The Protein Interaction Networks of Catalytically-Active and Catalytically-Inactive PqsE in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a human pathogen that relies on quorum sensing to establish infections. The PqsE quorum-sensing protein is required for P. aeruginosa virulence factor production and infection. PqsE has a reported enzymatic function in the biosynthesis of the quorum-sensing autoinducer called PQS. However, this activity is redundant because, in the absence of PqsE, this role is fulfilled by alternative thioesterases. Rather, PqsE drives P. aeruginosa pathogenic traits via a protein-protein interaction with the quorum-sensing receptor/transcription factor, RhlR, an interaction that enhances affinity of RhlR for target DNA sequences. PqsE catalytic activity is dispensable for interaction with RhlR. Thus, PqsE virulence function can be decoupled from its catalytic function. Here, we present an immunoprecipitation-mass spectrometry method employing eGFP-PqsE fusions to define the protein interactomes of wildtype PqsE and the catalytically inactive PqsE(D73A) variant in P. aeruginosa and their dependence on RhlR. Several proteins were identified to have specific interactions with wildtype PqsE, while not forming associations with PqsE(D73A). In the {Delta}rhlR strain, an increased number of specific PqsE interactors were identified, including the partner autoinducer synthase to RhlR, called RhlI. Collectively, these results suggest that specific protein-protein interactions depend on PqsE catalytic activity and that RhlR may prevent proteins from interacting with PqsE, possibly due to competition between RhlR and other proteins for PqsE binding. Our results provide a foundation for the identification of the in vivo PqsE catalytic function and, potentially, new proteins involved in P. aeruginosa quorum sensing. IMPORTANCEPseudomonas aeruginosa causes hospital-borne infections in vulnerable patients, including in immunocompromised individuals, burn victims, and cancer patients undergoing chemotherapy. There are no effective treatments for P. aeruginosa infections, which are usually broadly resistant to antibiotics. Animal models show that to establish infection and to cause illness, P. aeruginosa relies on an interaction between two proteins: PqsE and RhlR. There could be additional protein-protein interactions involving PqsE, which, if defined, could be exploited for the design of new therapeutic strategies to combat P. aeruginosa. Here, we reveal previously unknown protein interactions in which PqsE participates that will be investigated for potential roles in pathogenesis.