Mechanism of stimulation of T cells by Streptococcus pyogenes: isolation of a major mitogenic factor, cytoplasmic membrane-associated protein.

Our previous studies established that heat-killed Streptococcus pyogenes, as well as other gram-positive cocci, when incubated with human peripheral blood leukocytes (PBLs) in culture, induced polyclonal activation of T lymphocytes. The activated T lymphocytes included CD4+ CD8- helper T cells and CD3+ and CD4- CD8- double-negative T cells with gamma delta T-cell receptors. In the present study, we isolated a major factor with this unique mitogenic activity against human T lymphocytes from S. pyogenes. This active fraction was found in the cytoplasmic membrane (CM) of the heat-killed organisms but not in other cellular fractions such as cell walls, peptidoglycan, lipoteichoic acids, or cytoplasmic soluble fractions. An active molecule(s) was further isolated from the CM by cholic acid extraction followed by Sephacryl S-200 chromatography. The molecule was protease labile but highly resistant to heat, had a pI of greater than or equal to 9.3 and a molecular weight of 10,000 to 15,000 according to gel filtration experiments, and was termed CM-associated protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protein purified by anion-exchange chromatography showed a single band with a molecular weight of 15,000, corresponding to mitogenically active regions. Purified CM-associated protein induced activation of T lymphocytes, which consisted of CD4+ CD8- T cells and CD4- CD8- double-negative T-cell receptor gamma/delta + T-cell populations, as did the whole cells of S. pyogenes.