Identification of functional antigenic segments of toxic shock syndrome toxin 1 by differential immunoreactivity and by differential mitogenic responses of human peripheral blood mononuclear cells, using active toxin fragments

Abstract

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.

Mutants of staphylococcal toxic shock syndrome toxin 1: mitogenicity and recognition by a neutralizing monoclonal antibody

“…Blomster-Hautamaa et al (4), Murphy et al (25), and Kokan-Moore and Bergdoll (17) showed that an internal cyanogen bromide fragment (amino acids 34 to 158) retained mitogenic activity and immunological reactivity to anti-TSST-1 MAbs. Edwin and Kass (13) showed that a papain fragment of TSST-1 corresponding to its carboxy terminus (amino acids 88 to 194) retained biological activities as well as reactivity with several anti-TSST-1 MAbs developed in our laboratory (7). On the basis of these observations, they postulated that the biologically active region of TSST-1 resides in a 71-amino-acid segment encompassed by residues 88 and 158.…”

Localization of a T-Cell Epitope of Superantigen Toxic Shock Syndrome Toxin 1 to Residues 125 to 158

“…Historically, empirical epitope localization of protein antigens has relied upon either enzymatic digestion or cyanogen bromide cleavage into successively smaller fragments retaining epitopic specificity (12). Later, advances in peptide synthesis technology paved the way for the generation of small fragments with overlapping sequences and the construction of synthetic peptides corresponding to different areas of the protein, which could be probed for reactivity with T or B cells.…”

Bacterial pyrogenic exotoxins as superantigens

“…Several groups have thoroughly investigated residues within superantigens, TCR, and class II molecules that are important for their physical and functional interaction (5,10,22,23,30,41,42,65,101,107,112,113,117,126,202,205,206,213,235,254,257,275). Studies investigating functional domains of the staphylococcal enterotoxin (SE) superantigens have localized mitogenically important regions to the amino-terminal part of the molecule (30,126,203), while others have suggested the importance of residues at the carboxy-terminal half (24,113).…”