PROJECT SUMMARY Pseudomonas aeruginosa poses a major threat to human health due to limited treatment options and its ability to become resistant to antibiotics. P. aeruginosa and other Gram-negative bacteria are particularly difficult to treat because their asymmetric outer membranes, comprising an electronegative matrix of lipopolysaccharide (LPS) in the outer leaflet, form an electrostatic barrier excluding most antibiotics. The candidate aims apply advanced genomic, genetic and chemical biological strategies to study this important human pathogen, both to develop novel therapeutic agents and to gain insights into the basic biology of vulnerable targets. In work in progress, recent target-focused, whole cell screening identified 128 small molecules hypothesized to kill P. aeruginosa by disrupting LPS transport to the outer membrane. In Aim 1, with small molecule hits in hand, the candidate proposes to develop these compounds by optimizing their activities and establishing their mechanisms of action. To this end, the candidate has already developed high- throughput gene expression profiling methods to identify and prioritize hits that induce transcriptional responses in LPS transport pathways. Preliminary data revealed one lead candidate, C0918, induced a transcriptional response remarkably similar to that of a known LPS transport inhibitor, demonstrating that mechanisms of action can be inferred by gene responses compared to those of known antibiotics. Drawing on his background in protein science, when putative target proteins emerge, the candidate outlines strategies for protein expression, purification, direct-binding studies, and structure determination by cryogenic electron microscopy. Lead compounds in Aim 1 will also serve as valuable molecular probes to investigate the regulatory pathways underpinning LPS biosynthesis and transport in Aim 2. The candidate will perform a genetic screen to discover LPS regulatory genes in P. aeruginosa by mutagenizing an engineered reporter strain, which encodes fluorescent proteins marking expression levels of key LPS synthesis and transport genes. To complemental screening efforts, the candidate will also characterize single and double mutants encoding regulated copies of these key genes in LPS biosynthesis and transport, aimed at determining phenotypic consequences when LPS biosynthetic intermediates buildup under conditions of high LPS synthesis but low transport. With the guidance of his mentor, Dr. Deb Hung, the candidate has developed a five-year training program to provide both the technical and didactic training necessary to become an independent physician-scientist focused on using small molecules to target LPS transport, while also gaining insights into its underlying regulatory machinery in P. aeruginosa. Importantly, this project will be overseen by a scientific advisory committee providing expertise in key areas of this proposal, including LPS biology, bacterial genetics, genomics, and chemic...