Báo cáo Y học: Structural analysis of the lipopolysaccharide from nontypeable Haemophilus influenzae strain 1003 potx

Eur J Biochem 269, 808–818 (2002) Ó FEBS 2002 Structural analysis of the lipopolysaccharide from nontypeable Haemophilus influenzae strain 1003 ˚ Martin Mansson1, Derek W Hood2, Jianjun Li3, James C Richards3, E Richard Moxon2 and Elke K H Schweda1 Clinical Research Centre, Karolinska Institutet and University College of South Stockholm, Huddinge, Sweden; Molecular Infectious Diseases Group and Department of Paediatrics, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK; 3Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada Structural analysis of the lipopolysaccharide (LPS) of nontypeable Haemophilus influenzae strain 1003 has been achieved by the application of high-field NMR techniques, ESI-MS, capillary electrophoresis coupled to ESI-MS, composition and linkage analyses on O-deacylated LPS and core oligosaccharide material It was found that the LPS contains the common structural element of H influenzae, L-a-D-Hepp-(1 fi 2)-[PEtn fi 6]-L-a-DHepp-(1 fi 3)-[b-D-Glcp-(1 fi 4)]-L-a-D-Hepp-(1 fi 5)[PP Etn fi 4]-a-Kdop-(2 fi 6)-Lipid A, in which the b-DGlcp residue is substituted by phosphocholine at O-6 and an acetyl group at O-4 A second acetyl group is located at O-3 of the distal heptose residue (HepIII) HepIII is chain elongated at O-2 by either a b-D-Glcp residue (major), lactose or sialyllactose (minor, i.e a-Neu5Ac-(2 fi 3)-b-DGalp-(1 fi 4)-b-D-Glcp), where a third minor acetylation site was identified at the glucose residue Disialylated species were also detected In addition, a minor substitution of ester-linked glycine at HepIII and Kdo was observed Haemophilus influenzae is an important cause of human disease worldwide and is found in both encapsulated (types a–f) and unencapsulated (nontypeable) forms Nontypeable H influenzae (NTHi) strains commonly colonize the nasopharynx of healthy carriers and are important causes of upper and lower respiratory tract infections [1] The lipopolysaccharide (LPS) molecule, an outer membrane component, has been shown to be important for colonization, bacterial persistence and survival in the circulatory system H influenzae LPS is composed of a membrane-anchoring lipid A moiety linked by a single 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residue to the oligosaccharide portion The carbohydrate regions provide targets for recognition by host immune responses and expression of certain oligosaccharide epitopes is implicated in virulence potential [2] H influenzae LPS has been found to have several epitopes in common with LPS from Neisseria gonorrhoeae, Neisseria meningitidis and Haemophilus ducreyi Some of these shared epitopes mimic human antigens, possibly allowing the bacteria to evade the host immune system [3–5] The oligosaccharide portion of H influenzae LPS is subject to high-frequency phase variation of terminal epitopes, which can lead to a very heterogeneous population of LPS molecules within a single strain [2] It is believed that phase variation provides an adaptive mechanism that is advantageous for survival of bacteria confronted by rapidly changing microenvironments in the host [6] The availability of the complete genome sequence of H influenzae strain Rd [7] has facilitated a comprehensive study of LPS biosynthetic loci in the type b strain Eagan (RM153) [8] and in the index sequenced strain, Rd– (RM118) [9] Gene functions have been identified that are responsible for most of the steps in the biosynthesis of the oligosaccharide portion of the LPS molecules Molecular structural studies of LPS from mutant and wild-type strains of H influenzae by us and others [10–19] have resulted in a structural model consisting of a conserved triheptosyl inner-core moiety (labelled HepI–HepIII) in which each of the heptose residues can provide a point for elongation by oligosaccharide chains or for attachment of noncarbohydrate substituents (Scheme 1) Glucose, galactose, L-glycero-D-manno-heptose, D-glycero-D-mannoheptose, Kdo, N-acetylglucosamine, N-acetylgalactosamine and N-acetylneuraminic acid are the sugar components found in H influenzae LPS Common noncarbohydrate substituents are phosphate, phosphoethanolamine, phosphocholine, acetate and glycine Our recent studies have focussed on the structural diversity of LPS expression and the genetic basis for that diversity in a representative set of Correspondence to E Schweda, University College of South Stockholm, Clinical Research Centre, NOVUM, S-141 86 Huddinge, Sweden Fax: + 46 8585 838 20, Tel.: + 46 8585 838 23, E-mail: elke.schweda@kfc.ki.se Abbreviations: CE, capillary electrophoresis; Kdo, 3-deoxy-D-mannooct-2-ulosonic acid; AnKdo-ol, reduced anhydro Kdo; Hep, L-glycero-D-manno-heptose; Hex, hexose; HexNAc, N-acetylhexosamine; HPAEC, high-performance anion-exchange chromatography; lipid A-OH, O-deacylated lipid A; LPS, lipopolysaccharide; LPS-OH, O-deacylated LPS; MS/MS, tandem mass spectrometry; Neu5Ac, N-acetylneuraminic acid; NTHi, nontypeable Haemophilus influenzae; OS, oligosaccharide; PCho, phosphocholine; PEtn, phosphoethanolamine; PPEtn, pyrophosphoethanolamine (Received 30 July 2001, revised 23 November 2001, accepted 28 November 2001) Keywords: Haemophilus; lipopolysaccharide; sialic acid; phosphocholine; CE-ESI-MS/ MS Ó FEBS 2002 Structural analysis of LPS from NTHi strain 1003 (Eur J Biochem 269) 809 PPEtn ↓ R1→4)-L-α-D-HepIp-(1→5)-α-Kdop-(2→6)-Lipid A ↑ R2→3)-L-α-D-HepIIp6←PEtn ↑ R3 →2/3)-L-α-D-HepIIIp Scheme (R1, R2, R3 = H or sugar residues) NTHi clinical isolates obtained from otitis media patients In the present study we report on the structural analysis of LPS from NTHi strain 1003 EXPERIMENTAL PROCEDURES Bacterial cultivation and preparation of LPS NTHi strain 1003 was obtained from the Finnish Otitis Media Cohort Study and is an isolate obtained from the middle ear Bacteria were grown in brain-heart infusion broth supplemented with haemin (10 lgỈmL)1), NAD (2 lgỈmL)1) and Neu5Ac (25 lgỈmL)1) LPS was extracted from lyophilized bacteria by using phenol/chloroform/light petroleum, as described by Galanos et al [20], but modified with a precipitation step of the LPS with diethyl ether/ acetone (1 : 5, v/v; vol.) LPS was purified by ultracentrifugation (82 000 g, °C, 12 h) Chromatography Gel filtration chromatography was performed using a BioGel P-4 column (2.5 · 80 cm) with pyridinium acetate (0.1 M, pH 5.3) as eluent and a differential refractometer as detector GLC was carried out on a Hewlett-Packard 5890 instrument with a DB-5 fused silica capillary column (25 m · 0.25 mm · 0.25 lm) and a temperature gradient of 160 °C (1 min) to 250 °C (1 min) at °CỈmin)1 Highperformance anion-exchange chromatography (HPAEC) was performed on a Dionex Series 4500i chromatography system (Dionex, Sunnyvale, USA) using a CarboPac PA1 column (4 · 250 mm) and pulsed amperometric detection Samples were eluted using a linear gradient of 0.1 M NaOH to 500 mM NaOAc in 0.1 M NaOH over 20 and a flow rate of mLỈmin)1 Preparation of oligosaccharides O-Deacylation of LPS with hydrazine O-Deacylation of LPS was achieved as previously described [21] Briefly, LPS (10 mg) was stirred in anhydrous hydrazine (0.5 mL) at 37 °C for h The reaction mixture was cooled after which cold acetone (4 mL) was slowly added to destroy excess hydrazine After h, precipitated O-deacylated LPS (LPSOH) was collected by centrifugation (48 200 g, 20 min) The pellet was washed twice with cold acetone and finally lyophilized from water, giving a yield of mg Mild acid hydrolysis of LPS Reduced core oligosaccharide (OS) material was obtained after mild acid hydrolysis of LPS (50 mg) with 1% aqueous acetic acid (pH 3.1, 50 mL) at 100 °C for h in the presence of borane-N-methylmorpholine complex (7.0 mg) The insoluble lipid A (28 mg) was separated by centrifugation and the watersoluble part was purified by gel filtration, giving one oligosaccharide-containing fraction (OS-1, 7.6 mg) Dephosphorylation of oligosaccharide Oligosaccharide material (0.5 mg) was dissolved in cold 48% aqueous HF (75 lL) in a polypropylene tube and stored at °C for 48 h, then aqueous HF was evaporated by a stream of air while the tube was kept in an ice-bath Neuraminidase treatment of O-deacylated LPS LPS-OH (0.2 mg) was treated with 20 milliunits of neuraminidase (from Arthrobacter ureafaciens, ICN, Costa Mesa, CA, USA) in 0.2 mL 10 mM NaOAc, pH 5.0, at 37 °C for h The reaction-mixture was subjected to HPAEC without further work-up The retention time for Neu5Ac was 21.8 The enzyme cleaves terminal neuraminic acids linked a-2,3, a-2,6 or a-2,8 to oligosaccharides [22] Mass spectrometry GLC-MS was carried out with a Delsi Di200 chromatograph equipped with a NERMAG R10–10H quadrupole mass spectrometer ESI-MS was performed with a VG Quattro Mass Spectrometer (Micromass, Manchester, UK) in the negative ion mode LPS-OH and oligosaccharide samples were dissolved in water/acetonitrile (1 : 1) to a concentration of mgỈmL)1 Sample solutions were injected via a loop into a running solvent of water/ acetonitrile (1 : 1) at a flow rate of 10 lLỈmin)1 CE-ESIMS/MS was carried out with a Crystal model 310 CE instrument (ATI Unicam, Boston, MA, USA) coupled to an API 300 mass spectrometer (Perkin Elmer/Sciex, Concord, Canada) via a MicroIonspray interface as described earlier [23] Mass spectra were acquired with dwell times of 3.0 ms per step of m/z unit in full-mass scan mode The MS/MS data were acquired in full scan mode using a dwell time of 2.0 ms per step of m/z unit which leads to a mass precision of ± Da Fragment ions formed by collisional activation of selected precursor ions with nitrogen in the RF-only quadrupole collision cell, were mass-analyzed by scanning the third quadrupole NMR spectroscopy NMR spectra were recorded for solutions in D2O at 25 °C (OS-1) or 20 °C (LPS-OH) The LPS-OH sample was solubilized by adding perdeutero-EDTA (2 mM) and perdeutero-SDS (10 mgỈmL)1) to the D2O solution [14] NMR spectra were obtained on a Varian UNITY 600 MHz spectrometer using standard pulse sequences for twodimensional homonuclear proton chemical shift correlation (DQF-COSY, TOCSY, NOESY), heteronuclear 1H-13C correlation (HSQC) and heteronuclear 1H-31P correlation (HMQC) experiments All experiments (except the HMQC experiments) were run in the phase-sensitive mode Mixing times of 50 ms and 180 ms were used for TOCSY Ó FEBS 2002 ˚ 810 M Mansson et al (Eur J Biochem 269) experiments and a mixing time of 250 ms was used in the NOESY experiments Chemical shifts are reported in p.p.m., using internal sodium 3-trimethylsilylpropanoated4 (d 0.00, 1H), external 1,4-dioxane in D2O (d 67.4, 13C) or external 85% aqueous phosphoric acid (d 0.0, 31P) as references Analytical methods Sugars were identified as their alditol acetates as previously described [24] Methylation analysis was performed as described earlier [19] Methylation analysis was also performed using methyl trifluoromethanesulphonate and 2,6di-tert-butylpyridine in trimethyl phosphate as described by Prehm [25] The methylated compounds were recovered on a SepPak C18 cartridge The relative proportions of the various alditol acetates and partially methylated alditol acetates obtained in sugar and methylation analyses correspond to the detector response of the GLC-MS The absolute configurations of the hexoses were determined by the method devised by Gerwig et al [26] The total content of fatty acids was analysed as previously described [27] Glycine was determined by HPAEC following treatment of LPS with 0.1 M NaOH at 20–22 °C for 30 The retention time for glycine was 12.5 RESULTS Characterization of LPS NTHi strain 1003 was cultivated in liquid media and the LPS was extracted using the phenol/chloroform/light petroleum method Compositional sugar analysis of the LPS sample indicated D-glucose (Glc), D-galactose (Gal), 2-amino-2-deoxy-D-glucose (GlcN) and L-glycero-D-mannoheptose (Hep) in the ratio 38 : 13 : : 46, as identified by GLC-MS of their corresponding alditol acetate and 2-butyl glycoside derivatives [26] As described earlier, the LPS contained Neu5Ac [28] and glycine [23] as evidenced by HPAEC, following treatment of samples with neuraminidase and 0.1 M NaOH, respectively Methylation analysis of LPS showed terminal Glc, terminal Gal, 4-substituted Glc, 3-substituted Gal, 2-substituted Hep, 3,4-disubstituted Hep and 6-substituted GlcN in the relative proportions 14 : 15 : 14 : : 28 : 24 : The data is consistent with biantennary structures, containing the common inner-core L-a-D-Hepp-(1 fi 2)-L-a-D-Hepp-(1 fi 3)-[b-Delement, Glcp-(1 fi 4)]-L-a-D-Hepp-(1 fi 5)-a-Kdop of H influenzae LPS The presence of this structural element was confirmed by subsequent ESI-MS and NMR analysis (see below) Linkage analysis of LPS with methylation under neutral conditions [25] was also performed in order to determine the positions of base-labile substituents (glycine, acetates, see below) Using this procedure, the same sugar derivatives were obtained as previously, except that 2-substituted Hep was not detected Instead, minor amounts of 2,3-disubstituted Hep and 2,3,4-trisubstituted Hep were observed Treatment of the LPS with anhydrous hydrazine under mild conditions afforded water-soluble O-deacylated material (LPS-OH) ESI-MS data (Table 1) indicated a heterogeneous mixture of glycoforms consistent with each molecular species containing a conserved PEtn-substituted triheptosyl inner-core moiety attached via a phosphorylated Kdo linker to the putative O-deacylated lipid A (lipid A-OH) The mass spectrum was dominated by triply and Table Negative ion ESI-MS data and proposed compositions for O-deacylated LPS (LPS-OH) and oligosaccharide preparation OS-1 derived from LPS of NTHi strain 1003 Average mass units were used for calculation of molecular mass values based on proposed compositions as follows: Hex, 162.14; Hep, 192.17; Kdo, 220.18; AnKdo-ol, 222.20; P, 79.98; PEtn, 123.05; PCho, 165.13; Ac, 42.04; Gly, 57.05 and Lipid A-OH, 953.02 Relative abundance was estimated from the area of molecular ion peak relative to the total area (expressed as percentage) Peaks representing less than 5% of the base peak are not included in the table Observed ions (m/z) Sample (M-4H)4– (M-3H)3– LPS-OHa 609.4 640.1 650.0 680.8 Molecular mass (Da) (M-2H)2– Observed Calculated Relative abundance (%) Proposed composition 725.2 746.4 753.6 767.4 774.9 795.4 806.3 827.4 834.7 848.3 855.9 876.4 2441.5 2564.4 2603.6 2727.0 1452.4 1494.8 1509.2 1536.8 1551.8 1592.8 1614.6 1656.8 1671.4 1698.6 1713.8 1754.8 2442.2 2565.2 2604.3 2727.3 1453.2 1495.2 1510.3 1537.3 1552.3 1594.3 1615.4 1657.4 1672.4 1699.4 1714.4 1756.5 30 30 17 23 11 30 3 11 18 PChHex2ỈHep3ỈPEtn1ỈP1ỈKdLipidA-OH PChHex2ỈHep3ỈPEtn2ỈP1ỈKdLipidA-OH PChHex3ỈHep3ỈPEtn1ỈP1ỈKdLipidA-OH PChHex3ỈHep3ỈPEtn2ỈP1ỈKdLipidA-OH PChAc1ỈHex2ỈHep3ỈPEtn1ỈAnKdo-ol PChAc2ỈHex2ỈHep3ỈPEtn1ỈAnKdo-ol PChAc1ỈGly1ỈHex2ỈHep3ỈPEtn1ỈAnKdo-ol PChAc3ỈHex2ỈHep3ỈPEtn1ỈAnKdo-ol PChAc2ỈGly1ỈHex2ỈHep3ỈPEtn1ỈAnKdo-ol PChAc3ỈGly1ỈHex2ỈHep3ỈPEtn1ỈAnKdo-ol PChAc1ỈHex3ỈHep3ỈPEtn1ỈAnKdo-ol PChAc2ỈHex3ỈHep3ỈPEtn1ỈAnKdo-ol PChAc1ỈGly1ỈHex3ỈHep3ỈPEtn1ỈAnKdo-ol PChAc3ỈHex3ỈHep3ỈPEtn1ỈAnKdo-ol PChAc2ỈGly1ỈHex3ỈHep3ỈPEtn1ỈAnKdo-ol PChAc3ỈGly1ỈHex3ỈHep3ỈPEtn1ỈAnKdo-ol 812.8 853.8 866.7 907.9 OS-1b,c a Minor amounts of sialylated Hex3 glycoforms were indicated b Minor amounts of Hex2 and Hex3 glycoforms lacking acetyl groups or containing a second glycyl group were indicated c Very minor amounts of Hex1 and Hex4 glycoforms were indicated Ó FEBS 2002 Structural analysis of LPS from NTHi strain 1003 (Eur J Biochem 269) 811 Fig Negative ion ESI-MS spectrum of O-deacylated LPS from NTHi strain 1003 showing quadruply charged ions The peak at m/z 609.4 corresponds to a glycoform with the composition PChHex2ỈHep3ỈPEtn1ỈP1ỈKdLipid A-OH Sodiated adduct ions are indicated by asterisks (*) Fig Negative ion ESI-MS spectrum of oligosaccharide preparation OS-1 derived from LPS of NTHi strain 1003 showing doubly charged ions The peaks at m/z 725.2 and 806.3 correspond to glycoforms with the compositions PChAc1ỈHex2)3ỈHep3ỈPEtn1ỈAnKdo-ol, respectively quadruply charged ions Major quadruply charged ions were observed at m/z 609.4/640.1 and at m/z 650.0/680.8 corresponding to glycoforms with the respective compositions PChHex2ỈHep3ỈPEtn1)2ỈP1ỈKdLipid A-OH and (Fig 1) PChHex3ỈHep3ỈPEtn1)2ỈP1ỈKdLipid A-OH Minor quadruply charged ions could also be observed at m/z 722.6/753.5 consistent with the Neu5Ac-containing compositions Neu5AcỈPChHex3ỈHep3ỈPEtn1)2ỈP1ỈKd Lipid A-OH The occurrence of Neu5Ac-containing glycoforms was confirmed by precursor ion monitoring for loss of m/z 290 in CE-ESI-MS/MS experiments and the presence of triply charged ions at m/z 964/1005 corresponded to the above mentioned compositions In addition, triply charged ions at m/z 1061/1103 of lesser abundance indicated disialylated species of the respective compositions Neu5Ac2 ỈPChHex3 ỈHep3 ỈPEtn1)2 ỈP1 ỈKdLipid A-OH Glycoforms containing a Neu5Ac-Neu5Ac element have previously been identified in other NTHi strains [29–31] These signals were not detectable above background in the direct ESI MS experiment (minor) corresponded to the respective compositions PChAc1)3ỈHex2ỈHep3ỈPEtn1ỈAnKdo-ol, while ions at m/z 806.3, 827.4 and 848.3 (minor) were consistent with PChHex3ỈHep3ỈPEtn1ỈAnKdo-ol containing 1–3 acetyl groups, respectively In addition, ions were observed at m/z 753.6 (minor), 774.9, 795.4 (minor) and at m/z 834.7 (minor), 855.9, 876.4 (minor) which corresponded to glycoforms containing glycine with the respective compositions PChAc1)3ỈGly1ỈHex2ỈHep3ỈPEtn1ỈAnKdo-ol and PChAc1)3ỈGly1ỈHex3ỈHep3ỈPEtn1ỈAnKdo-ol Minor amounts of Hex2 and Hex3 glycoforms lacking acetyl groups or containing a second glycyl group were also indicated (data not shown) In addition to the major Hex2 and Hex3 glycoforms, trace amounts of Hex1 and Hex4 glycoforms were also observed (data not shown) On treatment of OS-1 with 1% aqueous NH3, ESI-MS showed complete removal of acetate and glycine, confirming glycine to be ester-linked Information on the location of the acetyl and glycyl groups as well as glycose sequence was provided by ESI tandem mass spectrometry (MS/MS) in the positive ion mode following on-line separation by capillary electrophoresis (CE) The product ion spectrum obtained from the doubly charged ion at m/z 858 (composition: PChAc2Ỉ Gly1ỈHex3ỈHep3ỈPEtn1ỈAnKdo-ol) and the proposed fragmentation pattern is shown in Fig 3A The spectrum contained an intense ion at m/z 370 corresponding to PChoAcHex to which additions of Hep (m/z 562), AnKdo-ol (m/z 784), HepPEtn (m/z 1099), Gly (m/z 1157) and AcGlyHep (m/z 1392) could be seen As observed earlier, marker ions at m/z 280 and 292 corresponded to GlyAnKdo-ol and AcGlyHep, respectively [23] and gave evidence that Gly is located mostly at HepIII (see Scheme 1) and to some extent at Kdo Furthermore, acetyl groups are located at the hexose residue linked to HepI and at HepIII The latter is corroborated by the ion at m/z 235 corresponding to the composition AcHep Information on the location of the third acetylation site was obtained from the CE-ESI-MS/MS spectra on m/z 770 and 851 (respective compositions: PChAc3ỈHex2)3ỈHep3ỈPEtn1ỈAnKdo-ol) of which the latter spectrum is shown in Fig 3B These spectra showed ions at m/z 235, 370, 562, 784 and 1099 described above without further additions of 42 Da corresponding to Characterization of oligosaccharides Partial acid hydrolysis of LPS with dilute aqueous acetic acid afforded an insoluble lipid A and core oligosaccharide material, which was purified by gel filtration chromatography, giving OS-1 Methylation analysis of OS-1 showed terminal Glc, terminal Gal, 4-substituted Glc, 2-substituted Hep and 3,4-disubstituted Hep in the relative proportions 25 : : 12 : 32 : 25 Methylation analysis of dephosphorylated OS-1 showed significantly increasing amounts of terminal Glc and 2-substituted Hep, thereby indicating phosphorylation of these residues in the native material The absence of 3-substituted Gal as compared with the methylation analysis of LPS, indicated the acid-labile Neu5Ac residue (see ESI-MS results below) to be attached to galactose at that position in the native material, i.e Neu5Ac-(2 fi 3)-Gal-(1 fi ESI-MS indicated OS-1 to contain O-acetylated Hex2 and Hex3 glycoforms, with the glycoforms containing two acetyl groups as more abundant (Table 1, Fig 2) In the negative ion mode ESI-MS spectrum of OS-1, doubly charged ions at m/z 725.2, 746.4 (major) and 767.4 ˚ 812 M Mansson et al (Eur J Biochem 269) Ó FEBS 2002 Fig 600 MHz 1H NMR spectra of O-deacylated LPS (A) and OS-1 (B) derived from LPS of NTHi strain 1003 (A) The spectrum was recorded in D2O containing mM perdeutero-EDTA and 10 mgỈmL)1 perdeutero-SDS at 20 °C (B) The spectrum was recorded in D2O at 25 °C Fig CE-ESI-MS/MS (positive mode) analysis of OS-1 derived from LPS of NTHi strain 1003 (A) Product ion spectrum of [M + 2H]2+ m/z 858 corresponding to the composition PChAc2ỈGly1ỈHex3Ỉ Hep3ỈPEtn1ỈAnKdo-ol; the proposed structure is shown in the inset The ion at m/z 280 is marked by an asterisk (*) (B) Product ion spectrum of m/z 851 corresponding to the composition PChAc3Ỉ Hex3ỈHep3ỈPEtn1ỈAnKdo-ol Selected fragment ions of structural significance are indicated an acetyl group However, the intense ion at m/z 205 observed in both spectra of the glycoforms containing three acetyl groups corresponded to the composition AcHex from which it could be concluded that the third acetylation site is at the hexose residue linked to HepIII Characterization of LPS-OH and OS-1 by NMR The 1H NMR resonances of LPS-OH and OS-1 were assigned by 1H-1H chemical shift correlation experiments (DQF-COSY and TOCSY) and the chemical shift data are shown in Tables and Subspectra corresponding to the individual glycosyl residues were identified on the basis of spin-connectivity pathways delineated in the 1H chemical shift correlation maps, the chemical shift values, and the vicinal coupling constants (measured from DQF-COSY spectrum) The 13C NMR resonances of LPS-OH and OS-1 were assigned by heteronuclear 1H-13C chemical shift correlation in the 1H detected mode (HSQC) and the data are presented in Tables and The chemical shift data are consistent with each D-sugar residue being present in the pyranosyl ring form Further evidence for this conclusion was obtained from NOE data (Table 4) which also served to confirm the anomeric configurations of the linkages and determine the sequence of the glycoses Characterization of the Kdo-lipid A-OH element The structure of the lipid A-OH region in several H influenzae strains has been shown to consist of a b-1,6-linked D-glucosamine disaccharide substituted by N-linked 3-hydroxytetradecanoic acid at C-2 and C-2¢ and phosphomonoester groups at C-1 and C-4¢ [10,13,14,19,27] In the present investigation, ESI-MS data (Table 1), fatty acid compositional analysis (yielding 3-hydroxytetradecanoic acid) and NMR experiments on LPS-OH (giving similar results as for NTHi strain 486 [19]) indicated the presence of the same lipid A-OH structure in NTHi strain 1003 As observed earlier [19], two spin-systems could be traced for the single a-linked Kdo residue, probably due to the partial occurrence of PEtn attached to the phosphate group at O-4 of Kdo [13,14,19] Structure of the core region In the 1H NMR spectrum of LPS-OH (Fig 4A), four separated signals of equal areas were observed between d 5.6 and 5.0 Three of these signals were anomeric resonances of the heptose residues (HepI– HepIII) in the inner-core region The Hep ring systems were identified on the basis of the observed small J1,2 values and their a-configurations were confirmed by the occurrence of single intraresidue NOE between the respective H-1 and H-2 resonances (Table 4) as observed earlier [32] In the 1H NMR spectrum of OS-1 (Fig 4B), anomeric resonances of the heptoses as well as one acetylation site were identified at d 5.69–5.59 (1H, not resolved) and d 5.14–5.02 (3H, not resolved) Intense signals from methyl protons of the O-acetyl groups were observed at d 2.21/2.20, which Ó FEBS 2002 Structural analysis of LPS from NTHi strain 1003 (Eur J Biochem 269) 813 Table 1H and 13C NMR chemical shifts for O-deacylated LPS of NTHi strain 1003 Data was recorded in D2O containing mM perdeuteroEDTA and 10 mg mL)1 perdeutero-SDS at 20 °C Signals corresponding to PCho methyl protons and carbons occurred at 3.22 and 54.8 p.p.m., respectively Signals corresponding to Neu5Ac methyl protons and carbons occurred at 2.01 and 22.7 p.p.m., respectively 3JH,H values for anomeric 1H resonances (H-1) are given in parentheses; n.r., not resolved (small coupling) Residue Glycose unit H-1/C-1 H-2/C-2 H-3/C-3 H-4/C-4 H-5/C-5 H-6 A C)1-6 H-6B GlcNI fi6)-a-D-GlcpN-(1 fi 5.46 (4.5) 94.5 3.82 54.7 3.93 72.4 3.75 70.7 – – a – – fi6)-b-D-GlcpN-(1 fi 4.60 (7.7) 102.6 3.88 55.8 3.81 73.9 4.02 – 3.70 – – – fi3,4)-L-a-D-Hepp-(1 fi 5.14 (n.r.) 100.4 4.14 71.0 4.04 74.6 4.19b 74.6 4.21 72.7 4.08b 69.4 5.59 (n.r.) 4.34 3.94 4.19 3.71 99.6 79.4 70.0 – 5.07 (n.r.) 100.2 4.20 78.6 3.96 72.3 3.72 67.8 C)1-7 H-7B – HepI A – GlcNII H-7 HepII fi2)-L-a-D-Hepp-(1 fi – – 4.54 3.67 3.83 71.7 74.4 62.2 – – – – › PEtn HepIII fi2)-L-a-D-Hepp-(1 fi – – – GlcI PCho fi 6)-b-D-Glcp-(1 fi 4.49 (7.6) 103.9 3.34 74.4 3.45 76.5 3.59 70.0 3.53 75.4 4.14 65.0 4.26 GlcII b-D-Glcp-(1 fi 4.60 (7.6) 102.6 3.31 73.5 3.52 76.3 3.39 70.5 3.52 76.3 3.71 61.4 3.89 4.64 (7.6) 102.4 3.35 73.1 3.67 75.0 3.66 79.3 3.67 75.0 3.80 60.8 3.97 4.43 (7.7) 103.8 3.52 71.7 3.65 73.2 3.90 69.3 3.72c 76.1 – – – 4.51 (7.7) 103.7 3.54 70.0 4.11 – 3.95 – – – – – – 1.79/2.74e – 3.67 – 3.84 – – – – – 1.99/2.40e – 4.73 – 4.30 72.0 3.85 – 3.73c 70.0 1.97/2.35e – 4.58 – 4.25 72.2 3.85 – 3.73c 70.0 GlcII* Gal Gal* Neu5Acd Kdof,g fi4)-b-D-Glcp-(1 fi b-D-Galp-(1 fi fi3)-b-D-Galp-(1 fi a-Neu5Ac-(2 fi fi4,5)-a-Kdop-(2 fi PEtn 4.10/4.06 62.6 3.27/3.29 40.7 PPEtn 4.24 63.2 3.34 40.9 PCho 4.37 60.2 3.68 66.7 a –, not obtained owing to the complexity of the spectrum b H-4/H-6 of HepI were identified at d 4.19/4.08 by NOE from GlcI c Tentative assignment from NOE data d H-8/C-8 and H-9A /H-9B/C-9 values of Neu5Ac not determined e Values corresponding to the axial proton and the equatorial proton, respectively f H-8A /H-8B/C-8 values of Kdo not determined g Several signals were observed for Kdo due to heterogeneity in the structure correlated to 13C signals at d 21.4 in the HSQC spectrum A crosspeak from the ester-linked glycine substituent was observed at d 4.00/41.0 (in the HSQC spectrum) due to correlation between the methylene proton and its carbon Several signals for methylene protons of AnKdo-ol were observed in the DQF-COSY and TOCSY spectra of OS-1 in the region d 2.20–1.66 As observed earlier [11], several anhydro-forms of Kdo are formed during the hydrolysis by elimination of phosphate or pyrophosphoethanolamine from the C-4 position, which causes both the signal splitting of the methylene protons and the appearance of several anomeric signals for HepI and HepII (Table 3) The monosaccharide sequence within the core region as indicated by CE-ESI-MS/MS (described above) was confirmed from transglycosidic NOE connectivities (Table 4) between anomeric and aglyconic protons on adjacent residues The occurrence of intense interresidue NOEs between the proton pairs HepIII H-1/HepII H-2, HepII H-1/HepI H-3 (LPS-OH and OS-1) and HepI H-1/Kdo H-5 and H-7 (LPS-OH) confirmed the sequence of the heptose-containing trisaccharide unit and the point of attachment to Kdo as L-a-D-Hepp-(1 fi 2)-L-a-D-Hepp-(1 fi 3)-L-a-D-Hepp(1 fi 5)-a-Kdop For the LPS-OH sample, relatively large J1,2 values of the anomeric resonances observed at d 4.64 (J 7.6 Hz), 4.60 (J 7.6 Hz), 4.51 (weak, J 7.7 Hz), 4.49 (J 7.6 Hz) and 4.43 (J 7.7 Hz) indicated each of the corresponding residues to have the b-anomeric configuration Further evidence for this was provided by the occurrence of intraresidue NOE between the respective H-1, H-3 and H-5 resonances On the Ó FEBS 2002 ˚ 814 M Mansson et al (Eur J Biochem 269) Table 1H and 13C NMR chemical shifts for oligosaccharide preparation OS-1 derived from LPS of NTHi strain 1003 Data was recorded in D2O at 25 °C Signals corresponding to PCho methyl protons and carbons occurred at 3.24 and 54.7 p.p.m., respectively Pairs of deoxyprotons of reduced AnKdo were identified in the DQF-COSY spectrum at 2.20–1.66 p.p.m Residue Glycose unit H-1/C-1 H-2/C-2 H-3/C-3 H-4/C-4 H-5/C-5 c H-6A/C-6 H-6B H-7A/C-7 H-7B HepII fi3,4)-L-a-D-Hepp-(1 fi 5.04–5.14a 4.00–4.07a 3.97–4.05a 4.24–4.26a,b – 4.15b – – fi2)-L-a-D-Hepp-(1 fi 97.5–99.0a 5.59–5.69a 71.3–71.4a 4.31–4.35a 72.8–73.0a 4.01–4.02a 74.7 – – 3.77 68.5 4.57 – 3.72 3.90 99.4–100.1a HepI 79.7–79.8a 69.9 – 72.2 75.2 63.0 5.02 4.39 5.08 4.02 – – – › PEtn HepIII fi2)-L-a-D-Hepp-(1 fi – › OAc 99.9 76.1 74.0 64.8 – – GlcI PCho fi6)-b-D-Glcp-(1 fi 4.51 103.8 3.39 74.3 3.46 76.4 3.61 69.9 3.54 75.4 4.16 64.9 4.27 – GlcI3–OAc PCho fi6)-b-D-Glcp-(1 fi 4.62 3.54 4.99 3.80 3.66 4.14 4.28 103.8 4.56 72.3 3.43 78.0 3.70 68.1 4.89 75.1 3.79 64.9 3.96 4.12 103.8 74.2 74.8 72.1 73.3 64.9 4.56 103.0 3.31 73.8 3.50 76.4 3.43 70.5 3.50 76.4 3.75 61.7 3.87 4.58 102.9 3.42 73.7 3.63 75.2 3.69 79.3 3.63 75.2 3.84 61.0 3.92 4.48 103.8 3.54 71.7 3.67 73.3 3.93 69.3 3.73d 76.1 – – – PEtn 4.14 62.7 3.28 40.9 PCho 4.37/4.31 60.2 3.70/3.68 66.8 › OAc GlcI4–OAc PCho fi6)-b-D-Glcp-(1 fi › OAc GlcII GlcII* Gal b-D-Glcp-(1 fi fi4)-b-D-Glcp-(1 fi b-D-Galp-(1 fi Ac 2.21 2.20 – 21.4 4.00 – 41.0 Gly a Several signals were observed for HepI and HepII due to heterogeneity in the AnKdo moiety b H-4/H-6 of HepI were identified at d 4.24– 4.26/4.15 by NOE from GlcI, GlcI3-OAc and GlcI4-OAc c –, not obtained owing to the complexity of the spectrum d Tentative assignment from NOE data basis of the chemical shift data and the large J2,3, J3,4 and J4,5 values ( Hz, measured from DQF-COSY spectrum), the residues with anomeric shifts of d 4.49, 4.60 and 4.64 could be attributed to the terminal Glc (GlcI and GlcII) and 4-substituted Glc (GlcII*) identified by methylation analysis On the basis of low J3,4 and J4,5 values (< Hz) and chemical shift data, the residues with anomeric resonances at d 4.43 and 4.51 were attributed to the terminal Gal (Gal) and 3-substituted Gal (Gal*) identified by linkage analysis Signals for the methyl protons of PCho were observed at d 3.22 (LPS-OH) and d 3.24 (OS-1) and spin-systems for ethylene protons from this residue and from PEtn were similar to those observed earlier [19] 1H-31P NMR corre- lation studies of LPS-OH and OS-1 confirmed PCho and PEtn to be located at GlcI and HepII, respectively In the spectrum of LPS-OH, intense 31P resonances from phosphomonoesters were observed at d )0.18 and )0.31 Correlations between the former signal and the signals from H-6 of HepII (d 4.54) and the methylene proton pair of PEtn (d 4.10/4.06) in the 1H-31P HMQC experiment confirmed substitution by PEtn at O-6 of HepII Correlations between the signal at d )0.31 and the signals from the H-6 protons of GlcI (d 4.26 and 4.14) and the methylene protons of PCho (d 4.37) established the PCho substituent to be attached to O-6 of this glucose residue An 1H-31P HMQC experiment on the OS-1 sample showed similar correlations (see below) Phosphorylation at O-6 of the Ó FEBS 2002 Structural analysis of LPS from NTHi strain 1003 (Eur J Biochem 269) 815 Table Proton NOE data for O-deacylated LPS and oligosaccharide preparation OS-1 derived from LPS of NTHi strain 1003 Measurements were made from NOESY experiments NR, not rationalized Observed proton Anomeric proton Intraresidue NOE Interresidue NOE a GlcNI GlcNIIa HepI HepII HepIII GlcI GlcI3–OAc GlcI4–OAc GlcII GlcII* Gal Gal*a H-2 H-3, H-5 H-2 H-2 H-2 b b H-3, H-3, H-3, H-3, H-3, H-3, H-3 H-5 H-5 H-5 H-5c H-5d H-5 NR NR H-5, H-7 of Kdoa H-3 of HepI; H-1 of HepIII H-1, H-2 of HepII; H-1 of GlcII; H-1 of GlcII* H-4, H-6 of HepI H-4, H-6 of HepI H-4, H-6 of HepI H-1, H-2 of HepIII H-1, H-2 of HepIII H-4 of GlcII*d,e H-4 of GlcII*d Observed only in the spectrum of O-deacylated LPS b Observed only in the spectrum of OS-1 c H-3, H-5 of GlcII were overlapping in LPS-OH (3.52 p.p.m.) and OS-1 (3.50 p.p.m.) d H-3, H-4 and H-5 of GlcII* were overlapping in LPS-OH (3.67 p.p.m.) H-3 and H-5 of GlcII* were overlapping in OS-1 (3.63 p.p.m.) e H-3 of Gal overlapped with H-4 of GlcII* a b-D-Glcp residue was in agreement with the significant downfield shifts for signals from H-6A, H-6B and C-6 of GlcI, compared to the corresponding chemical shifts for the monosaccharide residue [33] Interresidue NOE were observed between H-1 of the terminal GlcI residue and H-4 and H-6 of HepI, confirming [12] the presence of the structural element PCho fi 6)b-D-Glcp-(1 fi 4)-L-a-D-Hepp-(1 fi Interresidue NOE between GlcII H-1 and HepIII H-1 and H-2 indicated substitution at O-2 of HepIII as b-D-Glcp-(1 fi 2)-L-a-DHepp-(1 fi The occurrence of transglycosidic NOE connectivities between the proton pairs Gal H-1/GlcII* H-4 and GlcII* H-1/HepIII H-1 and H-2 established the sequence of a disaccharide unit and its attachment point to HepIII as b-D-Galp-(1 fi 4)-b-D-Glcp-(1 fi 2)-L-a-DHepp-(1 fi Weak but characteristic signals from the H-3 methylene protons of Neu5Ac were observed at d 1.79 (H-3ax, J3ax,3eq ¼ 12.3 Hz) and d 2.74 (H-3eq, J3eq,4 ¼ 4.3 Hz) in the 1H NMR spectrum of LPS-OH As chemical shift data (Table 2) were similar to those of reported structures containing a-Neu5Ac-(2 fi 3)-b-D-Galp-(1 fi [19], it was established that Neu5Ac was 2,3-linked to galactose as indicated by the methylation analyses Chemical shift values for several HepIII resonances in OS-1 differed considerably from the corresponding values in LPS-OH (Tables and 3) Downfield shifts were obtained for the signals from H-3 (+1.12 p.p.m.), H-2 (+0.19 p.p.m.), H-4 (+0.30 p.p.m.) and C-3 (+1.7 p.p.m.) of HepIII in OS-1, while the signals from C-2 ()2.5 p.p.m.) and C-4 ()3.0 p.p.m.) were shifted upfield, which indicated HepIII to be acetylated at O-3 [34] This was consistent with the 2,3-disubstituted Hep observed in the methylation analysis under neutral conditions described above Furthermore, glycine was indicated to be located at O-4 of HepIII based on the occurrence of 2,3,4-trisubstituted Hep, although this could not be confirmed by NMR experiments In OS-1, the spin-system for GlcI was characterized by rather weak cross-peaks However, two other spin-systems were observed which were indicated to be monoacetylated GlcI-residues (GlcI3–OAc, GlcI4–OAc) from the chemical shift differences compared to the GlcI residue For GlcI3–OAc, downfield shifts were obtained for the signals from H-3 (+1.53 p.p.m.), H-2 (+0.15 p.p.m.), H-4 (+0.19 p.p.m.) and C-3 (+1.6 p.p.m.), while the signals from C-2 ()2.0 p.p.m.) and C-4 ()1.8 p.p.m.) were shifted upfield, indicating acetylation at O-3 [34] For GlcI4–OAc, chemical shift values were consistent with acetylation at O-4 as downfield shifts were obtained for the signals from H-4 (+1.28 p.p.m.), H-3 (+0.24 p.p.m.), H-5 (+0.25 p.p.m.) and C-4 (+2.2 p.p.m.), while the signals from C-3 ()1.6 p.p.m.) and C-5 ()2.1 p.p.m.) were shifted upfield [34] The peak area of the H-4 resonance of GlcI4–OAc (d 4.89) was considerably larger than the peak area of the H-3 resonance of GlcI3–OAc (d 4.99), indicating a higher degree of acetylation at O-4 than at O-3 Two spin-systems could be observed for PCho in OS-1 at d 4.31/3.68 and 4.37/3.70 The former spin-system arose from PCho linked to O-6 of GlcI4–OAc as evidenced by correlations between a 31P resonance at d )0.53 and the signals from the H-6 protons of GlcI4–OAc (d 4.12 and 3.96) and the signal at d 4.31 in the 1H-31P HMQC spectrum of OS-1 Correspondingly, correlations could be seen between a 31P resonance at d )0.41 and the signals from the H-6 protons of GlcI3–OAc and GlcI (d 4.28/4.27 and 4.14/4.16) and the signal at d 4.37 The occurrence of acetylation at O-3 of GlcI was probably a result of acetyl group migration from the O-4 position Repeated NMR experiments on oligosaccharide samples showed that GlcI4–OAc decreased in favour of GlcI3–OAc during storage The ESI-MS data of OS-1 (Table 1) indicated the major glycoforms to contain two acetyl groups which can be located at HepIII (at O-3) and GlcI (at O-4) as evidenced from CE-ESI-MS/MS (Fig 3) and NMR data (Table 3) CE-ESI-MS/MS also indicated GlcII/GlcII* to carry an O-acetyl substituent but the location could not be confirmed by NMR, probably due to low abundance of glycoforms containing this acetyl substitution pattern As mentioned above, the LPS can also contain glycoforms substituted with glycine at HepIII or Kdo From the combined data, Scheme is proposed for the sialylated and fully acylated Hex3 glycoform DISCUSSION NTHi strain 1003 is an isolate obtained from a child with otitis media as part of an epidemiological study in Finland [29] The present study indicates that this strain elaborates two major LPS glycoform populations containing two or three hexose residues attached to the common tri-heptosyl inner-core structure The Hex3 glycoform contains a lactose unit attached to HepIII that can also carry a Neu5Ac residue Earlier investigations have shown that modification of the LPS with Neu5Ac residues is widespread among H influenzae strains [4,29] Recently, the Neu5Ac level in LPS from 24 different NTHi strains (including NTHi strain 1003) was determined by HPAEC [28] In NTHi strain Ó FEBS 2002 ˚ 816 M Mansson et al (Eur J Biochem 269) Scheme Proposed structure of sialylated and fully acylated Hex3 glycoform 1003, Neu5Ac occupies the same molecular environment as in strain RM118 (Rd–) and NTHi strains 375 and 486, that is, attached to b-galactose of a lactose moiety [a-Neu5Ac(2 fi 3)-b-D-Galp-(1 fi 4)-b-D-Glcp-(1 fi ] [19,29,30] But as for NTHi strain 486, glycoforms showing elongation of lactose with a terminal a-D-Galp residue were not observed, in contrast to the pk epitope [a-D-Galp-(1 fi 4)-b-D-Galp(1 fi 4)-b-D-Glcp-(1 fi ] expressed by strain Rd– and NTHi strain 375 Minor amounts of disialylated glycoforms were also detected as for NTHi strains 375 and 176 [29–31], where the two Neu5Ac residues were found to be linked to each other Recently, the phase-variable gene, lic3A, was shown to encode an a-2,3-sialyltransferase that adds Neu5Ac to terminal lactose [30] The core oligosaccharide of NTHi strain 1003 is highly acetylated The O-3 position of HepIII is occupied by an O-acetyl group, which is the same linkage position for acetyl groups that recently have been reported for several type b strains [17] Another O-acetyl group was found to be linked to O-4 of the PCho-substituted b-D-Glcp residue attached to HepI, a structural feature not reported before A minor, third acetylation site was found at the b-D-Glcp residue attached to HepIII From a survey of 24 NTHi strains we have recently reported the presence of glycine in all LPS preparations which can occur at different sites in the inner-core region [23] For NTHi strain 1003, glycoforms containing glycine at HepIII and Kdo were identified based on the occurrence of marker ions in CE-ESI-MS/MS spectra In addition, glycine is tentatively assigned to O-4 of HepIII based on methylation analysis data This investigation provides the first example of a NTHi strain with PCho attached to O-6 of a terminal b-D-Glcp residue at HepI (Scheme 1; R1 ¼ PCho fi 6)-b-D-Glcp) Preliminary results from our laboratory indicate this to be a very common molecular environment for the PCho epitope in NTHi It is noteworthy that the same molecular environment for PCho has been found in the H influenzae strain Rd– [14], the strain on which the complete genome has been sequenced The PCho decoration of the LPS appears to favour colonization of respiratory tract epithelia in an experimental rat model of H influenzae infection, whereas its absence may confer relative resistance to host factors such as complement mediated killing [35] From a survey of 24 NTHi strains, PCho was found to be present in the LPS of all of these strains (data not shown) Structural analyses have shown that PCho can be linked to terminal hexose residues attached to either one of the three inner-core heptose residues of H influenzae LPS [14,17,19] The lic1 locus of H influenzae controls both the expression and phase variation of the PCho epitope [36] The ability of this organism to link PCho to different oligosaccharide components was recently demonstrated to be associated with DNA sequence polymorphism in lic1D, a gene encoding a putative diphosphonucleoside choline transferase [37] It was further suggested that C-reactive protein binds more effectively to glycoforms bearing PCho on hexoses attached to HepIII, rather than to glycoforms bearing PCho on hexoses attached to HepI [37] During the course of investigation several growths of bacteria were obtained for NTHi strain 1003 which did not differ in the basic carbohydrate backbone structures of the LPS However, a hydroxybensylic substituent was found in the inner-core of the LPS in one growth Structural details of this finding are under investigation ACKNOWLEDGEMENTS The authors would like to thank sincerely the members of the Finnish Otitis Media Study Group at the National Public Health Institute in Finland for the provision of NTHi strains from the middle ear fluid, obtained as part of the Finnish Otitis Media Cohort Study The Swedish NMR centre (Goteborg, Sweden) is acknowledged for ă providing access to their 600 MHz facilities Mary Deadman and Shannon Walsh are acknowledged for culturing of H influenzae strains REFERENCES Murphy, T.F & Apicella, M.A (1987) Nontypeable Haemophilus influenzae: a review of clinical aspects, surface antigens, and the human immune response to infection Rev Infect Dis 9, 1–15 Kimura, A & Hansen, E.J (1986) Antigenic and phenotypic variations of Haemophilus influenzae type b lipopolysaccharide and their relationship to virulence Infect Immun 51, 69–79 Ó FEBS 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Schweda, E.K.H (2001) A new structural type for Haemophilus influenzae lipopolysaccharide Structural analysis of the lipopolysaccharide from nontypeable Haemophilus influenzae strain 486 Eur J Biochem... FEBS 2002 Structural analysis of LPS from NTHi strain 1003 (Eur J Biochem 269) 811 Fig Negative ion ESI-MS spectrum of O-deacylated LPS from NTHi strain 1003 showing quadruply charged ions The peak... sensitive procedure for determination of 5-N-acetyl neuraminic acid in lipopolysaccharides of Haemophilus influenzae: a survey of 24 nontypeable H influenzae strains Carbohydr Res 335, 251–260 Hood, D.W.,