Structural insights into a novel family of integral membrane siderophore reductases.

Gram-negative bacteria take up the essential ion Fe3+ as ferric-siderophore complexes through their outer membrane using TonB-dependent transporters. However, the subsequent route through the inner membrane differs across many bacterial species and siderophore chemistries and is not understood in detail. Here, we report the crystal structure of the inner membrane protein FoxB (from Pseudomonas aeruginosa) that is involved in Fe-siderophore uptake. The structure revealed a fold with two tightly bound heme molecules. In combination with in vitro reduction assays and in vivo iron uptake studies, these results establish FoxB as an inner membrane reductase involved in the release of iron from ferrioxamine during Fe-siderophore uptake.

Pseudomonas aeruginosa and its multiple strategies to access iron.

Pseudomonas aeruginosa is a ubiquitous bacterium found in many natural and man-made environments. It is also a pathogen for plants, animals, and humans. As for almost all living organisms, iron is an essential nutrient for the growth of P. aeruginosa. The bacterium has evolved complex systems to access iron and maintain its homeostasis to survive in diverse natural and dynamic host environments. To access ferric iron, P. aeruginosa is able to produce two siderophores (pyoverdine and pyochelin), as well as use a variety of siderophores produced by other bacteria (mycobactins, enterobactin, ferrioxamine, ferrichrome, vibriobactin, aerobactin, rhizobactin and schizokinen). Furthermore, it can also use citrate, in addition to catecholamine neuromediators and plant-derived mono catechols, as siderophores. The P. aeruginosa genome also encodes three heme-uptake pathways (heme being an iron source) and one ferrous iron acquisition pathway. This review aims to summarize current knowledge concerning the molecular mechanisms involved in all the iron and heme acquisition strategies used by P. aeruginosa.

Vectorization via Siderophores Increases Antibacterial Activity of K(RW)3 Peptides against Pseudomonas aeruginosa.

A series of new conjugates comprised from a small synthetic antimicrobial peptide (AMP) and a siderophore-type vector component was designed and tested for activity on P. aeruginosa PAO1 and several genetically modified strains. As AMP, the well-established arginine-tryptophane combination K(RW)3 (P1) was chosen with an added lysine for siderophore attachment. This peptide is easy to prepare, modify, and possesses good anti-bacterial activity. On the vector part, we examined several moieties: (i) the natural siderophore deferoxamine (DFO); (ii) bidentate iron chelators based on the hydroxamate building block (4 a-c) ; (iii) the non-siderophore chelators deferasirox (DFX) and deferiprone-carboxylate (DFP-COOH). All conjugates were prepared by solid phase synthesis techniques and fully characterized by HPLC and mass spectrometry (including HR-MS). 55 Fe uptake assays indicate a receptor-mediated uptake for 4 a-c, DFP-COOH and DFO, which is dependent on the outer membrane transporter FoxA in the case of DFO. All conjugates showed increased antibacterial activity against P. aeruginosa compared to the parent peptide P1 alone when investigated in iron-depleted medium. MIC values were as low as 2 µM (for P1-DFP) on wild type P. aeruginosa. The activity of P1-DFO and P1-DFP was even better on genetically mutated strains unable to produce siderophores (down to 0.5 µM). Although the DFX vector on its own was not able to transport iron inside the bacterial cell as shown by 55 Fe uptake studies, the P1-DFX conjugate had excellent antibacterial activity compared to P1 (2 µM, and as low as 0.25 µM on a receptor-deficient strain unable to produce siderophores), suggesting that the conjugates were indeed recognized and internalized by an (unknown) transporter. Control experiments with an equimolar mixture of P1 and DFX confirm that the observed activity is intrinsic to vectorization. This work thus demonstrates the power of linking small AMPs covalently to siderophores for a new class of Trojan Horse antibiotics, with P1-DFP and P1-DFX being the most potent conjugates.

In cellulo FRET-FLIM and single molecule tracking reveal the supra-molecular organization of the pyoverdine bio-synthetic enzymes in Pseudomonas aeruginosa.

The bio-synthesis of pyoverdine (PVD) in Pseudomonas aeruginosa involves multiple enzymatic steps including the action of non-ribosomal peptide synthetases (NRPSs). One hallmark of NRPS is their ability to make usage of non-proteinogenic amino-acids synthesized by co-expressed accessory enzymes. It is generally proposed that different enzymes of a secondary metabolic pathway assemble into large supra-molecular complexes. However, evidence for the assembly of sequential enzymes in the cellular context is sparse. Here, we used in cellulo single-molecule tracking and Förster resonance energy transfer measured by fluorescence lifetime microscopy (FRET-FLIM) to explore the spatial partitioning of the ornithine hydroxylase PvdA and its interactions with NRPS. We found PvdA was mostly diffusing bound to large complexes in the cytoplasm with a small exchangeable trapped fraction. FRET-FLIM clearly showed that PvdA is physically interacting with PvdJ, PvdI, PvdL, and PvdD, the four NRPS involved in the PVD pathway in Pseudomonas aeruginosa PAO1. The binding modes of PvdA were strikingly different according to the NRPS it is interacting with, suggesting that PvdA binding sites have co-evolved with the enzymatic active sites of NRPS. Our data provide evidence for strongly organized multi-enzymatic complexes responsible for the bio-synthesis of PVD and illustrate how binding sites have evolved to finely control the co-localization of sequential enzymes and promote metabolic pathway efficiency.

Opportunistic use of catecholamine neurotransmitters as siderophores to access iron by Pseudomonas aeruginosa.

Iron is an essential nutrient for bacterial growth and the cause of a fierce battle between the pathogen and host during infection. Bacteria have developed several strategies to access iron from the host, the most common being the production of siderophores, small iron-chelating molecules secreted into the bacterial environment. The opportunist pathogen Pseudomonas aeruginosa produces two siderophores, pyoverdine and pyochelin, and is also able to use a wide panoply of xenosiderophores, siderophores produced by other microorganisms. Here, we demonstrate that catecholamine neurotransmitters (dopamine, l-DOPA, epinephrine and norepinephrine) are able to chelate iron and efficiently bring iron into P. aeruginosa cells via TonB-dependent transporters (TBDTs). Bacterial growth assays under strong iron-restricted conditions and with numerous mutants showed that the TBDTs involved are PiuA and PirA. PiuA exhibited more pronounced specificity for dopamine uptake than for norepinephrine, epinephrine and l-DOPA, whereas PirA specificity appeared to be higher for l-DOPA and norepinephrine. Proteomic and qRT-PCR approaches showed pirA transcription and expression to be induced in the presence of all four catecholamines. Finally, the oxidative properties of catecholamines enable them to reduce iron, and we observed ferrous iron uptake via the FeoABC system in the presence of l-DOPA.

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, 55 Fe 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.

Phenotypic Adaption of Pseudomonas aeruginosa by Hacking Siderophores Produced by Other Microorganisms.

Bacteria secrete siderophores to access iron, a key nutrient poorly bioavailable and the source of strong competition between microorganisms in most biotopes. Many bacteria also use siderophores produced by other microorganisms (exosiderophores) in a piracy strategy. Pseudomonas aeruginosa, an opportunistic pathogen, produces two siderophores, pyoverdine and pyochelin, and is also able to use a panel of exosiderophores. We first investigated expression of the various iron-uptake pathways of P. aeruginosa in three different growth media using proteomic and RT-qPCR approaches and observed three different phenotypic patterns, indicating complex phenotypic plasticity in the expression of the various iron-uptake pathways. We then investigated the phenotypic plasticity of iron-uptake pathway expression in the presence of various exosiderophores (present individually or as a mixture) under planktonic growth conditions, as well as in an epithelial cell infection assay. In all growth conditions tested, catechol-type exosiderophores were clearly more efficient in inducing the expression of their corresponding transporters than the others, showing that bacteria opt for the use of catechol siderophores to access iron when they are present in the environment. In parallel, expression of the proteins of the pyochelin pathway was significantly repressed under most conditions tested, as well as that of proteins of the pyoverdine pathway, but to a lesser extent. There was no effect on the expression of the heme and ferrous uptake pathways. Overall, these data provide precise insights on how P. aeruginosa adjusts the expression of its various iron-uptake pathways (phenotypic plasticity and switching) to match varying levels of iron and competition.

1,2,3-Triazole-gold(I)-triethylposphine derivatives active against resistant Gram-positive pathogens.

Innovative organogold(I) antibacterial compounds were synthesized by click chemistry with triethylphosphine-gold(I) azides and an alkyne derivative. The resulting organo-gold(I) compounds exhibit high levels of antibacterial activity against Gram-positive pathogens, with particularly low MICs against Clostridium difficile.

The Esterase PfeE, the Achilles' Heel in the Battle for Iron between Pseudomonas aeruginosa and Escherichia coli.

Bacteria access iron, a key nutrient, by producing siderophores or using siderophores produced by other microorganisms. The pathogen Pseudomonas aeruginosa produces two siderophores but is also able to pirate enterobactin (ENT), the siderophore produced by Escherichia coli. ENT-Fe complexes are imported across the outer membrane of P. aeruginosa by the two outer membrane transporters PfeA and PirA. Iron is released from ENT in the P. aeruginosa periplasm by hydrolysis of ENT by the esterase PfeE. We show here that pfeE gene deletion renders P. aeruginosa unable to grow in the presence of ENT because it is unable to access iron via this siderophore. Two-species co-cultures under iron-restricted conditions show that P. aeruginosa strongly represses the growth of E. coli as long it is able to produce its own siderophores. Both strains are present in similar proportions in the culture as long as the siderophore-deficient P. aeruginosa strain is able to use ENT produced by E. coli to access iron. If pfeE is deleted, E. coli has the upper hand in the culture and P. aeruginosa growth is repressed. Overall, these data show that PfeE is the Achilles' heel of P. aeruginosa in communities with bacteria producing ENT.

An overview of siderophore biosynthesis among fluorescent Pseudomonads and new insights into their complex cellular organization.

Siderophores are iron-chelating molecules produced by bacteria to access iron, a key nutrient. These compounds have highly diverse chemical structures, with various chelating groups. They are released by bacteria into their environment to scavenge iron and bring it back into the cells. The biosynthesis of siderophores requires complex enzymatic processes and expression of the enzymes involved is very finely regulated by iron availability and diverse transcriptional regulators. Recent data have also highlighted the organization of the enzymes involved in siderophore biosynthesis into siderosomes, multi-enzymatic complexes involved in siderophore synthesis. An understanding of siderophore biosynthesis is of great importance, as these compounds have many potential biotechnological applications because of their metal-chelating properties and their key role in bacterial growth and virulence. This review focuses on the biosynthesis of siderophores produced by fluorescent Pseudomonads, bacteria capable of colonizing a large variety of ecological niches. They are characterized by the production of chromopeptide siderophores, called pyoverdines, which give the typical green colour characteristic of fluorescent pseudomonad cultures. Secondary siderophores are also produced by these strains and can have highly diverse structures (such as pyochelins, pseudomonine, yersiniabactin, corrugatin, achromobactin and quinolobactin).

Synthesis of conjugates between oxazolidinone antibiotics and a pyochelin analogue.

Pseudomonas aeruginosa is a Gram-negative pathogenic bacterium responsible for severe infections, and it is naturally resistant to many clinically approved antibiotic families. Oxazolidinone antibiotics are active against many Gram-positive bacteria, but are inactive against P. aeruginosa. Increasing the uptake of oxazolidinones through the bacterial envelope could lead to an increased antibiotic effect. Pyochelin is a siderophore of P. aeruginosa which delivers external iron to the bacterial cytoplasm and is a potential vector for the development of Trojan Horse oxazolidinone conjugates. Novel pyochelin-oxazolidinone conjugates were synthesized using an unexpectedly regioselective peptide coupling between an amine functionalized pyochelin and oxazolidinones functionalized with a terminal carboxylate.

An overview of the biological metal uptake pathways in Pseudomonas aeruginosa.

Biological metal ions, including Co, Cu, Fe, Mg, Mn, Mo, Ni and Zn ions, are necessary for the survival and the growth of all microorganisms. Their biological functions are linked to their particular chemical properties: they play a role in structuring macromolecules and/or act as co-factors catalyzing diverse biochemical reactions. These metal ions are also essential for microbial pathogens during infection: they are involved in bacterial metabolism and various virulence factor functions. Therefore, during infection, bacteria need to acquire biological metal ions from the host such that there is competition for these ions between the bacterium and the host. Evidence is increasingly emerging of "nutritional immunity" against pathogens in the hosts; this includes strategies making access to metals difficult for infecting bacteria. It is clear that biological metals play key roles during infection and in the battle between the pathogens and the host. Here, we summarize current knowledge about the strategies used by Pseudomonas aeruginosa to access the various biological metals it requires. P. aeruginosa is a medically significant Gram-negative bacterial opportunistic pathogen that can cause severe chronic lung infections in cystic fibrosis patients and that is responsible for nosocomial infections worldwide.

Metallome of Pseudomonas aeruginosa: a role for siderophores.

In this paper, we describe the total metal composition (metallome) of Pseudomonas aeruginosa. Inductively coupled plasma atomic emission spectroscopy analyses showed that P. aeruginosa cells concentrate each metal of the metallome from the extracellular media with different efficiencies. Growth in nutrient-restricted media did not substantially affect the overall profile of the metallome; however, the uptake of some metals was strongly stimulated, showing the high potential of some metal acquisition pathways to adapt to changing growth conditions. We also investigated the role of the two major siderophores produced by P. aeruginosa, pyoverdine and pyochelin, in iron uptake and more generally in metallome homeostasis. In addition to their role in iron acquisition, siderophore production also significantly prevented the accumulation of toxic metals in P. aeruginosa cells, thus preserving the equilibrium of the metallome in a polluted environment.

Catechol siderophores repress the pyochelin pathway and activate the enterobactin pathway in Pseudomonas aeruginosa: an opportunity for siderophore-antibiotic conjugates development.

Previous studies have suggested that antibiotic vectorization by siderophores (iron chelators produced by bacteria) considerably increases the efficacy of such drugs. The siderophore serves as a vector: when the pathogen tries to take up iron via the siderophore, it also takes up the antibiotic. Catecholates are among the most common iron-chelating compounds used in synthetic siderophore-antibiotic conjugates. Using reverse transcription polymerase chain reaction and proteomic approaches, we showed that the presence of catecholate compounds in the medium of Pseudomonas aeruginosa led to strong activation of the transcription and expression of the outer membrane transporter PfeA, the ferri-enterobactin importer. Iron-55 uptake assays on bacteria with and without PfeA expression confirmed that catechol compounds imported iron into P. aeruginosa cells via PfeA. Uptake rates were between 0.3 × 10(3) and 2 × 10(3) Fe atoms/bacterium/min according to the used catechol siderophore in iron-restricted medium, and remained as high as 0.8 × 10(3) Fe atoms/bacterium/min for enterobactin, even in iron-rich medium. Reverse transcription polymerase chain reaction and proteomic approaches showed that in parallel to this switching on of PfeA expression, a repression of the expression of pyochelin (PCH) pathway genes (PCH being one of the two siderophores produced by P. aeruginosa for iron acquisition) was observed.

Cell-cell contacts confine public goods diffusion inside Pseudomonas aeruginosa clonal microcolonies.

The maintenance of cooperation in populations where public goods are equally accessible to all but inflict a fitness cost on individual producers is a long-standing puzzle of evolutionary biology. An example of such a scenario is the secretion of siderophores by bacteria into their environment to fetch soluble iron. In a planktonic culture, these molecules diffuse rapidly, such that the same concentration is experienced by all bacteria. However, on solid substrates, bacteria form dense and packed colonies that may alter the diffusion dynamics through cell-cell contact interactions. In Pseudomonas aeruginosa microcolonies growing on solid substrate, we found that the concentration of pyoverdine, a secreted iron chelator, is heterogeneous, with a maximum at the center of the colony. We quantitatively explain the formation of this gradient by local exchange between contacting cells rather than by global diffusion of pyoverdine. In addition, we show that this local trafficking modulates the growth rate of individual cells. Taken together, these data provide a physical basis that explains the stability of public goods production in packed colonies.

A cell biological view of the siderophore pyochelin iron uptake pathway in Pseudomonas aeruginosa.

Pyochelin (PCH) is a siderophore produced and secreted by Pseudomonas aeruginosa for iron capture. Using (55) Fe uptake and binding assays, we showed that PCH-Fe uptake in P. aeruginosa involves, in addition to the highly studied outer membrane transporter FptA, the inner membrane permease FptX, which recognizes PCH-(55) Fe with an affinity of 0.6 ± 0.2 nM and transports the ferri-siderophore complex from the periplasm into the cytoplasm: fptX deletion inhibited (55) Fe accumulation in the bacterial cytoplasm. Chromosomal replacement was used to generate P. aeruginosa strains producing fluorescent fusions with FptX, PchR (an AraC regulator), PchA (the first enzyme involved in the PCH biosynthesis) and PchE (a non-ribosomic peptide-synthetase involved in a further step). Fluorescence imaging and cellular fractionation showed a uniform repartition of FptX in the inner membrane. PchA and PchE were found in the cytoplasm, associated to the inner membrane all over the bacteria and also concentrated at the bacterial poles. PchE clustering at the bacterial poles was dependent on PchA expression, but on the opposite PchA clustering and membrane association was PchE-independent. PchA and PchE cellular organization suggests the existence of a siderosome for PCH biosynthesis as previously proposed for pyoverdine biosynthesis (another siderophore produced by P. aeruginosa).

Correction: synthesis and antibiotic activity of oxazolidinone-catechol conjugates against Pseudomonas aeruginosa.

Correction for 'Synthesis and antibiotic activity of oxazolidinone-catechol conjugates against Pseudomonas aeruginosa' by Aurélie Paulen, et al., Org. Biomol. Chem., 2016, DOI: 10.1039/c5ob01859e.

Synthesis and antibiotic activity of oxazolidinone-catechol conjugates against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative pathogenic bacterium responsible for severe infections in which resistance to most of the approved families of antibiotics is increasing. Oxazolidinone antibiotics are active against many Gram-positive bacteria, but are only weakly active against Gram-negative pathogens. We describe the synthesis of conjugates between a catechol moiety and oxazolidinone antibiotics. These conjugates were significantly more active against P. aeruginosa (218-1024 µM) than linezolid (MIC > 1024 µM), the reference molecule from the oxazolidinone family. Antibiotic activity was slightly higher in medium depleted of iron, suggesting the involvement of a bacterial iron uptake system in this biological activity. The bacterial iron uptake pathway involved in the transport is still to be addressed, but the present data excluded a contribution of the enterobactin transporter PfeA.

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 ((55)Fe 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.