Mutations affecting the activity of toxic shock syndrome toxin-1.
Toxic shock syndrome toxin-1 (TSST-1), the potent staphylococcal exoprotein linked to most cases of the toxic shock syndrome, is a V beta-restricted T-cell mitogen (a so-called "superantigen"). TSST-ovine (TSST-O) is a natural variant of TSST-1, and is produced by certain ovine mastitis-associated strains of Staphylococcus aureus. Compared to TSST-1, TSST-O is only weakly mitogenic for leporine or murine splenocytes. 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; 169, W80, K132, and T140 in TSST-O) are in part responsible for their discrepant mitogenic properties. In order to explore further the functional consequences of altering TSST-1 at residues 132 and 140, we engineered point mutants of TSST-1 at those positions. The mutant proteins were purified to homogeneity from culture supernants of a nontoxigenic strain of S. aureus using a combination of ultrafiltration, liquid-phase isoelectric focusing, and ion-exchange chromatography. The mutants retained global structural integrity as evidenced by circular dichroism spectroscopy, their preserved resistance to trypsin digestion, and their preserved binding to a neutralizing murine monoclonal antibody. The mutants were then tested for mitogenicity for human T-cells: The mutant I140T was approximately as active as wild-type TSST-1, while the mutant E132D was about 10-fold attenuated. On the other hand, the mutants E132A or E132K were each at least 1000-fold attenuated.(ABSTRACT TRUNCATED AT 250 WORDS)
Deresiewicz
R L
RL
Channing Laboratory, Boston, Massachusetts 02115.
Woo
J
J
Chan
M
M
Finberg
R W
RW
Kasper
D L
DL
D001427:Bacterial Toxins; D001483:Base Sequence; D002942:Circular Dichroism; D004768:Enterotoxins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D013211:Staphylococcus aureus / Q000235:genetics; D013329:Structure-Activity Relationship; D018089:Superantigens; D014169:Transformation, Bacterial; D014357:Trypsin / Q000378:metabolism
D001427:Bacterial Toxins; D004768:Enterotoxins; D018089:Superantigens; C030107:enterotoxin F, Staphylococcal; D014357:Trypsin