Staphylococcal alpha-toxin: repair of a calcium-impermeable pore in the target cell membrane.
Staphylococcal alpha-toxin forms heptameric pores that render membranes permeable for monovalent cations. The pore is formed by an amphipathic beta-barrel encompassing amino acid residues 118-140 of each subunit of the oligomer. Human fibroblasts are susceptible to alpha-toxin but are able to repair the membrane lesions. Thereby, toxin oligomers remain embedded in the plasma membrane and exposed to the extracellular medium. In this study, we sought to detect structural changes occurring in the pore-forming sequence during lesion repair. Single cysteine substitution mutants were labelled with the environmentally sensitive fluorochrome acrylodan and, after mixing with wild-type toxin, incorporated into hybrid heptamers on fibroblast membranes. Formation of the lipid-inserted beta-barrel was accompanied by characteristic fluorescence emission shifts. After lesion repair, the environment of the residues at the outer surface of the beta-barrel remained unchanged, indicating continued contact with lipids. However, the labelled residues oriented towards the channel lumen underwent a green to blue shift in fluorescence, indicating reduced exposure to water. Pore closure proceeded in the presence of calmodulin inhibitors and of microtubule disruptors; however, it was prevented by cytochalasin D and by inhibitors of lipid metabolism. Our findings reveal the existence of a novel mechanism of membrane repair that may consist in constriction of the inserted proteinaceous pore within the lipid bilayer.
Valeva
A
A
Institutes of Medical Microbiology and Hygiene, and Pharmacology, University of Mainz, Hochhaus am Augustusplatz, D-55101 Mainz, Germany. avaleva@mail.unimainz.de
Walev
I
I
Gerber
A
A
Klein
J
J
Palmer
M
M
Bhakdi
S
S
D001427:Bacterial Toxins / Q000633:toxicity*; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects* / Q000502:physiology; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D005347:Fibroblasts / Q000187:drug effects; D006460:Hemolysin Proteins / Q000633:toxicity*; D006801:Humans; D008214:Lymphocytes / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013210:Staphylococcus / Q000378:metabolism*
D001427:Bacterial Toxins; D006460:Hemolysin Proteins; C021091:staphylococcal alpha-toxin; D002118:Calcium