Two novel superantigens found in both group A and group C Streptococcus

Abstract

Two novel streptococcal superantigen genes (speL(Se) and speM(Se)) were identified from the Streptococcus equi genome database at the Sanger Center. Genotyping of 8 S. equi isolates and 40 Streptococcus pyogenes isolates resulted in the detection of the orthologous genes speL and speM in a restricted number of S. pyogenes isolates (15 and 5%, respectively). Surprisingly, the novel superantigen genes could not be found in any of the analyzed S. equi isolates. The results suggest that both genes are located on a mobile element that enables gene transfer between individual isolates and between streptococci from different Lancefield groups. S. equi pyrogenic exotoxin L (SPE-L(Se))/streptococcal pyrogenic exotoxin L (SPE-L) and SPE-M(Se)/SPE-M are most closely related to SMEZ, SPE-C, SPE-G, and SPE-J, but build a separate branch within this group. Recombinant SPE-L (rSPE-L) and rSPE-M were highly mitogenic for human peripheral blood lymphocytes, with half-maximum responses at 1 and 10 pg/ml, respectively. The results from competitive binding experiments suggest that both proteins bind major histocompatibility complex class II at the beta-chain, but not at the alpha-chain. The most common targets for both toxins were human Vbeta1.1 expressing T cells. Seroconversion against SPE-L and SPE-M was observed in healthy blood donors, suggesting that the toxins are expressed in vivo. Interestingly, the speL gene is highly associated with S. pyogenes M89, a serotype that is linked to acute rheumatic fever in New Zealand.

FIG. 1.

Family tree of streptococcal and staphylococcal SAgs. The tree was created using ClustalW (39) and the TreeView computer program (30). The novel SAgs belong to group A, together with SPE-C, SPE-G, SPE-J, and SMEZ, but build a separate branch within this group.

FIG. 2.

Multiple alignment of streptococcal SAg protein sequences. The protein sequences of mature proteins were aligned using the ClustalX computer program. The clear box near the C terminus represents a primary zinc binding motif (H-X-D), a feature of all streptococcal toxins except SPE-A and SSA. Structural elements of SMEZ-2 and SPE-C, respectively, are shown below.

FIG. 3.

Stimulation of human T cells with recombinant toxins. PBLs were isolated from human blood samples and incubated with various concentrations of recombinant toxin (in duplicates). After 3 days, 0.1 μCi of [³H]thymidine was added, and cells were incubated for another 24 h before being harvested and counted on a scintillation counter.

FIG. 4.

Competition binding studies with rSPE-L and rSPE-M. LG-2 cells were incubated in duplicate with 1 ng of ¹²⁵I-labeled recombinant toxin and increasing amounts of unlabeled toxins. After 1 h cells were washed and counted. (A) Competition assay with labeled rSPE-L; (B) competition assay with labeled rSPE-M.