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.

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.

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.

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.

Diphenyl-benzo[1,3]dioxole-4-carboxylic acid pentafluorophenyl ester: a convenient catechol precursor in the synthesis of siderophore vectors suitable for antibiotic Trojan horse strategies.

Catechols are components of many metal-chelating compounds, including siderophores that are naturally occurring iron(III) chelators excreted by microorganisms. Catechol derivatives are poorly soluble in organic media and the synthesis of catechol-containing molecules requires the use of protected catechol precursors with improved organic solubility. We therefore developed 2,2-diphenyl-benzo[1,3]dioxole-4-carboxylic acid pentafluorophenyl ester. This activated ester reacts with an amine functionalized scaffold to generate chelators in which the catechol functions are protected in the form of diphenyl-benzodioxole moieties. The catechol can subsequently be deprotected, at the end of the synthesis, with trifluoroacetic acid (TFA). This strategy was applied to the synthesis of two catechol compounds functionalized with a terminal propargyl extension. These two compounds were shown to promote iron uptake in Escherichia coli and Pseudomonas aeruginosa. These two compounds are suitable for use as vectors in antibiotic Trojan horse approaches, as they could be conjugated with azide-functionalized antibiotics using the Huisgen dipolar 1,3-cycloaddition.

Functional analysis of pyochelin-/enantiopyochelin-related genes from a pathogenicity island of Pseudomonas aeruginosa strain PA14.

Genomic islands are foreign DNA blocks inserted in so-called regions of genomic plasticity (RGP). Depending on their gene content, they are classified as pathogenicity, symbiosis, metabolic, fitness or resistance islands, although a detailed functional analysis is often lacking. Here we focused on a 34-kb pathogenicity island of Pseudomonas aeruginosa PA14 (PA14GI-6), which is inserted at RGP5 and carries genes related to those for pyochelin/enantiopyochelin biosynthesis. These enantiomeric siderophores of P. aeruginosa and certain strains of Pseudomonas protegens are assembled by a thiotemplate mechanism from salicylate and two molecules of cysteine. The biochemical function of several proteins encoded by PA14GI-6 was investigated by a series of complementation analyses using mutants affected in potential homologs. We found that PA14_54940 codes for a bifunctional salicylate synthase/salicyl-AMP ligase (for generation and activation of salicylate), that PA14_54930 specifies a dihydroaeruginoic acid (Dha) synthetase (for coupling salicylate with a cysteine-derived thiazoline ring), that PA14_54910 produces a type II thioesterase (for quality control), and that PA14_54880 encodes a serine O-acetyltransferase (for increased cysteine availability). The structure of the PA14GI-6-specified metabolite was determined by mass spectrometry, thin-layer chromatography, and HPLC as (R)-Dha, an iron chelator with antibacterial, antifungal and antitumor activity. The conservation of this genomic island in many clinical and environmental P. aeruginosa isolates of different geographical origin suggests that the ability for Dha production may confer a selective advantage to its host.

Engineering Siderophore Biosynthesis and Regulation Pathways to Increase Diversity and Availability.

Siderophores are small metal chelators synthesized by numerous organisms to access iron. These secondary metabolites are ubiquitously present on Earth, and because their production represents the main strategy to assimilate iron, they play an important role in both positive and negative interactions between organisms. In addition, siderophores are used in biotechnology for diverse applications in medicine, agriculture and the environment. The generation of non-natural siderophore analogs provides a new opportunity to create new-to-nature chelating biomolecules that can offer new properties to expand applications. This review summarizes the main strategies of combinatorial biosynthesis that have been used to generate siderophore analogs. We first provide a brief overview of siderophore biosynthesis, followed by a description of the strategies, namely, precursor-directed biosynthesis, the design of synthetic or heterologous pathways and enzyme engineering, used in siderophore biosynthetic pathways to create diversity. In addition, this review highlights the engineering strategies that have been used to improve the production of siderophores by cells to facilitate their downstream utilization.

Structure, function and binding selectivity and stereoselectivity of siderophore-iron outer membrane transporters.

To get access to iron, microorganisms produce and release into their environment small organic metal chelators called siderophores. In parallel, they produce siderophore-iron outer membrane transporters (also called TonB-Dependent Transporters or TBDT) embedded in the outer membrane; these proteins actively reabsorb the siderophore loaded with iron from the extracellular medium. This active uptake requires energy in the form of the proton motive force transferred from the inner membrane to the outer membrane transporter via the inner membrane TonB complex. Siderophores produced by microorganisms are structurally very diverse with molecular weights of 150 up to 2000Da. Siderophore-iron uptake from the extracellular medium by TBDTs is a highly selective and sometimes even stereoselective process, with each siderophore having a specific TBDT. Unlike the siderophores, all TBDTs have similar structures and belong to the outer membrane ß-barrel protein superfamily. The way in which the siderophore-iron complex passes through the TBDT is still unclear. In some bacteria, TBDTs are also partners of signaling cascades regulating the expression of proteins involved in siderophore biosynthesis and siderophore-iron acquisition.

Plant-Derived Catechols Are Substrates of TonB-Dependent Transporters and Sensitize Pseudomonas aeruginosa to Siderophore-Drug Conjugates.

Pseudomonas aeruginosa is an opportunistic pathogen responsible for acute and chronic infections in immunocompromised hosts. This organism is known to compete efficiently against coinfecting microorganisms, due in part to the secretion of antimicrobial molecules and the synthesis of siderophore molecules with high affinity for iron. P. aeruginosa possess a large repertoire of TonB-dependent transporters for the uptake of its own, as well as xenosiderophores released from other bacteria or fungi. Here, we show that P. aeruginosa is also capable of utilizing plant-derived polyphenols as an iron source. We found that exclusively plant-derived phenols containing a catechol group (i.e., chlorogenic acid, caffeic acid, quercetin, luteolin) induce the expression of the TonB-dependent transporters PiuA or PirA. This induction requires the two-component system PirR-PirS. Chlorogenic acid in its Fe(III)-loaded form was actively transported by PiuA and PirA and supported growth under iron-limiting conditions. Coincidentally, PiuA and PirA are also the main TonB transporters for the recently approved siderophore-drug conjugate cefiderocol. Surprisingly, quercetin supplementation increased the susceptibility of P. aeruginosa to siderophore-drug conjugates, due to induction of piuA and pirA expression mediated by the PirR-PirS two-component system. These findings suggest a potential novel therapeutic application for these biologically active dietary polyphenols. IMPORTANCE Iron is an essential element for living organisms. Most bacteria synthesize species-specific iron chelators, called siderophores, able to capture iron from their host or the environment. Pseudomonas aeruginosa, an opportunistic pathogen, produces two endogenous siderophores but is able to acquire iron also via xenosiderophores, produced by other bacteria or fungi, using a set of conserved TonB transporters. Here, we show that P. aeruginosa is also able to use plant metabolites, like quercetin and chlorogenic acid, as siderophores. These metabolites possess an iron-chelating catechol group and are recognized and transported by the TonB transporters PirA and PiuA. Since these transporters also promote the specific uptake of siderophore-drug conjugates, P. aeruginosa exposed to these plant catechols becomes hypersusceptible to this novel class of antibiotics. This unexpected finding suggests a potential therapeutic application for quercetin and chlorogenic acid, which were mainly investigated for their antioxidant and anti-inflammatory properties.

How the Presence of Hemin Affects the Expression of the Different Iron Uptake Pathways in Pseudomonas aeruginosa Cells.

Iron is an essential nutriment for almost all organisms, but this metal is poorly bioavailable. During infection, bacteria access iron from the host by importing either iron or heme. Pseudomonas aeruginosa, a gram-negative pathogen, secretes two siderophores, pyoverdine (PVD) and pyochelin (PCH), to access iron and is also able to use many siderophores produced by other microorganisms (called xenosiderophores). To access heme, P. aeruginosa uses three distinct uptake pathways, named Has, Phu, and Hxu. We previously showed that P. aeruginosa expresses the Has and Phu heme uptake systems and the PVD- and PCH-dependent iron uptake pathways in iron-restricted growth conditions, using proteomic and RT-qPCR approaches. Here, using the same approaches, we show that physiological concentrations of hemin in the bacterial growth medium result in the repression of the expression of the proteins of the PVD- and PCH-dependent iron uptake pathways, leading to less production of these two siderophores. This indicates that the pathogen adapts its phenotype to use hemin as an iron source rather than produce PVD and PCH to access iron. Moreover, the presence of both hemin and a xenosiderophore resulted in (i) the strong induction of the expression of the proteins of the added xenosiderophore uptake pathway, (ii) repression of the PVD- and PCH-dependent iron uptake pathways, and (iii) no effect on the expression levels of the Has, Phu, or Hxu systems, indicating that bacteria use both xenosiderophores and heme to access iron.

Hijacking of the Enterobactin Pathway by a Synthetic Catechol Vector Designed for Oxazolidinone Antibiotic Delivery in Pseudomonas aeruginosa.

Enterobactin (ENT) is a tris-catechol siderophore used to acquire iron by multiple bacterial species. These ENT-dependent iron uptake systems have often been considered as potential gates in the bacterial envelope through which one can shuttle antibiotics (Trojan horse strategy). In practice, siderophore analogues containing catechol moieties have shown promise as vectors to which antibiotics may be attached. Bis- and tris-catechol vectors (BCVs and TCVs, respectively) were shown using structural biology and molecular modeling to mimic ENT binding to the outer membrane transporter PfeA in Pseudomonas aeruginosa. TCV but not BCV appears to cross the outer membrane via PfeA when linked to an antibiotic (linezolid). TCV is therefore a promising vector for Trojan horse strategies against P. aeruginosa, confirming the ENT-dependent iron uptake system as a gate to transport antibiotics into P. aeruginosa cells.

Past mastering of metal transformation enabled physicians to increase their therapeutic potential.

BACKGROUND: Metals are trace elements, vital in some instances or toxic in others. Due to this toxicity, they have been used since ancient time as antimicrobials, and prescribed when plant-only remedies were not efficient enough. These remedies could still contain secrets that may lead to the discovery of new therapeutically interesting combinations. The objective of this study was to give a proof of concept that such remedies combining metals and plants are worth studying again. METHODS: We exploited 4 medical formularies (aqrabadhin), from three Arab authors from the 9-12th century. We reproduced a remedy, and analyzed the role of each ingredient. We further looked for the minimum inhibitory concentration against three pathogenic bacteria, and we analyzed toxic and inflammatory effects of this remedy on macrophages. RESULTS: Even if plants were extensively used (almost 80 % of all ingredients), more than 36 different minerals have been found in these 4 aqrabadhin. When it came to remedies against infections that could be applied externally, the use of metals grew to 70 %. We focused on a remedy, containing mainly metals. We have been able to attribute a role for each ingredient, to show that this skin remedy helped to combat the infection and to resorb the wound, and to highlight the mastering of metal transformation by these physicians. CONCLUSIONS: With a very simple recipe, mainly composed of metals, these past physicians designed a complete and synergistic remedy to combat abscesses, while restricting the toxic effect of metals to the site of infection. It is a first example showing that different metal manufactures were evolved to improve their therapeutic potentials. The knowledge acquired by these physician should deserve more attention, and unexpected features, original organo-metallic compounds or therapeutic synergy could still be found from such research.

Pyochelin enantiomers and their outer-membrane siderophore transporters in fluorescent pseudomonads: structural bases for unique enantiospecific recognition.

Pyochelin (Pch) and enantiopyochelin (EPch) are enantiomeric siderophores, with three chiral centers, produced under iron limitation conditions by Pseudomonas aeruginosa and Pseudomonas fluorescens , respectively. After iron chelation in the extracellular medium, Pch-Fe and EPch-Fe are recognized and transported by their specific outer-membrane transporters: FptA in P. aeruginosa and FetA in P. fluorescens . Structural analysis of FetA-EPch-Fe and FptA-Pch-Fe, combined with mutagenesis and docking studies revealed the structural basis of the stereospecific recognition of these enantiomers by their respective transporters. Whereas FetA and FptA have a low sequence identity but high structural homology, the Pch and EPch binding pockets do not share any structural homology, but display similar physicochemical properties. The stereospecific recognition of both enantiomers by their corresponding transporters is imposed by the configuration of the siderophore's C4'' and C2'' chiral centers. This recognition involves specific hydrogen bonds between the Arg91 guanidinium group and EPch-Fe for FetA and between the Leu117-Leu116 main chain and Pch-Fe for FptA. FetA and FptA are the first membrane receptors to be structurally described with opposite binding enantioselectivities for their ligands, giving insights into the structural basis of their enantiospecificity.

Synthesis and biological properties of conjugates between fluoroquinolones and a N3''-functionalized pyochelin.

Pyochelin is a siderophore common to Pseudomonas aeruginosa and several other pathogenic bacteria. A pyochelin functionalized at the N3'' position with a propyl-amine extension was previously synthesized. In the present work we proved that this analog binds FptA, the pyochelin outer membrane receptor, and transports iron(III) efficiently into bacteria. This functionalized pyochelin seemed to be a good candidate for antibiotic vectorization in the framework of a Trojan horse prodrug strategy. In this context, conjugates between pyochelin and three fluoroquinolones (norfloxacin, ciprofloxacin and N-desmethyl-ofloxacin) were synthesized with a spacer arm that was either stable or hydrolyzable in vivo. Some pyochelin-fluoroquinolone conjugates had antibacterial activities in growth inhibition experiments on several P. aeruginosa strains. However, these activities were weaker than those of the antibiotic alone. These properties appeared to be related to both the solubility and bioavailability of conjugates and to the stability of the spacer arm used.

Identification of the fatty acid coenzyme-A ligase FadD1 as an interacting partner of FptX in the Pseudomonas aeruginosa pyochelin pathway.

Pseudomonas aeruginosa is one of the most important nosocomial bacteria emerging as a highly multidrug-resistant pathogen. P. aeruginosa produces two siderophores including pyochelin (PCH) to fulfil its need for iron during infections. We know that both outer and inner membrane proteins FptA and FptX are involved in the ferri-PCH uptake, but this process requires increasing molecular and biochemical knowledge. Here, using bacterial two-hybrid and copurification assays we identified the fatty acid coenzyme-A ligase FadD1 as a novel interacting partner of the inner membrane transporter FptX and found that FadD1 may play a role in PCH production. We managed to purify the FadD1-FptX inner membrane complex and obtained low-resolution 3D models, opening the way for future high-resolution structures.