A multifunctional ATP-binding cassette transporter system from Vibrio cholerae transports vibriobactin and enterobactin

Vibrio cholerae uses the catechol siderophore vibriobactin for iron transport under iron-limiting conditions. We have identified genes for vibriobactin transport and mapped them within the vibriobactin biosynthetic gene cluster. Within this genetic region we have identified four genes, viuP, viuD, viuG and viuC, whose protein products have homology to the periplasmic binding protein, the two integral cytoplasmic membrane proteins, and the ATPase component, respectively, of other iron transport systems. The amino-terminal region of ViuP has homology to a lipoprotein signal sequence, and ViuP could be labeled with [(3)H]palmitic acid. This suggests that ViuP is a membrane lipoprotein. The ViuPDGC system transports both vibriobactin and enterobactin in Escherichia coli. In the same assay, the E. coli enterobactin transport system, FepBDGC, allowed the utilization of enterobactin but not vibriobactin. Although the entire viuPDGC system could complement mutations in fepB, fepD, fepG, or fepC, only viuC was able to independently complement the corresponding fep mutation. This indicates that these proteins usually function as a complex. V. cholerae strains carrying a mutation in viuP or in viuG were constructed by marker exchange. These mutations reduced, but did not completely eliminate, vibriobactin utilization. This suggests that V. cholerae contains genes in addition to viuPDGC that function in the transport of catechol siderophores.     

Catecholate Siderophores Protect Bacteria from Pyochelin Toxicity

Background

Bacteria produce small molecule iron chelators, known as siderophores, to facilitate the acquisition of iron from the environment. The synthesis of more than one siderophore and the production of multiple siderophore uptake systems by a single bacterial species are common place. The selective advantages conferred by the multiplicity of siderophore synthesis remains poorly understood. However, there is growing evidence suggesting that siderophores may have other physiological roles besides their involvement in iron acquisition.

Methods and Principal Findings

Here we provide the first report that pyochelin displays antibiotic activity against some bacterial strains. Observation of differential sensitivity to pyochelin against a panel of bacteria provided the first indications that catecholate siderophores, produced by some bacteria, may have roles other than iron acquisition. A pattern emerged where only those strains able to make catecholate-type siderophores were resistant to pyochelin. We were able to associate pyochelin resistance to catecholate production by showing that pyochelin-resistant Escherichia coli became sensitive when biosynthesis of its catecholate siderophore enterobactin was impaired. As expected, supplementation with enterobactin conferred pyochelin resistance to the entE mutant. We observed that pyochelin-induced growth inhibition was independent of iron availability and was prevented by addition of the reducing agent ascorbic acid or by anaerobic incubation. Addition of pyochelin to E. coli increased the levels of reactive oxygen species (ROS) while addition of ascorbic acid or enterobactin reduced them. In contrast, addition of the carboxylate-type siderophore, citrate, did not prevent pyochelin-induced ROS increases and their associated toxicity.

Conclusions

We have shown that the catecholate siderophore enterobactin protects E. coli against the toxic effects of pyochelin by reducing ROS. Thus, it appears that catecholate siderophores can behave as protectors of oxidative stress. These results support the idea that siderophores can have physiological roles aside from those in iron acquisition.     

The Vibrio cholerae VctPDGC system transports catechol siderophores and a siderophore-free iron ligand

Vibrio cholerae, the causative agent of cholera, has an absolute requirement for iron. It transports the catechol siderophores vibriobactin, which it synthesizes and secretes, and enterobactin. These siderophores are transported across the inner membrane by one of two periplasmic binding protein-dependent ABC transporters, VctPDGC or ViuPDGC. We show here that one of these inner membrane transport systems, VctPDGC, also promotes iron acquisition in the absence of siderophores. Plasmids carrying the vctPDGC genes stimulated growth in both rich and minimal media of a Shigella flexneri mutant that produces no siderophores. vctPDGC also stimulated the growth of an Escherichia coli enterobactin biosynthetic mutant in low iron medium, and this effect did not require feoB, tonB or aroB. A tyrosine to phenylalanine substitution in the periplasmic binding protein VctP did not alter enterobactin transport, but eliminated growth stimulation in the absence of a siderophore. These data suggest that the VctPDGC system has the capacity to transport both catechol siderophores and a siderophore-free iron ligand. We also show that VctPDGC is the previously unidentified siderophore-independent iron transporter in V. cholerae, and this appears to complete the list of iron transport systems in V. cholerae.     

How pathogenic bacteria evade mammalian sabotage in the battle for iron

Many bacteria, including numerous human pathogens, synthesize small molecules known as siderophores to scavenge iron. Enterobactin, a siderophore produced by enteric bacteria, is surprisingly ineffective as an iron-scavenging agent for bacteria growing in animals because of its hydrophobicity and its sequestration by the mammalian protein siderocalin, a component of the innate immune system. However, pathogenic strains of Escherichia coli and Salmonella use enzymes encoded by the iroA gene cluster to tailor enterobactin by glycosylation and linearization. The resulting modified forms of enterobactin, known as salmochelins, can evade siderocalin and are less hydrophobic than enterobactin, restoring this siderophore's iron-scavenging ability in mammals.     

Secretion, but not overall synthesis, of catecholate siderophores contributes to virulence of extraintestinal pathogenic Escherichia coli

Extraintestinal pathogenic Escherichia coli (ExPEC) use siderophores to sequester iron during infection. Enterobactin and salmochelins are catecholate siderophores produced by some ExPEC strains and other pathogenic enterobacteria. Siderophore export and synthesis mutants of avian ExPEC strain χ7122 were tested in a chicken infection model. In single-strain infections, siderophore-negative (ΔentDΔiuc), ΔentS and ΔentSΔiroC export mutants were attenuated in tissues and blood, whereas the ΔiroC export mutant was only attenuated in blood. Interestingly, the ΔentD mutant, producing only aerobactin, retained full virulence, and loss of entD in the ΔentSΔiroC mutant restored virulence. LC-MS/MS quantification of siderophores in export mutants demonstrated that loss of entS impaired enterobactin and mono-glucosylated enterobactin secretion, whereas loss of iroC impaired di- and tri-glucosylated enterobactin secretion. Loss of entS and/or iroC resulted in intracellular accumulation and increased secretion of siderophore monomers. Catecholate siderophore export mutants also demonstrated decreased fitness in a co-challenge infection model. By contrast, catecholate siderophore synthesis mutants (ΔentD and ΔiroB) competed as well as the wild-type strain. Results establish that EntS and IroC mediate specific export of catecholate siderophores and the role of these exporters for ExPEC virulence is contingent on enterobactin synthesis, which is not required when other siderophores like aerobactin are functional.     

The Pseudomonas aeruginosa pirA gene encodes a second receptor for ferrienterobactin and synthetic catecholate analogues

Actively secreted iron chelating agents termed siderophores play an important role in the virulence and rhizosphere competence of fluorescent pseudomonads, including Pseudomonas aeruginosa which secretes a high affinity siderophore, pyoverdine, and the low affinity siderophore, pyochelin. Uptake of the iron-siderophore complexes is an active process that requires specific outer membrane located receptors, which are dependent of the inner membrane-associated protein TonB and two other inner membrane proteins, ExbB and ExbC. P. aeruginosa is also capable of using a remarkable variety of heterologous siderophores as sources of iron, apparently by expressing their cognate receptors. Illustrative of this feature are the 32 (of which 28 putative) siderophore receptor genes observed in the P. aeruginosa PAO1 genome. However, except for a few (pyoverdine, pyochelin, enterobactin), the vast majority of P. aeruginosa siderophore receptor genes still remain to be characterized. Ten synthetic iron chelators of catecholate type stimulated growth of a pyoverdine/pyochelin deficient P. aeruginosa PAO1 mutant under condition of severe iron limitation. Null mutants of the 32 putative TonB-dependent siderophore receptor encoding genes engineered in the same genetic background were screened for obvious deficiencies in uptake of the synthetic siderophores, but none showed decreased growth stimulation in the presence of the different siderophores. However, a double knock-out mutant of ferrienterobactin receptor encoding gene pfeA (PA 2688) and pirA (PA0931) failed to be stimulated by 4 of the tested synthetic catecholate siderophores whose chemical structures resemble enterobactin. Ferric-enterobactin also failed to stimulate growth of the double pfeA-pirA mutant although, like its synthetic analogues, it stimulated growth of the corresponding single mutants. Hence, we confirmed that pirA represents a second P. aeruginosa ferric-enterobactin receptor. The example of these two enterobactin receptors probably illustrates a more general phenomenon of siderophore receptor redundancy in P. aeruginosa.     

Cloning of a Vibrio cholerae vibriobactin gene cluster: identification of genes required for early steps in siderophore biosynthesis.

Vibrio cholerae secretes the catechol siderophore vibriobactin in response to iron limitation. Vibriobactin is structurally similar to enterobactin, the siderophore produced by Escherichia coli, and both organisms produce 2,3-dihydroxybenzoic acid (DHBA) as an intermediate in siderophore biosynthesis. To isolate and characterize V. cholerae genes involved in vibriobactin biosynthesis, we constructed a genomic cosmid bank of V. cholerae DNA and isolated clones that complemented mutations in E. coli enterobactin biosynthesis genes. V. cholerae homologs of entA, entB, entC, entD, and entE were identified on overlapping cosmid clones. Our data indicate that the vibriobactin genes are clustered, like the E. coli enterobactin genes, but the organization of the genes within these clusters is different. In this paper, we present the organization and sequences of genes involved in the synthesis and activation of DHBA. In addition, a V. cholerae strain with a chromosomal mutation in vibA was constructed by marker exchange. This strain was unable to produce vibriobactin or DHBA, confirming that in V. cholerae VibA catalyzes an early step in vibriobactin biosynthesis.     

Computer-aided design of novel siderophores: pyridinochelin

Pyridinochelin, a novel tetradentate catecholate-type siderophore, has been designed on the basis of the active analog enterobactin and was then synthesized. Growth promotion tests indicate that this synthetic siderophore feeds various pathogenic bacteria most effectively with iron even though it lacks one catecholate group compared to enterobactin. The superposition of the mentioned siderophore structures suggests that the structure of the skeleton connecting the catecholate groups might be an important factor for the iron transport.     

Ferric enterobactin binding and utilization by Neisseria gonorrhoeae

FetA, formerly designated FrpB, an iron-regulated, 76-kDa neisserial outer membrane protein, shows sequence homology to the TonB-dependent family of receptors that transport iron into gram-negative bacteria. Although FetA is commonly expressed by most neisserial strains and is a potential vaccine candidate for both Neisseria gonorrhoeae and Neisseria meningitidis, its function in cell physiology was previously undefined. We now report that FetA functions as an enterobactin receptor. N. gonorrhoeae FA1090 utilized ferric enterobactin as the sole iron source when supplied with ferric enterobactin at approximately 10 microM, but growth stimulation was abolished when an omega (Omega) cassette was inserted within fetA or when tonB was insertionally interrupted. FA1090 FetA specifically bound 59Fe-enterobactin, with a Kd of approximately 5 microM. Monoclonal antibodies raised against the Escherichia coli enterobactin receptor, FepA, recognized FetA in Western blots, and amino acid sequence comparisons revealed that residues previously implicated in ferric enterobactin binding by FepA were partially conserved in FetA. An open reading frame downstream of fetA, designated fetB, predicted a protein with sequence similarity to the family of periplasmic binding proteins necessary for transporting siderophores through the periplasmic space of gram-negative bacteria. An Omega insertion within fetB abolished ferric enterobactin utilization without causing a loss of ferric enterobactin binding. These data show that FetA is a functional homolog of FepA that binds ferric enterobactin and may be part of a system responsible for transporting the siderophore into the cell.     

Galline Ex-FABP is an antibacterial siderocalin and a lysophosphatidic acid sensor functioning through dual ligand specificities

Galline Ex-FABP was identified as another candidate antibacterial, catecholate siderophore binding lipocalin (siderocalin) based on structural parallels with the family archetype, mammalian Siderocalin. Binding assays show that Ex-FABP retains iron in a siderophore-dependent manner in both hypertrophic and dedifferentiated chondrocytes, where Ex-FABP expression is induced after treatment with proinflammatory agents, and specifically binds ferric complexes of enterobactin, parabactin, bacillibactin and, unexpectedly, monoglucosylated enterobactin, which does not bind to Siderocalin. Growth arrest assays functionally confirm the bacteriostatic effect of Ex-FABP in?vitro under iron-limiting conditions. The 1.8?? crystal structure of Ex-FABP explains the expanded specificity, but also surprisingly reveals an extended, multi-chambered cavity extending through the protein and encompassing two separate ligand specificities, one for bacterial siderophores (as in Siderocalin) at one end and one specifically binding copurified lysophosphatidic acid, a potent cell signaling molecule, at the other end, suggesting Ex-FABP employs dual functionalities to explain its diverse endogenous activities.     

Binding of ferric enterobactin by the Escherichia coli periplasmic protein FepB

The periplasmic protein FepB of Escherichia coli is a component of the ferric enterobactin transport system. We overexpressed and purified the binding protein 23-fold from periplasmic extracts by ammonium sulfate precipitation and chromatographic methods, with a yield of 20%, to a final specific activity of 15,500 pmol of ferric enterobactin bound/mg. Periplasmic fluid from cells overexpressing the binding protein adsorbed catecholate ferric siderophores with high affinity: in a gel filtration chromatography assay the K(d) of the ferric enterobactin-FepB binding reaction was approximately 135 nM. Intrinsic fluorescence measurements of binding by the purified protein, which were more accurate, showed higher affinity for both ferric enterobactin (K(d) = 30 nM) and ferric enantioenterobactin (K(d) = 15 nM), the left-handed stereoisomer of the natural E. coli siderophore. Purified FepB also adsorbed the apo-siderophore, enterobactin, with comparable affinity (K(d) = 60 nM) but did not bind ferric agrobactin. Polyclonal rabbit antisera and mouse monoclonal antibodies raised against nearly homogeneous preparations of FepB specifically recognized it in solid-phase immunoassays. These sera enabled the measurement of the FepB concentration in vivo when expressed from the chromosome (4,000 copies/cell) or from multicopy plasmids (>100,000 copies/cell). Overexpression of the binding protein did not enhance the overall affinity or rate of ferric enterobactin transport, supporting the conclusion that the rate-limiting step of ferric siderophore uptake through the cell envelope is passage through the outer membrane.     

New synthetic catecholate-type siderophores based on amino acids and dipeptides

New analogs of bacterial siderophores with one, two or three catecholate moieties were synthesized using various mono- and diamino acid and dipetide scaffolds, respectively. In addition to 2,3-dihydroxybenzoyl siderophore analogs and their acylated derivatives, 3,4-dihydroxybenzoyl derivatives were prepared. Furthermore, the synthesis of a new triscatecholate serving as an intimate model for enterobactin is reported. Most of the new compounds gave a positive CAS-test and were active as siderophores tested by growth promotion assays with a set of siderophore indicator mutants under iron limitation. Structure-activity-correlations have also been studied.     

Siderocalin/Lcn2/NGAL/24p3 Does Not Drive Apoptosis Through Gentisic Acid Mediated Iron Withdrawal in Hematopoietic Cell Lines

Siderocalin (also lipocalin 2, NGAL or 24p3) binds iron as complexes with specific siderophores, which are low molecular weight, ferric ion-specific chelators. In innate immunity, siderocalin slows the growth of infecting bacteria by sequestering bacterial ferric siderophores. Siderocalin also binds simple catechols, which can serve as siderophores in the damaged urinary tract. Siderocalin has also been proposed to alter cellular iron trafficking, for instance, driving apoptosis through iron efflux via BOCT. An endogenous siderophore composed of gentisic acid (2,5-dihydroxybenzoic acid) substituents was proposed to mediate cellular efflux. However, binding studies reported herein contradict the proposal that gentisic acid forms high-affinity ternary complexes with siderocalin and iron, or that gentisic acid can serve as an endogenous siderophore at neutral pH. We also demonstrate that siderocalin does not induce cellular iron efflux or stimulate apoptosis, questioning the role siderocalin plays in modulating iron metabolism.     

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.     

Salmochelin, the long-overlooked catecholate siderophore of Salmonella

Salmochelin is a C-glucosylated enterobactin produced by Salmonella species, uropathogenic and avian pathogenic Escherichia coli strains, and certain Klebsiella strains. It was the first glucosylated siderophore described. The glucosylation has been interpreted as a bacterial evasion mechanism against the mammalian catecholate siderophore-binding protein siderocalin (NGAL-lipocalin). The synthesis, excretion, and uptake of salmochelin requires five genes, iroBCDEN, and also the enterobactin biosynthesis and utilization system. Some salmochelin-producing strains also secrete microcins, which possess a C-terminal, linear glucosyl-enterobactin moiety. These microcins recognize the catecholate siderophore receptors IroN, Cir, Fiu, and FepA, and may inhibit the growth of competitors for catecholate siderophores.     

Role of molybdate and other transition metals in the accumulation of protochelin by Azotobacter vinelandii

Both molybdate and iron are metals that are required by the obligately aerobic organism Azotobacter vinelandii to survive in the nutrient-limited conditions of its natural soil environment. Previous studies have shown that a high concentration of molybdate (1 mM) affects the formation of A. vinelandii siderophores such that the tricatecholate protochelin is formed to the exclusion of the other catecholate siderophores, azotochelin and aminochelin. It has been shown previously that molybdate combines readily with catecholates and interferes with siderophore function. In this study, we found that the manner in which each catecholate siderophore interacted with molybdate was consistent with the structure and binding potential of the siderophore. The affinity that each siderophore had for molybdate was high enough that stable molybdo-siderophore complexes were formed but low enough that the complexes were readily destabilized by Fe(3+). Thus, competition between Fe(3+) and molybdate did not appear to be the primary cause of protochelin accumulation; in addition, we determined that protochelin accumulated in the presence of vanadate, tungstate, Zn(2+), and Mn(2+). We found that all five of these metal ions partially inhibited uptake of (55)Fe-protochelin and (55)Fe-azotochelin complexes. Also, each of these metal ions partially inhibited the activity of ferric reductase, an enzyme important in the deferration of ferric siderophores. Our results suggest that protochelin accumulates in the presence of molybdate because protochelin uptake and conversion into its component parts, azotochelin and aminochelin, are inhibited by interference with ferric reductase.     

Iron(III) complexation by Vanchrobactin, a siderophore of the bacterial fish pathogen Vibrio anguillarum

The bacterial fish pathogen Vibrio anguillarum serotype O2 strain RV22 produces the mono catecholate siderophore Vanchrobactin (Vb) under conditions of iron deficiency. Vb contains two potential bidentate coordination sites: catecholate and salicylate groups. The iron(III) coordination properties of Vb is investigated in aqueous solutions using spectrophotometric and potentiometric methods. The stepwise equilibrium constants (log?K) for successive addition of Vb dianion to a ferric ion are 19.9; 13.3, and 9.5, respectively, for an overall association constant of 42.7. Based on the previous results, we estimated the equilibrium concentration of free iron(III) under physiological conditions for pH 7.4 solution containing 10(-6) M total iron and 10(-5) M total Vb as pFe = 20 (=-log[Fe(3+)]). The Vb model compounds catechol (Cat) and 2,4-dihydroxy-N-(2-hydroxyethyl)benzamide (Dhb) have also been examined, and the obtained results show that the interaction of the whole system of Vb that contains the ferric-chelating groups of both Dhb and Cat, is synergically greater than the separate parts; i.e. Vb is the best chelating agent either in acid or basic media. In summary, bacteria employing Vb-mediated iron transport thus are able to compete effectively for iron with other microorganisms within which they live.     

Mechanisms of siderophore iron transport in enteric bacteria.

Uptake of 55Fe- and 3H-labeled siderophores and their chronic analogues have been studied in Salmonella typhimurium LT-2 and Escherichia coli K-12. In S. typhimurium LT-2, at least two different mechanisms for siderophore iron transport may be operative. Uptake of 55Fe- and 3H-labeled ferrichrome and kinetically inert lambda-cis-chromic [3H]deferriferrichrome by the S. typhimurium LT-2 enb7 mutant, which is defective in the production of its native siderophore, enterobactin, appears to occur by two concurrent mechanisms. The first mechanism is postulated to involve either rapid uptake of iron released from the ferric complex by cellular reduction without penetration of the complex or ligand or dissociation of the complex and simultaneous uptake of both ligand and iron coupled with simultaneous expulsion of the ligand. The second mechanism appears to consist of slower uptake of the intact ferric complex.