New Evidence That the Tyr-157 and Tyr-159 Residues of Staphylococcal Exfoliative Toxin B Are Essential for Its Toxicity

To determine the active site of exfoliative toxin B (sETB) of Staphylococcus aureus, the etb gene was cloned from an S. aureus SU strain obtained from a patient with impetigo. We prepared a frame shift mutant protein from a recombinant plasmid with a BglII linker inserted into the Tyr-155 codon of the ETB gene (pETB/BglIIL). The recombinant mutant protein (ETB/BglIIL) obtained from Escherichia coli containing pETB/BglIIL showed no toxicity in neonatal mice and no agglutination activity. The 20-kDa ETB/BglIIL contained 185 amino acid residues. Site-directed mutagenesis was used to introduce mutations at either Tyr-155, Tyr-157, Tyr-159, or Tyr-162. Substitution of any of the Tyr residues decreased exfoliative activity compared with that of native sETB (4,000 EU/ml). Substitution of Tyr-155 with a Phe (ETB/Y155) decreased activity 5-fold (800 EU/ml). Substitution of Tyr-157 with Leu (ETB/Y157) decreased activity 80-fold (50 EU/ml) and decreased agglutination titer 5-fold compared with that of native sETB (400,000). Substitution of Tyr-159 with Leu (ETB/Y159)decreased activity 4-fold (1,000 EU/ml). When both Tyr-157 and Tyr-159 were mutated (ETB/Y157-159), both toxicity and antigenicity were completely lost. On an immunodiffusion test, ETB/Y157 showed a faint precipitation line, but ETB/BglIIL and ETB/Y157-159 had no activity, showing that the Tyr-157 and Tyr-159 residues are essential for the toxicity and antigenicity of ETB.