Báo cáo khoa học: Structural analysis of lipopolysaccharides from Haemophilus influenzae serotype f Structural diversity observed in three strains ppt

Structural analysis of lipopolysaccharides from Haemophilus influenzae serotype f Structural diversity observed in three strains Ha ˚ kan H. Yildirim 1 , Derek W. Hood 2 , E. Richard Moxon 2 and Elke K. H. Schweda 1 1 Clinical Research Centre, Karolinska Institutet and University College of South Stockholm, Sweden; 2 Molecular Infectious Diseases Group, University of Oxford Department of Pediatrics, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK Structural elucidation of the lipopolysaccharide (LPS) from threeserotypefHaemophilus influenzae clinical isolates RM6255, RM7290 and RM6252 has been achieved using NMR spectroscopy techniques and ESI-MS on O-deacyl- ated LPS and core oligosaccharide material (OS) as well as ESI-MS n on permethylated dephosphorylated OS. This is the first study to report structural details on LPS from sero- type f strains. We found that the LPSs of all strains were highly heterogeneous mixtures of glycoforms expressing the common H. influenzae structural element L -a- D -Hepp- (1fi2)-[PEtnfi6]- L -a- D -Hepp-(1fi3)-[b- D -Glcp-(1fi4)]- L -a- D - Hepp-(1fi5)-[PPEtnfi4]-a-Kdo-(2fi6)-lipid A with variable length of OS chains linked to each of the heptoses. The terminal heptose (HepIII) in RM6255 is substituted at the O-3 position by a b- D -Glcp residue whereas HepIII in strains RM7290 and RM6252 is substituted at O-2 by the globoside unit (a- D -Galp-(1fi4)-b- D -Galp-(1fi4)-b- D -Glc) or trun- cated versions thereof. The central heptose (HepII) is substituted by an a- D -Galp-(1fi4)-b- D -Galp-(1fi4)-b- D - Glcp-(1fi4)-a- D -Glcp unit in RM7290 and RM6252 or truncated versions thereof. Strain RM6255 does not express galactose in its LPS and only shows a cellobiose unit elon- gating from HepII (b- D -Glcp-(1fi4)-a- D -Glcp). ESI-MS n on dephosphorylated and permethylated OS provided infor- mation on the existence of additional minor isomeric glycoforms. Keywords: Haemophilus influenzae; lipopolysaccharide; NMR; ESI-MS. Haemophilus influenzae is an important cause of human disease worldwide and exists in encapsulated (type a through f) and unencapsulated (nontypeable) forms. Type b capsular strains are associated with invasive bacteraemic diseases, including meningitis and epiglottis, while acapsular or nontypeable strains of H. influenzae (NTHi) are primary pathogens in otitis media and lower respiratory tract infections [1]. Lipopolysaccharide (LPS) is an essential surface component of these pathogens and is implicated as a major virulence factor. LPS of H. influenzae can mimic host glycolipids and has a propensity for reversible switching of terminal epitopes (phase variation) of the oligosaccharide portion. Molecular structural studies of LPS from H. influ- enzae type b, d and NTHi strains have shown that the conserved part of LPS consists of a triheptosyl inner-core moiety linked to lipid A via 3-deoxy- D -manno-oct-2-ulo- sonic acid (Kdo). Each of the heptose residues (designated HepI, HepII and HepIII) can provide a point for elongation by oligosaccharide chains or for attachment of noncarbo- hydrate substituents (Structure 1). Correspondence to E. Schweda, University College of South Stock- holm, 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: Ac, acetate; AnKdo-ol, reduced anhydro Kdo; CE, capillary electrophoresis; gHSQC, gradient heteronuclear single quantum coherence; gHMQC, gradient selected heteronuclear mul- tiple quantum coherence; gHMBC, gradient selected heteronuclear multiple-bond correlation; GPC, gel permeation chromatography; Hep, heptose; L , D -Hep, L -glycero- D -manno-heptose;Hex,hexose; Hib, H. influenzae serotype b; Hif, H. influenzae serotype f; Kdo, 3-deoxy- D -manno-oct-2-ulosonic acid; lipid A-OH, O-deacylated lipid A; LPS, lipopolysaccharide; LPS-OH, O-deacylated lipopoly- saccharide; PAD, pulsed amperometric detection; MS n , multiple step tandem MS; Neu5Ac, N-acetyl neuraminic acid; NTHi, nontypeable Haemophilus influenzae; OS, oligosaccharide; PCho, phosphocholine; PEtn, phosphoethanolamine; PPEtn, pyrophosphoethanolamine. (Received 14 April 2003, revised 21 May 2003, accepted 27 May 2003) Eur. J. Biochem. 270, 3153–3167 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03693.x To date, HepI has been found to be substituted by glucose (R 1 ¼ b- D -Glcp) in all strains investigated. This glucose can provide a point for further chain extension. There is no chain extension from HepII in the type d-derived strain Rd [2], however, in three type b strains and a number of NTHi strains HepII is substituted by a glucose residue (R 2 ¼ a- D -Glcp) which can provide a point for further chain extension. HepIII is substituted by galactose {R 3 ¼ b- D -Galp-(1fi2) in type b strains [3]; R 3 ¼ b- D -Galp-(1fi3), NTHi strain 176 [4]} or oligosac- charides extending from glucose {R 3 ¼ b- D -Glcp-(1fi2), strain Rd, NTHi strain 1003 [5]; R 3 ¼ b- D -Glcp-(1fi3), NTHi strain 486 [6]}. In addition, N-acetyl neuraminic acid has been found to be a common constituent of LPS in H. influenzae [7]. Prominent noncarbohydrate substituents are phosphate (P), pyrophosphoethanolamine (PPEtn), phosphoethanolamine (PEtn), phosphocholine (PCho), acetate (Ac) and glycine (Gly). The oligosaccharide portion of H. influenzae LPS is known to be subject to high-frequency phase variation of terminal epitopes, which can lead to a very heterogeneous population of LPS molecules within a single strain [8]. Phase variation is thought to provide an adaptive mechanism which is advantageous for survival of bacteria confronted by the differing microenvironments and immune responses of the host [9]. A genetic mechanism contributing to LPS phase variation has been identified in five chromosomal loci; lic1, lic2, lic3, lgtC and lex2 [10–12]. It has been demon- strated that expression of PCho substituents in H. influen- zae LPS is subject to phase variation mediated by the lic1 locus [13]. Genes comprising the lic2 locus have been shown to be required for chain extension from HepII and, together with lgtC, in the phase variable expression of the p k epitope [a- D -Galp-(1fi4)-b- D -Galp-(1fi4)-b- D -Glcp-(1fi] [12,14,15]. The lic3 locus has been shown to encode an a-2,3-sialyltransferase that is responsible for addition of sialic acid (N-acetylneuraminic acid or Neu5Ac) to terminal lactose [16]. The phase-variable lex2 locus has been shown to encode a glycosyltransferase important in further oligo- saccharide extension from HepI in the inner core [17]. A comprehensive study of LPS biosynthetic loci has been undertaken in the type b strain Eagan (RM153) and in strain Rd – (RM118) [18,19]. H. influenzae serotype f (Hif) has its own structurally and serologically distinct capsular polysaccharide distinguishing it from the other five sero- types and the nontypeable strains [20]. Historically, Hif strains have been a rare cause of disease in children and adults [21,22]. However, septic arthritis, meningitis, sepsis, otitis media, pneumonia, cellulitis, and bacteremia are diseases associated with Hif infections in children who quite often have a predisposing medical condition, including documented immunodeficiency [20, 23–25]. Countries engaged in global vaccination programs against H. influenzae serotype b (Hib) experienced a rapid decline in diseases caused by Hib strains. In the UK, Hib infection decreased 98% in children younger than 5 years of age [22], thus resulting in a greater proportion of infections due to serotype f strains in the Hib postvaccination era [26]. The capsular polysaccharide of type f strains is known to be composed of b- D -GalpNAc(1fi4)-a- D -GalpNAc(1fiPO 4 repeating units [27]. However, no data on LPS structures are known. To gain further insight into the LPS structures expressed by H. influenzae, we report detailed structural analyses on three epidemiologically distinct type f strains. In these strains we identify a novel combination of oligosac- charide extensions not seen before in H. influenzae LPS. Experimental procedures Bacterial strains, growth conditions and LPS extraction H. influenzae type f strains RM6255, RM7290 and RM6252 are epidemiologically distinct clinical isolates which are representative of the limited genetic diversity observed within type f isolates. RM6252 was isolated in 1966 from the nasal pus of a 59-year-old male from Newcastle, England, who suffered from subacute sinusitis. RM6255 was isolated in 1967 from the sputum of a 72-year-old male from Newcastle, England, who suffered from a postoperative chest infection. RM7290 was isolated in 1974 from the sputum of a 30-year-old female from Kuala Lumpur, Malaysia, who suffered from respiratory infection and malnutrition. Bacteria were grown in brain–heart infusion broth supplemented with haemin (10 lgÆmL )1 )andNAD (2 lgÆmL )1 ). LPS was extracted from lyophilized bacteria using phenol/chloroform/light petroleum as described pre- viously but with the modification that the LPS was precipitated with 6 vols diethyl ether/acetone (1 : 5, v/v) [28]. LPS was purified by ultracentrifugation (82 000 g, 4 °C, 12 h). 11, 49 and 56 mg LPS was extracted from 1.6, 5.3, 3.4 g lyophilized bacteria from RM6255, RM7290 and RM6252, respectively. Chromatography Gel permeation chromatography (GPC) was performed using a Bio-Gel P4 column (2.5 · 80 cm) with pyridinium acetate (0.1 M , pH 5.3) as eluent and a differential refrac- tometer as detector. GLC was carried out using a Hewlett- Packard 5890 instrument with a DB-5 fused silica capillary column (25 m · 0.25 mm; 0.25 lminternaldiameter)and a temperature gradient of 160 °C(1min)fi 250 °Cat 3 °Cmin )1 . HPAEC was performed on a Dionex Series 4500i chromatography system using a CarboPac PA1 column (4 · 250 mm) and pulsed amperometric detection. Samples were eluted using a linear gradient of 0–500 m M NaOAc in 0.1 M NaOH over 20 min and a flow rate of 1 mLÆmin )1 . Preparation of oligosaccharides O-Deacylation of LPS with hydrazine. O-Deacylation of LPS was achieved as previously described [29]. Briefly, LPS (1 mg) was mixed with anhydrous hydrazine (0.1 mL) and stirred at 37 °C for 1 h. The reaction mixture was cooled and cold acetone (1 mL) was added to destroy excess hydrazine. The precipitated O-deacylated LPS (LPS-OH) was centrifuged (48 200 g,20min),thepelletwaswashed twice with cold acetone, once with diethyl ether, and then dissolved in water followed by lyophilization. Mild acid hydrolysis of LPS. Core oligosaccharide frac- tions were obtained from LPS (10 mg RM6255, 22 mg RM7290, 30 mg RM6252) following mild acid hydrolysis 3154 H. H. Yildirim et al. (Eur. J. Biochem. 270) Ó FEBS 2003 (1% acetic acid, pH 3.1, 100 °C, 2 h). The insoluble lipid A was separated from the hydrolysis mixtures by centrifuga- tion. The reducing agent, borane-N-methylmorpholine complex (1, 2 and 3 mg, respectively, for RM6255, RM7290 and RM6252), was included in the hydrolysis mixture. Following purification by GPC on the Biogel P4 column oligosaccharide (OS) fractions OS6255 (2.5 mg), OS7290 (4.0 mg) and OS6252 (9.7 mg) were obtained and investigated in this study. Dephosphorylation. OS6255, OS7290 and OS6252 (1 mg each) were incubated with 48% hydrogen fluoride (0.1 mL) for 48 h at 4 °C. Then, the samples were placed into an ice bath and HF was evaporated under a stream of nitrogen gas. The samples, OS6255-P, OS7290-P and OS6252-P, were dissolved in water and lyophilized. Mass spectrometry. GLC-MS was carried out with a Hewlett-Packard 5890 chromatograph connected to a NERMAG R10-10H quadrupole mass spectrometer using the same conditions for GLC as described above. ESI-MS on LPS-OH and OS samples was recorded on a VG Quattro triple quadrupole mass spectrometer (Micromass, Man- chester, UK) in the negative ion mode. The samples were dissolved in a mixture of water/acetonitrile (1 : 1, v/v). Sample solutions were injected via a syringe pump into a running solvent of H 2 O/CH 3 CN (1 : 1) at a flow rate of 10 lLÆmin )1 . Multiple step tandem ESI-MS (ESI-MS n ) experiments were performed on a Finnigan LCQ iontrap mass spectrometer (Finnigan-MAT, San Jose, CA, USA). Samples were dissolved in 1 m M sodium acetate in 70% MeOH/30% H 2 O. The applied flow rate was 10 lLÆmin )1 . All experiments were in the positive mode. Capillary electrophoresis (CE)-ESI-MS n was carried out with a Crystal model 310 CE instrument (ATI Unicam, Boston, MA, USA) coupled to an API 3000 mass spectrometer (Perkin-Elmer/Sciex, Concord, Canada) via a MicroIon- spray interface as described previously [30]. Analytical methods Sugars were identified by GLC-MS as their alditol acetates as previously described [31]. Methylation was performed with methyl iodide in dimethylsulfoxide in the presence of lithium methylsulfinylmethanide [32]. The methylated com- pounds were recovered using a SepPak C18 cartridge and subjected to sugar analysis or ESI-MS n .Therelative proportions of the various alditol acetates and partially methylated alditol acetates obtained in sugar- and methyl- ation analyses, discussed below, correspond to the detector response of the GLC-MS. The absolute configuration of glycoses was determined as previously described [33]. The presence of glycine was determined by HPAEC following treatment of LPS with 0.1 M NaOH at 20–22 °C for 30 min. N-acetylneuraminic acid was determined by treating LPS- OH (0.2 mg) with 20 mU 1 of neuraminidase in 0.2 mL 10 m M NaOAc, pH 5.0, at 37 °C for 4 h. One unit will liberate 1.0 lmol of Neu5Ac per min at pH 5.0 at 37 °C. The reaction mixture was analyzed by HPAEC as previ- ously described without further work-up [30]. The enzyme cleaves terminal Neu5Ac residues linked a-2,3, a-2,6 or a-2,8 to oligosaccharides. Fatty acids were identified as described previously [34]. NMR spectroscopy NMR spectra were recorded for OS samples in deuterium oxide (D 2 O) at 30 °C. Spectra were acquired on JEOL 500 MHz and Varian UNITY 600 MHz spectrometers using standard pulse sequences. The LPS-OH samples were solubilized by adding perdeutero-EDTA (2 m M ) and per- deutero-SDS (10 mgÆmL )1 )totheD 2 O solution. Chemical shifts were reported in p.p.m., and externally referenced to sodium 3-trimethylsilylpropanoate-d4 (d 0.00, 1 H) and acetone (d 29.8, 13 C). COSY, TOCSY with a mixing time of 180 ms, gradient selected heteronuclear single quantum coherence (gHSQC), gradient selected heteronuclear mul- tiple quantum coherence (gHMQC), and gradient selected heteronuclear multiple-bond correlation (gHMBC) experi- ments were performed according to standard pulse sequences. For interresidue correlation, two-dimensional NOESY experiments with a mixing time of 250 ms were used. Results Characterization of LPS H. influenzae type f strains RM6255, RM7290 and RM6252 were grown in liquid culture and the LPS was isolated by phenol/chloroform/light petroleum extraction [6]. Compositional analysis of the LPS samples for strains RM7290 and RM6252 identified D -glucose (Glc), D -galactose (Gal), 2-amino-2-deoxyglucose (GlcN) and L -glycero- D -manno-heptose (Hep) as the constituent sugars by GLC-MS of the derived alditol acetates and 2-butyl Table 1. Sugar analysis data for LPS-OH and OS samples derived from H. influen zae strains RM6255, RM7290 and RM6252. Relative detector response percentage Sugar residue a RM6255 RM7290 RM6252 OS LPS-OH OS LPS-OH OS LPS-OH Glc 81 64 59 43 51 40 Gal 32 23 37 33 Hep 19 20 9 21 11 14 GlcNAc 16 14 15 a Sugars were identified by GLC-MS as their alditol acetates. Ó FEBS 2003 Structural diversity of LPS in H. influenzae (Eur. J. Biochem. 270) 3155 glycoside derivatives on oligosaccharide material (Table 1). Strain RM6255 showed the same sugars except for Gal. In addition, glycine was detected as a substituent of each LPS by HPAEC with pulsed amperometric detection (HPAEC-PAD) as described earlier [30]. Neu5Ac can be detected in H. influenzae LPS by a method involving analysis by HPAEC-PAD of terminal N-acetyl neuraminic 2 acid residues released by neuraminidase treatment [7]. However, by applying this method in our study, N-acetyl neuraminic acid was not detected in any strain. O-Deacyla- tion of LPS by treatment with anhydrous hydrazine under mild conditions afforded water-soluble material (LPS-OH), which was subjected to ESI-MS. The ESI-MS spectra of the samples (negative mode) revealed abundant molecular peaks corresponding to triply and quadruply deprotonated ions. The MS data (Table 2), pointed to the presence of glycoforms in which each molecular species contains the conserved PEtn-substituted triheptosyl inner-core moiety attached via a phosphorylated Kdo linked to the O-deacylated lipid A (structure 1). Two populations of glycoforms were observed which differed by 123 Da (i.e. the mass of a PEtn group). As observed for other H. influenzae strains, this would be consistent with either phosphate or pyrophosphoethanolamine (PPEtn) substitu- tion at the O-4 position of the Kdo residue [2,3,35]. Thus, for strain RM6255, abundant quadruply/triply charged ions were observed at m/z 649.4/866.1 and 680.1/ 907.0 indicating Hex4 glycoforms with the respective compositions Hex 4 ÆHep 3 ÆPEtn 1,2 ÆP 1 ÆKdo 1 ÆLipidA-OH. Signals corresponding to minor glycoforms with composi- tions Hex 2 ÆHep 3 ÆPEtn 1,2 ÆP 1 ÆKdo 1 ÆLipidA-OH and Hex 3 Æ Hep 3 ÆPEtn 1,2 ÆP 1 ÆKdo 1 ÆLipidA-OH were observed at m/z 568.2/758.2, 599.2/799.1, 608.5/811.7, and 639.5/853.5, respectively. The ESI-MS spectrum of LPS-OH from RM7290 revealed major quadruply/triply charged ions at m/z 649.4/866.2, 730.5/974.3, and 761.3/1015.3 which indi- cated the occurrence of glycoforms with compositions Hex 4 ÆHep 3 ÆPEtn 1 ÆP 1 ÆKdo 1 ÆLipidA-OH, and Hex 6 ÆHep 3 Æ PEtn 1,2 ÆP 1 ÆKdo 1 ÆLipidA-OH, respectively. Less abundant quadruply charged ions at m/z 608.8, 639.6, 680.2, 690.0, 811.8 and 842.4 together with their triply charged counter- parts corresponded to glycoforms with the following compositions Hex 3 ÆHep 3 ÆPEtn 1,2 ÆP 1 ÆKdo 1 ÆLipidA-OH, Hex 4 ÆHep 3 ÆPEtn 2 ÆP 1 ÆKdo 1 ÆLipidA-OH, Hex 5 ÆHep 3 ÆPEtn 1 Æ P 1 ÆKdo 1 ÆLipidA-OH, and Hex 8 ÆHep 3 ÆPEtn 1,2 ÆP 1 ÆKdo 1 Æ LipidA-OH, respectively. The ESI-MS spectrum (negative mode) of LPS-OH from strain RM6252 showed ions corresponding to the same glycoforms as observed for strain RM7290. However, additional ions corresponding to Hex7 glycoforms with compositions Hex 7 ÆHep 3 ÆPEtn 1,2 ÆP 1 ÆKdo 1 ÆLipidA-OH were observed at m/z 1028.2 and 1068.8. In general, the ions corresponding to the higher glycoforms (Hex6 to Hex8) were of greater abundance in the spectrum of RM6252 than in the one of RM7290. From ESI-MS data it was also evident that PCho was not a major substituent in the LPS of the type f strains. Characterization of the Kdo-lipid A-OH element The structure of the lipid A-OH region in all H. influenzae strains investigated so far has been found 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¢ [2,3,5,6,34,36]. In this investigation, ESI-MS data (Table 2) and fatty acid compositional analysis yielding 3-hydroxytetradeca- noic acid indicated the presence of the same lipid A-OH structure in Hif. In addition, in a 1 H– 31 P coupled gHMQC experiment, a 31 Psignalatd )0.5 correlated to 1 H d 5.48, which was assigned to the anomeric 1 Hshift of the a-linked GlcN (GlcNI) in the lipid A. In the same spectrum, a 31 Psignalatd 3.4correlatedtoa 1 Hsignalat d 4.05 which was identified as the H-4 signal of the b-GlcN residue (GlcNII). Characterization of oligosaccharides Mild acid hydrolysis of LPS with dilute aqueous acetic acid afforded insoluble lipid A and core oligosaccharide, which after purification by gel filtration resulted in oligosaccharide samples OS6255, OS7290 and OS6252. Sugar analysis of the OS samples shown in Table 1 revealed D -glucose (Glc), L -glycero- D -manno-heptose in allthreestrainsaswellas D -galactose (Gal) in RM7290 and RM6252. The absence of 2-amino-2-deoxyglucose (GlcN) in the OS samples confirmed this sugar to be part of lipid A. After dephosphorylation of OS samples with 48% HF, methylation analysis of the resulting material (OS6255-P) showed terminal Glc, 4-substituted Glc, 3-substituted-Hep, 2,3-disubstituted Hep and 3,4-disubsti- tuted Hep as the major sugar components; linkage analysis of OS7290-P and OS6252-P revealed terminal Glc, terminal Gal, 4-substituted Glc, 4-substituted Gal, 3,4-disubstituted Hep, 2,3-disubstituted Hep and 2-substituted Hep in the relative proportions as shown in Table 3. The data was consistent with triantennary structures, containing the common inner-core element, L -a- D -Hepp-(1fi2)- L -a- D - Hepp-(1fi3)-[b- D -Glcp-(1fi4)]- L -a- D -Hepp-(1fi5)-a-Kdop of H. influenzae LPS. The presence of 3-substituted Hep in OS6255-P indicated substitution at the O-3 position of HepIII whereas in strains RM7290 and RM6252 substitu- tion at O-2 of HepIII was indicated (structure 1). ESI-MS on OS samples (Table 2) indicated OS6255 to be acetylated and OS7290 and OS6252 to be both acetylated and glycylilated. Thus in the ESI-MS spectrum of OS6255 (negative mode) major doubly charged ions were observed at m/z 783.8 and 804.7 corresponding to glycoforms with respective compositions Hex 4 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol and Ac 1 ÆHex 4 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol. Minor doubly charged ions at m/z 621.6, 642.8, 702.8, and 723.8 corresponded to Hex 2 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol, Ac 1 ÆHex 2 ÆHep 3 ÆPEtn 1 Æ AnKdo-ol, Hex 3 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol and Ac 1 ÆHex 3 Æ Hep 3 ÆPEtn 1 ÆAnKdo-ol, respectively. The ESI-MS spectra of OS7290 and OS6252 indicated 15 and 22 glycoforms, respectively. The Hex6 glycoforms were the most predom- inant ones in OS7290, comprising 64% of the OS molecules. They were detected as doubly charged ions at m/z 946.1, 966.9, and 995.3 corresponding to the respective composi- tions Hex 6 ÆHep 3 ÆPEtn 1 ÆP 1 ÆKdo 1 ÆAnKdo-ol, Ac 1 ÆHex 6 ÆHep 3 Æ PEtn 1 ÆP 1 ÆKdo 1 ÆAnKdo-ol, and Ac 1 ÆGly 1 ÆHex 6 ÆHep 3 ÆPEtn 1 Æ P 1 ÆKdo 1 ÆAnKdo-ol. The most predominant doubly charged ions in the ESI-MS spectrum of OS6252 at m/z 804.9 and 1129.2 corresponded to Hex4 and Hex8 glycoforms 3156 H. H. Yildirim et al. (Eur. J. Biochem. 270) Ó FEBS 2003 with compositions Ac 1 ÆHex 4 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol and Ac 1 ÆHex 8 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol, respectively. ESI-MS/MS Some information on the location of the acylation sites in OS7290 and OS6252 was provided by ESI MS/MS following on-line separation by CE in the positive mode [30,36]. Thus, product ion spectra obtained from the doubly charged ions at m/z 998 in OS7290 (composition: Ac 1 ÆGly 1 ÆHex 6 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol) and m/z 1160 in OS6252 (composition: Ac 1 ÆGly 1 ÆHex 8 ÆHep 3 ÆPEtn 1 Æ AnKdo-ol) showed ions at m/z 204 and 235 corresponding to AcÆHex and AcÆHep, respectively, indicating mixed acetylation at either a hexose residue or a heptose. No ions indicating glycylilation could be observed in the spectrum of OS6252. However, the spectrum of OS7290 showed ions at m/z 280 and 292 corresponding to GlyÆAnKdo-ol and AcÆGlyÆHep, respectively, indicating glycylilation at either Kdo or a heptose residue. No more detailed information could be obtained from the spectra which showed very random fragmentation patterns. Table 2. Negative ion ESI-MS data and proposed compositions for LPS-OH and OS samples of H. influenzae f-type strains RM6255, RM7290 and RM6252. Average mass units were used for calculation of molecular mass values based on proposed composition as follows: Hex, 162.14; Hep, 192.17; Kdo, 220.18; AnKdo-ol, 222.18; P, 79.98; PEtn, 123.05; Ac, 42.04; Gly, 57.05; and LipidA-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 intensity are not included in the table. Sample Observed ions (m/z) Molecular mass (Da) Relative abundance (%) (M-4H) 4– (M-3H) 3– (M-2H) 2– Observed Calculated 6255 7290 6252 Proposed composition LPS-OH – a 758.2 b 2277.6 2277.0 2 Hex 2 ÆHep 3 ÆPEtn 1 ÆP 1 ÆKdo 1 ÆLipidA-OH 599.2 b 799.1 b 2400.6 2400.1 3 Hex 2 ÆHep 3 ÆPetn 2 ÆP 1 ÆKdo 1 ÆLipidA-OH 608.9 811.5 2438.6 2439.2 6 6 6 Hex 3 ÆHep 3 ÆPEtn 1 ÆP 1 ÆKdo 1 ÆLipidA-OH 639.7 853.5 2563.2 2562.2 3 2 4 Hex 3 ÆHep 3 ÆPEtn 2 ÆP 1 ÆKdo 1 ÆLipidA-OH 649.4 866.1 2601.5 2601.4 37 16 16 Hex 4 ÆHep 3 ÆPEtn 1 ÆP 1 ÆKdo 1 ÆLipidA-OH 680.3 906.7 2724.2 2724.4 49 4 6 Hex 4 ÆHep 3 ÆPEtn 2 ÆP 1 ÆKdo 1 ÆLipidA-OH 689.9 920.1 2763.5 2763.4 5 11 Hex 5 ÆHep 3 ÆPEtn 1 ÆP 1 ÆKdo 1 ÆLipidA-OH 720.7 960.8 2886.1 2886.5 3 Hex 5 ÆHep 3 ÆPEtn 2 ÆP 1 ÆKdo 1 ÆLipidA-OH 730.4 973.5 2924.6 2925.6 37 8 Hex 6 ÆHep 3 ÆPEtn 1 ÆP 1 ÆKdo 1 ÆLipidA-OH 761.3 1015.1 3048.8 3048.6 23 7 Hex 6 ÆHep 3 ÆPEtn 2 ÆP 1 ÆKdo 1 ÆLipidA-OH 771.0 1028.3 3088.0 3087.7 10 Hex 7 ÆHep 3 ÆPEtn 1 ÆP 1 ÆKdo 1 ÆLipidA-OH 801.9 1068.8 3210.5 3210.8 6 Hex 7 ÆHep 3 ÆPEtn 2 ÆP 1 ÆKdo 1 ÆLipidA-OH 811.5 1082.2 3249.6 3249.9 12 Hex 8 ÆHep 3 ÆPEtn 1 ÆP 1 ÆKdo 1 ÆLipidA-OH 842.3 1123.1 3372.8 3372.9 2 13 Hex 8 ÆHep 3 ÆPEtn 2 ÆP 1 ÆKdo 1 ÆLipidA-OH OS 621.6 b 1245.2 1246.0 6 Hex 2 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 642.8 b 1287.6 1288.0 3 Ac 1 ÆHex 2 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 702.8 b 1407.6 1408.2 5 Hex 3 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 723.5 1449.0 1450.2 5 2 3 Ac 1 ÆHex 3 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 745.0 1492.0 1492.2 1 Ac 2 ÆHex 3 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 752.7 1507.0 1507.2 2 7 Ac 1 ÆGly 1 ÆHex 3 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 783.9 1569.8 1570.3 40 1 2 Hex 4 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 805.1 1611.8 1612.3 42 8 11 Ac 1 ÆHex 4 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 826.0 1653.0 1654.3 7 6 Ac 2 ÆHex 4 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 833.6 1669.2 1669.4 2 1 Ac 1 ÆGly 1 ÆHex 4 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 854.1 1710.2 1711.4 6 2 Ac 2 ÆGly 1 ÆHex 4 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 865.2 1732.8 1732.4 1 1 Hex 5 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 886.0 1774.0 1774.4 3 7 Ac 1 ÆHex 5 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 907.0 1815.8 1816.4 6 Ac 2 ÆHex 5 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 913.6 1829.2 1831.5 1 2 Ac 1 ÆGly 1 ÆHex 5 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 936.0 1873.6 1873.5 2 Ac 2 ÆGly 1 ÆHex 5 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 945.7 1893.8 1894.6 4 1 Hex 6 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 967.0 1936.8 1936.6 36 6 Ac 1 ÆHex 6 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 995.9 1993.8 1993.6 24 1 Ac 1 ÆGly 1 ÆHex 6 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 1027.0 2055.8 2056.7 2 Hex 7 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 1048.1 2098.0 2098.7 9 Ac 1 ÆHex 7 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 1076.8 2156.8 2155.8 4 Ac 1 ÆGly 1 ÆHex 7 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 1108.0 2218.2 2218.9 2 Hex 8 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 1129.1 2260.4 2260.9 2 14 Ac 1 ÆHex 8 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 1157.6 2318.1 2317.9 1 9 Ac 1 ÆGly 1 ÆHex 8 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol a Trace amounts of a signal at m/z 568.2 were detected. b The observed m/z-values correspond to spectra from RM6255. All other m/z-values correspond to spectra from RM6252. Observed values in RM7290 and RM6255 varied ± 0.8 Da. Ó FEBS 2003 Structural diversity of LPS in H. influenzae (Eur. J. Biochem. 270) 3157 Sequence analysis of OS6255-P, OS7290-P and OS6522-P by MS n OS6255-P, OS7290-P and OS6252-P were permethylated and analyzed by ESI-MS n in order to determine the sequence and branching details of the various glycoforms. This method was introduced by Reinhold et al. and has been applied by us recently [36–38]. In the ESI-MS spectrum of OS6255-P (positive mode), shown in Fig. 1A, four major sodiated parent ions were observed at m/z 1264.3, 1468.1, 1672.4 and 1876.6 corresponding to the dephosphorylated and permethylated Hex1 to Hex4 glycoforms (Hex 1)4 ÆHep 3 Æ AnKdo-ol). These ions were further fragmented in MS 2 experiments, and some of the resulting product ions underwent further fragmentation (MS 3 ) to confirm the existence of the proposed glycoforms shown in Table 4. Two isomeric Hex1 glycoforms were identified by performing MS 2 on parent ion m/z 1264.3. Two product ions at m/z 797.4 and 737.3 corresponding to the neutral losses of tHexÆHepIII and HepIÆAnKdo-ol, respectively, confirmed the presence of a Hex1 glycoform in which HepIII is substituted. The glycoform with a hexose elonga- ting from HepI was confirmed by product ions m/z 1001.4 and 753.3, due to losses of t-HepIII and tHepIIIÆHepII, respectively. Performing MS 2 on ion m/z 1468.3, and subsequent MS 3 on the resulting product ions determined four isomeric Hex2 structures. The ion corresponding to the loss of t-HepIII at m/z 1205.5 was further fragmented to give the ion at m/z 957.5 corresponding to the loss of an unsubstituted HepII. This confirmed the structure of the glycoform in which a disaccharide moiety substitutes HepI. Furthermore, in the same MS 3 spectrum an ion was detected at m/z 753.5 due to the loss of a tHex– HepII unit thus evidencing a structure with one hexose residue substituted to both HepI and HepII. The ion at m/z 548.9 corresponded to the loss of tHex–Hex–HepII. Finally, a structure with one hexose residue each linked to HepI and HepIII could be determined when the product ion, m/z 1001.5, corresponding to the loss of a terminal hexose linked to HepIII, was further fragmented to give an ion at m/z 753.3 due to the loss of HepII. Five isomeric Hex3 glycoforms were determined by performing MS 2 on the parent ion m/z 1672.4 and subsequent MS 3 on resulting product ions at m/z 1409.6 and 1205.6 due to losses of a terminal heptose and a terminal Hex–Hep residue, respectively. When the ion at m/z 1409.6 was further fragmented in a MS 3 experiment it gave, inter alia, product ions at m/z 883.4 and 753.4 due to Table 3. Methylation analysis data of the dephosphorylated OS samples derived from H. Influenzae type f strains RM6255, RM7290, and RM6252. Methylated sugar a T gm b Linkage assignment Relative abundance OS6255-P OS6252-P OS7290-P 2,3,4,6-Me 4 -Glc 1.00 D -Glcp-(1- 54 10 15 2,3,4,6-Me 4 -Gal 1.05 D -Galp-(1- 14 23 2,3,6-Me 3 -Gal 1.16 )4)- D -Galp-(1- 13 6 2,3,6-Me 3 -Glc 1.18 )4)- D -Glcp-(1- 18 31 36 3,4,6,7-Me 4 -Hep 1.43 )2)- L , D -Hepp(1- 5 12 2,4,6,7-Me 4 -Hep 1.45 )3)- L , D -Hepp-(1- 10 2,6,7-Me 3 -Hep 1.50 )3,4)- L , D -Hepp-(1- 3 13 14 4,6,7-Me 3 -Hep 1.55 )2,3)- L , D -Hepp-(1- 13 13 8 a 2,3,4,6-Me 4 -Glc represents 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl- D -glucitol-1-d 1 , etc. b Retention times (T gm ) are reported relative to 2,3,4,6-Me 4 -Glc. Fig. 1. ESI-MS spectra (positive mode) of the permethylated (A) OS6255-P (B) OS7290-P and (C) OS6252-P. The peaks marked by asterisks 5 indicate glycoforms with mono-methylated phosphate groups due to glycoforms resulting from incomplete dephosphorylation. 3158 H. H. Yildirim et al. (Eur. J. Biochem. 270) Ó FEBS 2003 losses of HepIÆAnKdo-ol and HexÆHexÆHepII elements, respectively. Thus two structures with terminal HepIII were identified: the first with a trisaccharide group linked to HepII and the second with a disaccharide group linked to HepII and a hexose linked to HepI. When the ion at m/z 1205.6 was further fragmented in a MS 3 experiment product ions at m/z 957.6, 753.4, and 549.3 were observed defining the losses of single substituted HepII, t-HexÆHepII, and HexÆHexÆHepII. A single Hex4 glycoform was identified in RM6255 comprising terminal hexose residues substituted to Table 4. Structures of glycoforms from OS6255-P, OS7290-P and OS6252-P elucidated by ESI-MS n . Subscripts denoted by the letters a, b and c indicated the number of hexose residues in the following structure: Relative abundance a (%) Structure 6255 Structure 7290 Structure 6252 Relative abundance b 6255 7290 6252 a b c a b c a b c 6255 7290 6252 Hex1 6.3 1 0 0 High 0 0 1 Low Hex2 17.5 1.2 3.3 2 0 0 2 0 0 2 0 0 Trace Trace Trace 1 1 0 1 1 0 1 1 0 Low Low Medium 0 2 0 0 2 0 0 2 0 Low Low Low 1 0 1 1 0 1 1 0 1 Medium Low High 0 1 1 Low 0 0 2 Low Hex3 23.3 2.4 12.4 1 2 0 1 2 0 1 2 0 Medium Low Low 2 0 1 1 1 1 1 1 1 Trace Medium Low 1 1 1 0 3 0 0 3 0 Medium Low Low 0 2 1 1 0 2 1 0 2 Low Medium Low 0 3 0 0 2 1 Low Low 2 0 1 Trace 2 1 0 Trace Hex4 52.9 25.5 27.7 1 2 1 1 2 1 1 2 1 High Low Medium 1 3 0 1 3 0 High Medium 1 1 2 1 1 2 Low Low 0 4 0 Trace 0 3 1 Trace 3 0 1 Trace 1 0 3 Low 2 0 2 Trace Hex5 6.1 16.5 1 4 0 1 4 0 Low Medium 1 3 1 1 3 1 Medium Low 0 3 2 0 3 2 Trace Trace 1 2 2 1 2 2 Low Low 2 3 0 1 1 3 Trace Trace Hex6 60.6 15.7 1 3 2 1 3 2 High Medium 2 3 1 Trace 1 4 1 Medium 1 2 3 Trace 0 3 3 Trace Hex7 1.2 10.3 1 3 3 1 3 3 Medium Medium 1 4 2 1 4 2 Medium Medium 0 4 3 Trace Hex8 3.0 14.0 1 4 3 1 4 3 High High a Relative abundance for each glycoform. Calculated from the intensity of the molecular ion peak relative to the total intensity of all molecular ion peaks in the MS spectrum expressed as percentage. b Relative abundance for structural isomers of each glycoform. Calculated from the intensity of the product ions in the MS 2 spectrum and indicated as follows: high (over 80%), medium (30–80%), low (2–30%), trace (below 2%). Ó FEBS 2003 Structural diversity of LPS in H. influenzae (Eur. J. Biochem. 270) 3159 both HepI and HepIII, and a HexÆHex unit substituted to HepII. This structure is defined by a MS 2 experiment on molecular ion m/z 1876.9 resulting in fragment ions m/z 1409.7 and 1145.5 due to the loss of tHexÆHepIII and tHexÆHepÆAnKdo-ol, respectively. This structure is further supported by the MS 3 experiment on m/z 1409.7 in which the resulting fragment ions m/z 753.4 and 679.4 are due to losses of tHexÆHexÆHepII and tHexÆHepIÆAnKdo-ol, respectively. In the ESI-MS spectra of permethylated OS7290-P and OS6252-P (Figs 1B and C) seven sodiated parent ions were observed corresponding to Hex2 to Hex8 glyco- forms (Hex 1)8 ÆHep 3 ÆAnKdo-ol). In order to obtain sequence and branching information, these molecular ions were further fragmented in MS 2 experiments, and if necessary MS 3 experiments were used. The fragment ions produced in MS 2 and MS 3 experiments were analyzed in analogy to OS6255-P described above and provided evidence for 22 structures present in OS7290-P and 33 structures present in OS6252-P as shown in Table 4. It was obvious that OS6252-P is a slightly more hetero- geneous mixture of glycoforms than OS7290-P. The Hex4 and Hex6 glycoforms are the most abundant in OS7290- P, while the abundance is almost equally distributed between Hex3 and Hex8 glycoforms in OS6252-P. The Hex6 glycoform is the most abundant in OS7290-P and comprises only one structure while the same glycoform has five isomers in OS6252-P. Thus, when the ion at m/z 2283.6 in OS7290-P was further fragmented in a MS 2 experiment (see Fig. 2A) it showed, inter alia,anionat m/z 1613.8 due to the loss of a t-Hex–Hex–HepIII unit. AMS 3 experiment on m/z 1613.8 revealed, inter alia, the ion at m/z 884.3 due to the loss of a tHex–HepI– AnKdo-ol unit, which gave evidence for the Hex6 glycoform in OS7290-P being substituted by a disacchar- ide element at HepIII and a trisaccharide unit at HepII (see Fig. 2B). Ions corresponding to this glycoform were identified in the same manner in OS6252-P. However, when the parent ion at m/z 2283.5 in OS6252-P was fragmented further in a MS 2 experiment it revealed additional ions at m/z 1817.8 and 1410.3 due to losses of tHex–HepIII and tHex–Hex–Hex-HepIII units, respect- ively (see Fig. 3A). The MS 3 experiment on m/z 1817.8 Fig. 2. The ESI-MS n analysis of permethyl- ated OS7290-P. (A) Product ion spectrum of m/z 2283.6 corresponding to the sodium adduct of the Hex6 glycoform. (B) The MS 3 spectrum of the fragment ion m/z 1613.8 resulting from the MS 2 of m/z 2283.6. The relevant fragmentation pathways are shown to the right of each spectrum. Fig. 3. The ESI-MS n analysis of permethylated OS6252-P. (A) Prod- uct ion spectrum of m/z 2283.6 corresponding to the sodium adduct of the Hex6 glycoform. The proposed fragmentations are shown beside the spectrum. The number of hexoses, Hex x , linked to HepI and HepII are determined by MS 3 .(B)MS 3 of the ions m/z 1410.3, 1613.9 and 1817.8 resulting from MS 2 of m/z 2283.5. The fragmentation pathways are shown at the bottom of the spectra. 3160 H. H. Yildirim et al. (Eur. J. Biochem. 270) Ó FEBS 2003 showed ions at m/z 1088.6 and 957.9 due to losses of tHex–HepI–AnKdo-ol and tHex–Hex–Hex–HepII, respect- ively. This defined two Hex6 glycoforms having either a trisaccharide or a tetrasaccharide unit linked to HepII (see Fig. 3B). When the ion at m/z 1410.3 was further fragmented in a MS 3 experiment it gave ions at m/z 884.4 and 679.9 due to losses of a HepI–AnKdo-ol and tHex– HepI–AnKdo-ol units, respectively. This gave evidence for two Hex6 glycoforms having either a disaccharide or a trisaccharide unit linked to HepII (see Fig. 3B). NMR spectroscopy on LPS-OH from RM6255 and O-deacylated OS7290 and OS6252 The 1 H NMR spectra were assigned using chemical shift correlation techniques (COSY, TOCSY, HMQC, HMBC and HSQC experiments). The chemical shift data corres- ponding to Hif 6255 is given in Table 5. NMR data corresponding to Hif 7290 and 6252 were found to be identical and are combined in Table 6. Subspectra corres- ponding to the individual glycosyl residues were identified on the basis of spin-connectivity pathways delineated in the 1 H chemical shift correlation maps, the chemical shift values, and the vicinal coupling constants. 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 7 and Fig. 4 (RM6255) and Fig. 5 (RM6252)], which also served to confirm the anomeric configurations of the linkages and the monosaccharide sequence. The Hep ring systems were identified on the basis of the small J 1,2 -values and their a-configurations were confirmed by the occurrence of single intraresidue NOE between the respective H-1 and H-2 resonances. The structure of the Hex4 glycoform in strain RM6255 was determined by examining the 1 H-NMR spectrum of its LPS-OH in detail. Anomeric 1 H/ 13 C NMR resonances of HepI, HepII and HepIII were identified at d 5.17/99.22, 5.78/97.6 and 5.08/100.1, respectively. Four anomeric signals corresponding to Glc residues I to IV were observed at d 4.57/101.7, 5.26/100.2, 4.54/102.4 and 4.54/99.3, respectively. In addition, the anomeric signals of the a-andb-linked GlcN residues of the lipid A part were observed at d 5.48/93.3 and 4.62/101.6, respectively. Chem- ical shift data are consistent with GlcI, III and IV being terminal residues and GlcII being substituted in O-4 in agreement with methylation analyses. The occurrence of interresidue NOESY connectivities between the proton pairs HepIII H-1/HepII H-2, HepII H-1/HepI H-3, and HepI H-1/Kdo H-5 confirmed the sequence of the heptose-containing trisaccharide unit in the inner-core region and the point of attachment to Kdo (structure 1). The occurrence of interresidue NOE between H-1 of GlcI and H-4/H-6 of HepI confirmed the 1,4 linkage between GlcI and HepI. Interresidue NOE connectivities between proton pairs GlcIII H-1/GlcII H-4/H-6 and GlcII H-1/HepII H-2/H-3 established the sequence of a disaccharide unit and its attachment point to HepII as b- D -Glcp-(1fi4)-a- D -Glcp-(1fi3)- L -a- D -Hepp- (1fi. Interresidue NOE between H-1 of GlcIV and H-3/ H-2 of HepIII gave evidence for the a b- D -Glcp-(1fi3)- L - a- D -Hepp-(1fi unit. Table 5. 1 Hand 13 C NMR chemical shifts for O-deacylated LPS of H. influenzae type f strain RM6255. Data was recorded in D 2 O containing 2 m M perdeutero-EDTA and 10 mgÆmL )1 perdeutero-SDS at 30 °C. 3 J H,H -values for anomeric 1 H resonances are given in parenthesis. ND, Not determined; NR, 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-6 H-6 B H-7 A /C-7 H-7 B GlcNI fi6)-a- D -GlcpN-(1fi 5.48 3.84 ND ND ND ND ND 93.3 53.40 GlcNII fi6)-b- D -GlcpN(1fi 4.62 3.88 ND 4.03 ND ND ND 101.6 55.6 72.0 HepI fi3,4)- L -a- D -Hepp(1fi I 5.17 (NR) 4.19 4.13 4.35 – 4.04 3.68 3.68 99.22 69.7 72.4 72.3 67.7 63.1 HepII fi2,3)- L -a- D -Hepp(1fi II 5.78 (NR) 4.28 4.08 ND 3.88 4.59 ND ND 97.6 78.1 78.3 ND 73.3 HepIII fi3)- L -a- D -Hepp(1fi III 5.08 (NR) 4.04 4.21 ND ND ND 3.73 3.86 100.1 67.6 76.5 63.1 GlcI b- D -Glcp (1fi IV 4.57 (8.0) 3.39 3.47 3.31 3.47 3.98 3.77 101.7 73.3 75.8 70.0 75.8 60.3 GlcII fi4)-a- D -Glcp(1fi V 5.26 (4.0) 3.59 3.86 3.70 3.89 3.97 3.89 100.0 71.2 71.3 78.6 70.8 59.4 GlcIII b- D -Glcp (1fi VI 4.54 (8.1) 3.41 3.52 3.44 3.52 3.95 3.78 102.4 72.5  75.0 69.2 75.5 60.3 GlcIV b- D -Glcp (1fi VII 4.54 (7.9) 3.42 3.44 3.41 3.44 3.93 3.76 99.3 72.5  75.0 69.3  75.0 60.3 Kdo fi4,5)-a-Kdop-(2fi 2.03/2.37 ND 4.29 ND 3.80 ND ND ND PPEtn 4.14 3.28 61.4 39.4 PEtn 4.08 3.26 60.6 39.5 Ó FEBS 2003 Structural diversity of LPS in H. influenzae (Eur. J. Biochem. 270) 3161 The ESI-MS data of OS6255 (Table 2) indicated that the most abundant glycoform in this strain was monoacetyl- ated. The acetylation site was identified by NMR on OS6255 (data not shown). It was observed that chemical shift values for several HepIII resonances in OS6255 differed considerably from the corresponding values in LPS-OH. Thus resonances for H-1/C-1, H-2/C-2 and H-3/C-3 were observed at d 5.13/97.6, 5.16/68.9 and 4.47/ 73.8, respectively. The chemical shift values were consistent with acetylation at O-2 as the H-1 (+ 0.05 p.p.m), H-2 (+ 1.2), H-3 (+ 0.26 p.p.m) and C-2 (+ 1.3 p.p.m) signals were shifted downfield, while C-1 () 2.5 p.p.m) and C-3 () 2.7 p.p.m) were shifted upfield [39]. From the combined data it could thus be concluded that the Hex4 glycoform of RM6255 has the structure 2. Structural details of the oligosaccharide epitopes in RM7290 and RM6252 were provided by 1 H-NMR and 13 C-NMR data of OS7290 and OS6252 (Table 6). Several signals for methylene protons of AnKdo-ol were observed in the COSY and TOCSY spectra in the region 2.25– 1.65 p.p.m. This is due to the fact that several anhydro- forms of Kdo are formed during the hydrolysis by elimination of phosphate or pyrophosphoethanolamine from the C-4 position [40]. Anomeric resonances of HepI, HepII and HepIII of OS7290 were identified at d 5.06–5.16, 5.68–5.72 and 5.08, respectively. As observed earlier, several anomeric signals for HepI and HepII appear due to the microheterogeneity caused by the anhydro-forms of Kdo [40]. Subspectra corresponding to GlcI to IV, GalI and GalII were identified in the two-dimensional COSY and TOCSY spectra at d 4.54, 5.33, 4.58, 4.46, 4.47, and 4.44, respectively. In addition, a minor spin system corresponding to a- D -Galp was identified at d 4.96.Chemicalshiftdataare consistent with GlcI and GalI being terminal residues in Table 6. 1 Hand 13 C NMR chemical shifts for OS7290 and OS6252. DatawasrecordedinD 2 Oat30°C. 3 J H,H -values for anomeric 1 H resonances are given in parenthesis. Signals corresponding to PCho methyl protons and carbons occurred at 3.20 and 53.6 p.p.m., respectively. Pairs of deoxy protons of reduced, AnKdo were identified in the DQF-COSY at d 1.91–2.30. 3 J H,H -values for anomeric 1 H resonances are given in parenthesis. NR, Not resolved (small coupling); ND, not determined. 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-6 H-6 B H-7 A /C-7 H-7 B Hex6 glycoform HepI fi3,4)- L -a- D -Hepp(1fi I 5.06–5.16 (NR) 4.01 4.06 4.15 ND 4.31 ND 96.2 69.7 76.8 74.2 72.8 HepII fi2,3)- L -a- D -Hepp(1fi II 5.68–5.72 (NR) 4.29 4.12 ND 3.73 4.56 3.79 3.90 97.9 77.8 77.4 71.2 73.9 61.7 HepIII fi2)- L -a- D -Hepp(1fi III 5.08 (NR) 4.01 4.08 ND ND ND ND 98.7 77.8 69.1 GlcI b- D -Glcp (1fi IV 4.54 (8.5) 3.35 3.47 3.41 3.47 3.77 3.96 102.2 73.3 76.0 72.0 76.0 60.6 GlcII fi4)-a- D -Glcp(1fi V 5.33 (4.8) 3.62 3.87 3.71 3.89 3.97 3.90 99.6 71.1 71.0 78.0 71.0 59.6 GlcIII fi4)-b- D -Glcp (1fi VI 4.58 (8.0) 3.39 3.67 3.71 3.71 3.84 4.00 102.0 72.0 74.4 78.0 71.0 59.6 GalI b- D -Galp(1fi VII 4.47 (8.4) 3.55 3.68 3.94 3.70 ND 102.5 76.5 72.0 68.0 74.9 GlcIV fi4)-b- D -Glcp (1fi VIII 4.46 (8.1) 3.42 3.70 3.69 3.69 3.85 4.00 101.5 69.6 74.3 78.0 70.9 59.6 GalII b- D -Galp(1fi IX 4.44 (8.4) 3.57 3.70 3.95 3.70 ND 102.5 76.5 72.0 68.0 74.9 PEtn 4.12 3.24 62.3 40.0 Hex8 glycoform GalI fi4)-b- D -Galp(1fi VII 4.54 (7.8) 3.59 3.76 4.07 3.81 ND 102.3 70.5 72.3 76.8 75.5 GalIII a- D -Galp(1fi X 4.98 (3.9) 3.84 3.90 4.05 4.37 3.72 3.72 99.8 68.0 68.7 72.2 70.5 60.0 ND GalII fi4)-b- D -Galp(1fi IX 4.52 (7.8) 3.61 3.76 4.07 3.81 ND 102.3 70.5 72.3 76.8 75.5 GalV a- D -Galp(1fi XI 4.96 (3.9) 3.86 3.92 4.05 4.37 3.72 3.72 99.8 68.0 68.8 72.2 70.6 60.0 ND 3162 H. H. Yildirim et al. (Eur. J. Biochem. 270) Ó FEBS 2003 [...]... diversity within the type f cluster Previous studies of LPS from H in uenzae have resulted in a structural model consisting of a conserved Ó FEBS 2003 Structural diversity of LPS in H in uenzae (Eur J Biochem 270) 3165 Fig 5 Selected region of the two-dimensional NOESY spectrum (mixing time 250 ms) of OS derived from LPS of RM6252 Cross-peaks that are characteristic for the Hex6 and Hex8 glycoforms are... GalII H-4 Fig 4 Selected region of the two-dimensional NOESY spectrum (mixing time 250 ms) of LPS-OH derived from LPS of RM6255 Cross-peaks of significant importance are labelled Discussion We report the first structural details for the LPS from H in uenzae type f strains Previous analyses on LPS from H in uenzae strains expressing a capsule have been limited to type b and a single strain derived from a... Population analysis of a large number of H in uenzae strains by Musser and colleagues using multilocus enzyme electrophoresis typing indicated that type f isolates form a relatively tight group of strains that is distinct from the type b and type d isolates [41] H in uenzae type f strains RM6255, RM7290 and RM6252 are three epidemiologically distinct isolates that were selected to be representative of the diversity. .. during in vitro growth was lower than was found in type b strains Analysis of large numbers of individual colonies of strain RM6255 and RM7290 by immunoblotting with monoclonal antibody TEPC 15 confirmed that PCho was variably expressed in each strain, albeit at a very low frequency (data not shown) The LPS of type f strains is substituted by acetates and glycine as has been observed for other strains. .. not shown) None of the strains expressed high levels of chain extension from HepI However, ESI-MSn on permethylated and dephosphorylated OS samples indicated minor glycoforms with disaccharide units elongating from HepI in all three strains RM6255, which does not contain lex2 might then express a b-D-Galp-(1fi4)b-D-Glcp-HepI unit as observed in H in uenzae 2019 whereas the other strains may express... a-2,3-sialyltransferase that adds Neu5Ac to a terminal lactose unit from HepIII Although each strain was found to express the lic3A gene (D Hood, unpublished results) none of the type f strains were found to contain detectable amounts of Neu5Ac in the LPS PCho is a prominent substituent in H in uenzae LPS Levels of expression have been shown to be high in NTHi and strain Rd, however, type b strains express... a minor extent [15] The presence of PCho has been shown to be important in colonization and virulence [43,44] The expression and phase variation of the PCho epitope on H in uenzae LPS require the four genes of the lic1 locus The three strains in our study contained the lic1 locus, however, PCho was detected only in the LPS from strain RM6252 in very low levels PCho expression in type f strains during... epitopes common for H in uenzae LPS However, chain extension by globoside units from both HepII and HepIII in the same glycoform has not been observed previously The availability of the complete genome sequence of H in uenzae strain Rd has facilitated a comprehensive study of LPS biosynthetic loci in the type b strain Eagan (RM153) and in a capsule deficient strain Rd– (RM118), derived from a type d isolate... lipopolysaccharide Structural analysis of the lipopoly- 17 18 19 20 21 22 saccharide from nontypeable Haemophilus in uenzae strain 486 Eur J Biochem 268, 2148–2159 Bauer, S.H., Mansson, M., Hood, D.W., Richards, J.C., Moxon, E.R & Schweda, E.K (2001) A rapid and sensitive procedure for determination of 5-N-acetyl neuraminic acid in lipopolysaccharides of Haemophilus in uenzae: a survey of 24 non-typeable H in uenzae. .. emergence of Haemophilus in uenzae types e and f as significant pathogens Clin Infect Dis 21, 1322–1324 Glatman-Freedman, A & Litman, N (1996) Septic arthritis caused by an unusual type of Haemophilus in uenzae J Infect 32, 143–145 Pincus, D.R & Robson, J.M (1998) Meningitis due to Haemophilus in uenzae type f J Paediatr Child Health 34, 95–96 Urwin, G., Krohn, J.A., Deaver-Robinson, K., Wenger, J.D., Farley, . Structural analysis of lipopolysaccharides from Haemophilus in uenzae serotype f Structural diversity observed in three strains Ha ˚ kan H proportion of infections due to serotype f strains in the Hib postvaccination era [26]. The capsular polysaccharide of type f strains is known to be composed of