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Characterisation of surface traits of Helicobacter pylori and their role in the infectious process
Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The surface appendages of bacteria determine the initial contact with host cells. Characterisation of functional organisation and spatial distribution of adhesive traits of outer membrane components of Gram-negative bacteria is a key issue in studies of the parasite-host cell interaction.

With focus on the enteropathogenic Helicobacter pylori, evidenced to cause chronic gastric infections in humans, detergent-digested freeze fracture replica labelling was applied for ultrastructural analyses of envelope distribution of the virulence factors blood group antigen binding adhesin (BabA), and the carbonic anhydrases (α-CA, ß-CA). In a preliminary study the methodology was also used to study the bacteria-host contact between phagocytosing human neutrophils and wild-type H. pylori.

In parallel, bacterial traits were analysed from a molecular and biochemical perspective. This included the specific roles of the BabA and the sialic acid-binding adhesin (SabA), and the neutrophil activating protein (HP-NAP) in neutrophilic stimulation and subsequent inflammatory process. It was concluded that SabA is crucial in the initiation of a neutrophilic response in the mediated inflammation.

This thesis has demonstrated the synergistic application of ultrastructural, molecular and cellular microbiology tools for delineating complex patterns in bacteria-host interactions, thus utilising the well-characterised and clinically important human pathogen H. pylori. This approach could be applicable to other Gram-negative species to clarify known and discern new virulence mechanisms in the multifaceted field of bacterial pathogenesis and bacterial interactions with human host cells.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2003. , 77 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 805
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-26665Local ID: 11231ISBN: 91-7373-490-X (print)OAI: oai:DiVA.org:liu-26665DiVA: diva2:247214
Public defence
2003-10-03, Elsa Brändströmsalen, Hälsouniversitetet, Linköping, 09:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-10-15Bibliographically approved
List of papers
1. A new method to visualize the helicobacter pylori-associated lewisb-binding adhesin utilizing SDS-digested freeze-fracture replica labeling
Open this publication in new window or tab >>A new method to visualize the helicobacter pylori-associated lewisb-binding adhesin utilizing SDS-digested freeze-fracture replica labeling
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2000 (English)In: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 48, no 6, 877-883 p.Article in journal (Refereed) Published
Abstract [en]

Freeze-fracture replica labeling has become a versatile tool to visualize both membrane components and other cell structures using SDS-replica cleaning before specific immunogold labeling of proteins or lipids. We report here for the first time the adoption and optimization of the method to studies of bacterial envelopes, as applied to structural analysis of the distribution of the unique BabA-adhesin of the gastric pathogen Helicobacter pylori. BabA is important for bacterial adherence to the human epithelial cell lining of the stomach. The adhesin was found to be distributed all over the bacterial cell surfaces. Our results suggest that the SDS-replica labeling allows assessment of protein localization to distinct cell compartments and analysis of co-localization with neighboring membrane structures.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26007 (URN)10.1177/002215540004800616 (DOI)10460 (Local ID)10460 (Archive number)10460 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
2. The blood group antigen binding activity of the Helicobacter pylori baba adhesin is regulated by local ph and redox potential
Open this publication in new window or tab >>The blood group antigen binding activity of the Helicobacter pylori baba adhesin is regulated by local ph and redox potential
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The blood group antigen hinding adhesin, BabA, which binds to fucosylated blood group antigens, such as the Lewis b (Leb) and H1 antigens constitutes one of the best recognized adhesin-receptor interactions that mediate adherence of Helicobacter pylori to the gastric epithelium. BabA belongs to a family of H. pylori outer membrane, proteins (HOPs), a group of some 30 proteins with most similar N- and C-terminal domains.

We previously identified the babA1 and babA2 genes, where babA2 was found to encode the BabA adhesin in strain CCUG17875. Here, we confirmed the identity of the BabA protein by immunoblot-analysis, followed by MALDIT-OF MS analysis, which also provided molecular weight of the BabA polypeptide and the unique peptide sequences for BabA. Surprisingly, the BabA protein was found to be expressed 50-fold higher compared to the number of calculated bacterial Leb-binding sites.

Furthermore, surface scan of the bacterial membrane by freeze fracture immuno-EM technique localized the BabA by immunogold labeling to the  bacterial surface in numbers similar to the predicted binding sites. To help explain the binding results, crosslinker-analyses were performed which revealed that BabA form supra-molecular complexes on the bacterial surfaces. In addition, binding to the Lewis b antigen was shown to be pH dependent and took place over a broad pH range but binding activity was reversibly lost when approaching pH 3, i.e. conditions similar to the acidic gastric juice.

The binding activity of the BabA adhesin was shown to be sensitive for reducing conditions, which suggests the presence of disulfide bond(s) close to the carbohydrate-binding domain. The dynamics of BabA in Leb-binding suggest that the bacterial adhesin is regulated by local variations in pH and redox potential, such as the pH gradient in the slimy mucus lining of the epithelium, and the reduced conditions of the inflamed gastric mucosa.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-84616 (URN)
Available from: 2012-10-15 Created: 2012-10-15 Last updated: 2012-10-15Bibliographically approved
3. Expression and localization of α- and β-carbonic anhydrase in Helicobacter pylori
Open this publication in new window or tab >>Expression and localization of α- and β-carbonic anhydrase in Helicobacter pylori
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2002 (English)In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1601, no 2, 192-199 p.Article in journal (Refereed) Published
Abstract [en]

Helicobacter pylori, the causative agent of peptic ulcer disease, expresses two different forms of the zinc-containing enzyme carbonic anhydrase (CA) (α and β), catalyzing the reversible hydration of CO2. Presumably, the high CO2 requirement of H. pylori implies an important role for this enzyme in the bacterial physiology. In this paper, expression of the CAs has been analyzed in three different strains of the bacterium, 26695, J99 and 17.1, and appears to be independent of CO2 concentration in the investigated range (0.1–10%). Presence of the potent and highly specific CA inhibitor, acetazolamide, in the medium does not seem to inhibit bacterial growth at the given sulfonamide concentration. Moreover, the localization and distribution of the α-CA was analyzed by immunonegative staining, while SDS-digested freeze-fracture immunogold labelling was used for the β-form of the enzyme. The latter method has the advantage of allowing assessment of protein localization to distinct cell compartments and membrane structures. The resulting electron microscopy images indicate a localization of the β-CA in the cytosol, on the cytosolic side of the inner membrane and on the outer membrane facing the periplasmic space. The α-enzyme was found attached to the surface of the bacterium.

Keyword
Carbonic anhydrase, Expression, Localization
National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-46779 (URN)10.1016/S1570-9639(02)00467-3 (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13Bibliographically approved
4. Helicobacter pylori sabA adhesin in persistent infection and chronic inflammation
Open this publication in new window or tab >>Helicobacter pylori sabA adhesin in persistent infection and chronic inflammation
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2002 (English)In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 297, no 5581, 573-578 p.Article in journal (Refereed) Published
Abstract [en]

Helicobacter pylori adherence in the human gastric mucosa involves specific bacterial adhesins and cognate host receptors. Here, we identify sialyl-dimeric-Lewis x glycosphingolipid as a receptor for H. pylori and show thatH. pylori infection induced formation of sialyl-Lewis x antigens in gastric epithelium in humans and in a Rhesus monkey. The corresponding sialic acid–binding adhesin (SabA) was isolated with the “retagging” method, and the underlyingsabA gene (JHP662/HP0725) was identified. The ability of many H. pylori strains to adhere to sialylated glycoconjugates expressed during chronic inflammation might thus contribute to virulence and the extraordinary chronicity of H. pylori infection.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26539 (URN)10.1126/science.1069076 (DOI)11100 (Local ID)11100 (Archive number)11100 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
5. Helicobacter pylori sabA adhesin evokes a strong inflammatory response in human neutrophils which is down-regulated by the neutrophil-activating protein
Open this publication in new window or tab >>Helicobacter pylori sabA adhesin evokes a strong inflammatory response in human neutrophils which is down-regulated by the neutrophil-activating protein
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2006 (English)In: Medical Microbiology and Immmunology, ISSN 0300-8584, E-ISSN 1432-1831, Vol. 195, no 4, 195-206 p.Article in journal (Refereed) Published
Abstract [en]

The human pathogen Helicobacter pylori expresses two dominant adhesins; the Lewis b blood group antigen binding adhesin, BabA, and the sialic acid-binding adhesin, SabA. These adhesins recognize specific carbohydrate moieties of the gastric epithelium, i.e. the Lewis b antigen, Leb, and the sialyl-Lewis x antigen, sLex, respectively, which promote infection and inflammatory processes in the gastroduodenal tract. To assess the contribution of each of BabA, SabA and the neutrophil activating protein (HP-NAP) in a local inflammation, we investigated the traits of H. pylori mutants in their capacity to interact with and stimulate human neutrophils. We thence found that the SabA adhesin was not only the key inducer of oxidative metabolism (Unemo et al. J Biol Chem 280:15390–15397, 2005), but also essential in phagocytosis induction, as evaluated by flow cytometry, fluorescence microscopy and luminol-enhanced chemiluminescence. The napA deletion resulted in enhanced generation of reactive oxygen species and impaired adherence to the host cells. In conclusion, the SabA adhesin stimulates human neutrophils through selectin-mimicry. Interestingly, HP-NAP modulates the oxidative burst, which could tune the impact of the H. pylori infection for establishment of balanced and chronic inflammation of the gastric mucosa.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-38156 (URN)10.1007/s00430-006-0018-x (DOI)42125 (Local ID)42125 (Archive number)42125 (OAI)
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2017-12-13Bibliographically approved
6. Advances in freeze-fracture replication for assessing immune interactions between Helicobacter pylori and human neurophils
Open this publication in new window or tab >>Advances in freeze-fracture replication for assessing immune interactions between Helicobacter pylori and human neurophils
(English)Manuscript (preprint) (Other academic)
Abstract [en]

A freeze-fracture replica labeling method adapted for studies of bacterial envelopes was recently introduced. This report describes a further development of this detergent-digested freeze-fracture replica labeling technique, enabling direct visualization of bacteria-host interaction, specifically between the gastric pathogen Helicobacter pylori and human neutrophils. The phagocytic process performed by the neutrophilic leukocytes represents a crucial element of the host defense system against invading microorganisms. The present methodology can be used as a tool that allows characterization of the molecular and ultrastructural events that take place between pathogenic microbes and professional phagocytes.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-84621 (URN)
Available from: 2012-10-15 Created: 2012-10-15 Last updated: 2012-10-15Bibliographically approved

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