Staphylococcus aureus α-Toxin Crystals on Lipid Layers: Effects of Trypsin Treatment

Abstract

α-toxin is a lethal, dermonecrotic, cytotoxic, and hemolytic protein which is secreted by most strains of Staphylococcus aureus . The biological effects are brought about by pertubation of membrane structures making them permeable to ions and small molecules. The structural basis for the mode of action is not known in detail. One hypothesis is that an oligomeric form of the toxin, observed both biochemically and by electron microscopy, circumscribe a hydrophilic pore through which transport can occur. Recent functional studies have demonstrated that oligomerization is a necessary but not a sufficient criterion for permeabilization. For example, trypsin treated α-toxin was, despite its lack of membrane damaging activity, able to form 200 kDa aggregates on mouse adrenocortical (Yl) tumor cells. In this work we have compared the structure of trypsin treated α-toxin with that of the intact toxin.α-toxin was purified from S. aureus strain wood 46 by isoelectric focussing and gel filtration. Tryptic toxin fragments (18 and 17 kD as determined by SDS-PAGE) were generated by digestion of native α-toxin (1 mg in 1.5 ml Tris buffered saline) with 30μg trypsin for 4 h at 37°C, pH 8.0. The reaction was terminated by the addition of 30μg lima beam trypsin inhibitor. Toxin specimens were applied to lipid layers pre-formed on carbon support films. Electron microscopy was performed after negative staining either with 1% Na-PTA (pH 7.0) or with a mixture of Na-PTA and glucose. Subsequent image processing of large coherent crystalline arrays produced correlation averaged projection maps. A three-dimensional model was obtained by collecting and combining data from tilted views.