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The antigenically variant M protein of Streptococcus pyogenes enhances virulence by promoting resistance to phagocytosis. The serum opacity factor (OF), produced by a subset of M serotypes, is also antigenically variant, and its antigenic variability exactly parallels that of M protein. OF-positive and OF-negative streptococci are also phenotypically distinguishable by a number of other criteria. In order to study the differences between OF-positive and OF-negative streptococci, we cloned and sequenced the type 49 M protein gene (emm49), the first to be cloned from an OF-positive strain. This gene showed evolutionary divergence from the OF-negative M protein genes studied previously. Furthermore, emm49 was part of a gene family, in contrast to the single-copy nature of previously characterized M protein genes.The M protein, a major virulence factor of Streptococcus pyogenes (group A streptococci), enhances the pathogenicity of the organism by blocking phagocytosis in the nonimmunized host (26,27). Antibodies to M protein develop in infected individuals and confer immunity (34). Streptococcal M protein, however, is antigenically variant, and opsonic antibodies to M protein are largely type specific. Thus, with 70 or more M protein serotypes (35), group A streptococci can successfully avoid immune surveillance.Molecular biological techniques have increased our knowledge of the structure of M protein and its antigenic variability. Five M protein genes have been cloned, and all have been at least partially sequenced (16,24,40,42,46). The M proteins encoded by these sequenced genes have a common framework which includes a conserved signal peptide, a hypervariable amino terminus, and a highly conserved C-terminus. Each gene also appears to contain some internal sequence repetitions, although the extent and degree of similarity of these repeats vary among the genes. Furthermore, regions preceding and following these M protein genes are conserved (16,42). This suggests that the genes are situated in a common expression site on the streptococcal chromosome.Our understanding of how the M protein hypervariable regions evolved is still incomplete. Duplication and deletion of intragenic repeats present in the variable regions of M molecules is a possible mechanism for the introduction of variant amino acids into the M protein sequences (25). Recent evidence shows that intragenic recombination can indeed introduce variant opsonic determinants into the repetitive regions of the M6 protein (28). Not all of the opsonic antigenic determinants of M proteins, however, are located in the repetitive regions of the molecules (25, 32). Thus, additional mechanisms must be involved in the generation of M protein antigenic diversity.A model explaining the generation of M protein antigenic diversity must account for the fact that other streptococcal proteins exhibit antigenic diversity in a nonrandom manner relative to the M type. Among these are the T antigen, a * Corresponding author. (18) and by genetic means (8). Clearly, the processe...

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Group A streptococci have traditionally been categorized into two broad groups based on the presence or absence of serum opacity factor (OF). Recent studies show that these two groups vary in a number of properties in addition to the OF phenotype, including sequence variations in the constant region of the antiphagocytic M protein genes, the presence or absence of immunoglobulin G Fc receptor proteins, and the presence or absence of multiple M protein-like genes situated in a tandem array. The M protein genes (emm) in OF- streptococcal strains are known to be part of a regulon of virulence-related genes controlled by the trans-acting positive regulatory gene, virR, situated just upstream of emm. In OF+ strains, however, the region adjacent to virR is occupied by an M protein-related, type IIa immunoglobulin G Fc receptor gene (fcrA), and the relative position of emm has not been determined. To further define the vir regulon in OF+ streptococci, we used the polymerase chain reaction to show that fcrA49 is situated immediately upstream of emm49 in the OF+ type 49 strain CS101. This result shows for the first time the separate identity and genetic linkage of these two genes in the vir regulon of an OF+ group A streptococcal strain and confirms our previous hypothesis that emm49 exists as the central gene in a trio of emm-like genes. Additionally, using DNA hybridizations, we found considerable sequence divergence between OF- and OF+ group A streptococci in virR and in the noncoding sequences between virR and the emm or fcrA expression site. We found, however, a high degree of sequence conservation in this region within each of the two groups of strains.

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