Microbial Iron Chelators Research Articles

Inhibition of lipid oxidation and hexanal production in cooked meats by microbial iron chelator deferriferrichrysin from rice wine

Lipid oxidation imparting off-flavor and decreasing the shelf life of meat is a significant concern of the global food industry. Here, the efficacy of a natural iron chelator, deferriferrichrysin (Dfcy), contained in sake (Japanese rice wine), against lipid oxidation of meat was investigated. Purified Dfcy and Dfcy-enhanced sake were prepared and added to three kinds of meat (chicken, pork, and beef) and heated at 70°C for 70 min, followed by storage at 4°C for 10 days and analysis of the efficacy to inhibit lipid oxidation. Dfcy reduced thiobarbituric acid-reactive substances (TBARS) values of meat products by over 50% with the addition of 100 mg/kg of Dfcy. Hexanal production was more efficiently reduced, that is, over 90% reduction with the addition of 50–100 mg/kg of Dfcy. The findings suggest Dfcy and Dfcy-containing sake as promising novel natural antioxidants to inhibit lipid oxidation in meat products. Novelty Impact Statement This study investigated the natural iron chelator deferriferrichrysin (Dfcy) contained in sake (Japanese rice wine) against the lipid oxidation of meat products. Purified Dfcy and Dfcy-containing sake inhibited hexanal production with over 90% reduction at 50–100 ppm in chicken, pork, and beef meat. Dfcy was shown to be an efficient inhibitor of lipid oxidation in meat products, and the strategy used in this study could be used to develop schemes for the production of other rice wines that contain iron chelator.

Magnitude and Mechanism of Siderophore-Mediated Competition at Low Iron Solubility in the Pseudomonas aeruginosa Pyochelin System.

A central question in microbial ecology is whether microbial interactions are predominantly cooperative or competitive. The secretion of siderophores, microbial iron chelators, is a model system for cooperative interactions. However, siderophores have also been shown to mediate competition by sequestering available iron and making it unavailable to competitors. The details of how siderophores mediate competition are not well understood, especially considering the complex distribution of iron phases in the environment. One pertinent question is whether sequestering iron through siderophores can indeed be effective in natural conditions; many natural environments are characterized by large pools of precipitated iron, and it is conceivable that any soluble iron that is sequestered by siderophores is replenished by the dissolution of these precipitated iron sources. Our goal here was to address this issue, and investigate the magnitude and mechanism of siderophore-mediated competition in the presence of precipitated iron. We combined experimental work with thermodynamic modeling, using Pseudomonas aeruginosa as a model system and ferrihydrite precipitates as the iron source with low solubility. Our experiments show that competitive growth inhibition by the siderophore pyochelin is indeed efficient, and that inhibition of a competitor can even have a stronger growth-promoting effect than solubilization of precipitated iron. Based on the results of our thermodynamic models we conclude that the observed inhibition of a competitor is effective because sequestered iron is only very slowly replenished by the dissolution of precipitated iron. Our research highlights the importance of competitive benefits mediated by siderophores, and underlines that the dynamics of siderophore production and uptake in environmental communities could be a signature of competitive, not just cooperative, dynamics.

Coordination Chemistry of Microbial Iron Transport.

ConspectusThis Account focuses on the coordination chemistry of the microbial iron chelators called siderophores. The initial research (early 1970s) focused on simple analogs of siderophores, which included hydroxamate, catecholate, or hydroxycarboxylate ligands. The subsequent work increasingly focused on the transport of siderophores and their microbial iron transport. Since these are pseudo-octahedral complexes often composed of bidentate ligands, there is chirality at the metal center that in principle is independent of the ligand chirality. It has been shown in many cases that chiral recognition of the complex occurs. Many techniques have been used to elucidate the iron uptake processes in both Gram-positive (single membrane) and Gram-negative (double membrane) bacteria. These have included the use of radioactive labels (of ligand, metal, or both), kinetically inert metal complexes, and Mössbauer spectroscopy. In general, siderophore recognition and transport involves receptors that recognize the metal chelate portion of the iron–siderophore complex. A second, to date less commonly found, mechanism called the siderophore shuttle involves the receptor binding an apo-siderophore.Since one of the primary ways that microbes compete with each other for iron stores is the strength of their competing siderophore complexes, it became important early on to characterize the solution thermodynamics of these species. Since the acidity of siderophores varies significantly, just the stability constant does not give a direct measure of the relative competitive strength of binding. For this reason, the pM value is compared. The pM, like pH, is a measure of the negative log of the free metal ion concentration, typically calculated at pH 7.4, and standard total concentrations of metal and ligand. The characterization of the electronic structure of ferric siderophores has done much to help explain the high stability of these complexes.A new chapter in siderophore science has emerged with the characterization of what are now called siderocalins. Initially found as a protein of the human innate immune system, these proteins bind both ferric and apo-siderophores to inactivate the siderophore transport system and hence deny iron to an invading pathogenic microbe. Siderocalins also can play a role in iron transport of the host, particularly in the early stages of fetal development. Finally, it is speculated that the molecular targets of siderocalins in different species differ based on the siderophore structures of the most important bacterial pathogens of those species.

Enhanced uptake and translocation of arsenic in Cretan brake fern (Pteris cretica L.) through siderophorearsenic complex formation with an aid of rhizospheric bacterial activity

Siderophores, produced by Pseudomonas aeruginosa, released slightly more Fe (53.6μmol) than that chelated by ethylenediaminetetraacetic acid (EDTA; i.e. 43.7μmol) in batch experiment using As-adsorbed ferrihydrite. More importantly, about 1.79μmol of As was found to be associated with siderophores in the aqueous phase due to siderophore–As complex formation when siderophores were used to release As from ferrihydrite. In contrast, As was not detected in the aqueous phase when EDTA was used, probably due to the readsorption of released As to ferrihydrite. A series of pot experiment was conducted to investigate the effect of siderophores as a microbial iron-chelator on As uptake by Cretan brake fern (Pteris cretica L.) during phtoextraction. Results revealed that P. cretica, a known As hyperaccumulator, grown in the siderophore-amended soil showed about 3.7 times higher As uptake (5.62mg-Asg−1-plant) than the plant grown in the EDTA-treated soil (1.51mg-Asg−1-plant). In addition, As taken up by roots of P. cretica in the presence of siderophores seemed to be favorably translocated to shoots (i.e. stems and leaves). About 79% of the accumulated As was detected in the shoots in the presence of siderophores after ten weeks. Fluorescence microscopic analysis confirmed that As in the roots was delivered to the leaves of P. cretica as a siderophore–As complex.

Exploiting Bacterial Iron Acquisition: Siderophore Conjugates

Siderophores are chelators synthesized by bacteria and fungi to sequester iron, which is essential for virulence and pathogenicity. Since the process involves active transport, which is highly regulated, remarkably efficient and often microbially selective, it has been exploited as a Trojan Horse method for development of microbe-selective antibiotics. Siderophores also have significant potential for the development of imaging contrast agents and diagnostics for pathogen-selective detection. These promising results demonstrate the versatility of natural and synthetic microbial iron chelators and their potential therapeutic applications.

Inactivation of tesA Reduces Cell Wall Lipid Production and Increases Drug Susceptibility in Mycobacteria

Phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs) are structurally related lipids noncovalently bound to the outer cell wall layer of Mycobacterium tuberculosis, Mycobacterium leprae, and several opportunistic mycobacterial human pathogens. PDIMs and PGLs are important effectors of virulence. Elucidation of the biosynthesis of these complex lipids will not only expand our understanding of mycobacterial cell wall biosynthesis, but it may also illuminate potential routes to novel therapeutics against mycobacterial infections. We report the construction of an in-frame deletion mutant of tesA (encoding a type II thioesterase) in the opportunistic human pathogen Mycobacterium marinum and the characterization of this mutant and its corresponding complemented strain control in terms of PDIM and PGL production. The growth and antibiotic susceptibility of these strains were also probed and compared with the parental wild-type strain. We show that deletion of tesA leads to a mutant that produces only traces of PDIMs and PGLs, has a slight growth yield increase and displays a substantial hypersusceptibility to several antibiotics. We also provide a robust model for the three-dimensional structure of M. marinum TesA (TesAmm) and demonstrate that a Ser-to-Ala substitution in the predicted catalytic Ser of TesAmm renders a mutant that recapitulates the phenotype of the tesA deletion mutant. Overall, our studies demonstrate a critical role for tesA in mycobacterial biology, advance our understanding of the biosynthesis of an important group of polyketide synthase-derived mycobacterial lipids, and suggest that drugs aimed at blocking PDIM and/or PGL production might synergize with antibiotic therapy in the control of mycobacterial infections.

Design, synthesis, and study of a mycobactin-artemisinin conjugate that has selective and potent activity against tuberculosis and malaria.

Although the antimalarial agent artemisinin itself is not active against tuberculosis, conjugation to a mycobacterial-specific siderophore (microbial iron chelator) analogue induces significant and selective antituberculosis activity, including activity against multi- and extensively drug-resistant strains of Mycobacterium tuberculosis. The conjugate also retains potent antimalarial activity. Physicochemical and whole-cell studies indicated that ferric-to-ferrous reduction of the iron complex of the conjugate initiates the expected bactericidal Fenton-type radical chemistry on the artemisinin component. Thus, this "Trojan horse" approach demonstrates that new pathogen-selective therapeutic agents in which the iron component of the delivery vehicle also participates in triggering the antibiotic activity can be generated. The result is that one appropriate conjugate has potent and selective activity against two of the most deadly diseases in the world.

Growth and siderophores production in Alcaligenes faecalis is regulated by metal ions

During stationary phase of growth under low stress of iron in succinic acid medium, Alcaligenes feacalis BCCM ID 2374 produced microbial iron chelators. Increase in iron concentration supported bacterial growth but suppressed siderophores production, 1 μM and 2 μM of iron was optimum for maximum siderophore yield, i.e. 354 and 360 μg/ml in untreated and deferrated medium, respectively. Threshold level of iron, which suppressed siderophores production in A. feacalis BCCM ID 2374, was 20 μM. Ten micromoles and above concentration of CuCl(2) and CoCl(2), and 20 μM of MgCl(2), MgSO(4), ZnCl(2) and ZnSO(4) severely affected siderophores production.

Vibriobactin Antibodies: A Vaccine Strategy

A new target strategy in the development of bacterial vaccines, the induction of antibodies to microbial outer membrane ferrisiderophore complexes, is explored. A vibriobactin (VIB) analogue, with a thiol tether, 1-(2,3-dihydroxybenzoyl)-5,9-bis[[(4S,5R)-2-(2,3-dihydroxyphenyl)-4,5-dihydro-5-methyl-4-oxazolyl]carbonyl]-14-(3-mercaptopropanoyl)-1,5,9,14-tetraazatetradecane, was synthesized and linked to ovalbumin (OVA) and bovine serum albumin (BSA). The antigenicity of the VIB microbial iron chelator conjugates and their iron complexes was evaluated. When mice were immunized with the resulting OVA-VIB conjugate, a selective and unequivocal antigenic response to the VIB hapten was observed; IgG monoclonal antibodies specific to the vibriobactin fragment of the BSA and OVA conjugates were isolated. The results are consistent with the idea that the isolated adducts of siderophores covalently linked to their bacterial outer membrane receptors represent a credible target for vaccine development.

Opposite Effects With and Without Iron

The lipocalin 24p3 binds to siderophores (microbial iron chelators) that bind iron and through this interaction can deliver iron to cells. Devireddy et al. identified the 24p3 receptor (24p3R), which resembles a 12-transmembrane cation transporter. Hela cells engineered to express 24p3R bound 24p3 and internalized it. When the cells were exposed to holo24p3 (complexed with iron-bound siderophore), intracellular iron concentrations were increased. When cells were exposed to apo24p3, which lacked iron, the intracellular concentration of iron decreased. When cells expressing 24p3R were labeled with radioactive iron, addition of apo24p3 resulted in the accumulation of iron in the culture medium that was coprecipitated with antibodies against 24p3. Thus, apo24p3 bound iron (presumably bound to an endogenous siderophore) and transported the iron out of the cell upon recycling of the receptor-holo24p3 complex. Consistent with transit through the recycling endosome, accumulation of labeled iron in the medium was blocked in cells expressing mutant versions of endosomal proteins (Rab11 and Rififylin) that inhibit recycling. 24p3 was released from cells regardless of whether the cells expressed the receptor. However, only 24p3 from the conditioned medium of cells expressing 24p3R was competent to bind radiolabeled iron, suggesting that newly synthesized 24p3 is not bound to an iron-binding protein but acquires this iron-binding protein after being internalized. Addition of apo24p3 to cells expressing the receptor resulted in increased abundance of the proapoptotic protein Bim and apoptosis, whereas holo24p3 did not induce apoptosis. Concomitant addition of transferrin to replenish the diminished cellular iron blocked apo24p3-induced apoptosis, indicating that apoptosis was the result of the decreased iron concentration. Finally, the authors show a connection between 24p3 and cancer caused by the BCR-ABL oncoprotein. In BCR-ABL cells, expression of 24p3R is repressed and the cells are refractory to the apoptotic effects of 24p3, despite showing an increase in 24p3 production. The chemotherapeutic agent imatinib, which inhibits BCR-ABL, inhibited the expression of the gene encoding 23p4 and increased the expression of the gene encoding 24p3R, thus resensitizing the cells to 23p4. The importance of iron in the apoptotic response of BCR-ABL to imatinib was confirmed by rescue of cells from imatinib-induced apoptosis by addition of holotransferrin. Thus, the 24p3 pathway appears to play a key role in iron homeostasis, and conditions that stimulate the production of apo24p3 result in loss of cellular iron and stimulation of apoptosis through increased production of proapoptotic Bim (see Richardson for commentary). L. R. Devireddy, C. Gazin, X. Zhu, M. R. Green, A cell-surface receptor for lipocalin 24p3 selectively mediates apoptosis and iron uptake. Cell 123 , 1293-1305 (2005). [PubMed] D. R. Richardson, 24p3 and its receptor: Dawn of a new iron age? Cell 123 , 1175-1177 (2005). [PubMed]

The determination of ferric iron in plants by HPLC using the microbial iron chelator desferrioxamine E

Common methods for plant iron determination are based on atomic absorption spectroscopy, radioactive measurements or extraction with subsequent spectrophotometry. However, accuracy is often a problem due to background, contamination and interfering compounds. We here describe a novel method for the easy determination of ferric iron in plants by chelation with a highly effective microbial siderophore and separation by high performance liquid chromatography (HPLC). After addition of colourless desferrioxamine E (DFE) to plant fluids, the soluble iron is trapped as a brown-red ferrioxamine E (FoxE) complex which is subsequently separated by HPLC on a reversed phase column. The formed FoxE complex can be identified due to its ligand-to-metal charge transfer band at 435 nm. Alternatively, elution of both, DFE and FoxE can be followed as separate peaks at 220 nm wavelength with characteristic retention times. The extraordinarily high stability constant of DFE with ferric iron of K = 10(32) enables extraction of iron from a variety of ferrous and ferric iron compounds and allows quantitation after separation by HPLC without interference by coloured by-products. Thus, iron bound to protein, amino acids, citrate and other organic acid ligands and even insoluble ferric hydroxides and phosphates can be solubilized in the presence desferrioxamine E. The "Ferrioxamine E method" can be applied to all kinds of plant fluids (apoplasmic, xylem, phloem, intracellular) either at physiological pH or even at acid pH values. The FoxE complex is stable down to pH 1 allowing protein removal by perchloric acid treatment and HPLC separation in the presence of trifluoroacetic acid containing eluents.

Studies on Effect of Analogs of Microbial Iron Chelators on Candida albican

Studies on Effect of Analogs of Microbial Iron Chelators on Candida albican

Microbial siderophore-mediated transport.

Microbial iron chelates, called siderophores, are synthesized by bacteria and fungi in response to low iron availability in the environment. The present review summarizes structural details of siderophore ligands with respect to their transport properties. This presentation is largely centred on the occurrence and function of siderophores in the various bacterial and fungal genera.

Siderophoregenic Bradyrhizobia boost yield of soybean.

After screening for siderophore (microbial iron chelator) production, of seven available cultures of soybean (Glycine max L.) root nodule bradyrhizobia, one strain, Bradyrhizobium japonicum NCIM 2746, was selected to confirm its phytopathogenic suppression and soybean growth promotion. Based on chromatographic and spectrophotometric studies, two different siderophores, a hydroxamate type (MW 734) and another catecholate type (MW 1000), were observed. Randomized block design (RBD) analysis of sick-pot studies (soil inoculated with phytopathogens) with an MACS 124 variety of soybean, bacterized with siderophoregenic B. japonicum, showed a marked increase in the percentage of germination, nodulation, chlorophyll, oil, protein content, and number of pods. Field trial study confirmed these pot results, which were evident from enhancement in shoot length, number of branches, chlorophyll content, number of nodules, root length, and number of pods. These results suggest the possibility of exploiting B. japonicum NCIM 2746 as a potential bioinoculant.

Significance of asymmetric sites in choosing siderophores as deferration agents.

The syntheses of the microbial iron chelators L-fluviabactin, its unnatural enantiomer, D-fluviabactin, L-homofluviabactin, and L-agrobactin, are described. The key steps involve the selective bis-acylation of the terminal nitrogens of norspermidine, spermidine, or homospermidine with 2,3-bis(benzyloxy)benzoic acid in the presence of 1,1-carbonyldiimidazole, followed by coupling of the N-hydroxysuccinimide ester of CBZ-protected L- or D-threonine with the central nitrogen. The effectiveness of each of these ligands in supporting the growth of Paracoccus denitrificans in a low-iron environment and the ability of these compounds to promote iron uptake are evaluated. The stereochemical configuration of the oxazoline ring is shown to be the major structural factor controlling both microbial growth stimulation and iron uptake. L-Fluviabactin, L-homofluviabactin, and L-agrobactin all promoted growth and iron uptake; D-fluviabactin was only marginally active. As with the microorganism's native siderophore, L-parabactin, all three ligands in the L-configuration investigated exhibited biphasic, i.e., both high-affinity and low-affinity, kinetics. The high-affinity system (iron concentration < 1 microM) yielded K(m) values between 0.11 and 0.23 microM and V(max) values from 157 to 129 pg-atoms Fe min(-1) (mg of protein)(-1), whereas the low-affinity scheme (iron concentration > 1 microM) gave K(m) values from 0.53 to 3.5 microM and V(max) values between 96 and 413 pg-atoms Fe min(-1) (mg of protein)(-1). Both L- and D-fluviabactin are very effective at clearing iron from the bile duct-cannulated rodent; when given subcutaneously at a dose of 150 micromol/kg, both ligands had iron clearing efficiencies of >13%, which is much greater than that of desferrioxamine in this model. Thus, by altering the stereochemistry of certain microbial siderophores, it is possible to generate deferration agents that are still effective at clearing iron from animals, yet do not promote microbial growth.

Studies and syntheses of siderophores, microbial iron chelators, and analogs as potential drug delivery agents.

Siderophores (microbial iron chelators) play an extremely important role in microbial pathogenicity. Microbial uptake of siderophore-iron complexes through active transport systems allow microbes to survive and proliferate even under iron deficient environments during invasion of a host. Due to their structural complexity, unique iron (III) chelation, acquisition properties, and their therapeutic potential, siderophores have attracted much attention in a broad range of disciplines. Tremendous progress has been made in siderophore syntheses, in determination of the structures and functions of outer membrane receptors (e.g. FhuA and FepA), and in the mechanistic insight into siderophore-iron-mediated active transport processes. One of the important practical applications of this active transport system is development of species-selective active drug transport (the Trojan Horse approach) to potentially treat infections due to drug resistant strains of microbes. Siderophore-drug conjugates have shown great potential in active drug delivery to target pathogenic microbes.

The importance of siderophores in iron nutrition of heterotrophic marine bacteria

Recent studies demonstrate that dissolved iron in seawater is bound to strong organic complexes that have stability constants comparable to those of microbial iron chelates. We examined iron acquisition by seven strains of heterotrophic marine bacteria from a number of siderophore–iron complexes, including desferrioxamine B (DFB) and marine siderophores partially purified from iron‐limited cultures. Hydroxamate siderophores were detected in the supernatants of four strains, one of which also produced a catechol. All strains transported iron bound to siderophores regardless of whether or not they produced their own, and the majority took up iron bound to DFB. Uptake rates of Fe siderophores were similar among iron‐limited strains and among ligands. Transport of FeDFB by strain Neptune was enhanced 20 times by iron limitation, whereas uptake of unchelated iron (Fe') did not saturate at the highest concentration tested and was not regulated by the iron nutritional status of the cells. The half‐saturation constant for uptake of FeDFB by Neptune was 15 nM, the lowest reported for an Fe siderophore in any microor ganism. Iron uptake by the catechol‐producing strain, LMG1, differed markedly in two respects from the other strains: LMG1 could not take up iron bound to DFB; furthermore, transport of Fe' by iron‐limited LMG1 was 10 times faster than the other strains and was upregulated 46 times compared to Fe‐sufficient cells. Experimental evidence suggests that iron transport by LMG1 may be mediated by surface‐associated catechol siderophores that scavenge inorganic ferric species as well as iron bound to weaker complexes, such as EDTA (ethylenediaminetetraacetic acid). The combined results of the study highlight the importance of siderophores in iron transport by heterotrophic marine bacteria and suggest, by inference, that bacteria may rely on siderophores to acquire iron in situ.

Synthesis of Fragments of the Peptide Component of Pseudobactin

Pseudobactin is a structurally complex and physiologically important siderophore (microbial iron chelator) from Pseudomonas putida- fluorescens. Various fragments of the unusual peptide component of pseudobactin listed below were prepared by solution-phase peptide synthesis. L-Lys· D-threo-β-OH Asp· L-Ala· D-allo-Thr· L-Ala L-Lys· D-threo-βOH Asp· L-Ala· D-allo-Thr D-threo-β-OH Asp· L-Ala· D- allo-Thr· L-Ala· D-N-OH-cycloOrn D-threo-β-OH-Asp· L-Ala· D-allo-Thr· L-Ala L-Ala· D-allo-Thr· L- Ala· D-N-OH-cycloOrn A class of related peptides named pseudomycins have shown promising antifungal activity. To examine if these peptide fragments above would elicit similar activity, the fragments were tested and found to have no antifungal activity in limited bioassays.

Synthesis of fragments of the peptide component of pseudobactin.

Pseudobactin is a structurally complex and physiologically important siderophore (microbial iron chelator] from Pseudomonas putida-fluorescens. Various fragments of the unusual peptide component of pseudobactin listed below were prepared by solution-phase peptide synthesis. L-Lys.D-threo-beta-OH Asp.L-Ala.D-allo-Thr.L-Ala L-Lys.D-threo-beta OH Asp.L-Ala.D-allo-Thr D-threo-beta-OH Asp.L-Ala.D-allo-Thr.L-Ala.D-N-OH-cycloOrn D-threo-beta-OH-Asp.L-Ala.D-allo-Thr.L-Ala L-Ala.D-allo-Thr.L-Ala.D-N-OH-cycloOrn A class of related peptides named pseudomycins have shown promising antifungal activity. To examine if these peptide fragments above would elicit similar activity, the fragments were tested and found to have no antifungal activity in limited bioassays.

Framework-Reactive Siderophore Analogs as Potential Cell-Selective Drugs. Design and Syntheses of Trimelamol-Based Iron Chelators.

Currently, the role of DNA-directed alkylating agents as potential anticancer/ antimicrobial drugs is of wide interest. Most of the alkylating agents used clinically as drugs damage DNA in cells without specificity, and this can lead to undesired toxicity problems. Minimizing serum residence time by targeting the drug to select pathogens or organs might diminish the effects of nonselective reactivity. This paper describes the syntheses and preliminary studies of analogs of siderophores (microbial iron chelators) 2 and 20 that incorporate centers within the siderophore framework capable of generating potent electrophiles (iminium ions), hopefully after directed cellular recognition and uptake. Formation of N-aminals from trimelamol (3) and substituted hydroxamic acid 4 or 5was critical for the design and synthesis of the targets. In preliminary biological testing, compound 2, a trimelamol-based siderophore analog, was active against Escherichia coli X580, illustrating the therapeutic potential of this new type of siderophore-mediated drug design and delivery.