| 1. |
- Bauer, S H J, et al.
(författare)
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A rapid and sensitive procedure for determination of 5-N-acetyl neuraminic acid in lipopolysaccharides of Haemophilus influenzae : a survey of 24 non-typeable H-influenzae strains
- 2001
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Ingår i: Carbohydrate Research. - 0008-6215 .- 1873-426X. ; 335:4, s. 251-260
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Tidskriftsartikel (refereegranskat)abstract
- In view of the importance of 5-N-acetyl neuraminic acid in bacterial pathogenesis, a sensitive, reproducible and reliable method for the determination of 5-N-acetyl neuraminic acid levels in lipopolysaccharide (LPS) is described and applied to 24 different non-typeable Haemophilus influenzae (NTHi) strains. The method involves analysis by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) of terminal 5-N-acetyl neuraminic acid residues released by neuraminidase treatment of O-deacylated LPS. The procedure is relatively fast and the instrumental effort is moderate. The results of the procedure were compared with data obtained by H-1 NMR and electrospray ionisation-mass spectrometry (ESI-MS). The analysis of LPS from 24 NTHi strains showed that 5-N-acetyl neuraminic acid was found to be a common constituent of LPS in NTHi. Only one strain (NTHi 432) did not show any sialylation. Molar ratios (LPS /5-N-acetyl neuraminic acid) ranged between 5/1 and 500/1. Several strains in which no 5-N-acetyl neuraminic acid could be determined by other methods including 1H NMR and ESI-MS were shown to contain 5-N-acetyl neuraminic acid by this HPAEC-PAD procedure. The method was applied to determine levels of terminal 5-N-acetyl neuraminic acid in LPS from NTHi strains grown under different conditions and mutant strains containing inactive LPS biosynthetic genes.
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| 2. |
- Bäckhed, Fredrik, 1973, et al.
(författare)
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Structural requirements for TLR4-mediated LPS signalling: a biological role for LPS modifications
- 2003
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Ingår i: Microbes Infect. - 1286-4579 .- 1769-714X. ; 5:12, s. 1057-63
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Tidskriftsartikel (refereegranskat)abstract
- Cells of the mucosal lining are the first to encounter invading bacteria during infection, and as such, they have developed numerous ways of detecting microbial intruders. Recently, we showed that epithelial cells recognize lipopolysaccharide (LPS) through the CD14-Toll-like receptor (TLR)-4 complex. Here, we identify the substructures of LPS that are recognized by the TLR4 receptor complex. In contrast to lipid A, the O-antigen does not mediate an inflammatory response; rather it interferes with the lipid A recognition. An Escherichia coli strain genetically modified to express penta-acylated lipid A not only showed reduced immunogenicity, but was also found to inhibit pro-inflammatory signalling induced by wild-type E. coli (hexa-acylated lipid A) as well as LPS from other bacteria of the Enterobacteriaceae family. Furthermore, penta-acylated LPS from Pseudomonas aeruginosa acted as an antagonist to hexa-acylated E. coli LPS, as did E. coli, as shown by its inhibitory effect on IL-8 production in stimulated cells. Hypo-acylated lipid A, such as that of P. aeruginosa, is found in several species within the gut microflora as well as in several bacteria causing chronic infections. Thus, our results suggest that the composition of the microflora may be important in modulating pro-inflammatory signalling in epithelial cells under normal as well as pathologic conditions.
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| 3. |
- 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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| 4. |
- 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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| 5. |
- Dzieciatkowska, Monika, et al.
(författare)
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Rapid method for sensitive screening of oligosaccharide epitopes in the lipooligosaccharide from Campylobacter jejuni strains isolated from Guillain-Barre syndrome and Miller Fisher syndrome patients
- 2008
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 46:10, s. 3429-3436
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Tidskriftsartikel (refereegranskat)abstract
- Campylobacter jejuni lipooligosaccharide (LOS) can trigger Guillain-Barre syndrome (GBS) due to its similarity to human gangliosides. Rapid and accurate structural elucidation of the LOS glycan of a strain isolated from a GBS patient could help physicians determine the spectrum of anti-ganglioside antibodies likely to be found and therefore provide valuable assistance in establishing an appropriate course of treatment. The ability of implemented mass spectrometry-based approaches in a clinical setting has been limited by the laborious and time-consuming nature of the protocols, typically 3 to 4 days, used to prepare LOS. In order to improve the analytical throughput, microwave-assisted enzymatic digestion was investigated. In this study, the bacterial cells were suspended in 50 mu l of 20 mM ammonium acetate buffer containing DNase and RNase and treated by direct microwave irradiation for 3 min. Then, proteinase K was added and the samples were again microwaved. The intact LOS samples were analyzed using electrophoresis-assisted open-tubular liquid chromatography-mass spectrometry. The reliability of the rapid, high-throughput technique was demonstrated through analysis of LOS glycans from 73 C. jejuni strains. The structure was elucidated using material from a single colony. The total time for sample preparation and MS analysis is less than 60 min.
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| 6. |
- 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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| 7. |
- 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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| 8. |
- 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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| 9. |
- Gulin, Sofia, et al.
(författare)
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Linking mass spectrometry and slab-polyacrylamide gel electrophoresis by passive elution of lipopolysaccharides from reverse-stained gels : Analysis of gel-purified lipopolysaccharides from Haemophilus influenzae strain Rd
- 2003
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 75:18, s. 4918-4924
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Tidskriftsartikel (refereegranskat)abstract
- Haemophilus influenzae is an important cause of human disease, and its lipopolysaccharide (LPS) is known to be a major virulence factor. H. influenzae produces short-chain LPS of which the heterogeneity is often visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using silver staining for detection. Individual bands have not previously been recovered by this method in quantities sufficient for mass spectrometry. In an attempt toward the development of sensitive mass spectrometrical strategies to be used in structural studies of H. influenzae LPS and LPS from other bacteria, we have applied here our previously described slab-PAGE-based micropurification method to obtain unmodified LPS fractions of high purity (>95%) from a crude LPS preparation of H. influenzae strain Rd. Two LPS-fractions were obtained which, after a procedure including mild acid hydrolysis, dephosphorylation, and permethylation of the resulting oligosaccharides, were subjected to tandem electrospray ionization mass spectrometry (ESI-MS/MS). The quantities of micropurified LPS fractions-the recovery of LPS in terms of total mass was 30%-were found sufficient to allow the characterization of LPS glycoforms. The ESI-MS spectra of the individual bands showed reduced heterogeneity. Furthermore, the integrity of the micropurified LPS was confirmed. The spectra-displayed molecular ions showed improved intensity, increased respective signal-to-noise ratios demonstrating the sensitivity of analysis. Consequently, both the direct determination of the molecular masses of the gel-separated LPS glycoforms and sequence analyses using ESI-MS/MS were possible.
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| 10. |
- Hecker, Andreas, et al.
(författare)
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Phosphocholine-Modified Macromolecules and Canonical Nicotinic Agonists Inhibit ATP-Induced IL-1 beta Release
- 2015
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Ingår i: Journal of Immunology. - : AMER ASSOC IMMUNOLOGISTS. - 0022-1767 .- 1550-6606. ; 195:5, s. 2325-2334
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Tidskriftsartikel (refereegranskat)abstract
- IL-1 beta is a potent proinflammatory cytokine of the innate immune system that is involved in host defense against infection. However, increased production of IL-1 beta plays a pathogenic role in various inflammatory diseases, such as rheumatoid arthritis, gout, sepsis, stroke, and transplant rejection. To prevent detrimental collateral damage, IL-1 beta release is tightly controlled and typically requires two consecutive danger signals. LPS from Gram-negative bacteria is a prototypical first signal inducing pro-IL-1 beta synthesis, whereas extracellular ATP is a typical second signal sensed by the ATP receptor P2X7 that triggers activation of the NLRP3-containing inflammasome, proteolytic cleavage of pro-IL-1 beta by caspase-1, and release of mature IL-1 beta. Mechanisms controlling IL-1 beta release, even in the presence of both danger signals, are needed to protect from collateral damage and are of therapeutic interest. In this article, we show that acetylcholine, choline, phosphocholine, phosphocholine-modified LPS from Haemophilus influenzae, and phosphocholine-modified protein efficiently inhibit ATP-mediated IL-1 beta release in human and rat monocytes via nicotinic acetylcholine receptors containing subunits alpha 7, alpha 9, and/or alpha 10. Of note, we identify receptors for phosphocholine-modified macromolecules that are synthesized by microbes and eukaryotic parasites and are well-known modulators of the immune system. Our data suggest that an endogenous anti-inflammatory cholinergic control mechanism effectively controls ATP-mediated release of IL-1 beta and that the same mechanism is used by symbionts and misused by parasites to evade innate immune responses of the host.
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| 11. |
- Hood, D W, et al.
(författare)
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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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Ingår i: Journal of Bacteriology. - 0021-9193 .- 1098-5530. ; 186:21, s. 7429-7439
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Tidskriftsartikel (refereegranskat)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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| 12. |
- 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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| 13. |
- Hood, D W, et al.
(författare)
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Genetic basis for expression of the major globotetraose-containing lipopolysaccharide from H-influenzae strain Rd (RM118)
- 2001
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 11:11, s. 957-967
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Tidskriftsartikel (refereegranskat)abstract
- A genetic basis for the biosynthetic assembly of the globotetraose containing lipopolysaccharide (LPS) of Haemophilus influenzae strain RM118 (Rd) was determined by structural analysis of LPS derived from mutant strains. We have previously shown that the parent strain RM118 elaborates a population of LPS molecules made up of a series of related glycoforms differing in the degree of oligosaccharide chain extension from the distal heptose residue of a conserved phosphorylated inner-core element, L-alpha -D-Hepp-(1-->2)-L-alpha -D-Hepp-(1-->3)-[beta -D-Glcp-(1-->4)-]-L-alpha -D-Hepp-(1-->5)-alpha -Kdo. The fully extended LPS glycoform expresses the globotetraose structure, beta -D-GalpNAc-(1-->3)-alpha -D-Galp(1-->4)-beta -D-Galp-(1-->4)-beta -D-Glcp. A fingerprinting strategy was employed to establish the structure of LPS from strains mutated in putative glycosyltransferase genes compared to the parent strain. This involved glycose and linkage analysis on intact LPS samples and analysis of O-deacylated LPS samples by electrospray ionization mass spectrometry and 1D H-1-nuclear magnetic resonance spectroscopy. Four genes, lpsA, lic2A, lgtC, and lgtD, were required for sequential addition of the glycoses to the terminal inner-core heptose to give the globotetraose structure. lgtC and lgtD were shown to encode glycosyltransferases by enzymatic assays with synthetic acceptor molecules. This is the first genetic blueprint determined for H. influenzae LPS oligosaccharide biosynthesis, identifying genes Involved in the addition of each glycose residue.
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| 14. |
- Houliston, R. Scott, et al.
(författare)
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A Haemophilus influenzae strain associated with fisher syndrome expresses a novel disialylated ganglioside mimic
- 2007
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 46:27, s. 8164-8171
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Tidskriftsartikel (refereegranskat)abstract
- The non-typeable Haemophilus influenzae strain DH1 was isolated from a 25 year old male patient with Fisher syndrome, a postinfectious autoimmune condition characterized by the presence of anti-GQ1b IgG antibodies that target and initiate damage to peripheral nerves. DH1 was found to display an alpha NeuAc(2-8)alpha NeuAc(2-3)beta Gal branch bound to the tetraheptosyl backbone core of its lipooligosaccharide (LOS). The novel sialylation pattern was found to be dependent on the activity of a bifunctional sialyltransferase, Lic3B, which catalyzes the addition of both the terminal and subterminal sialic acid residues. Patient serum IgGs bind to DH1 LOS, and the reactivity is significantly influenced by the presence of sialylated glycoforms. The display by DH1, of a surface glycan that mimics the terminal trisaccharide portion of disialosyl-containing gangliosides, provides strong evidence for its involvement in the development of Fisher syndrome.
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| 15. |
- Juntti-Berggren, Lisa, et al.
(författare)
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Dihydroxyacetone-induced oscillations in cytoplasmic free Ca2+ and the ATP/ADP ratio in pancreatic beta-cells at substimulatory glucose.
- 2003
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 278:42, s. 40710-40716
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Tidskriftsartikel (refereegranskat)abstract
- Glucose stimulation of pancreatic beta-cells causes oscillatory influx of Ca2+, leading to pulsatile insulin secretion. We have proposed that this is due to oscillations of glycolysis and the ATP/ADP ratio, which modulate the activity of ATP-sensitive K+ channels. We show here that dihydroxyacetone, a secretagogue that feeds into glycolysis below the putative oscillator phosphofructokinase, could cause a single initial peak in cytoplasmic free Ca2+ ([Ca2+]i) but did not by itself cause repeated oscillations in [Ca2+]i in mouse pancreatic beta-cells. However, in the presence of a substimulatory concentration of glucose (4 mm), dihydroxyacetone induced [Ca2+]i oscillations. Furthermore, these oscillations correlated with oscillations in the ATP/ADP ratio, as seen previously with glucose stimulation. Insulin secretion in response to dihydroxyacetone was transient in the absence of glucose but was considerably enhanced and somewhat prolonged in the presence of a substimulatory concentration of glucose, in accordance with the enhanced [Ca2+]i response. These results are consistent with the hypothesized role of phosphofructokinase as the generator of the oscillations. Dihydroxyacetone may affect phosphofructokinase by raising the free concentration of fructose 1,6-bisphosphate to a critical level at which it activates the enzyme autocatalytically, thereby inducing the pulses of phosphofructokinase activity that cause the metabolic oscillations.
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| 16. |
- Kumar Kondadi, Pradeep, et al.
(författare)
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Identification and Characterization of a Lipopolysaccharide alpha,2,3-Sialyltransferase from the Human Pathogen Helicobacter bizzozeronii
- 2012
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Ingår i: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; 194:10, s. 2540-2550
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Tidskriftsartikel (refereegranskat)abstract
- Terminal sialic acid in the lipopolysaccharides (LPSs) of mucosal pathogens is an important virulence factor. Here we report the characterization of a Helicobacter sialyltransferase involved in the biosynthesis of sialylated LPS in Helicobacter bizzozeronii, the only non-pylori gastric Helicobacter species isolated from humans thus far. Starting from the genome sequences of canine and human strains, we identified potential sialyltransferases downstream of three genes involved in the biosynthesis of N-acetylneuraminic acid. One of these candidates showed monofunctional alpha,2,3-sialyltransferase activity with a preference for N-acetyllactosamine as a substrate. The LPSs from different strains were shown by SDS-PAGE and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) to contain sialic acid after neuraminidase treatment. The expression of this sialyltransferase and sialyl-LPS appeared to be a phase-variable characteristic common to both human and canine H. bizzozeronii strains. The sialylation site of the LPSs of two H. bizzozeronii strains was determined to be NeuAc-Hex-HexNAc, suggesting terminal 3-sialyl-LacNAc. Moreover, serological typing revealed the possible presence of sialyl-Lewis X in two additional strains, indicating that H. bizzozeronii could also mimic the surface glycans of mammalian cells. The expression of sialyl-glycans may influence the adaptation process of H. bizzozeronii during the host jump from dogs to humans.
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| 17. |
- Landerholm, Malin K, et al.
(författare)
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Characterization of novel structural features in the lipopolysaccharide of nondisease associated nontypeable Haemophilus influenzae
- 2004
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 271:5, s. 941-953
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Tidskriftsartikel (refereegranskat)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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| 18. |
- Langereis, Jeroen D, et al.
(författare)
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Modified Lipooligosaccharide Structure Protects Nontypeable Haemophilus influenzae from IgM-Mediated Complement Killing in Experimental Otitis Media
- 2012
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Ingår i: mBio. - : American Society for Microbiology: mBio / American Society for Microbiology. - 2161-2129 .- 2150-7511. ; 3:4
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Tidskriftsartikel (refereegranskat)abstract
- Nontypeable Haemophilus influenzae (NTHi) is a Gram-negative, human-restricted pathogen. Although this bacterium typically colonizes the nasopharynx in the absence of clinical symptoms, it is also one of the major pathogens causing otitis media (OM) in children. Complement represents an important aspect of the host defense against NTHi. In general, NTHi is efficiently killed by complement-mediated killing; however, various resistance mechanisms have also evolved. We measured the complement resistance of NTHi isolates isolated from the nasopharynx and the middle ear fluids of OM patients. Furthermore, we determined the molecular mechanism of NTHi complement resistance. Complement resistance was strongly increased in isolates from the middle ear, which correlated with decreased binding of IgM. We identified a crucial role for the R2866_0112 gene in complement resistance. Deletion of this gene altered the lipooligosaccharide (LOS) composition of the bacterium, which increased IgM binding and complement-mediated lysis. In a novel mouse model of coinfection with influenza virus, we demonstrate decreased virulence for the R2866_0112 deletion mutant. These findings identify a mechanism by which NTHi modifies its LOS structure to prevent recognition by IgM and activation of complement. Importantly, this mechanism plays a crucial role in the ability of NTHi to cause OM. less thanbrgreater than less thanbrgreater thanIMPORTANCE Nontypeable Haemophilus influenzae (NTHi) colonizes the nasopharynx of especially young children without any obvious symptoms. However, NTHi is also a major pathogen in otitis media (OM), one of the most common childhood infections. Although this pathogen is often associated with OM, the mechanism by which this bacterium is able to cause OM is largely unknown. Our study addresses a key biological question that is highly relevant for child health: what is the molecular mechanism that enables NTHi to cause OM? We show that isolates collected from the middle ear fluid exhibit increased complement resistance and that the lipooligosaccharide (LOS) structure determines IgM binding and complement activation. Modification of the LOS structure decreased NTHi virulence in a novel NTHi-influenza A virus coinfection OM mouse model. Our findings may also have important implications for other Gram-negative pathogens harboring LOS, such as Neisseria meningitidis, Moraxella catarrhalis, and Bordetella pertussis.
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| 19. |
- Li, J J, et al.
(författare)
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Electrophoretic and mass spectrometric strategies for profiling bacterial lipopolysaccharides
- 2005
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Ingår i: MOLECULAR BIOSYSTEMS. - : Royal Society of Chemistry (RSC). - 1742-206X .- 1742-2051. ; 1:1, s. 46-52
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Tidskriftsartikel (refereegranskat)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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| 20. |
- Li, J J, et al.
(författare)
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Glycine is a common substituent of the inner core in Haemophilus influenzae lipopolysaccharide
- 2001
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 11:12, s. 1009-1015
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Tidskriftsartikel (refereegranskat)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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| 21. |
- 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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| 22. |
- 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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| 23. |
- 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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| 24. |
- 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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| 25. |
- 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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