Recombinant expression and characterisation of putative streptococcal pyrogenic exotoxins SPEGdys, SPELdys, and SPEMdys from Streptococcus dysgalactiae, subsp. equisimilis
Abstract number: o368
Schmidt K.H., Sachse S., Rödiger S., Gerlach D., Straube E., Rödel J.
Besides human pathogenic Streptococcus pyogenes (GAS) group C (GCS) and G (GGS) streptococci can be pathogenic for different mammalians. Human pathogenic GCS and GGS (hpGCS/GGS, Streptococcus dysgalactiae, subsp. equisimilis) can cause, like GAS, wound infections, otitis media, pharyngitis and streptococcal toxic shock syndrome. Recently, we found a superantigen (SAG) like gene, speGdys in hpGGS. Later genes encoding SPELdys and SPEMdys were also detected in hpGGS. The sequences of the encoding proteins (SPE's) were highly homologeous but not fully identical to the corresponding genes from group A streptococci.
Methods and Results:
We cloned all three genes derived from hpGGS in E. coli. In all constructs the putative signal peptides were omitted. All three recombinant proteins were expressed as inclusion bodies and remained soluble in urea buffers at a minimum concentration of 6 M. Renaturation succeeded by stabilisation with 15 mg/ml albumin following stepwise reduction of the urea concentration by dialysis against 0.05 M Tris, 0.15 M NaCl buffer, pH 8.0. The preparations were tested in the lymphocyte transformation test (LTT). The culture filtrates of the original hpGGS strains did not show human T cell stimulating activity. In the same manner, the in E. coli expressed recombinant proteins rSPEGdys as well as rSPELdys did not stimulate human T lymphocytes. rSPEMdys is still under investigation. The absence of biological activity was surprising, because each of the recombinant putative SPE's from hpGGS contain zinc binding region (H-F-D) and other binding sites like SPEG and SPEA from GAS. These recombinant proteins, cloned from GAS, stimulated T cells. It seems that the few differences in the AA sequences between the hpGGS putative SAG's and the GAS SAG's were responsible for the decreased biological activity of the proteins derived from hpGGS.
Until now, an active mitogen for human T cells has not been isolated from hpGCS and hpGGS. Active mitogens, so far, came from group C and group G streptococci isolated from horse or other animals. Instancing SPEGdys, in this case we found gene transfer from hpGGS to GAS. We suggest that after the gene transfer of SPE's from GGS to GAS, the genes in the receiving strains were altered. In GAS they may have changed resulting in production of active SAG's. Using the putative SPE's from hpGGS, LTT experiments with T cells from blood of animals, susceptible to GCS/GGS infection, are in progress.
|Session name:||XXIst ISTH Congress|
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