[Experimental studies on the mechanism of the action of staphylococcal epidermolytic toxin A utilizing recombinant toxin].

Abstract

The pathogenic role of proteinases and Ca++ in the action of staphylococcal epidermolytic toxin A (ETA) was investigated using recombinant ETA (rETA). rETA was released from the periplasmic space of E. coli transformed with the plasmid carrying ETA gene and purified by high performance liquid chromatography. The epidermolytic activity of the purified rETA was 5,000 epidermolytic unit per mg of protein. Pieces of newborn mouse skin were cultured in minimum essential medium containing rETA. Various concentrations of alpha 2-macroglobulin, N-ethylmaleimide, leupeptin, L-transepoxysuccynyl-leucylamide (4-guanidino) butane, phenylmethylsulfonyl fluroide, pepstatin A, ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis (2-aminoethylether) tetraacetic acid (EGTA) and 8-(N,N-diethylamino) octyl 3,4,5-trimethoxybenzoate (TMB-8) were added to the medium. Splitting in the upper epidermis occurred after 4 hr of incubation in the presence of 10 micrograms/ml rETA and was not inhibited by the proteinase inhibitors except EDTA and EGTA. EDTA, EGTA and TMB-8 inhibited the splitting completely at concentrations of 0.1-1 mM. The inhibitions caused by these agents were restored by the addition of Ca++ to the medium. These results strongly suggest that the action of ETA is mediated by the increase in cytoplasmic Ca++ concentration resulting from Ca++ influx and/or intracellular Ca++ mobilization.