Characterization of the RofA regulon in the pandemic M1global and emergent M1UK lineages of Streptococcus pyogenes

The standalone regulator RofA is a positive regulator of the pilus locus in Streptococcus pyogenes. Found in only certain emm genotypes, RofA has been reported to regulate other virulence factors, although its role in the globally dominant emm1 S. pyogenes is unclear. Given the recent emergence of a new emm1 (M1 UK) toxigenic lineage that is distinguished by three non-synonymous SNPs in rofA, we characterized the rofA regulon in six emm1 strains that are representative of the two contemporary major emm1 lineages (M1 (global) and M1 (UK)) using RNAseq analysis, and then determined the specific role of the M1 (UK)-specific rofA SNPs. Deletion of rofA in three M1 (global) strains led to altered expression of 14 genes, including six non-pilus locus genes. In M1 (UK) strains, deletion of rofA led to altered expression of 16 genes, including nine genes that were unique to M1 (UK). 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 three SNPs into rofA did not impact gene expression in an M1 (global) strain, reversal of three SNPs in an M1 (UK) strain 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 that interactions with a key histidine residue in the PRD domain of RofA would differ between M1 (UK) and M1 (global). RofA is a positive regulator of the pilus locus in all emm1 strains but effects on other genes are strain- and lineage-specific, with no clear, common DNA binding motif. The SNPs in rofA that characterize M1 (UK) may impact regulation of RofA; whether they alter phosphorylation of the RofA PRD domain requires further investigation.