Characterization of emergent toxigenic M1 and associated sublineages.

genotype 1 is a successful, globally distributed epidemic clone that is regarded as inherently virulent. An 1 sublineage, M1, that produces increased levels of SpeA toxin was associated with increased scarlet fever and invasive infections in England in 2015/2016. Defined by 27 SNPs in the core genome, M1 is now dominant in England. To more fully characterize M1, we undertook comparative transcriptomic and proteomic analyses of M1 and contemporary non-M1 1 strains (M1). Just seven genes were differentially expressed by M1 compared with contemporary M1 strains. In addition to , five genes in the operon that includes glycerol dehydrogenase were upregulated in M1 (, and phosphotransferase system IIC and IIB components), while aquaporin () was downregulated. M1 strains have a stop codon in . Deletion of in M1 abrogated glycerol dehydrogenase activity, and recapitulated upregulation of gene expression within the operon that includes , consistent with a feedback effect. Phylogenetic analysis identified two intermediate 1 sublineages in England comprising 13/27 (M1) and 23/27 SNPs (M1), respectively, that had failed to expand in the population. Proteomic analysis of invasive strains from the four phylogenetic 1 groups highlighted sublineage-specific changes in carbohydrate metabolism, protein synthesis and protein processing; upregulation of SpeA was not observed in chemically defined medium. In rich broth, however, expression of SpeA was upregulated ~10-fold in both M1 and M1 sublineages, compared with M1 and M1. We conclude that stepwise accumulation of SNPs led to the emergence of M1. While increased expression of SpeA is a key indicator of M1 and undoubtedly important, M1 strains have outcompeted M1 and other types that produce similar or more superantigen toxin. We speculate that an accumulation of adaptive SNPs has contributed to a wider fitness advantage in M1 on an inherently successful 1 streptococcal background.