RhlR-Regulated Acyl-Homoserine Lactone Quorum Sensing in a Cystic Fibrosis Isolate of Pseudomonas aeruginosa

Abstract

The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of airway infection in cystic fibrosis (CF) patients. P. aeruginosa employs several hierarchically arranged and interconnected quorum sensing (QS) regulatory circuits to produce a battery of virulence factors such as elastase, phenazines, and rhamnolipids. The QS transcription factor LasR sits atop this hierarchy and activates the transcription of dozens of genes, including that encoding the QS regulator RhlR. Paradoxically, inactivating lasR mutations are frequently observed in isolates from CF patients with chronic P. aeruginosa infections. In contrast, mutations in rhlR are rare. We have recently shown that in CF isolates, the QS circuitry is often rewired such that RhlR acts in a LasR-independent manner. To begin understanding how QS activity differs in this rewired background, we characterized QS activation and RhlR-regulated gene expression in P. aeruginosa E90, a LasR-null, RhlR-active chronic infection isolate. In this isolate, RhlR activates the expression of 53 genes in response to increasing cell density. The genes regulated by RhlR include several that encode virulence factors. Some, but not all, of these genes are present in the QS regulon described in the well-studied laboratory strain PAO1. We also demonstrate that E90 produces virulence factors at similar concentrations as PAO1, and in E90, RhlR plays a significant role in mediating cytotoxicity in a three-dimensional lung epithelium cell model. These data illuminate a rewired LasR-independent RhlR regulon in chronic infection isolates and suggest further investigation of RhlR as a possible target for therapeutic development in chronic infections. IMPORTANCE Pseudomonas aeruginosa is a prominent cystic fibrosis (CF) pathogen that uses quorum sensing (QS) to regulate virulence. In laboratory strains, the key QS regulator is LasR. Many isolates from patients with chronic CF infections appear to use an alternate QS circuitry in which another transcriptional regulator, RhlR, mediates QS. We show that a LasR-null CF clinical isolate engages in QS through RhlR and remains capable of inducing cell death in an in vivo-like lung epithelium cell model. Our findings support the notion that LasR-null clinical isolates can engage in RhlR QS and highlight the centrality of RhlR in chronic P. aeruginosa infections.

Intraspecies Signaling between Common Variants of Pseudomonas aeruginosa Increases Production of Quorum-Sensing-Controlled Virulence Factors

“…RhlR plays other important roles in host interactions ( 75 ) which may benefit P. aeruginosa LasR− strains. The observation that rhlR mutants are rare in natural isolates and that LasR− strains with active RhlR are virulent ( 25 , 76 ) underscores the relevance of this mechanism and highlights the importance of understanding how microbial interactions influence RhlR activity.…”

From genotype to phenotype: adaptations of Pseudomonas aeruginosa to the cystic fibrosis environment

“…Damkiaer and colleagues observed that a single rpoD mutation induced alginate overproduction only in a particular genetic background of the DK2 lineage and, thus, was positively selected [62]. Genic alterations of mexT were shown to compensate the effects of lasR inactivation, suggesting that positive selection of this mutation may be promoted in lasR-negative isolates [63][64][65][66]. In the same way, mutations reverting the mutator phenotype might be positively selected only after alteration of the genes from DNA repair systems [8].…”

Pseudomonas aeruginosa PA80 is a cystic fibrosis isolate deficient in RhlRI quorum sensing

“…Deletion of either LasRI and RhlRI systems can be complemented by restoring a functional protein or in the case of the synthases protein (lasI and rhlI) by exogenous autoinducer molecules 48,49 . Recent studies have elegantly demonstrated evolutionary trajectories for LasR null mutants where a functional RhlRI system independent of LasRI quickly emerges [48][49][50] . This was an interesting finding considering the QS hierarchy puts lasR at the top of the QS system with the LasRI activated complex inducing expression of the rhlR regulator.…”