The conserved coordination of acyl-homoserine lactone and PqsE signaling defines the RhlR-dependent quorum-sensing network in Pseudomonas aeruginosa clinical isolates

Abstract

Quorum sensing (QS) enables Pseudomonas aeruginosa to coordinate virulence and biofilm formation through cell density-dependent signaling. In clinical isolates from patients with cystic fibrosis (pwCF), mutations in canonical QS systems such as lasR and rhlI often lead to altered signaling hierarchies that complicate our understanding of QS regulation during chronic infection. Here, we dissect the relative contributions of the autoinducer N-butyryl-L-homoserine lactone (C4HSL) and the protein binding partner PqsE to RhlR-dependent transcription in CF clinical isolates. Using site-directed mutagenesis to generate RhlR and PqsE variants incapable of responding to C4HSL (RhlR A44M) or dimerizing to interact with RhlR (PqsENI), we show that both inputs are essential for the full expression of QS-regulated virulence factors, including pyocyanin and rhamnolipids. Transcriptomic analyses revealed that C4HSL and PqsE co-regulate a conserved set of 28 RhlR-dependent genes, encompassing canonical virulence loci as well as uncharacterized genes that are likely important for adaptation to the CF airway environment. These findings establish that clinical isolates maintain functional QS circuitry reliant on dual activation of RhlR by both C4HSL and PqsE, revealing a conserved regulatory module that underpins pathogenic behavior across genetically diverse isolates. AUTHOR SUMMARYUnderstanding quorum-sensing regulation in clinical isolates of Pseudomonas aeruginosa is essential to determine how the pathogen persists and adapts within the cystic fibrosis lung. While most studies have focused on laboratory strains, chronic isolates exhibit distinct genetic and regulatory adaptations that complicate our ability to generalize quorum sensing function. Our work defines the coordinated roles of C4HSL and PqsE in activating RhlR-dependent gene expression and virulence factor production in isolates from patients with cystic fibrosis. We identify a conserved core of quorum-sensing-regulated genes that remain dependent on both signals despite extensive genomic divergence. These findings highlight that, even within the evolutionary landscape of chronic infection, quorum-sensing signaling through RhlR remains a central and conserved determinant of virulence. By resolving the dual contributions of acyl-homoserine lactone and PqsE-mediated activation, this work provides a mechanistic foundation for future efforts to therapeutically target quorum-sensing pathways in clinical P. aeruginosa infections.