IDENTIFICATION AND PURIFICATION OF A NEW STAPHYLOCOCCAL-ENTEROTOXIN, H

A staphylococcal enterotoxin which elicited an emetic response in monkeys but did not share antigenic determinants with any of the identified enterotoxins was identified and purified from Staphylococcus aureus FRI-569. The emetic activity of this new enterotoxin was neutralized only by antibodies specific to it and not by antibodies to enterotoxins A, B, C, D, and E or toxic shock syndrome toxin 1. Immunodiffusion assays did not detect cross-reactivity between this new and all the other identified enterotoxins, The purification procedure involved removal of the enterotoxin from culture supernatant fluids by batch adsorption with CG-50 resin, CM-Sepharose FL ion-exchange chromatography, and Sephacryl 100 WR and Bio-Gel P-30 gel filtration, The molecular weight of this enterotoxin;in, 27,300, determined by gel filtration on Sephacryl 100 HR agreed with the molecular weight, 28,500, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The apparent migration of this enterotoxin determined by SDS-PAGE did not shift in the presence of a disulfide reducing agent, indicating that it is composed of a single-chain protein, The N-terminal amino acid sequence of the enterotoxin was determined to be Glu-Asp-Leu-His-Asp-Lys-Ser-Glu-Leu-Thr-Asp-Leu-Ala-Leu-Ala-Asn- Ala-Tyr-Gly-Gln-Tyr-Asn-His-Pro-Phe-Ile-Lys-Glu-Asn-Ile, which did not match the N-terminal sequences of any known proteins. The isoelectric point of the enterotoxin determined by isoelectric focusing was about 5.7, Western blot (immunoblot) analysis showed that this enterotoxin was not produced by S. aureus FRI-572 (MJB8O1), recently found to possess a gene (seg(+)) encoding an unidentified enterotoxin, G. We have designated this newly characterized enterotoxin enterotoxin H.