Project summary: Enterococci are leading causes of multidrug resistant hospital acquired infection – the first E in the ESKAPE acronym. Most E. faecium isolates at major centers are vancomycin resistant (VRE), and daptomycin is often the last line bactericidal drug for treatment. Unfortunately, daptomycin resistance is increasingly common, and can arise during treatment. Here we discovered a new vulnerability of VRE that appears to be related to production of membrane anchored lipoteichoic acid (LTA). A naturally occurring E. faecium mutant in this pathway, isolated from an infected urinary tract, exhibited hypersusceptibility to daptomycin, with a 20-fold lower MIC to this key drug. We developed preliminary data to show that this stemmed from a mutation in a gene termed lafB, which encodes an enzyme that make the immediate precursor glycopeptide onto which LTA is built. When selecting for revertants to the wild type level of daptomycin susceptibility or greater, we observed that lafB was always the first to mutate back, indicating that it may be dominant to other mutations that result in daptomycin resistance. If so, inhibition of lafB would have the potential to undo daptomycin resistance by other mechanisms, or at minimum, prevent those mutations from arising during treatment. The following proposal outlines a rigorous set of experiments to explore this new determinant of daptomycin susceptibility.