Báo cáo khoa học: Structural diversity in lipopolysaccharide expression in nontypeable Haemophilus influenzae Identification of L-glycero -D-manno-heptose in the outer-core region in three clinical isolates potx

Structural diversity in lipopolysaccharide expression in nontypeable Haemophilus influenzae Identification of L - glycero - D - manno -heptose in the outer-core region in three clinical isolates Martin Ma ˚ nsson 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, Huddinge, Sweden; 2 Molecular Infectious Diseases Group and Department of Paediatrics, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK Structural elucidation of the lipopolysaccharide (LPS) from three nontypeable Haemophilus influenzae clinical isolates, 1209, 1207 and 1233 was achieved using NMR spectro- scopy and ESI-MS on O-deacylated LPS and core oligo- saccharide (OS) material as well as ESI-MS n on permethylated dephosphorylated OS. It was found that the organisms expressed a tremendous heterogeneous glyco- form mixture resulting from the variable length of the OS chains attached to the common structural element of H. influenzae, 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. Notably, the O-6 position of the b- D -Glcp residue could either be occupied by PCho or L -glycero- D -manno-heptose ( L , D –Hep), which is a location for L , D –Hep that has not been seen previously in H. influenzae LPS. The outer-core L , D –Hep residue was further chain elongated at the O-6 position by the struc- tural element b- D -GalpNAc-(1fi3)-a- D -Galp-(1fi4)-b- D - Galp, or sequentially truncated versions thereof. The distal heptose residue in the inner-core was found to be chain elongated at O-2 by the globotetraose unit, b- D -GalpNAc- (1fi3)-a- D -Galp-(1fi4)-b- D -Galp-(1fi4)-b- D -Glcp,or sequentially truncated versions thereof. Investigation of LPS from an lpsA mutant of isolate 1233 and a lic1 mutant of isolate 1209 was also performed, which aside from confirming the functions of the gene products, simplified elucidation of the OS extending from the proximal heptose (the lpsA mutant), and showed that the organism exclu- sively expresses LPS glycoforms comprising the outer-core L , D –Hep residue when PCho is not expressed (the lic1 mutant). Keywords: Haemophilus; lipopolysaccharide; L -glycero- D -manno-heptose; phase variation; ESI-MS n . Haemophilus influenzae is an important cause of human disease worldwide and is found in both encapsulated (types a–f) and unencapsulated (nontypeable) forms. Nontypeable H. influenzae (NTHi) strains routinely colonize the naso- pharynx of healthy carriers and cause otitis media and respiratory tract infections [1]. The outer membrane com- ponent lipopolysaccharide (LPS) can influence each stage of the pathogenesis of H. influenzae infection. H. influenzae LPS is composed of a membrane-bound lipid A moiety connected to the core oligosaccharide (OS) via a single phosphorylated 3-deoxy- D -manno-oct-2-ulosonic acid (Kdo) residue. The carbohydrate regions provide targets for recognition by host immune responses and expression of certain OS epitopes can alter the virulence of the pathogen [2]. Some of these OS epitopes have been found to mimic human antigens, possibly allowing the bacteria to evade the host immune system [3,4]. The OS portion of H. influenzae LPS is subject to high-frequency phase variation (on/off switching of expression) of terminal epitopes, contributing to the vast LPS heterogeneity usually found within a single strain [2]. This heterogeneity may be an advantage to the bacteria, allowing them to better confront different host compartments and microenvironments and to survive the host immune response [5]. The availability of the complete genome sequence of H. influenzae strain Rd [6] has facili- tated a comprehensive study of LPS biosynthetic loci in the homologous strain RM118 [7] and in the type b strains Eagan (RM153) and RM7004 [8]. Gene functions have been identified that are responsible for most of the steps in the biosynthesis of the OS portion of their LPS molecules. Correspondence to E. Schweda, University College of South Stockholm, Clinical Research Centre, NOVUM, S-141 86 Huddinge, Sweden. Fax: + 46 8585 838 20, Tel.: + 46 8585 838 23, E-mail: elke.schweda@kfc.ki.se Abbreviations: CE, capillary electrophoresis; Kdo, 3-deoxy- D -manno- oct-2-ulosonic acid; AnKdo-ol, reduced anhydro Kdo; Hep, heptose; D , D -Hep, D -glycero- D -manno-heptose; L , D -Hep, L -glycero- D -manno-heptose; Hex, hexose; HexNAc, N-acetylhexos- amine; HMBC, heteronuclear multiple-bond correlation; lipid A-OH, O-deacylated lipid A; LPS, lipopolysaccharide; LPS-OH, O-deacylated LPS; MS n , multiple step tandem mass spectrometry; Neu5Ac, N-acetylneuraminic acid; NTHi, nontypeable Haemophilus influenzae; OS, oligosaccharide; PCho, phosphocholine; PEtn, phosphoethanolamine; PPEtn, pyrophosphoethanolamine. (Received 5 September 2002, revised 19 November 2002, accepted 26 November 2002) Eur. J. Biochem. 270, 610–624 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03399.x Molecular structural studies of LPS from H. influenzae strains [9–19] have resulted in a structural model consisting of a conserved phosphoethanolamine (PEtn)-substituted triheptosyl inner-core moiety (labelled HepI–HepIII) in which each of the heptose residues can provide a point for attachment of OS chains or noncarbohydrate substituents (Scheme 1). HepIII and the b- D -Glcp residue that is linked to HepI (labelled GlcI) have an especially wide range of alternatives in the substitution-pattern. HepIII has been found to be substituted by a b- D -Glcp residue either at O-2 [12] or O-3 [15]. Alternatively, substitution can occur by a b- D -Galp residue either at O-2 [10] or O-3 [18]. Analysis of LPS from lpsA mutants established in a number of strain backgrounds supports a role for LpsA in each of the alternative glycose substitutions of HepIII. HepIII has also been found to be substituted by the noncarbohydrate substituents Ac (either at O-2 [15] or O-3 [14]), Gly [15,20], P (at O-4 [11]) and PEtn [9]. For GlcI, the O-4 position has been found to be substituted by a b- D -Galp residue [9] or a b- D -Glcp residue [10], while in other strains, O-6 was substituted by phosphocholine (PCho) [12] or D -glycero- D -manno-heptose ( D , D –Hep) [13]. In NTHi strains SB 33 and 176 [16,18], disubstitution by b- D -Glcp (at O-4) and PCho (at O-6) occurs, while in strain RM118 [19], disubstitution by b- D -Galp (atO-4)andPCho (at O-6) was found. In NTHi strain 1003 [17], disubstitution by Ac (at O-4) and PCho (at O-6) was shown. In each strain analysed, PCho addition has been shown to be directed by the products of the lic1 locus. Our recent studies have focussed on the structural diversity of LPS expression and the genetic basis for that diversity in a representative set consisting of 24 NTHi clinical isolates obtained from otitis media patients. In the present study we report on the structural analysis of LPS from three of these isolates (1209, 1207 and 1233) which introduces a new glycosyl substituent on GlcI. Experimental procedures Bacterial strains used in this study NTHi middle ear isolates 1209, 1207 and 1233 were obtained from the Finnish Otitis Media Study Group [20a]. Isolates 1209 and 1207 are from a single patient on the same day but from different ears. 1233 was isolated from a different patient on a different date. Molecular epidemiological data follow- ing DNA sequence analysis showed that 1233 is identical to 1209 and 1207 except for one nucleotide change in one of the housekeeping genes investigated (unpublished results). Strains 1233lpsA and 1209lic1 were constructed by transformation of the designated isolate with plasmid clones containing the relevant gene(s) interrupted by an antibiotic resistance cassette, as described previously [7,8]. Bacterial cultivation and preparation of LPS Bacteria were grown in brain-heart infusion broth supple- mented with haemin (10 lgÆmL )1 )andNAD(2lgÆmL )1 ). LPS was extracted from lyophilized bacteria by using phenol/chloroform/light petroleum, as described by Galanos et al. [21], but modified with a precipitation step of the LPS with diethyl ether/acetone (1 : 5, v/v; 6 vol.). LPS was purified by ultracentrifugation (82 000 g,4°C, 12 h). Chromatography Gel filtration chromatography and GLC were carried out as described previously [15]. Preparation of OS material O-Deacylation of LPS. O-Deacylation of LPS was achi- eved with anhydrous hydrazine as described previously [15,22]. Mild acid hydrolysis of LPS. Reduced core OS material was obtained after mild acid hydrolysis (1% aqueous acetic acid, pH 3.1, 100 °C, 2 h) and simultaneous reduction (borane-N-methylmorpholine complex) of LPS from 1209 (120 mg), 1207, 1233, 1233lpsA (75 mg) and 1209lic1.The insoluble lipid A was separated by centrifugation and the water-soluble part was purified by gel filtration, giving one major OS-containing fraction from 1209 (OS-1, 22.5 mg), 1207, 1233, 1233lpsA (OS-2, 8.0 mg) and 1209lic1.AnNH 3 - treatedpartofOS-1(OS-1¢) was repeatedly chromato- graphed, giving a major (OS-1¢-A, 4.4 mg) and a minor (OS-1¢-B, 1.0 mg) fraction. OS-2 was rechromatographed, resulting in i.a. OS-2-A (3.0 mg) and OS-2-B (1.8 mg). Dephosphorylation of OS. Dephosphorylation of OS material was performed with 48% aqueous HF as described previously [17]. Mass spectrometry GLC-MS was carried out with a Hewlett-Packard 5890 chromatograph equipped with a NERMAG R10–10H quadrupole mass spectrometer. ESI-MS was performed as described previously [15]. ESI-MS n on permethylated dephosphorylated OS was performed on a Finnigan-MAT LCQ ion trap mass spectrometer (Finnigan-MAT, San Jose, CA, USA) in the positive ion mode. The samples were dissolved in methanol/water (7 : 3) containing 1 m M NaOAc to a concentration of about 1 mgÆmL )1 ,andwere injected into a running solvent of identical composition at 10 lLÆmin )1 . Scheme 1. R 1 ¼ H, PCho or D , D –Hep, R 2 ,R 4 ,R 5 ¼ H, Glc, Gal or Ac, R 3 ¼ HorGlc,Y¼ Gly, P or PEtn. Ó FEBS 2003 L , D –Hep in the outer-core region of NTHi LPS (Eur. J. Biochem. 270) 611 NMR spectroscopy NMR spectra were obtained at 22 °CforOSandLPS-OH samples either on a Varian UNITY 600 MHz spectrometer as described previously [15,17] or on a JEOL JNM-ECP500 spectrometer using the previously described experiments [15,17], except that a mixing time of 200 ms was used in all NOESY experiments. Analytical methods Sugars were identified as their alditol acetates as previously described [23]. Methylation analysis was performed as described earlier [15]. The relative proportions of the various alditol acetates and partially methylated alditol acetates obtained in sugar- and methylation analyses correspond to the detector response of the GLC-MS. Permethylation of dephosphorylated OS was performed in the same way as in the methylation analyses [15] but without the prior acety- lation step. The absolute configurations of the hexoses were determined by the method devised by Gerwig et al.[24].The total content of fatty acids was analysed as described previously [25]. Results NTHi isolates 1209, 1207 and 1233 and selected mutant strains were cultivated in liquid media and the LPS was extracted using the phenol/chloroform/light petroleum method. Characterization of LPS from NTHi isolate 1209 Compositional sugar analysis of the LPS sample indicated D -glucose (Glc), D -galactose (Gal), 2-amino-2-deoxy- D -glucose (GlcN) and L -glycero- D -manno-heptose ( L , D – Hep) in the ratio 31 : 43 : 1 : 25, as identified by GLC- MS of their corresponding alditol acetate and 2-butyl glycoside derivatives [24]. As described earlier, the LPS contained ester-linked glycine [20] and a low level of N-acetylneuraminic acid (Neu5Ac) [26] as shown by high- performance anion-exchange chromatography, following treatment of samples with 0.1 M NaOH and neuraminidase, respectively. Treatment of the LPS with anhydrous hydrazine under mild conditions afforded water-soluble O-deacylated material (LPS-OH). ESI-MS data (Table 1) indicated a heterogeneous mixture of glycoforms consistent with each molecular species containing a conserved PEtn-substituted triheptosyl inner-core moiety attached via a phosphorylated Kdo linker to the putative O-deacylated lipid A (lipid A-OH). Quadruply charged ions were observed at m/z 650.1/680.8 (major) and at m/z 690.4/721.2 corresponding to glycoforms with the respective compositions PCho• Hex 3 •Hep 3 •PEtn 1)2 •P 1 •Kdo•Lipid A-OH and PCho• Hex 4 •Hep 3 •PEtn 1)2 •P 1 •Kdo•Lipid A-OH (Fig. 1). Quad- ruply charged ions were also observed at m/z 697.2/728.0 and at m/z 737.7/768.6 (minor) indicating the presence of glycoforms containing four heptose residues with the compositions Hex 4 •Hep 4 •PEtn 1)2 •P 1 •Kdo•Lipid A-OH and Hex 5 •Hep 4 •PEtn 1)2 •P 1 •Kdo•Lipid A-OH, respect- ively. Quadruply charged ions of very low abundance could also be observed at m/z 609.4/640.4 and at m/z 741.2/772.1 consistent with the respective compositions PCho•Hex 2 • Hep 3 •PEtn 1)2 •P 1 •Kdo•Lipid A-OH and PCho•HexNAc 1 • Hex 4 •Hep 3 •PEtn 1)2 •P 1 •Kdo•Lipid A-OH. Thus, ESI-MS data indicated the presence of two subpopulations of glycoforms; a major subpopulation in which the glycoform compositions comprised three heptoses and PCho (Hep3- glycoforms), and a minor subpopulation with compositions comprising four heptoses but lacking PCho (Hep4-glyco- forms). NTHi LPS glycoforms with four heptoses have previously been observed ([13], M. Ma ˚ nsson, E. R. Moxon and E. K. H. Schweda, unpublished results), and in those cases the fourth heptose has the D -glycero- D -manno-confi- guration and is situated in the outer-core region of the LPS. As D , D –Hep was completely absent in the sugar analysis, it was concluded that the fourth heptose here has the L -glycero- D -manno-configuration. Characterization of OS from NTHi isolate 1209 Partial acid hydrolysis of LPS with dilute aqueous acetic acid afforded an insoluble lipid A and core OS material, Table 1. Negative ion ESI-MS data and proposed compositions for LPS-OH of NTHi isolate 1209. 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; P, 79.98; PEtn, 123.05; PCho, 165.13 and Lipid A-OH, 953.02. Relative abundance was estimated from the area of molecular ion peak relative to the total area (expressed as percentage). Peaks representing less than 5% of the base peak are not included in the table. Very minor amounts of glycoforms with the compositions PCho•Hex 2 •Hep 3 •PEtn 1)2 •P 1 •Kdo•Lipid A-OH and PCho•HexNAc 1 •Hex 4 •Hep 3 •PEtn 1)2 •P 1 •Kdo•LipidA-OHwerealsoindicatedby quadruply charged ions at m/z 609.4/640.4 and at m/z 741.2/772.1. Observed ions (m/z) Molecular mass (Da) Relative abundance (%) Proposed composition (M-4H) 4– (M-3H) 3– Observed Calculated 650.1 866.9 2604.0 2604.3 20 PCho•Hex 3 •Hep 3 •PEtn 1 •P 1 •Kdo•Lipid A-OH 680.8 908.0 2727.1 2727.3 43 PCho•Hex 3 •Hep 3 •PEtn 2 •P 1 •Kdo•Lipid A-OH 690.4 920.7 2765.4 2766.4 8 PCho•Hex 4 •Hep 3 •PEtn 1 •P 1 •Kdo•Lipid A-OH 721.2 962.0 2888.9 2889.5 7 PCho•Hex 4 •Hep 3 •PEtn 2 •P 1 •Kdo•Lipid A-OH 697.2 930.0 2792.9 2793.5 6 Hex 4 •Hep 4 •PEtn 1 •P 1 •Kdo•Lipid A-OH 728.0 970.9 2915.8 2916.5 12 Hex 4 •Hep 4 •PEtn 2 •P 1 •Kdo•Lipid A-OH 737.7 983.7 2954.4 2955.6 2 Hex 5 •Hep 4 •PEtn 1 •P 1 •Kdo•Lipid A-OH 768.6 1025.0 3078.2 3078.7 2 Hex 5 •Hep 4 •PEtn 2 •P 1 •Kdo•Lipid A-OH 612 M. Ma ˚ nsson et al. (Eur. J. Biochem. 270) Ó FEBS 2003 which was purified by gel filtration chromatography, giving OS-1. ESI-MS indicated OS-1 to contain O-acetylated (0–3 Ac) and/or O-glycylated glycoforms (0–2 Gly), where the most abundant peaks within each subpopulation of glyco- forms corresponded to compositions comprising two acetyl groups but lacking glycine (data not shown). On treatment of OS-1 with 1% aqueous NH 3 (giving OS-1¢), ESI-MS showed major doubly charged ions at m/z 785.3 and 879.9 corresponding to the respective compositions PCho•Hex 3 • Hep 3 •PEtn 1 •AnKdo-ol and Hex 4 •Hep 4 •PEtn 1 •AnKdo-ol (Table 2). In order to obtain sequence and branching information, OS-1 was dephosphorylated and permethylated and sub- jected to ESI-MS n [27,28]. Due to the increased MS response obtained by permethylation in combination with added sodium acetate [27], several glycoforms were observed in the MS spectra (positive mode) that were not detected in underivatized samples (Fig. 2A). Sodiated adduct ions were identified corresponding to the composi- tions Hex 1)6 •Hep 3 •AnKdo-ol, HexNAc 1 •Hex 4)5 •Hep 3 • AnKdo-ol, Hex 2)6 •Hep 4 •AnKdo-ol and HexNAc 1 • Hex 5)6 •Hep 4 •AnKdo-ol (Tables 3 and 4). The monosac- charide sequence and branching for the different glycoforms were obtained following collision-induced dissociation (CID) of the glycosidic bonds [27,28]. Through the ion mass distinction between reducing, nonreducing and inter- nal fragments resulting from the bond ruptures [27], the topology could be determined for all compositions found in the MS profiling spectrum (Tables 3 and 4). For most compositions, the presence of several (2–3) isomeric com- pounds were revealed by identifying product ions in the MS 2 spectra resulting from glycosidic cleavage between the heptose residues. MS 3 experiments were employed when necessary to confirm the structures. Fig. 1. Negative ion ESI-MS spectrum of O-deacylated LPS from NTHi isolate 1209 showing quadruply charged ions. The peak at m/z 650.1 corresponds to a glycoform with the composition PCho•Hex 3 •Hep 3 •PEtn 1 •P 1 •Kdo•Lipid A-OH. The peak at m/z 697.2 corresponds to a glycoform with the composition Hex 4 •Hep 4 •PEtn 1 •P 1 •Kdo•Lipid A-OH. Sodiated adduct ions are indicated by asterisks (*). Table 2. Negative ion ESI-MS data and proposed compositions for NH 3 -treated OS preparations OS-1¢,OS-1¢-A and OS-1¢-B derived from LPS of NTHi isolate 1209. Average mass units were used for calculation of molecular mass values based on proposed compositions as follows: Hex, 162.14; HexNAc, 203.19; Hep, 192.17; AnKdo-ol, 222.20; PEtn, 123.05 and PCho, 165.13. Relative abundance was estimated from the area of molecular ion peak relative to the total area (expressed as percentage). ND, not detected. Observed ions (m/z) (M-2H) 2– Molecular mass (Da) Relative abundance (%) Proposed composition Observed Calculated OS-1¢ OS-1¢-A OS-1¢-B 623.4 1248.8 1249.0 Trace a ND 5 PCho•Hex 1 •Hep 3 •PEtn 1 •AnKdo-ol 704.3 1410.6 1411.2 3 1 6 PCho•Hex 2 •Hep 3 •PEtn 1 •AnKdo-ol 785.3 1572.6 1573.3 70 80 30 PCho•Hex 3 •Hep 3 •PEtn 1 •AnKdo-ol 866.4 1734.8 1735.4 9 11 2 PCho•Hex 4 •Hep 3 •PEtn 1 •AnKdo-ol 968.2 1938.4 1938.6 Trace 2 ND PCho•HexNAc 1 •Hex 4 •Hep 3 •PEtn 1 •AnKdo-ol 718.0 1438.0 1438.2 ND ND 1 Hex 2 •Hep 4 •PEtn 1 •AnKdo-ol 798.8 1599.6 1600.4 Trace ND 3 Hex 3 •Hep 4 •PEtn 1 •AnKdo-ol 879.9 1761.8 1762.5 16 5 47 Hex 4 •Hep 4 •PEtn 1 •AnKdo-ol 960.9 1923.8 1924.6 2 1 6 Hex 5 •Hep 4 •PEtn 1 •AnKdo-ol a Trace amounts, defined as peaks representing less than 1% of the base peak. Ó FEBS 2003 L , D –Hep in the outer-core region of NTHi LPS (Eur. J. Biochem. 270) 613 For the major Hep3-glycoform with the composition Hex 3 •Hep 3 •AnKdo-ol ([M + Na] + 1671.8 Da), ion selec- tion and collisional activation of the precursor ion at m/z 1671.8 provided the MS 2 spectrum shown in Fig. 3. The ions at m/z 1453.6 and 1249.5 indicated losses of a single nonreducing terminal-Hex (t-Hex) and the nonreducing fragment t-Hex-Hex, respectively. The fragment at m/z 1001.5 (loss of t-Hex-Hex–Hep) and its trisaccharide counterpart at m/z 693.4 resulting from cleavage between HepII and HepIII, indicated the dihexose moiety to be attached to HepIII. The ions at m/z 753.4 (t-Hex–Hep- AnKdo-ol) and 941.4 (the counterpart) resulting from cleavage between HepI and HepII, finally, showed a terminal Hex residue to be linked to HepI. Loss of the terminal AnKdo-ol residue was also observed (as in almost every MS 2 spectrum) from the ion at m/z 1393.5. For the Hep3-glycoform with the composition Hex 4 • Hep 3 •AnKdo-ol ([M + Na] + 1875.9 Da), three isomers were shown to be present [Table 3, o ¼ 3, q ¼ 1(I),o¼ 2, q ¼ 2 (II), o ¼ 1, q ¼ 3 (III)]. Ions in the MS 2 spectrum at m/z 1657.7, 1453.6 and 1249.5 corresponded to losses of t-Hex (from I, II and III), t-Hex-Hex (I, II and III) and t-Hex-Hex-Hex (I and III), respectively. Isomer III could be derived from the ion pairs at m/z 753.3/1145.3 (cleavage HepI–HepII) and 1001.5/897.5 (cleavage HepII–HepIII). Isomer II was derived from the fragment pairs at m/z 957.5/ 941.3 and 1205.5/693.3 and isomer I, finally, from the ion pair at m/z 1161.4/737.4 and from the ion at m/z 1409.9. MS 3 experiments on selected product ions confirmed the assigned structures. For the structures containing HexNAc residues, it was observed that cleavage of the glycosidic bonds were highly favoured on the reducing side of the HexNAc residues [27]. To obtain unambiguous results, it was often necessary to perform MS 3 experiments on the product ion after loss of t-HexNAc or t-Hex-HexNAc. Fig. 2. ESI-MS spectra of permethylated dephosphorylated OS derived from LPS of NTHi isolates 1209 (A) and 1233 (B) showing singly charged ions, [M + Na] + . (A) Ions corresponding to selected Hep3- glycoforms are labelled. (B) Ions corresponding to selected Hep4- glycoforms are labelled. Table 3. Structures of the Hep3-glycoforms of NTHi isolates 1209 and 1233 as indicated by ESI-MS n on permethylated dephosphorylated OS. Subscripts denoted by the letters m, n, o, p and q indicate the number of glycose residues in the following structure: ND, not determined. Relative abun- dance a (%) Structure Relative abundance b Glycoform 1209 1233 m n o p q 1209 1233 Hex1 0.5 – 0 0 1 0 0 High 0 0 0 0 1 Trace Hex2 3.6 2.1 0 0 2 0 0 Low Medium 0 0 1 0 1 High Medium 0 0 0 0 2 Trace Trace Hex3 57.5 13.3 0 0 300– Low 0 0201– Trace 0 0 1 0 2 High Medium 0 0003– Trace Hex4 10.4 30.1 0 0 3 0 1 Trace Trace 0 0202Medium Low 0 0103Medium High Hex5 1.9 3.2 0 0 3 0 2 High Medium 0 0 2 0 3 Trace Medium Hex6 0.7 3.2 0 0 3 0 3 High High Hex7 – 0.8 ND HexNAc1Hex4 1.9 1.5 0 0 1 1 3 High High HexNAc1Hex5 0.7 0.9 0 0 213Medium Medium 1 1202Medium Medium HexNAc1Hex6 – 1.0 1 1 2 0 3 High HexNAc1Hex7 – 0.4 ND HexNAc2Hex6 – 0.2 ND a Relative abundance for each glycoform. Estimated from the area of molecular ion peak relative to the total area in the MS spectrum (expressed as percentage). b Relative abundance for the isomers of each glycoform. Estimated from the intensity of the fragments in MS 2 experiments and indicated as follows: high (over 80%), medium (30–80%), low (2–30%), trace (below 2%). 614 M. Ma ˚ nsson et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Elucidation of the Hep4-glycoforms introduced an addi- tional level of complexity, as ions resulting from cleavages of outer-core glycosidic linkages in some cases could not be mass differentiated from fragments resulting from HepII– HepIII ruptures. It was thus necessary to identify product ions resulting from cleavages between HepI and HepII with unique masses due to the AnKdo-ol moiety. For the major Hep4-glycoform with the composition Hex 4 •Hep 4 •AnKdo- ol ([M + Na] + 2124.0 Da), ions in the MS 2 spectrum (precursor ion [M + 2Na] 2+ 1073.5 Da) (Fig. 4A) at m/z 1905.9, 1701.7 (counterpart at m/z 445.3) and 1453.6 (counterpart at m/z 693.3) corresponded to losses of t-Hex, t-Hex-Hex and t-Hex-Hex–Hep. The fragments at m/z 1205.6 (loss of t-Hex-Hex–Hep–Hep) and 941.5 (the counterpart) indicated a dihexose moiety to be linked to HepIII. An MS 3 experiment on m/z 1205.6 (Fig. 4B) resul- tedinfragmentsatm/z 987.5 (loss of t-Hex), 739.3 (loss of t-Hex–Hep) and 693.3 (corresponding to t-Hex–Hep-Hex), which showed the trisaccharide element Hex–Hep-Hex to be attached to HepI. The topology of the other glycoforms were determined in a similar manner (data not shown). Table 4. Structures of the Hep4-glycoforms of NTHi isolates 1209 and 1233 as indicated by ESI-MS n on permethylated dephosphorylated OS. Subscripts denoted by the letters m, n, o and p indicate the number of glycose residues in the following structure: ND, not determined. Glycoform Relative abundance a (%) Structure Relative abundance b 1209 1233 m n o p 1209 1233 Hex1 – 0.3 0 0 0 0 High Hex2 1.6 0.8 0 1 0 0 High Medium 0 0 0 1 Low Medium Hex3 2.4 2.1 0 2 0 0 Low Medium 0 1 0 1 Medium Medium 0 0 0 2 Medium Medium Hex4 14.9 7.2 0 2 0 1 – Low 0 1 0 2 High Medium 0 0 0 3 – Medium Hex5 2.7 12.6 0 2 0 2 Medium Medium 0 1 0 3 Medium Medium Hex6 0.5 15.2 0 2 0 3 High High Hex7 – 1.5 ND Hex8 – 0.5 ND HexNAc1Hex4 – 0.7 1 2 0 1 Medium 0 0 1 3 Medium HexNAc1Hex5 0.4 0.7 1 2 0 2 Medium Medium 0 1 1 3 Medium Medium HexNAc1Hex6 0.3 1.2 1 2 0 3 Medium Medium 0 2 1 3 Medium Medium HexNAc1Hex7 – 0.3 ND HexNAc1Hex8 – 0.1 ND HexNAc2Hex6 – 0.1 1 2 1 3 High a Relative abundance for each glycoform. Estimated from the area of molecular ion peak relative to the total area in the MS spectrum (expressed as percentage). b Relative abundance for the isomers of each glycoform. Estimated from the intensity of the fragments in MS 2 experiments and indicated as follows: high (over 80%), medium (30–80%), low (2–30%), trace (below 2%). Fig. 3. ESI-MS 2 analysis of permethylated dephosphorylated OS derived from LPS of NTHi isolate 1209. Product ion spectrum of [M + Na] + m/z 1671.8 corresponding to a glycoform with the com- position Hex 3 •Hep 3 •AnKdo-ol. The proposed structure is shown in the inset. Fig. 4. ESI-MS n analysis of permethylated dephosphorylated OS derived from LPS of NTHi isolate 1209. (A) MS 2 spectrum of [M + 2Na] 2+ m/z 1073.5 corresponding to a glycoform with the composition Hex 4 •Hep 4 •AnKdo-ol. The proposed structure is shown in the inset. (B) MS 3 spectrum of the fragment ion at m/z 1205.6. Ó FEBS 2003 L , D –Hep in the outer-core region of NTHi LPS (Eur. J. Biochem. 270) 615 A large number of structural types were observed for the Hep3-glycoforms (15 found) and Hep4-glycoforms (13 found), all of which can be represented by structures I and II (Hep3-glycoforms) and structure III (Hep4-glycoforms) in Scheme 2. Structural characterization of the major glycoforms was achieved by NMR spectroscopy (see below). In order to decrease the OS heterogeneity and thereby simplify the elucidation by NMR, OS-1¢ was repeatedly chromato- graphed on a P-4 column with intermediate selection for the Hep3- and Hep4-glycoforms (by ESI-MS). The final result wasamajorfraction(OS-1¢-A, 4.4 mg) in which the Hep3- glycoforms accounted for about 95%, and a minor fraction (OS-1¢-B, 1.0 mg) in which the Hep4-glycoforms accounted for about 60% (Table 2). Sugar analysis of OS-1¢-A and OS-1¢-B indicated Glc, Gal and L , D –Hep in ratios of 26 : 48 : 26 and 29 : 37 : 34, respectively. Methylation analysis of OS-1¢-A (Table 5) indicated terminal-Gal (t-Gal), 4-substituted-Gal (4-Gal), 4-Glc, 3-Gal, 6-Glc, 4,6- disubstituted-Glc (4,6-Glc), 2–Hep, 3,4–Hep and 2,6–Hep in the relative proportions 27 : 7 : 37 : 2 : 2 : 2 : 17 : 4 : 2. The methylation analysis of OS-1¢-B showed significantly higher levels of 6-Glc and 6–Hep, of which the latter sugar derivative indicated the substitution-pattern for the fourth heptose residue (shown below). Characterization of OS fractions and LPS-OH from NTHi isolate 1209 by NMR The 1 H NMR resonances of OS material and LPS-OH were assigned by 1 H- 1 H chemical shift correlation experiments (DQF-COSY and TOCSY). Subspectra corresponding to the individual glycosyl residues were identified on the basis of spin-connectivity pathways delineated in the 1 Hchemical shift correlation maps, the chemical shift values, and the vicinal coupling constants. From the glycoform composi- tions of the different oligosaccharide fractions determined by ESI-MS (Table 2), the spin-systems could more easily be identified as originating from either the Hep3- or the Hep4- glycoform population. The 13 C NMR resonances of OS fractions and LPS-OH were assigned by heteronuclear 1 H- 13 C chemical shift correlation in the 1 H detected mode (HSQC). The chemical shift data obtained for the Hep4- glycoforms are summarized in Table 6 and are consistent Scheme 2. Structures representing the various Hep3- and Hep4- glycoforms in isolates 1209 and 1233. Truncated structures are indicated by ÔÆÆÆÆÕ. Table 5. Linkage analysis data for OS preparations derived from LPS of NTHi isolates 1209 and 1233. Trace amounts (defined as peaks representing less than 3% of the base peak) of 2,3,4,6-Me 4 -Glc [assigned as D -Glcp-(1fi], 2,3,4,6,7-Me 5 –Hep [ L , D –Hepp-(1fi], 2,3,4,6-Me 4 -GalN [ D -GalpNAc-(1fi] and 2,3,6-Me 3 -GlcN [fi4)- D -GlcpNAc-(1fi] were also indicated. Methylated sugar a T gm b Relative detector response (%) Linkage assignment OS-1¢-A OS-1¢-B 1233 OS 2,3,4,6-Me 4 -Gal 1.04 27 42 17 D -Galp-(1fi 2,3,6-Me 3 -Gal 1.16 7 1 22 fi4)- D -Galp-(1fi 2,3,6-Me 3 -Glc 1.17 37 28 13 fi4)- D -Glcp-(1fi 2,4,6-Me 3 -Gal 1.19 2 Trace 5 fi3)- D -Galp-(1fi 2,3,4-Me 3 -Glc 1.20 2 6 5 fi6)- D -Glcp-(1fi 2,3-Me 2 -Glc 1.34 2 Trace 1 fi4,6)- D -Glcp-(1fi 2,3,4,7-Me 4 –Hep 1.41 Trace 4 3 fi6)- L , D –Hepp-(1fi 3,4,6,7-Me 4 –Hep 1.42 17 17 19 fi2)- L , D –Hepp-(1fi 2,6,7-Me 3 –Hep 1.48 4 2 14 fi3,4)- L , D –Hepp-(1fi 3,4,7-Me 3 –Hep 1.53 2 Trace 1 fi2,6)- L , D –Hepp-(1fi 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 (T gm 1.00). 616 M. Ma ˚ nsson et al. (Eur. J. Biochem. 270) Ó FEBS 2003 with each D -sugar residue being present in the pyranosyl ring form. Further evidence for this conclusion was obtained from NOE data which also served to confirm the anomeric configurations of the linkages and, together with an HMBC experiment on OS-1¢, determined the monosaccharide sequence. Characterization of the Kdo-lipidA-OH element. ESI-MS data (Table 1), fatty acid compositional analysis (yielding 3-hydroxytetradecanoic acid) and NMR experiments on LPS-OH (data not shown, giving similar results as for NTHi strains 486 [15] and 1003 [17]) indicated the presence of the usual Kdo-lipid A-OH element in isolate 1209. As observed earlier [15,17], two spin-systems could be traced for the single a-linked Kdo residue, probably due to the partial occurrence of PEtn attached to the phosphate group at O-4 of Kdo [11,12,15]. Structure of the core region of the Hep3-glycoforms. In the 1 H NMR spectra of OS-1¢-A (Fig. 5A) and OS-1¢ (Fig. 5B), anomeric resonances corresponding to the inner- core region and GlcI could be observed at d 5.70–5.63 (HepII), 5.15–5.04 (HepI), 5.12 (HepIII) and 4.51 (GlcI), respectively. In addition, anomeric signals corresponding to a globotetraose unit and sequentially truncated versions thereof linked to HepIII, were identified at d 4.96 (t-a- D - Galp), 4.93 (weak, 3-a- D -Galp), 4.70 (4-b- D -Glcp), 4.64 (weak, t-b- D -GalpNAc), 4.52 (4-b- D -Galp)and4.46(t-b- D - Galp) as previously described for strain RM118 [12]. Spin- systems for the additional glycose residues shown by MS n (see Scheme 2) could not be rationalized due to weak and overlapping signals. Signals for methyl protons of PCho were observed at d 3.25 and spin-systems for ethylene protons from this residue and from PEtn were similar to those observed earlier [15,17]. 1 H- 31 P NMR correlation studies (data not shown) demonstrated PChotobelocated at O-6 of GlcI and PEtn to substitute HepII at O-6, as previously observed in strain RM118 [12] and NTHi strain 1003 [17]. The positions of the O-acetyl groups were determined from NMR experiments on OS-1. Intense signals from methyl protons of the O-acetyl groups were observed at d 2.20/2.18, which correlated to 13 C signals at d 21.1 in the HSQC spectrum. For GlcI, a spin-system was found where characteristic downfield shifts were obtained for the signals from H-4 and C-4, consistent with acetylation at O-4 as previously described for NTHi strain 1003 [17]. HepIII was indicated to be acetylated at O-3 as character- istic downfield shifts were obtained for the signals from H-3 and C-3, as previously described for strain 1003 [17]. A crosspeak from the ester-linked glycine substituent was also observed at d 3.99/40.7 (in the HSQC spectrum) due to correlation between the methylene proton and its carbon. From the combined data, the structure in Scheme 3 is proposed for the globotetraose-containing Hep3-glycoform (HexNAc1Hex4) of NTHi isolate 1209. Structure of the core region of the Hep4-glycoforms. In the 1 H NMR spectrum of OS-1¢-B (Fig. 5C), anomeric resonances of the three heptose residues (HepI–HepIII) in the inner-core region were observed at d 5.73–5.62 (1H, not resolved), 5.18–5.05 (1H, not resolved) and 4.99 (1H, not resolved). The Hep ring systems were identified on the basis Table 6. 1 H and 13 C NMR chemical shifts for Hep4-glycoforms of OS-1¢-B derived from LPS of NTHi isolate 1209. Data was recorded in D 2 Oat 22 °C. Pairs of deoxyprotons of reduced AnKdo were identified in the DQF-COSY spectrum at 2.19–1.55 p.p.m. Residue Glycose unit H-1/C-1 H-2/C-2 H-3/C-3 H-4/C-4 H-5/C-5 H-6 A /C-6 H-6 B H-7 A /C-7 H-7 B HepI fi3,4)- L -a- D –Hepp-(1fi 5.05–5.18 a 4.00–4.06 a 4.01–4.04 a 4.25 b – c 4.13 b –– 97.4–98.8 a 71.1–71.2 a 73.8 74.3 – 68.5 – HepII fi2)- L -a- D –Hepp-(1fi 5.62–5.73 a 4.18 3.94 3.96 3.77 4.57 3.71 3.89 6› PEtn 99.2–99.5 a 79.6 69.9 67.0 72.5 75.3 62.9 HepIII fi2)- L -a- D –Hepp-(1fi 4.99 4.16 4.00 3.78 – – – – 100.3 79.9 70.1 67.6 – – – GlcI fi6)-b- D -Glcp-(1fi 4.51 3.40 3.44 3.52 3.59 3.87 3.96 103.9 74.3 77.3 70.8 74.2 66.0 HepIV fi6)- L -a- D –Hepp-(1fi 4.96 4.10 3.80 4.01 – 4.21 3.75 3.93 100.0 70.5 – 66.6 – 79.3 63.1 GalI b- D -Galp-(1fi 4.51 3.62 3.69 3.94 3.74 d –– 104.8 71.7 73.2 69.2 75.5 – GlcII fi4)-b- D -Glcp-(1fi 4.54 3.41 3.72 3.71 3.71 3.84 4.00 103.1 73.1 75.1 79.0 75.1 60.8 GalII b- D -Galp-(1fi 4.46 3.54 3.68 3.94 3.74 d –– 103.7 71.6 73.2 69.2 76.0 – GalII* fi4)-b- D -Galp-(1fi 4.52 3.59 3.75 4.05 3.79 d –– 103.7 71.6 72.8 78.0 76.0 – GalIII a- D -Galp-(1fi 4.96 3.84 3.91 4.04 4.37 – – 101.0 69.3 69.9 69.6 71.5 – PEtn 4.13 3.27 62.7 40.8 a Several signals were observed for HepI and HepII due to heterogeneity in the AnKdo moiety. b H-4/H-6 of HepI were identified at d 4.25/ 4.13 by NOE from GlcI. c –, not obtained owing to the complexity of the spectrum. d Tentative assignment from NOE data. Ó FEBS 2003 L , D –Hep in the outer-core region of NTHi LPS (Eur. J. Biochem. 270) 617 of the observed 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 as observed earlier [15,17]. Several signals for methylene protons of AnKdo-ol were observed in the DQF-COSY and TOCSY spectra of OS-1¢-B in the region d 2.19–1.55. As previously observed [10,15,17], several anhydro-forms of Kdo are formed during the hydrolysis by elimination of phosphate or pyrophosphoethanolamine from the C-4 position, which causes both the signal splitting of the methylene protons and the appearance of several anomeric signals for HepI and HepII (Table 6). The occurrence of intense transglycosidic NOE connectivities between the proton pairs HepIII H-1/HepII H-2, HepII H-1/HepI H-3 (LPS-OH and OS-1¢-B) and HepI H-1/Kdo H-5 and H-7 (LPS-OH) confirmed the sequence of the heptose-contain- ing trisaccharide unit and the point of attachment to Kdo as L -a- D –Hepp-(1fi2)- L -a- D –Hepp-(1fi3)- L -a- D –Hepp-(1fi5)- a-Kdop. 1 H- 31 P NMR correlation studies demonstrated PEtntobelinkedtoO-6ofHepIIasa 31 P resonance at d 0.03 correlated to the signals from H-6 of HepII (d 4.57) and the methylene proton pair of PEtn (d 4.13). Fig. 5. 600 MHz 1 HNMRspectraofOS-1¢-A (A), OS-1¢ (B) and OS-1¢-B (C) derived from LPS of NTHi isolate 1209 showing the anomeric regions. (A) Anomeric resonances that are characteristic for the Hep3-glycoforms are labelled. Also indicated is an ethylene proton signal from PCho at d 4.38. (C) Anomeric resonances that are characteristic for the Hep4-glycoforms are labelled. 618 M. Ma ˚ nsson et al. (Eur. J. Biochem. 270) Ó FEBS 2003 In OS-1¢-B, relatively large J 1,2 -values (about 7.7 Hz) of the anomeric resonances observed at d 4.54, 4.52 (weak), 4.51, 4.51 and 4.46 indicated each of the corresponding residues to have the b-anomeric configuration. The residues with anomeric signals at d 4.96 (not resolved) and 4.96 (weak, J  4.0 Hz) were identified as having the a-anomeric configuration. Further evidence for the anomeric configu- rations was obtained from the occurrence of intraresidue NOE between the respective H-1, H-3 and H-5 resonances (b-configuration) or between H-1 and H-2 (a-configur- ation). On the basis of the chemical shift data and the large J 2,3 , J 3,4 and J 4,5 -values (9 Hz), the residues with anomeric shifts of d 4.51 and 4.54 could be attributed to the 6-Glc (GlcI) and 4-Glc (GlcII) identified by methylation analysis (Table 5). On the basis of low J 3,4 and J 4,5 -values (< 4 Hz) and chemical shift data, the residues with anomeric resonances at d 4.51, 4.46, 4.96 (weak, J  4.0 Hz) and 4.52 were attributed to the t-Gal (GalI, GalII and GalIII) and 4-Gal (GalII*) identified by linkage analysis. The residue with anomeric signal at d 4.96 (not resolved) was attributed to the 6–Hep (HepIV) identified in the methyla- tion analysis, on the basis of the small J 1,2 -value and chemical shift data. Interresidue NOE were observed between the proton pairs GalI H-1/HepIV H-6, HepIV H-1/GlcI H-6A, H-6B and GlcI H-1/HepI H-4 and H-6 (Fig. 6) which established the presence of the tetrasaccharide unit b- D -Galp-(1fi6)- L - a- D –Hepp-(1fi6)-b- D -Glcp-(1fi4)- L -a- D –Hepp-(1fi.This monosaccharide sequence was also confirmed by transgly- cosidic correlations in an HMBC experiment, where corre- lations were seen between GalI C-1/HepIV H-6, GalI H-1/ HepIV C-6 and HepIV H-1/GlcI C-6. The occurrence of interresidue NOE connectivities between the proton pairs GalII H-1/GlcII H-4 and GlcII H-1/HepIII H-1 and H-2 (Fig. 6) established the sequence of a disaccharide unit and its attachment point to HepIII as b- D -Galp-(1fi4)-b- D - Glcp-(1fi2)- L -a- D –Hepp-(1fi. The lactose element was shown to be further chain extended by an a- D -Galp residue as transglycosidic NOE between GalIII H-1/GalII* H-4 and GalII* H-1/GlcII H-4 were observed. Spin-systems for Scheme 3. Structure proposed for a Hep3-glycoform (HexNAc1Hex4) of NTHi isolate 1209. Fig. 6. Selected regions from the 600 MHz 2D NOESY spectrum (mixing time 200 ms) of OS-1¢-B derived from LPS of NTHi isolate 1209. Both regions were plotted at the same contour levels. Cross-peaks that are characteristic for the Hep4-glycoforms are labelled. Ó FEBS 2003 L , D –Hep in the outer-core region of NTHi LPS (Eur. J. Biochem. 270) 619 [...]... valuable tool for profiling the extensive degree of LPS heterogeneity in the H in uenzae strains The extreme variability observed in the OS structures is likely to have important implications for the biology and virulence of these isolates The 38 variant OS structures found in 1233 exemplifies the combinatorial power of multiple phase variable biosynthesis genes in maximizing the number of potential LPS antigens... mutant of NTHi isolate 1233 It was recently shown that the lpsA gene is involved in adding a b-D-Glcp residue in a 1,2-linkage to HepIII of H in uenzae strain RM118 [7] By construction of an lpsA mutant of NTHi isolate 1233 it was therefore expected that its LPS would not express any chain elongation at HepIII, but otherwise be structurally identical to the LPS of the clinical isolate, thereby facilitating... Characterization of the phosphocholine substituted oligosaccharide in lipopolysaccharides of type b Haemophilus in uenzae Eur J Biochem 267, 1–12 ˚ Mansson, M., Bauer, S.H.J., Hood, D.W., Richards, J.C., Moxon, E.R & Schweda, 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... From the combined data, the structure in Scheme 4 is proposed for the Hex5 glycoform in the Hep4-glycoform population of NTHi isolate 1209 Characterization of LPS and OS from NTHi isolate 1207 NTHi isolates 1207 and 1209 were obtained from the same patient on the same day (left and right ear isolates) Previous investigations indicated the LPS from these isolates to contain similar levels of Neu5Ac [26]... for expression of the major globotetraose-containing lipopolysaccharide from H in uenzae strain Rd (RM118) Glycobiology 11, 957–967 Hood, D.W., Deadman, M.E., Allen, T., Masoud, H., Martin, A., Brisson, J.R., Fleischmann, R., Venter, J.C., Richards, J.C & Moxon, E.R (1996) Use of the complete genome sequence information of Haemophilus in uenzae strain Rd to investigate lipopolysaccharide biosynthesis... well as glycine [20] From the results (data not shown) of sugar (of LPS) and methylation (of LPS-OH) analysis, ESI-MS (of LPS-OH and OS), 1H NMR (of LPS-OH) and ESI-MSn (of permethylated dephosphorylated OS) there was no indication of any structural difference between the LPS from these two isolates ESI-MS indicated a preponderance of higher molecular mass glycoforms in 1207 Correspondingly, the methylation... FEBS 2003 L,D–Hep in the outer-core region of NTHi LPS (Eur J Biochem 270) 623 expressed in these isolates In agreement with this, trace amounts of 4-substituted GlcN was also detected in the methylation analyses (Table 5) ESI-MSn analysis of permethylated OS material has been shown to be a powerful method for elucidating sequence, branching and linkage information [27,28] In the present investigation,... ear isolates) and 1233 obtained from another patient on a different date The structural relationship is in agreement with molecular epidemiological data following DNA sequence analysis which showed that 1233 is identical to 1209 and 1207 except for one nucleotide change in one of the housekeeping genes investigated (unpublished results) All three isolates express L,D–Hep in the outer-core region of the. .. saccharide binding domain for the mAb 2C7 established for Neisseria gonorrhoeae LOS by ES-MS and MSn Glycobiology 9, 157–171 Weiser, J.N., Shchepetov, M & Chong, S.T.H (1997) Decoration of lipopolysaccharide with phosphorylcholine: a phasevariable characteristic of Haemophilus in uenzae Infect Immun 65, 943–950 Holst, O (1999) Chemical structure of the core region of lipopolysaccharides In Endotoxin in Health... criticism of the manuscript 7 8 9 10 11 12 13 References 1 Murphy, T.F & Apicella, M.A (1987) Nontypeable Haemophilus in uenzae: a review of clinical aspects, surface antigens, and the human immune response to infection Rev Infect Dis 9, 1–15 2 Kimura, A & Hansen, E.J (1986) Antigenic and phenotypic variations of Haemophilus in uenzae type b lipopolysaccharide and their relationship to virulence Infect . Structural diversity in lipopolysaccharide expression in nontypeable Haemophilus in uenzae Identification of L - glycero - D - manno -heptose in the outer-core. Hep3-glycoform (HexNAc1Hex4) of NTHi isolate 1209. Structure of the core region of the Hep4-glycoforms. In the 1 H NMR spectrum of OS-1¢-B (Fig. 5C), anomeric resonances of the three