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Helicobacter pylori: molecular insights into regulation of adhesion properties
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. (Anna Arnqvist)
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Helicobacter pylori infects the human stomach and triggers an inflammatory response that damages the gastric tissue. This host-pathogen interplay has dire consequences as up to 20 % of infected individuals develop peptic ulcer disease or gastric cancer. Given that half of the world’s population is infected, the number of afflicted humans is staggering and also tells that H. pylori is extremely efficient in spreading and maintaining infection. To enable persistent infection many factors play a role, but one important feature of H. pylori is its impressive ability to adhere to the slimy gastric mucus layer and the underlying epithelial cells. This occurs mainly via the BabA and SabA proteins that bind ABO/Leb- and sLex/sLea-antigens. I have in my thesis studied how these two proteins are utilized and regulated.

H. pylori transcription is in part controlled by two-component systems (TCSs) that use a sensor protein and a DNA-binding response regulator. We have studied how these systems control sabA and to some extent babA and indeed found a better map of how sabA and babA is regulated at the transcriptional level. We also found that variations in a polynucleotide T-tract located in the sabA promotor could fine-tune SabA expression/ sLex-binding. Thus we have exposed how strict regulation by TCSs combined with stochastic processes together shapes attachment in the bacterial population.

As the buffering mucus layer is constantly exfoliated, placing H. pylori in bactericidal acid, we hypothesized that low pH should abrogate adhesion. SabA expression was indeed repressed in low pH, however BabA expression remained unaffected. The BabA/ Leb-binding was instead directly reversibly hampered by low pH and the degree of pH sensitivity was strain dependent and encoded in the BabA sequence. We believe that the pH dependent loss of binding is one key factor H. pylori utilizes to maintain persistent infection.

BabA is divided in generalists that bind ABO antigens and specialists that only bind blood group (bg) O. We co-crystalized BabA bound to these receptors and established the structural basis for generalist vs. specialist discrimination. We furthermore found a disulfide-clasped loop (CL2) in the center of the binding domain crucial for binding. Breaking CL2 with N-Acetylcysteine (NAC) disrupted binding and H. pylori infection mice experiments revealed inflammatory reduction upon NAC-treatment.

In sum, I have in my thesis dissected how H. pylori controls its adhesive abilities and how intrinsic properties in binding can be exploited for therapeutic purposes.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2016. , 53 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1813
Keyword [en]
Helicobacter pylori
National Category
Microbiology in the medical area
Research subject
Medical Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-120466ISBN: 978-91-7601-493-6 (print)OAI: oai:DiVA.org:umu-120466DiVA: diva2:928853
Public defence
2016-06-09, N300, Umeå universitet, Naturvetarhuset, Umeå, 09:00 (English)
Opponent
Supervisors
Funder
The Kempe Foundations
Available from: 2016-05-19 Created: 2016-05-16 Last updated: 2016-05-26Bibliographically approved
List of papers
1. A Repetitive DNA Element Regulates Expression of the Helicobacter pylori Sialic Acid Binding Adhesin by a Rheostat-like Mechanism
Open this publication in new window or tab >>A Repetitive DNA Element Regulates Expression of the Helicobacter pylori Sialic Acid Binding Adhesin by a Rheostat-like Mechanism
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2014 (English)In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 10, no 7, e1004234Article in journal (Refereed) Published
Abstract [en]

During persistent infection, optimal expression of bacterial factors is required to match the ever-changing host environment. The gastric pathogen Helicobacter pylori has a large set of simple sequence repeats (SSR), which constitute contingency loci. Through a slipped strand mispairing mechanism, the SSRs generate heterogeneous populations that facilitate adaptation. Here, we present a model that explains, in molecular terms, how an intergenically located T-tract, via slipped strand mispairing, operates with a rheostat-like function, to fine-tune activity of the promoter that drives expression of the sialic acid binding adhesin, SabA. Using T-tract variants, in an isogenic strain background, we show that the length of the T-tract generates multiphasic output from the sabA promoter. Consequently, this alters the H. pylori binding to sialyl-Lewis x receptors on gastric mucosa. Fragment length analysis of post-infection isolated clones shows that the T-tract length is a highly variable feature in H. pylori. This mirrors the host-pathogen interplay, where the bacterium generates a set of clones from which the best-fit phenotypes are selected in the host. In silico and functional in vitro analyzes revealed that the length of the T-tract affects the local DNA structure and thereby binding of the RNA polymerase, through shifting of the axial alignment between the core promoter and UP-like elements. We identified additional genes in H. pylori, with T- or A-tracts positioned similar to that of sabA, and show that variations in the tract length likewise acted as rheostats to modulate cognate promoter output. Thus, we propose that this generally applicable mechanism, mediated by promoter-proximal SSRs, provides an alternative mechanism for transcriptional regulation in bacteria, such as H. pylori, which possesses a limited repertoire of classical trans-acting regulatory factors.

National Category
Physiology Physical Chemistry
Identifiers
urn:nbn:se:umu:diva-91641 (URN)10.1371/journal.ppat.1004234 (DOI)000340551000026 ()24991812 (PubMedID)
Available from: 2014-08-13 Created: 2014-08-13 Last updated: 2017-12-05Bibliographically approved
2. Structural Insights into Polymorphic ABO Glycan Binding by Helicobacter pylori
Open this publication in new window or tab >>Structural Insights into Polymorphic ABO Glycan Binding by Helicobacter pylori
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2016 (English)In: Cell Host and Microbe, ISSN 1931-3128, E-ISSN 1934-6069, Vol. 19, no 1, 55-66 p.Article in journal (Refereed) Published
Abstract [en]

The Helicobacter pylori adhesin BabA binds mucosal ABO/Le b blood group (bg) carbohydrates. BabA facilitates bacterial attachment to gastric surfaces, increasing strain virulence and forming a recognized risk factor for peptic ulcers and gastric cancer. High sequence variation causes BabA functional diversity, but the underlying structural-molecular determinants are unknown. We generated X-ray structures of representative BabA isoforms that reveal a polymorphic, three-pronged Le(b) binding site. Two diversity loops, DL1 and DL2, provide adaptive control to binding affinity, notably ABO versus O bg preference. H. pylori strains can switch bg preference with single DL1 amino acid substitutions, and can coexpress functionally divergent BabA isoforms. The anchor point for receptor binding is the embrace of an ABO fucose residue by a disulfide-clasped loop, which is inactivated by reduction. Treatment with the redox-active pharmaceutic N-acetylcysteine lowers gastric mucosal neutrophil infiltration in H. pylori-infected Le(b)-expressing mice, providing perspectives on possible H. pylori eradication therapies.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-117839 (URN)10.1016/j.chom.2015.12.004 (DOI)000369839900010 ()26764597 (PubMedID)
Available from: 2016-04-04 Created: 2016-03-04 Last updated: 2017-11-30Bibliographically approved
3. Acid Responsive Helicobacter pylori Adherence: Implications for Chronic Infection and Disease
Open this publication in new window or tab >>Acid Responsive Helicobacter pylori Adherence: Implications for Chronic Infection and Disease
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(English)Manuscript (preprint) (Other academic)
Keyword
Helicobacter pylori
National Category
Microbiology in the medical area
Research subject
Medical Biochemistry
Identifiers
urn:nbn:se:umu:diva-120299 (URN)
Funder
Swedish Research CouncilSwedish Cancer SocietyThe Kempe FoundationsKnut and Alice Wallenberg Foundation
Available from: 2016-05-14 Created: 2016-05-14 Last updated: 2016-05-27
4. The Helicobacter pylori sialic acid binding adhesin SabA is regulated via a network of two-component systems
Open this publication in new window or tab >>The Helicobacter pylori sialic acid binding adhesin SabA is regulated via a network of two-component systems
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The acid-responsive signaling system ArsRS plays a key role in regulating factors important for survival in acidic conditions during infection of the human stomach by Helicobacter pylori. In addition, ArsRS was suggested to control the disease-associated attachment protein SabA, however, mechanistic data is still lacking. We show that the repressing effect of the ArsRS system on SabA expression occurs both at acidic and neutral conditions and is mediated at the transcriptional level. Purified His6-ArsR binds PsabA DNA at several sites, with varying affinity and independent of phosphorylation status and H. pylori strains showed unique cognate PsabA sequences to tweak the ArsR binding ability, resulting in strain-dependent repression of SabA expression. By site-directed mutagenesis we reveal key amino acids for the binding activity of ArsR. Finally, we show that that ArsR binds to A/T-rich DNA as dimers or larger multimers, suggesting that ArsR has affinity for DNA structures rather than to a specific promoter DNA sequence. SabA expression is further influenced by the FlgRS and CrdRS two-component systems, illustrating a complicated crosstalk among regulatory networks in H. pylori.

Keyword
Helicobacter pylori
National Category
Microbiology in the medical area
Research subject
Medical Biochemistry
Identifiers
urn:nbn:se:umu:diva-120293 (URN)
Funder
Swedish Research Council, K2009-56X-20037-04-3 (VR/NT)Swedish Cancer Society, 09 0641The Kempe Foundations
Available from: 2016-05-14 Created: 2016-05-14 Last updated: 2016-05-18

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