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New Evidence That the Tyr‐157 and Tyr‐159 Residues of Staphylococcal Exfoliative Toxin B Are Essential for Its Toxicity
Author(s) -
Sakurai Susumu,
Suzuki Hitoshi,
Saito Shinobu,
Konishi Yasuhiko,
Machida Katsuhiko,
Kohno Minoru
Publication year - 1998
Publication title -
microbiology and immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.664
H-Index - 70
eISSN - 1348-0421
pISSN - 0385-5600
DOI - 10.1111/j.1348-0421.1998.tb02358.x
Subject(s) - antigenicity , biology , microbiology and biotechnology , recombinant dna , biochemistry , gene , antibody , immunology
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 Bgl II linker inserted into the Tyr‐155 codon of the ETB gene (pETB/ Bgl IIL). The recombinant mutant protein (ETB/ Bgl IIL) obtained from Escherichia coli containing pETB/ Bgl IIL showed no toxicity in neonatal mice and no agglutination activity. The 20‐kDa ETB/ Bgl IIL 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/ Bgl IIL 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.