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 chemical biology. Throughout the career development award period, the candidate will expand
his knowledge base with complete didactic and hands-on training. The candidate will complete coursework in
bioinformatics and statistics to help with analyzing genomic-wide datasets. This proposal therefore
provides the necessary training and scientific foundation to achieve Dr. Romano's ultimate goal
of becoming a RO1-funded physician-scientist who applies advanced genomic and chemical
biological techniques to study and treat bacterial pathogens.