Identification of functionally active fragments of staphylococcal enterotoxin B

Abstract

It has been found that staphylococcal enterotoxin B contains a proteolysis-sensitive sequence in the cysteine loop formed by two half-cystines located in the middle of the toxin polypeptide chain. Fragments of the enterotoxin formed as a result of its digestion in this region have been isolated, their N-terminal scyuences have been determined and sites of proteolysis have been identified. It has been demonstrated that the N-terminal fragment of staphylococcal enterotoxin B is capable of activating T cell proliferation in the culture of human mononuclear cells practically to the same degree as the intact enterotoxin. The toxin's C-terminal fragment possesses an ability to activate calmodulindependent enzymes and is probably the toxicogenic part of the enterotoxin.Staphylococcal enterotoxins (S. enterotoxins) represent a group of proteins with molecular masses ranging between 27 -30 kDa which are secreted by Staphylococcus aureus and cause food poisoning [l]. At present, sevcn S. enterotoxins are known, namely A, B. C1, C2, C3, D and E [2]. Their amino acid sequences have been determined [3] and it was shown that all are single-chain polypeptides containing one disulfide bond formed by two half-cystines located in the middle of the polypeptide chain which form the so-called cysteine loop. S. enterotoxins are known to be most potent T cell mitogens [4]. T cell activation accompanied by induction of interleukin 2 and interferon [5] is conditioned by high-affinity interaction of S. enterotoxins with class 11 main histocompatibility complex (MHC) molecules [6] and subsequent presentation of the complex formed to a variable region of the subunit of the T cell receptor (V,TCR) [7]. From the character of receptor interactions and the nature of T cell proliferative response, the mechanism of S. enterotoxins' action on cells of the immune system is completely identical to that of MIS antigens [8, 91; on this basis, the foregoing proteins were united in the family of the so-called 'superantigens'. Despite the fact that interaction of S. enterotoxins with immunocompetent cclls has been studied in sufficient detail, there are no precise data explaining the interrelationship between immunomodulatory properties and the possible role of these toxins in pathogenesis of staphylococcal infections. There are also no data about the structural and functional organization of S. cnterotoxins and the regions of their moelcules responsible for receptor interactions and toxicogenic functions. .I).The present work is devoted to the study of the domain structure of S. enterotoxin B. We have found that it can be easily digested in the cysteine loop by contaminating proteases that are present in the toxin preparation. The fragments formed were isolated and their N-terminal amino acid sequences were determined, which allowed these fragments to be identified in the S . enterotoxin B structure. Studying the functional properties of the fragments obtained also allowed us to identify a region in the S. enterotoxin B structure that is responsible...

Biological activity of toxic shock syndrome toxin 1 and a site-directed mutant, H135A, in a lipopolysaccharide-potentiated mouse lethality model

“…These mutant molecules offer an excellent opportunity for dissecting the structure-function properties of staphylococcal superantigens by using different in vitro and in vivo techniques. Experiments with peptide fragments from superantigens also help to delineate regions of these toxins that are important for biological activity (3,19,20,29,34,58). The H135A mutant of TSST-1 is particularly intriguing since it has very little biological activity in various in vitro or in vivo assays (10,11).…”

Generation, Characterization, and Epitope Mapping of Neutralizing and Protective Monoclonal Antibodies against Staphylococcal Enterotoxin B-induced Lethal Shock

“…The major advantage of mAbs is that they are biochemically defined reagents that can be readily manufactured in unlimited supply. Although some mAbs have been generated for SEB, most of these studies demonstrate only efficacy or binding in vitro (33)(34)(35). In other studies mAbs were generated by vaccination with SEB fragments that recognize the MHC II or V␤ TCR binding site on SEB (13).…”

Assessment of the Functional Regions of the Superantigen Staphylococcal Enterotoxin B

“…Several approaches have been used to determine the regions of the bacterial superantigens involved in the mitogenic activity of these toxins. These include the use of synthetic peptides corresponding to regions of staphylococcal enterotoxin A (SEA) to block the activity of the native toxin [11,12], the analysis of the activity of proteolytic digestion fragments of several of the toxins including SEA, SEB, SEC1, SEC2, and TSST-1 [13,14,15,16,17,18,19], the use of monoclonal antibodies specific for identified epitopes to neutralize superantigen activity [16,17,18], the characterization of recombinant mutant SEA, SEB, TSST-1, and streptococcal pyrogenic exotoxin A (SPEA) containing amino acid substitutions [20,21,22,23,24], and the analysis of toxin chimeras to localize regions involved in the TcR Vβ allele selectivity [25,26]. Results from experiments carried out to determine the location of epitopes responsible for the biological activities of the superantigens have frequently appeared contradictory.…”