Evolution of PqsE as aPseudomonas aeruginosa-specific regulator of LuxR-type receptors: insights fromPseudomonasandBurkholderia

Abstract

Pseudomonas aeruginosais a Gram-negative opportunistic pathogen that poses a significant public health threat, particularly in healthcare settings. A key determinant ofP. aeruginosavirulence is the regulated synthesis and release of extracellular products, which is controlled by a cell density-dependent signaling system known as quorum sensing (QS).P. aeruginosauses a complex QS network, including two systems that rely on diffusible N-acylhomoserine lactone (AHL) signal molecules. The LuxR-type receptor RhlR is unique in that it requires not only its cognate AHL but also the accessory protein PqsE to maximally bind to promoter DNA and to initiate transcription. Our group demonstrated that PqsE physically interacts with RhlR, enhancing its affinity for target promoters across theP. aeruginosagenome. Although LuxR-type receptors are widespread in Gram-negative bacteria and important for pathogenesis, PqsE orthologs are restricted toPseudomonasandBurkholderiaspecies. This study explored the conservation of PqsE and examined PqsE ortholog structure-function across different species. Our results show that PqsE inPseudomonasretain their functional interactions with RhlR homologs, unlike PqsE orthologs inBurkholderiaspp., which do not interact with their respective LuxR-type receptors. Additionally, we assessed the AHL preferences of different receptors and hypothesized that the PqsE-RhlR interaction evolved to stabilize the inherently unstable RhlR, preventing its degradation. Indeed, we observe higher levels of RhlR protein turnover in a strain lackingpqsEcompared to WT, which can be rescued in a strain of lacking the Lon protease.IMPORTANCEPseudomonas aeruginosa, a major pathogen for patients with cystic fibrosis and a primary constituent of healthcare-associated infections, relies on a complex quorum-sensing (QS) network to coordinate virulence factor production. Central to this system is the interaction between two proteins, PqsE and RhlR, which drive gene expression essential for pathogenesis. Our study investigates the conservation of the PqsE-RhlR interaction across related bacterial species, revealing that PqsE inPseudomonascan enhance RhlR activity, while orthologs inBurkholderialack this capacity. These findings offer new insights into the specificity and evolution of QS mechanisms, highlighting the PqsE-RhlR interaction as a potentially selective target for treatingP. aeruginosainfections.