MITOGENIC ACTIVITIES OF AMINO-ACID SUBSTITUTION MUTANTS OF STAPHYLOCOCCAL-ENTEROTOXIN-B IN HUMAN AND MOUSE LYMPHOCYTE-CULTURES

Site-directed mutagenesis has been used to introduce amino acid substi tutions at specific residues of the staphylococcal enterotoxin B (SEB) gene cloned from Staphylococcus aureus 10-275. The mitogenic activiti es of these derivatives were determined in two assay systems: (i) mous e spleen cells and (ii) a mixture of human peripheral blood mononuclea r cells and lymphocytes. Substitution of either His-12, His-32, His-12 1, His-166, Lys-152, or Gly-205 did not significantly alter the mitoge nic activity from that of the wild-type toxin in either proliferation assay. Substitution of either residue Asn-23, Phe-44, or Cys-93 reduce d the mitogenicity of SEB by a degree that depended upon the assay sys tem used. Similar to the results reported by others measuring toxin ac tivation of mouse lymphoid cells, we found that substitutions of these three residues of SEB caused at least 800-fold reductions of mitogeni c activity from that of the wild-type toxin. When tested for toxicity in vivo in D-galactosamine-treated mice, the reduced activities of the se mutant toxins, however, were not as pronounced, In contrast, when t ested in the human cell mitogenicity assay, these mutant toxins were a ctive. Small alterations in activity (two- to fivefold reduction) were observable only at low concentrations. Our findings reveal the import ance of using human lymphocytes in addition to the traditional mouse s pleen cell assay when assessing biological activities of staphylococca l enterotoxins.