Characterization of the zinc-metalloprotease activity of Helicobacter pylori HomB and its possible role in driving inflammatory responses

• Applicant: Professor Dr. Franco Harald Falcone
• Subject Area: Medical Microbiology and Mycology, Hygiene, Molecular Infection Biology ; Immunology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 504974429

H. pylori is a widespread bacterium which causes gastric inflammation, ranging from subclinical to gastric ulcers and cancer. HomB has been suggested to be a virulence factor associated with peptic ulcer disease and gastric cancer. It is known that HomB-deficient bacterial strains have a reduced ability to induce the key pro-inflammatory cytokine Interleukin-8 (IL-8) from epithelial cells. It is also known that H. pylori can elicit IL-8 via the protease activated receptor 2 (PAR-2), but the molecular identity of the underlying proteases is unknown. We have obtained robust preliminary evidence indicating that HomB is a metalloprotease which can induce IL-8 from gastric epithelial cells. Furthermore, it has been suggested that HomB is involved in adhesion to gastric epithelial cells, but no ligands have been suggested.Our aim is to close the knowledge gaps by fully characterizing the metzincin metalloprotease activity of HomB and its paralogs, and by assessing the ability of recombinant HomB to bind to gastric cell lines via glycans. This will be achieved by:- Recombinant expression of HomA and HomB wildtype and active site mutants - Full biochemical characterization of inhibition by metalloprotease and other protease inhibitors - Demonstration of PAR-2 activation by creating a PAR-2 stable transfectant cell line - In silico screening of MCCC chemical compound libraries and in vitro assessment of ability to inhibit IL-8 induction- Clarification of adhesin capability and identification of the putative ligand on epithelial cells using glycan arrays, CaR-ESI/MS and ELISA-type assessments, and assessment of binding to gastric epithelial cell lines using fluorescently labelled HomB

• DFG Programme: Research Grants
• International Connection: Canada
• Cooperation Partner: Professor Dr. John S. Klassen