MODE OF PRIMARY BINDING TO TARGET MEMBRANES AND PORE FORMATION INDUCED BY VIBRIO-CHOLERAE CYTOLYSIN (HEMOLYSIN)

Vibrio cholerae cytolysin (VCC) is produced by many non-choleratoxigen ic strains of V. cholerae, and possibly represents a relevant pathogen icity determinant of these bacteria. The protein is secreted as a pro- toxin that is proteolytically cleaved to yield the active toxin with a molecular mass of approximately 63 kDa. We here describe a simple pro cedure for preparative isolation of mature VCC from bacterial culture supernatants, and present information on its mode of binding and pore formation in biological membranes. At low concentrations, toxin monome rs interact with a high-affinity binding site on highly susceptible ra bbit erythrocytes. This as yet unidentified binding site is absent on human erythrocytes, which are less susceptible to the toxin action. At higher concentrations, binding of the toxin occurs to both rabbit and human erythrocytes in a non-saturable manner. Cell-bound toxin monome rs oligomerize to form supramolecular structures that are seen in the electron microscope as apparently hollow funnels, and oligomerization correlates functionally with the appearance of small transmembrane por es. Osmotic protection experiments indicate that the toxin channels ar e of finite size with a diameter of 1-2 nm. The mode of action of VCC closely resembles that of classical pore-forming toxins such as staphy lococcal a-toxin and the aerolysin of Aeronomonas hydrophila.