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

葡萄球菌α毒素是由致病性金黄色葡萄球菌分泌的溶细胞的33 K-Da多肽。它与靶细胞膜结合并组装成六聚体，表现出跨膜通道活性。以磷脂酰胆碱（PC）构成的多层脂质体为模型膜，研究了膜流动性对毒素组装过程的影响。我们的研究结果表明，当PC膜通过从凝胶到液晶态的相变或通过在PC膜中包含>30 mol %的胆固醇而流化时，α-毒素通过瞬时的非溶血单体形式组装成六聚体形式。从14个不同杂交瘤株获得的单克隆抗体根据其对α-毒素的结合和溶血活性的抑制作用分为3组。目前正在分析被每种单克隆抗体识别的表位。

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.