An enzyme moonlights as a chaperone to control virulence of Pseudomonas aeruginosa

Abstract Pseudomonas aeruginosa is a major cause of nosocomial infections and also leads to severe exacerbations in cystic fibrosis or chronic obstructive pulmonary disease. Three intertwined quorum sensing systems control virulence of P. aeruginosa, with the rhl circuit playing the leading role in late and chronic infections. The majority of traits controlled by rhl transcription factor RhlR depend on PqsE, a dispensable thioesterase in Pseudomonas Quinolone Signal (PQS) biosynthesis4 that interferes with RhlR through an enigmatic mechanism likely involving direct interaction of both proteins. Here we show that PqsE and RhlR form a weakly interacting, probably transient 2:2 protein complex that, together with the RhlR agonist N-butanoyl-L-homoserine lactone (C4-HSL), stabilizes RhlR to a soluble form, thereby rendering the otherwise unstable transcription factor active. By using a PqsE-RhlR fusion protein first, we determined a crystal structure of the complex that allows us to identify residues essential for the interaction. To corroborate our hypothesis of chaperone-like activity of PqsE, we designed stability-optimized variants of RhlR that bypass the need for C4-HSL and PqsE in activating PqsE/RhlR-controlled processes of P. aeruginosa. Together, our data provide insight into the unique regulatory role of PqsE and lay groundwork for developing new P. aeruginosa-specific pharmaceuticals.