No abstract

Quorum sensing (QS) allows bacteria to assess their local cell density using chemical signals and plays a prominent role in the ability of common pathogens to infect a host. Non-native molecules capable of attenuating bacterial QS represent useful tools to explore the role of this pathway in virulence. As individual bacterial species can have multiple QS systems and/or reside in mixed communities with other bacteria capable of QS, chemical tools that are either selective for one QS system or “pan-active” and target all QS pathways are of significant interest. Herein we outline the analysis of a set of compounds reported to target one QS system in Pseudomonas aeruginosa for their activity in two other QS circuits in this pathogen and the discovery of molecules with novel activity profiles, including subsets that agonize all three QS systems, agonize one but antagonize the other two, or strongly antagonize just one.

Cell-to-cell signaling, or quorum sensing (QS), in Gram-negative bacteria is governed by small molecule signals (N-acyl L-homoserine lactones, AHLs) and their cognate intracellular receptors (LuxR-type proteins). The mechanistic underpinnings of QS in these bacteria are severely limited due to the challenges of isolating and manipulating most LuxR-type proteins. Quantitative assays to characterize the direct binding of ligands to these receptors are largely non-existent. We report herein a Förster Resonance Energy Transfer (FRET) assay that leverages (i) conserved endogenous tryptophans located in the LuxR-type protein ligand-binding site and synthetic fluorophore-AHL conjugates, and (ii) isolation/stabilization of the proteins bound to weak agonists. The FRET assay permits straightforward measurement of ligand-binding affinities with receptor—either in vitro or in cells—and was shown to be compatible with six LuxR-type receptors. These methods will advance fundamental investigations of the mechanisms of LuxR-type proteins and the development of small molecule modulators of QS.