Toxic-shock-syndrome toxin-1 (TSST-1), a 22-kilodalton (kDa) polypeptide, was proteolyzed by papain, generating three distinct fragments, identified as 16, 12, and 10 kDa (based on molecular masses estimated from the predicted amino acid sequence). The NH2-terminal sequence analysis of the fragments indicated that the peptide bonds between Tyr-52 and Ser-53 and between Gly-87 and Val-88 were cleaved. Functional activity, evaluated through enzyme-linked immunosorbent and inhibition assays, was demonstrated only with the 16- and 12-kDa fragments. The presence of homologous and heterologous antigenic determinants on the fragments was demonstrated by immunoblotting. In in vitro stimulation of human peripheral blood mononuclear cells, the 12-kDa fragment was significantly (P = .003) more active than the 16-kDa fragment. The former composed 75% of the latter and occupied the COOH-terminal portion of the holotoxin. The functional domains were located on two-thirds of the TSST-1 molecule, toward the COOH-terminal end, and mitogenicity apparently was separable from serological activity.
When toxic shock syndrome toxin 1 was subjected to papain hydrolysis, two serologically active fragments of 16.3 kilodaltons (16K fragment) and 12.4 kilodaltons (12K fragment) were generated, whereas a third fragment of 9.7 kilodaltons (1OK fragment) was inactive. The biologic activities of the fragments were evaluated in vitro by determining their ability to promote nonspecific proliferation of human peripheral blood mononuclear cells. The 12K fragment was significantly (P c 0.013) more stimulatory than the 16K fragment. When human peripheral blood mononuclear cells were preincubated for a period of 24 h with various concentrations of the 16K fragment, followed by incubation with a constant amount (2 x 10-2 ng/ml) of whole toxin, the level of DNA synthesis induced by the holotoxin was reduced by approximately 60% when compared with that of controls exposed to whole toxin alone. The 12K fragment did not demonstrate a similar blocking effect. Immunoblots of the toxic shock syndrome toxin 1 digest, which were exposed to monoclonal antibodies (MAbs) developed against native toxin, depicted the presence of two different antigenic regions (epitopes). One MAb, 8-5-7, which has been shown previously to inhibit the biologic activity of the holotoxin in vitro and in vivo, reacted primarily with the 12K fragment. A second MAb, 10-6-1, that did not neutralize interleukin-1 production reacted primarily with the 16K fragment. On the basis of the differential mitogenic responses and the identification of heterologous epitopes, it was concluded that the functional region of the holotoxin can be partitioned into at least two functional segments encompassed between amino acid residues 53 and 87 and between amino acid residues 88 and 194 on the polypeptide chain.
A 21-mer synthetic peptide (KGEKVDLNTKRTKKSQHTSEG), designated TSST-1(58-78), was constructed from the primary structure of the toxic shock syndrome toxin 1 (TSST-1). The peptide reacted with a panel of neutralizing monoclonal antibodies (MAbs) to whole TSST-1 in solid-phase immunoassays. TSST-1(58-78) promoted the in vitro proliferation of human peripheral blood mononuclear cells (PBMC) in a dose-dependent manner. Minimum dose required for stimulation (P less than or equal to .05 microM) was 0.75 microM peptide. This mitogenic effect was abrogated by incubation of the peptide with MAbs to whole TSST-1 before addition to PBMC. The ability of TSST-1(58-78) to stimulate the proliferation of highly purified resting human T cells was analyzed. Significant proliferation (P less than or equal to .01) was observed only in the presence of increasing populations of monocytes added to the cultures. Adherent human monocytes exposed to TSST-1(58-78) released tumor necrosis factor. Thus, some of the immunoregulatory properties attributed to TSST-1 are demonstrated by the region of the toxin represented by the peptide TSST-1(58-78).
Spleen cells from mice immunized with staphylococcal enterotoxin A were successfully fused with NS-1 mouse myeloma cells. Two of the four clones studied produced monoclonal antibodies to staphylococcal enterotoxin A in growth medium which showed titers of >106 to 107 when tested by the indirect enzyme-linked immunosorbent assay. These monoclonal antibodies showed reactivity with enterotoxins A and E in the enzyme-linked immunosorbent assay. However, the reactivity was higher with enterotoxin A than with enterotoxin E. Nanogram quantities of crude staphylococcus enterotoxin A from Staphylococcus aureus growth were detected by the monoclonal antibodies in electroimmunoblots via autoradiography.
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