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␤2, V␤4, and V␤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-cellstimulating activity. Although several crude preparations of M5 protein were associated with mitogenicity for V␤2 and V␤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.

M protein of a Streptococcus dysgalactiae human wound isolate shows multiple binding to different plasma proteins and shares epitopes with keratin and human cartilage

“…The C repeat region was not necessary for this interaction. The majority of M proteins bind to ¢brinogen [1,25,30,31] and the Nterminal half, which represents the variable region in these molecules, seems to be the ¢brinogen binding site. However, by sequence comparisons of the Nterminal variable segments of M protein sequences accessible in the EMBL database a consensus sequence for ¢brinogen binding could so far not be identi¢ed.…”

“…The N-terminal half of MC, including the regions A and B, was found to bind ¢brinogen. This con-¢rms the variable N-terminal part of M proteins as ¢brinogen binding epitope [1,25,30]. However, by database sequence comparisons with the N-terminal parts of known M protein amino acid sequences a characteristic sequence motif for ¢brinogen binding could not be identi¢ed.…”

“…Vimentin (bovine) was obtained from Sigma Chemical Co. The N-terminal half of M5 protein, pepM5, was produced as described recently [30]. Antiserum against MC was raised in rabbits with aliquots of 100 Wg of the entire puri¢ed protein for each immunisation step by the protocol of Louvard et al [17].…”

“…MC was found at the cell surface as well as in the culture supernatant of strain 25287. Native MC was isolated from mutanolysin extract of harvested cells from a 5-l streptococcal culture [30] and from the supernatant of this culture [34]. The proteins were precipitated with ammonium sulfate at 70% saturation in the culture supernatant.…”

“…The resulting precipitate was resuspended in distilled water and dialysed against 0.02 M ammonium hydrogen carbonate. Dialysed material was centrifuged and the supernatant containing the crude protein was puri¢ed by a¤nity chromatography on ¢brinogen Sepharose [30,32]. The column was equilibrated and washed with 0.05 M Tris, 0.15 M NaCl, pH 7.5.…”