Profiling Helicobacter pylori proteases

Helicobacter pylori (H. pylori) is one of the most successful pathogen that colonizes the human stomach of about 40-50% of the worlds population lifelong. Those persistent infections with H. pylori can lead to severe clinically relevant disorders ranging from ulceration, chronic gastritis, MALT (mucosa-associated lymphoid tissue) lymphomas and gastric cancer in human patients. H. pylori expresses a wide range of well-characterized virulence factors; however, data are accumulating that the activity of proteolytic active enzymes originated from H. pylori actively contributes to pathogenesis. In this project, we will investigate a different set of uncharacterized proteins which has a predicted proteolytic activity and extracellular localization allowing direct modifications of host cell surface molecules. We will define the role of predicted proteases in the bacterial pathogenesis by generation of deletion mutants. Consequences of protease activities on bacterial binding, crossing the epithelium, changes in the gene expression and H. pylori-mediated cell migration will be investigated in state-of-art methods using sophisticated infection models. The proteolytic activity and cleavage sites of recombinant proteins will be investigated and employed in the development of novel assays allowing qualitative and quantitative analyses. Finally, inhibitor design will be applied to block specific protease activity which strongly promotes novel strategies in drug intervention in H. pylori-associated diseases in vivo. Altogether, the proposed project will provide important information on novel mechanisms in H. pylori-induced pathogenesis.

Helicobacter pylori (H. pylori) is one of the most successful pathogens in the human body. Without antibiotic treatment, H. pylori infects the stomach for life and can cause serious disease, including gastric and duodenal ulcers, gastritis and gastric cancer. H. pylori produces a broad spectrum of well-characterized virulence factors, but recent studies indicate a significant influence of H. pylori proteases with a direct impact on disease development. In this project, we investigated a broad spectrum of these enzymes for which a protein-cleaving activity as well as extracellular localization had been predicted, thus allowing a direct interaction with host surface molecules. Further analysis yielded a final list of 19 protease candidates. To investigate their activity and their functional consequences on H. pylori pathogenesis, deletion mutants, recombinant proteins and antibodies were generated. These tools were used to analyze the effects of the proteases on important processes in H. pylori pathogenesis. Based on these data from a comprehensive screening, special focus was put on the characterization of Hp1012, Hp1350, Hp0657 and HtrA. We developed highly sensitive FRET-based protease activity assays, which will be used for high throughput (inhibitor-) screening in the future. Our studies have also shown that the proteases are not only secreted into the environment but are also transported into infected cells via pinched off periplasmatic content in the form of OMVs (outer membrane vesicles). Both, secreted proteases and the OMV-transported proteases are important for the pathogenesis of H. pylori and cleave target molecules of host cells, strongly affecting the epithelial polarity of gastric tumor cells and gastroid-derived primary epithelial cells. The results of this project show that bacterial proteases play a crucial role in H. pylori pathogenesis and represent easily druggable targets for protease inhibitors and thus very attractive candidates for pathoblockers.