SUMMARY Methicillin-resistant S. aureus (MRSA) are leading causes of serious community and hospital-associated infection. In addition to acquiring antibiotic resistance, S. aureus converts to a persistent antibiotic-tolerant form in which traditional treatments are powerless. Antibiotic-tolerant persister cells are responsible for recalcitrant chronic infections. To address the urgent need for new anti-MRSA therapeutics, we developed a C. elegans- MRSA screening platform to identify compounds with activity against MRSA. The assay identifies compounds with both in vivo efficacy and low host toxicity. We combined a whole animal in vivo screen of ~82,000 synthetic compounds for ones that exhibit potential anti-MRSA activity with in vitro screens for compounds with the ability to kill the stubborn persister subpopulation. We have focused on four compounds, CD437, nTZDpa, bithionol, and PQ401, all four of which eradicate both actively growing MRSA and stationary phase MRSA persister cells by disrupting their membrane lipid bilayers. These compounds exhibit fast killing rates, synergism with other antibiotics, extremely low probabilities of resistance selection, and high selectivity for bacterial over mammalian membranes. Using bithionol and nTZDpa and analogs of these compounds, we found that anti-persister activity of these compounds positively correlates with their ability to increase membrane fluidity. All four compounds have been previously studied as potential therapeutics and bithionol is a former FDA-approved anthelmintic. Despite the potential of membrane-disrupting compounds to kill persisters, it is generally thought that their therapeutic applications are limited due to a lack of selectivity for bacterial over mammalian membranes. However, our results show that the presence of cholesterol in mammalian but not in bacterial membranes provides an important opportunity to identify/design non-toxic anti-persister membrane-disrupting compounds. We therefore propose the following two aims to identify features of membrane-disrupting compounds that correlate with their ability to kill persisters but not disrupt mammalian membranes. In Aim 1, we will utilize biochemical, physiological, biophysical, and cryo-EM techniques to 1) further investigate the correlation between the ability of compounds to kill persisters and their ability to increase membrane fluidity, and 2) to identify the mechanisms of action and targets of membrane-disrupting compounds. We also propose to test the efficacy of membrane-disrupting compounds in a murine S. aureus subcutaneous abscess infection model in collaboration with the Hooper lab and determine whether the compounds also target E. faecalis cell membranes. In Aim 2, we will utilize high throughput microfluidics technology developed in the Paulsson laboratory (Core B) and referred to as “mother machines” to: 1) isolate rare MRSA persister cells in growing cultures, and 2) in collaboration with the Walker lab, use Tn-seq ...