Functional characterization of streptococcal pyrogenic exotoxin J, a novel superantigen.

Streptococcus pyogenes (group A streptococcus) produces a variety of exotoxins, including the streptococcal pyrogenic exotoxins (SPEs; scarlet fever toxins) that have been implicated in severe invasive streptococcal diseases such as streptococcal toxic shock syndrome (STSS) and scarlet fever (reviewed in references 29, 37, 51, and 54). The SPEs belong to a larger group of pyrogenic toxin superantigens (PTSAgs) (5) that stimulate T cells by binding both invariant regions on major histocompatibility complex (MHC) class II molecules and specific Vβ chains of the T-cell receptor; this activity has been termed superantigenicity (36). The ability of PTSAgs to bind to the T-cell receptor (TCR) is independent of the peptide contained in the groove of MHC class II molecules, and since there are relatively few TCR Vβ regions, the frequency of T cells responding to PTSAg exposure exceeds that of conventional peptide antigens by several orders of magnitude (reviewed in reference 35). Extensive T-cell proliferation results in massive cytokine release, which is believed to contribute to the most severe effects of STSS. Structural and immunological characterization of the SPEs have revealed that they are low-molecular-weight proteins (24,000 to 28,000) that are relatively heat and protease resistant. Although only SPEs A and C are generally considered to be associated with STSS (7–9, 16, 17, 41, 45, 55, 58), distinct SPE serotypes are now known to include serotypes A (23, 63), B (14), C (12), F (46, 64), G (48), and H (48), as well as the streptococcal superantigen (SSA) (40) and multiple streptococcal mitogenic exotoxin Z (SMEZ) serotypes (25, 48, 49). However, unlike the other SPE serotypes, SPEs B and F have enzymatic activity (protease and DNase, respectively) (13, 18, 27), neither has sequence homology to the other SPEs, and SPE B is known to not share structural similarity with the other SPE serotypes (24). It has been shown that several clinical strains of S. pyogenes from patients with STSS produce neither SPE A nor SPE C (6, 10, 25, 42, 44, 62). This observation raises the possibility that uncharacterized PTSAgs, related to SPE A or SPE C, are capable of causing STSS. Consistent with this hypothesis, it has been demonstrated that novel streptococcal superantigens are in fact made by virulent streptococci (1, 40). Furthermore, selective depletion of certain Vβ T-cell subsets from patients with STSS that did not correlate with Vβ patterns from known superantigens has been demonstrated (62). Importantly, this study also determined that many of the strains did not possess the genes for SPE A, SPE C, or SSA. Two novel streptococcal superantigens (SPE G and SPE H) were recently identified and characterized by Proft et al. (48), aided by the S. pyogenes genomic database at the University of Oklahoma. These authors also identified SMEZ-2, a novel superantigen very similar to the previously described SMEZ (25). The crystal structures of SPE H and SMEZ-2 were also recently reported, and both conformed to the generic bacterial superantigen folding pattern (1). This group demonstrated that SMEZ, unlike other superantigens (at least to our current knowledge), is highly polymorphic in that 22 different smez alleles were identified. This research also revealed that although the polymorphisms in the SMEZ serotypes (now serotypes 1 through 22) maintained their Vβ T-cell subset stimulatory profiles (Vβ4 and Vβ8), they resulted in antigenic variants (48). During the characterization of SPEs G, H, and SMEZ-2, a partial gene sequence of a putative PTSAg was also identified. This putative gene was named speJ, although it was not characterized further (48). The present study describes the cloning, expression, and immunological characterization of this novel PTSAg, termed SPE J, and we show that this toxin has properties similar to those of already-characterized PTSAgs, including mitogenic activity for rabbit and human T cells, Vβ-specific superantigenic activity, and lethal activity in two models of STSS. SPE J represents a seventh streptococcal superantigen, most similar to SPE C, and raises interesting questions about the evolution of the streptococcal superantigens.