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

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

Bacterial Iron Uptake Pathways: Gates for the Import of Bactericide Compounds

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Conception, synthèse et vectorisation d'inhibiteurs potentiels de la protéine bactérienne TonB

La multiplication des résistances aux antibiothérapies actuelles et l utilisation potentielle de bactéries pathogènes dans le cadre d attentats bioterroristes rendent nécessaire la recherche de nouvelles cibles biologiques et la découverte de nouvelles stratégies antibiotiques. Dans ce contexte, les mécanismes d assimilation du fer chez les bactéries à Gram négatif sont des cibles particulièrement prometteuses. Le fer est en effet un élément essentiel à la vie, mais peu biodisponible. Les bactéries ont donc développé des mécanismes efficaces pour subvenir à leurs besoins en fer. Ces mécanismes de transport nécessitent un apport d énergie fourni par une machinerie bactérienne complexe, la machinerie TonB. La protéine TonB, qui joue un rôle central dans le fonctionnement de cette machinerie, est la cible de notre approche. Nous souhaitons séquestrer cette protéine dans le périplasme grâce à des composés peptidiques fonctionnalisés par des hétérocycles de type isoindole ou 1,2,4-triazine. La conception et la synthèse de ces molécules sont présentées dans ce manuscrit, ainsi que leurs perspectives de vectorisation en utilisant une stratégie dite du "cheval de Troie". Notre contribution à la mise au point d un test d affinité in vitro est également abordée.The increasing resistances to the current antibiotherapies, and the potential use of pathogenic bacteria as biological weapons led us to the absolute necessity of discovering new biological targets and new antibiotic strategies. In this context, iron uptake pathways of Gram negative bacteria are promising targets. Indeed, iron is an essential nutrient, but it has a low bioavailability. Bacteria have developed efficient iron uptake pathways in order to proliferate. Iron is transported in the bacterial cell by specific outer membrane transporters and thanks to the energy provided by a complex molecular machinery, called TonB. The TonB protein, which is the keystone of this machinery, is a key target for the development of new antibiotics. We would like to sequester this protein in the periplasm thanks to molecules constituted of a peptidic moiety and a heterocyclic moiety such as isoindole or 1,2,4-triazine. The conception and the synthesis of these compounds are presented in this document, as well as their possibilities to be vectorized using a Trojan Horse strategy. Our contribution to the development of an in vitro test of affinity is presented as well.STRASBOURG-Bib.electronique 063 (674829902) / SudocSudocFranceF

Vectorisation d'antibiotiques par des sidérophores et synthèse d'inhibiteurs potentiels de l'assimilation du fer chez Pseudomonas aeruginosa et Burkholderia cepacia

La pyochéline est un siderophore commun à Pseudomonas aeruginosa et Burkholderia cepacia, bactéries responsables d infections graves chez les patients atteints de mucoviscidose. Le système d assimilation du fer pyochéline-dépendant, faisant intervenir plusieurs protéines dont son récepteur spécifique FptA, est une cible de choix dans le cadre de nouvelles stratégies antibiotiques. Dans ce contexte, nous avons synthétisé une pyochéline fonctionnalisée en position N3" qui se lie au récepteur FptA et transporte le fer en utilisant la voie de transport pyochéline-dépendante. Dans une stratégie prodrogue de type cheval de Troie , cette pyochéline fonctionnalisée a ensuite été utilisée comme motif d adressage d'antibiotiques de la famille des fluoroquinolones. Malgré une activité moindre que l antibiotique testé seul, la synthèse de conjugués entre la pyochéline fonctionnalisée et le groupement fluorescent 4-nitro-2,1,3-benzoxadiazole (NBD) a montré que la pyochéline synthétique est capable de vectoriser efficacement un xénobiotique en utilisant le système de transport du fer pyochéline-dépendant. En parallèle, nous avons conçu des inhibiteurs potentiels de la protéine membranaire FptA. Plusieurs de ces composés se lient fortement à la protéine cible mais sans parvenir à inhiber le transport du fer. Ces molécules ont cependant permis d améliorer nos connaissances sur les relations structure-activité liant la pyochéline à son récepteur de membrane externe, notamment grâce à la synthèse de l énantiopyochéline, un sidérophore de Pseudomonas fluorescens, qui a mis en évidence les fondements structuraux de la stéréospécificité de reconnaissance du récepteur FptA.The pyochelin is a common siderophore of Pseudomonas aeruginosa and Burkholderia cepacia , two pathogenic Gram negative bacteria responsible for severe infections in the case of cystic fibrosis affected patients. The pyochelin-dependant iron uptake, involving several proteins like its specific receptor FptA, is a promising target to develop new antibiotic strategies. In this context, we synthesized a pyochelin analog, functionalized on the N3" position, which binds to the FptA receptor and efficiently promotes iron uptake using the pyochelin-dependant iron uptake. The functionalized pyochelin was then conjugated to fluoroquinolone antibiotics in a Trojan horse prodrug strategy. Despite a lower biological activity compared to the antibiotics tested alone, the synthesis of conjugates between the N3" functionalized pyochelin and the 4-nitro-2,1,3-benzoxadiazole (NBD) fluorophore proved that our synthetic siderophore analogue is able to efficiently transport a xenobiotic molecule using the pyochelin-dependant iron uptake system. In parallel, we designed potential inhibitors of FptA receptor. Several molecules were able to efficiently bind to the target receptor without inhibiting the iron uptake. Nevertheless, these molecules brought invaluable information on the structure-activity relationships between the siderophore and its specific outer membrane receptor. In this context, the synthesis of enantiopyochelin, a siderophore produced by Pseudomonas fluorescens, brought new insights on the structural basis underlying the high stereospecificity of the FptA receptor binding site.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Interactions between Aspergillus fumigatus and Pulmonary Bacteria: Current State of the Field, New Data, and Future Perspective

Aspergillus fumigatus and Pseudomonas aeruginosa are central fungal and bacterial members of the pulmonary microbiota. The interactions between A. fumigatus and P. aeruginosa have only just begun to be explored. A balance between inhibitory and stimulatory effects on fungal growth was observed in mixed A. fumigatus–P. aeruginosa cultures. Negative interactions have been seen for homoserine-lactones, pyoverdine and pyochelin resulting from iron starvation and intracellular inhibitory reactive oxidant production. In contrast, several types of positive interactions were recognized. Dirhamnolipids resulted in the production of a thick fungal cell wall, allowing the fungus to resist stress. Phenazines and pyochelin favor iron uptake for the fungus. A. fumigatus is able to use bacterial volatiles to promote its growth. The immune response is also differentially regulated by co-infections

Synthesis of conjugates between oxazolidinone antibiotics and a pyochelin analogue

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Dipyridylamine-acetamide (Dpaa): A primary amine protecting group orthogonally cleavable under acidic conditions in the presence of t-butyloxycarbonyl (Boc) and t-butylester

International audienceWe show that dipyridylamine-acetamide (Dpaa), can be cleaved under mild acidic conditions (30% formic acid in dichloromethane). The release of the amine function is orthogonal to other acid-labile protecting groups. Calculations suggest that the ease of Dpaa cleavage relies on activation of the carbonyl function by the protonated dipyridylamine nitrogen and activation of a water molecule by a hydrogen-bond network

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