Treatment of mouse embryo fibroblasts (MEF) grown in vitro with purified staphylococcal exfoliative toxin (ET) increased the concanavalin A (Con A) agglutinability of MEF 3.5-fold over control cells. Possible explanations for this phenomenon were investigated. ET lacked proteolytic activity on denatured casein. Con A, however, was found to interact directly with ET as evidenced by the formation of precipitation in an agar gel diffusion plate and in increased turbidity in solution. This

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... on was inhibited by a-methyl-D-glucopyranoside. The ability of Con A to precipitate with ET suggests that the toxin contains a carbohydrate component and that the carbohydrate associated with ET is branched rather than linear. An analysis of a purified preparation of ET indicated the presence of 9% carbohydrate and no lipid. Bacteriophage group II Staphylococcus aureus have been associated with a spectrum of clinical manifestations termed the staphococcal scalded skin syndrome (14, 15 ). This syndrome is characterized by exfoliation of the superficial layer of the epidermis caused by disruption of the desmosomes at their intercellular contact sites within the granular cell layer (13). The extracellular staphylococcal product responsible for epidermal exfoliation has been isolated and purified (1, 11, 12, 16) and termed exfoliative toxin (ET) in our laboratory (16). When ET is injected subcutaneously or intraperitoneally into neonatal mice, a typical Nikolsky sign develops which is followed by widespread exfoliation of the epidermis (13, 14). The toxin can be separated by ammonium sulfate precipitation followed by isoelectric focusing (16) or column chromotography on diethylaminoethyl-cellulose, followed by polyacrylamide gel electrophoresis (12) or isoelectric focusing, followed by ammonium sulfate precipitation (1). Although ET has been considered to be a protein with a molecular weight of approximately 24,000, some of its physical and chemical properties suggest the presence of components other than amino acids. ET is acid labile, but does not lose biological activity on storage at 4 C for 5 months, heating at 100 C for 20 min, or treatment with Pronase (0.01 mg/ml) or trypsin (0.33 mg/ml) for 2 h (11, 12, 16). The 'Present address: University of Teheran, Faculty of Veterinary Medicine, Teheran, Iran. capacity of the toxin to cause exfoliation, however, is partially destroyed by more rigorous exposure to proteolytic enzymes and heat. Both the specific structure and the mechanism of action of this staphylococcal toxin remain poorly defined at the present time. The purpose of the present study is to determine if the ET produced by phage group II staphylococci is a glycoprotein and to initiate studies concerning possible mechanisms by which the toxin causes separation of granular cells in the epidermis of both humans and experimental animals. MATERIALS AND METHODS Isolation and purification of ET. ET was isolated from the phage group II S. aureus strain UT 0002 (19). The toxin-producing culture was grown in separate 250-ml shake flasks (Bellco) containing 50 ml of heart infusion broth (Difco). The flasks were incubated with shaking at 37 C on a New Brunswick model G 76 gyrotory water-bath shaker covered by a Plexiglas hood, which was flushed with 100% CO2 twice daily. When approximately 48 h of growth was completed, the cells were then centrifuged at 10,000 x g for 15 min at 4 C and discarded. Ammonium sulfate was added to the cell-free supernatant to a final concentration of 80%, and ET was purified from the resulting crude material by the electrofocusing procedure described by Melish et al. (16). The purified preparations of ET utilized in these experiments were shown to be free of alpha-toxin activity by their inability to lyse rabbit erythrocytes and to give a single band on polyacrylamide gel which could be eluted and shown to produce exfoliation in newborn mice. The poly-1260 on May 5, 2020 by guest http://iai.asm.org/ Downloaded from