A multiplexed chemical screen identifies a novel, species-specific Pseudomonas aeruginosa inhibitor that targets LPS interaction with the outer membrane protein, OprH

The surge of antimicrobial resistance in recent decades threatens efficacy of current antibiotics, particularly against Pseudomonas aeruginosa, a highly resistant gram-negative pathogen. The asymmetric outer membrane of P. aeruginosa combined with its array of efflux pumps provide a barrier to xenobiotic intracellular accumulation, thus making the discovery of novel drugs with whole cell antibacterial activity very challenging. We adapted PROSPECT, a genome-wide, target-based, whole cell screening strategy, to take a focused approach to discover small molecule probes with specific activity against engineered P. aeruginosa mutants depleted for essential proteins localized at the outer membrane. We identified BRD1401, a small molecule that has specific activity against a P. aeruginosa mutant depleted for the essential lipoprotein, OprL. Genetic studies identified a novel link between OprL and the non-essential, outer membrane β barrel protein, OprH, to modulate BRD1401 activity. BRD1401 directly bound to OprH to disrupt the known interaction between OprH and lipopolysaccharide (LPS) in vitro and in whole bacteria. OprH also biochemically interacted with OprL, thus providing a link between outer membrane and peptidoglycan in P. aeruginosa. Thus, a whole cell, multiplexed screen against P. aeruginosa identified a species-specific inhibitor and probe molecule that revealed novel pathogen biology. P. aeruginosa strains, either wildtype PA14 or PAO1 strains, or engineered oprL-hypomorph or oprL-KD strains, or BRD1401-resistant strains, were exposed to either varying salt in the growth medium in the presence of either 0.5% DMSO vehicle control or 125 or 250 µM BRD1401 or to varying doses of arabinose in the presence of either 0.5% DMSO vehicle control or 125 µM BRD1401.