Characterization of natural product inhibitors of quorum sensing reveals competitive inhibition of
Pseudomonas aeruginosa
RhlR by
ortho
-vanillin
Authors: Woods Kathryn E. , Akhter Sana , Rodriguez Blanca , Townsend Kade A. , Smith Nathan , Smith Ben , Wambua Alice , Craddock Vaughn , Abisado-Duque Rhea G. , Santa Emma E. , Manson Daniel E. , Oakley Berl R. , Hancock Lynn E. , Miao Yinglong , Blackwell Helen E. , Chandler Josephine R. 
Published: July 2024

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 cause 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 bacterial pathogenesis and has attracted much interest as a target for anti-virulence therapies over the past 30 years, for example, antagonists of the LasR and RhlR QS receptors in
Pseudomonas aeruginosa
. Potent and selective QS inhibitors 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
P. aeruginosa
QS control. The results and insights provided herein will advance the design of chemical tools to study QS with improved activities and selectivities.