The multiple molecular structure of the M proteins of group A streptococci.

Abstract

The M proteins are the antigens conferring serological specificity to the 45 or 50 types of group A streptococci. IMnore importantly, these proteins play a predominant role in virulence by rendering the streptococci refractory to phagocytosis in the absence of type-specific antibody. Owing to unusual stability at low pH and high temperature,' the M proteins are best obtained from whole cells or streptococcal cell walls by solubilization at pH 2 and 1000. Recent studies have shown that the M proteins may be solubilized from cell walls with a muralytic enzyme released during bacteriophage lysis of group C streptococci. Materials and Methods.-Streptococci and antisera: The group A streptococci, types 12, 14, and 24, culture methods, immunodiffusion techniques, and methods for the preparation of adsorbed and unadsorbed typing sera have been previously described.5 6Reagents: Crude lytic enzyme of the group C streptococcal bacteriophage system was prepared as previously described.6 The crude enzyme was concentrated from the cell lysate by precipitation at 00 in 70% ammonium sulfate. One ,ug of enzyme per ml decreased the optical density of a suspension of whole streptococci from 0.20 to 0.10 in 30 min. Buffered saline containing 0.01 M potassium phosphate pH 7.0 was used as the antigen diluent for serological procedures and Sephadex chromatography.Preparation of M proteins: Streptococci harvested after growth for 18 hr in enriched Difco Todd-Hewitt broth5 were washed and ruptured with glass beads in a Gifford-Wood Eppenbach Micro-Mill according to the method of Markowitz and Lange.7 For smaller amounts of cells the Braun MSK homogenizer was used to obtain cell walls according to the method of Bleiweis et al.8The following is a typical protocol using 130 gm of wet cells broken in the Eppenbach mill. After removal of the glass beads with a coarse sintered glass funnel, the debris composed of cell walls and membranes was washed twice in 2-liter volumes of buffered saline by centrifugation at 9,000 X g for 30 min. All operations were carried out at 0-5'. The cell walls containing the M protein were separated from the membranes by sedimenting the walls at 5,000 X g for 30 min in 2 liters of buffered saline; this step was repeated three times. At each washing the walls were evenly resuspended with a Waring Blendor; octyl alcohol was added to prevent foaming. After the

The Relationship between M-antigen and Opacity Factor in Group A Streptococci

“…The first peak emerged as the pH of the buffer approached 5-5, and the second as the pH changed from 5-5 to 7. This was in agreement with the results of Fox & Wittner (1965, 1969 for other M-antigens. The first peak contained M-protein but the E260,280 ratio for this peak was 1-7 indicating possible contamination with residual nucleic acids.…”

“…The P 60 fractions of the M-positive type 49 and type 2 strains were subjected to CMC chromatography with a stepwise pHgradient from 4.0 to 7.0 (Fox & Wittner, 1965). The resultant chromatogram (Fig.…”

“…was added and the column eluted with 0-1 M-acetate buffer pH 5-5 until the pH of the effluent reached 5.5. Elution was continued with 0.1 M-K phosphate buffer pH 7 until the effluent pH reached 7 ( Fox & Wittner, 1965).…”

Amino acid sequence and physicochemical similarities between streptococcal M protein and mammalian tropomyosin.

“…RESULTS AND DISCUSSION M proteins characteristically contain unusually high levels of lysine, aspartate or asparagine, glutamate or glutamine, leucine, and alanine but no cysteine and only negligible amounts of glycine, proline, and aromatic amino acids (7,(13)(14)(15)(16). Such a distribution of amino acids is typical of a fibrous proteins in general (17); of these, skeletal muscle tropomyosin bears the greatest resemblance in composition to M proteins.…”

“…In comparing the sequence of the M-24 peptides (7) with that of rabbit skeletal tropomyosin (25), it was noted that M-24 peptides CB-3 through CB-7 all contain a hexapeptide, GluAla-Glu-Lys-Ala-Ala (residues [14][15][16][17][18][19], which is identical to segment 115-120 of tropomyosin. Although this hexapeptide occurs five times in the M-24 molecule, it is found in no other protein sequence listed in the Protein Segment Dictionary 76 (27).…”