DEVELOPMENT OF MUTANTS OF STAPHYLOCOCCAL TOXIC SHOCK SYNDROME TOXIN-1

Toxic shock syndrome toxin-l (TSST-1) is the main etiology of toxic sh ock syndrome and is secreted as mature protein 194 amino acids in leng th. To investigate the structure function relationship of TSST-1 with the host immune system, we constructed staphylococcal shuttle plasmid encoding TSST-1 using polymerase chain reaction, which is site-directe d mutagenesis. Point mutants of TSST-1 at residues 132 and 140 were al so constructed by oligonucleotide directed mutagenesis. Wild type and mutant TSST-1 were prepared from culture supernatants of Staphylococcu s aureus containing shuttle plasmid encoding TSST-1 grown in brain hea rt infusion (BHI) dialysate. The toxin was purified by a combination o f dye affinity chromatography and ultrafiltration. Toxins were detecte d by immunoblotting of SDS-polyacrylamide gels using TSST-1 specific p olyclonal antibodies. The effects of the wild type and mutant type of TSST-1 on human peripheral blood mononuclear cells were studied. The m utant protein E132K produced 10 times less tumor necrosis factor-alpha (TNF-alpha) than that of wild type TSST-1 and 1140T. Thus residue 132 appears to be critical for the expression of biological activities of TSST-1 in vitro.