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Characterization of Helicobacter pylori vesicles and their cognate properties for intimate host interactions
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. (Arnqvist)
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. (Arnqvist)
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. (Schleucher)
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
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(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:umu:diva-25737OAI: oai:DiVA.org:umu-25737DiVA: diva2:233438
Available from: 2009-09-01 Created: 2009-09-01 Last updated: 2017-05-22Bibliographically approved
In thesis
1. NMR studies of host-pathogen interactions
Open this publication in new window or tab >>NMR studies of host-pathogen interactions
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes the use of Nuclear Magnetic Resonance (NMR) for characterizing two host-pathogen interactions: The behavior of a regulatory RNA of the Hepatitis B virus (HBV) and the attachment of Helicobacter pylori (H. pylori) to the gastric mucosa. NMR is a powerful tool in biomedicine, because molecules ranging from small ligands to biomacromolecules can be studied with atomic resolution. Different NMR experiments are designed to determine structures, or to monitor interactions, folding, stability or motion.

Paper I describes the analysis of the motions of a regulatory RNA of HBV. The NMR structure of the RNA had revealed before that several well-conserved nucleotides adopt multiple conformations. Therefore an analysis of possible underlying motions was undertaken using two different NMR techniques, one of which (off-resonance ROESY) was applied to nucleic acids for the first time. The observed motions suggest an explanation why the structurally poorly defined nucleotides are highly conserved.

In paper II we improved the ROESY NMR experiment, which is used to measure internuclear distances for structure determination of medium-sized molecules. Using a small protein and an organometallic complex as examples, we demonstrated that the new EASY ROESY experiment yields clean spectra that can directly be integrated to derive interatomic distances.

H. pylori, the bacterium involved in peptic ulcer disease and gastric cancer, survives in the harsh acidic environment of the stomach. It possesses many membrane proteins which mediate adherence, raising the question, if their activity is related to membrane composition. In paper III & IV we analyzed therefore the phospholipid composition of H. pylori membranes.

In paper III, an advanced method for the analysis of the phospholipid composition of biological membranes was developed. The two-dimensional semi-constant-time 31P,1H-COSY experiment combines information from phosphorus and hydrogen atoms of phospholipids for their unambiguous identification. Furthermore, the high resolution of the two-dimensional experiment allows the quantification of phospholipids where conventional methods fail.

In paper IV we applied the new experiment to analyze the lipid composition of whole H. pylori cells, their inner and outer membranes, and of vesicles shed by the bacterium. The goal of this study was to characterize the vesicles which are suggested to play a role in the inflammation process. We established that the outer membrane and the vesicles have similar phospholipid compositions, suggesting that the vesicles are largely derived from the outer membrane.

The NMR results presented here elucidate details of molecular systems engaged in pathogenicity, as basis for therapeutic strategies against these pathogens.

Place, publisher, year, edition, pages
Umeå: , 2009. 48 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1275
Keyword
NMR, regulatory RNA, HBV, H.pylori, phospholipids, ROESY
National Category
Biophysics
Research subject
Biochemistry
Identifiers
urn:nbn:se:umu:diva-25710 (URN)978-91-7264-816-6 (ISBN)
Distributor:
Medicinsk kemi och biofysik, 901 85, Umeå
Public defence
2009-09-26, KB3A9, plan 3, KBC-huset, Umeå Universität, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2009-09-08 Created: 2009-08-31 Last updated: 2009-09-22Bibliographically approved
2. Helicobacter pylori: molecular mechanisms for variable adherence properties
Open this publication in new window or tab >>Helicobacter pylori: molecular mechanisms for variable adherence properties
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

More than half of all people worldwide are infected with H. pylori. The infection always cause a gastric inflammation that may develop into peptic ulcer disease or gastric cancer. Attachment proteins, adhesins, mediate specific adherence of H. pylori to receptor structures on the human gastric mucosa. The best-characterized H. pylori adhesin-receptor interactions are the BabA adhesin and the binding to the fucosylated blood group antigens ABO/Lewis b (Leb) and the SabA adhesin and its binding to the inflammation associated sialyl-Lewis x antigen. During H. pylori infection the availability of receptor structures on the human gastric mucosa changes as a consequence of the host inflammatory and immune responses. Consequently the bacterial population need to adjust its adherence properties to stay colonized. This thesis describes mechanisms that generate H. pylori populations with variable adherence properties and mechanisms for adjustment of adhesin expression levels.In H. pylori strains devoid of Leb-binding, we found bacterial cells with Leb-binding. Isolation of such H. pylori clones demonstrated that the change in receptor binding phenotype was obtained via the mechanisms of homologous recombination and slipped strand mispairing (SSM). Disease presentation in relation to BabA expression was studied in H. pylori infected Mongolian gerbils. We showed that BabA was not essential for colonization but caused severe injury to the gastric mucosa and was turned off during long-term infection by nucleotide changes within the babA gene. Gerbils infected with BabA-weak-expressing strains maintained BabA expressing clones for a longer period than gerbils that were infected with BabA-high-expressing strains. Studies of the gerbil gastric mucosal glycosylation showed that gerbils respond in a similar way as humans and Rhesus monkeys which support gerbils to be a model suitable for studying H. pylori infection and disease outcome in relation to adherence.We studied the SSM mechanism of SabA phase variation and the cognate shift in sLex-binding phenotype and we show sLex-binding activity to be growth phase dependent. H. pylori vesicles were characterized for the major phosholipid and protein components. Virulence factors e.g., VacA, and CagA were identified and both the BabA and the SabA adhesins was shown to be located on the vesicle surface and to mediate specific binding to their cognate receptors present on the human gastric mucosa. H. pylori generate bacterial cells with different receptor binding phenotypes via the mechanisms of homologous recombination, SSM and nucleotide changes. These mechanisms will probably contribute to bacterial fitness by the generation of quasi species populations where some of the clones will be better adapted to the environmental chances during persistent infection.

Place, publisher, year, edition, pages
Umeå: Umeå university, 2009. 57 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1280
Keyword
H. pylori, adherence, BabA, SabA, Leb, sLex, phase variation, recombination, vesicles
Identifiers
urn:nbn:se:umu:diva-25931 (URN)978-91-7264-820-3 (ISBN)
Distributor:
Oral mikrobiologi, 901 87, Umeå
Public defence
2009-10-09, KBC3A9, plan 3, KBC-huset, Umeå Universitet, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2009-09-21 Created: 2009-09-13 Last updated: 2017-05-24Bibliographically approved

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Olofsson, AnnelieVallström, AnnaPetzold, KatjaSchleucher, JürgenCarlsson, SvenNyunt Wai, SunGröbner, GerhardArnqvist, Anna
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