Identification & characterisation of the two novel streptococcal pyrogenic exotoxins SPE-L & SPE-M.

BACKGROUND & OBJECTIVES The streptococcal pyrogenic exotoxins (SPEs) are produced by Streptococcus pyogenes and belong to the family of bacterial superantigens, a group of highly mitogenic proteins. The aim of this study was to search unfinished streptococcal genomes for novel superantigens, to generate recombinant proteins from potential open reading frames (ORFs) and to analyse them for superantigen activity. METHODS The microbial genome database was searched using a TBLASTN search programme. Genotyping of S. equi and S. pyogenes isolates was done using the specific primer pairs. The spe-l and spe-m genes were amplified by PCR. RESULTS Two novel streptococcal superantigen genes (sepe-l and sepe-m) were identified from the Streptococcus equi genomic database at the Sanger Centre. Genotyping of S. pyogenes isolates resulted in the detection of the orthologous genes spe-l and spe-m in a restricted number of S. pyogenes isolates and revealed a link of spe-l to the M89 serotype. Recombinant SPE-L and rSPE-M were highly mitogenic for human peripheral blood lymphocytes with half maximum responses at 1 pg/ml and 10 pg/ml, respectively. The results from competitive binding experiments suggest that both proteins bind MHC class II at the beta-chain, but not at the alpha-chain. The most common target for both toxins were human Vbetal.1 expressing T cells. Seroconversion against SPE-L and SPE-M was observed in healthy blood donors. INTERPRETATION & CONCLUSION The two novel ORFs identified in both, S. equi and S. pyogenes, code for proteins that show typical superantigen features. The seroconversion seen in some blood donors suggest that the proteins are indeed produced by the bacteria. Interestingly, the spe-l gene is highly associated with S. pyogenes M89, which is linked to acute rheumatic fever in New Zealand.