| 1. |
- 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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| 2. |
- 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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| 3. |
- 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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| 4. |
- 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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