| 1. |
- 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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| 2. |
- 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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| 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. |
- Schweda, Elke K. H., et al.
(författare)
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Profiling structural elements of short-chain lipopolysaccharide of non-typeable Haemophilus influenzae
- 2008
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Ingår i: Innate Immunity. - : SAGE Publications. - 1753-4259 .- 1753-4267. ; 14:4, s. 199-211
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Tidskriftsartikel (refereegranskat)abstract
- Lipopolysaccharide (LPS) is a major Virulence determinant of the human bacterial pathogen Hoemophilus influenzae. A characteristic feature of H. influenzae LPS is the extensive intra- and inter-strain heterogeneity of glycoform structure which is key to the role of the molecule in both commensal and disease-causing behaviour of the bacterium. The chemical composition of non-typeable Haemophilus influenzae (NTHi) LPS is hi-lily diverse. It contains a number of different monosaccharides (Neu5Ac, L-glycero-D-manno heptose, D-glycero-D-manno heptose, Kdo, D-Glc, D-Gal, D-GlcNAc, D-GaINAc) and non-carbohydrate substituents. Prominent non-carbohydrate components are O-acetyl groups, glycine and phosphates. We now know that sialic acid (N-acetylneuraminic acid or Neu5Ac) and certain oligosaccharide extensions are important in the pathogenesis of NTHi: however, the biological implications for many of the various features are Still unknown. Electrospray ionization mass spectrometry ill combination with separation techniques like CE and HPLC is an indispensable tool in profiling glycoform populations ill heterogeneous LPS samples. Mass Spectrometry is characterized by its extreme sensitivity. Trace amounts of glycoforms expressing important Virulence determinants can be detected and characterized on minute amounts of material. The present review focuses oil LPS structures and mass spectrometric methods which enable us to profile these in complex mixtures.
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| 5. |
- Twelkmeyer, Brigitte
(författare)
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Structural studies on lipopolysaccharides from haemophilus species
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background: Carbohydrates are indispensable mediators for a variety of cellular interactions. In biological systems carbohydrates are usually linked to a carrier as e.g. proteins or lipids. One example is the molecule lipopolysaccharide (LPS). LPS is one of the major outer membrane constituents found in Gram-negative bacteria and they play key roles in the biology of these organisms. Notably, they have been found to be important virulence factors in pathogenic species. Aims: In this thesis the biosynthesis and the molecular structures of LPS expressed by Haemophilus influenzae and Haemophilus parainfluenzae were investigated. These two bacteria colonize the human nasopharynx. H. influenzae with capsule type b is involved in invasive diseases such as meningitidis and epiglottis while non-encapsulated H. influenzae (non-typeable, NTHi) often cause otitis media, and acute and chronic lower respiratory tract infections in infants. Though, closely related H. parainfluenzae is a commensal. Materials and Methods: Structural elucidation of LPS involves initial de-lipidation to obtain water-soluble material that is suitable for subsequent analyses by chemical, nuclear magnetic resonance (NMR) and mass spectrometric (MS) methods. Results: The function of the gene lic2B in H. influenzae type b strain Eagan was investigated. The LPS expressed by the mutant strain Eaganlic2Blic2C+ was analyzed and found to encode for a glucosyltransferase responsible for the addition of β-D-Glcp to O-4 of α-D-Glcp-(1→ elongating from the middle inner core heptose. Further, the LPS structures of NTHi strains 1247 and 1008 were determined. NTHi strain 1247 expressed globotetraose elongating from the phosphocholine bearing GlcI, [β-DGalpNAc-(1→3)-α-D-Galp-(1→4)-β-D-Galp-(1→4)-β-D-Glcp-(1→4)-[PCho→6]-β-DGlcIp-(1→], or truncated versions thereof. Globotetraose was also found to elongate from the distal heptose in NTHi 1247. The lpsA mutant of strain 1247 allowed the identification of a novel disialyllactose epitope, [α-Neu5Ac-(2→8)-α-Neu5Ac-(2→3)-β-D-Galp-(1→4)-β-D-Glcp-(1→], from the proximal inner core heptose. Alternatively, a globotetraose was found to elongate from GlcI. NTHi strain 1008 showed almost identical glycoforms to NTHi strain 1247 but lacked terminal N-acetyl galactoseamine. All of the three investigated H. parainfluenzae strains were shown to express the same lipid A and inner core as NTHi. H. parainfluenzae genome strain T3T1 and strain 22 were shown to express rough-type LPS having novel outer core structures elongating from GlcI that were [α-Neu5,9Ac2-(2→6)-β-D-GalpNAc-(1→4)-β-D-Galp-(1→3)-β-DFucpNAc4N-(1→] in strain T3T1 and [Neu5Ac-(2→6)-α-D-Galp-(1→6)-β-D-Glcp-(1→3)-β-D-FucpNAc4N-(1→] in strain 22. H. parainfluenzae strain 13 expressed an Orepeating chain with the structure [→6)-[Ac→3]-β-D-Galf-(1→3)-[PEtn→6]-β-DGlcpNAc-(1→]. Conclusion: It was showed that the H. parainfluenzae LPS investigated here lacks all virulence determining LPS attributes expressed by H. influenzae such as phosphocholine and phase variable expression of outer core structures. This may provide further insight into the factors relating to commensal vs. pathogenic behavior inside the host.
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| 6. |
- Young, Rosanna E. B., et al.
(författare)
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Haemophilus parainfluenzae expresses diverse lipopolysaccharide O-antigens using ABC transporter and Wzy polymerase-dependent mechanisms
- 2013
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Ingår i: International Journal of Medical Microbiology. - : Elsevier. - 1438-4221 .- 1618-0607. ; 303:8, s. 603-617
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Tidskriftsartikel (refereegranskat)abstract
- Lipopolysaccharide O-antigens are the basis of serotyping schemes for Gram negative bacteria and help to determine the nature of host-bacterial interactions. Haemophilus parainfluenzae is a normal commensal of humans but is also an occasional pathogen. The prevalence, diversity and biosynthesis of O-antigens were investigated in this species for the first time. 18/18 commensal H. parainfluenzae isolates contain a O-antigen biosynthesis gene cluster flanked by glnA and pepB, the same position as the hmg locus for tetrasaccharide biosynthesis in Haemophilus influenzae. The O-antigen loci show diverse restriction digest patterns but fall into two main groups: (1) those encoding enzymes for the synthesis and transfer of Fuc-NAc4N in addition to the Wzy-dependent mechanism of O-antigen synthesis and transport and (2) those encoding galactofuranose synthesis/transfer enzymes and an ABC transporter. The other glycosyltransferase genes differ between isolates. Three H. parainfluenzae isolates fell outside these groups and are predicted to synthesise O-antigens containing ribitol phosphate or deoxytalose. Isolates using the ABC transporter system encode a putative O-antigen ligase, required for the synthesis of O-antigen-containing LPS glycoforms, at a separate genomic location. The presence of an O-antigen contributes significantly to H. parainfluenzae resistance to the killing effect of human serum in vitro. The discovery of O-antigens in H. parainfluenzae is striking, as its close relative H. influenzae lacks this cell surface component.
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