Mutants of staphylococcal toxic shock syndrome toxin 1: mitogenicity and recognition by a neutralizing monoclonal antibody.
Toxic shock syndrome toxin 1 (TSST-1), a 22-kilodalton protein made by strains of Staphylococcus aureus harboring the chromosomal toxin gene, may elicit toxic shock syndrome in humans. In vitro, TSST-1 induces T cells to proliferate and macrophages to secrete interleukin-1. To conduct a structure-function analysis, point mutations on the TSST-1 gene were generated by site-directed mutagenesis to identify amino acids critical for activity of the toxin. Specific tyrosine and histidine residues were replaced by alanines. Wild-type and mutant TSST-1 gene constructs were expressed in Escherichia coli, and the products were tested for their mitogenic potential and reactivity with a TSST-1 neutralizing monoclonal antibody (MAb 8-5-7). Four of the mutants were similar to the wild type; i.e., the mutant toxins stimulated murine T cells and reacted with MAb 8-5-7 equally as well as the wild type. Two mutants exhibited a decrease in mitogenic activity, but one of these retained the capacity to bind with MAb 8-5-7 while the other was no longer recognized by the same antibody. One double mutant demonstrated minimal mitogenic activity and did not react in enzyme-linked immunosorbent and immunoblot assays with MAb 8-5-7. The data show that specific residues near the carboxy terminus of TSST-1 are essential for mitogenic activity and in forming the epitope recognized by neutralizing MAb 8-5-7.
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati Medical Center, Ohio 45267-0524.
L
L
Blanco
E M
EM
Choi
K
K
Connolly
M R
MR
Thompson
P F
PF
Bonventre
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001427:Bacterial Toxins; D001483:Base Sequence; D001667:Binding, Competitive; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins / Q000235:genetics* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D051379:Mice; D008934:Mitogens / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D009154:Mutation; D009500:Neutralization Tests; D013154:Spleen / Q000166:cytology / Q000276:immunology; D018089:Superantigens
D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D004768:Enterotoxins; D000939:Epitopes; D008934:Mitogens; D018089:Superantigens; C030107:enterotoxin F, Staphylococcal