Characterisation of emergent toxigenic M1UK Streptococcus pyogenes and associated sublineages

Emm 1 Streptococcus pyogenes is a successful, globally-distributed epidemic clone that is regarded as inherently invasive. An emm 1 sublineage, M1UK, that expresses increased SpeA toxin, was associated with increased scarlet fever and invasive infections in England in 2015/2016. Defined by 27 SNPs in the core genome, M1UK is now dominant in England. To more fully characterise M1UK, we undertook comparative transcriptomic and proteomic analyses of M1UK and contemporary non-M1UK emm 1 strains (M1global). Just seven genes were differentially expressed by M1UK compared with contemporary M1global strains. In addition to speA, five genes in the operon that includes glycerol dehydrogenase were upregulated in M1UK (gldA, mipB/talC, pflD, and pts system IIC and IIB components), while aquaporin (glpF2) was downregulated. M1UK strains have a stop codon in gldA. Deletion of the gldA gene in M1global abrogated glycerol dehydrogenase activity, and recapitulated upregulation of gene expression within the operon that includes gldA, consistent with a feedback effect. Phylogenetic analysis identified two intermediate emm 1 sublineages in England comprising 13/27 (M113SNPs) and 23/27 SNPs (M123SNPs) respectively, that had failed to expand in the population. Proteomic analysis of these four major phylogenetic emm 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, transcription and secretion of SpeA was upregulated ~10-fold in both M123SNPs and M1UK sublineages, compared with M113SNPs and M1global. We conclude that stepwise accumulation of SNPs led to the emergence of M1UK. While increased expression of SpeA is a key indicator of M1UK and undoubtedly important, M1UK strains have outcompeted M123SNPs and other emm types that produce similar or more superantigen toxin. We speculate that an accumulation of adaptive SNPs has contributed to a wider fitness advantage in M1UK on an inherently successful emm 1 streptococcal background. Data availability RNAseq. All new RNAseq data are uploaded to the European Nucleotide Archive under project reference PRJEB58303 Genomic data. All genomes listed are available on the European Nucleotide Archive using accession numbers as listed in the appendix, Proteomes. Proteomic data are available on FigShare 10.6084/m9.figshare.21777809 and will be uploaded to PRIDE Impact Summary Although the major Streptococcus pyogenes reservoir is in children with pharyngitis and skin infections, S. pyogenes can lead to rarer, invasive infections that are rapidly progressive and associated with high mortality and morbidity. Emm 1 S. pyogenes strains are the single most frequent genotype to cause invasive infections in high income countries and are established worldwide as an epidemic clone. The M1UK S. pyogenes emm 1 sublineage which is defined by 27 new SNPs in the core genome, and characterised by increased scarlet fever toxin SpeA production, emerged and rose to dominance over a period of 5-6 years since initial recognition, outcompeting other emm 1 strains in England. Increased dominance of emm 1 among invasive infections this winter, on a background of already-increased numbers of S. pyogenes infections, points to a key shift in host-pathogen interaction. We hypothesize that a combination of pathogen fitness, virulence, and host susceptibility have coalesced to account for the excess of circulating S. pyogenes and emm 1 invasive infections. In this paper we undertake a systems-based evaluation of M1UK in comparison to older non-M1UK emm 1 strains, and identify a number of pathways that are altered in addition to the previously-reported increased SpeA expression. The emergence of a new sublineage within an already virulent clone requires ongoing surveillance, and more detailed investigation of the likely mechanisms leading to increased fitness. The capacity of S. pyogenes to cause outbreaks at national scale highlights a potential need to consider strain-specific public health guidance, underlining the inherent virulence of this exclusively human pathogen. ### Competing Interest Statement The authors have declared no competing interest.