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Staphylococcal scalded-skin syndrome (SSSS) is an exfoliative dermatitis of infants and children that results from infection with exfoliative-toxin-producing Staphylococcus aureus (1, 15). SSSS is characterized by the formation of large bullae without inflammatory cell infiltrate and separation of extended areas of the epidermis at the stratum granulosum leaving the keratinocytes intact. Two biologically and serologically distinct S. aureus exotoxins are responsible for the skin manifestations of SSSS in humans, exfoliative toxin A (ETA) and exfoliative toxin B (ETB) (25). ETA (26.9 kDa) is encoded on the bacterial chromosome and shares 40% amino acid identity with the plasmid-borne ETB (27.3 kDa) (2, 13, 19). Despite extensive studies, the exact mechanism responsible for the skin disruption is not known.

X-ray crystallographic structures of ETA and ETB (5, 20, 23, 24) suggest that the toxins are members of the trypsin-like serine protease family. Protease activity has not been demonstrated for either toxin in vitro, but both ETA and ETB have intrinsic esterase activity, which is associated with serine proteases (3). Thus, it is likely that both toxins are proteases. In addition to having possible protease activity, both ETA and ETB are reported to be bacterial superantigens (14, 16, 17, 24). Bacterial superantigens are a family of proteins able to bind simultaneously to the major histocompatibility complex and to the T-cell receptor (TCR), resulting in stimulation of a large number of T cells expressing specific V subsets of the TCR repertoire (14).

Previously, Fleischer and Bailey reported that recombinant ETA expressed in a superantigen-free S. aureus background does not have mitogenic activity (7). They conclude that the activity seen by others is due to contamination of the toxin preparations with other superantigens. However, since this report, additional literature has addressed the superantigenic activity of ETA and ETB. Here we demonstrate that recombinant exfoliative toxins produced in an Escherichia coli background do not act as T-cell mitogens and thus are not bacterial superantigens.

Isolation, expression, and purification of recombinant exfoliative toxins. DNA fragments encoding ETA and ETB were obtained by utilizing PCR and DNA from exfoliative-toxin-producing bacterial strains. Toxins were expressed and purified using the Novagen (Madison, Wis.) pET expression system. In brief, a 780-bp DNA fragment encoding mature ETA was obtained using the oligonucleotide primers ETA-3 (5'-GCGCCTCGAGGTTTCAGCAGAAGAAATAAAA-3') and ETA-4 (5' - GCGCC T CGAG T ATAAAACATCCACGGAT T T T - 3') and chromosomal DNA of an ETA-producing S. aureus strain. A 775-bp DNA fragment encoding mature ETB was amplified from plasmid pIJ002 (generously provided by Peter McNamara) using oligonucleotide primers ETB-3 (5'-GCGCCATATGAAAGAATACAGCGCA-3') and ETB-4 (5'-CGCGGGATCCATATTGAAATATTAA-3'). The amplifications were performed using Taq DNA polymerase (Bethesda Research Laboratories, Gaithersburg, Md.) or Pfu Turbo DNA polymerase (Stratagene, La Jolla, Calif.). Primers were designed to amplify the coding sequences for the mature proteins without the amino-terminal signal sequences. They were also designed to contain restriction endonuclease sites that facilitated insertion of the fragments into the E. coli expression vector pET-15b (Novagen). The DNA sequences of both the ETA- and ETB-encoding fragments were determined and shown to be identical to the GenBank database sequences (eta, accession numbers L25372 and M20371; etb, accession numbers M17348 and M13775). In order to generate an appropriate negative control, the ETA-encoding fragment was mutated to code for mature ETA with an alanine residue replacing the putative active-site serine residue at position 195 (21, 22), using overlap extension mutagenesis (10). The complete nucleotide sequence of both strands of the mutated fragment was determined to confirm the presence of the desired mutation.

Recombinant exfoliative toxins produce the symptoms of SSSS in the neonatal mouse assay. The recombinant exfoliative toxins were tested for activity in neonatal mice (15). One-day-old BALB/c mice were injected subcutaneously at the nape of the neck with increasing concentrations of rETA, rETB, rETA-S195A, PBS (negative control), or ETA-producing S. aureus (positive control) and observed at various times postinjection for symptoms of SSSS. All mice were returned to lactating mothers and were observed at hourly intervals for gross appearance. They were graded on appearance and tactile examination (Table 1). Mice died during the course of the experiment or were sacrificed at the end of the 24-h observation period. After only 2 h, the mice injected with the highest dose of rETA (25 µg/g of body weight) showed obvious signs of exfoliation (Table 1). rETB and rETA exhibited similar activities, while rETA-S195A did not cause any visible signs of exfoliation even at 10 times the dosage that elicited a positive response with either rETA or rETB.

View larger version (139K): [in this window] [in a new window]   FIG. 1.   Histological examination of neonatal mouse skin exposed to rETA and PBS. Sections are from the bases of the tails of 2-day-old mice sacrificed at 24 h postinjection and were stained with hematoxylin and eosin. Magnification, ×40. (A) Control mouse injected with PBS showing intact skin; (B) mouse injected with 5 µg of rETA/g of body weight showing the characteristic splitting at the stratum granulosum.

Determination of superantigen activity. rETA, rETB, and rETA-S195A were assayed for mitogenic activity using human peripheral blood mononuclear cells (PBMCs). Mitogenicity assays were performed as described previously (12). Briefly, heparinized whole blood from human adults was fractionated on Ficoll-Paque (Pharmacia Biotech, Piscataway, N.J.) and the PBMCs were harvested. Cells (10⁵) were added to 96-well U-bottom plates in RPMI 1640 supplemented with 10% fetal calf serum and were incubated for 72 h with various concentrations of one of the following: rETA, rETB, rETA-S195A, rSpeA1 (purified in this laboratory as described previously [12]), staphylococcal V8 protease (Promega, Madison, Wis.), and PBS. Staphylococcal V8 protease is a serine protease structurally similar to the exfoliative toxins and was used as a negative control; the streptococcal superantigen rSpeA1 was used as a positive control. One microcurie of [³H]thymidine (ICN Biochemicals, Costa Mesa, Calif.) was added to each well, and cells were incubated for an additional 24 h and harvested; the [³H]thymidine uptake was then quantified. For each toxin or control, three to five distinct donors were used. Assays were performed a minimum of three times per recombinant toxin with different donor cells each time. The human PBMCs responded as expected to both rSpeA1 (Fig. 2) and the anti-CD3 monoclonal antibody OKT3, which served as a nonspecific T-cell mitogen (data not shown). However, there was no detectable mitogenic activity when PBS, rETA, rETB, rETA-S195A, or V8 protease was added to the cells. To determine if the lack of activity was due to the age of the donor, freshly isolated human umbilical cord blood was prepared as described above, as a source of neonatal mononuclear cells. Neonatal mononuclear cells had the same mitogenic response to rETA, rSpeA1, OKT3, and PBS as adult PBMCs (data not shown).

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Mitogenic activities of rETA, rETB, rETA-S195A, staphylococcal V8 protease (V8), and rSpeA1. Shown are the results obtained from one representative experiment. All experiments had similar results.