Rising infection rates with multidrug-resistant pathogens calls for antibiotics with novel modes of action. Herein, we identify the inner membrane protein TonB, a motor of active uptake in ...Gram-negative bacteria, as a novel target in antimicrobial therapy. The interaction of the TonB box of outer membrane transporters with TonB is crucial for the internalization of essential metabolites. We designed TonB box peptides and coupled them with synthetic siderophores in order to facilitate their uptake into bacteria in up to 32 synthetic steps. Three conjugates repressed the growth of Pseudomonas aeruginosa cells unable to produce their own siderophores, with minimal inhibitory concentrations between 0.1 and 0.5 μM. The transporters mediating uptake of these compounds were identified as PfeA and PirA. The study illustrates a variant of cellular suicide where a transporter imports its own inhibitor and demonstrates that artificial siderophores can import cargo with molecular weights up to 4 kDa.
The pyoverdine siderophore is produced by Pseudomonas aeruginosa to access iron. Its synthesis involves the complex coordination of four nonribosomal peptide synthetases (NRPSs), which are ...responsible for assembling the pyoverdine peptide backbone. The precise cellular organization of these NRPSs and their mechanisms of interaction remain unclear. Here, we used a combination of several single-molecule microscopy techniques to elucidate the spatial arrangement of NRPSs within pyoverdine-producing cells. Our findings reveal that PvdL differs from the three other NRPSs in terms of localization and mobility patterns. PvdL is predominantly located in the inner membrane, while the others also explore the cytoplasmic compartment. Leveraging the power of multicolor single-molecule localization, we further reveal co-localization between PvdL and the other NRPSs, suggesting a pivotal role for PvdL in orchestrating the intricate biosynthetic pathway. Our observations strongly indicates that PvdL serves as a central orchestrator in the assembly of NRPSs involved in pyoverdine biosynthesis, assuming a critical regulatory function.
Siderophores are iron chelators produced by bacteria to access iron, an essential nutriment. Pyoverdine (PVDI), the major siderophore produced by Pseudomonas aeruginosa PAO1, consists of a ...fluorescent chromophore linked to an octapeptide. The ferric form of PVDI is transported from the extracellular environment into the periplasm by the outer membrane transporter, FpvA. Iron is then released from the siderophore in the periplasm by a mechanism that does not involve chemical modification of the chelator but an iron reduction step. Here, we followed the kinetics of iron release from PVDI, in vitro and in living cells, by monitoring its fluorescence (as apo PVDI is fluorescent, whereas PVDI-Fe(III) is not). Deletion of the inner membrane proteins fpvG (PA2403) and fpvH (PA2404) affected 55Fe uptake via PVDI and completely abolished PVDI-Fe dissociation, indicating that these two proteins are involved in iron acquisition via this siderophore. PVDI-Fe dissociation studies, using an in vitro assay, showed that iron release from this siderophore requires the presence of an iron reducer (DTT) and an iron chelator (ferrozine). In this assay, DTT could be replaced by the inner membrane protein, FpvG, and ferrozine by the periplasmic protein, FpvC, suggesting that FpvG acts as a reductase and FpvC as an Fe2+ chelator in the process of PVDI-Fe dissociation in the periplasm of P. aeruginosa cells. This mechanism of iron release from PVDI is atypical among Gram-negative bacteria but seems to be conserved among Pseudomonads.
Bacteria use small molecules called siderophores to scavenge iron. Siderophore-Fe
complexes are recognised by outer-membrane transporters and imported into the periplasm in a process dependent on the ...inner-membrane protein TonB. The siderophore enterobactin is secreted by members of the family Enterobacteriaceae, but many other bacteria including Pseudomonas species can use it. Here, we show that the Pseudomonas transporter PfeA recognises enterobactin using extracellular loops distant from the pore. The relevance of this site is supported by in vivo and in vitro analyses. We suggest there is a second binding site deeper inside the structure and propose that correlated changes in hydrogen bonds link binding-induced structural re-arrangements to the structural adjustment of the periplasmic TonB-binding motif.
Summary
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.
Pyoverdine I (PVDI) and pyochelin (PCH) are the two major siderophores produced by Pseudomonas aeruginosa PAO1 to import iron. The biochemistry of the biosynthesis of these two siderophores has been ...described in detail in the literature over recent years. PVDI assembly requires the coordinated action of seven cytoplasmic enzymes and is followed by a periplasmic maturation before secretion of the siderophore into the extracellular medium by the efflux system PvdRT-OpmQ. PCH biosynthesis also involves seven cytoplasmic enzymes but no periplasmic maturation. Recent findings indicate that the cytoplasmic enzymes involved in each of these two siderophore biosynthesis pathways can form siderophore-specific multi-enzymatic complexes called siderosomes associated with the inner leaflet of the cytoplasmic membrane. This organization may optimize the transfer of the siderophore precursors between the various participating enzymes and avoid the diffusion of siderophore precursors, able to chelate metals, throughout the cytoplasm. Here, we describe these recently published findings and discuss the existence of these siderosomes in P. aeruginosa.
This review focuses on recent findings indicating that siderophore biosynthesis pathways in bacteria have a high cellular organization. The enzymes involved in such processes can form siderophore-specific multi-enzymatic complexes called siderosomes associated with the inner leaflet of the cytoplasmic membrane. This cellular organization probably ensures the efficiency of siderophore biosynthesis. Display omitted
•Siderophore biosynthesis pathways are complex and involve different enzymes.•These biosynthesis pathways have a specific cellular organization in siderosomes.
Bacteria access iron, a key nutrient, by producing siderophores or using siderophores produced by other microorganisms. The pathogen
produces two siderophores but is also able to pirate enterobactin ...(ENT), the siderophore produced by
. ENT-Fe complexes are imported across the outer membrane of
by the two outer membrane transporters PfeA and PirA. Iron is released from ENT in the
periplasm by hydrolysis of ENT by the esterase PfeE. We show here that
gene deletion renders
unable to grow in the presence of ENT because it is unable to access iron
this siderophore. Two-species co-cultures under iron-restricted conditions show that
strongly represses the growth of
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
strain is able to use ENT produced by
to access iron. If
is deleted,
has the upper hand in the culture and
growth is repressed. Overall, these data show that PfeE is the Achilles' heel of
in communities with bacteria producing ENT.
Iron is an essential nutrient for the survival and virulence of Pseudomonas aeruginosa. The pathogen expresses at least 15 different iron‐uptake pathways, the majority involving small iron chelators ...called siderophores. P. aeruginosa produces two siderophores, but can also use many produced by other microorganisms. This implies that the bacterium expresses appropriate TonB‐dependent transporters (TBDTs) at the outer membrane to import the ferric form of each of the siderophores used. Here, we show that the two α‐carboxylate‐type siderophores rhizoferrin‐Fe and staphyloferrin A‐Fe are transported into P. aeruginosa cells by the TBDT ActA. Among the mixed α‐carboxylate/hydroxamate‐type siderophores, we found aerobactin‐Fe to be transported by ChtA and schizokinen‐Fe and arthrobactin‐Fe by ChtA and another unidentified TBDT. Our findings enhance the understanding of the adaptability of P. aeruginosa and hold significant implications for developing novel strategies to combat antibiotic resistance.
Iron is a key nutrient for the growth of almost all bacteria. The pathogen Pseudomonas aeruginosa is able to express at least 15 different iron acquisition pathways, each involving a specific outer membrane transporter. Most of these iron‐uptake pathways rely on small iron chelators (siderophores) produced by other microorganisms. We identified the outer membrane transporters involved in the uptake of iron via two α‐carboxylate siderophores and three mixed α‐carboxylate/hydroxamate siderophores.
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.
Enterobactin (ENT) is a siderophore (iron-chelating compound) produced by Escherichia coli to gain access to iron, an indispensable nutrient for bacterial growth. ENT is used as an exosiderophore by ...Pseudomonas aeruginosa with transport of ferri-ENT across the outer membrane by the PfeA transporter. Next to the pfeA gene on the chromosome is localized a gene encoding for an esterase, PfeE, whose transcription is regulated, as for pfeA, by the presence of ENT in bacterial environment. Purified PfeE hydrolyzed ferri-ENT into three molecules of 2,3-DHBS (2,3-dihydroxybenzoylserine) still complexed with ferric iron, and complete dissociation of iron from ENT chelating groups was only possible in the presence of both PfeE and an iron reducer, such as DTT. The crystal structure of PfeE and an inactive PfeE mutant complexed with ferri-ENT or a nonhydrolyzable ferri-catechol complex allowed identification of the enzyme binding site and the catalytic triad. Finally, cell fractionation and fluorescence microscopy showed periplasmic localization of PfeE in P. aeruginosa cells. Thus, the molecular mechanism of iron dissociation from ENT in P. aeruginosa differs from that previously described in E. coli. In P. aeruginosa, siderophore hydrolysis occurs in the periplasm, with ENT never reaching the bacterial cytoplasm. In E. coli, ferri-ENT crosses the inner membrane via the ABC transporter FepBCD and ferri-ENT is hydrolyzed by the esterase Fes only once it is in the cytoplasm.
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