Saturday, May 24, 2025
5/24/2025
Delineation of mechanisms underlying response regulator activation in group A Streptococcus - PROJECT SUMMARY: Two component gene regulatory systems (TCS), which generally consist of a membrane-embedded sensor kinase which controls the phosphorylation of its cognate response regulator, are a central mechanism by which bacteria modulate gene expression in response to external stimuli. The control of virulence regulator/sensor kinase (CovRS) of the major human pathogen group A Streptococcus (GAS) has long been a model for understanding how TCS influence bacterial infectivity. Phosphorylated CovR (CovR~P) primarily serves to repress virulence factor production, and CovS can either phosphorylate or dephosphorylate CovR at D53 in response to extracellular factors. Recent completion of global CovR binding/transcriptome analyses (ChIP- seq/RNAseq) showed that CovR~P repressed virulence factor encoding genes have the expected increased CovR promoter binding and decreased transcript levels at high CovR~P levels. However, these studies also identified a large number of directly CovR regulated genes with increased transcript quantity at higher CovR~P levels (i.e. CovR~P activated). By ChIP-seq analyses, these CovR~P activated genes had higher CovR promoter occupancy at lower CovR~P levels suggesting that non-phosphorylated CovR (CovR-NP) may be the predominant CovR isoform binding/acting at these promoters. Additionally, we have identified that ska, which encodes the critical GAS virulence factor streptokinase, is CovR~P repressed in emm1 strains but CovR~P activated in acapsular emm28, emm87, and emm89 strains. It is the goal of this proposal to elucidate mechanisms underlying “CovR~P activation”. In Specific Aim 1, we propose to perform comparative ChIP-exo analyses of wild-type (~80% CovR~P) and CovR-D53A (100% CovR-NP) emm1 and emm89 strains to better define the contribution of CovR-NP to global CovR binding. ChIP-exo provides an advance over ChIP-seq because of an exonuclease digestion step which greatly improves identification of the precise DNA binding location of transcriptional regulators. Additionally, we will perform focused in vitro transcription assays and luminescence-based plasmid reporter studies to definitively test the comparative function of CovR-NP and CovR~P at specific CovR~P activated promoters. In specific aim 2, we will seek to determine the mechanism by which ska is CovR~P repressed in emm1 but CovR~P activated in emm89 GAS. Specifically, we will perform in vitro transcription assays using CovR~P and CovR-NP as well as in vivo promoter activity assays using emm1 and emm89 ska promoter DNA in low and high CovR~P emm1 and emm89 strains. Moreover, we will swap the emm1 and emm89 promoters (i.e. place the emm1 promoter into an emm89 strain) to discern the role of strain background on observed transcript level variation as well as assess the role of the non-coding ska regulatory RNA fasX. Completion of the proposed research will markedly improve mechanistic understanding of how a model prokaryotic response regulator activates gene expression.
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