Chaperone activity of serine protease HtrA of Helicobacter pylori as a crucial survival factor under stress conditions
| Autor: | Urszula Zarzecka, Joanna Skorko-Glonek, Aileen Harrer, Anna Zawilak-Pawlik, Steffen Backert |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
Stress endurance
medicine.medical_treatment Virulence lcsh:Medicine Chaperone Protein aggregation E- cadherin Biochemistry complex mixtures Virulence factor 03 medical and health sciences chemistry.chemical_compound Stress Physiological ddc:570 medicine Tumor Cells Cultured Humans lcsh:QH573-671 Molecular Biology 030304 developmental biology Serine protease 0303 health sciences Protease biology Secreted proteins Helicobacter pylori 030306 microbiology Chemistry lcsh:Cytology Research lcsh:R Cell Biology Naturwissenschaftliche Fakultät Hydrogen-Ion Concentration bacterial infections and mycoses Protein quality control system HtrA Secretory protein Puromycin Chaperone (protein) biology.protein bacteria Serine Proteases Molecular Chaperones Signal Transduction |
| Zdroj: | Cell Communication and Signaling, Vol 17, Iss 1, Pp 1-18 (2019) Cell Communication and Signaling : CCS |
| Popis: | Background Serine protease HtrA exhibits both proteolytic and chaperone activities, which are involved in cellular protein quality control. Moreover, HtrA is an important virulence factor in many pathogens including Helicobacter pylori, for which the crucial stage of infection is the cleavage of E-cadherin and other cell-to-cell junction proteins. Methods The in vitro study of H. pylori HtrA (HtrAHp) chaperone activity was carried out using light scattering assays and investigation of lysozyme protein aggregates. We produced H. pylori ∆htrA deletion and HtrAHp point mutants without proteolytic activity in strain N6 and investigated the survival of the bacteria under thermal, osmotic, acidic and general stress conditions as well as the presence of puromycin or metronidazole using serial dilution tests and disk diffusion method. The levels of cellular and secreted proteins were examined using biochemical fraction and Western blotting. We also studied the proteolytic activity of secreted HtrAHp using zymography and the enzymatic digestion of β-casein. Finally, the consequences of E-cadherin cleavage were determined by immunofluorescence microscopy. Results We demonstrate that HtrAHp displays chaperone activity that inhibits the aggregation of lysozyme and is stable under various pH and temperature conditions. Next, we could show that N6 expressing only HtrA chaperone activity grow well under thermal, pH and osmotic stress conditions, and in the presence of puromycin or metronidazole. In contrast, in the absence of the entire htrA gene the bacterium was more sensitive to a number of stresses. Analysing the level of cellular and secreted proteins, we noted that H. pylori lacking the proteolytic activity of HtrA display reduced levels of secreted HtrA. Moreover, we compared the amounts of secreted HtrA from several clinical H. pylori strains and digestion of β-casein. We also demonstrated a significant effect of the HtrAHp variants during infection of human epithelial cells and for E-cadherin cleavage. Conclusion Here we identified the chaperone activity of the HtrAHp protein and have proven that this activity is important and sufficient for the survival of H. pylori under multiple stress conditions. We also pinpointed the importance of HtrAHp chaperone activity for E- cadherin degradation and therefore for the virulence of this eminent pathogen. |
| Databáze: | OpenAIRE |
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