| 1. |
- Lundström, Susanna L., et al.
(författare)
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Application of capillary electrophoresis mass spectrometry and liquid chromatography multiple-step tandem electrospray mass spectrometry to profile glycoform expression during Haemophilus influenzae pathogenesis in the chinchilla model of experimental otitis media
- 2008
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Ingår i: Infection and Immunity. - 0019-9567 .- 1098-5522. ; 76:7, s. 3255-3267
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Tidskriftsartikel (refereegranskat)abstract
- Otitis media caused by nontypeable Haemophilus influenzae (NTHi) is a common and recurrent bacterial infection of childhood. The structural variability and diversity of H. influenzae lipopolysaccharide (LPS) glycoforms are known to play a significant role in the commensal and disease-causing behavior of this pathogen. In this study, we determined LPS glycoform populations from NTHi strain 1003 during the course of experimental otitis media in the chinchilla model of infection by mass spectrometric techniques. Building on an established structural model of the major LPS glycoforms expressed by this NTHi strain in vitro (M. Mansson, W. Hood, J. Li, J. C. Richards, E. R. Moxon, and E. K. Schweda, Eur. J. Biochem. 269:808-818, 2002), minor isomeric glycoform populations were determined by liquid chromatography multiple-step tandem electrospray mass spectrometry (LC-ESI-MSn). Using capillary electrophoresis ESI-MS (CE-ESI-MS), we determined glycoform profiles for bacteria from direct middle ear fluid (MEF) samples. The LPS glycan profiles were essentially the same when the MEF samples of 7 of 10 animals were passaged on solid medium (chocolate agar). LC-ESI-MSn provided a sensitive method for determining the isomeric distribution of LPS glycoforms in MEF and passaged specimens. To investigate changes in LPS glycoform distribution during the course of infection, MEF samples were analyzed at 2, 5, and 9 days postinfection by CE-ESI-MS following minimal passage on chocolate agar. As previously observed, sialic acid-containing glycoforms were detected during the early stages of infection, but a trend toward more-truncated and less-complex LPS glycoforms that lacked sialic acid was found as disease progressed.
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| 2. |
- Deadman, Mary E., et al.
(författare)
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Specific amino acids of the glycosyltransferase LpsA direct the addition of glucose or galactose to the terminal inner core heptose of Haemophilus influenzae lipopolysaccharide via alternative linkages
- 2006
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 281:40, s. 29455-29467
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Tidskriftsartikel (refereegranskat)abstract
- Lipopolysaccharide is the major glycolipid of the cell wall of the bacterium Haemophilus influenzae, a Gram-negative commensal and pathogen of humans. Lipopolysaccharide is both a virulence determinant and a target for host immune responses. Glycosyltransferases have high donor and acceptor substrate specificities that are generally limited to catalysis of one unique glycosidic linkage. The H. influenzae glycosyltransferase LpsA is responsible for the addition of a hexose to the distal heptose of the inner core of the lipopolysaccharide molecule and belongs to the glycosyltransferase family 25. The hexose added can be either glucose or galactose and linkage to the heptose can be either beta 1-2 or beta 1-3. Each H. influenzae strain uniquely produces only one of the four possible combinations of linked sugar in its lipopolysaccharide. We show that, in any given strain, a specific allelic variant of LpsA directs the anomeric linkage and the added hexose, glucose, or galactose. Site-directed mutagenesis of a single key amino acid at position 151 changed the hexose added in vivo from glucose to galactose or vice versa. By constructing chimeric lpsA gene sequences, it was shown that the 3' end of the gene directs the anomeric linkage (beta 1-2 or beta 1-3) of the added hexose. The lpsA gene is the first known example where interstrain variation in lipopolysaccharide core structure is directed by the specific sequence of a genetic locus encoding enzymes directing one of four alternative possible sugar additions from the inner core.
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| 3. |
- Dzieciatkowska, Monika, et al.
(författare)
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Characterization of intact lipopolysaccharides from the Haemophilus influenzae strain RM 118 using electrophoresis-assisted open-tubular liquid chromatography-mass spectrometry
- 2008
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 29:10, s. 2171-2181
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Tidskriftsartikel (refereegranskat)abstract
- We have applied an electrophoresis-assisted open-tubular LC-MS method for analyzing intact lipopolysaccharides (LPSs) from Haemophilus influenzae strain RM 118 (Rd). We were able to obtain structural information on both core oligosaccharides (OSs) and the lipid A moiety including the sialylation, glycylation, and the distribution of fatty acid residues on the disaccharide backbone of lipid A. The fragmentation patterns of sodiated and protonated LPS molecules were investigated for determining the location of sialic acid. It was found that the tandem mass spectra of sodiated ions provided unambiguous evidence of both sialylated lactose and sialylated lacto-N-neotetraose. In contrast, the fragment ions of protonated ions only offered the evidence for the existence of sialylated lacto-N-neotetraose. The lipid A of Gram-negative bacteria, as the principal endotoxic component of LP S, plays a major role in the pathogenesis of bacterial infections. We have previously characterized lipid. A species after mild acid hydrolysis of LPS during which lipid A precipitates. In this study, intact LPS was directly introduced to a tandem mass spectrometer. In-source dissociation strategy was employed, followed by multiple-stage MS/MS on the ions originating from the lipid part to obtain structural information. This is the first time that the structure of lipid A of H. influenzae was characterized by MS/MS on intact LPS molecules without any prior chemical modifications. In the same way information on the OS can be obtained by MS/MS by focusing on ions originating from core OS.
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| 4. |
- Field, Dawn, et al.
(författare)
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The minimum information about a genome sequence (MIGS) specification.
- 2008
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Ingår i: Nature biotechnology. - : Springer Science and Business Media LLC. - 1546-1696 .- 1087-0156. ; 26:5, s. 541-7
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Tidskriftsartikel (refereegranskat)abstract
- With the quantity of genomic data increasing at an exponential rate, it is imperative that these data be captured electronically, in a standard format. Standardization activities must proceed within the auspices of open-access and international working bodies. To tackle the issues surrounding the development of better descriptions of genomic investigations, we have formed the Genomic Standards Consortium (GSC). Here, we introduce the minimum information about a genome sequence (MIGS) specification with the intent of promoting participation in its development and discussing the resources that will be required to develop improved mechanisms of metadata capture and exchange. As part of its wider goals, the GSC also supports improving the 'transparency' of the information contained in existing genomic databases.
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| 5. |
- Finn, Robert D, et al.
(författare)
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Pfam : clans, web tools and services.
- 2006
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Ingår i: Nucleic Acids Res. - : Oxford University Press (OUP). - 1362-4962 .- 0305-1048. ; 34:Database issue, s. D247-51
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Tidskriftsartikel (refereegranskat)
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| 6. |
- Fox, Kate L., et al.
(författare)
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Duplicate copies of lic1 direct the addition of multiple phosphocholine residues in the lipopolysaccharide of Haemophilus influenzae
- 2008
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Ingår i: Infection and Immunity. - 0019-9567 .- 1098-5522. ; 76:2, s. 588-600
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Tidskriftsartikel (refereegranskat)abstract
- The genes of the lic1 operon (lic1A to lic1D) are responsible for incorporation of phosphocholine (PCho) into the lipopolysaccharide (LPS) of Haemophilus influenzae. PCho plays a multifaceted role in the commensal and pathogenic lifestyles of a range of mucosal pathogens, including H. influenzae. Structural studies of the LPS of nontypeable H. influenzae (NTHI) have revealed that PCho can be linked to a hexose on any one of the oligosaccharide chain extensions from the conserved inner core triheptosyl backbone. In a collection of NTHI strains we found several strains in which there were two distinct but variant lic1D DNA sequences, genes predicted to encode the transferase responsible for directing the addition of PCho to LPS. The same isolates were also found to express concomitantly two PCho residues at distinct positions in their LPS. In one such NTHI isolate, isolate 1158, structural analysis of LPS from lic1 mutants confirmed that each of the two copies of lic1D directs the addition of PCho to a distinct location on the LPS. One position for PCho addition is a novel heptose, which is part of the oligosaccharide extension from the proximal heptose of the LPS inner core. Modification of the LPS by addition of two PCho residues resulted in increased binding of C-reactive protein and had consequential effects on the resistance of the organism to the killing effects of normal human serum compared to the effects of glycoforms containing one or no PCho. When bound, C-reactive protein leads to complement-mediated killing, indicating the potential biological significance of multiple PCho residues.
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| 7. |
- Hood, Derek W., et al.
(författare)
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Genes required for the synthesis of heptose-containing oligosaccharide outer core extensions in Haemophilus influenzae lipopolysaccharide
- 2010
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Ingår i: Microbiology. - : Microbiology Society. - 1350-0872 .- 1465-2080. ; 156, s. 3421-3431
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Tidskriftsartikel (refereegranskat)abstract
- Heptose-containing oligosaccharides (OSs) are found in the outer core of the lipopolysaccharide (LPS) of a subset of non-typable Haemophilus influenzae (NTHi) strains. Candidate genes for the addition of either L-glycero-D-manno-heptose (LD-Hep) or D-glycero-D-manno-heptose (DD-Hep) and subsequent hexose sugars to these OSs have been identified from the recently completed genome sequences available for NTHi strains. losA1/losB1 and losA2/losB2 are two sets of related genes in which losA has homology to genes encoding glycosyltransferases and losB to genes encoding heptosyltransferases. Each set of genes is variably present across NTHi strains and is located in a region of the genome with an alternative gene organization between strains that contributes to LPS heterogeneity. Dependent upon the strain background, the LPS phenotype, structure and serum resistance of strains mutated in these genes were altered when compared with the relevant parent strain. Our studies confirm that losB1 and losB2 usually encode DD-heptosyl- and LD-heptosyl transferases, respectively, and that losA1 and losA2 encode glycosyltransferases that play a role in OS extensions of NTHi LPS.
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| 8. |
- Li, Jianjun, et al.
(författare)
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Structural characterization of sialylated glycoforms of H-influenzae by electrospray mass spectrometry : fragmentation of protonated and sodiated O-deacylated lipopolysaccharides
- 2007
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Ingår i: Rapid Communications in Mass Spectrometry. - : Wiley. - 0951-4198 .- 1097-0231. ; 21:6, s. 952-960
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Tidskriftsartikel (refereegranskat)abstract
- Sialylated lipopolysaccharide (LPS) glycoforms from Haemophilus influenzae were characterized by tandem mass spectrometry using a new generation hyphenated mass spectrometer which combines a triple quadrupole and a linear ion trap (Q-Trap). The fragmentation of both protonated and sodiated molecular ions from O-deacylated LPS (LPS-OH) obtained in MS2 experiments in the positive mode was studied. The MS2 spectra of protonated ions provided unambiguous evidence for the presence and sequence of sialylated lactosamine present in lacto-N-neotetraose oligosaccharide extensions but not for sialyl-lactose structures whilst fragmentation of sodiated adducts, [M+Na](+), afforded information diagnostic of mono- and disialylated lactose extensions. To study this we used a highly sialylated LPS from a H. influenzae strain capable of sialyl-lactose expression only. We then applied the method to the H. influenzae genome strain, Rd, in which glycoforms containing both sialyl-lactose and sialyl-lacto-N-neotetraose were detected from diagnostic B-ions at m/z 638.2 ([Neu5Ac(1) Hex(2)+ Na](+)) and 657.2 ([Neu5Ac(1) Hex(1) HexNAc(1)+H](+)). Unique fragmentation patterns provided the locations and sequences of these oligosaccharide extensions. This is the first time both sialylated lactose and sialylated lacto-N-neotetraose units have been detected and characterized by tandem mass spectrometry in the same molecule. This methodology is of general applicability for determination of common sialylated oligosaccharide extension in bacterial LPS.
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| 9. |
- Lundström, Susanna L., et al.
(författare)
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Novel globoside-like oligosaccharide expression patterns in nontypeable Haemophilus influenzae lipopolysaccharide
- 2007
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 274:18, s. 4886-4903
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Tidskriftsartikel (refereegranskat)abstract
- 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 [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 terminal heptose (HepIII) and the corresponding structures with an alpha-D-Glcp as the reducing sugar linked to the middle heptose (HepII) in the same LPS molecule. Previously these motifs had been found linked only to either the proximal heptose (HepI) or HepIII of the triheptosyl inner-core moiety l-alpha-D-Hepp-(1 -> 2)-[PEtn -> 6]-l-alpha-D-Hepp-(1 -> 3)-l-alpha-D-Hepp-(1 -> 5)-[PPEtn -> 4]-alpha-Kdo-(2 -> 6)-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 oligosaccharide 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.
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| 10. |
- Lundström, Susanna L., et al.
(författare)
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Structural analysis of the lipopolysaccharide from nontypeable Haemophilus influenzae strain R2846
- 2008
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 47:22, s. 6025-6038
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Tidskriftsartikel (refereegranskat)abstract
- We here report the lipopolysaccharide (LPS) structures expressed by nontypeable Haemophilus influenzae R2846, a strain whose complete genome sequence has recently been obtained. Results were obtained by using NMR techniques and ESI-MS on O-deacylated LPS and core oligosaccharide material (OS) as well as ESI-MSn on permethylated dephosphorylated OS. A beta-D-Glcp-(1 -> 4)-D-alpha-D-Hepp-(1 -> 6)-beta-D-Glcp-(1 -> 4) unit was found linked to the proximal heptose (HepI) of the conserved triheptosyl inner-core moiety, L-alpha-D-Hepp-(1 -> 2)-[PEtn -> 6]-L-alpha-D-Hepp-(1 -> 3)-L-alpha-D-Hepp-(1 -> 5)-[PPEtn -> 4]-alpha-Kdo-(2 -> 6)-lipid A. The beta-D-Glcp (GlcI) linked to HepI was also branched with oligosaccharide extensions from O-4 and O-6. O-4 of GlcI was substituted with sialyllacto-N-neotetraose [alpha-Neu5Ac-(2 -> 3)-beta-D-Galp-(1 -> 4)-beta-GlcpNAc-(1 -> 3)-beta-D-Galp-(1 -> 4)-beta-D-Glcp-(1 ->] and the related structure [(PEtn -> 6)-alpha-D-GalpNAc-(1 -> 6)-beta-D-Galp-(1 -> 4)-beta-D-GlcpNAc-(1 -> 3)-beta-D-Galp-(1 -> 4)-beta-Glcp-(1-]. The distal heptose (HepIII) was substituted at O-2 by beta-D-Gal. Phosphate, phosphoethanolamine, phosphocholine, acetate, and glycine were found to substitute the core oligosaccharide. Two heptosyltransferase genes, losB1 and losB2, have been identified from the R2846 genome sequence and are candidates to add the noncore heptose to the LPS. Mutant strain R2846losB1 did not show DD-heptose in the extension from HepI but still contained minor quantities of LD-heptose at the same position, indicating that the losB1 gene is required to add DD-heptose to Glcl. The LPS from strain R2846losB1/losB2 expressed no noncore heptose, consistent with losB2 directing the addition of LD-heptose.
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