Abstract
BackgroundThe serine protease HtrA is an important factor for regulating stress responses and protein quality control in bacteria. In recent studies, we have demonstrated that the gastric pathogen Helicobacter pylori can secrete HtrA into the extracellular environment, where it cleaves-off the ectodomain of the tumor suppressor and adherens junction protein E-cadherin on gastric epithelial cells.ResultsE-cadherin cleavage opens cell-to-cell junctions, allowing paracellular transmigration of the bacteria across polarized monolayers of MKN-28 and Caco-2 epithelial cells. However, rapid research progress on HtrA function is mainly hampered by the lack of ΔhtrA knockout mutants, suggesting that htrA may represent an essential gene in H. pylori. To circumvent this major handicap and to investigate the role of HtrA further, we overexpressed HtrA by introducing a second functional htrA gene copy in the chromosome and studied various virulence properties of the bacteria. The resulting data demonstrate that overexpression of HtrA in H. pylori gives rise to elevated rates of HtrA secretion, cleavage of E-cadherin, bacterial transmigration and delivery of the type IV secretion system (T4SS) effector protein CagA into polarized epithelial cells, but did not affect IL-8 chemokine production or the secretion of vacuolating cytotoxin VacA and γ-glutamyl-transpeptidase GGT.ConclusionsThese data provide for the first time genetic evidence in H. pylori that HtrA is a novel major virulence factor controlling multiple pathogenic activities of this important microbe.
Highlights
The serine protease High temperature requirement protein A (HtrA) is an important factor for regulating stress responses and protein quality control in bacteria
In order to study the function of htrA in more detail, we aimed to overexpress the protein by introduction of a second htrA gene copy of strain 26695 in the chromosome of H. pylori
Hp wt and Hp htrA26695 were grown for 24 h in brain heart infusion (BHI) liquid broth medium containing 10% FCS in the presence or absence of 1 mM or 2 mM IPTG, respectively, and the resulting lysates were checked for expression of HtrA and other well-known H. pylori proteins using Western blotting
Summary
The serine protease HtrA is an important factor for regulating stress responses and protein quality control in bacteria. We have demonstrated that the gastric pathogen Helicobacter pylori can secrete HtrA into the extracellular environment, where it cleaves-off the ectodomain of the tumor suppressor and adherens junction protein E-cadherin on gastric epithelial cells. VacA is an autotransporter and secreted into the extracellular space, where it induces multiple responses including cell vacuolation, alteration of endo-lysosomal trafficking, immune cell inhibition and apoptosis [5, 18]. Other pathogenicityassociated processes comprise urease-triggered neutralization of acidic pH, flagella-mediated motility, expression of multiple adhesins (BabA/B, SabA, AlpA/B, HopQ, HopZ, OipA and others), inhibition of T cell proliferation by secreted γ-glutamyl-transpeptidase GGT, and secretion of proteases such as HtrA [3, 19,20,21]
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