Structure of the RhlR-PqsE complex from Pseudomonas aeruginosa reveals mechanistic insights into quorum-sensing gene regulation.

Pseudomonas aeruginosa is an opportunistic pathogen that is responsible for thousands of deaths every year in the United States. P. aeruginosa virulence factor production is mediated by quorum sensing, a mechanism of bacterial cell-cell communication that relies on the production and detection of signal molecules called autoinducers. In P. aeruginosa, the transcription factor receptor RhlR is activated by a RhlI-synthesized autoinducer. We recently showed that RhlR-dependent transcription is enhanced by a physical interaction with the enzyme PqsE via increased affinity of RhlR for promoter DNA. However, the molecular basis for complex formation and how complex formation enhanced RhlR transcriptional activity remained unclear. Here, we report the structure of ligand-bound RhlR in complex with PqsE. Additionally, we determined the structure of the complex bound with DNA, revealing the mechanism by which RhlR-mediated transcription is enhanced by PqsE, thereby establishing the molecular basis for RhlR-dependent virulence factor production in P. aeruginosa.

The PqsE-RhlR Interaction Regulates RhlR DNA Binding to Control Virulence Factor Production in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes disease in immunocompromised individuals and individuals with underlying pulmonary disorders. P. aeruginosa virulence is controlled by quorum sensing (QS), a bacterial cell-cell communication mechanism that underpins transitions between individual and group behaviors. In P. aeruginosa, the PqsE enzyme and the QS receptor RhlR directly interact to control the expression of genes involved in virulence. Here, we show that three surface-exposed arginine residues on PqsE comprise the site required for interaction with RhlR. We show that a noninteracting PqsE variant [PqsE(NI)] possesses catalytic activity, but is incapable of promoting virulence phenotypes, indicating that interaction with RhlR, and not catalysis, drives these PqsE-dependent behaviors. Biochemical characterization of the PqsE-RhlR interaction coupled with RNA-seq analyses demonstrates that the PqsE-RhlR complex increases the affinity of RhlR for DNA, enabling enhanced expression of genes encoding key virulence factors. These findings provide the mechanism for PqsE-dependent regulation of RhlR and identify a unique regulatory feature of P. aeruginosa QS and its connection to virulence. IMPORTANCE Bacteria use a cell-cell communication process called quorum sensing (QS) to orchestrate collective behaviors. QS relies on the group-wide detection of molecules called autoinducers (AI). QS is required for virulence in the human pathogen Pseudomonas aeruginosa, which can cause fatal infections in patients with underlying pulmonary disorders. In this study, we determine the molecular basis for the physical interaction between two virulence-driving QS components, PqsE and RhlR. We find that the ability of PqsE to bind RhlR correlates with virulence factor production. Since current antimicrobial therapies exacerbate the growing antibiotic resistance problem because they target bacterial growth, we suggest that the PqsE-RhlR interface discovered here represents a new candidate for targeting with small molecule inhibition. Therapeutics that disrupt the PqsE-RhlR interaction should suppress virulence. Targeting bacterial behaviors such as QS, rather than bacterial growth, represents an attractive alternative for exploration because such therapies could potentially minimize the development of resistance.