| 1. |
- Hood, D W, et al.
(author)
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Biosynthesis of cryptic lipopolysaccharide glycoforms in Haemophilus influenzae involves a mechanism similar to that required for O-antigen synthesis
- 2004
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In: Journal of Bacteriology. - 0021-9193 .- 1098-5530. ; 186:21, s. 7429-7439
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Journal article (peer-reviewed)abstract
- It is generally thought that mucosal bacterial pathogens of the genera Haemophilus, Neisseria, and Moraxella elaborate lipopolysaccharide (LPS) that is fundamentally different from that of enteric organisms that express O-specific polysaccharide side chains. Haemophilus influenzae elaborates short-chain LPS that has a role in the pathogenesis of H. influenzae infections. We show that the synthesis of LPS in this organism can no longer be as clearly distinguished from that in other gram-negative bacteria that express an O antigen. We provide evidence that a region of the H. influenzae genome, the hmg locus, is involved in the synthesis of glycoforms in which tetrasaccharide units are added en bloc, not stepwise, to the normal core glycoforms, similar to the biosynthesis of an O-antigen.
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| 2. |
- Landerholm, Malin K, et al.
(author)
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Characterization of novel structural features in the lipopolysaccharide of nondisease associated nontypeable Haemophilus influenzae
- 2004
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In: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 271:5, s. 941-953
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Journal article (peer-reviewed)abstract
- Nontypeable Haemophilus influenzae (NTHi) is a common commensal of the human upper respiratory tract and is associated with otitis media in children. The structures of the oligosaccharide portions of NTHi lipopolysaccharide (LPS) from several otitis media isolates are now well characterized but it is not known whether there are structural differences in LPS from colonizing, nondisease associated strains. Structural analysis of LPS from nondisease associated NTHi strains 11 and 16 has been achieved by the application of high-field NMR techniques, ESI-MS, ESI-MSn, capillary electrophoresis coupled to ESI-MS, composition and linkage analyses on O-deacylated LPS and core oligosaccharide material. This is the first study to report structural details on LPS from strains taken from the nasopharynx from healthy individuals. Both strains express identical structures and contain the common element of H. influenzae LPS, L-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-Kdop-(2-->6)-lipid A, in which each heptose is elongated by a single hexose residue with no further oligosaccharide extensions. In the major Hex3 glycoform, the terminal Hepp residue (HepIII) is substituted at the O-2 position by a beta-D-Galp residue and the central Hepp residue (HepII) is substituted at O-3 by a alpha-D-Glcp residue. Notably, the strains express two phosphocholine (PCho) substituents, one at the O-6 position of alpha-D-Glcp and the other at the O-6 position of beta-D-Galp. Major acetylation sites were identified at O-4 of Gal and O-3 of HepIII. Additionally, both strains express glycine, and strain 11 also expresses detectable amounts of N-acetylneuraminic acid.
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| 3. |
- Li, J J, et al.
(author)
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Electrophoretic and mass spectrometric strategies for profiling bacterial lipopolysaccharides
- 2005
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In: MOLECULAR BIOSYSTEMS. - : Royal Society of Chemistry (RSC). - 1742-206X .- 1742-2051. ; 1:1, s. 46-52
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Journal article (peer-reviewed)abstract
- Capillary electrophoresis (CE) is a high-resolution separation technique that has been widely used for trace analysis in biological samples. On-line capillary electrophoresis-electro spray mass spectrometry (CE-MS) was developed for the analysis of lipopolysaccharide (LPS) glycoforms from the gram-negative bacteria, Haemophilus influenzae. In this paper, we report on the application of CE-MS to characterize structural differences in O-deacylated LPS samples from H. influenzae strains Rd 11.7 and 375.1. The resolution capability of on-line CE-MS was first demonstrated by analysis of a complex LPS mixture from H. influenzae strain Rd 11.7. This strain contains a mixture of isomeric glycoforms differing in the number and positions of hexose moieties. Sialic acid containing glycoforms were also determined. Structural features of LPS from a lic1 mutant of H. influenzae strain 375 (375.1) were studied using on-line CE-MS/MS. With the separation provided by CE, two isomeric glycoforms differing in the location of phosphoethanolamine substituents were characterized by tandem mass spectrometry.
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| 4. |
- Li, J J, et al.
(author)
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Glycine is a common substituent of the inner core in Haemophilus influenzae lipopolysaccharide
- 2001
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In: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 11:12, s. 1009-1015
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Journal article (peer-reviewed)abstract
- A survey of both typeable and nontypeable strainsof Haemophilus influenzae indicated that they contain glycine (Gly) in their lipopolysaccharide (LPS). Significant amounts (30-250 pmol Gly/mug LPS) were determined by high-performance anion-exchange chromatography using pulsed amperometric detection after treatment of the LPS with mild alkali. Oligosaccharides obtained from LPS after mild acid hydrolysis and gel filtration chromatography were investigated by electrospray ionization mass spectrometry (ESI-MS) and capillary electrophoresis (CE) ESI-MS. In all cases, molecular ions corresponding to the major glycoforms were identified and were accompanied by ions differing by 57 Da, thus indicating the presence of glycine. The position of glycine in these glycoforms was determined by CE-ESI-MS/MS analyses. It was found that, depending on strain, glycine can substitute each of the heptoses of the inner-core element, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1-->3)-L-alpha-D-Hepp- (1-->5)-alpha-Kdo of H. influenzae LPS as well as Kdo. In some strains, mixtures of monosubstituted Gly-containing glycoforms having different substitution patterns were identified.
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| 5. |
- Månsson, Martin, et al.
(author)
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Structural analysis of the lipopolysaccharide from nontypeable Haemophilus influenzae strain 1003
- 2002
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In: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 269:3, s. 808-818
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Journal article (peer-reviewed)abstract
- Structural analysis of the lipopolysaccharide (LPS) of nontypeable Haemophilus influenzae strain 1003 has been achieved by the application of high-field NMR techniques, ESI-MS. capillary electrophoresis coupled to ESI-MS. composition and linkage analyses on O-deacylated LPS and core oligosaccharide material. 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)-[PP Etn --> 4]-alpha-Kdop-(2 --> 6)-Lipid A. in which the beta-D-Glcp residue is substituted by phosphocholine at O-6 and an acetyl group at O-4. A second acetyl group is located at O-3 of the distal heptose residue (HepIII). HepIII is chain elongated at O-2 by either a beta-D-Glcp residue (major), lactose or sialyllactose (minor, i.e. alpha-Neu5Ac-(2 --> 3)-beta-D-Galp-(1 --> 4)-beta-D-Glcp), where a third minor acetylation site was identified at the glucose residue. Disialylated species were also detected. In addition. a minor substitution of ester-linked glycine at HepIII and Kdo was observed.
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| 6. |
- Schweda, Elke K H, et al.
(author)
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Structural profiling of lipopolysaccharide glycoforms expressed by non-typeable Haemophilus influenzae : phenotypic similarities between NTHi strain 162 and the genome strain Rd
- 2003
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In: Carbohydrate Research. - 0008-6215 .- 1873-426X. ; 338:23, s. 2731-2744
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Journal article (peer-reviewed)abstract
- Non-typeable Haemophihis influenzae (NTHi) is a significant cause of otitis media in children. We have employed single and multiple step electrospray ionization mass spectrometry (ESIMS) and NMR spectroscopy to profile and elucidate lipopolysaccharide (LPS) structural types expressed by NTHi strain 162, a strain obtained from an epidemiological study in Finland. ESIMS on O-deacylated LPS (LPS-OH) and core oligosaccharide (OS) samples of LPS provided information on the composition and relative abundance of glycoforms differing in the number of hexoses linked to the conserved inner-core element, L-alpha-D-Hepp-(1--> 2)-[PEtn --> 6]-L-alpha-D-Hepp-(1 --> 3)-L-alpha-D-Hepp-(1 --> 5)(.)[PPEtn --> 4]-alpha-Kdop-(2 --> 6)-Lipid A of H. influenzae LPS. The strain examined was found to elaborate Hex2 to Hex5 LPS glycoform populations having structures identical to those observed for H. influenzae strain Rd [Risberg, A.; Masoud, H.; Martin, A.; Richards, J.C.; Moxon, E.R.; Schweda, E.K.H. Eur. J Biochem. 1999, 261, 171-180], the strain for which the complete genome has been sequenced. In addition, sialyllactose-containing glycoforms previously identified in strain Rd as well as several NTHi strains, were identified as minor components. Multiple step tandem ESIMS (MS") on dephosphorylated and permethylated OS provided information on the arrangement of glycoses within the major population of glycoforms and on the existence of additional isomeric glycoforms. Minor Hex1 and Hex6 glycoforms were detected and characterized where the Hex6 glycoform was comprised of a dihexosamine-containing pentasaccharide chain attached at the proximal heptose residue of the inner-core unit. LPS structural motifs present in the NTHi strain 162 are expressed by a genetically diverse set of disease causing isolates, providing the basis for a vaccine strategy against NTHi otitis media.
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| 7. |
- Yildirim, Håkan H., et al.
(author)
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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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In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 44:13, s. 5207-5224
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Journal article (peer-reviewed)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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| 8. |
- Yildirim, Håkan H, et al.
(author)
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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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In: Carbohydrate Research. - : Elsevier BV. - 0008-6215 .- 1873-426X. ; 340:17, s. 2598-2611
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Journal article (peer-reviewed)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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| 9. |
- Schweda, Elke K H, et al.
(author)
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Structural analysis of lipopolysaccharide oligosaccharide epitopes expressed by non-typeable Haemophilus influenzae strain 176
- 2002
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In: Carbohydrate Research. - 0008-6215 .- 1873-426X. ; 337:5, s. 409-420
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Journal article (peer-reviewed)abstract
- The structure of the lipopolysaccharide (LPS) from non-typeable Haemophilus influenzae strain 176 has been investigated. Electrospray ionization-mass spectrometry (ESIMS) on O-deacylated LPS (LPS-OH) and core oligosaccharide (OS) samples obtained after mild-acid hydrolysis of LPS provided information on the composition and relative abundance of the glycoforms. ESIMS tandem-mass spectrometry on LPS-OH confirmed the presence of minor sialylated and disialylated glycoforms. Oligosaccharide samples were studied in detail using high-field NMR techniques. It was found that the LPS contains the common inner-core element of H. influenzae. L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1-->3)-[beta-D-Glep-(1 -->4)]-L-alpha-D-Hepp-(1-->5)[PPEtn-->4]-alpha-Kdop-(2-->6)-Lipid A having glycosyl substitution at the O-3 position of the terminal heptose as recently observed for non-typeable H. influenzae strain 486 [Mansson, M.: Bauer. S. H. J.: Hood, D. W.; Richards, J. C. Moxon, E. R. Schweda. E. K. H., Eur. J. Biochem. 2001. 268. 2148-2159]. The following LPS structures were identified as the major glycoforms. the most significant being indicated with an asterisk (*) (glycoforms are partly substituted with Gly at the terminal Hep): [GRAPHICS].
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| 10. |
- Valle, M., Zavialov, A., Li, W., Stagg, S.M., Sengupta, J., Nielsen, R., Nissen, P., Harvey, S., Ehrenberg, M. and Frank, J.
(author)
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Incorporation of aminoacyl-tRNA in the ribosome as seen by cryo-EM.
- 2003
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In: Nature Structural Biology. ; 10, s. 899-906
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Journal article (peer-reviewed)
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