Novel globoside-like oligosaccharide expression patterns in nontypeable Haemophilus influenzae lipopolysaccharide Susanna L. Lundstro ¨ m 1 , Brigitte Twelkmeyer 1 , Malin K. Sagemark 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, Huddinge, Sweden 2 Institute for Biological Sciences, National Research Council of Canada, Ottawa, Canada 3 Molecular Infectious Diseases Group and Department of Paediatrics, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK Haemophilus influenzae is an important cause of human disease worldwide and exists in encapsulated (type a–f) and unencapsulated (nontypeable) forms. Type b capsular strains are associated with invasive bacteraemic diseases, including meningitis, epiglottitis, cellulitis and pneumonia, whereas acapsular or non- typeable strains of H. influenzae (NTHi) are primary pathogens in otitis media and cause both acute and chronic lower respiratory tract infections [1,2]. The potential of H. influenzae to cause disease depends Keywords globoside; globotetraose; Haemophilus influenzae; lipopolysaccharide; sialyllactose Correspondence E. Schweda, University College of South Stockholm, Clinical Research Centre, Novum, S-141 86 Huddinge, Sweden Fax: +46 85 858 3820 Tel: +46 85 858 3823 E-mail: Elke.Schweda@crc.ki.se (Received 29 May 2007, revised 18 July 2007, accepted 25 July 2007) doi:10.1111/j.1742-4658.2007.06011.x We report the novel pattern of lipopolysaccharide (LPS) expressed by two disease-associated nontypeable Haemophilus influenzae strains, 1268 and 1200. The strains express the common structural motifs of H. influenzae; globotetraose [b-d-GalpNAc-(1fi3)-a-d-Galp-(1fi4)-b-d-Galp-(1fi4)-b-d- Glcp] and its truncated versions globoside [a-d-Galp-(1fi4)-b-d-Galp-(1fi4)- b-d-Glcp] and lactose [b-d-Galp-(1fi4)-b-d-Glcp] linked to the terminal heptose (HepIII) and the corresponding structures with an a-d-Glcp as the reducing sugar linked to the middle heptose (HepII) in the same LPS mole- cule. Previously these motifs had been found linked only to either the proxi- mal heptose (HepI) or HepIII of the triheptosyl inner-core moiety l-a-d- Hepp-(1fi2)-[PEtnfi6]-l- a-d-Hepp-( 1fi3)-l-a-d-Hepp-(1fi5)-[PPEtnfi4]- a-Kdo-(2fi6)-lipid A. This novel finding was obtained by structural studies of LPS using NMR techniques and ESI-MS on O-deacylated LPS and core oligosaccharide material, as well as electrospray ionization-multiple-step tandem mass spectrometry on permethylated dephosphorylated oligosaccha- ride material. A lpsA mutant of strain 1268 expressed LPS of reduced complexity that facilitated unambiguous structural determination. Using capillary electrophoresis-ESI-MS ⁄ MS we identified sialylated glycoforms that included sialyllactose as an extension from HepII, this is a further novel finding for H. influenzae LPS. In addition, each LPS was found to carry phosphocholine and O-linked glycine. Nontypeable H. influenzae strain 1200 expressed identical LPS structures to 1268 with the difference that strain 1200 LPS had acetates substituting HepIII, whereas strain 1268 LPS has glycine at the same position. Abbreviations AnKdo-ol, reduced anhydro Kdo; CE, capillary electrophoresis; Hep, L-glycero-D-manno-heptose; Hex, hexose; HexNAc, N-acetylhexosamine; Kdo, 3-deoxy- D-manno-oct-2-ulosonic acid; lipid A-OH, O-deacylated lipid A; LPS, lipopolysaccharide; LPS-OH, O-deacylated lipopolysaccharide; MS n , multiple-step tandem mass spectrometry; Neu5Ac, N-acetyl neuraminic acid; NTHi, nontypeable Haemophilus influenzae; OS, oligosaccharide; PCho, phosphocholine; PEtn, phosphoethanolamine; PPEtn, pyrophosphoethanolamine; tHep, terminal heptose. 4886 FEBS Journal 274 (2007) 4886–4903 ª 2007 The Authors Journal compilation ª 2007 FEBS upon its surface-expressed carbohydrate antigens, cap- sular polysaccharide [3] and lipopolysaccharide (LPS) [4]. LPS is an essential and characteristic surface com- ponent of H. influenzae. This bacterium has been found to express short-chain LPS, lacking O-specific polysaccharide chains and is often referred to as lipo- oligosaccharide. Extensive structural studies of LPS from H. influenzae by us and others have led to the identification of a conserved glucose-substituted trihep- tosyl inner-core moiety l-a-d-Hepp-(1fi2)-[PEtnfi6]- l-a-d-Hepp-(1fi3)-[b-d-Glcp-(1fi4)]-l-a-d-Hepp linked to lipid A via 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) 4-phosphate. This inner-core unit provides the template for attachment of oligosaccharides and non- carbohydrate substituents [5]. The outer core region of NTHi LPS mimics host glycolipids and the expression of terminal epitopes is subject to high-frequency phase variation, leading to a very heterogeneous population of LPS molecules within a single strain. Phase varia- tion is thought to provide an adaptive mechanism which is advantageous for the survival of bacteria con- fronted by the differing microenvironments and the immune responses of the host. Several structures mim- icking the globoside series of mammalian glycolipids have been identified in NTHi LPS such as globotetraose [b-d-GalpNAc-(1fi3)-a-d-Galp-(1fi4)-b-d-Galp-(1fi4)- b-d-Glcp-(1fi], globoside [a-d -Galp-(1fi4)-b-d-Galp- (1fi4)-b-d-Glcp], lactose [b-d-Galp-(1fi4)-b-d-Glcp] and sialyllactose [a-Neu5Ac-(2fi3)-b-d-Galp-(1fi4)-b- d-Glcp(1fi] [5]. Biosynthesis of these oligosaccharide extensions has been shown to proceed in a stepwise fashion [6]. It has also been shown that H. influenzae can express sialyllacto -N-neotetraose [a-Neu5Ac-(2fi3)- b-d-Galp-(1fi4)-b-d-GlcpNAc-(1fi3)-b-d-Galp-(1fi4)- b-d-Glcp-(1fi] or the related structure, ( PEtnfi6)-a -d- GalpNAc-(1fi6)-b-d-Galp-(1fi4)-b-d-GlcpNAc-(1fi3)- b-d-Galp-(1fi4)-b-d-Glcp-(1fi, both linked to l-gly- cero-d-manno-heptose (Hep)I. Biosynthesis of these ter- minal tetrasaccharide moieties has been found to resemble that of the O-antigen repeating unit, with the tetrasaccharide being added en bloc [7]. Noncarbo- hydrate substituents such as pyrophosphoethanolamine (PPEtn), phosphoethanolamine (PEtn), phosphocholine (PCho), acetate (Ac) and glycine (Gly) are common in NTHi LPS [5]. Our previous studies have focused on the conserva- tion and variability of patterns of LPS expressed in a representative set of NTHi clinical isolates obtained from otitis media patients [8–16] and relating this to the role of LPS in commensal and virulence behaviour. Recently, we demonstrated that oligosaccharides containing terminal sialic acid epitopes are essential virulence determinants in experimental otitis media [17]. In this study, we present novel LPS structures expressed in NTHi strains 1268 and 1200. The strains were previously shown to be very closely related [18]. Herein, we demonstrate that the two strains, as pre- dicted, have almost identical LPS structures, the only difference being the presence of O-acetyl groups in strain 1200. Both strains were found to express LPS glycoforms containing globoside and globoside-like epitopes extending simultaneously from HepIII and HepII, respectively. These LPS glycoforms have not previously been found in H. influenzae. In order to unambiguously establish this, we made use of a geneti- cally defined isogenic mutant strain, NTHi 1268lpsA, which had oligosaccharide extensions from HepI and HepII only. The mutant strain also allowed us to iden- tify sialyllactose units substituting HepII. This is the first time that sialyllactose has been detected in that molecular environment. The presence of sialylated gly- coforms likely contributes to the resistance of the strain to killing by normal human serum. Results NTHi wild-type strains 1268 and 1200 and mutant strain 1268lpsA NTHi strains 1268 and 1200 are clinical isolates origi- nating from the Finnish Otitis Media Study Group. The strains have the same ribotype, and by multilocus sequence typing had identical nucleotide sequences in three of seven LPS alleles [18]. Because of the hetero- geneous mixtures of LPS glycoforms typical of wild-type NTHi strains, a lpsA mutant strain of 1268 was made to facilitate the elucidation of its structure. It has previ- ously been shown that the lpsA gene is responsible for addition of a hexose (Hex) to the distal heptose (HepIII) of the inner-core of the Hi LPS molecule [6]. By disrupt- ing the lpsA gene, we sought to construct a mutant (1268lpsA) lacking any chain elongation from HepIII, but otherwise identical to the wild-type 1268 strain. The two NTHi wild-type and the 1268lpsA mutant strains were grown in liquid media, the bacteria harvested and desiccated, the LPS was then isolated by extraction using the phenol ⁄ chloroform ⁄ light petroleum method. Characterization of LPS from NTHi strains 1268, 1200 and 1268lpsA In earlier investigations it was found that the LPS of NTHi strains 1268 and 1200 contained ester-linked glycine and Neu5Ac, as shown by high-performance anion-exchange chromatography with pulsed ampero- metric detection following treatment of samples with S. L. Lundstro ¨ m et al. LPS structure of NTHi strains 1268 and 1200 FEBS Journal 274 (2007) 4886–4903 ª 2007 The Authors Journal compilation ª 2007 FEBS 4887 0.1 m NaOH and neuraminidase [19,20]. Furthermore, the lipid A backbone of the respective LPS has been described by Helander et al. [21,22]. LPS from all strains was treated with anhydrous hydrazine under mild conditions to give the water-sol- uble O-deacylated lipopolysaccharide (LPS-OH) which was subjected to compositional and linkage analyses as well as ESI-MS. Compositional sugar analysis of LPS-OH from the wild-type strain (1268) indicated d-glucose (Glc), d-galactose (Gal), 2-amino-2-deoxy-d-glucose (GlcN), 2-amino-2-deoxy-d-galactose (GalN) and l-glycero-d- manno-heptose (Hep) in a ratio of 32 : 28 : 21 : 9 : 10, as identified by GLC-MS of their corresponding alditol acetate and 2-butyl glycoside derivatives (Table S1). Sugar analysis of LPS-OH from strain 1200 revealed the presence of the same sugars as in 1268 in compara- ble amounts (Table S1). LPS-OH samples were dephosphorylated with 48% hydrogen fluoride prior to methylation analysis. Mate- rial from 1268 showed terminal Glc, terminal Gal, 4-substituted Gal, 4-substituted Glc, 3-substituted Gal, terminal Hep, 2-substituted Hep, 3,4-substituted Hep, terminal GalN, 2,3-substituted Hep, 4-substituted GlcN and 6-substituted GlcN in the relative amounts of 16 : 4 : 7 : 14 : 3 : 11 : 6 : 14 : 2 : 19 : 2 : 2. Meth- ylation analysis on dephosphorylated LPS-OH from NTHi strain 1200 revealed the presence of the same sugars as 1268 in comparable amounts (Table S2). The methylation analysis data were consistent with trian- tennary structures in NTHi 1268 and 1200, containing the common inner-core element, l-a-d-Hepp-(1fi2)- l-a-d-Hepp-(1fi3)-[b-d-Glcp-(1fi4)]-l-a-d-Hepp-(1fi5)- a-Kdop of H. influenzae LPS. The ESI-MS spectrum of LPS-OH from 1268 revealed abundant molecular peaks corresponding to triply and quadruply deprotonated ions (Table 1). The MS data indicated the presence of heterogeneous mixtures of glycoforms, consistent with each molecular species con- taining the conserved PEtn substituted triheptosyl inner-core moiety attached via a phosphorylated Kdo linked to the O-deacylated lipid A (lipid A-OH). As a characteristic feature, populations of glycoforms were observed that differed by 123 Da (i.e. a PEtn group), consistent with either phosphate or PPEtn substitution at the O-4 position of the Kdo residue [23–25]. For clar- ity, glycoforms containing five Hex with no N-acetyl- hexosamine (HexNAc) residue are referred to as Hex5 glycoforms. Glycoforms containing five Hex including a HexNAc residue are referred to as HexNAcHex4 glyco- forms. In the ESI-MS spectrum (negative mode) major quadruply charged ions were observed at m ⁄ z 609.4 and 640.2 corresponding to glycoforms with respective com- positions PChoÆHex 2 ÆHep 3 ÆPEtnÆPÆKdoÆlipid A-OH and PChoÆHex 2 ÆHep 3 ÆPEtn 2 ÆPÆKdoÆlipid A-OH. Ions corresponding to HexNAc containing glycoforms with respective compositions PChoÆHexNAcÆHex 4 ÆHep 3 Æ PEtnÆPÆKdoÆlipid A-OH and PChoÆHexNAcÆHex 4 ÆHep 3 Æ PEtn 2 ÆPÆKdoÆlipid A-OH, and PChoÆHexNAcÆHex 5 Æ Hep 3 ÆPEtnÆPÆKdoÆlipid A-OH and PChoÆHexNAcÆHex 5 Æ Hep 3 ÆPEtn 2 ÆPÆKdoÆlipid A-OH were detected at m ⁄ z 741.3 ⁄ 772.1 and 781.8 ⁄ 812.8. Furthermore, glycoforms with compositions PChoÆHex 5 ÆHep 3 ÆPEtnÆPÆKdoÆlipid A-OH and PChoÆHex 5 ÆHep 3 ÆPEtn 2 ÆPÆKdoÆlipid A-OH were indicated at m ⁄ z 731.1 and 761.7, respectively. Peaks of low intensity corresponding to a minor Hex- NAcHex4 glycoform without a PCho substituent were also identified. The ESI-MS spectrum of LPS-OH from strain 1200 showed the same ions as 1268 except for those corresponding to HexNAcHex4 glycoforms with- out a PCho substituent (Table 1). ESI-MS data of LPS-OH from 1268lpsA showed less heterogeneity with no indications of Hex5 or HexNAc- Hex5 glycoforms. Ions corresponding to the HexNAc- Hex4 glycoforms lacking PCho were moderately higher in abundance in 1268lpsA than in 1268 (Table 1). Ions corresponding to sialylated glycoforms were not unambiguously identified in the full ESI-MS spec- tra of LPS-OH samples due to extensive overlap with those corresponding to major, nonsialylated glyco- forms, and ⁄ or low abundance. However, their presence was confirmed for LPS-OH of 1268lpsA in precursor ion monitoring tandem mass spectrometry experiments by scanning for loss of m ⁄ z 290 (Neu5Ac, negative ion mode) or m ⁄ z 274 (Neu5Ac-H 2 O, positive mode) fol- lowing capillary electrophoresis (CE)-ESI-MS ⁄ MS. The data are shown in Fig. 1 and summarized in Table S3. In the precursor negative-mode ion-scan spectrum (Fig. 1A) quadruply and triply charged ions corresponding to a complex mixture of sialylated gly- coforms containing three to six hexose residues were observed. The major ion at m ⁄ z 909.5 corresponded to a Hex3 glycoform with the composition Neu5AcÆ Hex 3 Æ Hep 3 ÆPEtnÆPÆKdoÆlipid A-OH. Particularly noteworthy are HexNAc-containing glycoforms detected at m ⁄ z 1086.0, 1127.5, 1180.5, 1207.0 and 1249.5 having the respective compositions, PChoÆNeu5AcÆHexNAcÆHex 4 Æ Hep 3 ÆPEtnÆPÆKdoÆlipid A-OH, PChoÆNeu5AcÆHexNAcÆ Hex 4 ÆHep 3 ÆPEtn 2 ÆPÆKdoÆlipid A-OH, PChoÆNeu5AcÆ HexNAcÆHex 5 ÆHep 3 ÆPEtn 2 ÆPÆKdoÆlipid A-OH, PChoÆ Neu5AcÆHexNAc 2 ÆHex 5 ÆHep 3 ÆPEtnÆ PÆKdoÆ lipid A-OH and PChoÆNeu5AcÆHexNAc 2 ÆHex 5 ÆHep 3 ÆPEtn 2 ÆPÆKdoÆ lipid A-OH. In the precursor ion scan spectrum obtained in the positive mode (Fig. 1B) ions corre- sponding to sialylated Hex3 glycoforms were not observed, whereas ions corresponding to sialylated LPS structure of NTHi strains 1268 and 1200 S. L. Lundstro ¨ m et al. 4888 FEBS Journal 274 (2007) 4886–4903 ª 2007 The Authors Journal compilation ª 2007 FEBS glycoforms containing HexNAc were readily detected confirming their presence. Characterization of oligosaccharides derived from NTHi strains 1268, 1200 and 1268lpsA Mild acid hydrolysis of LPS with dilute aqueous acetic acid afforded insoluble lipid A and core oligosaccharide material (OS), which after purification by gel-filtration chromatography resulted in OS samples from the vari- ous strains. Strains 1268 and 1200 gave leading fractions of higher molecular mass referred to as 1268OS and 1200OS which were investigated in detail. Strain 1268lpsA gave lpsAOS. Sugar analyses (Table S1) performed on 1268OS, lpsAOS and 1200OS were consistent with the data obtained on LPS-OH for both the wild-type and mutant strains, revealing the presence of Glc, Gal, Table 1. Negative ion ESI-MS data and proposed compositions for LPS-OH and OS of strains 1268, 1200 and 1268lpsA. Average mass units were used to calculate molecular mass based on proposed compositions as follows: Hex, 162.14; HexNAc, 203.19; Hep, 192.17; Kdo, 220.18; P, 79.98; PEtn, 123.05; PCho, 165.13; AnKdo-ol, 222.20; Gly, 57.05; Ac, 42.04; lipid A-OH, 953.02. All LPS-OH and OS glycoforms contain Hep 3 ÆPEtnÆPÆKdoÆlipid A-OH or Hep 3 ÆPEtnÆAnKdo-ol, respectively. nr, not rationalized. Sample Observed ions (m ⁄ z) Molecular mass (Da) Relative Abundance (%) Proposed composition(M-4H) 4– (M-3H) 3– (M-2H) 2– Obs Calc 1268 1268lpsA 1200 LPS-OH 609.4 812.8 2441.5 2442.2 17 36 16 PChoÆHex 2 640.2 854.1 2565.1 2565.2 30 45 34 PChoÆHex 2 ÆPEtn 680.9 2727.6 2727.3 6 PChoÆHex 3 ÆPEtn 700.1 933.7 2804.3 2804.5 2 5 HexNAcÆHex 4 731.1 974.6 2927.6 2927.5 2 3 HexNAcÆHex 4 ÆPEtn 721.3 2889.2 2889.5 7 PChoÆHex 4 ÆPEtn 731.1 975.2 2928.5 2928.6 3 4 PChoÆHex 5 741.3 988.8 2969.3 2969.6 5 5 PChoÆHexNAcÆHex 4 761.7 1015.9 3050.8 3051.6 12 8 PChoÆHex 5 ÆPEtn 772.1 1029.6 3092.1 3092.7 12 6 7 PChoÆHexNAcÆHex 4 ÆPEtn 781.8 1042.7 3131.2 3131.8 6 2 PChoÆHexNAcÆHex 5 812.8 1083.9 3255.0 3254.8 11 16 PChoÆHexNAcÆHex 5 ÆPEtn OS 540.6 1083.2 1083.9 2 Hex 621.6 1245.2 1246.0 1 Hex 2 682.7 1367.4 – 13 nr 704.3 1410.6 1411.1 4 71 1 PChoÆHex 2 725.2 1452.4 1453.1 5 PChoÆAcÆHex 2 732.9 1467.8 1468.1 7 2 1 PChoÆGlyÆHex 2 746.3 1494.6 1495.2 4 PChoÆAc 2 ÆHex 2 754.2 1510.4 1510.2 1 PChoÆGlyÆAcÆHex 2 774.6 1551.2 1552.2 1 PChoÆGlyÆAc 2 ÆHex 2 783.8 1569.6 1570.3 3 Hex 4 785.6 1573.2 1573.2 3 PChoÆHex 3 806.9 1615.8 1615.3 2 PChoÆAcÆHex 3 866.5 1735.0 1735.4 4 1 2 PChoÆHex 4 885.5 1773.0 1773.5 3 HexNAcÆHex 4 887.5 1777.0 1777.4 4 PChoÆAcÆHex 4 895.0 1792.0 1792.4 1 PChoÆGlyÆHex 4 947.6 1897.2 1897.5 12 4 PChoÆHex 5 645.2 968.1 1938.3 1938.6 22 5 8 PChoÆHexNAcÆHex 4 968.3 1938.6 1939.5 2 PChoÆAcÆHex 5 976.0 1954.0 1954.6 2 5 PChoÆGlyÆHex 5 988.9 1979.8 1980.6 9 PChoÆAcÆHexNAcÆHex 4 988.9 1979.8 1981.6 2 PChoÆAc 2 ÆHex 5 664.5 996.3 1995.6 1995.6 8 6 PChoÆGlyÆHexNAcÆHex 4 1009.6 2021.2 2022.6 4 PChoÆAc 2 ÆHexNAcÆHex 4 1017.2 2036.4 2037.7 9 PChoÆGlyÆAcÆHexNAcÆHex 4 699.3 1049.2 2100.7 2100.7 33 12 PChoÆHexNAcÆHex 5 1069.9 2141.8 2142.7 15 PChoÆAcÆHexNAcÆHex 5 718.3 1077.5 2157.5 2157.8 7 PChoÆGlyÆHexNAcÆHex 5 S. L. Lundstro ¨ m et al. LPS structure of NTHi strains 1268 and 1200 FEBS Journal 274 (2007) 4886–4903 ª 2007 The Authors Journal compilation ª 2007 FEBS 4889 Hep, GalN and GlcN. The considerable decrease in GlcN in the OS samples confirmed this sugar to be part of lipid A but also indicated traces of glycoforms that contain GlcN. 1268OS, lpsAOS and 1200OS were dephosphorylated with 48% hydrogen fluoride prior to methylation anal- ysis. Methylation analysis (Table S2) on the resulting material from 1268OS showed terminal Glc, terminal Gal, 4-substituted Gal, 4-substituted Glc, 3-substituted Gal, terminal Hep, 2-substituted Hep, 3,4-substituted Hep, terminal GalN, 2,3-substituted Hep and 4-substi- tuted GlcN. Methylation analysis performed on nonde- phosphorylated 1268OS revealed the same sugars but with a decrease in terminal Glc, 4-substituted Glc and 2,3-substituted Hep, which indicated phosphorylation on those sugars (data not shown). Methylation analy- sis on dephosphorylated lpsAOS gave the same sugar derivatives as 1268OS but showing a significant increase in terminal Hep and the absence of 2-substi- tuted Hep. Moreover, a decrease of 4-substituted Gal, 4-substituted Glc and 3-substituted Gal was observed in the methylation analysis of this OS sample. Methyl- ation analysis data for 1200OS was comparable with the data obtained for 1268OS (Table S2). ESI-MS on OS samples (Table 1) indicated all strains to be glycylated. In addition, OS samples from NTHi 1200 showed ions corresponding to acetylated glycoforms. ESI-MS on 1268OS and 1200OS revealed major HexNAcHex4 and HexNAcHex5 glycoforms. Lower molecular mass glycoforms were minor, in agreement with OS samples being leading fractions after GPC. Glycoforms, of which the O-glycylated ones were of minor abundance, were evidenced as doubly negatively charged ions as follows: PChoÆ Hex 2 ÆHep 3 ÆPEtnÆAnKdo-ol and PChoÆGlyÆHex 2 ÆHep 3 Æ PEtnÆAnKdo-ol (m ⁄ z 704.3 ⁄ 732.9), PChoÆHex 5 ÆHep 3 Æ PEtnÆAnKdo-ol and PChoÆGlyÆHex 5 ÆHep 3 ÆPEtnÆ AnKdo-ol (m ⁄ z 947.6 ⁄ 976.0), PChoÆHexNAcÆHex 4 Æ Hep 3 ÆPEtnÆAnKdo-ol and PChoÆGlyÆHexNAcÆHex 4 Æ Hep 3 ÆPEtnÆAnKdo-ol (m ⁄ z 968.1 ⁄ 996.3), and PChoÆ HexNAcÆHex 5 ÆHep 3 ÆPEtnÆAnKdo-ol and PChoÆGlyÆ HexNAcÆHex 5 ÆHep 3 ÆPEtnÆAnKdo-ol (m ⁄ z 1049.2 ⁄ 1077.5). In addition, ions at m ⁄ z 866.5 ⁄ 895.0 indicated the glycoforms PChoÆHex 4 ÆHep 3 ÆPEtnÆAnKdo-ol and PChoÆGlyÆHex 4 ÆHep 3 ÆPEtnÆAnKdo-ol. The glycoforms indicated in lpsAOS were in agreement with those found in the equivalent LPS-OH and showed major Hex2 glycoforms. ESI-MS data of 1200OS revealed the presence of glycoforms substituted by up to two acetate groups. Information on the location of Ac was provided by ESI multiple-step tandem mass spectrometry (MS n )inthe positive-ion mode. The product ion spectrum obtained from the molecular ion at m ⁄ z 1496.4 (composition: PChoÆAc 2 ÆHex 2 ÆHep 3 ÆPEtnÆAnKdo-ol) (Fig. 2A) con- tained, inter alia,theionatm ⁄ z 919.1 resulting from the loss of Hex-HepI-AnKdo-ol. MS 3 performed on this ion revealed a prominent ion at m ⁄ z 643.3 (composition: PChoÆHexÆHepIIÆPEtn) (Fig. 2B) resulting from the loss of a diacetylated heptose subunit indicative of HepIII being substituted with two acetates. These experiments also confirmed that PCho substituted the hexose linked Fig. 1. CE-ESI-MS ⁄ MS spectra of LPS-OH derived from NTHi 1268lpsA. The indicated compositions include the PÆKdoÆlipid A-OH element. (A) Precursor ion spectrum (nega- tive mode) using m ⁄ z 290 as the fragment ion for identification of sialylated compo- nents in 1268lpsA. (B) Precursor ion spec- trum (positive mode) using m ⁄ z 274 as the fragment ion for identification of sialylated components in 1268lpsA. LPS structure of NTHi strains 1268 and 1200 S. L. Lundstro ¨ m et al. 4890 FEBS Journal 274 (2007) 4886–4903 ª 2007 The Authors Journal compilation ª 2007 FEBS to HepII and that PEtn substituted HepII. MS 3 experi- ments on ions corresponding to monoacetylated glycoforms revealed the same substitution pattern (data not shown). Sequence analysis on dephosphorylated and permethylated oligosaccharide samples using ESI-MS n Sequence and branching details of the various glyco- forms in 1268OS, lpsAOS and 1200OS were obtained using ESI-MS n in the positive mode on dephosphoryl- ated and permethylated material [13,26]. Because of the increased MS response obtained by permethylation in combination with added sodium acetate, several gly- coforms were observed in the MS spectra that were not detected in underivatized samples. Thus, the ESI- MS mass spectrum of 1268OS (positive mode) (Fig. 3A) showed sodiated singly charged adduct ions ([M+Na] + ) corresponding to the glycoforms Hex 2 Æ Hep 3 ÆAnKdo-ol, Hex 3 ÆHep 3 ÆAnKdo-ol, Hex 4 ÆHep 3 Æ AnKdo-ol and Hex 5 ÆHep 3 ÆAnKdo-ol (m ⁄ z 1467.9, 1672.4, 1875.8 and 2080.1), HexNAcÆ Hex 4 ÆHep 3 Æ AnKdo-ol and HexNAcÆHex 5 ÆHep 3 ÆAnKdo-ol (m ⁄ z 2120.8 and 2325.3) and HexNAc 2 ÆHex 4 ÆHep 3 ÆAnKdo-ol (m ⁄ z 2366.7). The HexNAc2Hex4 glycoform was not detected in the underivatized samples due to low abundance. In order to obtain sequence and branching informa- tion, these molecular ions were further fragmented in MS 2 and MS 3 experiments. For most glycoforms the presence of several isomeric compounds was revealed by identifying product ions in MS 2 spectra (Table S4). MS 3 experiments were used when necessary to confirm structures. Two isomeric Hex2 glycoforms were identified in 1268OS by fragmenting the molecular ion m ⁄ z 1467.9. The resulting spectrum revealed ions at m ⁄ z 1206.1 (major) and 1002.0 (minor) corresponding to loss of terminal (t)Hep and tHex-Hep. The ion at m ⁄ z 754.3 corresponded to the fragment tHex-HepI-AnKdo-ol. Thus in the major Hex2 isomer terminal hexoses substituted both HepI and HepII. In the minor Hex2 glycoform both HepI and HepIII were substituted with terminal hexose residues. Performing MS 2 on the ion m ⁄ z 1672.4 and subsequent MS 3 on the resulting Fig. 3. ESI-MS n analysis of permethylated OS of strain 1268. (A) Full-scan spectrum (positive mode) on permethylated dephos- phorylated 1268OS. (B) Product ion spectrum of [M+Na] + m ⁄ z 2120.8 corre- sponding to the HexNAcHex4 glycoform. Proposed key fragments are indicated in the structure. (C) MS 3 of the ion at m ⁄ z 1859.0 from MS 2 of m ⁄ z 2120.8. Proposed key fragments are indicated in the structure. Fig. 2. ESI-MS n analysis of OS derived from NTHi strain 1200. (A) Product ion spectrum of [M+H] + m ⁄ z 1496.4 corresponding to the PChoÆAc 2 ÆHex 2 ÆHep 3 ÆPEtnÆAnKdo-ol glycoform. The proposed fragmentation is shown beside the spectrum. (B) MS 3 on fragment ion m ⁄ z 919.1, corresponding to the loss of Hex-HepI-AnKdo-ol. The proposed fragmentation is shown beside the spectrum. S. L. Lundstro ¨ m et al. LPS structure of NTHi strains 1268 and 1200 FEBS Journal 274 (2007) 4886–4903 ª 2007 The Authors Journal compilation ª 2007 FEBS 4891 product ions determined two major and two minor Hex3 isomeric glycoforms. The ion corresponding to the loss of tHepIII at m ⁄ z 1409.9 was further frag- mented to give the ion at m ⁄ z 753.5 due to the loss of a tHex-Hex-HepII unit, thus evidencing a structure with one hexose residue substituted to HepI and a disaccharide moiety substituting HepII. Furthermore, in the same MS 3 spectrum a minor ion was detected at m ⁄ z 957.3 corresponding to the loss of tHex-HepII. This ion confirmed the structure of a glycoform in which a disaccharide moiety substitutes HepI and one hexose substitutes HepII. The ion at m ⁄ z 1206.0 corre- sponded to the loss of tHex-HepIII from the molecular ion. It was further fragmented to give the ion at m ⁄ z 753.4 which originated from the loss of a tHex-HepII unit, thus indicating a structure with one hexose resi- due substituting each heptose residue. Finally, a minor structure with one hexose residue linked to HepI and two hexoses linked to HepIII could be determined when the product ion, m ⁄ z 1001.5, corresponding to the loss of tHex-Hex-HepIII, was further fragmented to give the ion at m ⁄ z 753.2 due to the loss of HepII. Four isomeric Hex4 glycoforms were identified by per- forming MS 2 on the parent ion at m ⁄ z 1875.8 and sub- sequent MS 3 on the resulting product ions at m ⁄ z 1614.0, 1206.1 and 1001.7 due to the losses of a termi- nal heptose, a terminal Hex-Hex-Hep residue and a terminal Hex-Hex-Hex-Hep residue, respectively. When the ion at m ⁄ z 1614.0 was further fragmented in aMS 3 experiment it gave product ions at m⁄ z 883.5 and 753.4 due to losses of the epitopes tHex-HepI- AnKdo-ol and tHex-Hex-Hex-HepII, respectively. Fur- thermore, a product ion at m⁄ z 1161.2 indicated the loss of tHex-HepII. Thus two structures with terminal HepIII were identified: the first with one hexose linked to HepI and a trisaccharide group linked to HepII and the second containing elongation of a trisaccharide group substituting HepI and one hexose on HepII. When the ion at m ⁄ z 1206.1 was further fragmented in MS 3 experiments a product ion at m ⁄ z 753.4 was observed defining the loss of tHex-HepII, which indi- cated a major glycoform containing a disaccharide unit on HepIII and one hexose residue on each of HepI and HepII. The product ion at m ⁄ z 1001.7 was further fragmented to give the ion at m ⁄ z 753.3 due to the loss of HepII, revealing a minor glycoform containing a tri- saccharide unit on HepIII and with one hexose on HepI. One isomeric Hex5 glycoform was observed by fragmenting the molecular ion at m ⁄ z 2080.1. The iso- mer was defined by the ions at m ⁄ z 1349.9 and 1206.2 corresponding to the loss of tHex-HepI-AnKdo-ol and tHex-Hex-Hex-HepIII which indicated HepI to be substituted by one hexose and HepIII to be elongated by three hexoses. The structure was confirmed in MS 3 experiments on m ⁄ z 1206.2 where the product ion at m ⁄ z 754.4 indicated the loss of tHex-HepII. The molecular ion at m ⁄ z 2120.8 corresponded to a glycoform with four hexoses and one hexosamine. One single isomer (Fig. 3B,C) was identified by fragmenting the molecular ion. In the resulting spectrum, fragment ions were observed at m ⁄ z 1862.4, 1859.0 and 1390.7 resulting from the loss of tHexNAc, tHep and tHex- HepI-AnKdo-ol, respectively. A MS 3 experiment on m ⁄ z 1859.0, showing the loss of tHex-HepI-AnKdo-ol (m ⁄ z 1129.4) confirmed that this glycoform contained a tHexNAc-Hex-Hex-Hex elongation from HepII and a single hexose substituting HepI. The molecular ion at m ⁄ z 2325.3 corresponded to a HexNAcHex5 glyco- form. When this ion was further fragmented it gave ions at m ⁄ z 2065.5, 1594.8 and 1205.9 resulting from the loss of tHexNAc, tHex-HepI-AnKdo-ol and tHex- NAc-Hex-Hex-Hex-Hep, respectively. The ion at m ⁄ z 1205.9 was further fragmented to give the ion at m ⁄ z 754.1 due to the loss of a tHex-HepII unit, thus evi- dencing a structure with one hexose residue substituted to each of HepI and HepII, and a tetrasaccharide moi- ety with terminal hexosamine substituting HepIII. The molecular ion at m ⁄ z 2366.7 corresponded to a glycoform with the composition HexNAc 2 ÆHex 4 Æ Hep 3 ÆAnKdo-ol. The single isomer of this glycoform was defined in the MS 2 spectrum by the ions at m ⁄ z 2108.0, 2105.0, 1903.5 and 1658.3 corresponding to the loss of tHexNAc, tHep, tHexNAc-Hex and tHexNAc- Hex-HexNAc. MS 3 performed on the ion at m ⁄ z 1658.3 indicated the loss of tHep (m ⁄ z 1396.1) and the fragment ion of -Hex-Hex-HepI-AnKdo-ol (m ⁄ z 944.1). Thus, this glycoform contained a tHexNAc- Hex-HexNAc-Hex-Hex- unit elongating from HepI and with one hexose substituting HepII. ESI-MS n data obtained from lpsAOS clearly indi- cated the absence of glycoforms expressing chain extension from HepIII. The major isoforms observed were otherwise equivalent to those found in the wild- type strain, except for an extra Hex1 glycoform (m ⁄ z 1264.1) containing one hexose substituent on HepI determined from the fragment ion at m ⁄ z 753.2 indi- cating the loss of tHepIII-HepII (Table S4). Strain 1200 contained virtually the same glycoforms as observed in strain 1268 except for those having elongations from HepI. However, traces of three other higher molecular mass forms; HexNAcÆHex 6 ÆHep 3 Æ AnKdo-ol, HexNAcÆHex 7 ÆHep 3 ÆAnKdo-ol and Hex- NAc 2 ÆHex 7 ÆHep 3 ÆAnKdo-ol at m ⁄ z 2528.9, 2732.9 and 2976.9, respectively, were observed and investigated. MS 2 of m ⁄ z 2528.9 gave fragment ions at m ⁄ z 2269.2, 1410.0 and 1799.5 corresponding to losses of LPS structure of NTHi strains 1268 and 1200 S. L. Lundstro ¨ m et al. 4892 FEBS Journal 274 (2007) 4886–4903 ª 2007 The Authors Journal compilation ª 2007 FEBS tHexNAc, tHexNAc-Hex-Hex-Hex-HepIII and tHex- HepI-AnKdo-ol, indicating HepI to be substituted by one hexose, HepII by two hexoses and HepIII by a tHexNAc-Hex-Hex-Hex unit. The same spectrum showed a second glycoform defined by the ion at m ⁄ z 1859.7 indicating that HepIII was elongated with two hexoses. This was confirmed by MS 3 on the ion at m ⁄ z 1799.5 giving the fragment ion at m ⁄ z 1128.9 corresponding to the loss of tHex-Hex-HepIII, thus indicating HepII to be substituted by a tHexNAc-Hex- Hex-Hex- unit. MS 2 of m ⁄ z 2732.9 gave fragment ions at m ⁄ z 2474.4, 1613.7 and 2002.9 corresponding to the losses of tHexNAc, tHexNAc-Hex-Hex-Hex-HepIII and tHex-HepI-AnKdo-ol, respectively, revealing HepI to be substituted by one hexose, HepII by three hexoses and HepIII by the tHexNAc-Hex-Hex-Hex unit. MS 2 of m ⁄ z 2976.9 gave the fragment ions m ⁄ z 2718.4 and 1858.5, indicating the loss of tHexNAc and tHexNAc-Hex-Hex-Hex-HepIII. The ion at m ⁄ z 1858.5 was further fragmented which gave the fragment ions at m ⁄ z 1599.7 and 1129.0 corresponding to the losses of tHexNAc and tHex-HepI-AnKdo-ol. This indicated the HexNAc2Hex7 isomer to be substituted by one hexose at HepI and tHexNAc-Hex-Hex-Hex units substituting both HepII and HepIII. Characterization of lpsAOS, 1268OS and 1200OS by NMR Major structures were elucidated by detailed 1 H, 13 C and 31 P NMR analyses. 1 H and 13 C NMR resonances were assigned using gradient chemical shift correlation techniques (COSY, TOCSY and HMQC experiments). The chemical shift data corresponding to 1268OS, lpsAOS and 1200OS are given in Table 2. Prior to NMR analyses the samples were treated with 1 m NH 3 to remove O-acyl groups. Subspectra corresponding to the individual glycosyl residues were identified on the basis of spin-connectivity pathways delineated in the 1 H chemical shift correlation maps, the chemical shift values, and the vicinal coupling constants. The mono- saccharide sequences of the major glycoforms were confirmed from transglycosidic NOE connectivities between anomeric and aglyconic protons on adjacent residues (Table S5). The chemical shift data are consis- tent with each sugar residue being present in the pyr- anosyl ring form. Further evidence for this conclusion was obtained from NOE data which also served to confirm the anomeric configurations of the linkages and the monosaccharide sequence. NOESY spectra of 1268OS, lpsAOS and 1200OS revealed inter-residue NOE connectivities between the anomeric protons of HepIII to HepII H-1 ⁄ H-2, HepII to HepI H-3, HepI to Kdo H-5 ⁄ H-7 and GlcIV to HepI H-4 ⁄ H-6, which confirmed the sequence of the conserved triheptosyl inner core unit. Several signals for methylene protons of An Kdo-ol were observed in the COSY and TOCSY spectra in the region d 1.87–2.18. This is due to the fact that several anhydro-forms of Kdo are formed during the hydrolysis by elimination of phosphate or pyrophosphoethanolamine from the C-4 position [27]. 1 H– 31 P correlation experiments indicated PEtn (d P 0.01) to be linked to O-6 of HepII. Structure of the Hex2, Hex4 and HexNAcHex4 glycoforms in lpsAOS Sequence analysis of lpsAOS by ESI-MS n revealed a predominant Hex2 glycoform having a triheptosyl inner-core from which chain elongation by hexoses only appeared from HepI and HepII (Table S4). In addition, glycoforms having further extensions from HepII by HexNAc-Hex-Hex-Hex or truncated versions thereof were detected. In the 1 H NMR spectrum of lpsAOS, anomeric resonances corresponding to the triheptosyl moiety (HepI–HepIII) were identified at d 5.05–5.16, 5.83 and 5.03, respectively. Subspectra cor- responding to the hexose residues were identified in the 2D COSY and TOCSY (Fig. 4A) spectra at d 5.28 (Glc residue V), 4.97 (Gal residue VII), 4.92 (Gal resi- due VII), 4.66 (GalNAc residue VIII), 4.57 ⁄ 4.64 (Gal residue VI) and 4.54 (Glc residue IV), respectively. The chemical shift data were consistent with VII (d H-1 4.97) and VIII being terminal residues. The terminal and 4-substituted forms of residue V could be distinguished by different H-2 and H-4 shifts (d H-2 3.54 ⁄ 3.59 and d H-4 3.50 ⁄ 3.80), which was also confirmed in COSY and 1 H) 13 C HMQC experiments (d C-4 69.8 ⁄ 76.3). The high H-6 A ⁄ B shifts of V (d 4.11 ⁄ 4.18) indicated this position to be substituted with a PCho subunit, which was confirmed in 1 H– 31 P correlation experiments showing a 31 P resonance at d )0.05 correlating to H-6 A ⁄ B of V and the methylene protons of PCho at d 4.35. The spin systems at d 4.57 and 4.64 could both be assigned to residue VI indicating the anomeric proton of this residue to be sensitive to changes in molecular environment due to the microheterogeneity of the sample. Because the oligosaccharide contains Hex4 glycoforms with and without PCho, we assume that the proton at d 4.57 corresponds to glycoforms substituted by PCho and the one at d 4.64 to those that do not. Inter-residue NOE between the proton pairs of V H-1 ⁄ II H-3 confirmed these residues to be linked to position O-3 in HepII. Inter-residue NOE were observed between the VII H-1 ⁄ VI H-4 confirming a a-d-Galp-(1fi4)-b-d-Galp unit within the extension from HepII. Inter-residue NOE from VI H-1 and S. L. Lundstro ¨ m et al. LPS structure of NTHi strains 1268 and 1200 FEBS Journal 274 (2007) 4886–4903 ª 2007 The Authors Journal compilation ª 2007 FEBS 4893 Table 2. 1 H and 13 C chemical shifts for 1268OS, lpsAOS and 1200OS. Prior to NMR analyses the samples were O-deacylated. Spectra were recorded in D 2 Oat25°C. Chemical shift values compared between the three strains could vary by up to ± 0.01 p.p.m. Signals originating from the Hex5 and HexNAcHex5 glycoforms were not observed in the lpsA mutant. Signals corresponding to PCho methyl protons and car- bons occurred at d 3.23 and 54.7, respectively. Pairs of deoxy protons of reduced AnKdo-ol were identified in COSY and TOCSY spectra at d 1.87–2.18. Signals corresponding to GalNAc methyl 1 H and 13 C occurred at d 2.05 and 23.02, respectively. a Observed from intense NOE signals. b –, not determined. c Observed in TOCSY of strain 1268 only. d Observed as intra-residue NOE from H-1 of d 4.64 only. e An extra terminal b-hexose was observed in strain 1200 and 1268 (weak) at d H-1,C-1 4.46, 102.7; d H-2,C-2 3.54,72.9; d H-3,C-3 3.69,72.7; d H-4 3.54 and d H-5 3.74, respectively. Also, intra-residue NOE signals from the anomeric proton to H-3 and H-5 were observed. No inter-residue NOE connections could be detected. LPS structure of NTHi strains 1268 and 1200 S. L. Lundstro ¨ m et al. 4894 FEBS Journal 274 (2007) 4886–4903 ª 2007 The Authors Journal compilation ª 2007 FEBS V H-3 were not observed, probably due to low abun- dance of the corresponding glycoforms. However, NMR data combined with data from methylation and tandem MS analysis corroborate the sequence of the extending glycose unit from HepII as [b-d-GalNAcp- (1fi3)-a-d-Galp-(1fi4)-b-d-Galp-(1fi4)-a-d-Glcp-(1fi]. The HexNAcHex4 glycoform in lpsAOS is shown in Fig. 5. Also indicated are the truncated Hex4 and Hex2 glycoforms. Structure of the Hex5 and HexNAcHex5 glycoforms in 1268OS and 1200OS Sequence analysis of 1268OS by ESI-MS n revealed in addition to HexNAcHex4 glycoforms, abundant Hex- NAcHex5 glycoforms having the structure observed for lpsAOS and also those with chain elongation from HepIII (Table S4). In the 1 H NMR spectrum of 1268OS (Fig. S1A), anomeric resonances correspond- ing to the triheptosyl moiety (HepI–HepIII) were iden- tified at d 5.05–5.16, 5.71 and 5.13, respectively. Spin systems corresponding to the hexose residues were identified in the COSY and TOCSY spectra. The occurrence of inter-residue NOESY connectivities between protons on contiguous residues in 1268OS confirmed an identical structural element as shown in Fig. 5. In addition, in the COSY and TOCSY spectra of 1268OS anomeric signals at d 4.43 and 4.52 could be attributed to fi4)-b-d-Glcp (IX) and fi4)-b-d-Galp (X) residues, respectively. Additional spin systems cor- responding to terminal GalNAc and Gal residues indi- cated by methylation analysis were not observed. It was reasonable to assume that these overlapped with the resonances of the corresponding sugars extending from HepII. Thus resonances at d 4.92 and 4.66 were assigned to correspond to residues XI and XII, respec- tively. Inter-residue NOE between X H-1 ⁄ IX H-4 and IX H-1 ⁄ III H-1,2 (Fig. 6A) gave evidence for the fi4)-b-d-Gal p-(1fi4)-b-d-Glcp-(1fi2)-l-a-d-HepIIIp-(1fi unit. Because inter-residue NOE between XII H-1 ⁄ XI H-3 and XI H-1 ⁄ X H-4 was observed we propose that a globotetraose unit is the full extension from HepIII in 1268OS. The HexNAcHex5 glycoform in 1268OS and 1200OS is shown in Fig. 7 as well as the truncated Hex5 glycoform. Fig. 4. Selected region of phase sensitive TOCSY spectra (mixing time 180 ms) of (A) lpsAOS and (B) 1200OS. Cross-peaks of impor- tance are labelled. See Table 2 for an explanation of the roman numerals. (A) Signals corresponding to structures with full exten- sion from HepII (Fig. 5) are indicated. (B) Signals corresponding to structures with full extension from HepIII (Fig. 7) are indicated. IV β- D-Glcp-(1→4)- L -α-D -HepIp-(1→5)-AnKdo-ol PCho 3 Hex4 Hex2 ↓↑ 6 1 β- D -GalNAc p-(1→3)- α - D-Galp-(1→4)-β- D -Galp -(1 → 4 )- α - D -Glc p-(1→3)- L -α - D -HepII p 6← PEtn 2 VIII VII VI V ↑ 1 L -α-D-HepIIIp Fig. 5. Structure proposed for the HexNAcHex4 glycoform in lpsAOS. Also indicated are the truncated Hex4 and Hex2 glycoforms. S. L. Lundstro ¨ m et al. LPS structure of NTHi strains 1268 and 1200 FEBS Journal 274 (2007) 4886–4903 ª 2007 The Authors Journal compilation ª 2007 FEBS 4895 [...]... strains has been shown to be associated with increased virulence in an in vivo model of H in uenzae infection [33] PCho substitution is a common feature of H in uenzae LPS that contributes to the ability of NTHi to colonize and persist within the human respiratory tract, at least in part by mediating bacterial adherence to and invasion of the host epithelia [34–36] A majority of H in uenzae strains, including... (1987) Nontypable Haemophilus in uenzae: a review of clinical aspects, surface antigens, and the human immune response to infection Rev Infect Dis 9, 1–15 3 Anderson P, Johnston RB Jr & Smith DH (1972) Human serum activities against Haemophilus in uenzae, type b J Clin Invest 51, 31–38 4 Zwahlen A, Rubin LG & Moxon ER (1986) Contribution of lipopolysaccharide to pathogenicity of Haemophilus in uenzae: comparative... Structural diversity in lipopolysaccharide expression in nontypeable Haemophilus in uenzae Identification of L-glycero-D-manno-heptose in the outer-core region in three clinical isolates Eur J Biochem 270, 610– 624 Schweda EK, Li J, Moxon ER & Richards JC (2002) Structural analysis of lipopolysaccharide oligosaccharide epitopes expressed by non-typeable Haemophilus in uenzae strain 176 Carbohydr Res... Glycine is a common substituent of the inner core in Haemophilus in uenzae lipopolysaccharide Glycobiology 11, 1009–1015 Helander IM, Lindner B, Brade H, Altmann K, Lindberg AA, Rietschel ET & Zahringer U (1988) Chemical ¨ structure of the lipopolysaccharide of Haemophilus in uenzae strain I-69 Rd- ⁄ b+ Description of a novel deeprough chemotype Eur J Biochem 177, 483–492 Mikhail I, Yildirim HH, Lindahl... Digalactoside expression in the lipopolysaccharide of Haemophilus in uenzae and its role in intravascular survival Infect Immun 73, 7022–7026 34 Weiser JN, Pan N, McGowan KL, Musher D, Martin A & Richards J (1998) Phosphorylcholine on the lipopolysaccharide of Haemophilus in uenzae contributes to persistence in the respiratory tract and sensitivity to serum killing mediated by C-reactive protein J Exp Med 187,... Structural profiling of lipopolysaccharide glycoforms expressed by non-typeable Haemophilus in uenzae: phenotypic similarities between NTHi strain 162 and the genome strain Rd Carbohydr Res 338, 2731–2744 Yildirim HH, Li J, Richards JC, Hood DW, Moxon ER & Schweda EK (2005) An alternate pattern for globoside oligosaccharide expression in Haemophilus in uenzae lipopolysaccharide: structural diversity in nontypeable. .. Schweda EK (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 ˚ Mansson M, Hood DW, Li J, Richards JC, Moxon ER & Schweda EK (2002) Structural analysis of the lipopolysaccharide from nontypeable Haemophilus in uenzae strain 1003 Eur J Biochem 269, 808–818 ˚ Mansson... addition of glucose or galactose to the terminal inner core heptose of Haemophilus in uenzae lipopolysaccharide via alternative linkages J Biol Chem 281, 29455–29467 30 Masoud H, Martin A, Thibault P, Moxon ER & Richards JC (2003) Structure of extended lipopolysaccharide glycoforms containing two globotriose units in Haemophilus in uenzae serotype b strain RM7004 Biochemistry 42, 4463–4475 31 Cox AD,... thus strains 1200 and 1268 are representative of this LPS structural motif in H in uenzae One glycoform containing full extension from both HepII and HepIII was identified in minor amounts in strain 1200 during permethylation analysis The proposed structure of this form is seen in Fig 8 From the structural detail obtained we can make some observations on the inclusion of epitopes in the LPS of strains 1200... Haemophilus in uenzae adhere to and invade human bronchial epithelial cells via an interaction of lipooligosaccharide with the PAF receptor Mol Microbiol 37, 13–27 36 Lysenko E, Richards JC, Cox AD, Stewart A, Martin A, Kapoor M & Weiser JN (2000) The position of phosphorylcholine on the lipopolysaccharide of Haemophilus in uenzae affects binding and sensitivity to C-reactive protein-mediated killing . strains, including NTHi strains and strain Rd, have been shown to carry PCho at O-6 of GlcI [13,23], in other strains, including H. in uenzae type b strains,. Novel globoside-like oligosaccharide expression patterns in nontypeable Haemophilus in uenzae lipopolysaccharide Susanna L.