| In the past two decades, the reemergence of severe invasive group A streptococcal (GAS) diseases has been associated with few strains, including an M1T1 substrain that has shown an unprecedented global spread and an unusual persistence. In susceptible hosts, clonal M1T1 isolates show a highly virulent phenotype characterized by evasion of host immunity and secretion of invasive proteins and superantigenic toxins. To understand the basis for M1T1's unusual predominance, we analyzed its genome and extracellular proteome to identify unique genetic and phenotypic features that set it off from other members of the same M1 serotype. Analysis of M1T1 secreted proteome revealed that the streptococcal cysteine protease, SpeB, degraded most extracellular GAS proteins, including several virulence factors. A SpeB+/SpeA -M1T1 GAS strain injected into murine subcutaneous chambers shifted to a stable SpeB-/SpeA+ phenotype that possessed enhanced lymphocyte-stimulating capacity. The absence or the inactivation of SpeB allowed proteomic identification of M1T1 proteins that are absent in the previously sequenced M1 SF370, including SpeA2, a known superantigen, and a novel chimeric phage-encoded streptodornase that we named Sda1. We determined sda1 nucleotide sequence and found that, while the protein shares two highly conserved domains with several DNases and putative DNases in streptococci, it possesses a unique C-terminal domain that was found to confer increased activity on the protein. The cloned and expressed Sda1 degraded streptococcal and mammalian DNA at physiological pH. Subsequently, we complemented our proteomic studies by screening for the unique genes of the M1T1 strain. Using differential microarry hybridization, we identified three prophages that we named SPhinX, MemPhiS, and PhiRamid. While MemPhiS is similar to phage 370.3 of the SF370 strain, SPhinX and PhiRamid are unique to M1T1. The genomes of these three prophages are highly mosaic, with different segments being related to distinct streptococcal phages. Bioinformatic and phylogenetic analyses revealed a highly conserved open reading frame (ORF) that we named 'paratox.' We found linkage disequilibrium between particular paratox alleles and specific toxin genes. Based on the conservation of paratox and other genes flanking the toxins, we propose a recombination-based model for toxin dissemination among prophages. |
