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

Abstract

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/BgIIIL). The recombinant mutant protein (ETB/BgIIIL) obtained from Escherichia coli containing pETB/BgIIIL showed no toxicity in neonatal mice and no agglutination activity. The 20-kDa ETB/BgIIIL 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/BgIIIL 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.

Exfoliative Toxins of Staphylococcus aureus

“…Based on the results of site-directed mutagenesis, Sakurai et al [60] concluded that the substitution of either Tyr 157 or 159 in ETB decreased exfoliative activity and the double mutation resulted in the complete loss of exfoliative activity and antigenicity. Interestingly, ETA does not possess either one of these tyrosines but contains Phe and His at these positions and in ETD these positions are occupied by Tyr and Thr.…”

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

“…ETA's structure indicates that it belongs to the chymotrypsin-like family of serine proteases and that it cleaves protein substrates at sites after acidic residues. On the other hand, Sakurai et al (16) reported that the Tyr-157 and Tyr-159 residues are involved in the toxicity of ETB. They also reported that ETA contained the same amino acid residues.…”