Characterization of novel structural features in the lipopolysaccharide of nondisease associated nontypeable Haemophilus influenzae Malin K. Landerholm 1 , Jianjun Li 2 , James C. Richards 2 , Derek W. Hood 3 , E. Richard Moxon 3 and Elke K. H. Schweda 1 1 Clinical Research Centre, Karolinska Institutet and University College of South Stockholm, NOVUM, Huddinge, Sweden; 2 Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada; 3 Molecular Infectious Diseases Group, University of Oxford, Department of Paediatrics, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK Nontypeable Haemophilus influenzae (NTHi) is a common commensal of the human upper respiratory tract and is associated with otitis media in children. The structures of the oligosaccharide portions of NTHi lipopolysaccharide (LPS) from several otitis media isolates are now well characterized but it is not known whether there are structural differences in LPS from colonizing, nondisease associated strains. Struc- tural analysis of LPS from nondisease associated NTHi strains 11 and 16 has been achieved by the application of high-field NMR techniques, ESI-MS, ESI-MS n , capillary electrophoresis coupled to ESI-MS, composition and link- age analyses on O-deacylated LPS and core oligosaccharide material. This is the first study to report structural details on LPS from strains taken from the nasopharynx from healthy individuals. Both strains express identical structures and contain the common element of H. influenzae LPS, L -a- D -Hepp-(1fi2)-[PEtnfi6]- L -a- D -Hepp-(1fi3)-[b- D -Glcp- (1fi4)]- L -a- D -Hepp-(1fi5)-[PPEtnfi4]-a-Kdop-(2fi6)- lipid A, in which each heptose is elongated by a single hexose residue with no further oligosaccharide extensions. In the major Hex3 glycoform, the terminal Hepp residue (HepIII) is substituted at the O-2 position by a b- D -Galp residue and the central Hepp residue (HepII) is substituted at O-3 by a a- D - Glcp residue. Notably, the strains express two phosphocho- line (PCho) substituents, one at the O-6 position of a- D -Glcp and the other at the O-6 position of b- D -Galp. Major acety- lation sites were identified at O-4 of Gal and O-3 of HepIII. Additionally, both strains express glycine, and strain 11 also expresses detectable amounts of N-acetylneuraminic acid. Keywords: carriage; ESI-MS; Haemophilus influenzae; lipopolysaccharide; NMR. Acapsular or nontypeable Haemophilus influenzae (NTHi) is a major bacterial cause of otitis media and respiratory tract infections in the first years of life. Otitis media is a childhood disease, which accounts for the highest frequency of paediatric visits in Western countries [1]. NTHi frequently colonize the nasopharynx. Exposure to H. influenzae begins after birth so that from infancy onward, carriage of one or more strains for periods of days to months is common. Epidemiological studies on nasopharyngeal carriage of NTHi have been performed with healthy children in day-care centers [2,3]. It was found that younger children acquire and eliminate a number of different strains, some of which are shared with other children, whereas other strains are restricted to a single host. High rates of carriage did not appear to correlate with occurrence of disease. However, it has been observed that colonization levels increase shortly before the onset of disease and otitis prone children tend to be more heavily colonized. Moreover, the number of times a child is colonized with H. influenzae has been found to be directly related to the frequency of otitis media [4]. NTHi causes otitis media when the bacteria opportunistically translocates from the nasopharynx to the middle ear via the eustachian tube, often in the presence of viral respiratory infections [5]. Whether all colonizing strains of H. influenzae are equally capable of causing otitis media or alternatively, some strains possess particular virulence factors necessary to cause otitis media, is still not known. Cell wall lipopolysaccharide (LPS) is an essential and characteristic surface component of H. influenzae and is implicated as a major virulence factor. H. influenzae elab- orates short-chain LPS which lacks O-specific polysaccha- ride chains and is often referred to as lipooligosaccharide. LPS oligosaccharide epitopes of H. influenzae can mimic host glycolipids. The molecule is heterogeneous due to a number of mechanisms including high frequency switching of the expression (phase variation) of a number of epitopes. 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; gHMQC, gradient selected heteronuclear multiple quantum coher- ence; Hep, L -glycero- D -manno-heptose; Hex, hexose; HexNAc, N-acetylhexosamine; HPAEC, high-performance anion-exchange chromatography; Kdo, 3-deoxy- D -manno-oct-2-ulosonic acid; LPS, lipopolysaccharide; LPS-OH, O-deacylated LPS; lipid A-OH, O-deacylated lipid A; Neu5Ac, N-acetylneuraminic acid; NTHi, nontypeable Haemophilus influenzae; OS, oligosaccharide; PCho, phosphocholine; PEtn, phosphoethanolamine; PPEtn, pyrophos- phoethanolamine; MS n , multiple step tandem mass spectrometry. (Received 7 October 2003, revised 18 December 2003, accepted 15 January 2004) Eur. J. Biochem. 271, 941–953 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.03996.x The availability of the complete genome sequence of H. influenzae strain Rd [6] has facilitated a comparative study of LPS biosynthetic loci from type b and d strains [7,8]. Gene functions have been identified in those strains that are responsible for most of the steps in the biosynthesis of the oligosaccharide portions of the LPS molecules. The inner core of H. influenzae LPS has been found to consist of the triheptosyl oligosaccharide moiety, L -a- D -Hepp- (1fi2)-[PEtnfi6]- L -a- D -Hepp-(1fi3)-[b- D -Glcp-(1fi4)]- L -a- D - Hepp-(1fi5)-a-Kdop, in which each of the three heptose residues can provide a point for elongation by oligosaccha- ride chains or for attachment of noncarbohydrate substit- uents. H. influenzae strains expressing LPS glycoform populations having biantennary [9–11] and triantennary [12–14] structures have been reported. Our previous studies have focused on the extent of conservation and variability of LPS expression in a repre- sentative set of clinical isolates of NTHi obtained from otitis media patients [10,11,13,15–20] and relating this to the role of the molecule in commensal and virulence behavior. Recently, we demonstrated that terminal sialic acid con- taining oligosaccharide epitopes are essential virulence determinants in experimental otitis media [21]. The clinical isolates investigated by us were human middle ear isolates that were characterized by ribotyping and that span a H. influenzae species-level dendrogram comprising > 400 strains of both typeable and NTHi strains [22,23]. To gain a better understanding between acquisition and development of disease caused by NTHi and the possible role of LPS epitopes in bacterial translocation, we have now extended our studies to include a number of nondisease associated isolates which were collected from the nasopharynx of healthy children in the same study (on the efficacy of a pneumococcal vaccine) referred to above. Here we report on structural analyses of two of these strains, referred to as NTHi carriage strains 11 and 16. Experimental procedures Bacterial strains, growth and LPS extraction Nondisease associated NTHi strains 11 and 16 are isolates from the nasopharynx of infants of 21 and 20 months of age, respectively, and are part of a series of studies aimed at evaluating conjugate pneumococcal vaccines conducted by the Finnish Otitis Media Study Group [24,25]. These two strains were selected from a group of 64 carriage NTHi isolates following phylogenetic analysis to identify isolates representative of the genetic diversity of these strains. Bacteria were grown in brain/heart infusion broth (Difco) (3.7%, w/v) containing NAD (2 lgÆmL )1 ), and hemin (10 lgÆmL )1 ). Bacteria from 5 L of culture were harvested and LPS extracted from lyophilized bacteria using the phenol/chloroform/light petroleum method involving pre- cipitation of the LPS with diethyl ether/acetone (1 : 5, v/v; 6 vols) as described previously [9]. Chromatography Gel filtration chromatography was performed on a Bio-Gel P4 column (2.6 · 90 cm; Bio-Rad, Hercules, CA, USA) using pyridinium acetate (0.1 M , pH 5.3) as eluent. Column eluents were monitored by a differential refractometer (Bischoff-chromatography, Leonberg, Germany) and frac- tions were collected by lyophilization. Gas liquid chroma- tography (GLC) analyses were carried out using an Agilent 6890 instrument with a DB-5 fused silica capillary column [Agilent Technologies, Palo Alto, CA, USA; 25 m · 0.25 mm (0.25 lm internal diameter)] and a temperature gradient of 160 °C (for 1 min) to 250 °Catarateof 3 °Cpermin. High performance anion-exchange chromatography (HPAEC) was performed on a Dionex Series 4500i chromatography system using a CarboPac PA1 column (4 · 250 mm; both Dionex Corporation, Sunnyvale, CA, USA) 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 [26]. Briefly, LPS (1 mg) was mixed with anhydrous hydrazine (0.2 mL) and stirred at 40 °C for 1 h. The reaction mixture was cooled and cold acetone (1.8 mL) was added drop-wise to destroy excess hydrazine. The precipitated O-deacylated LPS (LPS- OH) was centrifuged (48 200 g,5min),thepelletwas washed twice with cold acetone, and then dissolved in water followed by lyophilization. Mild acid hydrolysis of LPS. Reduced core oligosaccha- ride (OS) fractions were obtained from LPS (50 mg, strain 11; 20 mg, strain 16) after mild acid hydrolysis (1% acetic acid, pH 3.1, 100 °C, 2 h) and simultaneous reduc- tion with borane-N-methylmorpholine. The insoluble lipid A (12 and 5.6 mg, respectively) was separated from the hydrolysis mixtures by centrifugation at 21 600 g for 15 min. Following purification by gel filtration on the Bio- Gel P4 column, three OS fractions, 11-OS-1 (14.7 mg), 11-OS-2 (2.4 mg) and 11-OS-3 (2.3 mg) were obtained from strain 11. From strain 16, two fractions, 16-OS-1 (6.7 mg) and 16-OS-2 (1 mg) were collected. O-Deacylation of OS samples was achieved with 1 M aqueous ammonia (18 h, 25 °C). The lipid A portions were purified by partition using chloroform/methanol/water (2 : 1 : 1; v/v/v). After centri- fugation at 48 000 g for 5 min, the chloroform phase was evaporated to dryness using a stream of nitrogen. Dephosphorylation of OS. Oligosaccharides 11-OS-1 and 16-OS-1 (1 mg each) were incubated with 48% aqueous HF (0.15 mL) for 48 h at 4 °C. Samples were then placed into an ice bath and HF was evaporated under a stream of nitrogen gas to give 11-OS-1HF and 16-OS-1HF, respect- ively, which were then dissolved in water and lyophilized. 11-OS-2 and 11-OS-3 (< 1 mg each) were dephosphory- latedinthesameway. Mass spectrometry Electrospray ionization mass spectrometry (ESI-MS) was recorded on a VG Quattro triple quadrupole mass spectro- meter (Micromass, Manchester, UK) in the negative ion 942 M. K. Landerholm et al.(Eur. J. Biochem. 271) Ó FEBS 2004 mode. LPS-OH and OS 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 water/ acetonitrile (1 : 1; v/v) at a flow rate of 10 lLÆmin )1 . Multiple step tandem ESI-MS (ESI-MS n ) experiments were performed in the positive ion mode on a Finnigan LCQ iontrap mass spectrometer (Finnigan-MAT, San Jose, CA, USA). Samples were dissolved in 1 m M sodium acetate in methanol/water (7 : 3; v/v). The applied flow rate was 10 lLÆmin )1 . Lipid A was investigated by ESI-MS in the negative ion mode on the Finnigan LCQ ion trap mass spectrometer as described by Kussak et al.[27].Briefly,the lipid A was dissolved in chloroform/methanol (1 : 1; v/v) and introduced into the mass spectrometer at a flow rate of 5 lLÆmin )1 . CE-ESI-MS and CE-ESI-MS/MS were carried out in the positive ion mode with a PrinCE-C 660 instrument (Prince Technologies, Emmen, the Netherlands) coupled to an API 3000 mass spectrometer (Perkin-Elmer/ Sciex, Concord, Canada) via a MicroIonspray interface as described previously [16]. GLC-MS was carried out with a Hewlett Packard 5890 chromatograph (Agilent Technolo- gies) equipped with a NERMAG R10-10H quadrupole mass spectrometer using the same conditions for GLC as described above except for the initial temperature for GLC- MS being 130 °C. NMR spectroscopy NMR spectra were recorded on solutions in deuterium oxide at 25 °C after several lyophilizations with D 2 O. Spectra were acquired on a JEOL Esquire 500 spectrometer (JEOL, Tokyo, Japan) using standard pulse sequences. Chemical shifts are reported in p.p.m. referenced to internal sodium 3-trimethylsilylpropanoate-d 4 (d 0.00, 1 H) and external acetone (d 29.8, 13 C). COSY, TOCSY with mixing times of 50 and 180 ms, gradient selected heteronuclear single quantum coherence and gradient selected hetero- nuclear multiple quantum coherence (gHMQC) experi- ments were performed using standard pulse sequences. For interresidue correlation, two-dimensional NOESY experi- ments with a mixing time of 200 ms were used. Analytical methods Sugars were identified as their alditol acetates as described previously [28]. Methylation analysis of LPS-OH was accomplished on acetylated material, which was obtained by treatment of the sample with acetic anhydride (0.2 mL) and 4-diethylaminopyridine (1 mg) at 20–22 °Cfor4h. Methylation was performed with methyl iodide in dimethyl- sulfoxide in the presence of lithium methylsulfinylmethanide [29]. The methylated compounds were recovered using a SepPak C18 cartridge (Waters, Milford, MA, USA) and subjected to sugar analysis or sequential analysis by multiple step tandem ESI-MS (ESI-MS n ). The relative proportions of the various alditol acetates and partially methylated alditol acetates obtained in sugar- and methylation-analyses are reported as the detector responses of the GLC-MS. Absolute configurations of glycoses were determined by the method of Gerwig et al. [30]. The presence of glycine was determined by HPAEC following treatment of LPS with 0.1 M NaOH at 20–22 °C for 30 min [16]. N-Acetylneu- raminic acid was determined by treating LPS-OH (0.2 mg) with 20 mU of neuraminidase in 0.2 mL 10 m M NaOAc, pH 5.0, at 37 °C for 4 h. The reaction mixture was analyzed by HPAEC as previously described without further purification [18]. The enzyme cleaves terminal N-acetylneu- raminic acid (Neu5Ac) residues linked a-2,3, a-2,6 or a-2,8 to oligosaccharides. Fatty acids were identified as their methyl esters, as described previously [31]. Results Characterization of LPS Nondisease associated NTHi strains 11 and 16 are naso- pharyngeal isolates obtained from the Finnish Otitis Media Study Group. The strains were grown in liquid culture and the respective LPSs were isolated by phenol/chloroform/ light petroleum extraction [9]. Compositional analysis of LPS samples indicated D -glucose (Glc), D -galactose (Gal), 2-amino-2-deoxy- D -glucose (GlcN) and L -glycero- D -manno- heptose (Hep) as the constituent sugars in the ratios Glc/Gal/GlcN/Hep 1.0 : 0.3 : 0.8 : 0.8 (w/w/w/w) (strain 11) and 1.0 : 0.2 : 0.6 : 1.4 (w/w/w/w) (strain 16) identified by GLC-MS of the derived alditol acetates and 2-butyl glycoside derivatives. In addition, small amounts of glycine and traces of N-acetyl neuraminic acid (Neu5Ac) were detected as substituents of LPS by HPAEC following treatment of 0.1 M NaOH and neuraminidase, respectively, with the exception that no Neu5Ac was found in strain 16 [16,18]. It was estimated from ESI-MS data (see below) that less than 10% of all glycoforms were substituted with glycine, their compositions are given in tables below. The presence of Neu5Ac was confirmed in a precursor ion monitoring MS/MS experiment (negative ion mode) by scanning for loss of m/z 290 (Neu5Ac) following CE-ESI- MS/MS (see below). The lipid A portions obtained from LPS of both strains after mild acid hydrolysis (see below) were investigated by ESI-MS in the negative ion mode after first partitioning in chloroform/methanol/water (2 : 1 : 1; v/v/v) [27]. The spectra were virtually identical revealing, inter alia,mole- cular ions for both a diphosphorylated (m/z 1825, minor) and a monophosphorylated (m/z 1744, major) lipid A moiety substituted with four 3-hydroxytetradecanoic and two tetradecanoic acids. O-Deacylation of LPS from both strains by treatment with anhydrous hydrazine under mild conditions afforded water soluble LPS-OH material, which was subjected to methylation analyses and analyses by mass spectrometric techniques. Methylation analysis revealed the presence of terminal Glc, 2-substituted-Hep, 3,4-disubstituted Hep and 2,3,6-trisubstituted-Hep as the major components in both strains (Table 1). The data is consistent with triantennary structures, containing the common inner core ele- ment, L -a- D -Hepp-(1fi2)-[PEtnfi6]- L -a- D -Hepp-(1fi3)-[b- D - Glcp-(1fi4)]- L -a- D -Hepp-(1fi5)-a-Kdop of H. influenzae LPS linked to the conserved lipid A consisting of a b-1,6- linked D -glucosamine disaccharide [9–14,17,19,20,31]. ESI-MS on LPS-OH. The ESI-MS spectrum of LPS-OH samples (negative ion mode) revealed abundant molecular signals corresponding to triply and quadruply deproto- Ó FEBS 2004 LPS glycoforms of nondisease associated NTHi strains (Eur. J. Biochem. 271) 943 nated ions. The MS data presented in Table 2 pointed to the presence of glycoforms in which each molecular species contains the conserved phosphoethanolamine (PEtn)-substituted triheptosyl inner core moiety attached via a phosphorylated Kdo linked to the O-deacylated lipid A. For strain 11, as observed earlier, populations of glycoforms were observed which differed by 123 Da (i.e. a PEtn group) which was consistent with either phosphate or pyrophosphoethanolamine (PPEtn) substitution at the O-4 position of the Kdo residue [12]. In the spectrum of LPS-OH derived from strain 11, abundant quadruply charged ions at m/z 691.4 and 722.0, together with their corresponding triply charged ions at m/z 922.1 and 963.0, indicated the presence of glycoforms with the respective compositions PCho 2 •Hex 3 •Hep 3 •PEtn 1,2 •P•Kdo•Lipid A- OH. In addition, minor ions corresponding to Hex3 glycoforms with one PCho were detected at m/z 650.1 and 681.1 and with no PCho at m/z 609.0 and 639.4. In the spectrumofLPS-OHfromstrain16,onemajorpeakwas observed at m/z 691.4 corresponding to a composition PCho 2 •Hex 3 •Hep 3 •PEtn 1 •P•Kdo•Lipid A-OH. In addi- tion, a minor triply charged ion signal at m/z 907.5 corresponded to a composition of PCho•Hex 3 •Hep 3 • PEtn 2 •P•Kdo•Lipid A-OH. The presence of additional trace amounts of PPEtn containing glycoforms in strain 16 (Table 2) could be confirmed in a precursor ion monitoring tandem mass spectrometry experiment (negative ion mode) by scanning for loss of m/z 220 (PPEtn) following on-line separation by capillary electro- phoresis (CE-ESI-MS/MS). Being of low abundance, ions corresponding to sialylated species indicated by HPAEC were not observed in the full MS spectra. However, their presence in strain 11 could be confirmed in a precursor ion monitoring tandem mass spectrometry experiment (negative ion mode) by scanning for loss of m/z 290 (Neu5Ac) following CE-ESI-MS/MS. The resulting spec- trum revealed a minor quadruply charged ion at m/z 754 corresponding to the glycoform Neu5Ac•PCho•Hex 3 • Hep 3 •PEtn 2 •P•Kdo•Lipid A-OH. In addition, major ions at m/z 786, 796 and 827 were observed corresponding to disialylated Hex3 glycoforms having the respective com- positions, Neu5Ac 2 •Hex 3 •Hep 3 •PEtn 2 •P•Kdo•Lipid A- OH and Neu5Ac 2 •PCho•Hex 3 •Hep 3 •PEtn 1,2 •P•Kdo• Lipid A-OH (data not shown). In agreement with compositional analysis (see above) no signals correspond- ing to sialylated compounds were detected for strain 16 in this experiment. Characterization of core oligosaccharides Partial acid hydrolysis of LPS from both strains with dilute acetic acid afforded insoluble lipid A and core oligosaccha- ride fractions which were separated by gel filtration to give one major (leading) and two minor fractions, 11-OS-1, 11-OS-2 and 11-OS-3 for strain 11 and one major and one minor fraction for strain 16, 16-OS-1 and 16-OS-2. The major oligosaccharides 11-OS-1/16-OS-1 were dephospho- rylated to give 11-OS-1HF/16-OS-1HF. Methylation ana- lysis of 11-OS-1 showed terminal Glc, 2-substituted-Hep, 3,4-disubstituted Hep and 2,3-substituted-Hep as the major components (Table 1). Methylation analysis of dephos- phorylated oligosaccharide 11-OS-1HF showed signifi- cantly increased amounts of terminal Glc, terminal Gal and 2,3-disubstituted Hep indicating phosphorylation of the corresponding sugar residues (Table 1). Methylation ana- lysis of dephosphorylated oligosaccharide 16-OS-1HF showed the same derivatives as observed for 11-OS-1HF. Methylation analyses of the minor fractions 11-OS-2, 11-OS-3 and 16-OS-2 showed the same major sugar derivatives. Sequence analyses of oligosaccharide samples. In order to obtain sequence and branching information, oligosac- charides were dephosphorylated and permethylated and subjected to ESI-MS n [11,14,19,20]. A representative ESI- MS spectrum is shown in Fig. 1A and the data is Table 1. Linkage analysis data for LPS derived samples from nontypeable H. influenzae carriage strains 11 and 16. Retention times (T gm )are reported relative to 2,3,4,6-Me 4 -Glc. 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. Methylated sugar T gm Relative detector response Linkage assignment LPS-OH (strain 11) LPS-OH (strain 16) 11-OS-1 11-OS-1HF 11-OS-2 11-OS-3 16-OS-1HF 16-OS-2 2,3,4,6-Me 4 -Glc 1.00 23 26 31 33 30 37 26 42 D -Glcp-(1- 2,3,4,6-Me 4 -Gal 1.04 1 – 1 18 8 12 25 11 D -Galp-(1- 3,4,6-Me 3 -Glc a 1.14 3 2 1 – 1 3 2 – ) 2)- D -Glcp-(1- 2,3,6-Me 3 -Gal a 1.15 – – – – 1 6 – – ) 4)- D -Galp-(1- 2,3,6-Me 3 -Glc a 1.16 – – – – 1 – – – ) 4)- D -Glcp-(1- 2,4,6-Me 3 -Gal a 1.19 – – – – – – 1 – ) 3)- D -Galp-(1- 2,3,4- Me 3 - Glc a 1.20 – – 1 – 3 6 2 – ) 6)- D -Glcp-(1- 3,6,7-Me 3 -Hep a 1.36 3 – 7 3 6 – 6 11 ) 2,4)- L , D -Hepp-(1- 3,4,6,7-Me 4 -Hep 1.42 16 25 33 13 12 9 14 13 ) 2)- L , D -Hepp(1- 2,6,7-Me 3 -Hep 1.49 33 18 16 16 34 21 7 23 ) 3,4)- L , D -Hepp-(1- 4,6,7-Me 3 -Hep 1.55 3 2 10 17 4 6 17 – ) 2,3)- L , D -Hepp-(1- 4,7-Me 2 -Hep 1.58 14 11 – – – – – – ) 2,3,6)- L , D -Hepp-(1- 2,3,4,-Me 4 -GlcN 1.69 4 16 – – – – – – ) 6)- D -GlcpNAc(1- a Not rationalized: probably due to undermethylation, as ESI-MS n did not reveal any Hex-Hex units. 944 M. K. Landerholm et al.(Eur. J. Biochem. 271) Ó FEBS 2004 Table 2. Negative ion ESI-MS data and proposed compositions for O-deacylated LPS (LPS-OH) and oligosaccharide (OS) preparations derived from nontypeable H. influenza e carriage strains 11 and 16. 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; a-Neu5Ac, 291.26; Ac, 42.04; Gly, 57.05 and Lipid A-OH, 953.02. Relative abundance was estimated from the area of the ions relative to the total area (expressed as percentage). Signals representing less than 5% of the base peak are not included in the table. Sample Observed ions (m/z) Molecular Mass (Da) Relative Abundance (%) Proposed Composition (M-4H) 4– (M-3H) 3– (M-2H) 2– Observed Calculated Strain 11 Strain 16 LPS-OH a 609.0 812.4 – 2440.0 2439.2 3 – Hex 3 •Hep 3 •PEtn 1 •P 1 •Kdo•Lipid A-OH 639.4 853.2 – 2561.6 2562.2 4 – b Hex 3 •Hep 3 •PEtn 2 •P 1 •Kdo•Lipid A-OH 650.1 867.0 – 2604.2 2604.3 7 – PCho•Hex 3 •Hep 3 •PEtn 1 •P 1 •Kdo•Lipid A-OH 681.1 907.5 – 2727.0 2727.3 5 9 PCho•Hex 3 •Hep 3 •PEtn 2 •P 1 •Kdo•Lipid A-OH 691.4 922.1 – 2769.4 2769.4 38 91 PCho 2 •Hex 3 •Hep 3 •PEtn 1 •P 1 •Kdo•Lipid A-OH 722.0 963.0 – 2892.0 2892.5 43 – b PCho 2 •Hex 3 •Hep 3 •PEtn 2 •P 1 •Kdo•Lipid A-OH 11-OS-1 11-OS-2 11-OS-3 16-OS-1 16-OS-2 OS – – 644.2 1290.4 1291.05 – – – 1 3 PCho•Ac•Hex 1 •Hep 3 •PEtn 1 •AnKdo-ol – – 665.3 1332.6 1333.09 – – – 1 2 PCho•Ac 2 •Hex 1 •Hep 3 •PEtn 1 •AnKdo-ol – – 686.2 1374.4 1375.13 – – – 1 – PCho•Ac• 3 Hex 1 •Hep 3 •PEtn 1 •AnKdo-ol – – 702.8 1407.7 1408.16 – – 8 – – Hex 3 •Hep 3 •PEtn 1 •AnKdo-ol – – 704.5 1411.0 1411.15 – 2 – – 4 PCho•Hex 2 •Hep 3 •PEtn 1 •AnKdo-ol – – 723.8 1449.7 1450.2 – – 25 – – Ac•Hex 3 •Hep 3 •PEtn 1 •AnKdo-ol – – 725.4 1452.9 1453.19 – 5 – 1 6 PCho•Ac•Hex 2 •Hep 3 •PEtn 1 •AnKdo-ol – – 744.8 1491.6 1492.24 – – 11 – – Ac 2 •Hex 3 •Hep 3 •PEtn 1 •AnKdo-ol – – 746.5 1494.9 1495.23 4 8 – 2 4 PCho•Ac 2 •Hex 2 •Hep 3 •PEtn 1 •AnKdo-ol – – 752.5 1507.0 1506.71 – – 9 – – Ac 1 •Gly•Hex 3 •Hep 3 •PEtn 1 •AnKdo-ol – – 765.8 1533.6 1534.28 – – 4 – – Ac 3 •Hex 3 •Hep 3 •PEtn 1 •AnKdo-ol – – 767.7 1537.4 1537.27 – 4 – 1 – PCho•Ac 3 •Hex 2 •Hep 3 •PEtn 1 •AnKdo-ol – – 773.2 1548.4 1548.75 – – 5 – – Ac 2 •Gly•Hex 3 •Hep 3 •PEtn 1 •AnKdo-ol – – 785.4 1572.8 1573.29 – 7 6 1 2 PCho•Hex 3 •Hep 3 •PEtn 1 •AnKdo-ol – – 806.5 1615.0 1615.33 – 21 6 1 8 PCho•Ac•Hex 3 •Hep 3 •PEtn 1 •AnKdo-ol – – 828.0 1657.9 1657.37 – 11 9 2 – PCho•Ac 2 •Hex 3 •Hep 3 •PEtn 1 •AnKdo-ol – – 846.5 1695.0 1695.31 – – – 1 – PCho•Ac•Hex 3 •Hep 3 •PEtn 1 •AnKdo-ol – – 848.5 1699.0 1699.41 – 3 – – – PCho•Ac 3 •Hex 3 •Hep 3 •PEtn 1 •AnKdo-ol – – 868.0 1738.0 1738.4 7 6 4 9 8 PCho 2 •Hex 3 Hep 3 •PEtn 1 •AnKdo-ol – – 889.0 1780.0 1780.44 31 12 5 27 27 PCho 2 •Ac 1 •Hex 3 •Hep 3 •PEtn 1 •AnKdo-ol – – 910.0 1822.0 1822.48 44 14 8 35 27 PCho 2 •Ac 2 •Hex 3 •Hep 3 •PEtn 1 •AnKdo-ol – – 917.4 1836.8 1837.49 2 – – – – PCho 2 •Ac 1 •Gly•Hex 3 •Hep 3 •PEtn 1 •AnKdo-ol – – 931.1 1864.2 1864.52 8 4 – 7 5 PCho 2 •Ac 3 •Hex 3 •Hep 3 •PEtn 1 •AnKdo-ol – – 938.2 1878.4 1879.53 4 3 – 2 – PCho 2 •Ac 2 •Gly•Hex 3 •Hep 3 •PEtn 1 •AnKdo-ol – – 952.2 1906.4 1906.56 – – – 4 4 PCho 2 •Ac 4 •Hex 3 •Hep 3 •PEtn 1 •AnKdo-ol – – 959.3 1920.6 1921.57 – – – 2 – PCho 2 •Ac 3 •Gly•Hex 3 •Hep 3 •PEtn 1 •AnKdo-ol – – 973.0 1948.0 1948.6 – – – 2 – PCho 2 •Ac 5 •Hex 3 •Hep 3 •PEtn 1 •AnKdo-ol a Traces of sialylated species could be identified in strain 11 by scanning for loss of m/z 290 (Neu5Ac) in a precursor ion monitoring tandem mass spectrometry experiment (negative ion mode) following online separation by capillary electrophoresis (CE-ESI-MS/MS). Quadruply charged ions at m/z 754, 786, 796 and 827 corresponded to proposed compositions Neu5Ac•PCho•Hex 3 •Hep 3 •PEtn 2 •Kdo•Lipid A-OH, Neu5Ac 2 •Hex 3 •Hep 3 •PEtn 2 •Kdo•Lipid A-OH, Neu5Ac 2 •PCho•Hex 3 •Hep 3 •PEtn 1 •Kdo•Lipid A-OH and Neu5Ac 2 •PCho•Hex 3 •Hep 3 •PEtn 2 •Kdo•Lipid A-OH, respectively. b Ions corresponding to proposed composition could be observed in a precursor ion monitoring tandem mass spectrometry experiment (negative ion mode) by scanning for loss of m/z 220 (PPEtn) following online separation by capillary electrophoresis (CE-ESI-MS/MS). Ó FEBS 2004 LPS glycoforms of nondisease associated NTHi strains (Eur. J. Biochem. 271) 945 summerized in Table 3. Sodiated molecular ions at m/z 1263.7, 1467.8 and 1671.9 corresponding to Hex 1)3 • Hep 3 •AnKdo-ol glycoforms, respectively, were identified. The Hex3 glycoform was dominant in both strains. MS 2 experiments were performed on all molecular ions and in some cases further fragmentation (MS 3 ) was employed. These data gave evidence for the glycoforms presented in Table 4. The isomeric glycoforms for strain 11 and strain 16 were found to be identical except for strain 16 having only one of the three Hex1 glycoforms expressed by strain 11. The shared isomeric Hex1 glycoform was identified in the MS 2 spectrum of parent ion m/z 1263.7 by ions m/z 797.3 and 737.4 due to losses of terminal Hex-Hep and Hep-AnKdo-ol units, respectively. No ion due to the loss of a terminal Hep was observed in the MS 3 experiment on m/z 737.4. The strain 11 exclusive isomeric Hex1 glycoform with a disubstituted HepI residue was defined by ions m/z 1001.5 and 753.1 corresponding to loss of a terminal Hep and terminal Hep-Hep unit from the parent ion. Analyses on 11-OS-2 and -3 revealed a third Hex1 isomeric glycoform being substituted at HepII. This Hex1 glycoform was identified in the MS 3 spectrum of ion m/z 737.4 which revealed an ion at m/z 475 due to the loss of a terminal Hep unit. Performing MS 2 on parent ion m/z 1467.8, and subse- quent MS 3 on the resulting product ions determined three isomeric Hex2 structures. The ion corresponding to the loss of t-Hex-Hep at m/z 1001.4 was further fragmented to give the ion at m/z 753.4 corresponding to the loss of a monosubstituted HepII. This confirmed the structure of the major glycoform in which a hexose moiety substitutes both HepI and HepIII. In the same MS 3 experiment an ion at m/z 474.9 was observed corresponding to the loss of a Hep-AnKdo-ol unit. This confirmed the structure of a glycoform in which a hexose moiety substitutes both HepII and HepIII. Furthermore, in the MS 2 spectrum the ion corresponding to the loss of t-Hex-Hep-AnKdo-ol at m/z 737.0 was further fragmen- tedtogivetheionatm/z 475.4 corresponding to the loss of terminal HepIII. This confirmed the structure of a glycoform in which a hexose moiety substitutes both HepI and HepII. No ions indicating a Hex-Hex unit were observed. One Hex3 glycoform was determined by performing MS 2 on the parent ion m/z 1671.8 to give ions m/z 1205.5 and 941.5.4 due to losses of terminal Hex-Hep and Hex-Hep- AnKdo-ol (Fig. 1B and Table 4). When the ion at m/z 941.4 was further fragmented in a MS 3 experiment (Fig. 1C) it Fig. 1. ESI-MS spectra (positive mode) of permethylated 11-OS-1HF. (A) Parent ion mass spectra of permethylated 11-OS-1HF. Ions cor- responding to [M+Na] + of the Hex1 to Hex3 glycoforms are indi- cated; (B) Product ion (MS 2 )spectrumof[M+Na] + m/z 1671.9 and its proposed fragmentation pattern; (C) MS 3 spectrum of fragment ion m/z 941.5 and proposed fragmentation pattern. Table 3. Positive ion ESI-MS data and proposed compositions for glycoforms of dephosphorylated and permethylated oligosaccharide fractions. Average mass units were used for calculation of molecular mass values based on proposed compositions as follows: Hex, 162.14; Hep, 192.17; AnKdo-ol, 222.20; Me, 14.03; Na, 22.99. [M + Na] + Molecular Mass (Da) Relative abundence (%) Proposed composition GlycoformObserved Calculated 11-OS-1 11-OS-2 11-OS-3 16-OS-1 1263.7 1240.7 1240.6 1 2 2 6 Hex 1 •Hep 3 •AnKdo-ol Hex1 1467.8 1444.8 1444.7 7 18 19 9 Hex 2 •Hep 3 •AnKdo-ol Hex2 1671.9 1648.9 1648.8 92 80 79 85 Hex 3 •Hep 3 •AnKdo-ol Hex3 946 M. K. Landerholm et al.(Eur. J. Biochem. 271) Ó FEBS 2004 gave, inter alia, a product ion at m/z 475.1 due to the loss of a t-Hex-Hep unit. This confirmed the structure of a glycoform with one hexose moiety substituting each Hep. There was no indication of any Hex-Hex units in this glycoform either. Electrospray-ionization mass spectrometry on OS sam- ples. ESI-MS data on underivatized oligosaccharide mate- rials demonstrated the heterogeneity in the oligosaccharide part due to various substituents, in particular acetates and glycine. The ESI-MS spectrum of 11-OS-1 (negative ion mode, Table 2) showed two major doubly charged ions at m/z 889.0 and 910.0 corresponding to Hex3 glycoforms with the respective compositions PCho 2 •Ac 1,2 •Hex 3 •Hep 3 • PEtn 1 •AnKdo-ol. In addition, minor ions at m/z 746.5, 868.0 and 931.1 corresponding to Hex2 and Hex3 glyco- forms with the respective compositions PCho 1 •Ac 2 •Hex 2 • Hep 3 •PEtn 1 •AnKdo-ol and PCho 2 •Ac 0,3 •Hex 3 •Hep 3 • PEtn 1 •AnKdo-ol were observed. The spectrum also showed ions at m/z 917.4 and 938.2 corresponding to glycine- substituted species. The ESI-MS spectra of 11-OS-2 and 11-OS-3 revealed ions corresponding to acylated Hex2 and Hex3 glycoforms with the difference that in 11-OS-2 glycoforms with one PCho predominated, while in 11-OS- 3 glycoforms with no PCho were most abundant (Table 2). The ESI-MS spectrum of 16-OS-1 was dominated by two doubly charged ions at m/z 889.0 and 910.0, corresponding to the same Hex3 glycoforms as in 11-OS-1. Additional minor ions corresponding to various acylated Hex1–3 glycoforms containing one or two PCho were observed. The ESI-MS spectrum of 16-OS-2 showed basically iden- tical ions as 16-OS-1 with increased abundance of ions corresponding to glycoforms with one PCho (Table 2). Information on the location of the acetyl groups and glycine in 11-OS-1 was provided by ESI tandem mass spectrometry (MS/MS) following online separation by capillary electrophoresis (CE). The product ion spectrum (positive mode) obtained from the doubly charged ion at m/z 891 (composition: PCho 2 •Ac•Hex 3 •Hep 3 •PEtn 1 • AnKdo-ol) and the proposed fragmentation pattern is shown in Fig. 2A. The spectrum contained an intense ion at m/z 370 corresponding to PChoAcHex to which additions of Hep (m/z 562) could be seen. Loss of the PChoAcHex- Hepfragmentresultedintheionatm/z 1219 giving evidence for the acetyl group to be located at the hexose linked to HepIII. The minor ion at m/z 643 showed consecutive losses due to PEtn (m/z 520) and Hep (m/z 328; composition PChoHex) confirming the PChoHexHepIIPEtn segment. Information on the location of a second acetyl group was obtained from the CE-ESI-MS/MS spectrum on m/z 912 (composition: PCho 2 •Ac 2 •Hex 3 •Hep 3 •PEtn 1 •AnKdo-ol; Fig. 2B). Additions of HepAc to the intense ion at m/z 370 corresponding to PChoAcHex could be seen at m/z 604 indicating that HepIII was acetylated. The presence of another acetylation site, however, was indicated by the ion at m/z 1219 to which a minor addition of 42 Da (m/z 1261) was observed. This and other minor acetylation sites were deduced from the CE-ESI-MS/MS spectrum on m/z 933 (composition: PCho 2 •Ac 3 •Hex 3 •Hep 3 •PEtn 1 •AnKdo-ol; data not shown) in which an addition of 42 Da to the major ion at m/z 370 giving m/z 412 indicated further acetylation of the hexose linked to HepIII. To the ions at m/z 562, 604 and 643 with compositions described above, additions of 42 Da resulted in m/z 604, 646 and 685, respectively, indicating also diacetylation of HepIII and/or Table 4. Hex1–Hex3 isomeric glycoforms observed in dephosphorylated oligosaccharides as identified by ESI-MS n after permethylation. Indicated are product ions of significant importance and the corresponding fragments. Glycoform Product ions a The Hex1 glycoform having a terminal distal Hep is only observed in 11-OS-1,2,3. b The Hex1 glycoform having a substituted HepII is only observed in 11-OS-2,3. Ó FEBS 2004 LPS glycoforms of nondisease associated NTHi strains (Eur. J. Biochem. 271) 947 acetylation of the hexose linked to HepII. The location of the major acetyl groups on the hexose linked to HepIII and HepIII itself could be confirmed and fully established by NMR (see below). Information on the location of glycine was obtained from the CE-ESI-MS/MS spectra on m/z 919 and 940 (compositions: PCho•Ac 1,2 •Gly•Hex 3 •Hep 3 • PEtn 1 •AnKdo-ol, respectively) showing, inter alia, ions at m/z 619 (composition: PChoAcHexHepGly; Fig. 2C) and 661 (composition: PChoAcHexHepAcGly; data not shown), respectively. These data indicated that glycine substituted HepIII. By analogy, the acylation sites in 16-OS- 1 were found to be the same as in 11-OS-1 (data not shown). NMR spectroscopy on OS samples The 1 H NMR resonances of OS material were assigned using chemical shift correlation techniques (DQF-COSY and TOCSY experiments) and the chemical shift data are presented in Table 5. The 13 C NMR chemical shifts were assigned by heteronuclear 1 H- 13 C chemical shift correlation experiments in the 1 H detected mode (Table 5). Subspectra corresponding to the individual glycosyl residues were identified on the basis of spin-connectivity pathways delineatedinthe 1 H chemical shift correlation maps, the chemical shift values, and the vicinal coupling constants. Spin systems for HepIII could not be delineated beyond H-2. 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 6), which also served to confirm the anomeric configurations of the linkages and the monosac- charide 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 only. Several signals for methylene protons of AnKdo-ol were observed in the COSY and TOCSY spectra in the region d 2.24–1.84. This is due to the fact that several anhydro-forms of Kdo are formed during the hydrolysis as observed previously [9–14,17,19,20]. The methyl protons of the O-acetyl groups in 11-OS-1 and 16-OS-1 were observed at d 2.19, which correlated to a 13 Csignalatd 21.4 in the gHMQC spectrum. A gHMQC cross peak was observed at d 4.02/41.3 which was assigned to the ester-linked glycine Fig. 2. CE-ESI-MS/MS (positive ion mode) analysis of 11-OS-1 derived from LPS of NTHi carriage strain 11. (A) Product ion (MS 2 ) spectrum of [M + 2H] 2+ m/z 891 corres- ponding to the composition PCho 2 •A- c•Hex 3 •Hep 3 •PEtn•AnKdo-ol (B) Product ion (MS 2 )spectrumof[M+2H] 2+ m/z 912 corresponding to the composition PCho 2 •A- c 2 •Hex 3 •Hep 3 •PEtn 1 •AnKdo-ol. (C) Product ion (MS 2 )spectrumof[M+2H] 2+ m/z 919 corresponding to the composition PCho 2 •Ac•Gly•Hex 3 •Hep 3 •PEtn•AnKdo- ol. The proposed structures are shown in the insets. 948 M. K. Landerholm et al.(Eur. J. Biochem. 271) Ó FEBS 2004 Table 5. 1 Hand 13 C chemical shifts for oligosaccharide preparations 11-OS-1 and 16-OS-1. Data was recorded in D 2 Oat22°C. 3 J H,H -values for anomeric 1 H resonances (H-1) are given in parentheses; n.r, not resolved (small coupling). Pairs of deoxyprotons of reduced AnKdo were identified in the DQF-COSY at 2.24–1.84 p.p.m. The signal corresponding to PCho methyl protons occurred at 3.24 p.p.m. OS*, O-deacylated samples: chemical shifts were identical for both strains. OS**, Selected chemical shifts for acetylated residues observed in samples prior to treatment with aqueous ammonia. –, result not obtained. Sample 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 OS* HepI 5.17–5.04 a 4.06–4.00 a 4.06 4.27 b – 4.16 b –– 97.3–99.8 (n.r)  70 73.4 74.6 – 66.6 – HepII 5.80–5.67 4.26 4.08 – 3.75 4.62 3.72 3.90 99.5 (n.r) 79.8 78.7 – 73.1 75.8 63.7 HepIII 5.12–5.10 3.98 – ––– – – 100.6 (n.r) 79.7 –––– – GlcI 4.52 3.35 3.46 3.46 3.46 3.80 3.97 104.2 (7.3) 75.2 77.3 77.3 77.3 62.3 GlcII 5.31 3.56 3.80 3.64 3.86 4.08 4.28 102.0 (3.2) 72.8 73.7 69.7 72.2 64.9 Gal 4.39 3.58 3.75 3.99 3.78 4.05 4.05 104.0 (7.8) 71.3 73.4 69.6 74.0 65.6 PEtn 4.14 3.29 62.8 40.7 PCho 4.35 3.70 60.4 66.9 OS** HepIII 3-OAc 5.11 4.20 5.09 4.0 – – – – – 100.1 (n.r) 76.6 74.3 – – – – Gal 4-OAc 4.32 3.59 3.93 5.37 – – – 104.5 (n.r) – – 71.4 (2.8) –– Gal 3-OAc 4.52 3.80 4.91 4.18 4.04 – – 104.0 (4.1) – 75.8 (n.r) ––– Gal 2-OAc 4.39 4.89 3.80 4.18 – – – 104.5 (n.r) 75.8 (n.r) – ––– a Several signals were observed for HepI and HepII due to heterogeneity in the AnKdo moiety. b H-4/H-6 of HepI are observed by NOE from H-1 of GlcI. Ó FEBS 2004 LPS glycoforms of nondisease associated NTHi strains (Eur. J. Biochem. 271) 949 substituents. The structure of the OS backbone in 11-OS-1 and 16-OS-1 was determined by detailed NMR analyses after O-deacylation of samples with 1 M aqueous ammonia. In the 1 H NMR spectra of the resulting material from both strains the characteristic signal for the methyl groups of PCho was observed at d 3.24. Anomeric resonances from HepI–HepIII were identified at d 5.17–5.04, 5.80–5.67 and 5.12–5.10, respectively. Subspectra corresponding to GlcI, GlcII and Gal were identified in the 2D COSY and TOCSY spectra where the anomeric proton signals were found at d 4.52, 5.31 and 4.39, respectively. From the downfield shifted signals of H-6 A , B of Gal at d 4.05 and H-6 A,B of GlcII at d 4.08 and 4.28 it was concluded that these residues were substituted with PCho at those positions which was in agreement with CE-ESI-MS/MS analysis (see above). Inter- residue NOE connectivities (Table 6) between proton pairs GlcI H-1/HepI H-4,6, HepIII H-1/HepII H-2, HepII H-1/ HepI H-3 confirmed the presence of the common inner core triheptosyl moiety substituted with a glucose residue at HepI. Interresidue NOE between GlcII H-1/HepII H-2,3 indicated substitution at O-3 of HepII as a- D -Glcp-(1fi3)- L - a- D -Hepp-(1fi. In addition interresidue NOE between Gal H-1 and HepIII H-1 and H-2 indicated substitution at O-2 of HepIII as b- D -Galp-(1fi2)- L -a- D -Hepp-(1fi. CE-ESI- MS/MS experiments indicated the major acetylation sites in the oligosaccharides to be at HepIII and Gal (see above). The points of substitution on these residues were identified by NMR on 11-OS-1 and 16-OS-1 prior to de-O-acylation. The chemical shifts of H-2, H-3, C-2 and C-3 of HepIII were observed at d 4.20, 5.09, 76.6 and 74.3, respectively. The significant downfield shifted H-3 and upfield shifted C-2 indicated O-3 of HepIII to be acetylated [32]. For the b- D - Galp residue, resonances were observed that corresponded to three mono-acetylated species designated Gal 4-OAc , Gal 3-OAc ,Gal 2-OAc , respectively. Thus, three spin-systems were observed at d 5.37 (J 3,4 2.8 Hz, 0.5 H), 4.91 (0.25 H) and 4.89 (0.25H) corresponding to H-4, H-3 and H-2 of Gal 4-OAc ,Gal 3-OAc ,Gal 2-OAc , respectively. For Gal 2-OAc , when compared to unsubstituted Gal, downfield shifts were obtained for the signals from H-2 (+1.31 p.p.m.), H-3 (+0.05 p.p.m.) and C-2 (+4.5 p.p.m.), indicating acetyla- tion at O-2. For Gal 3-OAc ,thesignalsofH-3 (+1.16 p.p.m.), H-2 (+0.22 p.p.m.), H-4 (+0.19 p.p.m.) and C-3 (+2.4 p.p.m.) were shifted downfield, indicating acetylation at O-3. Acetylation at O-4 was indicated from the downfield shifted signals of H-4 (+1.38 p.p.m.), H-3 (+0.18 p.p.m) and C-4 (+1.8 p.p.m.). Substitution sites for the other, minor acyl groups indicated by CE-ESI-MS could not be identified. From the combined data the structure in Fig. 3 is proposed for the major di-O-acetylated Hex3 LPS glyco- form of NTHi strains 11 and 16. Discussion As part of our ongoing studies on the role of H. influenzae LPS in disease pathogenesis, we have undertaken a system- atic analysis of LPS from a genetically diverse set of human isolates of NTHi. The majority of strains collected were from patients with otitis media, but a number of isolates were also obtained from healthy, asymptomatic individuals. Until now, no structural data has been available on LPS glycoform patterns from H. influenzae strains that were not associated with disease. Structural studies have revealed that every NTHi strain investigated to date [10,11,13, 17,19,20,33–35] produces LPS containing the conserved triheptosyl inner core moiety (Fig. 4). In addition, gene Table 6. Proton NOE data for oligosaccharide preparation 11-OS-1 derived from LPS of NT H . infl uenzae carriage strain 11. Measure- mentsweremadefromNOESYexperiments. Anomeric proton Observed proton Intraresidue NOE Interresidue NOE HepI H-2 H-5 of Kdo HepII H-2 H-3 of HepI; H-1 of HepIII HepIII H-2 H-1, H-2 of HepII GlcI H-3, H-5 H-4, H-6 of HepI GlcII H-2 H-2, H-3 of HepII Gal H-3, H-5 H-1, H-2 of HepIII Fig. 3. Structure proposed for the core oligosaccharide of the major Hex3 LPS glycoform of NTHi carriage strains 11 and 16. Fig. 4. Structural representation of the lipopolysaccharide from H. influenzae. R 1 ,R 2 ,R 3 ¼ H or sugar residues. Lipid A is composed of a b-1,6-linked D -glucosamine disaccharide substituted by phos- phomonoester groups at C-1 and C-4¢ and 3-hydroxytetradecanoic and tetradecanoic as described previously [31]. 950 M. K. Landerholm et al.(Eur. J. Biochem. 271) Ó FEBS 2004 [...]... structural feature on surface structures of pathogens residing in the human respiratory tract, including H in uenzae Expression of PCho has been associated with persistence of H in uenzae in the respiratory tract in an experimental rat model of infection, and with relative sensitivity to host factors such as complement mediated killing [36] Structural analyses of a majority of H in uenzae strains including... glycine has been shown to be a prominent substituent in the core region of H in uenzae LPS All strains investigated so far express minor amounts of this amino acid [16] The most prominent site for this substituent is HepIII, but it can also been found at HepII or Kdo In the strains investigated here the position of glycine is indicated at HepIII The biological significance of glycine on the role of LPS... the lic1 locus resulting in two unique lic1D sequences within these bacteria A proponderance of glycoforms containing two PCho units may enhance the ability of these strains to colonize the nasopharynx Modification of LPS by addition of terminal Neu5Ac is widespread among H in uenzae strains [15,18] and has recently been shown to be crucial for infection in the chinchilla model of experimental otitis... role in the procurement of PCho and its positioning within the LPS molecule [40] Lic1D is the PCho transferase and depending on the sequence of the lic1D gene, the translated transferase specifically adds PCho to a hexose residue attached to one of the heptose units of the inner core moiety [40] Thus one would predict that in NTHi strains 11 and 16 there exists some duplication of all or part of the. .. E.K.H (2001) A new structural type for Haemophilus in uenzae lipopolysaccharide Structural analysis of the lipopolysaccharide from nontypeable Haemophilus in uenzae strain 486 Eur J Biochem 268, 2148–2159 Yildirim, H.H., Hood, D.W., Moxon, E.R & Schweda, E.K.H (2003) Structural analysis of lipopolysaccharides from Haemophilus in uenzae serotype f Structural diversity observed in three strains Eur J Biochem... Acknowledgements The authors would like to thank 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 nasopharynx, obtained as part of the Finnish Otitis Media Cohort Study Gaynor Randle is acknowledged for culture of strains Financial assistance from Aventis Pasteur and the Karolinska Institutet (KI-fonder) is gratefully... PChofi6)-a-D-Glcp) in NTHi strain 486 [13] The present investigation provides the first example of H in uenzae strains expressing two PCho substituents; one at the a-D-Glcp residue from HepII (Fig 4; R2 ¼ PChofi6)-a-D-Glcp) and the other at the terminal b-D-Galp residue at HepIII (Fig 4; R3 ¼ PChofi6)-b-D-Galp) The expression of the PCho epitope on the H in uenzae LPS is phase variable and dependent upon the lic1... contain detectable amounts of sialylated Hex3 glycoforms Interestingly, no glycoforms with disaccharide units composed of hexoses could be identified in this study, indicating that Neu5Ac might be added to the LPS in a novel molecular environment Approximately 50% of all H in uenzae strains investigated by us express acetylated LPS structures Information on the location of the acetates has been provided... LPS of H in uenzae is not clear In conclusion, we have obtained structural details for the first time for LPS from H in uenzae isolates not associated with disease and show that they express structural motifs Ó FEBS 2004 952 M K Landerholm et al (Eur J Biochem 271) observed for other NTHi strains However, the precise combination of these motifs has not been seen before In particular, there is a novel. .. Thibault, P., Martin, A., Richards, J.C & Moxon, E.R (1999) Sialic acid in the lipopolysaccharide of Haemophilus in uenzae: strain distribution, in uence on serum resistance and structural characterization Mol Microbiol 33, 679–692 ˚ Li, J., Bauer, S.H.J., Mansson, M., Moxon, E.R., Richards, J.C & Schweda, E.K.H (2001) Glycine is a common substituent of the inner-core in Haemophilus in uenzae lipopolysaccharide . Characterization of novel structural features in the lipopolysaccharide of nondisease associated nontypeable Haemophilus in uenzae Malin K. Landerholm 1 ,. tract, including H. in uenzae. Expression of PCho has been associated with persistence of H. in uenzae in the respirat- ory tract in an experimental rat model of infection,