Dataset Information

Interaction of TonB with the outer membrane receptor FpvA of Pseudomonas aeruginosa.

ABSTRACT: Pyoverdine-mediated iron uptake by the FpvA receptor in the outer membrane of Pseudomonas aeruginosa is dependent on the inner membrane protein TonB1. This energy transducer couples the proton-electrochemical potential of the inner membrane to the transport event. To shed more light upon this process, a recombinant TonB1 protein lacking the N-terminal inner membrane anchor (TonB(pp)) was constructed. This protein was, after expression in Escherichia coli, purified from the soluble fraction of lysed cells by means of an N-terminal hexahistidine or glutathione S-transferase (GST) tag. Purified GST-TonB(pp) was able to capture detergent-solubilized FpvA, regardless of the presence of pyoverdine or pyoverdine-Fe. Targeting of the TonB1 fragment to the periplasm of P. aeruginosa inhibited the transport of ferric pyoverdine by FpvA in vivo, indicating an interference with endogenous TonB1, presumably caused by competition for binding sites at the transporter or by formation of nonfunctional TonB heterodimers. Surface plasmon resonance experiments demonstrated that the FpvA-TonB(pp) interactions have apparent affinities in the micromolar range. The binding of pyoverdine or ferric pyoverdine to FpvA did not modulate this affinity. Apparently, the presence of either iron or pyoverdine is not essential for the formation of the FpvA-TonB complex in vitro.

SUBMITTER: Adams H

PROVIDER: S-EPMC1540090 | biostudies-literature | 2006 Aug

REPOSITORIES: biostudies-literature

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Interaction of TonB with the outer membrane receptor FpvA of Pseudomonas aeruginosa.

Adams Hendrik H Zeder-Lutz Gabrielle G Schalk Isabelle I Pattus Franc F Celia Hervé H

Journal of bacteriology 20060801 16

Pyoverdine-mediated iron uptake by the FpvA receptor in the outer membrane of Pseudomonas aeruginosa is dependent on the inner membrane protein TonB1. This energy transducer couples the proton-electrochemical potential of the inner membrane to the transport event. To shed more light upon this process, a recombinant TonB1 protein lacking the N-terminal inner membrane anchor (TonB(pp)) was constructed. This protein was, after expression in Escherichia coli, purified from the soluble fraction of ly ...[more]

PMID: 16885443

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Project description:The Pseudomonas quinolone signal (PQS) is an important quorum-sensing molecule in Pseudomonas aeruginosa that also mediates its own packaging and transport by stimulating outer membrane vesicle (OMV) formation. Because OMVs have been implicated in many virulence-associated behaviors, it is critical that we understand how they are formed. Our group proposed the bilayer-couple model for OMV biogenesis, where PQS intercalates into the outer membrane, causing expansion of the outer leaflet and consequently inducing curvature. In accordance with the model, we hypothesized that PQS must be transported from the cytoplasm to the outer membrane before it can initiate OMV formation. We initially examined two laboratory strains of P. aeruginosa and found significant strain-dependent differences. PQS export correlated strongly with OMV production, even though equivalent amounts of total PQS were produced by both strains. Interestingly, we discovered that poor OMV producers sequestered the majority of PQS in the inner membrane, which appeared to be the result of early saturation of the export pathway. Further analysis showed that strain-specific PQS export and OMV biogenesis patterns were stable once established but could be significantly altered by changing the growth medium. Finally, we demonstrated that the associations described for laboratory strains also held for three clinical strains. These results suggest that factors controlling the export of PQS dictate OMV biogenesis. This work provides new insight into PQS-controlled virulence in P. aeruginosa and provides important tools to further study signal export and OMV biogenesis.IMPORTANCE Bacterial secretion has been recognized as an essential facet of microbial pathogenesis and human disease. Numerous virulence factors have been found to be transported within outer membrane vesicles (OMVs), and delivery using these biological nanoparticles often results in increased potency. OMV biogenesis is an important but poorly understood process that is ubiquitous among Gram-negative organisms. Our group seeks to understand the biochemical mechanisms behind the formation of OMVs and has developed a model of small-molecule-induced membrane curvature as an important driver of this process. With this work, we demonstrate that PQS, a known small-molecule OMV inducer, must be exported to promote OMV biogenesis in both lab-adapted and clinical strains of Pseudomonas aeruginosa In supporting and expanding the bilayer-couple model of OMV biogenesis, the current work lays the groundwork for studying environmental and genetic factors that modulate OMV production and, consequently, the packaging and delivery of many bacterial factors.

Dataset Information

Interaction of TonB with the outer membrane receptor FpvA of Pseudomonas aeruginosa.

Publications

Interaction of TonB with the outer membrane receptor FpvA of Pseudomonas aeruginosa.

Similar Datasets

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