Analysis of the superantigenic activity of mutant and allelic forms of streptococcal pyrogenic exotoxin A

Abstract

Infections with Streptococcus pyogenes (group A streptococcus) can result in the recently described streptococcal toxic shock syndrome (STSS), which is characterized by rashes, hypotension, multiorgan failure, and a high mortality rate. S. pyogenes isolates associated with STSS usually produce streptococcal pyrogenic exotoxin A (SpeA), a bacterial superantigen capable of stimulating host immune cells. Most of the symptoms of STSS are believed to result from cytokine release by the stimulated cells. To better understand the pathogenesis of STSS, we began studies on the SpeA-immune cell interaction. We generated 20 mutant forms of SpeA1 (SpeA encoded by allele 1), and the mutant toxins were analyzed for mitogenic stimulation of human peripheral blood mononuclear cells, affinity for class II major histocompatibility complex molecules (DQ), and disulfide bond formation. Residues necessary for each of these functions were identified. There are four alleles of speA, and STSS strains usually contain either allele 2 or allele 3. The product of allele 2, SpeA2, had slightly higher affinity for the class II MHC molecule compared with SpeA1 but not significantly greater mitogenic activity. SpeA3, however, was significantly increased in mitogenic activity and affinity for class II MHC compared with SpeA1. Thus, we have evidence that the toxin encoded by some of the highly virulent S. pyogenes STSSassociated isolates is a more active form of SpeA.

Structural basis for the recognition of superantigen streptococcal pyrogenic exotoxin A (SpeA1) by MHC class II molecules and T-cell receptors

“…Previous studies have identified SpeA1 residues required for high affinity binding to HLA-DQ (Kline and Collins, 1996). Substitution of each of the SpeA1 residues Leu42, Asp45, Leu46, Ile47, Tyr48 and Tyr83 with alanine resulted in a significant decrease in affinity of the mutant toxin for HLA-DQ.…”

“…Thus, SpeA3 exhibits the highest affinity for the DQ molecule. In addition, SpeA3 is the most mitogenic of the three allelic forms to human peripheral blood mononuclear cells (PBMC) (Kline and Collins, 1996). In the SpeA1 structure, Val76 is part of the β4 strand at the interface of the N-and C-terminal domains, at the bottom of the molecule (Figure 2B and C).…”

Analysis of the interaction between the bacterial superantigen streptococcal pyrogenic exotoxin A (SpeA) and the human T‐cell receptor

“…Except where noted in parentheses, all substituted residues were changed to alanine. Mutant toxins were generated as previously described (Kline andCollins, 1996). et al, 1991;Braun et al, 1993;Leonard et al, 1991;Tomai et al, 1992).…”

“…Activity of the allelic forms of SpeA rSpeA3 is significantly more mitogenic than rSpeA1 or rSpeA2 in vitro (Kline and Collins, 1996). Therefore, it was expected that rSpeA3 would stimulate V␤2-, 12-, and 14-expressing T cells to levels greater than or equal to those observed with rSpeA1.…”

“…Previously we generated 22 forms of recombinant SpeA (rSpeA) (Kline and Collins, 1996). Each form was analysed for the ability to activate human peripheral blood mononuclear cells (PBMCs) and the ability to bind the class II MHC molecule.…”