PROJECT SUMMARY Invasive methicillin-resistant Staphylococcus aureus (MRSA) infections are responsible for 30% of all deaths due to antibiotic-resistant infections in the United States. MRSA infections are particularly frightening because they are readily transmitted both in hospital settings and in the community, often to otherwise healthy individuals, and they are typically resistant not only to beta-lactams, but to other major classes of antibiotics. There is an ongoing need to identify vulnerabilities in S. aureus that can be targeted through new therapeutic strategies. This project leverages previous technological and conceptual innovations by the Project Team that combined novel screening paradigms with state-of-the-art genomics technologies to identify promising pathways for inhibition as well as compounds that inhibit those pathways. Aim I builds on our discovery, made using a TnSeq data-mining approach, of a protein that acts as a positive regulator of lipoteichoic acid (LTA) synthesis. This protein forms a complex with the essential signal peptidase SpsB, which inactivates LTA synthase. We will elucidate how the protein regulator functions and will investigate the mechanisms that cause lethality when SpsB is inhibited. The work may lead to new strategies to target SpsB in combination with other compounds. Aim II builds on our discovery, made using an innovative screening platform and target identification strategy, of D-alanylation inhibitors that may be useful as antibiotic potentiators and anti-virulence agents. To provide methods to characterize these compounds, we will elucidate the pathway for D-alanylation. The D-alanylation pathway is broadly conserved in firmicutes and related pathways are widespread in bacteria, yet how they mediate transfer of amino acids and acyl groups across the membrane is not understood. This work will therefore have broad implications for understanding bacterial physiology. Aim III will use TnSeq to elucidate the synthetic lethal network for a gene important for LTA assembly, and we will then use that knowledge to identify targets for compounds we previously discovered that kill bacteria lacking that gene. In addition to providing potentially useful compound-target pairs for antibacterial therapy, this work will provide a genetic interaction network for each target we discover, leading to a comprehensive understanding of how different components of the cell envelope cooperate to create a robust, functional barrier between the organism and its environment, and potentially allowing future prediction of compound combinations effective for preventing the spread of resistance. We will also synergize with other subprojects by supporting their efforts to use Johan Paulsson’s microfluidics platform (Core B) for massively parallel image analysis of individual mutant lineages from pooled transposon libraries. We will provide MRSA and MSSA transposon libraries, individual mutants, training and advice on using the l...