The RhlR quorum-sensing receptor controls Pseudomonas aeruginosa pathogenesis and biofilm development independently of its canonical homoserine lactone autoinducer

Abstract

Quorum sensing (QS) is a bacterial cell-to-cell communication process that relies on the production, release, and response to extracellular signaling molecules called autoinducers. QS controls virulence and biofilm formation in the human pathogen Pseudomonas aeruginosa. P. aeruginosa possesses two canonical LuxI/R-type QS systems, LasI/R and RhlI/R, which produce and detect 3OC12-homoserine lactone and C4-homoserine lactone, respectively. Here, we use biofilm analyses, reporter assays, RNA-seq studies, and animal infection assays to show that RhlR directs both RhlI-dependent and RhlI-independent regulons. In the absence of RhlI, RhlR controls the expression of genes required for biofilm formation as well as genes encoding virulence factors. Consistent with these findings, Δ rhlR and Δ rhlI mutants have radically different biofilm phenotypes and the Δ rhlI mutant displays full virulence in animals whereas the Δ rhlR mutant is attenuated. The Δ rhlI mutant cell-free culture fluids contain an activity that stimulates RhlR-dependent gene expression. We propose a model in which RhlR responds to an alternative ligand, in addition to its canonical C4-homoserine lactone autoinducer. This alternate ligand promotes a RhlR-dependent transcriptional program in the absence of RhlI.

Author summary

Quorum sensing (QS) is a cell-to-cell communication process that bacteria use to coordinate group behaviors. QS is essential for virulence and biofilm formation in many bacteria including the human pathogen Pseudomonas aeruginosa. P. aeruginosa has high clinical relevance because it has acquired resistance to commonly used antibiotics, and is a priority pathogen on the CDC ESKAPE pathogen list. The urgent need for new antimicrobials to combat P. aeruginosa infections makes targeting QS for interference an attractive approach. Here, we investigate P. aeruginosa under biofilm conditions that mimic authentic P. aeruginosa lifestyles in environmental and medical contexts rather than in traditional laboratory conditions. This strategy enabled us to find that P. aeruginosa uses a novel QS signal molecule that controls biofilm formation and virulence. The new signal molecule acts together with the long-known QS receptor RhlR. Using physiologic, genetic, and molecular studies, combined with animal models of infection, we characterize the roles of QS components in biofilm formation and virulence. We find that RhlR and the putative new signal molecule are crucial for both traits. Our work suggests that targeting RhlR with small molecule inhibitors could provide an exciting path forward for the development of novel antimicrobials.