Development of validated probes for the bacterial type III secretion system

With increasing incidence of antibiotic resistance, development of new therapies against bacterial pathogens isessential for global public health. The bacterial type III secretion system (T3SS) represents an excellent drugtarget because it is externally accessible to small molecules and enables virulence of Pseudomonas,Salmonella, Chlamydia, and numerous other important pathogens. We have developed a high throughputscreening pipeline to discover T3SS inhibitors and have shown the robustness of our approach through pilotscreens identifying three classes of compounds active against the T3SS. We now propose to broaden ourscope and screen three unique libraries comprising ~58,000 natural product fractions developed by membersof our consortium as well as two commercial synthetic chemical libraries. According to the CDC, every yearover 50,000 healthcare-associated Pseudomonas aeruginosa infections occur in the U.S., >6,000 of which arecaused by multidrug resistant strains. To identify Pseudomonas T3SS inhibitors and potential futuretherapeutics, we will carry out the following aims. In Aim 1, we will implement our primary screen andcounterscreens to identify natural product fractions and synthetic compounds with specific T3SS inhibitoryactivity. In Aim 2, we will validate hits identified in Aim 1, using three orthogonal distinct approaches. Theidentity and structure of bioactive natural products will be determined and initial structure activity analysisperformed on identified synthetic scaffolds. Prioritized compounds will be purified or synthesized and evaluatedfor off target activity, if any, as well as breadth of activity against T3SSs in multiple relevant pathogens. In Aim3, mode of action will be determined, using parallel genetic and biochemical approaches. This rigorousstrategy will provide ~10 T3SS inhibitor chemical probes with identified molecular targets active against P.aeruginosa and potentially other pathogens.