Characterization of Helicobacter pyloriHP0231 (DsbK): Role in disulfide bond formation, redox homeostasis and production of Helicobacter cystein-rich protein HcpE

Abstract

Summary: Helicobacter pylori is a human gastric pathogen that colonizes ~50% of the world's population. It can cause gastritis, gastric or duodenal ulcers and also gastric cancer. The numerous side effects of available treatments and the emergence of antibiotic resistant strains are severe concerns that justify further research into H.pylori's pathogenic mechanisms. H.pylori produces secreted proteins that may play a role in virulence, including the Helicobacter cysteine-rich protein HcpE (aka HP0235). We demonstrate herein that HcpE is secreted in the culture supernatant both as a soluble protein and in association with outer membrane vesicles. We show that the structure of HcpE comprises an organized array of disulfide bonds. We identify DsbK (aka HP0231) as a folding factor necessary for HcpE production and secretion in H.pylori and show that recombinant DsbK can interact with and refold unprocessed, reduced HcpEin vitro. These experiments highlight the first biologically relevant substrate for DsbK. Furthermore, we show that DsbK has disulfide bond (Dsb) forming activity on reduced lysozyme and demonstrate a DsbA-type of activity for DsbK upon expression in E.coli, despite its similarity with DsbG. Finally, we show a role of DsbK in maintaining redox homeostasis in H.pylori. We identified DsbK (HP0231) as a folding factor necessary for the production and secretion of the Helicobacter cysteine rich protein HcpE of the gastric human pathogen Helicobacter pylori and showed that recombinant DsbK can interact with and refold unprocessed, reduced HcpE in vitro. We showed that DsbK has disulfide bond forming activity on reduced lysozyme and has a DsbA-type of activity upon expression in E. coli. We also showed a role of DsbK in maintaining redox homeostasis in H. pylori.