. and a minor signal at 5.50 p.p.m. were determined to be gluco- pyranose amino sugars, from the appearance of their spin systems and the fact that the H-2 resonances of 3.89 and 3.92 p.p.m. correlated in a 13 C- 1 H heteronuclear 1760 A. D. Cox et al.(Eur. J. Biochem. 270) Ó FEBS 2003 Table 1. CE-ESI-MS data and proposed compositions of O-deacylated LPS and core oligosaccharide (OS) from N. meningitidis strains 1000, 425/93 and NM115. Average mass units were used for calculation of molecular mass based on proposed composition as follows: Sial, 291.00; Hex, 162.15; Hep, 192.17; HexNAc, 203.19; Kdo, 220.18; OAc, 42.00; Gly, 57.00. The average molecular mass of the O-deacylated lipid A (Lipid A-OH) is as indicated. Lipid A-OH consists of two glucosamine residues each bearing an N-linked 3-OH C 14:0 fatty acid and a phosphate group. Variation in lipid A-OH sizes observed are due to the presence of an additional phosphate group (1032), and additional PEtn moiety (1075), an additional phosphate group and an additional PEtn moiety (1155) and one additional phosphate group and two additional PEtn moieties (1278). Relative intensity of each glycoform/phosphoform is expressed relative to the largest peak. Strain Observed ions (m/z) Molecular mass (Da) Relative intensity Lipid A a Core OS Proposed composition(M)3H) 3– (M)2H) 2– (M + H) + Observed Calculated 1000wt 943.0 1415.3 – 2832.6 2831.7 1.0 952 1880 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH O-deac – 1334.0 – 2669.8 2669.5 0.2 952 1718 3Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH 1000 galE – 1151.3 – 2304.5 2304.5 1.0 952 1352 2Hex, HexNAc, 2Hep, 2Kdo, Lipid A-OH O-deac 1000 galE core – – 1133.0 1132.0 1132.0 0.2 – – 2Hex, HexNAc, 2Hep, aKdo OS – – 1151.0 1150.0 1150.0 0.3 – – 2Hex, HexNAc, 2Hep, Kdo – – 1175.0 1174.0 1174.0 0.5 – – 2Hex, HexNAc, 2Hep, aKdo, OAc – – 1190.0 1189.0 1189.0 0.1 – – 2Hex, HexNAc, 2Hep, aKdo, Gly – – 1193.0 1192.0 1192.0 1.0 – – 2Hex, HexNAc, 2Hep, Kdo, OAc – – 1208.0 1207.0 1207.0 0.1 – – 2Hex, HexNAc, 2Hep, Kdo, Gly – – 1232.0 1231.0 1231.0 0.6 – – 2Hex, HexNAc, 2Hep, aKdo, OAc, Gly – – 1250.0 1249.0 1249.0 0.6 – – 2Hex, HexNAc, 2Hep, Kdo, OAc, Gly 425/93 889 – – 2670 2669.5 0.2 952 1718 3Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH O-deac 916 – – 2750 2759.5 0.1 1032 1718 3Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH 930 – – 2793 2792.5 0.2 1075 1718 3Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH 957 – – 2873 2872.5 0.1 1155 1718 3Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH 998 – – 2996 2995.5 0.1 1278 1718 3Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH 943 – – 2832 2831.7 0.2 952 1880 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH 970 – – 2912 2911.7 0.2 1032 1880 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH 984 – – 2955 2954.7 0.6 1075 1880 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH 1011 – – 3035 3034.7 0.6 1155 1880 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH 1052 – – 3158 3157.7 0.2 1278 1880 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH 986 – – 2961 2960.5 0.1 952 2009 Sial, 3Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH 1013 – – 3041 3040.5 0.1 1032 2009 Sial, 3Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH 1027 – – 3084 3083.5 0.2 1075 2009 Sial, 3Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH 1054 – – 3164 3163.5 0.2 1155 2009 Sial, 3Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH 1095 – – 3287 3286.5 0.1 1278 2009 Sial, 3Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH 1040 – – 3123 3122.7 0.4 952 2171 Sial, 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH 1067 – – 3203 3202.7 0.5 1032 2171 Sial, 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH 1081 – – 3246 3245.7 0.8 1075 2171 Sial, 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH 1108 – – 3326 3325.7 1.0 1155 2171 Sial, 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH 1149 – – 3449 3448.7 0.3 1278 2171 Sial, 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH Ó FEBS 2003 Novel inner-core structure in meningococcal LPS (Eur. J. Biochem. 270) 1761 single quantum coherence (HSQC) experiment with 13 C resonances of  56.0 p.p.m., this 13 C chemical shift being diagnostic of amino-substituted carbons. The signals at 5.39 and 5.50 p.p.m. were attributable to the a-glucos- amine residue of lipid A. The heterogeneity of this residue was probably due to variation in phosphate substitution patterns in the lipid A region of the molecule (unpublished data). There were no other residues in the a-anomeric region, and of particular interest was the absence of an a-glucose residue that is often found substituting the 3 position of the Hep II residue in mAb B5 nonreactive strains such as immunotype strains L2 [5] and L5 [8] and clinical strain BZ157 [10]. The remainder of the anomeric resonances in the low-field region (4.45– 6.00 p.p.m.) of the spectrum were all attributable to b-linked residues by virtue of their chemical shifts and, in the case of resolved residues, their high J 1,2 ( 8Hz) coupling constants. Three of these resonances at 4.72 (GlcNAc), 4.57 (Glc I) and 4.50 (Glc II) p.p.m. were assigned to the gluco-configuration from the appearance of their spin systems. The resonance at 4.72 p.p.m. was attributed to an amino sugar because its H-2 resonance correlatedina 13 C- 1 H HSQC experiment with a 13 C resonance of  55 p.p.m. The remaining two resonances in the low-field region at 4.55 (Gal II) and 4.45 (Gal I) p.p.m. were assigned to galacto-pyranosyl residues from the appearance of their characteristic spin systems to the H-4 resonance in a TOCSY experiment. The sequence of glycosyl residues of the LPS-OH from strain 425/93 was determined from interresidue 1 H- 1 H NOE measurements between anomeric and aglyconic protons on adjacent glycosyl residues (Table 2). Thus a lacto-N-neotetraose oligosaccharide unit attached to the proximal heptose residue was readily identified, as was the inner-core diheptosyl moiety substituted with N-acetylglucosamine. These are common structural motifs which have been identified in most N. meningitidis immunotypes. Intriguingly, a novel NOE contact was observed between the anomeric resonance of the Glc II residue at 4.50 p.p.m. and the H-2 resonance of the Hep I residue at 4.20 p.p.m. (Fig. 3A). Similarly there was a NOE connectivity between the H-1 resonance of the Hep I residue at 5.45 p.p.m. and the H-1 resonance of the Glc II resonance at 4.50 p.p.m. (Fig. 3B). Taken together these NOE data suggested that the Hep I residue was also substituted at the 2 position by the Glc II residue. This behaviour has been observed previ- ously for a b-Glc residue replacing a heptose residue at the 2 position [19], because of the proximity of the heptose H-1 proton enabling a NOE effect between the anomeric protons. This structural arrangement has not been previously observed in meningococcal LPS. Con- firmatory data for this novel linkage were obtained from a2D 13 C- 1 H HSQC experiment in which the 1 H resonance for the H-2 proton of Hep I at 4.20 p.p.m. gave a 13 C cross-peak at 78.0 p.p.m. consistent with substitution at the 2 position of this Hep I residue (Fig. 4), when compared with a 13 C chemical shift of  69–71 p.p.m. for the H-2 resonance of the Hep I residue from the meningococcal immunotype strains LPS [6,7]. Additional evidence for a novel substitution pattern at Hep I was provided by an alteration in the inter-NOE Table 1. (Continued). Strain Observed ions (m/z) Molecular mass (Da) Relative intensity Lipid A a Core OS Proposed composition (M)3H) 3– (M)2H) 2– (M + H) + Observed Calculated NM115 943 – – 2832 2831.7 0.4 952 1880 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH O-deac 970 – – 2912 2911.7 0.3 1032 1880 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH 984 – – 2955 2954.7 0.8 1075 1880 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH 1011 – – 3035 3034.7 0.3 1155 1880 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH 1052 – – 3158 3157.7 0.2 1278 1880 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH 1040 – – 3123 3122.7 0.7 952 2171 Sial, 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH 1067 – – 3203 3202.7 0.4 1032 2171 Sial, 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH 1081 – – 3246 3245.7 1.0 1075 2171 Sial, 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH 1108 – – 3326 3325.7 0.4 1155 2171 Sial, 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH 1149 – – 3449 3448.7 0.1 1278 2171 Sial, 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH a As determined by MS-MS analysis. 1762 A. D. Cox et al.(Eur. J. Biochem. 270) Ó FEBS 2003 contacts observed from the H-1 resonance of the Glc I residue. When Glc I substitutes Hep I at the 4 position, NOE contacts are usually observed between the anomeric proton resonance of the Glc I residue and the H-4 and H-6 proton resonances of the Hep I residue [10]. In the present structure, a NOE contact was only observed to the H-4 resonance, suggesting that a change in confor- mation has occurred at Hep I caused by the presence of the Glc II residue at the 2 position. It was also possible to discriminate between the 4 position of Hep I and the 2 position of Hep II, as the location of the Glc I residue, because of the absence of characteristic H-1 to H-1 NOE contacts normally observed for substitution of a heptose residue at the 2 position [19]. Almost identical results were obtained from the LPS-OH of strain 1000 and strain NM115. Chemical shifts for the Gal II residue of strain 1000 were different because in this strain, Gal II is a terminal residue whereas in strain 425/93 and NM115 it is substituted by sialic acid at the 3 position, as evidenced by the differences in the chemical shifts for the H-3 proton resonances (Table 2). The 1 H-NMR data for strain 1000 and NM115 are summarized in Table 2, confirming that each strain had the same LPS structure, the major 4 Hex glycoform of which is depicted in Fig. 5. To confirm the novel linkage pattern at Hep I, methy- lation analysis was carried out on the core oligosaccharide from the galE mutant of strain 1000. As expected, 1,2,3,4,5- O-acetyl-6,7-di-O-methylheptitol was identified (data not shown), consistent with the presence of the 2,3,4-trisubsti- tuted Hep I residue. Discussion Strains 425/93 and NM115 were initially identified in a collection of meningococcal serogroup B isolates by virtue of their lack of reactivity with mAbs specific for defined meningococcal LPS inner-core epitopes (unpublished data). Strain 425/93 came from a collection of carriage isolates [14] whereas the NM115 strain was isolated from patients with meningococcal sepsis [15]. Strain 1000 was initially identified in a collection of meningococcal clinical isolates by virtue of its lack of reactivity with mAbs identified to require PEtn at the distal heptose residue (Hep II) in the inner core ([12]; unpublished data). All previous structural studies on meningococcal LPS had revealed the same substitution pattern at the proximal heptose residue (Hep I), wherein Hep II substituted Hep I at the 3 position, and the first glucose residue (Glc I) of the oligosaccharide chain substituted Hep I at the 4 position. Structural analysis of LPS from meningococcal strains 1000, 425/93 and NM115 revealed the same arrangement. However, an additional glucose residue (Glc II) was also identified and found to substitute Hep I at the 2 position. This organization at Fig. 2. b-Anomeric region of the TOCSY spectrum from the O-deac- ylated LPS of N. meningitidis strain 425/93. The spectrum was recor- dedinD 2 Oat25°C. Fig. 1. Anomeric region of the 1D 1 H-NMR spectrum of the O-deacylated LPS from N. meningitidis strain 425/93. The spectrum was recorded in D 2 Oat25°C. Ó FEBS 2003 Novel inner-core structure in meningococcal LPS (Eur. J. Biochem. 270) 1763 Hep I has not been observed previously in meningococcal LPS. The observed lack of reactivity with several inner-core mAbs would suggest that this novel substitution pattern either alters the inner-core conformation or masks inner- core epitopes. Molecular modelling studies are underway to determine if the 2,3,4-trisubstituted Hep I residue adopts a unique conformation because of the constraints of substi- tution at the three ring positions. A unique conformation would also be consistent with the observed differences in the NOE contacts for the Glc I to Hep I linkage. In all three strains examined, in which the Hep I residue of the LPS bears the additional Glc II residue at the 2 position, only an interresidue NOE to the H-4 proton of Hep I is observed from Glc I. The well-established NOE contacts between the anomeric proton resonance of the Glc I residue and the H4 and H6 proton resonances of the Hep I residue [10], observed in previous studies of meningococcal LPS, were not observed, consistent with an altered pattern of substi- tution at Hep I, suggesting that a change in conformation has occurred caused by the presence of the Glc II residue at the 2 position of Hep I. Experiments will be initiated to attempt to identify the gene encoding the glucosyltrans- ferase responsible for the addition of this b-glucose residue to the 2 position of Hep I. Trisubstitution of the Hep I residue of LPS has been observed in other bacterial species. An additional glucose residue has been identified previously at the 6 position of the Hep I residue of LPS from Vibrio cholerae O22 [20] and Mannheimia haemolytica [21]. Other residues have been identified as substituents at the 2 position of Hep I with a b-configured glucuronic acid residue in the LPS of Vibrio parahaemolyticus O12 [22], and an a-configured galactose residue in the LPS from Campylo- bacter lari [23,24]. The arrangement of residues at Hep I in the LPS of V. parahaemolyticus O12 is identical with that identified here except that in the meningococcal strains investigated here it is a glucose residue at the 2 position. It is intriguing that only a small number of the meningococcal strains so far examined elaborate this LPS structure, and it will be interesting to see how common this substitution pattern is and, perhaps more crucially, how many men- ingococcal strains possess the genetic machinery required to elaborate this novel structure. These analyses have therefore revealed further potential for variation in the inner-core LPS of meningococcal strains. The potential to vary the degree of substitution at the Hep I residue provides N. meningitidis with additional mechanisms to alter the conformation of its LPS epitopes and possibly affect its interaction with the host. Table 2. 1 H-NMR chemical shifts (recorded at 25 °C, in D 2 O relative to HOD at 4.78 p.p.m) and NOE data for the LPS-OH from strains (i) 1000, (ii) 425/93, and (iii) NM115. ND, not determined. Residue Strain H-1 H-2 H-3 H-4 H-5 NOEs Hep I i 5.42 4.21 4.12 4.15 ND Kdo H-5, Glc II H-1 ii 5.45 4.20 4.10 4.14 ND Kdo H-5, Glc II H-1 5.42 4.20 ND ND ND Kdo H-5, Glc II H-1 iii 5.48 4.18 4.11 4.13 ND Kdo H-5, Glc II H-1 Hep II i 5.51 4.14 ND ND ND Hep-I H-3 ii 5.50 4.15 ND ND ND Hep-I H-3 iii 5.50 4.15 ND ND ND Hep-I H-3 a-GlcNAc i 5.06 3.89 3.80 3.55 ND Hep-II H-2, Hep-II H-1 ii 5.07 3.89 3.80 3.55 ND Hep-II H-2, Hep-II H-1 iii 5.06 3.89 3.80 3.55 ND Hep-II H-2, Hep-II H-1 a-GlcN (lipid A) i ND ND ND ND ND – ii 5.39 3.92 3.80 ND ND – 5.50 3.97 3.84 ND ND – iii 5.42 3.80 ND ND ND – b-Glc (Glc I) i 4.55 3.44 3.64 3.64 3.64 Hep-I H-4 ii 4.57 3.44 3.64 3.64 3.64 Hep-I H-4 iii 4.57 3.44 3.64 3.64 3.64 Hep-I H-4 b-Gal (Gal I) i 4.45 3.60 3.77 4.16 ND Glc-I H-4 ii 4.45 3.57 3.76 4.16 ND Glc-I H-4 iii 4.45 3.60 3.76 4.16 ND Glc-I H-4 b-GlcNAc i 4.72 3.81 3.75 3.75 3.59 Gal-I H-3, Gal-I H-4 ii 4.72 3.82 3.75 3.75 3.58 Gal-I H-3, Gal-I H-4 iii 4.72 3.82 3.75 3.75 3.58 Gal-I H-3, Gal-I H-4 b-Gal (Gal II) i 4.48 3.55 3.68 3.93 ND GlcNAc H-4 ii 4.55 3.60 4.12 3.96 ND GlcNAc H-4 iii 4.55 3.50 4.11 3.96 ND GlcNAc H-4 b-Glc (Glc II) i 4.50 3.39 3.49 ND ND Hep-I H-2, Hep-I H-1 ii 4.50 3.38 3.42 3.68 3.55 Hep-I H-2, Hep-I H-1 iii 4.50 3.36 3.42 3.68 3.55 Hep-I H-2, Hep-I H-1 b-GlcN (lipid A) i ND ND ND ND ND ND ii 4.65 3.80 ND ND ND ND iii 4.62 3.80 ND ND ND ND 1764 A. D. Cox et al.(Eur. J. Biochem. 270) Ó FEBS 2003 Acknowledgements We are grateful to P. Kriz (National Institute of Public Health, Prague) and M. Maiden (Oxford) who kindly provided strain 425/93 from a collection of carriage strains from the Czech Republic. We gratefully acknowledge the contribution of O. Harrison, C. Ison and R. Heyder- man (Imperial College, London) who provided strain NM115. We thank J. Li for CE-MS-MS studies and D. W. Hood for valuable discussions. References 1. Kahler, C.M. & Stephens, D.S. (1998) Genetic basis for bio- synthesis, structure, and function of meningococcal lipooligo- saccharide (endotoxin). Crit Rev. Microbiol. 24, 281–334. 2.Scholten,R.J.,Kuipers,B.,Valkenburg,H.A.,Dankert,J., Zollinger, W.D. & Poolman, J.T. (1994) Lipo-oligosaccharide immunotyping of Neisseria meningitidis byawhole-cellELISA with monoclonal antibodies. J. Med. Microbiol. 41, 236–243. 3. Di Fabio, J.L., Michon, F., Brisson, J. & Jennings, H.J. 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