Figure 1.

The ropB polymorphisms in serotype M3 group A streptococci (GAS) strains demonstrate a pattern of diversifying selection and significantly alter RopB regulatory activity. A, The ropB gene was sequenced in 1178 serotype M3 GAS strains collected from patients with different disease manifestations in diverse geographic regions and time periods. The numbers of strains with a wild-type (dark brown bar) or variant (light brown bar) ropB allele are shown. B, Gene sequencing identified 84 ropB alleles. Relative to the wild-type sequence, every polymorphism alters the RopB protein. The number of variant ropB alleles encoding each type of amino acid change is shown. C, Most of the 83 variant ropB alleles were present in only a few strains, with 54 alleles being specific to a single strain. D, RopB is the major regulator of secreted streptococcal cysteine protease B (SpeB), a proven virulence factor implicated in invasive disease. Most strains with a variant ropB allele demonstrated markedly reduced or absent SpeB expression as measured by Western immunoblot assay of culture supernatants. Representative strains are shown. Multiple immunoblots were performed simultaneously and processed identically, with lanes reordered such that ropB alleles were shown left to right with respect to the amino acid sequence. Recombinant SpeB zymogen and supernatant from a GAS strain with the gene encoding SpeB deleted (designated ΔspeB) were used as a positive and negative control, respectively. E, Similarly, most strains with a variant ropB allele demonstrated markedly reduced or absent SpeB secreted protease activity, as measured by casein hydrolysis in milk plates.