PROJECT SUMMARY: The ability of bacteria to modify gene expression in adaptation to environmental cues is a key component of bacterial pathogenesis. Two component gene regulatory systems (TCS), which generally consist of a membrane-embedded sensor kinase that alters phosphorylation and dimerization status of its cognate response regulator in reaction to various stimuli, are a central mechanism by which bacteria adjust expression of a diverse array of genes. The control of virulence regulator (CovR) of the major human pathogen group A Streptococcus (GAS) is a model system for understanding how TCS proteins influence bacterial infectivity. Phosphorylated CovR (CovR~P) primarily serves to repress virulence factor production such that hypervirulent GAS strains with decreased CovR~P levels emerge during human infection and following experimental mouse challenge. It was recently shown that inactivation of the control of virulence sensor kinase (CovS) reduces CovR~P and that CovR~P levels vary between GAS strains of different M serotypes. The goals of this project are to delineate how changes in CovR~P levels impact GAS pathogenesis and to determine the molecular mechanisms by which differences in CovR~P levels alter GAS global gene expression. In aim 1, the effect of CovR~P variation on GAS virulence and the emergence of hypervirulent GAS strains will be evaluated in two murine models using a series of isoallelic strains that have defined CovR~P levels due to engineered single amino acid substitutions in either CovR or CovS. Identified variation in virulence will be correlated with global gene expression profiles to determine mechanisms by which CovR~P alterations influence GAS infectivity. Specific Aim 2 will seek to decipher the specific role of CovR phosphorylation and dimerization in the regulation of CovR-controlled genes that differ in their response to varying CovR~P levels. To this end, we will employ in vivo protein-protein interaction assays to identify co-factors that interact with CovR isoforms that have defined phosphorylation and dimerization characteristics. The effect of these interactions on CovR-mediated expression for genes belonging to various sub-groups will be verified using in vitro transcription assays. In aim 3, we will generate the first in vivo analysis of CovR-DNA interaction via a ChIP-Seq approach. Actual visualization of CovR-DNA complexes using atomic force microscopy will be used to test the hypothesis that CovR~P differentially interacts with distinct arrangements of cis-regulatory elements found in the promoter regions of virulence factor encoding genes belonging to the different subgroups of CovR-regulated genes noted in Specific Aim 2. Given that CovR belongs to the large OmpR/PhoB family of bacterial transcription factors, completion of the proposed research will significantly augment understanding of the mechanistic basis by which TCS impact bacterial pathogenesis.