Purification of Protease from a Mixture of Exfoliative Toxin and Newborn-Mouse Epidermis

Abstract

Although the role of exfoliative toxin in staphylococcal scalded-skin syndrome has been suggested to be that of a serine protease, it has not been demonstrated to show proteolytic activity. Our purpose was to purify a proteolytic enzyme from a mixture of exfoliative toxin and newborn-mouse epidermis. We used gel filtration and ion-exchange and hydroxyapatite chromatography with a high-pressure liquid chromatography system. A casein-hydrolyzing enzyme was isolated from the mixture. The molecular mass of the enzyme was confirmed to be 20 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Subcutaneous injection of the purified enzyme into newborn mice reproduced the epidermal splitting that is seen in staphylococcal scalded-skin syndrome. These results suggest that exfoliative toxin does not work as a protease itself but that some reaction between exfoliative toxin and an epidermal component(s) first produces a protease, after which epidermal splitting occurs.

Extracellular Proteases of Staphylococcus spp.

“…The closely related cadherins such as desmoglein 3 were not affected. These data contradicts the suggestions by Ninomiya et al (2000), that the interaction of ET with epidermal component(s) induces proteolytically active enzyme. In addition, both toxins were shown to cleave α-melanocyte-stimulating hormone but only ETA cleaved β-melanocyte-stimulating hormone.…”

“…The epidermolytic toxins have an intrinsic esterase activity, while ETA S195G active site serine mutant is inactive (Bailey and Redpath, 1992) and completely devoid of biological activity (Prévost et al, 1991;Redpath et al, 1991). On the other hand, Ninomiya et al (2000) reported the isolation of a protease corssreacting with anti-ET antibodies and readily hydrolyzing casein, but with molecular weight lower than the toxins, from the mixture of exfoliative toxin and newborn-mouse epidermis. Neither could the epidermal splitting in the mouse bioassay be completely inhibited by DFP, although the treatment prolonged the time needed for epidermolysis to occur (Dancer et al, 1990).…”

“…ETA esterase activity was significantly inactivated by di-isopropyl phosphofluoridate (DFP), but even after a long incubation time the inhibition was incomplete (Bailey and Redpath, 1992). Based on these results it was speculated that interaction of ETs with epidermal component(s) induced the proteolytically active enzyme, but little was reported on the activation mechanism, or its effect on enzyme structure (Ninomiya et al, 2000). In contrast, the ETB esterase activity was completely inhibited by DFP and the loss of esterase activity correlated with the loss of epidermolytic activity.…”

Staphylococcus aureus exfoliative toxins: How they cause disease.

“…In other studies ETA has been shown to cleave ␣-and ␤-melanocyte stimulating hormones after a lengthy incubation period ; however, it is unclear how this might result in or contribute to SSSS or BI. Also, other investigators studying ETB have purified a distinct 20 kDa protease from a supernatant of preparations of neonatal mouse epidermis treated with ETB which was able to reproduce epidermolysis similar to that of SSSS when injected into neonatal mice (Ninomiya et al, 2000). These results would suggest the presence of an additional target for the ETB versus ETA, as Dsg1 has been shown to be cleaved by both ETB and ETA (Amagai et al, 2000;Amagai et al, 2002).…”

Recent developments in staphylococcal scalded skin syndrome

“…The authors suggest that these proteins may be the target for the toxins. On the other hand, a Japanese group isolated and purified a distinct 20 kDa protease from a supernatant of exfoliative toxin with neonatal mouse epidermis and showed that this protease could reproduce the mid‐epidermal splitting of SSSS when injected subcutaneously into newborn mice [43].…”