3D imaging of the 58 kDa cell binding subunit of the Helicobacter pylori cytotoxin

Pathogenic strains of Helicabacter pylori produce a potent exotoxin, VacA, which intoxicates gastric epithelial cells and leads to peptic ulcer. The t oxin is released from the bacteria as a high molecular mass homo-oligomer o f a 95 kDa polypeptide which undergoes specific proteolytic cleavage to 37 kDa and 58 kDa subunits. We have engineered a strain of H. pylori to delete the gene sequence coding for the 37 kDa subunit. The remaining 58 kDa subu nit is expressed efficiently and exported as a soluble dimer that is non-to xic but binds target cells in a manner similar to the holotoxin. A 3D recon struction of the molecule from electron micrographs of quick-freeze, deep-e tched preparations reveals the contribution of each building block to the s tructure and permits the reconstruction of the oligomeric holotoxin startin g from individual subunits. In this model P58 subunits are assembled in a r ing structure with P37 subunits laying on the top. The data indicate that t he 58 kDa subunit is capable of folding autonomously into a discrete struct ure recognizable within the holotoxin and containing the cell binding domai n. (C) 1999 Academic Press.