Characterization of the RofA regulon in the pandemic M1globaland emergent M1UKlineages ofStreptococcus pyogenes

Abstract

AbstractBackground & AimsThe standalone regulator RofA is a positive regulator of the pilus locus inStreptococcus pyogenes. Found in only certainemmgenotypes, RofA has been reported to regulate other virulence factors, although its role in the globally dominantemm1S. pyogenesis unclear. Given the recent emergence of a newemm1 (M1UK) toxigenic lineage that is distinguished by three non-synonymous SNPs inrofA, we characterized therofAregulon in sixemm1 strains, that are representative of the two contemporary majoremm1 lineages (M1globaland M1UK) using RNAseq analysis, and then determined the specific role of the M1UK-specificrofASNPs.ResultsDeletion ofrofAin three M1globalstrains led to altered expression of 14 genes, including six non-pilus locus genes. In M1UKstrains, deletion ofrofAled to altered expression of 16 genes, including 9 genes that were unique to M1UK. Only the pilus locus genes were common to the RofA regulons of both lineages, while transcriptomic changes varied between strains even within the same lineage. Although introduction of the 3 SNPs intorofAdid not impact gene expression in an M1globalstrain, reversal of 3 SNPs in an M1UKstrain led to an unexpected number of transcriptomic changes that in part recapitulated transcriptomic changes seen when deleting RofA in the same strain. Computational analysis predicted interactions with a key histidine residue in the PRD domain of RofA would differ between M1UKand M1global.SummaryRofA is a positive regulator of the pilus locus in allemm1 strains but effects on other genes are strain- and lineage-specific, with no clear, common DNA binding motif. The SNPs inrofAthat characterize M1UKmay impact regulation of RofA; whether they alter phosphorylation of the RofA PRD domain requires further investigation.Author summaryRofA belongs to the group of “mga-like” bacterial regulatory proteins that comprise a DNA binding domain as well as a phosphorylation domain (PRD) that is responsive to changes in sugar availability. In certainemmgenotypes ofStreptococcus pyogenes,rofAsits upstream of the pilus locus, to act as a positive regulator. The recent emergence of a SpeA exotoxin-producing sublineage ofemm1S. pyogenes, (M1UK) has focused attention on the role of RofA; M1UKand its associated sublineages are characterized by 3 non-synonymous SNPs inrofA,that include adjacent SNPs in the PRD domain. Here, we determine the impact ofrofAdeletion and the 3rofASNPs in both the widely disseminated M1globalclone and the newly emergent M1UKclone. While production of SpeA undoubtedly contributes to infection pathogenesis, the evolution of M1UKpoints to a role for metabolic regulatory rewiring in success of this lineage.