Binding properties of pyochelin and structurally related molecules to FptA of Pseudomonas aeruginosa

Pyochelin (Pch) is a siderophore that is produced in iron-limited conditions, by both Pseudomonas aeruginosa and Burkholderia cepacia. This iron uptake pathway could therefore be a target for the development of new antibiotics. Pch is (4'R,2'R/S,4'R)-2'-(2-hydroxyphenyl)-3'-methyl-4',5',2',3',4',5'-hexahydro-[4',2']bithiazolyl-4'-carboxylic acid, and has three chiral centres located at positions C4', C2' and C4'. In P.aeruginosa, this siderophore chelates iron in the extracellular medium and transports it into the cells via a specific outer membrane transporter FptA. Docking experiments using the X-ray structure of FptA-Pch-Fe showed that iron-loaded or unloaded Pch diastereoisomers could bind to FptA. This was confirmed by in vivo binding assays. These binding properties and the iron uptake ability were not affected by removal of the C4' chiral centre. After removal of both the C4' and C2' chiral centres, the molecule still bound to FptA but was unable to transport iron. The overall binding mode of this iron-complexed analogue was inverted. These findings describe the first antagonist of the Pch/FptA iron uptake pathway. Pch also complexes with iron in conjunction with other bidentate ligands such as cepabactin (Cep) or ethylene glycol. Docking experiments showed that such complexes bind to FptA via the Pch molecule. The mixed Pch-Fe-Cep complex was also recognized by FptA, having an affinity intermediate between that for Pch(2)-Fe and Cep(3)-Fe. Finally, the iron uptake properties of the different Pch-related molecules suggested a mechanism for FptA-Pch-Fe complex formation similar to that of the FpvA/Pvd uptake system. All these findings improve our understanding of specificity of the interaction between FptA and its siderophore.     

Synthesis and biological properties of thiazole-analogues of pyochelin,a siderophore of Pseudomonas aeruginosa

Pyochelin is a siderophore common to all strains of Pseudomonas aeruginosa utilized by this Gram-negative bacterium to acquire iron(III). FptA is the outer membrane transporter responsible of ferric-pyochelin uptake in P. aeruginosa. We describe in this Letter the synthesis and the biological properties (⁵⁵Fe uptake, binding to FptA) of several thiazole analogues of pyochelin. Among them we report in this Letter the two first pyochelin analogues able to bind FptA without promoting any iron uptake 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.     

TonB-dependent transporters and their occurrence in cyanobacteria

Background  

Different iron transport systems evolved in Gram-negative bacteria during evolution. Most of the transport systems depend on outer membrane localized TonB-dependent transporters (TBDTs), a periplasma-facing TonB protein and a plasma membrane localized machinery (ExbBD). So far, iron chelators (siderophores), oligosaccharides and polypeptides have been identified as substrates of TBDTs. For iron transport, three uptake systems are defined: the lactoferrin/transferrin binding proteins, the porphyrin-dependent transporters and the siderophore-dependent transporters. However, for cyanobacteria almost nothing is known about possible TonB-dependent uptake systems for iron or other substrates.     

TonB-dependent receptors—structural perspectives

Plants, bacteria, fungi, and yeast utilize organic iron chelators (siderophores) to establish commensal and pathogenic relationships with hosts and to survive as free-living organisms. In Gram-negative bacteria, transport of siderophores into the periplasm is mediated by TonB-dependent receptors. A complex of three membrane-spanning proteins TonB, ExbB and ExbD couples the chemiosmotic potential of the cytoplasmic membrane with siderophore uptake across the outer membrane. The crystallographic structures of two TonB-dependent receptors (FhuA and FepA) have recently been determined. These outer membrane transporters show a novel fold consisting of two domains. A 22-stranded antiparallel β-barrel traverses the outer membrane and adjacent β-strands are connected by extracellular loops and periplasmic turns. Located inside the β-barrel is the plug domain, composed primarily of a mixed four-stranded β-sheet and a series of interspersed α-helices. Siderophore binding induces distinct local and allosteric transitions that establish the structural basis of signal transduction across the outer membrane and suggest a transport mechanism.     

TonB-dependent receptors-structural perspectives

Plants, bacteria, fungi, and yeast utilize organic iron chelators (siderophores) to establish commensal and pathogenic relationships with hosts and to survive as free-living organisms. In Gram-negative bacteria, transport of siderophores into the periplasm is mediated by TonB-dependent receptors. A complex of three membrane-spanning proteins TonB, ExbB and ExbD couples the chemiosmotic potential of the cytoplasmic membrane with siderophore uptake across the outer membrane. The crystallographic structures of two TonB-dependent receptors (FhuA and FepA) have recently been determined. These outer membrane transporters show a novel fold consisting of two domains. A 22-stranded antiparallel beta-barrel traverses the outer membrane and adjacent beta-strands are connected by extracellular loops and periplasmic turns. Located inside the beta-barrel is the plug domain, composed primarily of a mixed four-stranded beta-sheet and a series of interspersed alpha-helices. Siderophore binding induces distinct local and allosteric transitions that establish the structural basis of signal transduction across the outer membrane and suggest a transport mechanism.     

Terbium, a fluorescent probe for investigation of siderophore pyochelin interactions with its outer membrane transporter FptA

Pyochelin (Pch) is a siderophore and FptA is its outer membrane transporter produced by Pseudomonas aeruginosa to import iron. The fluorescence of the element terbium is affected by coordinated ligands and it can therefore be used as a probe to investigate the pyochelin-iron uptake pathway in P. aeruginosa. At pH 8.0, terbium fluorescence is greatly enhanced in the presence of pyochelin indicating chelation of the metal by the siderophore. Titration curves showed a 2:1 (Pch:Tb³⁺) stoichiometry and an affinity of K =( 2 ± - 1 )× 10¹¹ M^− 2 was determined. Pch-Tb interaction with the transporter FptA could be followed in vitro and in vivo in P. aeruginosa cells, by Fluorescence Resonance Energy Transfer (FRET) between three partners: the tryptophans of FptA (donor), Pch (acceptor for the Trps and donor for Tb³⁺) and Tb³⁺ (acceptor). Pch-Tb binds to the Pch-Fe outer membrane transporter FptA with a dissociation constant (K_d) of 4.6 μM. This three-partner FRET is a potentially valuable tool for investigation of the interactions between FptA and its siderophore Pch.     

Fate of ferrisiderophores after import across bacterial outer membranes: different iron release strategies are observed in the cytoplasm or periplasm depending on the siderophore pathways

Siderophore production and utilization is one of the major strategies deployed by bacteria to get access to iron, a key nutrient for bacterial growth. The biological function of siderophores is to solubilize iron in the bacterial environment and to shuttle it back to the cytoplasm of the microorganisms. This uptake process for Gram-negative species involves TonB-dependent transporters for translocation across the outer membranes. In Escherichia coli and many other Gram-negative bacteria, ABC transporters associated with periplasmic binding proteins import ferrisiderophores across cytoplasmic membranes. Recent data reveal that in some siderophore pathways, this step can also be carried out by proton-motive force-dependent permeases, for example the ferrichrome and ferripyochelin pathways in Pseudomonas aeruginosa. Iron is then released from the siderophores in the bacterial cytoplasm by different enzymatic mechanisms depending on the nature of the siderophore. Another strategy has been reported for the pyoverdine pathway in P. aeruginosa: iron is released from the siderophore in the periplasm and only siderophore-free iron is transported into the cytoplasm by an ABC transporter having two atypical periplasmic binding proteins. This review presents recent findings concerning both ferrisiderophore and siderophore-free iron transport across bacterial cytoplasmic membranes and considers current knowledge about the mechanisms involved in iron release from siderophores.     

TonB or not TonB: is that the question?

Bacteria are able to survive in low-iron environments by sequestering this metal ion from iron-containing proteins and other biomolecules such as transferrin, lactoferrin, heme, hemoglobin, or other heme-containing proteins. In addition, many bacteria secrete specific low molecular weight iron chelators termed siderophores. These iron sources are transported into the Gram-negative bacterial cell through an outer membrane receptor, a periplasmic binding protein (PBP), and an inner membrane ATP-binding cassette (ABC) transporter. In different strains the outer membrane receptors can bind and transport ferric siderophores, heme, or Fe3+ as well as vitamin B12, nickel complexes, and carbohydrates. The energy that is required for the active transport of these substrates through the outer membrane receptor is provided by the TonB/ExbB/ExbD complex, which is located in the cytoplasmic membrane. In this minireview, we will briefly examine the three-dimensional structure of TonB and the current models for the mechanism of TonB-dependent energy transduction. Additionally, the role of TonB in colicin transport will be discussed.     

The heterologous siderophores ferrioxamine B and ferrichrome activate signaling pathways in Pseudomonas aeruginosa

Pseudomonas aeruginosa secretes two siderophores, pyoverdine and pyochelin, under iron-limiting conditions. These siderophores are recognized at the cell surface by specific outer membrane receptors, also known as TonB-dependent receptors. In addition, this bacterium is also able to incorporate many heterologous siderophores of bacterial or fungal origin, which is reflected by the presence of 32 additional genes encoding putative TonB-dependent receptors. In this work, we have used a proteomic approach to identify the inducing conditions for P. aeruginosa TonB-dependent receptors. In total, 11 of these receptors could be discerned under various conditions. Two of them are only produced in the presence of the hydroxamate siderophores ferrioxamine B and ferrichrome. Regulation of their synthesis is affected by both iron and the presence of a cognate siderophore. Analysis of the P. aeruginosa genome showed that both receptor genes are located next to a regulatory locus encoding an extracytoplasmic function sigma factor and a transmembrane sensor. The involvement of this putative regulatory locus in the specific induction of the ferrioxamine B and ferrichrome receptors has been demonstrated. These results show that P. aeruginosa has evolved multiple specific regulatory systems to allow the regulation of TonB-dependent receptors.     

Mutual inhibition of cobalamin and siderophore uptake systems suggests their competition for TonB function.

Vitamin B12 (CN-Cbl) and iron-siderophore complexes are transported into Escherichia coli in two energy-dependent steps. The first step is mediated by substrate-specific outer membrane transport proteins and the energy-coupling TonB protein complex, and the second step uses separate periplasmic permeases for transport across the cytoplasmic membrane. Genetic and biochemical evidence suggests that the TonB-dependent outer membrane transporters contact TonB directly, and thus they might compete for limiting amounts of functional TonB. The transport of iron-siderophore complexes, such as ferrichrome, causes a partial decrease in the rate of CN-Cbl transport. Although CN-Cbl uptake does not inhibit ferrichrome uptake in wild-type cells, in which the amount of the outer membrane ferrichrome transporter FhuA far exceeds that of the cobalamin transporter BtuB, CN-Cbl does inhibit ferrichrome uptake when BtuB is overexpressed from a multicopy plasmid. This inhibition by CN-Cbl is increased when the expression of FhuA and TonB is repressed by growth with excess iron and is eliminated when BtuB synthesis is repressed by CN-Cbl. The mutual inhibition of CN-Cbl and ferrichrome uptake is overcome by increased expression of TonB. Additional evidence for interaction of the Cbl and iron transport systems is provided by the strong stimulation of the BtuB- and TonB-dependent transport of CN-Cbl into a nonexchangeable, presumably cytoplasmic pool by preincubation of cells with the iron(II) chelator 2,2'-dipyridyl. Other metal ion chelators inhibited CN-Cbl uptake across the outer membrane. Although the effects of chelators are multiple and complex, they indicate competition or interaction among TonB-dependent transport systems.     

A role for PchHI as the ABC transporter in iron acquisition by the siderophore pyochelin in Pseudomonas aeruginosa

Iron is an essential nutrient for bacterial growth but poorly bioavailable. Bacteria scavenge ferric iron by synthesizing and secreting siderophores, small compounds with a high affinity for iron. Pyochelin (PCH) is one of the two siderophores produced by the opportunistic pathogen Pseudomonas aeruginosa. After capturing a ferric iron molecule, PCH-Fe is imported back into bacteria first by the outer membrane transporter FptA and then by the inner membrane permease FptX. Here, using molecular biology, ⁵⁵Fe uptake assays, and LC–MS/MS quantification, we first find a role for PchHI as the heterodimeric ABC transporter involved in the siderophore-free iron uptake into the bacterial cytoplasm. We also provide the first evidence that PCH is able to reach the bacterial periplasm and cytoplasm when both FptA and FptX are expressed. Finally, we detected an interaction between PchH and FptX, linking the ABC transporter PchHI with the inner permease FptX in the PCH-Fe uptake pathway. These results pave the way for a better understanding of the PCH siderophore pathway, giving future directions to tackle P. aeruginosa infections.     

Crystal structure at high resolution of ferric-pyochelin and its membrane receptor FptA from Pseudomonas aeruginosa

Pyochelin is a siderophore and virulence factor common to Burkholderia cepacia and several Pseudomonas strains. We describe at 2.0 A resolution the crystal structure of the pyochelin outer membrane receptor FptA bound to the iron-pyochelin isolated from Pseudomonas aeruginosa. One pyochelin molecule bound to iron is found in the protein structure, providing the first three-dimensional structure at the atomic level of this siderophore. The pyochelin molecule provides a tetra-dentate coordination of iron, while the remaining bi-dentate coordination is ensured by another molecule not specifically recognized by the protein. The overall structure of the pyochelin receptor is typical of the TonB-dependent transporter superfamily, which uses the proton motive force from the cytoplasmic membrane through the TonB-ExbB-ExbD energy transducing complex to transport ferric ions across the bacterial outer membrane: a transmembrane 22 beta-stranded barrel occluded by a N-terminal domain that contains a mixed four-stranded beta-sheet. The N-terminal TonB box is disordered in two crystal forms, and loop L8 is found to point towards the iron-pyochelin complex, suggesting that the receptor is in a transport-competent conformation.     

Alr0397 is an outer membrane transporter for the siderophore schizokinen in Anabaena sp. strain PCC 7120

Iron uptake in proteobacteria by TonB-dependent outer membrane transporters represents a well-explored subject. In contrast, the same process has been scarcely investigated in cyanobacteria. The heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 is known to secrete the siderophore schizokinen, but its transport system has remained unidentified. Inspection of the genome of strain PCC 7120 shows that only one gene encoding a putative TonB-dependent iron transporter, namely alr0397, is positioned close to genes encoding enzymes involved in the biosynthesis of a hydroxamate siderophore. The expression of alr0397, which encodes an outer membrane protein, was elevated under iron-limited conditions. Inactivation of this gene caused a moderate phenotype of iron starvation in the mutant cells. The characterization of the mutant strain showed that Alr0397 is a TonB-dependent schizokinen transporter (SchT) of the outer membrane and that alr0397 expression and schizokinen production are regulated by the iron homeostasis of the cell.     

Iron-free pyoverdin binds to its outer membrane receptor FpvA in Pseudomonas aeruginosa: a new mechanism for membrane iron transport

Under iron limitation, Pseudomonas aeruginosa secretes a fluorescent siderophore called pyoverdin, which, after complexing iron, is transported back into the cell via its outer membrane receptor FpvA. Previous studies demonstrated co-purification of FpvA with iron-free PaA and reported similar binding affinities of iron-free pyoverdin and ferric-pyoverdin to purified FpvA. The fluorescence resonance energy transfer between iron-free PaA and the FpvA receptor here reveals the existence of an FpvA-pyoverdin complex in P. aeruginosa in vivo, suggesting that the pyoverdin-loaded FpvA is the normal state of the receptor in the absence of iron. Using tritiated ferric-pyoverdin, it is shown that iron-free PaA binds to the outer membrane but is not taken up into the cell, and that in vitro and, presumably, in vivo ferric-pyoverdin displaces the bound iron-free pyoverdin on FpvA-PaA to form FpvA-PaA-Fe complexes. In vivo, the kinetics of formation of this FpvA-PaA-Fe complex are more than two orders of magnitude faster than in vitro and depend on the presence of TonB. In P. aeruginosa, two tonB genes have been identified (tonB1 and tonB2). TonB1 is directly involved in ferric-pyoverdin uptake, and TonB2 seems to be able partially to replace TonB1 in its role in iron acquisition. However, no effect of TonB1 or TonB2 on the apparent affinity of free pyoverdin to FpvA was observed, and a 17-fold difference was measured between the affinities of the two forms of pyoverdin (PaA and PaA-Fe) to FpvA in the absence of TonB1 or TonB2. The mechanism of iron uptake in P. aeruginosa via the pyoverdin pathway is discussed in view of these new findings.     

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 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.