Characterisation of emergent toxigenic M1_UKStreptococcus pyogenesand associated sublineages

AbstractEmm1Streptococcus pyogenesis a successful, globally-distributed epidemic clone that is regarded as inherently invasive. Anemm1 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, M1UKis now dominant in England. To more fully characterise M1UK, we undertook comparative transcriptomic and proteomic analyses of M1UKand contemporary non-M1UKemm1 strains (M1global).Just seven genes were differentially expressed by M1UKcompared with contemporary M1globalstrains. 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. M1UKstrains have a stop codon in gldA. Deletion of the gldA gene in M1globalabrogated glycerol dehydrogenase activity, and recapitulated upregulation of gene expression within the operon that includes gldA, consistent with a feedback effect.Phylogenetic analysis identified two intermediateemm1 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 phylogeneticemm1 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 M123SNPsand M1UKsublineages, compared with M113SNPsand M1global.We conclude that stepwise accumulation of SNPs led to the emergence of M1UK. While increased expression of SpeA is a key indicator of M1UKand undoubtedly important, M1UKstrains have outcompeted M123SNPsand otheremmtypes that produce similar or more superantigen toxin. We speculate that an accumulation of adaptive SNPs has contributed to a wider fitness advantage in M1UKon an inherently successfulemm1 streptococcal background.Data availabilityRNAseq. All new RNAseq data are uploaded to the European Nucleotide Archive under project reference PRJEB58303Genomic 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 PRIDEImpact SummaryAlthough the majorStreptococcus pyogenesreservoir is in children with pharyngitis and skin infections,S. pyogenescan lead to rarer, invasive infections that are rapidly progressive and associated with high mortality and morbidity.Emm1S. pyogenesstrains are the single most frequent genotype to cause invasive infections in high income countries and are established worldwide as an epidemic clone. The M1UKS. pyogenes emm1 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 otheremm1 strains in England. Increased dominance ofemm1 among invasive infections this winter, on a background of already-increased numbers ofS. pyogenesinfections, 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 circulatingS. pyogenesandemm1 invasive infections. In this paper we undertake a systems-based evaluation of M1UKin comparison to older non-M1UKemm1 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 ofS. pyogenesto 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.