Inhibitors of the PqsR Quorum-Sensing Receptor Reveal Differential Roles for PqsE and RhlI in Control of Phenazine Production

<i>Pseudomonas aeruginosa</i> is a leading cause of hospital-acquired infections and it is resistant to many current antibiotic therapies, making development of new antimicrobial treatments imperative. The cell-to-cell communication process called quorum sensing controls <i>P. aeruginosa</i> pathogenicity. Quorum sensing relies on the production, release, and group-wide detection of extracellular signal molecules called autoinducers. Quorum sensing enables bacteria to synchronize group behaviors. <i>P. aeruginosa</i> possesses multiple quorum-sensing systems that control overlapping regulons, including some required for virulence and biofilm formation. Interventions that target <i>P. aeruginosa</i> quorum-sensing receptors are considered a fruitful avenue to pursue for new therapeutic advances. Here, we developed a <i>P. aeruginosa</i> strain that carries a bioluminescent reporter fused to a target promoter that is controlled by two <i>P. aeruginosa</i> quorum-sensing receptors. The receptors are PqsR, which binds and responds to the autoinducer called PQS (2-heptyl-3-hydroxy-4(1<i>H</i>)-quinolone) and RhlR, which binds and responds to the autoinducer called C4-HSL (C4-homoserine lactone). We used this reporter strain to screen >100,000 compounds with the aim of identifying inhibitors of either or both the PqsR and RhlR quorum-sensing receptors. We report results for 30 PqsR inhibitors from this screen. All of the identified compounds inhibit PqsR with IC₅₀ values in the nanomolar to low micromolar range and they are readily docked into the autoinducer binding site of the PqsR crystal structure, suggesting they function competitively. The majority of hits identified are not structurally related to previously reported PqsR inhibitors. Recently, RhlR was shown to rely on the accessory protein PqsE for full function. Specifically, RhlR controls different subsets of genes depending on whether or not it is bound to PqsE, however, the consequences of differential regulation on the quorum-sensing output response have not been defined. PqsR regulates <i>pqsE</i>. That feature of the system enabled us to exploit our new set of PqsR inhibitors to show that RhlR requires PqsE to activate the biosynthetic genes for pyocyanin, a key <i>P. aeruginosa</i> virulence factor, while C4-HSL is dispensable. These results highlight the promise of inhibition of PqsR as a possible <i>P. aeruginosa</i> therapeutic to suppress production of factors under RhlR-PqsE control.