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Background High temperature requirement A (HtrA) is an active serine protease secreted by the group-I carcinogen Helicobacter pylori (H. pylori). The human cell adhesion protein and tumor suppressor E-cadherin (hCdh1) expressed on the surface of gastric epithelial cells was identified as the first HtrA substrate. HtrA-mediated hCdh1 cleavage and subsequent disruption of intercellular adhesions are considered as important steps in H. pylori pathogenesis. In this study, we performed a proteomic profiling of H. pylori HtrA (HpHtrA) to decipher the complex mechanism of H. pylori interference with the epithelial barrier integrity. Results Using a proteomic approach we identified human desmoglein-2 (hDsg2), neuropilin-1, ephrin-B2, and semaphorin-4D as novel extracellular HpHtrA substrates and confirmed the well characterized target hCdh1. HpHtrA-mediated hDsg2 cleavage was further analyzed by in vitro cleavage assays using recombinant proteins. In infection experiments, we demonstrated hDsg2 shedding from H. pylori-colonized MKN28 and NCI-N87 cells independently of pathogen-induced matrix-metalloproteases or ADAM10 and ADAM17. Conclusions Characterizing the substrate specificity of HpHtrA revealed efficient hDsg2 cleavage underlining the importance of HpHtrA in opening intercellular junctions.

Helicobacter pylori (H. pylori) secretes the chaperone and serine protease high temperature requirement A (HtrA) that cleaves gastric epithelial cell surface proteins to disrupt the epithelial integrity and barrier function. First inhibitory lead structures have demonstrated the essential role of HtrA in H. pylori physiology and pathogenesis. Comprehensive drug discovery techniques allowing high-throughput screening are now required to develop effective compounds. Here, we designed a novel fluorescence resonance energy transfer (FRET) peptide derived from a gel-based label-free proteomic approach (direct in-gel profiling of protease specificity) as a valuable substrate for H. pylori HtrA. Since serine proteases are often sensitive to metal ions, we investigated the influence of different divalent ions on the activity of HtrA. We identified Zn++ and Cu++ ions as inhibitors of H. pylori HtrA activity, as monitored by in vitro cleavage experiments using casein or E-cadherin as substrates and in the FRET peptide assay. Putative binding sites for Zn++ and Cu++ were then analyzed in thermal shift and microscale thermophoresis assays. The findings of this study will contribute to the development of novel metal ion-dependent protease inhibitors, which might help to fight bacterial infections. (VLID)5311015

Background High temperature requirement A (HtrA) is a widely expressed chaperone and serine protease in bacteria. HtrA proteases assemble and hydrolyze misfolded proteins to enhance bacterial survival under stress conditions. Listeria monocytogenes (L. monocytogenes) is a foodborne pathogen that induces listeriosis in humans. In previous studies, it was shown that deletion of htrA in the genome of L. monocytogenes increased the susceptibility to cellular stress and attenuated virulence. However, expression and protease activity of listerial HtrA (LmHtrA) were never analyzed in detail. Results In this study, we cloned LmHtrA wildtype (LmHtrAwt) and generated a proteolytic inactive LmHtrASA mutant. Recombinant LmHtrAwt and LmHtrASA were purified and the proteolytic activity was analyzed in casein zymography and in vitro cleavage assays. LmHtrA activity could be efficiently blocked by a small molecule inhibitor targeting bacterial HtrA proteases. The expression of LmHtrA was enhanced in the stationary growth phase of L. monocytogenes and significantly contributed to bacterial survival at high temperatures. Conclusions Our data show that LmHtrA is a highly active caseinolytic protease and provide a deeper insight into the function and mechanism, which could lead to medical and biotechnological applications in the future. (VLID)4599705

<> proteolytic activity of "Listeria monocytogenes" HtrA Carmen M. Abfalter, Sabine Bernegger, Miroslaw Jarzab, Gernot Posselt, Karthe Ponnuraj and Silja Wessler Identification of Desmoglein-2 as a novel target of "Helicobacter pylori" HtrA in epithelial cells Sabine Bernegger, Robert Vidmar, Marko Fonovic, Gernot Posselt, Boris Turk, Silja Wessler <> novel FRET peptide assay reveals efficient "Helicobacter pylori" HtrA inhibition through zinc and copper binding Sabine Bernegger, Cyrill Brunner, Matej Vizovišek, Marko Fonovic, Gaetano Cuciniello, Flavia Giordano, Vesna Stanojlovic, Miroslaw Jarzab, Philip Simister, Stephan M. Feller, Gerhard Obermeyer, Gernot Posselt, Boris Turk, Chiara Cabrele, Gisbert Schneider & Silja Wessler submitted by Sabine Bernegger Literaturverzeichnis: Blatt 135-162 Kumulative Dissertation aus drei Artikeln Dissertation Paris-Lodron University of Salzburg 2021 Abstract/Zusammenfassung in deutscher und englischer Sprache