MUTATIONS AFFECTING THE ACTIVITY OF TOXIC SHOCK SYNDROME TOXIN-1

Toxic shock syndrome toxin-1 (TSST-1), the potent staphylococcal exopr otein linked to most cases of the toxic shock syndrome, is a V-beta-re stricted T-cell mitogen (a so-called ''superantigen''). TSST-ovine (TS ST-O) is a natural variant of TSST-1, and is produced by certain ovine mastitis-associated strains of Staphylococcus aureus. Compared to TSS T-1, TSST-O is only weakly mitogenic for leporine or murine splenocyte s. It differs from TSST-1 at 7 amino acid residues over its 194 amino acid length. Terminus shuffling between the two proteins has suggested that their C-terminal differences (T69, Y80, E132, and I140 in TSST-1 ; I69, W80, K132, and T140 in TSST-O) are in part responsible for thei r discrepant mitogenic properties. In order to explore further the fun ctional consequences of altering TSST-1 at residues 132 and 140, we en gineered point mutants of TSST-1 at those positions. The mutant protei ns were purified to homogeneity from culture supernatants of a nontoxi genic strain of S. aureus using a combination of ultrafiltration, liqu id-phase isoelectric focusing, and ion-exchange chromatography. The mu tants retained global structural integrity as evidenced by circular di chroism spectroscopy, their preserved resistance to trypsin digestion, and their preserved binding to a neutralizing murine monoclonal antib ody. The mutants were then tested for mitogenicity for human T-cells: The mutant I140T was approximately as active as wild-type TSST-1, whil e the mutant E132D was about 10-fold attenuated. On the other hand, th e mutants E132A or E132K were each at least 1000-fold attenuated. We c onclude that the mitogenic activity of TSST-1 for human T-cells depend s critically on the presence and precise position of the negative char ge at residue 132.