The LiaFSR transcriptome reveals an interconnected regulatory network in group A Streptococcus

The mechanisms by which bacteria sense the host environment and alter gene expression are poorly understood. LiaFSR is a gene regulatory system unique to Gram-positive bacteria including group A Streptococcus (GAS) and responds to cell envelope stress. We previously showed that LiaF acts as an inhibitor to LiaFSR activation in GAS. To better understand gene regulation associated with LiaFSR activation, we performed RNA-sequencing on isogenic deletion mutants fixed in a LiaFSR “always on” (DliaF) or “always off” (DliaR) state. Transcriptome analyses of DliaF and DliaR in GAS showed near perfect inverse correlation including the gene encoding the global transcriptional regulator SpxA2. In addition, mutant transcriptomes included genes encoding multiple virulence factors and showed substantial overlap with the CovRS regulon. Chromatin immunoprecipitation quantitative PCR demonstrated direct spxA2 gene regulation following activation of the response regulator, LiaR. High SpxA2 levels as a result of LiaFSR activation were directly correlated with increased CovR-regulated virulence gene transcription. Further, consistent with known virulence gene repression by phosphorylated CovR, elevated SpxA2 levels were inversely correlated with CovR phosphorylation. Despite increased transcription of several virulence factors, DliaF (high SpxA2) exhibited a paradoxical virulence phenotype in both in vivo mouse and ex vivo human blood models of disease. Likewise, despite decreased virulence factor transcription in DliaR (low SpxA2), increased virulence was observed in an in vivo mouse model of disease – a phenotype attributable, in part, to known SpxA2-associated speB transcription. Our findings provide evidence of a critical role of LiaFSR in sensing the host environment and suggest a potential mechanism for gene regulatory system crosstalk shared by many Gram-positive pathogens. Comparison of wild-type to mutants lacking the response regulator (LiaR) or the accessory protein (LiaF) of the LiaFSR system in group A Streptococcus