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Mitogenicity of M5 protein extracted from Streptococcus pyogenes cells is due to streptococcal pyrogenic exotoxin C
K H Schmidt1, D Gerlach, L Wollweber
1Institute of Experimental Microbiology, Friedrich Schiller University, Jena, Federal Republic of Germany.
Abstract:
M proteins of Streptococcus pyogenes are virulence factors which impede phagocytosis, bind to many plasma proteins, and induce formation of cross-reactive autoimmune antibodies. Recently, it has been reported that some M proteins, extracted with pepsin from streptococci (pep M), are superantigens. One of these, pep M5, was investigated in detail and was shown to stimulate human T cells bearing V beta 2, V beta 4, and V beta 8. In the present study, we extracted and purified M5 protein by different biochemical methods from two M type 5 group A streptococcal strains. The crude extracts were fractionated by affinity chromatography and ion-exchange chromatography. All fractions were tested in parallel for M protein by immunoblotting and for T-cell-stimulating activity. Although several crude preparations of M5 protein were associated with mitogenicity for V beta 2 and V beta 8 T cells, the M5 proteins, irrespective of the extraction method, could be purified to the extent that they were no longer mitogenic. The mitogenic activity was not destroyed during the purification procedures but was found in fractions separated from M protein. In these fractions, streptococcal pyrogenic exotoxin C and mitogenic factor MF could be detected by protein blotting and enzyme-linked immunosorbent assay. Moreover, anti-M protein sera did not inhibit the mitogenic activity of crude extracts, but antisera which contained anti-streptococcal pyrogenic exotoxin C antibodies showed inhibition. The inability of M5 protein to stimulate T cells was confirmed with recombinant pep M5 produced in Escherichia coli. Our data strongly suggest that the mitogenic activity in M protein preparations is caused by traces of streptococcal superantigens different from M protein.
Insights
M5 protein from Streptococcus pyogenes does not directly stimulate T cells. Instead, crude M protein preparations contain superantigens, like streptococcal pyrogenic exotoxin C, which cause T cell mitogenicity.
Area of Science:
- Microbiology
- Immunology
- Molecular Biology
Background:
- M proteins are key virulence factors in Streptococcus pyogenes, mediating immune evasion and autoimmune responses.
- Some pepsin-extracted M proteins (pep M) have been identified as superantigens, capable of stimulating T cells.
Purpose of the Study:
- To investigate the T-cell stimulating activity of purified M5 protein from Streptococcus pyogenes.
- To determine if M5 protein itself is mitogenic or if the activity originates from contaminating factors.
Main Methods:
- Extraction and purification of M5 protein using affinity and ion-exchange chromatography.
- Testing T-cell mitogenicity of M5 protein fractions using human T cells.
- Detection of streptococcal pyrogenic exotoxin C and mitogenic factor (MF) using immunoblotting and ELISA.
- Confirmation with recombinant pep M5 protein.
Main Results:
- Purified M5 protein lacked T-cell mitogenic activity, regardless of extraction method.
- Mitogenic activity in crude extracts was associated with fractions separate from M5 protein.
- Streptococcal pyrogenic exotoxin C and MF were identified in the mitogenic fractions.
- Antibodies against streptococcal pyrogenic exotoxin C, but not M protein, inhibited mitogenicity.
Conclusions:
- The mitogenic activity observed in crude M protein preparations is attributed to contaminating streptococcal superantigens, not the M5 protein itself.
- M5 protein from Streptococcus pyogenes is not inherently mitogenic for T cells.
- Accurate identification of bacterial virulence factors requires rigorous purification to exclude contaminating superantigens.