The binding mechanism of pyoverdin with the outer membrane receptor FpvA in Pseudomonas aeruginosa is dependent on its iron-loaded status

In iron-deficient conditions, Pseudomonas aeruginosa secretes a major fluorescent siderophore named pyoverdin (Pvd), which after chelating iron(III) is transported back into the cell via its outer membrane receptor FpvA. FpvA is a TonB-dependent transport protein and has the ability to bind Pvd in its apo- or iron-loaded form. The fluorescence properties of Pvd were used to determine the binding kinetics of metal-free and metal-loaded Pvd to FpvA and showed two major features. First, the kinetics of formation of the FpvA-Pvd complex, in vivo and in vitro, are markedly slower compared to those observed for FpvA-Pvd-metal. Second, apo-Pvd and Pvd-metal absorbed with biphasic kinetics to FpvA: the bimolecular step (association of the ligand with the receptor) is followed by a slower step (t(1/2) values of 5 and 34 min for Pvd-metal and Pvd, respectively) that presumably leads to a more stable complex. The most likely explanation for this second step is that the binding of the ligand to the receptor induces a conformational change on FpvA, which may be different, depending on the loading status of Pvd. Analysis of the dissociation of metal-free Pvd from FpvA revealed an energy and a TonB dependency. The dissociation of iron-free Pvd from FpvA in the absence of the TonB protein occurs with slow kinetics in the range of hours, but it can be highly activated by the protonmotive force and TonB to reach a kinetic with a t(1/2) of 1 min. Apparently, under iron-limited conditions, TonB activates the FpvA receptor, resulting in a fast release of iron-free Pvd and generating an unloaded FpvA receptor, competent for binding extracellular Pvd-Fe.     

Copurification of the FpvA ferric pyoverdin receptor of Pseudomonas aeruginosa with its iron-free ligand: implications for siderophore-mediated iron transport.

The Pseudomonas aeruginosa FpvA receptor is a TonB-dependent outer membrane transport protein that catalyzes uptake of ferric pyoverdin across the outer membrane. Surprisingly, FpvA expressed in P. aeruginosa grown in an iron-deficient medium copurifies with a ligand X that we have characterized by UV, fluorescence, and mass spectrometry as being iron-free pyoverdin (apo-PaA). PaA was absent from FpvA purified from a PaA-deficient P. aeruginosa strain. The properties of ligand binding in vitro revealed very similar affinities of apo-PaA and ferric-PaA to FpvA. Fluorescence resonance energy transfer was used to study in vitro the formation of the FpvA-PaA-Fe complex in the presence of PaA-Fe or citrate-Fe. The circular dichroism spectrum of FpvA indicated a 57% beta-structure content typical of porins and in agreement with the 3D structures of the siderophore receptors FhuA and FepA. In the absence of the protease's inhibitors, a truncated form of FpvA lacking 87 amino acids at its N-terminus was purified. This truncated form still bound PaA, and its beta-sheet content was conserved. This N-terminal region displays significant homology to the N-terminal periplasmic extensions of FecA from Escherichia coli and PupB from Pseudomonas putida, which were previously shown to be involved in signal transduction. This suggests a similar function for FpvA. The mechanism of iron transport in P. aeruginosa via the pyoverdin pathway is discussed in the light of all these new findings.     

The pyoverdin receptor FpvA, a TonB-dependent receptor involved in iron uptake by Pseudomonas aeruginosa (review)

Iron is an important element, essential for the growth of almost all living cells. Because of the high insolubility of iron(III) in aerobic conditions, many gram-negative bacteria produce, under iron limitation, small iron-chelating compounds called siderophores, together with new outer-membrane proteins, which function as receptors for the ferrisiderophores. Pseudomonas aeruginosa, an important human opportunistic pathogen, produces at least three known siderophores when grown in iron-deficient conditions: pyochelin, salicylate and pyoverdin. This review focuses on pyoverdin and on the ability of FpvA to bind iron-free and ferric-PaA pyoverdin, in the light of recent information gained from biochemical and biophysical studies and of the recently solved 3D-structures of the related ferrichrome FhuA and enterobactin FepA receptors in Escherichia coli.     

The pyoverdin receptor FpvA,a TonB-dependent receptor involved in iron uptake by Pseudomonas aeruginosa (Review)

Iron is an important element, essential for the growth of almost all living cells. Because of the high insolubility of iron(III) in aerobic conditions, many gram-negative bacteria produce, under iron limitation, small iron-chelating compounds called siderophores, together with new outer-membrane proteins, which function as receptors for the ferrisiderophores. Pseudomonas aeruginosa, an important human opportunistic pathogen, produces at least three known siderophores when grown in irondeficient conditions: pyochelin, salicylate and pyoverdin. This reviewfocuses on pyoverdin and on the ability of FpvA to bind iron-free and ferric-PaA pyoverdin, in the light of recent information gained from biochemical and biophysical studies and of the recently solved 3D-structures of the related ferrichrome FhuA and enterobactin FepA receptors in Escherichia coli.     

A new mechanism for membrane iron transport in Pseudomonas aeruginosa

Various biochemical and biophysical studies have demonstrated the existence of a novel iron-uptake mechanism in Pseudomonas aeruginosa, different from that generally described for ferrichrome and ferric-enterobactin in Escherichia coli. This new iron-uptake mechanism involves all the proteins generally reported to be involved in the uptake of ferric-siderophore complexes in Gram-negative bacteria (i.e. the outer membrane receptor, periplasmic binding protein and ATP-binding-cassette transporter), but differs in the behaviour of the siderophore. One of the key features of this process is the binding of iron-free pyoverdin to the outer membrane receptor FpvA in conditions of iron deficiency.     

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

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.     

Recycling of pyoverdin on the FpvA receptor after ferric pyoverdin uptake and dissociation in Pseudomonas aeruginosa

Under iron-limiting conditions, Pseudomonas aeruginosa secretes a fluorescent siderophore called pyoverdin (PaA), which, after complexing iron, is transported back into the cells via its outer membrane receptor FpvA. The recent finding that all FpvA receptors on the bacterial cell surface are loaded with iron-free PaA under iron limiting conditions has raised questions about the mechanism by which P. aeruginosa transports efficiently iron. We used [(3)H]PaA' [(55)Fe]PaA-Fe, and a kinetically stable chromium-PaA complex to show that iron loading of the receptor occurs through a siderophore displacement mechanism in vivo. Moreover, the fluorescence properties of iron-free PaA revealed that, after PaA-Fe uptake and dissociation, the PaA molecule is recycled into the extracellular medium. We used fluorescence resonance energy transfer (FRET) between the PaA chromophore and the FpvA tryptophans in vivo to monitor the kinetics of PaA displacement by PaA-Fe at the cell surface, the dissociation of iron from the siderophore, and the recycling of PaA back to the receptor on the outer membrane of the bacteria in real time. The loading status of FpvA (PaA versus PaA-Fe) was shown to depend on the relative concentration of the two forms of pyoverdin in the growth medium.     

The interaction between pyoverdin and its outer membrane receptor in Pseudomonas aeruginosa leads to different conformers: a time-resolved fluorescence study

In iron limitation conditions, Pseudomonas aeruginosa secretes a major fluorescent siderophore named pyoverdin (PaA). PaA has an extremely high affinity for Fe(3+) but also chelates other ions such as Al(3+) and Ga(3+) with a lower affinity. The transfer of PaA-Fe(3+) across the outer membrane of the bacteria is mediated by the receptor FpvA, a TonB-dependent outer membrane transport protein. FpvA binds the iron-free and iron-loaded forms of pyoverdin with similar affinities, but only PaA-Fe(3+) is taken up by the cell, suggesting that FpvA adopts different conformations depending on its loading status. We used time-resolved fluorescence spectroscopy to characterize the different forms of FpvA-PaA in vitro. We showed that the FpvA-PaA complex adopts two different conformations depending on how it was prepared (formed in vitro or in vivo prior to purification). The dihydroquinoline moiety of both conformers is fully protonated, or coordinated by protein charged groups, but the polarity of its environment, its solvent accessibility, and its rotational dynamics are much slower when the FpvA-PaA complex is formed in vivo than in vitro. In the presence of Ga(3+) or Al(3+) ions, the solvent accessibility and mobility of the dihydroquinoline moiety in the two FpvA-PaA complexes are intermediate between those observed for the metal-free ones. In addition, the F?rster resonance energy transfer kinetics from FpvA tryptophan residues to the PaA chromophore differs from one complex to the other, revealing differences in one or more of the donor-acceptor topologies.     

The crystal structure of the pyoverdine outer membrane receptor FpvA from Pseudomonas aeruginosa at 3.6 angstroms resolution

The pyoverdine outer membrane receptor FpvA from Pseudomonas aeruginosa translocates ferric-pyoverdine across the outer membrane via an energy consuming mechanism that involves the inner membrane energy transducing complex of TonB-ExbB-ExbD and the proton motive force. We solved the crystal structure of FpvA loaded with iron-free pyoverdine at 3.6 angstroms resolution. The pyoverdine receptor is folded in two domains: a transmembrane 22-stranded beta-barrel domain occluded by an N-terminal domain containing a mixed four-stranded beta-sheet (the plug). The beta-strands of the barrel are connected by long extracellular loops and short periplasmic turns. The iron-free pyoverdine is bound at the surface of the receptor in a pocket lined with aromatic residues while the extracellular loops do not completely cover the pyoverdine binding site. The TonB box, which is involved in intermolecular contacts with the TonB protein of the inner membrane, is observed in an extended conformation. Comparison of this first reported structure of an iron-siderophore transporter from a bacterium other than Escherichia coli with the known structures of the E.coli TonB-dependent transporters reveals a high structural homology and suggests that a common sensing mechanism exists for the iron-loading status in all bacterial iron siderophore transporters.     

In vivo incorporation of selenomethionine in proteins using Pseudomonas aeruginosa as expression host: case study-the outer membrane receptor FpvA

The number of protein structures solved using multiwavelength anomalous diffraction methods coupled with selenomethionine substitution has grown dramatically over the last years. We show using the outer membrane pyoverdin receptor FpvA that Pseudomonas aeruginosa can be used for producing proteins with a high level of selenomethionine incorporation. To circumvent problems encountered with mass spectroscopy analysis of purified membrane proteins, in-gel trypsin digestion of FpvA coupled with MALDI mass spectrometry analysis of the resulting peptides was used to determine the extent of selenomethionine incorporation. Selenomethionine incorporation greater than 95% was achieved using P. aeruginosa as an overexpression system.     

FpvA receptor involvement in pyoverdine biosynthesis in Pseudomonas aeruginosa

Alignment of the Pseudomonas aeruginosa ferric pyoverdine receptor, FpvA, with similar ferric-siderophore receptors revealed that the mature protein carries an extension of ca. 70 amino acids at its N terminus, an extension shared by the ferric pseudobactin receptors of P. putida. Deletion of fpvA from the chromosome of P. aeruginosa reduced pyoverdine production in this organism, as a result of a decline in expression of genes (e.g., pvdD) associated with the biosynthesis of the pyoverdine peptide moiety. Wild-type fpvA restored pvd expression in the mutant, thereby complementing its pyoverdine deficiency, although a deletion derivative of fpvA encoding a receptor lacking the N terminus of the mature protein did not. The truncated receptor was, however, functional in pyoverdine-mediated iron uptake, as evidenced by its ability to promote pyoverdine-dependent growth in an iron-restricted medium. These data are consistent with the idea that the N-terminal extension plays a role in FpvA-mediated pyoverdine biosynthesis in P. aeruginosa.     

Pyoverdine-mediated iron uptake in Pseudomonas aeruginosa: the Tat system is required for PvdN but not for FpvA transport

Under iron-limiting conditions, Pseudomonas aeruginosa PAO1 secretes a fluorescent siderophore called pyoverdine (Pvd). After chelating iron, this ferric siderophore is transported back into the cells via the outer membrane receptor FpvA. The Pvd-dependent iron uptake pathway requires several essential genes involved in both the synthesis of Pvd and the uptake of ferric Pvd inside the cell. A previous study describing the global phenotype of a tat-deficient P. aeruginosa strain showed that the defect in Pvd-mediated iron uptake was due to the Tat-dependent export of proteins involved in Pvd biogenesis and ferric Pvd uptake (U. Ochsner, A. Snyder, A. I. Vasil, and M. L. Vasil, Proc. Natl. Acad. Sci. USA 99:8312-8317, 2002). Using biochemical and biophysical tools, we showed that despite its predicted Tat signal sequence, FpvA is correctly located in the outer membrane of a tat mutant and is fully functional for all steps of the iron uptake process (ferric Pvd uptake and recycling of Pvd on FpvA after iron release). However, in the tat mutant, no Pvd was produced. This suggested that a key element in the Pvd biogenesis pathway must be exported to the periplasm by the Tat pathway. We located PvdN, a still unknown but essential component in Pvd biogenesis, at the periplasmic side of the cytoplasmic membrane and showed that its export is Tat dependent. Our results further support the idea that a critical step of the Pvd biogenesis pathway involving PvdN occurs at the periplasmic side of the cytoplasmic membrane.     

The metal dependence of pyoverdine interactions with its outer membrane receptor FpvA

To acquire iron, Pseudomonas aeruginosa secretes the fluorescent siderophore pyoverdine (Pvd), which chelates iron and shuttles it into the cells via the specific outer membrane transporter FpvA. We studied the role of iron and other metals in the binding and transport of Pvd by FpvA and conclude that there is no significant affinity between FpvA and metal-free Pvd. We found that the fluorescent in vivo complex of iron-free FpvA-Pvd is in fact a complex with aluminum (FpvA-Pvd-Al) formed from trace aluminum in the growth medium. When Pseudomonas aeruginosa was cultured in a medium that had been treated with a metal affinity resin, the in vivo formation of the FpvA-Pvd complex and the recycling of Pvd on FpvA were nearly abolished. The accumulation of Pvd in the periplasm of Pseudomonas aeruginosa was also reduced in the treated growth medium, while the addition of 1 microM AlCl(3) to the treated medium restored the effects of trace metals observed in standard growth medium. Using fluorescent resonance energy transfer and surface plasmon resonance techniques, the in vitro interactions between Pvd and detergent-solubilized FpvA were also shown to be metal dependent. We demonstrated that FpvA binds Pvd-Fe but not Pvd and that Pvd did not compete with Pvd-Fe for FpvA binding. In light of our finding that the Pvd-Al complex is transported across the outer membrane of Pseudomonas aeruginosa, a model for siderophore recognition based on a metal-induced conformation followed by redox selectivity for iron is discussed.     

Enolase-like protein present on the outer membrane of Pseudomonas aeruginosa binds plasminogen

Pseudomonas aeruginosa is one of the pathogenic bacteria which utilize binding of the host plasminogen (Plg) to promote their invasion throughout the host tissues. In the present study, we confirmed that P. aeruginosa exhibits binding affinity for human plasminogen. Furthermore, we showed that the protein detected on the cell wall of P. aeruginosa and binding human plasminogen is an enolase-like protein. The hypothesis that alpha-enolase, a cytoplasmatic glycolytic enzyme, resides also on the cell surface of the bacterium was supported by electron microscopy analysis. The plasminogen-binding activity of bacterial cell wall outer membrane enolase-like protein was examined by immunoblotting assay.     

Adaptive resistance to polymyxin in Pseudomonas aeruginosa due to an outer membrane impermeability mechanism

The isolated outer membrane from cells of a Pseudomonas aeruginosa strain exhibiting adaptive resistance to polymyxin was not affected by polymyxin treatment, as monitored by electron microscopy of negatively stained preparations. This was in sharp contrast with extensive disruption by polymyxin of the outer membranes of the parent polymyxin-sensitive strain and the resistant strain following reversion to greater polymyxin sensitivity. The isolated cytoplasmic membrane of the polymyxin-resistant strain, on the other hand, remained sensitive to the disruptive effects of polymyxin treatment. The permeability characteristics of the resistant strains appear to be altered, as indicated by differences in minimal inhibitory concentrations for a variety of antibiotics between the polymyxin-sensitive and polymyxin-resistant strains. No evidence was found for a polymyxin-inactivating enzyme in osmotic shock fluid from the polymyxin-resistant strain. No evidence for a cytoplasmic membrane repair mechanism was found in the polymyxin-resistant strain. These observations suggest that the mechanism of adaptive polymyxin resistance in this model system is the alteration of the outer membrane so that it excludes polymyxin from reaching the still sensitive cytoplasmic membrane.     

Pseudomonas aeruginosa outer membrane: peptidoglycan-associated proteins.

The Pseudomonas aeruginosa outer membrane was isolated with attached peptidoglycan and fractionated with Triton X-100, ethylenediaminetetraacetate, and lysozyme. The data suggest that major outer membrane proteins F, H2, and I are noncovalently associated with the peptidoglycan.     

Enterobactin-mediated iron transport in Pseudomonas aeruginosa.

A pyoverdine-deficient strain of Pseudomonas aeruginosa was unable to grow in an iron-deficient minimal medium in the presence of the nonmetabolizable iron chelator ethylene diamine-di(omega-hydroxyphenol acetic acid) (EDDHA), although addition of enterobactin to EDDHA-containing minimal media did restore growth of the pyoverdine-deficient P. aeruginosa. Consistent with the apparent ability of enterobactin to provide iron to P. aeruginosa, enterobactin-dependent 55Fe3+ uptake was observed in cells of P. aeruginosa previously grown in an iron-deficient medium containing enterobactin (or enterobactin-containing Escherichia coli culture supernatant). This uptake was energy dependent, was observable at low concentrations (60 nM) of FeCl3, and was absent in cells cultured without enterobactin. A novel protein with a molecular weight of approximately 80,000 was identified in the outer membranes of cells grown in iron-deficient minimal medium containing enterobactin, concomitant with the induction of enterobactin-dependent iron uptake. A Tn501 insertion mutant lacking this protein was isolated and shown to be deficient in enterobactin-mediated iron transport at 60 nM FeCl3, although it still exhibited enterobactin-dependent growth in iron-deficient medium containing EDDHA. It was subsequently observed that the mutant was, however, capable of enterobactin-mediated iron transport at much higher concentrations (600 nM) of FeCl3. Indeed, enterobactin-dependent iron uptake at this concentration of iron was observed in both the mutant and parent strains irrespective of whether they had been cultured in the presence of enterobactin. Apparently, at least two uptake systems for ferrienterobactin exist in P. aeruginosa: one of higher affinity which is specifically inducible by enterobactin under iron-limiting conditions and the second, of lower affinity, which is also inducible under iron-limiting conditions but is independent of enterobactin for induction.     

Inositol polyphosphate-mediated iron transport in Pseudomonas aeruginosa

It has previously been shown that myo-inositol hexakisphosphate (myo-InsP6) mediates iron transport into Pseudomonas aeruginosa and overcomes iron-dependent growth inhibition. In this study, the iron transport properties of myo-inositol trisphosphate and tetrakisphosphate regio-isomers were studied. Pseudomonas aeruginosa accumulated iron (III) at similar rates whether complexed with myo-Ins(1,2,3)P3 or myo-InsP6. Iron accumulation from other compounds, notably D/L myo-Ins(1,2,4,5)P4 and another inositol trisphosphate regio-isomer, D-myo-Ins(1,4,5)P3, was dramatically increased. Iron transport profiles from myo-InsP6 into mutants lacking the outer membrane porins oprF, oprD and oprP were similar to the wild-type, indicating that these porins are not involved in the transport process. The rates of reduction of iron (III) to iron (II) complexed to any of the compounds by a Ps. aeruginosa cell lysate were similar, suggesting that a reductive mechanism is not the rate-determining step.     

Mechanism of ferripyoverdine uptake by Pseudomonas aeruginosa outer membrane transporter FpvA: no diffusion channel formed at any time during ferrisiderophore uptake

To get access to iron, Pseudomonas aeruginosa produces the siderophore pyoverdine (PVD), composed of a fluorescent chromophore linked to an octapeptide, and its corresponding outer membrane transporter FpvA. This transporter is composed of three domains: a β-barrel inserted into the membrane, a plug that closes the channel formed by the barrel, and a signaling domain in the periplasm. The plug and the signaling domain are separated by a sequence of five residues called the TonB box, which is necessary for the interaction of FpvA with the inner membrane TonB protein. Genetic deletion of the plug domain resulted in the presence of a β-barrel in the outer membrane unable to bind and transport PVD-Fe. Expression of the soluble plug domain with the TonB box inhibited PVD-(55)Fe uptake most likely through interaction with TonB in the periplasm. A reconstituted FpvA in the bacterial outer membrane was obtained by the coexpression of separately encoded plug and β-barrel domains, each endowed with a signal sequence and a signaling domain. This resulted in polypeptide complementation after secretion across the cytoplasmic membrane. The reconstituted FpvA bound PVD-Fe with the same affinity as wild-type FpvA, indicating that the resulting transporter is correctly folded and reconstituted in the outer membrane. PVD-Fe uptake was TonB-dependent but 75% less efficient compared to wild-type FpvA. These data are consistent with a gated mechanism in which no open channel with a complete removal of the plug domain for PVD-Fe diffusion is formed in FpvA at any point during the uptake cycle.     

Permeability of Pseudomonas aeruginosa outer membrane to hydrophilic solutes.

Pseudomonas aeruginosa is usually resistant to a wide variety of antibacterial agents, and it has been inferred, on the basis of indirect evidence, that this was due to the low permeability of its outer membrane. We determined the permeability of P. aeruginosa outer membrane directly, by measuring the rates of hydrolysis of cephacetrile, cephaloridine, and various phosphate esters by hydrolytic enzymes located in the periplasm. The permeability to these compounds was about 100-fold lower than in the outer membrane of Escherichia coli K-12. Also, we found that the apparent Km values for active transport of various carbon and energy source compounds were typically higher than 20 microM in P. aeruginosa, in contrast to E. coli in which the values are usually lower than 5 microM. These results also are consistent with the notion that the P. aeruginosa outer membrane indeed has a low permeability to most hydrophilic compounds and that this membrane acts as a rate limiting step in active transport processes with high Vmax values.