Helicobacter pylori cell surface interactions with glycosaminoglycans. Identification and characterisation of proteins binding to heparin/heparan sulphate
Sammanfattning: Helicobacter pylori is a gastric pathogen which cause chronic type B gastritis and peptic ulcer disease. H. pylori produces virulence factors such as urease, vacuolating cytotoxin VacA, cag pathogenicity island-associated proteins, flagella and adhesins. These proteins interact with several host cell molecules such as sialylated cell surface glycoproteins, some of glycolipids and mucins. Glycosaminoglycans are widely distributed molecules in human cells and extracellular matrix and interact with H. pylori cell surface proteins. Heparan sulphate shows a pH-dependent binding to all strains, which is inhibited by sulphated glycosaminoglycans and other polysulphated molecules such as dextran sulphate and fucoidan. Using bioinformatics methods, the binding of H. pylori VacA cytotoxin to heparin and heparan sulphate was predicted. Specific peptides were synthesised and their interactions with surface-immobilised heparan sulphate studied in a BIAcore biosensor. Synthetic peptides bound to heparan sulphate with a moderate affinity. It was shown that native toxin interacts with immobilised heparin. Heparan sulphate was proposed as a receptor/co-receptor for VacA cytotoxin binding to host cell surfaces. H. pylori urease binds to surface-immobilised heparin and heparan sulphate. The interaction was characterised using a BIAcore biosensor. The binding was a thousand-fold higher at pH 5.5 compared to pH 6.5. The binding epitopes were identified by SELDI-TOF MS technology. Using a proteomic technology, four immunogenic heparin-binding proteins were identified: cell binding factor 2, urease, one outer membrane protein and one hypothetical protein. The binding of three of these proteins to heparin was demonstrated for the first time. The cell binding factor was identified as a specific and highly immunogenic H. pylori surface protein. 2-DE and 2-D immunoblotting allow efficient separation and identification of immunogenic proteins. Understanding how GAGs interact with pathogens, will allow the design of new experimental putative anti-microbial compounds based on sulphated polysaccharide structures.
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