title: Mitogenicity of M5 protein extracted from Streptococcus pyogenes cells is due to streptococcal pyrogenic exotoxin C and mitogenic factor MF.
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.
authors.affiliation: Institute of Experimental Microbiology, Friedrich Schiller University, Jena, Federal Republic of Germany.
authors.forename: K H
authors.initials: KH
authors.lastname: Schmidt
authors.forename: D
authors.initials: D
authors.lastname: Gerlach
authors.forename: L
authors.initials: L
authors.lastname: Wollweber
authors.forename: W
authors.initials: W
authors.lastname: Reichardt
authors.forename: K
authors.initials: K
authors.lastname: Mann
authors.forename: J H
authors.initials: JH
authors.lastname: Ozegowski
authors.forename: B
authors.initials: B
authors.lastname: Fleischer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D001483:Base Sequence; D002352:Carrier Proteins; D005098:Exotoxins / Q000302:isolation & purification / Q000494:pharmacology*; D006801:Humans; D008565:Membrane Proteins; D008934:Mitogens / Q000302:isolation & purification / Q000494:pharmacology*; D008969:Molecular Sequence Data; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology; D013045:Species Specificity; D013297:Streptococcus pyogenes / Q000502:physiology*; D018089:Superantigens / Q000302:isolation & purification
chemical_list: D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D005098:Exotoxins; D008565:Membrane Proteins; D008934:Mitogens; D011994:Recombinant Proteins; C117980:SpeA protein, Streptococcus pyogenes; D018089:Superantigens; C027951:erythrogenic toxin; C083356:mitogenic factor, Streptococcus; C014689:streptococcal M protein