Chaperone activity of serine protease HtrA of Helicobacter pylori as a crucial survival factor under stress conditions

Abstract

BackgroundSerine 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.MethodsThe 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.ResultsWe 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.ConclusionHere 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.

Pan-genome analysis and ancestral state reconstruction of class halobacteria: probability of a new super-order

“…Chaperone serine proteases and monothiol glutaredoxin were also acquired. A very recent research on serine protease in helicobacter pyori has claimed to function under stress conditions 58 . Membrane trafficking genes such as ArsR family transcriptional regulator, arsenate/arsenite/antimonite-responsive transcriptional repressor and CU + /H + antiporter were also acquired giving them resistance to high metal toxicity.…”

Helicobacter pylori Virulence Factors—Mechanisms of Bacterial Pathogenicity in the Gastric Microenvironment

“…Moreover, excessive amounts of calcium ions seem to prevent the HtrA-related E-cadherin cleavage along with the additional stabilization of the junctions [ 358 ]. Furthermore, H. pylori strains that are deprived of HtrA are much more sensitive to particular conditions, including elevated temperature, osmotic shock, or treatment with puromycin [ 359 , 360 ]. Indeed, HtrA constitutes a crucial protective factor that prevents H. pylori from numerous stress conditions, enabling effective bacterial colonization within the gastric mucosa.…”

A novel FRET peptide assay reveals efficient Helicobacter pylori HtrA inhibition through zinc and copper binding

“…6A, left panel, lane 2), suggesting direct binding of Zn ++ to HpHtrA. Using a thermal shift assay, we first determined the melting temperature of about 75 °C for HpHtrA wt, which is quite high and indicates an enhanced stability that enables survival under stress conditions 30,49 . Mutation of D 165 and S 166 resulted in an increase in the thermal denaturation of HpHtrA, while S 164 and D 168 mutations did not alter stability ( Fig.…”

“…The development of a substrate-derived peptide inhibitor further emphasized the significance of HtrA activity in H. pylori pathogenesis 11 . Importantly, bacterial HtrA also functions as a chaperone that refolds and degrades misfolded proteins under stress conditions 30 . Due to this important function in bacterial physiology, HtrA expression is essential for H. pylori.…”