From the periplasmic signaling domain to the extracellular face of an outer membrane signal transducer of Pseudomonas aeruginosa: crystal structure of the ferric pyoverdine outer membrane receptor

The pyoverdine outer membrane receptor, FpvA, from Pseudomonas aeruginosa translocates ferric pyoverdine across the outer membrane through an energy consuming mechanism using the proton motive force and the TonB-ExbB-ExbD energy transducing complex from the inner membrane. We solved the crystal structure of the full-length FpvA bound to iron-pyoverdine at 2.7 A resolution. Signal transduction to an anti-sigma protein of the inner membrane and to TonB-ExbB-ExbD involves the periplasmic domain, which displays a beta-alpha-beta fold composed of two alpha-helices sandwiched by two beta-sheets. One iron-pyoverdine conformer is bound at the extracellular face of FpvA, revealing the conformer selectivity of the binding site. The loop that contains the TonB box, involved in interactions with TonB, and connects the signaling domain to the plug domain of FpvA is not defined in the electron density following the binding of ferric pyoverdine. The high flexibility of this loop is probably necessary for signal transduction through the outer membrane.     

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.     

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.     

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.     

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.     

FpvA bound to non-cognate pyoverdines: molecular basis of siderophore recognition by an iron transporter

The first step in the specific uptake of iron via siderophores in Gram-negative bacteria is the recognition and binding of a ferric siderophore by its cognate receptor. We investigated the molecular basis of this event through structural and biochemical approaches. FpvA, the pyoverdine–Fe transporter from Pseudomonas aeruginosa ATCC 15692 (PAO1 strain), is able to transport ferric–pyoverdines originating from other species, whereas most fluorescent pseudomonads are only able to use the one they produce among the more than 100 known different pyoverdines. We solved the structure of FpvA bound to non-cognate pyoverdines of high- or low-affinity and found a close correlation between receptor–ligand structure and the measured affinities. The structure of the first amino acid residues of the pyoverdine chain distinguished the high- and low-affinity binders while the C-terminal portion of the pyoverdines, often cyclic, does not appear to contribute extensively to the interaction between the siderophore and its transporter. The specificity of the ferric–pyoverdine binding site of FpvA is conferred by the structural elements common to all ferric–pyoverdines, i.e. the chromophore, iron, and its chelating groups.     

Pyoverdine-mediated regulation of FpvA synthesis in Pseudomonas aeruginosa: involvement of a probable extracytoplasmic-function sigma factor,FpvI

A search of the pvd pyoverdine biosynthesis locus of Pseudomonas aeruginosa identified an open reading frame, PA2387, whose product exhibited a sequence similar to those of a number of so-called extracytoplasmic- function sigma factors responsible for siderophore-dependent expression of iron-siderophore receptors in Escherichia coli and Pseudomonas putida. Deletion of this gene, dubbed fpvI, compromised pyoverdine-dependent FpvA ferric pyoverdine receptor production and fpvA gene expression, while the cloned gene stimulated fpvA expression. A Fur-binding site was identified immediately upstream of fpvI, consistent with the observed iron-regulated expression of fpvI and fpvA.     

The PvdRT-OpmQ efflux pump controls the metal selectivity of the iron uptake pathway mediated by the siderophore pyoverdine in Pseudomonas aeruginosa

Pyoverdine (PVD) is the major siderophore produced by Pseudomonas aeruginosa for iron acquisition. PvdRT-OpmQ is an ATP-dependent efflux pump involved in the secretion of newly synthesized pyoverdine (PVD) and of PVD that has transported and released its iron into the bacterium from the periplasm into the extracellular medium. This iron uptake pathway also involves an outer membrane transporter, FpvA, for PVD-Fe uptake from the extracellular medium into the periplasm. In binding assays, FpvA bound PVD in complex with many different metals, with affinities from 2.9?nM for PVD-Fe to 13?μM for PVD-Al. Uptake assays with various FpvA and PvdRT-OpmQ mutants, monitored by inductively coupled plasma-atomic emission spectrometry (ICP-AES) for metal detection, and by fluorescence for PVD detection, showed that both metals and PVD accumulated in P.?aeruginosa, due to the uptake of these compounds via the FpvA/PVD pathway. Higher levels of accumulation were observed in the absence of PvdRT-OpmQ expression. Thus, FpvA has a broad metal specificity for both the binding and uptake of PVD-metal complexes, and the PvdRT-OpmQ efflux pump exports unwanted metals complexed with PVD from the bacterium. This study provides the first evidence of efflux pump involvement in the export of unwanted siderophore-metal complexes and insight into the molecular mechanisms involved controlling the metal selectivity of siderophore-mediated iron uptake pathways.     

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.     

A beta strand lock exchange for signal transduction in TonB-dependent transducers on the basis of a common structural motif

Transport of molecules larger than 600 Da across the outer membrane involves TonB-dependent receptors and TonB-ExbB-ExbD of the inner membrane. The transport is energy consuming, and involves direct interactions between a short N-terminal sequence of receptor, called the TonB box, and TonB. We solved the structure of the ferric pyoverdine (Pvd-Fe) outer membrane receptor FpvA from Pseudomonas aeruginosa in its apo form. Structure analyses show that residues of the TonB box are in a beta strand which interacts through a mixed four-stranded beta sheet with the periplasmic signaling domain involved in interactions with an inner membrane sigma regulator. In this conformation, the TonB box cannot form a four-stranded beta sheet with TonB. The FhuA-TonB or BtuB-TonB structures show that the TonB-FpvA interactions require a conformational change which involves a beta strand lock-exchange mechanism. This mechanism is compatible with movements of the periplasmic domain deduced from crystallographic analyses of FpvA, FpvA-Pvd, and FpvA-Pvd-Fe.     

Distribution and evolution of ferripyoverdine receptors in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium, which is also able to cause severe opportunistic infections in humans. The colonization of the host is importantly affected by the production of the high-affinity iron (III) scavenging peptidic siderophore pyoverdine. The species P. aeruginosa can be divided into three subgroups ('siderovars'), each characterized by the production of a specific pyoverdine and receptor (FpvA). We used a multiplex PCR to determine the FpvA siderovar on 345 P. aeruginosa strains from environmental or clinical origin. We found about the same proportion of each type in clinical strains, while FpvA type I was slightly over-represented (49%) in environmental strains. Our multiplex PCR also detected the presence or absence of an additional receptor for type I pyoverdine (FpvB). The fpvB gene was in fact present in the vast majority of P. aeruginosa strains (93%), regardless of their siderovar or their origin. Finally, molecular analyses of fpvA and fpvB genes highlighted a complex evolutionary history, probably linked to the central role of iron acquisition in the ecology and virulence of P. aeruginosa .     

Identification of residues of FpvA involved in the different steps of Pvd-Fe uptake in Pseudomonas aeruginosa

FpvA is an outer membrane transporter involved in iron uptake by the siderophore pyoverdine (Pvd) in Pseudomonas aeruginosa. This transporter, like all other proteins of the same family, consists of a transmembrane 22 beta-stranded barrel occluded by a plug domain. The beta-strands of the barrel are connected by large extracellular loops and short periplasmic turns. Site-directed mutagenesis was carried out on FpvA to identify the extracellular loops or parts of these loops involved in the various stages of Pvd-Fe uptake. The G286C, W362C, and W434C mutations in loops L1, L3, and L4, respectively, disturbed the binding of the apo siderophore, as shown by time-resolved fluorescence spectroscopy. Iron uptake experiments followed by fluorescence resonance energy transfer (FRET) or using 55Fe indicated that residues W434 and G701 and, therefore, loops L4 and L9 must be involved in Pvd-Fe uptake by FpvA. The two corresponding mutants incorporated smaller than normal amounts of 55Fe into cells, and no Pvd recycling on FpvA was observed after iron release. Surprisingly, the S603C mutation in loop L7 increased the amount of Pvd-Fe transported. Our results suggest that W434 (L4), S603 (L7), and G701 (L9) are involved in the mechanism of Pvd-Fe uptake.     

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.     

Loss of a pyoverdine secondary receptor in Pseudomonas aeruginosa results in a fitter strain suitable for population invasion

The rapid emergence of antibiotic resistant bacterial pathogens constitutes a critical problem in healthcare and requires the development of novel treatments. Potential strategies include the exploitation of microbial social interactions based on public goods, which are produced at a fitness cost by cooperative microorganisms, but can be exploited by cheaters that do not produce these goods. Cheater invasion has been proposed as a ‘Trojan horse’ approach to infiltrate pathogen populations with strains deploying built-in weaknesses (e.g., sensitiveness to antibiotics). However, previous attempts have been often unsuccessful because population invasion by cheaters was prevented by various mechanisms including the presence of spatial structure (e.g., growth in biofilms), which limits the diffusion and exploitation of public goods. Here we followed an alternative approach and examined whether the manipulation of public good uptake and not its production could result in potential ‘Trojan horses’ suitable for population invasion. We focused on the siderophore pyoverdine produced by the human pathogen Pseudomonas aeruginosa MPAO1 and manipulated its uptake by deleting and/or overexpressing the pyoverdine primary (FpvA) and secondary (FpvB) receptors. We found that receptor synthesis feeds back on pyoverdine production and uptake rates, which led to strains with altered pyoverdine-associated costs and benefits. Moreover, we found that the receptor FpvB was advantageous under iron-limited conditions but revealed hidden costs in the presence of an antibiotic stressor (gentamicin). As a consequence, FpvB mutants became the fittest strain under gentamicin exposure, displacing the wildtype in liquid cultures, and in biofilms and during infections of the wax moth larvae Galleria mellonella, which both represent structured environments. Our findings reveal that an evolutionary trade-off associated with the costs and benefits of a versatile pyoverdine uptake strategy can be harnessed for devising a Trojan-horse candidate for medical interventions.Subject terms: Molecular evolution, Bacterial genetics, Clinical microbiology     

Mutational analysis of a bifunctional ferrisiderophore receptor and signal-transducing protein from Pseudomonas aeruginosa

The FpvA protein of Pseudomonas aeruginosa strain PAO1 mediates uptake of a siderophore, ferripyoverdine. It is also a component of a signal transduction pathway that controls production of an exotoxin, a protease, pyoverdine, and FpvA itself. The purpose of the research described here was to dissect these different functions of FpvA. Signaling involves an N-terminal domain of FpvA, and it was shown that this domain is probably located in the periplasm, as expected. Short peptides were inserted at 36 sites within FpvA by linker insertion mutagenesis. The effects of these mutations on the presence of FpvA in the outer membrane, on FpvA-mediated uptake of ferripyoverdine, and on pyoverdine synthesis and gene expression were determined. Five of the mutations resulted in the absence of FpvA from the outer membrane of the bacteria. All of the remaining mutations eliminated either the transport or signaling function of FpvA and most affected both functions. Three mutations prevented transport of ferripyoverdine but had no effect on the signal transduction pathway showing that transport of ferripyoverdine is not required for the trans-membrane signaling process. Conversely, eight mutations affected pyoverdine-mediated signaling but had no effect on transport of ferripyoverdine. These data show that insertions throughout FpvA resulted in loss of function and that signaling and transport are separate and discrete functions of FpvA.     

Siderophore-mediated cell signalling in Pseudomonas aeruginosa: divergent pathways regulate virulence factor production and siderophore receptor synthesis

Under iron-limiting conditions, Pseudomonas aeruginosa produces a siderophore called pyoverdine. Pyoverdine is secreted into the extracellular environment where it chelates iron, and the resulting ferri-pyoverdine complexes are transported back into the bacteria by a cell surface receptor protein FpvA. Pyoverdine also acts as a signalling molecule inducing the production of three secreted virulence factors. Binding of ferri-pyoverdine to FpvA transduces a signal to the periplasmic part of the membrane-spanning antisigma factor FpvR. The signal is transmitted to the cytoplasmic part of FpvR, which controls the activity of an extracytoplasmic family (ECF) sigma factor protein PvdS. This results in the production of the virulence factors pyoverdine, exotoxin A and PrpL endoprotease. Here, we show that a second divergent branch of this signalling pathway regulates the production of the FpvA protein. FpvR negatively regulates the activity of a second ECF sigma factor, FpvI, which is required for the synthesis of FpvA, and the presence of ferri-pyoverdine greatly increases the activity of FpvI so that production of FpvA is induced. To the best of our knowledge, this is the first example of a branched signalling system of this sort and the first example of an antisigma factor protein (FpvR) that directly regulates the activities of two different ECF sigma factor proteins (PvdS and FpvI).     

Evidence for diversifying selection at the pyoverdine locus of Pseudomonas aeruginosa

Pyoverdine is the primary siderophore of the gram-negative bacterium Pseudomonas aeruginosa. The pyoverdine region was recently identified as the most divergent locus alignable between strains in the P. aeruginosa genome. Here we report the nucleotide sequence and analysis of more than 50 kb in the pyoverdine region from nine strains of P. aeruginosa. There are three divergent sequence types in the pyoverdine region, which correspond to the three structural types of pyoverdine. The pyoverdine outer membrane receptor fpvA may be driving diversity at the locus: it is the most divergent alignable gene in the region, is the only gene that showed substantial intratype variation that did not appear to be generated by recombination, and shows evidence of positive selection. The hypothetical membrane protein PA2403 also shows evidence of positive selection; residues on one side of the membrane after protein folding are under positive selection. R', previously identified as a type IV strain, is clearly derived from a type III strain via a 3.4-kb deletion which removes one amino acid from the pyoverdine side chain peptide. This deletion represents a natural modification of the product of a nonribosomal peptide synthetase enzyme, whose consequences are predictive from the DNA sequence. There is also linkage disequilibrium between the pyoverdine region and pvdY, a pyoverdine gene separated by 30 kb from the pyoverdine region. The pyoverdine region shows evidence of horizontal transfer; we propose that some alleles in the region were introduced from other soil bacteria and have been subsequently maintained by diversifying selection.     

Aromatic components of two ferric enterobactin binding sites in Escherichia coli FepA

Ferric enterobactin is a catecholate siderophore that binds with high affinity (Kd approximately 10-10 M) to the Escherichia coli outer membrane protein FepA. We studied the involvement of aromatic amino acids in its uptake by determining the binding affinities, kinetics and transport properties of site-directed mutants. We replaced seven aromatic residues (Y260, Y272, Y285, Y289, W297, Y309 and F329) in the central part of FepA primary structure with alanine, individually and in double combinations, and determined the ability of the mutant proteins to interact with ferric enterobactin and the protein toxins colicins B and D. All the constructs showed normal expression and localization. Among single mutants, Y260A and F329A were most detrimental, reducing the affinity between FepA and ferric enterobactin 100- and 10-fold respectively. Double substitutions involving Y260, Y272 and F329 impaired (100- to 2500-fold) adsorption of the iron chelate more strongly. For Y260A and Y272A, the drop in adsorption affinity caused commensurate decreases in transport efficiency, suggesting that the target residues primarily act in ligand binding. F329A, like R316A, showed greater impairment of transport than binding, intimating mechanistic involvement during ligand internalization. Furthermore, immunochemical studies localized F329 in the FepA ligand binding site. The mutagenesis results suggested the existence of dual ligand binding sites in the FepA vestibule, and measurements of the rate of ferric enterobactin adsorption to fluoresceinated FepA mutant proteins confirmed this conclusion. The initial, outermost site contains aromatic residues and probably functions through hydrophobic interactions, whereas the secondary site exists deeper in the vestibule, contains both charged and aromatic residues and probably acts through hydrophobic and electrostatic bonds.