Using Chemical Synthetic Lethality to Identify New Antimicrobial Targets in Gram-Negative Bacterial Pathogens

Project Summary & Abstract Infectious disease is one of the most pressing health challenges of the new century. The emergence of multidrug-resistant pathogens is on the rise and new methodologies and molecular targets are needed for the development of antimicrobial agents. In this proposal, we will address both shortcomings by implementing an approach that identifies new molecular targets through the use of drug combination therapies against two difficult-to-treat Gram-negative pathogens, Burkholderia pseudomallei and Pseudomonas aeruginosa. Our approach is based on chemical synthetic lethality, a phenomenon whereby two molecules are innocuous individually but deadly in combination. The selection of the appropriate combination of molecules is based on the effects of subinhibitory doses of antibiotics, which do not exert growth defects, but can expose genetic and/or metabolic vulnerabilities that are then exploited by a second inhibitor. We have demonstrated proof-of-concept for this method and identified a new target in B. pseudomallei with additional ones to be validated as part of our proposed research. In the first Aim, we will identify a small molecule inhibitor of methionine synthase, one of the recently identified targets, which is essential in the presence of low-dose trimethoprim. Validating the chemical synthetic lethality of this inhibitor in conjunction with low-dose trimethoprim through cellular assays will yield an antimicrobial cocktail against B. pseudomallei. In the second Aim, the strategy will be applied to the notorious ESKAPE pathogen P. aeruginosa. Here we will establish both forward chemical genetics and forward classical genetics screens in the presence of low- dose antibiotics to find new chemical synthetic lethal combinations as well as novel antimicrobial targets. This approach will thus facilitate the discovery of a drug cocktail against P. aeruginosa. With our proof-of-concept in hand and well-defined projects goals, we are poised to successfully complete the two independent yet related Aims of this proposal in the planned two-year period. The results will add new antimicrobial targets to the existing repertoire and advance drug combination therapies against two recalcitrant pathogens.