Optimization of Artificial Siderophores as 68Ga-Complexed PET Tracers for In Vivo Imaging of Bacterial Infections.

The diagnosis of bacterial infections at deep body sites benefits from noninvasive imaging of molecular probes that can be traced by positron emission tomography (PET). We specifically labeled bacteria by targeting their iron transport system with artificial siderophores. The cyclen-based probes contain different binding sites for iron and the PET nuclide gallium-68. A panel of 11 siderophores with different iron coordination numbers and geometries was synthesized in up to 8 steps, and candidates with the best siderophore potential were selected by a growth recovery assay. The probes [68Ga]7 and [68Ga]15 were found to be suitable for PET imaging based on their radiochemical yield, radiochemical purity, and complex stability in vitro and in vivo. Both showed significant uptake in mice infected with Escherichia coli and were able to discern infection from lipopolysaccharide-triggered, sterile inflammation. The study qualifies cyclen-based artificial siderophores as readily accessible scaffolds for the in vivo imaging of bacteria.

Deciphering the interplay between the genotoxic and probiotic activities of Escherichia coli Nissle 1917.

Although Escherichia coli Nissle 1917 (EcN) has been used therapeutically for over a century, the determinants of its probiotic properties remain elusive. EcN produces two siderophore-microcins (Mcc) responsible for an antagonistic activity against other Enterobacteriaceae. EcN also synthesizes the genotoxin colibactin encoded by the pks island. Colibactin is a virulence factor and a putative pro-carcinogenic compound. Therefore, we aimed to decouple the antagonistic activity of EcN from its genotoxic activity. We demonstrated that the pks-encoded ClbP, the peptidase that activates colibactin, is required for the antagonistic activity of EcN. The analysis of a series of ClbP mutants revealed that this activity is linked to the transmembrane helices of ClbP and not the periplasmic peptidase domain, indicating the transmembrane domain is involved in some aspect of Mcc biosynthesis or secretion. A single amino acid substitution in ClbP inactivates the genotoxic activity but maintains the antagonistic activity. In an in vivo salmonellosis model, this point mutant reduced the clinical signs and the fecal shedding of Salmonella similarly to the wild type strain, whereas the clbP deletion mutant could neither protect nor outcompete the pathogen. The ClbP-dependent antibacterial effect was also observed in vitro with other E. coli strains that carry both a truncated form of the Mcc gene cluster and the pks island. In such strains, siderophore-Mcc synthesis also required the glucosyltransferase IroB involved in salmochelin production. This interplay between colibactin, salmochelin, and siderophore-Mcc biosynthetic pathways suggests that these genomic islands were co-selected and played a role in the evolution of E. coli from phylogroup B2. This co-evolution observed in EcN illustrates the fine margin between pathogenicity and probiotic activity, and the need to address both the effectiveness and safety of probiotics. Decoupling the antagonistic from the genotoxic activity by specifically inactivating ClbP peptidase domain opens the way to the safe use of EcN.

Nocardamin glucuronide, a new member of the ferrioxamine siderophores isolated from the ascamycin-producing strain Streptomyces sp. 80H647.

A new siderophore glucuronide, nocardamin glucuronide (1), was isolated together with nocardamin (2) by applying the one strain-many compounds (OSMAC) approach to the ascamycin-producing strain, Streptomyces sp. 80H647, and performing multivariate analysis using mass spectral data. Structure elucidation was accomplished by a combination of NMR and MS analyses. The absolute configuration of the glucuronic acid moiety was found to be ß-D-GlcA by hydrolysis using ß-glucuronidase, subsequent derivatization of the hydrolysate, and comparison with standards. The siderophore activity of 1 was evaluated through the chrome azurol S assay and was comparable to that of 2 and deferoxamine (IC50 13.4, 9.5, and 6.3 µM, respectively). Nocardamin glucuronide (1) is the first example of a siderophore glucuronide.

Deferoxamine-induced electronegative ERG responses.

PURPOSE: To report a case of deferoxamine-induced retinopathy characterized by electroretinography (ERG), optical coherence tomography angiography (OCT-A), and other multimodal imaging. METHODS: This is an observational case report of one patient. Full-field ERG was performed. OCT-A, spectral-domain optical coherence tomography (SD-OCT), color fundus photography, and fundus autofluorescence were used to characterize the retinopathy induced by deferoxamine use. RESULTS: A 64-year-old man with a history of ß-thalassemia intermedia presented with worsening visual acuity, nyctalopia, and electronegative ERG. OCT-A revealed atrophy of the choriocapillaris in areas of hypoautofluorescence, corresponding to regions of retinal atrophy. SD-OCT showed disruption of the ellipsoid zone, granular hyperreflective deposits within the retinal pigment epithelium, thinning of the retinal layers, and extensive choroidal sclerosis and atrophy of the retinal pigment epithelium. CONCLUSION: Deferoxamine-induced retinopathy can manifest with electronegative maximal ERG responses, and OCT-A can be used to detect deferoxamine toxicity.

Effect of Pseudomonas fluorescens and pyoverdine on the phytoextraction of cesium by red clover in soil pots and hydroponics.

With the aim of improving the phytoextraction rate of cesium (Cs), the effect of Pseudomonas fluorescens ATCC 17400 and its siderophore pyoverdine (PVD) on the uptake of Cs by red clover was studied in soil pots. This work also provides a mechanistic understanding of the Cs-bacteria (or PVD)-illite-plant interactions by using a simplified experimental design, i.e., hydroponics with either Cs in solution or Cs-spiked illite in suspension. For soil spiked with 11.2 mmol kg-1 (1480 mg kg-1) of Cs, 0.43% of total Cs was taken up by red clover in 12 days (119 µmol g-1 (16 mg g-1) of Cs dry matter in roots and 40 µmol g-1 (5 mg g-1) in shoots). In hydroponics with Cs in solution (0.1 mmol L-1 or 13 mg L-1), 75% of Cs was taken up vs. only 0.86% with Cs-spiked illite suspension. P. fluorescens and PVD did not increase Cs concentrations in aboveground parts and roots of red clover and even decreased them. The damaging effect of PVD on red clover growth was demonstrated with the biomass yielding 66% of the control in soil pots (and 100% mortality after 12 days of exposition) and only 56% in hydroponics (78% with illite in suspension). Nonetheless, PVD and, to a lesser extent, P. fluorescens increased the translocation factor up to a factor of 2.8. This study clearly showed a direct damaging effect of PVD and to a lower extent the retention of Cs by biofilm covering both the roots and illite, both resulting in the lower phytoextraction efficiency.

Safety and pharmacokinetics of the oral iron chelator SP-420 in ß-thalassemia.

Our phase I, open-label, multi-center, dose-escalation study evaluated the pharmacokinetics (PK) of SP-420, a tridentate oral iron chelating agent of the desferrithiocin class, in patients with transfusion dependent ß-thalassemia. SP-420 was administered as a single dose of 1.5 (n = 3), 3 (n = 3), 6 (n = 3), 12 (n = 3), and 24 (n = 6) mg/kg or as a twice-daily dose of 9 mg/kg (n = 6) over 14-28 days. There was a near dose-linear increase in the mean plasma SP-420 concentrations and in the mean values for Cmax and AUC0-τ over the dose range evaluated. The median tmax ranged from 0.5 to 2.25 h and was not dose dependent. The study was prematurely terminated by the sponsor due to renal adverse events (AE) including proteinuria, increase in serum creatinine, and one case of Fanconi syndrome. Other adverse effects included hypersensitivity reactions and gastrointestinal disturbances. Based on current dose administration, the renal AE observed outweighed the possible benefits from chelation therapy. However, additional studies assessing efficacy and safety of lower doses or less frequent dosing of SP-420 over longer durations with close monitoring would be necessary to better explain the findings of our study and characterize the safety of the study drug.

Inhibition of P-glycoprotein stimulates cell death under Hypoxia-mimicking conditions.

The most common drug resistance mechanism in tumor cells is expression on their surface of the energy-dependent pump like P-glycoprotein (P-gp) that expels chemotherapeutic agents from the interior. An imitation of the hypoxic condition by the iron chelator deferoxamine caused Hypoxia-inducible factor 1-alpha (HIF-1α) stabilization and inhibition of doxorubicin-induced apoptosis in colon cancer НСТ116 cells. P-gp blocker verapamil suppressed doxorubicin accumulation leading to cell death induction. Considering these results, P-gp may be used as a potential target to stimulate chemotherapeutic drugs activity that will contribute to more efficient tumor cells elimination.

Mitophagy confers resistance to siderophore-mediated killing by Pseudomonas aeruginosa.

In the arms race of bacterial pathogenesis, bacteria produce an array of toxins and virulence factors that disrupt core host processes. Hosts mitigate the ensuing damage by responding with immune countermeasures. The iron-binding siderophore pyoverdin is a key virulence mediator of the human pathogen Pseudomonas aeruginosa, but its pathogenic mechanism has not been established. Here we demonstrate that pyoverdin enters Caenorhabditis elegans and that it is sufficient to mediate host killing. Moreover, we show that iron chelation disrupts mitochondrial homeostasis and triggers mitophagy both in C. elegans and mammalian cells. Finally, we show that mitophagy provides protection both against the extracellular pathogen P. aeruginosa and to treatment with a xenobiotic chelator, phenanthroline, in C. elegans. Although autophagic machinery has been shown to target intracellular bacteria for degradation (a process known as xenophagy), our report establishes a role for authentic mitochondrial autophagy in the innate immune defense against P. aeruginosa.

Deferoxamine promotes MDA-MB-231 cell migration and invasion through increased ROS-dependent HIF-1α accumulation.

BACKGROUND/AIM: Deferoxamine (DFO), an iron chelator, has been reported to induce hypoxia-inducible factor-1α (HIF-1α) expression. HIF-1α plays a critical role in promoting tumor metastasis. However, the molecular mechanisms underlying induction of HIF-1α in breast cancer cells remain unknown. Our aim was to ascertain whether DFO enhanced cancer metastasis in MDA-MB-231 cells. METHODS: Cellular reactive oxygen species (ROS) was measured by flow cytometry. Cell migration was determined by wound healing and transwell assays. Protein and mRNA expression were detected by western blotting and RT-PCR, respectively. RESULTS: DFO treatment enhanced cell migration and invasion, while HIF-1α expression was significantly up-regulated at the post-transcriptional level. However, treatment with a NADPH oxidase inhibitor, diphenyleneiodonium (DPI), strongly inhibited ROS generation and HIF-1α expression, as well as cell migration and invasion. Notably, DFO treatment increased extracellular signal-regulated kinase (ERK)1/2 phosphorylation. Inhibition of ROS production with DPI attenuated DFO-induced ERK1/2 activation. Moreover, a MEK1 inhibitor, PD98059, suppressed DFO-induced cell migration and invasion. CONCLUSION: DFO-induced HIF-1α expression involves a cascade of signaling events including ROS generation, activation of ERK signaling, and subsequent promotion of cell migration and invasion. These findings indicate a risk associated with DFO and other iron chelators for treatment of tumors with invasive potential.

Adipokine expression in brown and white adipocytes in response to hypoxia.

BACKGROUND: Adipose tissue has emerged as an important endocrine regulator by secreting hormones referred to as adipokines. Recent studies showed that adipose tissue considerably responds to hypoxia. Although the impact of white adipose tissue on regulative processes is established, the importance of brown adipose tissue in adults has emerged just recently. METHODS: Brown (BA) and white adipocytes (WA) were cultured either in the presence of chemical hypoxia-mimetics or under hypoxic atmosphere of 1% oxygen. Expression of hypoxia-inducible factor 1α (HIF- 1α) was assessed by western blot. The expression levels of several known HIF-1α-regulated proteins [vascular endothelial growth factor (VEGF), leptin, adiponectin, and angiotensinogen (AGT)] were quantified. RESULTS: Both chemical hypoxia-mimetics and physical hypoxia led to increased nuclear HIF-1α expression and to decreased cytoplasmatic adiponectin in both cell types. In contrast, VEGF and AGT expression did not change upon hypoxic stimulation. Leptin was exclusively detectable in WA, while uncoupling-protein 1 (UCP-1) was expressed in BA only. CONCLUSIONS: WA and BA are sensitive to hypoxia, in which HIF-1α expression is induced. Protein expression of adiponectin is hypoxia-dependent, whereas AGT, VEGF, leptin, and UCP-1 expression do not change secondary to hypoxia.

Leishmania chagasi: effect of the iron deficiency on the infection in BALB/c mice.

Iron deficiency and visceral leishmaniasis are serious problems of public health. The aim of this study was to evaluate the effect of iron deficiency, induced by the iron chelator desferrioxamine, on the course of the infection by Leishmania chagasi in BALB/c mice. Our data show that the iron chelator caused significant reduction in hemoglobin concentration of treated mice and reduction in parasite load in spleen and liver. Significant differences were not observed in the production of IFN-gamma and IL-4 among the experimental groups. In conclusion, the data reported in this paper suggest that iron deficiency may favor the host. If there is not enough iron available to the parasite, its multiplication may be reduced and infection attenuated.

Autophagy after experimental intracerebral hemorrhage.

Autophagy contributes to ischemic brain injury, but it is not clear if autophagy occurs after intracerebral hemorrhage (ICH). This study examined whether ICH-induced cell death is partly autophagic. It then examined the role of iron in inducing this form of cell death after ICH. Male, adult Sprague-Dawley rats received an infusion of autologous whole blood or ferrous iron into the right basal ganglia. Control rats (sham) had a needle insertion. The rats were killed at 1, 3, 7, or 28 days later. Some rats were treated with either deferoxamine or vehicle after ICH. Microtubule-associated protein light chain-3 (LC3), a biomarker of autophagosome, and cathepsin D, a lysosomal biomarker, were measured by Western blot analysis and immunohistochemistry. Immunofluorescent double-labeling was used to identify the cell types expressing cathepsin D. Electron microscopy was performed to examine the cellular ultrastructure changes after ICH. We found that conversion of LC3-I to LC3-II, cathepsin D expression, and vacuole formation are increased in the ipsilateral basal ganglia after ICH. Intracerebral infusion of iron also resulted in enhanced conversion of LC3-I to LC3-II and increased cathepsin D levels. Deferoxamine (an iron chelator) treatment significantly reduced the conversion of LC3-I to LC3-II and cathepsin D levels after ICH. Our results demonstrated that autophagy occurs after ICH, and iron has a key role in ICH-induced autophagy. This also suggests that iron-induced autophagy may play a role in brain injury in other diseases associated with iron overload.

[Retinal pigment epithelium--desferal]. / Rétine et epithélium pigmentaire--le desferal.

Deferoxamine mesylate (Desferal) is a chelating agent used in hemosiderosis and aluminium overload consecutive to renal dialysis. This drug is the most efficacious for treating iron overload but is associated with ocular toxicity: dose and duration related symptomatic optic neuropathy on the one hand, reversible if treatment stopped, and acute retinal involvement followed by irreversible paucisymptomatic pigmentary changes on the other hand. Toxic risk factors are intravenous mode of administration, high doses, small iron or aluminium overload, diabetes and young age. Hence, dosis should be adapted to the amount of overload and ophthalmological follow-up should be instaured. Indeed, if treatment is stopped at the beginning of the toxic effect, ocular involvement is reversible. The baseline ophthalmological examination should include visual acuity measurement, color vision, visual fields, slit lamp and fundus. In case of risk factors, electrophysiology and fluoangiography should be added.

Desferrioxamine (DFX) has genotoxic effects on cultured human lymphocytes and induces the p53-mediated damage response.

Desferrioxamine (DFX), which is an iron chelator, mimics hypoxia by enhancing HIF1-alpha accumulation and upregulating inflammatory mediators. DFX is usually beneficial, with preventive effects related primarily to its ability to scavenge reactive oxygen species. However, toxic effects on skeletal and ocular organs have been reported. The cytokinesis block micronucleus test and alkaline single-cell gel (Comet) assay were used to evaluate the genotoxic effects of DFX on human blood lymphocytes. Cultured human lymphocytes treated with 130microM DFX for various periods of time showed significant differences in the incidence of micronucleated binucleate cells, as well as in the length and moment of the comet tail. Western blot analysis using antibodies to proteins involved in the p53-mediated response to DNA damage revealed that p53 was accumulated and DNA damage checkpoint kinases were activated in lymphocytes treated with DFX. On the other hand, the p53 downstream target proteins p21 and bax were not affected, which indicates that DFX does not promote the transactivational activity of p53. Apoptosis assays demonstrated DFX-induced apoptosis of lymphocytes via the caspase cascade. The observed increase in the sub-G1 fraction and enhanced caspase-3 activity indicate that DFX can promote apoptosis in human lymphocytes, and these results were confirmed by protein immunoblot analysis. As apoptotic cell death is preceded by the collapse of the mitochondrial membrane potential, we also measured the mitochondrial membrane potential (Deltapsi(m)) using DiOC6, which is a fluorescent membrane potential probe. The fluorescence intensity of DiOC6 in lymphocytes was significantly reduced in a time-dependent manner after DFX treatment. Taken together, these results indicate that DFX activates p53-mediated checkpoint signals and induces apoptosis via mitochondrial damage in human peripheral blood lymphocytes.

Effects of synthetic siderophores on proliferation of Plasmodium falciparum in infected human erythrocytes.

Because iron is essential for Plasmodium falciparum, we investigated the in vitro potential of various synthetic siderophores to kill P. falciparum in infected human erythrocytes. The substances with the most promising profile, i.e., low 50% lethal doses for plasmodia and minimum toxicity towards mammalian cells, were siderophores with an acylated monocatecholate or a triscatecholate as substituent.

Siderophore activity of chemically synthesized dihydroxybenzoyl derivatives of spermidines and cystamide.

Chemically synthesized dihydroxybenzoyl derivatives of spermidine and cystamide containing two-, three- and four-bidentates with the hydroxyl groups in 2,3 or 3,4 position were examined in cross-feeding tests using Gram-negative siderophore indicator strains carrying different iron-related markers, and two Mycobacterium spp. The catecholates were unable to feed tonB mutants of E. coli and S. typhimurium as well as the fepA, fiu, cir mutant of E. coli, pointing to a tonB- and fepA, cir, fiu-dependent transport. Bis(2,3-dihydroxybenzoyl)derivatives promoted Salmonella spp, E. coli, K. pneumoniae and P. aeruginosa strains significantly better than did 3,4-dihydroxybenzoyl derivatives. N4-substituted spermidines acted more effectively than non-substituted derivatives. Bis(2,3-dihydroxybenzoyl) cystamide was superior to the other catecholates tested in growth promotion of Gram-negative bacteria. The two four-bidentates and the tri-bidentate reacted to K. pneumoniae in an inhibitory mode. The position of the hydroxyl groups did not significantly influence the growth promotion of M. smegmatis and M. fortiutum in the cases of substituted spermidines and of cystamides.

Hemoglobin exacerbates the ocular inflammatory response to endotoxin.

BACKGROUND: There is a clinical impression that bleeding into sites of inflammation exacerbates the inflammatory response. It has been hypothesized that hemoglobinic iron (Fe) contributes to this response by catalyzing free radical reactions. In the present study, the effects of autologous hemoglobin on the inflammatory response to endotoxin was determined. In addition, the possible contributions of Fe to this response was assessed by co-injection of either transferrin or desferrioxamine. METHODS: A mild ocular inflammation was induced in rabbits by intravitreal injection of 0.25 ng endotoxin. In some animals apotransferrin, hemoglobin, hemoglobin + apotransferrin or hemoglobin + desferrioxamine were co-injected. Twenty-four hours later, anterior uveitis was quantified by slit-lamp examination and determination of protein concentration and infiltration of white cells into the aqueous humor. RESULTS: Co-injection of autologous hemoglobin with endotoxin greatly exacerbated the ocular inflammatory response to endotoxin, especially the infiltration of white cells, which was increased 15-fold. Both apotransferrin, which binds Fe at high affinity, and desferrioxamine, which chelates Fe, greatly decreased the cellular response to the co-injection. CONCLUSIONS: It is likely that hemoglobinic Fe is responsible for the increased infiltration of white cells caused by the co-injection of autologous hemaglobin and endotoxin.

[Pseudomonas fluorescens: production of pyoverdine in human blood at 4 degrees C and cytotoxic effect of the pigment]. / Pseudomonas fluorescens: producción de pioverdina en sangre humana a 4 grados C y efecto citotóxico del pigmento.

Pseudomonas fluorescens PAB strain produced pyoverdine in a synthetic medium, this pigment was purified by solvent extraction and ion exchange, then sterilized by filtration. Where cytotoxic effect on human leukocytes was assayed, death and lysis was observed. Sublytic doses decreased leukocytes phagocytosis and chemotaxis. Bacteria grew and produced pigment in blood stored a 4 degrees C, with a pyoverdine production of 0.13 mg/ml of serum after 5 days of incubation.

Production of the phytotoxic metabolite, ferricrocin, by the fungus Colletotrichum gloeosporioides.

A siderophore was isolated as a non-specific phytotoxic compound from a culture of Colletotrichum gloeosporioides isolated from infected blackberry. This siderophore was identified as ferricrocin by NMR, IR, MS, and CD spectra. The phytotoxic activities of ferricrocin and deferriferricrocin were compared.

An investigation of desferrithiocin metabolism.

The hydrolyses of (S)-desferrithiocin (DFT, 1), (R)-desmethyl-DFT (2), and (R)-desazadesmethyl-DFT (3) were studied at pH 2.5 and 7.2 in order to access the stability of the thiazolines at the pH of the stomach and the serum. At 37 degrees C and pH 2.5, DFT (1) (t1/2 = 18.6 h), desmethyl-DFT (2) (t1/2 = 8.74 h), and desazadesmethyl-DFT (3) (t1/2 = 31.7 h) were shown to open principally to the thiol amides with trace amounts of the corresponding thioesters, < or = 2%. The thiazolines were resistant to hydrolysis at pH 7.2. Iron(III) stabilized significantly the thiazolines in the complexes 16a/b of 3 in regard to hydrolysis at pH 2.5 (t1/2 > 20 days). The iron(III) complexes 16a/b were shown to be stable at pH 7.2. While the thiol amides 13 and 14 of 1 and 2 were isolated from the hydrolysis of the parent desferrithiocins, the thioester 4 and the thiol amide 5 of 3 were synthesized and their stability in aqueous solution, iron-clearance properties, and toxicity were evaluated. Thioester 4 was shown to rearrange to thiol amide 5 at pH 2.5 and 37 degrees C with a half-life of 4.18 h and instantaneously at pH 7.2. Thiol amide 5 is in equilibrium with 4 (5/4 = 49:1) at pH 2.5 and was shown to be stable at pH 7.2. Thioester 4 and thiol amide 5 demonstrated neither iron-clearance activity in iron-overloaded rats nor toxic side effects in mice. Hydrolysis products of the drug, which might be generated in the stomach, seem unlikely to be the source of the drug's toxicity or iron-clearing properties.