Characterization of natural product inhibitors of quorum sensing in Pseudomonas aeruginosa reveals competitive inhibition of RhlR by ortho-vanillin

Quorum sensing (QS) is a cell-cell signaling system that enables bacteria to coordinate population density-dependent changes in behavior. This chemical communication pathway is mediated by diffusible N-acyl L-homoserine lactone signals and cytoplasmic signal-responsive LuxR-type receptors in Gram-negative bacteria. As many common pathogenic bacteria use QS to regulate virulence, there is significant interest in disrupting QS as a potential therapeutic strategy. Prior studies have implicated the natural products salicylic acid, cinnamaldehyde and other related benzaldehyde derivatives as inhibitors of QS in the opportunistic pathogen Pseudomonas aeruginosa, yet we lack an understanding of the mechanisms by which these compounds function. Herein, we evaluate the activity of a set of benzaldehyde derivatives using heterologous reporters of the P. aeruginosa LasR and RhlR QS signal receptors. We find that most tested benzaldehyde derivatives can antagonize LasR or RhlR reporter activation at micromolar concentrations, although certain molecules also caused mild growth defects and nonspecific reporter antagonism. Notably, several compounds showed promising RhlR or LasR specific inhibitory activities over a range of concentrations below that causing toxicity. ortho-Vanillin, a previously untested compound, was the most promising within this set. Competition experiments against the native ligands for LasR and RhlR revealed that ortho-vanillin can interact competitively with RhlR but not with LasR. Overall, these studies expand our understanding of benzaldehyde activities in the LasR and RhlR receptors and reveal potentially promising effects of ortho-vanillin as a small molecule QS modulator against RhlR. IMPORTANCE Quorum sensing (QS) regulates many aspects of pathogenesis in bacteria and has attracted interest as a target for anti-virulence therapies. As QS is regulated by low molecular weight chemical signals, the development of chemical strategies that can interfere with this cell-cell communication pathway has seen considerable scrutiny over the past 25 years. Much of this research has focused on common human pathogens, including the LasR and RhlR QS receptors in Pseudomonas aeruginosa. Potent and selective chemical agents capable of blocking the activity of these receptors remain relatively scarce, however. Natural products have provided a bounty of chemical scaffolds with anti-QS activities, but their molecular mechanisms are poorly characterized. The current study serves to fill this void by examining the activity of an important and wide-spread class of natural product QS modulators, benzaldehydes and related derivatives, in LasR and RhlR. We demonstrate that ortho-vanillin can act as a competitive inhibitor of RhlR, a receptor that has emerged and may supplant LasR in certain settings as a target for QS control in P. aeruginosa. The results and insights provided herein will advance the design of chemical tools to study QS with improved activities and selectivities.