Involvement of thePseudomonas aeruginosaMexAB-OprM efflux pump in the secretion of the metallophore pseudopaline
Authors: Gomez Nicolas Oswaldo , Tetard Alexandre , Ouerdane Laurent , Laffont Clémentine , Brutesco Catherine , Ball Geneviève , Lobinski Ryszard , Denis Yann , Plésiat Patrick , Llanes Catherine , Arnoux Pascal , Voulhoux Romé 
Published: May 2020

ABSTRACTThe ability for all organisms to acquire metals from their environment is essential for life. To overcome the metal restriction imposed by the host’s nutritional immunity, bacterial pathogens exploits the use of small high metal affinity molecules called metallophores. Metallophores are first synthetized in the cytoplasm, then secreted into the medium where they sequester the metal. The metal-metallophore complex is then imported into the bacterium following binding to dedicated cell surface receptors. Recently, a new family of metallophores has been discovered in pathogenic bacteria called staphylopine inStaphylococcus aureusand pseudopaline inPseudomonas aeruginosa. Here, we are expending the molecular understanding of pseudopaline secretion/recovery cycle across the double-membraned envelope of the Gram-negative bacteriumPseudomonas aeruginosa. We first revealed that pseudopaline is secreted in a two-step process including export across the inner membrane by the CntI exporter followed by a specific transport across the outer membrane by the MexAB-OprM efflux pump. Such involvement of MexAB-OprM in pseudopaline secretion, reveal a new natural function that extends its spectrum of functions and therefore reasserts its interest as antibacterial target. We then addressed the fate of the recovered metal-loaded pseudopaline by combiningin vitroreconstitution experiments using radio-labeled pseudopaline subjected to bacterial lysates, andin vivophenotyping in absence of pseudopaline transporters. Our data support the existence of a pseudopaline degradation/modification mechanism, possibly involved in metal release following pseudopaline recovery. All together our data allowed us to provide an improved molecular model of secretion, recovery and fate of this important metallophore byP. aeruginosa.IMPORTANCEPseudopaline is a broad spectrum metallophore produced and used byPseudomonas aeruginosato supply the bacterium in metal in metal scarce environments. Here we are investigating the pseudopaline transport/recovery cycle across the bacterial envelope. We are first demonstrating that pseudopaline secretion in the medium is achieved by a specific efflux pump, usually dedicated to the release of toxic compounds such as antibiotics, thus revealing a new natural function for this efflux pump reasserting its interest as antibacterial target. Additional experiments also revealing the existence of an intracellular pseudopaline degradation mechanism providing new clues to another obscure step of the pseudopaline cycle which is the intracellular metal liberation from the imported metal-pseudopaline complex. All together our data allowed us to disclose important aspects of the secretion, recovery and fate of this essential molecule used byP. aeruginosato survive during infections thus constituting new potential targets for antibacterial development.