Genotype emm4 Streptococcus pyogenes constitute a major disease-causing emm-type worldwide, and have been associated with scarlet fever. Genotype emm4 are lysogenised by three toxigenic prophage, and an atoxigenic SpyCI, thought to regulate the DNA mismatch repair operon by growth-phase dependent excision. Phylogenetic analyses determined that emm4 in our collection and internationally, clustered with either reference genomes MGAS10750 or MEW427, the latter of which were found to have undergone substantial gene loss within prophage-encoding regions. Gene loss affected modules pertaining to lysogeny, genetic regulation, replication, and encapsidation, but not cognate toxins. Corresponding elements of MGAS10750-like emm4 were full-length and undegraded. The lineages associated with either MGAS10750 or MEW427 were thus denoted M4complete and M4degraded, respectively. Prophage of M4degraded isolates were shown to be cryptic, and incapable of excision, whereas prophage of M4complete isolates were inducible. Unexpectedly, this had little effect on cognate toxin expression, which was independent of induction and replication. Further, the emm4 SpyCI was found to be immobile, irrespective of gene content, and appears not to regulate the DNA MMR operon, as had been reported. A number of assays did not demonstrate lineage-specific phenotypes, however, M4complete isolates were significantly more virulent than M4degraded isolates in a Galleria mellonella infection model. Intriguingly, efforts to explore this response in more infection-relevant conditions revealed that co-culture with human tonsil cells greatly enhanced expression of the prophage-associated superantigen ssa, among both M4complete and M4degraded isolates, and this was also observed among emm3 and emm12. There may also be a hitherto unappreciated role for major bacterial regulators in the control and elicitation of this response. These data suggest that the relationship between S. pyogenes, their viral parasites and the human host may be much more complex than had been anticipated, and present a number of exciting avenues for further study
