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

Sabine Bernegger,Miroslaw Jarzab,Marko Fonovic,Gerhard Obermeyer,Vesna Stanojlovic,Gaetano Cuciniello,Chiara Cabrele,Cyrill Brunner,Philip Simister,Silja Wessler,Boris Turk,Matej Vizovišek,Gisbert Schneider,Gernot Posselt,Stephan M Feller,Flavia Giordano

doi:10.1038/s41598-020-67578-2

Abstract

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.

Highlights

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
The detected characteristic specificity profile AR/QRV↓AY corresponds well with the signature site [VITA]↓[VITA]-x-x-D-[DN] previously identified in the HtrA substrate E-cadherin and verifies the preference of HpHtrA to cleave between hydrophobic amino a cids[11]
The sequence AR/QRV↓AY resembles the AQPVEA linker region between the EC5 and the transmembrane domain (TMD) of E-cadherin that has already been suggested as a preferred cleavage site in E-cadherin for HpHtrA during the infection

Summary

Introduction

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. The finding that the serine protease high temperature requirement A (HtrA) expressed by H. pylori targets cell surface proteins of infected host cells added an important aspect to the model of H. pylori pathogenesis. The EC domain consists of the five tandem repeats EC1–EC5 with interspaced calcium-binding motifs, which are required for functional homophilic cis and trans interactions of E-cadherin between epithelial cells[6]. These sites have been identified as preferred signature motifs for H. pylori HtrA11. HtrA paves the intercellular way for H. pylori to transmigrate across the epithelial layer and to facilitate β1-integrinmediated delivery of the bacterial oncoprotein cytotoxin-associated gene A (CagA)[4,13]

Methods

Results

Conclusion