The emergence of antibiotic resistant bacteria is one of the greatest threats to human health in the 21st century. Among these bacteria, vancomycin-resistant enterococci (VRE) are one of the most challenging organisms in clinical settings. Indeed, vancomycin-resistant Enterococcus faecium have been designated by the Infectious Disease Society of America as one of the superbugs against which new therapies are urgently needed. Currently, the only antibiotic with in vitro bactericidal activity against VRE is daptomyci (DAP), a lipopeptide antibiotic whose target is the bacterial cell membrane (CM). DAP has become the key front-line antibiotic against these organisms due to the paucity of other options. However, the main challenge when using DAP against VRE is the development of resistance during therapy. We have identified a cluster of genes in enterococci (designated liaFSR) encoding a three-component regulatory system involved in the cell envelope bacterial response to antibiotics (which is highly conserved in most Gram-positive pathogens). Our preliminary data indicate that LiaR, which encodes the response regulator of the system, is the master regulator of the CM response to antimicrobials and antimicrobial peptides in VRE. Indeed, deletion of liaR reversed resistance to DAP in several strains of E. faecalis and E. faecium, independent of the genetic background. We hypothesize that interfering with the LiaR response will provide a novel strategy to restore and preserve the activity of anti-VRE antimicrobials (e.g., DAP). We seek to develop proof-of concept that this anti- adaptation strategy targeting LiaR could be applied to VRE and, potentially, other multidrug-resistant Gram- positive organisms. In Specific Aim I, we will characterize LiaR from E. faecium by i) deleting liaR in 2 additional DAP-R E. faecium with unusual genotypes to confirm the universal role of LiaR on DAP-R, ii) determine the minimal level of LiaR required to produce the DAP-R phenotype by cloning liaR under the control of a nisin-inducible promoter, iii) identify the LiaR regulon by specific and global comparative expression analysis using qRT-PCR and RNASeq analyses, and iv) investigate of the effect of the liaR deletion in selection of DAP-R and the distribution of CM cardiolipin (CL) microdomains (using 10-N-nonyl acridine orange). Specific Aim II will be directed to performing biochemical and structural characterization of the response regulator LiaR and a mutant protein harboring a Trp73?Cys substitution in the receiver domain. We will i) perform DNA footprinting analysis of LiaR and LiaRW73C on the putative promoters, ii) assess the ability of LiaRW73C to bind to target DNA vs wild-type, using microscale thermophoresis and, iii) determine the oligomerization kinetics of LiaR and LiaRW73C using analytical ultracentrifugation (AUC). We will also pursue high resolution structures of the LiaR, LiaRW73C and LiaR:DNA complexes by X-ray crystallography. We expect that our findings would provide the rationale for development of novel anti-adaptation antibiotics that would preserve the life of currently used drugs or prevent developing of resistance during therapy.