Study on the Membrane-damaging Action of Staphylococcal Alpha-toxin by use of Multilamellar Liposomes.

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Staphylococcal alpha-toxin is a cytolytic 33K-Da polypeptide secreted by pathogenic Staphylococcus aureus. It binds to the membrane of target cells and assembles to hexamer, exhibiting transmembrane-channel activitiy. The toxin is considered to be a prototype for transmembrane-pore-forming proteins including streptolysin O, complement and perforin.By use of multilamellar liposome composed of Phosphatidyl-Choline (PC) as a model membrane, we studied influence of membrane fluidity on the assembly process of the toxin. Our results indicated that alpha-toxin assembled into hexameric form via a transient, nonhemolytic-monomer form, when PC membrane was fluidized either by the phase transition from gel to liquid crystalline state or by the inclusion of >30 mol % cholesterol in the PC membrane.To determine the domain of alpha-toxin which is involved in each step of the assembly process, we prepared monoclonal antibodies to the toxin. Monoclonal antibodies obtained from 14 different hybridoma strains were categorized to 3 groups according to their inhibitory action on binding and hemolytic activity of alpha-toxin. Analysis of the epitope which is recognized by each of the monoclonal antibodies is now in progress.