The oxylipin-dependent quorum-sensing system (ODS) of Pseudomonas aeruginosa relies on the production and sensing of two extracellular oxylipins, 10S-hydroxy-(8E)-octadecenoic acid (10-HOME) and 7S,10S-dihydroxy-(8E)-octadecenoic acid (7,10-DiHOME). Here, we implemented a genetic screen of P. aeruginosa strain PAO1 aimed to identify genes required for 10-HOME and 7,10-DiHOME production. Among the 14 genes identified, four encoded previously known components of the ODS and 10 encoded parts of the Xcp type II secretion system (T2SS). We subsequently created a clean xcpQ deletion mutant, which encodes the necessary outer membrane component of Xcp, and found it recapitulated the impaired functionality of the T2SS transposon mutants. Further studies showed that the ΔxcpQ mutant was unable to secrete the oxylipin synthase enzymes across the outer membrane. Specifically, immunoblotting for OdsA, which is responsible for the generation of 10-HOME from oleic acid, detected the enzyme in supernatants from wild-type PAO1 but not ΔxcpQ cultures. Likewise, chromatography of supernatants found that 10-HOME was not in supernatants collected from the ΔxcpQ mutant. Accordingly, diol synthase activity was increased in the periplasm of ΔxcpQ mutant consistent with a stoppage in its transport. Importantly, after exposure of the ΔxcpQ mutant to exogenous 10-HOME and 7,10-DiHOME, the ODS effector genes become active; thus, the sensing component of the ODS does not involve the T2SS. Finally, we observed that Xcp contributed to robust in vitro and in vivo biofilm formation in oleic acid availability- and ODS-dependent manner. Thus, T2SS-mediated transport of the oxylipin synthase enzymes to outside the bacterial cell is required for ODS functionality. IMPORTANCE We previously showed that the ODS of P. aeruginosa produces and responds to oxylipins derived from host oleic acid by enhancing biofilm formation and virulence. Here, we developed a genetic screen strategy to explore the molecular basis for oxylipins synthesis and detection. Unexpectedly, we found that the ODS autoinducer synthases cross the outer membrane using the Xcp type 2 secretion system (T2SS) of P. aeruginosa, and so the biosynthesis of oxylipins occurs extracellularly. T2SS promoted biofilm formation in the presence of oleic acid as a result of ODS activation. Our results identify two new T2SS secreted proteins in P. aeruginosa and reveal a new way by which this important opportunistic pathogen interacts with the host environment.
Keywords: Pseudomonas aeruginosa; autoinducer; oleic acid; oxylipin; quorum sensing; type 2 secretion system.