Involvement of the Pseudomonas aeruginosa MexAB–OprM efflux pump in the secretion of the metallophore pseudopaline

Abstract

The 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 in Staphylococcus aureus and pseudopaline in Pseudomonas aeruginosa. Here, we are expending the molecular understanding of pseudopaline secretion/recovery cycle across the double-membraned envelope of the Gram-negative bacterium Pseudomonas 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 combining in vitro reconstitution experiments using radio-labeled pseudopaline subjected to bacterial lysates, and in vivo phenotyping 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 by P. aeruginosa.

Systematic identification of molecular mediators of interspecies sensing in a community of two frequently coinfecting bacterial pathogens

“…aeruginosa produces a similar secreted opine metallophore, pseudopaline (PsP), which is also important for zinc uptake, and is produced by CntL and CntM with the addition of a terminal -ketoglutarate instead of pyruvate (Fig 3A) [52,[55][56][57]. PsP is exported by CntI in the inner membrane and MexAB-OprM in the outer membrane and is imported back into the cell by CntO [56,58]. The entire cntOLMI operon was up-regulated upon exposure to S. aureus supernatant (Tables A and B in S1 File).…”

Structure and metal-binding properties of PA4063, a novel player in periplasmic zinc trafficking byPseudomonas aeruginosa

“…This is possible because this microbe is endowed with other proven or putative metaluptake systems regulated by Zur, such as ZrmABCD, HmtA, PA2911-PA2914 and PA4063-PA4066 (Pederick et al, 2015), which are briefly presented below. The most studied among these is ZrmABCD, which is involved in the synthesis and management of pseudopaline, a low-molecular-weight chelating agent that mediates the import of zinc in a siderophore-like manner (Mastropasqua et al, 2017;Lhospice et al, 2017;McFarlane & Lamb, 2017;Hermansen et al, 2018;Gomez et al, 2021). The importance of this system is shown by the fact that the inactivation of both ZnuABC and ZrmABCD reduces the ability of P. aeruginosa to proliferate in zinc-poor environments and drastically reduces its ability to cause disease in mice, possibly due to a significant reduction of the expression of several virulence features such as biofilm formation, swimming ability, extracellular protease production and siderophore release (D'Orazio et al, 2015;Mastropasqua et al, 2017Mastropasqua et al, , 2018.…”

Systematic identification of molecular mediators underlying sensing ofStaphylococcus aureusbyPseudomonas aeruginosa

“…3A) (49,(52)(53)(54). PsP is exported by CntI in the inner membrane and MexAB-OprM in the outer membrane and is imported back into the cell by CntO (52,55). The entire cntOLMI operon was upregulated upon exposure to S. aureus supernatant (Table S1 and S2).…”