PROJECT SUMMARY The continued and inevitable emergence of antibiotic resistance demands a vigorous and sustained effort to identify fundamentally new targets and strategies for innovative antimicrobial therapeutics. Antibiotic-resistant enterococci such as vancomycin-resistant enterococci (VRE) are major causes of hospital-acquired infections, and there are few good therapeutic options to treat VRE infections. A great deal is known about the genetic and biochemical mechanisms of vancomycin resistance provided by the horizontally acquired vanA and vanB gene clusters, which reprogram the native enterococcal peptidoglycan synthesis pathway to produce and use peptidoglycan precursors containing D-lactate (instead of the natural D-alanine) at the terminal position of the peptide side chain. These modified precursors must be acted on by multiple proteins in the native enterococcal peptidoglycan synthesis pathway to successfully build the essential peptidoglycan, yet the role of enterococcal host factors in mediating vancomycin resistance is poorly understood. Our data indicate that host factors encoded in the core enterococcal genome are in fact required for phenotypic vancomycin resistance conferred by horizontally acquired van gene clusters. In particular, a cell-wall- stress sensing system known as CroS/R responds to vancomycin-induced cell wall stress in VRE and drives transcription of host enterococcal genes essential for vancomycin resistance. The first major knowledge gaps that must be overcome to understand how enterococcal host factors drive vancomycin resistance, and to subsequently exploit such factors as targets for new therapeutics that sensitize VRE to vancomycin, are (i) to identify the specific CroS/R-dependent enterococcal host factors that are required for vancomycin resistance; and (ii) to demonstrate that CroS/R-dependent host factors are required for vancomycin resistance in all types of clinically relevant VRE. The research proposed here will fill these gaps to establish a solid foundation for the future development of new therapeutics that disable vancomycin resistance and potentiate the activity of vancomycin against VRE.

Project Narrative Antibiotic-resistant bacteria, such as vancomycin-resistant enterococci (VRE) and methicillin- resistant Staphylococcus aureus (MRSA), are major causes of hospital-acquired infections and contribute to an escalating healthcare crisis. The research proposed here promises to reveal new insights into the biological processes driving antibiotic resistance that will facilitate the development of new treatments for infections caused by drug-resistant pathogens. In particular, this work promises to define new targets for innovative therapeutics with potentially unique modes of action.