Molecular Characterization of the Group A <i>Streptococcus</i> Virulence‐Regulatory System <scp>FasBCAX</scp>

By regulating the assortment and abundance of its virulence factors at different anatomic sites, the group A Streptococcus (GAS) can cause a range of human diseases. The Fas regulatory system is encoded by a four-gene locus, fasBCAX, with fasX encoding the FasX small regulatory RNA effector molecule. FasX post-transcriptionally regulates target mRNAs through well-characterized mechanisms. Less characterized are the layers of regulation that occur upstream of FasX activity, such as how the products of the fasBCA genes enhance FasX abundance 100-fold. Here, we present data consistent with FasBCA forming a three-component regulatory system, with FasBC being sensor kinase-like proteins that, upon recognizing one or more signals, heterodimerize and phosphorylate FasA, with phosphorylated FasA binding to the fasX promoter and inducing transcription. We identified key amino acids involved in phosphate flow, including H246 of FasC and D60 of FasA, and demonstrated that certain domains (e.g., the kinase domain of FasC) are dispensable for activity. Additionally, we show that a proteinaceous factor within human plasma activates the Fas system. This work represents the first molecular analysis of the Fas proteins which, by modulating FasX levels, play a critical role in the ability of GAS to coordinately regulate virulence factor production.