Cloning, expression, and characterization of the superantigen streptococcal pyrogenic exotoxin G from Streptococcus dysgalactiae

Abstract

We identified seven novel variants of streptococcal pyrogenic exotoxin G (SPEGG), a superantigen, in Streptococcus dysgalactiae subsp. dysgalactiae or equisimilis isolates from clinical cases of infection in humans and animals. Phylogenetic analysis of the SPEGG variants indicated two clades in the dendrogram: one composed of variants derived from the bacteria isolated from the humans and the other composed of variants from the bacteria isolated from the animals. Bovine peripheral blood mononuclear cells (PBMCs) were stimulated effectively by recombinant SPEGGs (rSPEGGs) expressed in Escherichia coli, while human PBMCs were not stimulated well by any of the rSPEGGs tested. SPEGGs selectively stimulated bovine T cells bearing Vbeta1,10 and Vbeta4. Bovine serum showed reactivity to the rSPEGG proteins. These results indicated that SPEGGs have properties as superantigens, and it is likely that SPEGGs play a pathogenic role in animals.

FIG. 1.

PFGE pattern of S. dysgalactiae isolates carrying spegg genes and a dendrogram constructed by computer-assisted comparison of PFGE patterns generated from all isolates used in this study. In the dendrogram, the isolates carrying spegg are shown in boldface. Isolates from humans are indicated with a dollar sign ($). The scale bar indicates percent similarity among the isolates.

FIG. 2.

Multiple alignment of SPE-C, SPE-G, and SPEGG amino acid sequences. The amino acid sequences were aligned using Clustal W (35). The bars above the sequences indicate the secondary structure based on the crystal structure of SPE-C (PDB identification no. 1AN8). Highly conserved regions and the zinc-binding motif are boxed with dotted lines. SPEGG variants derived from isolates from animals (SPEGG6 to SPEGG8) are separated with a dotted line from those from humans, and the residues commonly mutated in SPEGG6 to SPEGG8 are indicated in boxes (also see Fig. 6). Corresponding residues of interest (see text for details) in the alignment are marked with Δ.

FIG. 3.

Phylogenetic tree of streptococcal and staphylococcal SAGs, including SPEGGs. This phylogenetic tree was constructed using Clustal W, and the GenBank accession numbers of the SAGs sequences are as follows: SEH, CAI77677; SePE-H, AAF72809; SPE-H, AAK33907; SPE-A, AAL97141; SEG, AAX11325; SSA, AAA65928; SEB, AAL04126; SEC3, AAA26624; SEC2, P34071; SED, P20723; SEJ, AAC78590; SEP, BAB43036; SEA, AAP37183; SEE, P12993; SePE-I, AAF72808; SPE-I, AAL31571; SEL, BAB58170; SEK, AAL04147; SEQ, AAL04146; SEM, AAG36925; TSST-1, BAB58173; SPE-C, AAA27017; SPE-J, AAZ50974; SPE-G, AAZ50801; SMEZ-2, AAD52087; SMEZ, CAD91900; SPE-L, BAC63752; SPE-Lse, CAH65000; SPE-Mse, CAH68555; SPE-M, AAL97849; and SDM, AB074529.

FIG. 4.

Stimulation of human and bovine PBMCs with rSPEGGs. PBMCs were isolated from bovine (A) and human (B) blood samples and incubated with various concentrations of rSPEGGs. After 2 days, 0.1 μCi of [³H]thymidine was added, and cells were incubated for a further 18 h before being harvested and counted for the thymidine uptake with a beta counter. These experiments are representative of four independent experiments. In panel C, bovine PBMCs (solid bars) and T-cell-depleted bovine PBMCs (open bars) were stimulated with 1 μg/ml of SPEGGs and SPE-G for 2 days and the mitogenic response was measured as described in Materials and Methods. Serial dilution was performed without SPEGG and used as a negative control (marked as “cell”). cont, control.

FIG. 5.

Reactivity of bovine serum to rSPEGGs. Serum samples obtained from 10 cows were diluted 1:1,000, and their reactivity to SPEGG was analyzed using ELISA. Fetal calf serum (FCS) was used as a negative control, and the threshold line is presented according to the absorbance (Abs.) obtained from FCS.

FIG. 6.

Molecular modeling of the SPEGG protein. SPEGG2 and -3 were modeled onto the crystal structures of SPE-C with MODELLER (32). Residues that differed between animal and human forms of SPEGG are shown in black.