Oligomerisation of cell-bound staphylococcal alpha-toxin in relation to membrane permeabilisation.
We have studied the kinetics of staphylococcal alpha-toxin oligomerisation in relation to membrane permeabilisation, using as targets cultured adrenocortical Y1 cells, rabbit red blood cells (RRBC), human platelets, and liposomes prepared of lipids extracted from platelets. After isolation of membranes from toxin-treated cells, oligomeric toxin was detected (i) by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography or Western blotting, and (ii) by electron microscopy of negatively stained specimens. alpha-Toxin was found to oligomerise on all membranes independently of the temperature. On RRBC and Y1 cells most of the membrane associated toxin appeared converted to the oligomeric form. Hexamers were always present along with membrane permeabilisation. However, hexamers were also detected at conditions when membrane permeabilisation did not occur; at low temperature, in the presence of high concentrations of Ca2+, and after pretreatment of cells with concanavalin A (Con A). Addition of a neutralising monoclonal antibody (MAb) to cell-bound toxin collected it into aggregates much larger than the hexamers. By contrast hexameric toxin remained after addition of a non-neutralising MAb. Our data suggest that the active toxin species is not monomeric, and support the hypothesis that alpha-toxin permeabilises membranes by forming hexameric protein-lined transmembrane channels.
Thelestam
M
M
Department of Bacteriology, Karolinska Institutet, Stockholm, Sweden.
Olofsson
A
A
Blomqvist
L
L
Hebert
H
H
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects*; D003208:Concanavalin A / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism; D006460:Hemolysin Proteins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D010446:Peptide Fragments / Q000378:metabolism; D011817:Rabbits; D013696:Temperature; D014407:Tumor Cells, Cultured
D000906:Antibodies; D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D046911:Macromolecular Substances; D010446:Peptide Fragments; C021091:staphylococcal alpha-toxin; D003208:Concanavalin A; D002118:Calcium