| 1. |
- Mikhail, Ivan, et al.
(författare)
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Structural characterization of lipid A from nontypeable and type f Haemophilus influenzae : Variability of fatty acid substitution
- 2005
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 340:2, s. 303-316
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Tidskriftsartikel (refereegranskat)abstract
- Lipid A isolated by mild acid hydrolysis from lipopolysaccharides of 22 nontypeable and 2 type f Haemophilus influenzae strains was investigated using electrospray ionization coupled to quadrupole ion trap mass spectrometry. The lengths, positions, and number of acyl chains in the lipid A molecule were determined using multiple-step tandem mass spectrometry (MSn). All of the analyzed strains showed a major lipid A molecule comprising beta-2-amino-2-deoxy-D-glucopyranose-(1 -> 6)-alpha-2-amino-2-deoxy-D-glucopyranose phosphorylated at the C4 ' and C1 positions. The C2/C2 ' and C3/C3 ' positions were substituted by amide-linked and esterlinked 3-hydroxytetradecanoic acid chains, respectively. The fatty acid chains oil C3 ' and C2 ' were further esterified by tetradecanoic acid chains. In all strains, minor amounts of lipid A molecules with different acylation patterns were identified. Thus, structures comprising the hexaacylated lipid A with the C2 or C3 position being Substituted by 3-hydroxydecanoic acid, and hexaacylated lipid A with the C3 and C3 ' positions being substituted by 3-hydroxydodecanoic or dodecanoyloxytetradecanoic acid, respectively, were found. In addition, lipid A with an acetyl group attached to the 3-hydroxytetradecanoic acid groups attached to the C2 or C3 position was detected in two nontypeable H. influenzae strains.
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| 2. |
- Engskog, Mikael K. R., et al.
(författare)
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A dual role for the lex2 locus : identification of galactosyltransferase activity in non-typeable Haemophilus influenzae strains 1124 and 2019
- 2009
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Ingår i: Carbohydrate Research. - : Elsevier BV. - 0008-6215 .- 1873-426X. ; 344:5, s. 632-641
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Tidskriftsartikel (refereegranskat)abstract
- Lipopolysaccharide (LPS) of Haemophilus influenzae comprises a conserved tri-L-glycero-D-manno-heptosyl inner-core moiety (L-alpha-D-Hepp-(1 -> 2)-[PEtn -> 6]-L-alpha-D-Hepp-(1 -> 3)-[beta-D-Glclp-(1 -> 4)]-L-alpha-D-Hepp-(1 -> 5)-alpha-Kdop) to which addition of beta-D-Glcp to O-4 of Glcl in serotype b strains is controlled by the gene lex2B. In non-typeable H. influenzae strains 1124 and 2019, however, a beta-D-Galp is linked to O-4 of Glcl. In order to test the hypothesis that the 1ex2 locus is involved in the expression Of beta-D-Galp-(1 -> 4-beta-D-Glcp-(1 -> - from Hepl, 1ex2B was inactivated in strains 1124 and 2019, and LPS glycoform populations from the resulting mutant strains were investigated. Detailed structural analyses using NMR techniques and electrospray-ionisation mass spectrometry (ESIMS) on O-cleacylated LPS and core oligosaccharide material (OS), as well as ESIMS" on permethylated dephosphorylated OS, indicated both lex2B mutant strains to express only beta-D-Glcp extensions from Hepl. This provides strong evidence that Lex2B functions as a galactosyltransferase adding a beta-D-Galp to O-4 of Glcl in these strains, indicating that allelic polymorphisms in the lex2B sequence direct alternative functions of the gene product.
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| 3. |
- Fox, K L, et al.
(författare)
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Novel lipopolysaccharide biosynthetic genes containing tetranucleotide repeats in Haemophilus influenzae, identification of a gene for adding O-acetyl groups
- 2005
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 58:1, s. 207-216
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Tidskriftsartikel (refereegranskat)abstract
- Many of the genes for lipopolysaccharide (LPS) biosynthesis in Haemophilus influenzae are phase variable. The mechanism of this variable expression involves slippage of tetranucleotide repeats located within the reading frame of these genes. Based on this, we hypothesized that tetranucleotide repeat sequences might be used to identify as yet unrecognized LPS biosynthetic genes. Synthetic oligonucleotides (20 bases), representing all previously reported LPS-related tetranucleotide repeat sequences in H. influenzae, were used to probe a collection of 25 genetically and epidemiologically diverse strains of non-typeable H. influenzae. A novel gene identified through this strategy was a homologue of oafA, a putative O-antigen LPS acetylase of Salmonella typhimurium, that was present in all 25 non-typeable H. influenzae, 19 of which contained multiple copies of the tetranucleotide 5'-GCAA. Using lacZ fusions, we showed that these tetranucleotide repeats could mediate phase variation of this gene. Structural analysis of LPS showed that a major site of acetylation was the distal heptose (HepIII) of the LPS inner-core. An oafA deletion mutant showed absence of O-acetylation of HepIII. When compared with wild type, oafA mutants displayed increased susceptibility to complement-mediated killing by human serum, evidence that O-acetylation of LPS facilitates resistance to host immune clearance mechanisms. These results provide genetic and structural evidence that H. influenzae oafA is required for phase variable O-acetylation of LPS and functional evidence to support the role of O-acetylation of LPS in pathogenesis.
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| 4. |
- Tinnert, A S, et al.
(författare)
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Structural investigation of lipopolysaccharides from nontypeable Haemophilus influenzae : investigation of inner-core phosphoethanolamine addition in NTHi strain 981
- 2005
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Ingår i: Carbohydrate Research. - : Elsevier BV. - 0008-6215 .- 1873-426X. ; 340:11, s. 1900-1907
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Tidskriftsartikel (refereegranskat)abstract
- LPS of NTHi comprises a conserved tri-L-glycero-D-manno-heptosyl inner-core moiety (L-alpha-D-Hepp-(1 -> 2)-[PEtn -> 6]-L-alpha-D-Hepp-(1 -> 3)-[beta-D-Glcp-(1 -> 4)]-L-alpha-D-Hepp-(1 -> 5)-alpha-Kdop) in which addition of PEtn to the central heptose (HepII) in strain Rd is controlled by the gene lpt6. It was recently shown that NTHi strain 981 contains an additional PEtn linked to O-3 of the terminal heptose of the inner-core moiety (HepIII). In order to establish whether lpt6 is also involved in adding PEtn to HepIII, lpt6 in strain 981 was inactivated. The structure of the LPS of the resulting mutant strain 981lpt6 was investigated by MS and NMR techniques by which it was confirmed that the lpt6 gene product is responsible for addition of PEtn to O-6 of HepII in strain 981. However, it is not responsible for adding PEtn to O-3 of HepIII since the 981lpt6 mutant still had full substitution with PEtn at HepIII
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| 5. |
- Yildirim, Håkan H., et al.
(författare)
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An alternate pattern for globoside oligosaccharide expression in Haemophilus influenzae lipopolysaccharide : Structural diversity in nontypeable strain 1124
- 2005
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 44:13, s. 5207-5224
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Tidskriftsartikel (refereegranskat)abstract
- Common structural motifs of Haemophilus influenzae lipopolysaccharide (LPS) are globotetraose [beta-D-GalpNAc-(1 -> 3)-alpha-D-Galp-(1 -> 4)-beta-D-Galp-(1 -> 4)-beta-D-Glcp] and its truncated versions globoside [alpha-D-Galp-(1 -> 4)-beta-D-Galp-(1 -> 4)-beta-D-Glcp] and lactose [beta-D-Galp-(1 -> 4)-beta-D-Glcp] linked to the tenninal heptose (HepIII) of the triheptosyl inner-core moiety L-alpha-D-Hepp-(1 -> 2)-[PEA -> 6]-L-alpha-D-Hepp-(1 -> 3)L-alpha-D-Hepp-(1 -> 5)-[PPEA -> 4]-alpha-Kdo-(2 -> 6)-lipid A. We report here structural studies of LPS from nontypeable H. influenzae strain 1124 expressing these motifs linked to both the proximal heptose (HepI) and HepIII at the same time. This novel finding was obtained by structural studies of LPS using NMR techniques and electrospray ionization mass spectrometry (ESI-MS) on O-deacylated LPS and core oligosaccharide material (OS) as well as ESI-MSn on permethylated dephosphorylated OS. The use of defined mutants allowed us to confirm structures unambiguously and understand better the biosynthesis of each of the globotetraose units. We found that lgtC is involved in the expression of beta-D-Galp-(1 -> 4)-beta-D-Galp in both extensions, whereas lic2A directs only the expression Of beta-D-Ga1p-(1 -> 4)-beta-D-Glcp when linked to HepIII. The LPS of NTHi strain 1124 contained sialylated glycoforms that were identified by CE-ESI-MS/MS. A common sialylated structure in H. influenzae LPS is sialyllactose linked to HepIII. This structure exists in strain 1124. However, results for the lpsA mutant indicate that sialyllactose extends from HepI as well, a molecular environment for sialyllactose in H. influenzae that has not been reported previously. In addition, the LPS was found to carry phosphoryleholine, O-linked glycine, and a third PEA group which was linked to O3 of HepIII.
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| 6. |
- Yildirim, Håkan H, et al.
(författare)
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Complex O-acetylation in non-typeable Haemophilus influenzae lipopolysaccharide : evidence for a novel site of O-acetylation
- 2005
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Ingår i: Carbohydrate Research. - : Elsevier BV. - 0008-6215 .- 1873-426X. ; 340:17, s. 2598-2611
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Tidskriftsartikel (refereegranskat)abstract
- The structure of the lipopolysaccharide (LPS) of non-typeable Haemophilus influenzae strain 723 has been elucidated using NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS) on O-deacylated LPS and core oligosaccharide material (OS), as well as ESI-MSn on permethylated dephosphorylated OS. It was found that the LPS contains the common structural element of H. influenzae, L-alpha-D-Hepp-(1 -> 2)-[PEtn -> 6]-L-alpha-D-Hepp-(1 -> 3)-[beta-D-Glcp-(1 -> 4)]-L-alpha-D-Hepp-(1 -> 5)-[PPEtn -> 4]-alpha-Kdo-(2 -> 6)-Lipid A, in which the beta-D-Glcp residue (GlcI) is substituted by phosphocholine at O-6 and the distal heptose residue (HepIII) by PEW at O-3, respectively. In a subpopulation of glycoforms O-2 of HepIII was substituted by beta-D-Galp-(1 -> 4)-beta-D-Glcp-(1 -> or beta-D-Glcp-(1 ->. Considerable heterogeneity of the LPS was due to the extent of substitution by O-acetyl groups (Ac) and ester-linked glycine of the core oligosaccharide. The location for glycine was found to be at Kdo. Prominent acetylation sites were found to be at GlcI, HepIII, and the proximal heptose (HepI) residue of the triheptosyl moiety. Moreover, GlcI was acetylated at O-3 and/or O-4 and HepI was acetylated at O-2 as evidenced by capillary electrophoresis ESI-MS" in combination with NMR analyses. This is the first study to show that an acetyl group can substitute HepI of the inner-core region of H. influenzae LPS.
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| 7. |
- Yildirim, Håkan H., et al.
(författare)
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Structural analysis of lipopolysaccharides from Haemophilus influenzae serotype f - Structural diversity observed in three strains
- 2003
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 270:15, s. 3153-3167
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Tidskriftsartikel (refereegranskat)abstract
- Structural elucidation of the lipopolysaccharide (LPS) from three serotype f Haemophilus influenzae clinical isolates RM6255, RM7290 and RM6252 has been achieved using NMR spectroscopy techniques and ESI-MS on O-deacylated LPS and core oligosaccharide material (OS) as well as ESI-MSn on permethylated dephosphorylated OS. This is the first study to report structural details on LPS from serotype f strains. We found that the LPSs of all strains were highly heterogeneous mixtures of glycoforms expressing the common H. influenzae structural element l-alpha-d-Hepp -(1-->2)-[P Etn-->6]-l-alpha-d-Hepp -(1-->3)-[beta-d-Glcp -(1-->4)]-l-alpha-d-Hepp -(1-->5)-[PP Etn-->4]-alpha-Kdo-(2-->6)-lipid A with variable length of OS chains linked to each of the heptoses. The terminal heptose (HepIII) in RM6255 is substituted at the O-3 position by a beta-d-Glcp residue whereas HepIII in strains RM7290 and RM6252 is substituted at O-2 by the globoside unit (alpha-d-Galp -(1-->4)-beta-d-Galp -(1-->4)-beta-d-Glc) or truncated versions thereof. The central heptose (HepII) is substituted by an alpha-d-Galp -(1-->4)-beta-d-Galp -(1-->4)-beta-d-Glcp -(1-->4)-alpha-d-Glcp unit in RM7290 and RM6252 or truncated versions thereof. Strain RM6255 does not express galactose in its LPS and only shows a cellobiose unit elongating from HepII (beta-d-Glcp -(1-->4)-alpha-d-Glcp ). ESI-MSn on dephosphorylated and permethylated OS provided information on the existence of additional minor isomeric glycoforms.
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| 8. |
- Yildirim, Håkan H.
(författare)
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Structural diversity of the lipid A and core oligosaccharide moieties of the lipopolysaccharides from nontypeable and serotype f Haemophilus influenzae
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis describes structural studies of the oligosaccharide and lipid A moieties of lipopolysaccharides (LPSs) isolated from disease-causing Haemophilus influenzae strains. The nontypeable strains were clinical isolates from the middle ear of children suffering from otitis media and the serotype f strains had been collected from three adults with respiratory tract infections. The LPS molecules are situated on the cell wall of H. influenzae strains and they play a very important role in colonization, infection, evasion of host immune system and inflammatory response. Previous studies have implicated the heterogeneous repertoire of LPS structures within a strain and mimicry of human cell wall structures to be involved in the diseasecausing behavior of this organism. Structural analysis of the oligosaccharide moieties with advanced applications of nuclear magnetic resonance (NMR) and various electrospray ionization mass spectrometry (ESI-MS) techniques revealed novel structural features in each of the investigated strains. All of the strains displayed a very complex mixture of LPS structures that differed between and within the pathogens. Moreover, all of the strains had the capacity to express mimics of human glycolipids. The genetic basis for LPS biosynthesis for H. influenzae is established for the strain of which the complete genome has been determined. In this thesis the function of the genes involved in the biosynthesis of LPS was investigated in a nontypeable strain by using the combination of genetic engineering and structural analysis. The synergy of genomics and analytical carbohydrate chemistry led to the identification of novel structural epitopes, and furthermore, enabled us to identify a new function for one of these genes. The most recent structural study of lipid A from H. influenzae was conducted in 1988 on a mutant strain. The results of that study established the presence of only one lipid A structure. in this thesis we investigated lipid A from both nontypeable and serotype wild type strains by performing tandem ESI-MS and the results confirmed earlier findings but also evidenced other lipid A structures previously not associated with H. influenzae. Moreover, all of the strains exhibited a heterogeneous population of lipid A molecules.
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