Cryptic specialized metabolites drive Streptomyces exploration and provide a competitive advantage during growth with other microbes.

Streptomyces bacteria have a complex life cycle that is intricately linked with their remarkable metabolic capabilities. Exploration is a recently discovered developmental innovation of these bacteria, that involves the rapid expansion of a structured colony on solid surfaces. Nutrient availability impacts exploration dynamics, and we have found that glycerol can dramatically increase exploration rates and alter the metabolic output of exploring colonies. We show here that glycerol-mediated growth acceleration is accompanied by distinct transcriptional signatures and by the activation of otherwise cryptic metabolites including the orange-pigmented coproporphyrin, the antibiotic chloramphenicol, and the uncommon, alternative siderophore foroxymithine. Exploring cultures are also known to produce the well-characterized desferrioxamine siderophore. Mutational studies of single and double siderophore mutants revealed functional redundancy when strains were cultured on their own; however, loss of the alternative foroxymithine siderophore imposed a more profound fitness penalty than loss of desferrioxamine during coculture with the yeast Saccharomyces cerevisiae. Notably, the two siderophores displayed distinct localization patterns, with desferrioxamine being confined within the colony area, and foroxymithine diffusing well beyond the colony boundary. The relative fitness advantage conferred by the alternative foroxymithine siderophore was abolished when the siderophore piracy capabilities of S. cerevisiae were eliminated (S. cerevisiae encodes a ferrioxamine-specific transporter). Our work suggests that exploring Streptomyces colonies can engage in nutrient-targeted metabolic arms races, deploying alternative siderophores that allow them to successfully outcompete other microbes for the limited bioavailable iron during coculture.

Biosynthesis of novel desferrioxamine derivatives requires unprecedented crosstalk between separate NRPS-independent siderophore pathways.

Iron is essential to many biological processes but its poor solubility in aerobic environments restricts its bioavailability. To overcome this limitation, bacteria have evolved a variety of strategies, including the production and secretion of iron-chelating siderophores. Here, we describe the discovery of four series of siderophores from Streptomyces ambofaciens ATCC23877, three of which are unprecedented. MS/MS-based molecular networking revealed that one of these series corresponds to acylated desferrioxamines (acyl-DFOs) recently identified from S. coelicolor. The remaining sets include tetra- and penta-hydroxamate acyl-DFO derivatives, all of which incorporate a previously undescribed building block. Stable isotope labeling and gene deletion experiments provide evidence that biosynthesis of the acyl-DFO congeners requires unprecedented crosstalk between two separate non-ribosomal peptide synthetase (NRPS)-independent siderophore (NIS) pathways in the producing organism. Although the biological role(s) of these new derivatives remain to be elucidated, they may confer advantages in terms of metal chelation in the competitive soil environment due to the additional bidentate hydroxamic functional groups. The metabolites may also find application in various fields including biotechnology, bioremediation, and immuno-PET imaging.IMPORTANCEIron-chelating siderophores play important roles for their bacterial producers in the environment, but they have also found application in human medicine both in iron chelation therapy to prevent iron overload and in diagnostic imaging, as well as in biotechnology, including as agents for biocontrol of pathogens and bioremediation. In this study, we report the discovery of three novel series of related siderophores, whose biosynthesis depends on the interplay between two NRPS-independent (NIS) pathways in the producing organism S. ambofaciens-the first example to our knowledge of such functional cross-talk. We further reveal that two of these series correspond to acyl-desferrioxamines which incorporate four or five hydroxamate units. Although the biological importance of these novel derivatives is unknown, the increased chelating capacity of these metabolites may find utility in diagnostic imaging (for instance, 89Zr-based immuno-PET imaging) and other applications of metal chelators.

Chelating Effect of Siderophore Desferrioxamine-B on Uranyl Biomineralization Mediated by Shewanella putrefaciens.

In contaminated water and soil, little is known about the role and mechanism of the biometabolic molecule siderophore desferrioxamine-B (DFO) in the biogeochemical cycle of uranium due to complicated coordination and reaction networks. Here, a joint experimental and quantum chemical investigation is carried out to probe the biomineralization of uranyl (UO22+, referred to as U(VI) hereafter) induced by Shewanella putrefaciens (abbreviated as S. putrefaciens) in the presence of DFO and Fe3+ ion. The results show that the production of mineralized solids {hydrogen-uranium mica [H2(UO2)2(PO4)2·8H2O]} via S. putrefaciens binding with UO22+ is inhibited by DFO, which can both chelate preferentially UO22+ to form a U(VI)-DFO complex in solution and seize it from U(VI)-biominerals upon solvation. However, with Fe3+ ion introduced, the strong specificity of DFO binding with Fe3+ causes re-emergence of biomineralization of UO22+ {bassetite [Fe(UO2)2(PO4)2·8(H2O)]} by S. putrefaciens, owing to competitive complexation between Fe3+ and UO22+ for DFO. As DFO possesses three hydroxamic functional groups, it forms hexadentate coordination with Fe3+ and UO22+ ions via these functional groups. The stability of the Fe3+-DFO complex is much higher than that of U(VI)-DFO, resulting in some DFO-released UO22+ to be remobilized by S. putrefaciens. Our finding not only adds to the understanding of the fate of toxic U(VI)-containing substances in the environment and biogeochemical cycles in the future but also suggests the promising potential of utilizing functionalized DFO ligands for uranium processing.

FoxR is an AraC-like transcriptional regulator of ferrioxamine uptake in Salmonella enterica.

Salmonella enterica spp. produce siderophores to bind iron with high affinity and can also use three xenosiderophores secreted by other microorganisms, ferrichrome, coprogen, and ferrioxamine. Here we focused on FoxA, a TonB-dependent transporter of ferrioxamines. Adjacent to foxA is a gene annotated as a helix-turn-helix (HTH) domain-containing protein, SL0358 (foxR), in the Salmonella enterica serovar Typhimurium SL1344 genome. FoxR shares homology with transcriptional regulators belonging to the AraC/XylS family. foxR is syntenic with foxA in the Enterobacteriaceae family, suggesting their functional relatedness. Both foxA and foxR are repressed by the ferric uptake regulator (Fur) under iron-rich growth conditions. When iron is scarce, FoxR acts as a transcriptional activator of foxA by directly binding to its upstream regulatory region. A point mutation in the HTH domain of FoxR abolished this binding, as did mutation of a direct repeat motif in the foxA upstream regulatory region. Desferrioxamine (DFOE) enhanced FoxR protein stability and foxA transcription but did not affect the affinity of FoxR binding to the foxA regulatory region. In summary, we have identified FoxR as a new member of the AraC/XylS family that regulates xenosiderophore-mediated iron uptake by S. Typhimurium and likely other Enterobacteriaceae members.

Ferroptosis inhibitor improves cardiac function more effectively than inhibitors of apoptosis and necroptosis through cardiac mitochondrial protection in rats with iron-overloaded cardiomyopathy.

Iron overload cardiomyopathy (IOC) is the leading cause of death in cases of iron overload in patients. Previous studies demonstrated that iron overload led to cardiomyocyte dysfunction and death through multiple pathways including apoptosis, necroptosis and ferroptosis. However, the dominant cell death pathway in the iron-overloaded heart needs clarification. We tested the hypothesis that ferroptosis, an iron-dependent cell death, plays a dominant role in IOC, and ferroptosis inhibitor exerts greater efficacy than inhibitors of apoptosis and necroptosis on improving cardiac function in iron-overloaded rats. Iron dextran was injected intraperitoneally into male Wistar rats for four weeks to induce iron overload. Then, the rats were divided into 5 groups: treated with vehicle, apoptosis inhibitor (z-VAD-FMK), necroptosis inhibitor (Necrostatin-1), ferroptosis inhibitor (Ferrostatin-1) or iron chelator (deferoxamine) for 2 weeks. Cardiac function, mitochondrial function, apoptosis, necroptosis and ferroptosis were determined. The increased expression of apoptosis-, necroptosis- and ferroptosis-related proteins, were associated with impaired cardiac and mitochondrial function in iron-overloaded rats. All cell death inhibitors attenuated cardiac apoptosis, necroptosis and ferroptosis in iron-overloaded rats. Ferrostatin-1 was more effective than the other drugs in diminishing mitochondrial dysfunction and Bax/Bcl-2 ratio. Moreover, both Ferrostatin-1 and deferoxamine reversed iron overload-induced cardiac dysfunction as indicated by restored left ventricular ejection fraction and E/A ratio, whereas z-VAD-FMK and Necrostatin-1 only partially improved this parameter. These results indicated that ferroptosis could be the predominant form of cardiomyocyte death in IOC, and that inhibiting ferroptosis might be a potential novel treatment for IOC.

Amelioration effects of the soybean lecithin-gallic acid complex on iron-overload-induced oxidative stress and liver damage in C57BL/6J mice.

CONTEXT: Gallic acid (GA) and lecithin showed important roles in antioxidant and drug delivery, respectively. A complex synthesized from GA and soybean lecithin (SL-GAC), significantly improved bioavailability of GA and pharmacological activities. However, the antioxidant activity of SL-GAC and its effect on iron-overload-induced liver injury remains unexplored. OBJECTIVE: This study investigates the antioxidant properties of SL-GAC in vitro and in mice, and its remediating effects against liver injury by iron-overloaded. MATERIALS AND METHODS: In vitro, free radical scavenging activity, lipid peroxidation inhibition, and ferric reducing power of SL-GAC were measured by absorbance photometry. In vivo, C57BL/6J mice were randomized into 4 groups: control, iron-overloaded, iron-overloaded + deferoxamine, and iron-overloaded + SL-GAC. Treatments with deferoxamine (150 mg/kg/intraperitioneally) and SL-GAC (200 mg/kg/orally) were given to the desired groups for 12 weeks, daily. Iron levels, oxidative stress, and biochemical parameters were determined by histopathological examination and molecular biological techniques. RESULTS: In vitro, SL-GAC showed DPPH and ABTS free radicals scavenging activity with IC50 values equal to 24.92 and 128.36 µg/mL, respectively. In C57BL/6J mice, SL-GAC significantly reduced the levels of serum iron (22.82%), liver iron (50.29%), aspartate transaminase (25.97%), alanine transaminase (38.07%), gamma glutamyl transferase (42.11%), malondialdehyde (19.82%), total cholesterol (45.96%), triglyceride (34.90%), ferritin light chain (18.51%) and transferrin receptor (27.39%), while up-regulated the levels of superoxide dismutase (24.69%), and glutathione (11.91%). CONCLUSIONS: These findings encourage the use of SL-GAC to treat liver injury induced by iron-overloaded. Further in vivo and in vitro studies are needed to validate its potential in clinical medicine.

Iron overload-induced ferroptosis of osteoblasts inhibits osteogenesis and promotes osteoporosis: An in vitro and in vivo study.

Growing evidence indicates that iron overload is an independent risk factor for osteoporosis. However, the mechanisms are not fully understood. The purpose of our study was to determine whether iron overload could lead to ferroptosis in osteoblasts and to explore whether ferroptosis of osteoblasts is involved in iron overload-induced osteoporosis in vitro and in vivo. Ferric ammonium citrate was used to mimic iron overload conditions, while deferoxamine and ferrostatin-1 were used to inhibit ferroptosis of MC3T3-E1 cells in vitro. The ferroptosis, osteogenic differentiation and mineralization of MC3T3-E1 cells were assessed in vitro. A mouse iron overload model was established using iron dextran. Immunohistochemical analysis was performed to determine ferroptosis of osteoblasts in vivo. Enzyme-linked immunosorbent assays and calcein-alizarin red S labelling were used to assess new bone formation. Dual x-ray absorptiometry, micro-computed tomography and histopathological analysis were conducted to evaluate osteoporosis. The results showed that iron overload reduced cell viability, superoxide dismutase and glutathione levels, increased reactive oxygen species generation, lipid peroxidation, malondialdehyde levels and ferroptosis-related protein expression, and induced ultrastructural changes in mitochondria. Iron overload could also inhibit osteogenic differentiation and mineralization in vitro. Inhibiting ferroptosis reversed the changes described above. Iron overload inhibited osteogenesis, promoted the ferroptosis of osteoblasts and induced osteoporosis in vivo, which could also be improved by deferoxamine and ferrostatin-1. These results demonstrate that ferroptosis of osteoblasts plays a crucial role in iron overload-induced osteoporosis. Maintaining iron homeostasis and targeting ferroptosis of osteoblasts might be potential measures of treating or preventing iron overload-induced osteoporosis.

Norepinephrine induces growth of Desulfovibrio vulgaris in an iron dependent manner.

Desulfovibrio spp. is a commensal sulfate reducing bacterium that is present in small numbers in the gastrointestinal tract. Increased concentrations of Desulfovibrio spp. (blooms) have been reported in patients with inflammatory bowel disease and irritable bowel syndrome. Since stress has been reported to exacerbate symptoms of these chronic diseases, this study examined whether the stress catecholamine norepinephrine (NE) promotes Desulfovibrio growth. Norepinephrine-stimulated growth has been reported in other bacterial taxa, and this effect may depend on the availability of the micronutrient iron. OBJECTIVES: This study tested whether norepinephrine exposure affects the in vitro growth of Desulfovibrio vulgaris in an iron dependent manner. METHODS: DSV was incubated in a growth medium with and without 1 µm of norepinephrine. An additional growth assay added the iron chelator deferoxamine in NE exposed DSV. Iron regulatory genes were assessed with and without the treatment of NE and Deferoxamine. RESULTS: We found that norepinephrine significantly increased growth of D. vulgaris. Norepinephrine also increased bacterial production of hydrogen sulfide. Additionally, norepinephrine significantly increased bacterial expression in three of the four tested iron regulatory genes. The iron chelator deferoxamine inhibited growth of D. vulgaris in a dose-dependent manner and reversed the effect of norepinephrine on proliferation of D. vulgaris and on bacterial expression of iron regulatory genes. CONCLUSION: The data presented in this work suggests that promotion of D. vulgaris growth by norepinephrine is iron dependent.

Topical bilirubin-deferoxamine hastens excisional wound healing by modulating inflammation, oxidative stress, angiogenesis, and collagen deposition in diabetic rats.

AIM OF THE STUDY: The study was performed to understand the detailed mechanism of diabetic wound healing by bilirubin-deferoxamine (DFO) combination on topical application. MATERIALS AND METHODS: There were two study groups, control, and treatment. The granulation tissues collected on different days (3, 7, 14, and 19) were studied in detail for inflammatory mediators, angiogenesis markers, epithelialization, and oxidative stress parameters. RESULTS: A significant increase in wound contraction percentage was observed from day 7 in the bilirubin-DFO treatment group. The combinatorial treatment significantly reduced tumour necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß), and enhanced IL-10 levels. Upregulated mRNAs of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1 alpha (HIF-1 α) along with CD31 immunohistochemistry showed the pro-angiogenesis potential of the combination. Hematoxylin and Eosin (H and E) staining and Masson's trichrome staining showed reduced inflammatory cell infiltration, enhanced fibroblast proliferation, well-organized collagen fibers, and the development of new blood vessels. Collagen deposition is further supported by immunohistochemistry studies and Masson's trichrome staining. Bilirubin-DFO combination also reduced lipid peroxidation and elevated antioxidative enzymes. CONCLUSION: Topical application of bilirubin-DFO showed immense potential in augmenting skin wound regeneration in diabetes by upregulating the antioxidant status as well as increasing angiogenesis, collagen deposition, and modulating cytokines.

Deferoxamine ameliorated Al(mal)3-induced neuronal ferroptosis in adult rats by chelating brain iron to attenuate oxidative damage.

Aluminum (Al), a neurotoxic element, can induce Alzheimer's disease-like (AD-like) changes by triggering neuronal death. Iron homeostasis disturbance has also been implicated in Alzheimer's disease (AD), and excess iron exacerbates oxidative damage and cognitive defects. Ferroptosis is a nonapoptotic form of cell death dependent upon intracellular iron. However, the involvement of neuronal death induced by aluminum maltolate (Al(mal)3) in the pathogenesis of AD remains elusive. In this study, the results of three different behavioral experiments suggested that the learning and memory ability deteriorated and autonomous activity declined of these rats that exposed Al(mal)3 were alleviated by deferoxamine (DFO). Transmission electron microscope observations showed that the membrane was ruptured, and the membrane density increased and ridge disappearance (the most prominent characteristic of ferroptosis) in the perinuclear and cytoplasmic compartments of the hippocampal neurons were perceived in the exposure group, while the DFO group and 18 µM/kg Al(mal)3+DFO group were alleviated compared with 18 µM/kg Al(mal)3. In addition, DFO prevented oxidative stress, such as increased glutathione (GSH) and decreased malondialdehyde (MDA) and reactive oxygen species (ROS), while the latter two indexes had the same changing tendency as the total iron of brain tissue. These data indicated that Al(mal)3 could cause ferroptosis in Sprague-Dawley (SD) rat neurons, which was inhibited by DFO via reducing the content of iron and increasing the ability of cells to resist oxidative damage.

Biotechnological and Ecological Potential of Micromonospora provocatoris sp. nov., a Gifted Strain Isolated from the Challenger Deep of the Mariana Trench.

A Micromonospora strain, isolate MT25T, was recovered from a sediment collected from the Challenger Deep of the Mariana Trench using a selective isolation procedure. The isolate produced two major metabolites, n-acetylglutaminyl glutamine amide and desferrioxamine B, the chemical structures of which were determined using 1D and 2D-NMR, including 1H-15N HSQC and 1H-15N HMBC 2D-NMR, as well as high resolution MS. A whole genome sequence of the strain showed the presence of ten natural product-biosynthetic gene clusters, including one responsible for the biosynthesis of desferrioxamine B. Whilst 16S rRNA gene sequence analyses showed that the isolate was most closely related to the type strain of Micromonospora chalcea, a whole genome sequence analysis revealed it to be most closely related to Micromonospora tulbaghiae 45142T. The two strains were distinguished using a combination of genomic and phenotypic features. Based on these data, it is proposed that strain MT25T (NCIMB 15245T, TISTR 2834T) be classified as Micromonospora provocatoris sp. nov. Analysis of the genome sequence of strain MT25T (genome size 6.1 Mbp) revealed genes predicted to responsible for its adaptation to extreme environmental conditions that prevail in deep-sea sediments.

Coupling MALDI-TOF mass spectrometry protein and specialized metabolite analyses to rapidly discriminate bacterial function.

For decades, researchers have lacked the ability to rapidly correlate microbial identity with bacterial metabolism. Since specialized metabolites are critical to bacterial function and survival in the environment, we designed a data acquisition and bioinformatics technique (IDBac) that utilizes in situ matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze protein and specialized metabolite spectra recorded from single bacterial colonies picked from agar plates. We demonstrated the power of our approach by discriminating between two Bacillus subtilis strains in <30 min solely on the basis of their differential ability to produce cyclic peptide antibiotics surfactin and plipastatin, caused by a single frameshift mutation. Next, we used IDBac to detect subtle intraspecies differences in the production of metal scavenging acyl-desferrioxamines in a group of eight freshwater Micromonospora isolates that share >99% sequence similarity in the 16S rRNA gene. Finally, we used IDBac to simultaneously extract protein and specialized metabolite MS profiles from unidentified Lake Michigan sponge-associated bacteria isolated from an agar plate. In just 3 h, we created hierarchical protein MS groupings of 11 environmental isolates (10 MS replicates each, for a total of 110 spectra) that accurately mirrored phylogenetic groupings. We further distinguished isolates within these groupings, which share nearly identical 16S rRNA gene sequence identity, based on interspecies and intraspecies differences in specialized metabolite production. IDBac is an attempt to couple in situ MS analyses of protein content and specialized metabolite production to allow for facile discrimination of closely related bacterial colonies.

The secreted metabolome of Streptomyces chartreusis and implications for bacterial chemistry.

Actinomycetes are known for producing diverse secondary metabolites. Combining genomics with untargeted data-dependent tandem MS and molecular networking, we characterized the secreted metabolome of the tunicamycin producer Streptomyces chartreusis NRRL 3882. The genome harbors 128 predicted biosynthetic gene clusters. We detected >1,000 distinct secreted metabolites in culture supernatants, only 22 of which were identified based on standards and public spectral libraries. S. chartreusis adapts the secreted metabolome to cultivation conditions. A number of metabolites are produced iron dependently, among them 17 desferrioxamine siderophores aiding in iron acquisition. Eight previously unknown members of this long-known compound class are described. A single desferrioxamine synthesis gene cluster was detected in the genome, yet different sets of desferrioxamines are produced in different media. Additionally, a polyether ionophore, differentially produced by the calcimycin biosynthesis cluster, was discovered. This illustrates that metabolite output of a single biosynthetic machine can be exquisitely regulated not only with regard to product quantity but also with regard to product range. Compared with chemically defined medium, in complex medium, total metabolite abundance was higher, structural diversity greater, and the average molecular weight almost doubled. Tunicamycins, for example, were only produced in complex medium. Extrapolating from this study, we anticipate that the larger part of bacterial chemistry, including chemical structures, ecological functions, and pharmacological potential, is yet to be uncovered.

Biosynthesis of desferrioxamine siderophores initiated by decarboxylases: A functional investigation of two lysine/ornithine-decarboxylases from Gordonia rubripertincta CWB2 and Pimelobacter simplex 3E.

Lysine is a precursor for desferrioxamine siderophore biosynthesis. The pathway is often initiated by lysine decarboxylases. However, little is known about those enzymes from Actinobacteria which represents a diverse class of desferrioxamine producers. In this study we focused on the genes grdesA form Gordonia rubripertincta CWB2 and psdesA from Pimelobacter simplex VkMAC-2033D that encode decarboxylases presumed to be involved in the synthesis of desferrioxamine siderophores. The corresponding proteins GrDesA and PsDesA, were heterologously produced in Escherichia coli and purified. PsDesA was isolated bound to the cofactor pyridoxal 5-phosphate and GrDesA was purified in its apo form. PsDesA showed a moderate substrate preference for lysine (Km = 0.17 mM, kcat = 0.26 s-1) compared to ornithine (Km = 0.13 mM, kcat = 0.14 s-1), while GrDesA exhibited specificity for lysine (Km = 0.13 mM, kcat = 1.2 s-1) compared to ornithine (Km = 2.9 mM, kcat = 0.18 s-1). The maximum decarboxylase activity of PsDesA was achieved at pH 7.5 at 35 °C, although PsDesA was stable up to 40°, its relative activity decreased significantly at 50 °C. The temperature optimum (40 °C) and thermostability of GrDesA were likewise, but it exhibited maximum activity at pH range 8.0-8.5, and sharply decreased outside of this range. The expression and characterization of these two decarboxylases provides insight into the biosynthetic pathway of desferrioxamines from G.rubripertincta and P. simplex and supports the functional annotation of related pathways.

Detecting TRA-1-60 in Cancer via a Novel Zr-89 Labeled ImmunoPET Imaging Agent.

TRA-1-60 (TRA) is a cell-surface antigen implicated in drug resistance, relapse, and recurrence. Its expression has been reported in breast, prostate, pancreatic, ovarian tumors, and follicular lymphoma, which paved the development of the therapeutic antibody, Bstrongomab (Bsg), and its drug conjugates. Because patient selection is critical to achieve clinical benefit, a noninvasive imaging agent to select TRA+ lesions in patients is needed. Herein, we report the development of the immunopositron emission tomography (immunoPET) radiotracer 89Zr-radiolabeled Bsg and its potential to delineate TRA+ tumors. Bsg was conjugated to the bifunctional chelator desferrioxamine (DFO) and radiolabeled with [89Zr]Zr-oxalate. [89Zr]Zr-DFO-Bsg was characterized in vitro and evaluated in vivo for uptake and specificity in high and low TRA-expressing BxPC-3 pancreatic and PC-3 prostate cancer models, respectively. Uptake was compared against [89Zr]Zr-DFO-IgG, a nonspecific control radiotracer. Immunohistochemical (IHC) staining of patient cancer tissues using Bsg was performed to explore its clinical significance. A specific activity of 0.18 ± 0.01 GBq/mg (4.8 ± 0.3 mCi/mg) was obtained for [89Zr]Zr-DFO-Bsg. BxPC-3 xenografts exhibited three-fold higher radiotracer uptake compared to [89Zr]Zr-DFO-IgG. Competitive saturation studies using BxPC-3 xenografts further confirmed tracer specificity. The TRA-specific probe had lower accumulation in PC-3 xenografts. Ex vivo autoradiographs correlated with TRA expression from the histopathology of the resected tumor xenografts. Additionally, patient cancer tissues demonstrated positive staining with Bsg with metastatic lesions exhibiting the highest staining. This study demonstrates the potential of [89Zr]Zr-DFO-Bsg as an imaging agent for noninvasive detection of TRA+ tumors.

Novel South African Rare Actinomycete Kribbella speibonae Strain SK5: A Prolific Producer of Hydroxamate Siderophores Including New Dehydroxylated Congeners.

In this paper, we report on the chemistry of the rare South African Actinomycete Kribbella speibonae strain SK5, a prolific producer of hydroxamate siderophores and their congeners. Two new analogues, dehydroxylated desferrioxamines, speibonoxamine 1 and desoxy-desferrioxamine D1 2, have been isolated, together with four known hydroxamates, desferrioxamine D1 3, desferrioxamine B 4, desoxy-nocardamine 5 and nocardamine 6, and a diketopiperazine (DKP) 7. The structures of 1-7 were characterized by the analysis of HRESIMS and 1D and 2D NMR data, as well as by comparison with the relevant literature. Three new dehydroxy desferrioxamine derivatives 8-10 were tentatively identified in the molecular network of K. speibonae strain SK5 extracts, and structures were proposed based on their MS/MS fragmentation patterns. A plausible spb biosynthetic pathway was proposed. To the best of our knowledge, this is the first report of the isolation of desferrioxamines from the actinobacterial genus Kribbella.

Cell Permeable Imidazole-Desferrioxamine Conjugates: Synthesis and In Vitro Evaluation.

Desferrioxamine (DFO), a clinically approved iron chelator used for iron overload, is unable to chelate labile plasma iron (LPI) because of its limited cell permeability. Herein, alkyl chain modified imidazolium cations with varied hydrophobicities have been conjugated with DFO. The iron binding abilities and the antioxidant properties of the conjugates were found to be similar to DFO. The degree of cellular internalization was much higher in the octyl-imidazolium-DFO conjugate (IV) compared with DFO, and IV was able to chelate LPI in vitro. This opens up a new avenue in using N-alkyl imidazolium salts as a delivery vector for hydrophilic cell-impermeable drugs.

Analogues of desferrioxamine B (DFOB) with new properties and new functions generated using precursor-directed biosynthesis.

Desferrioxamine B (DFOB) is a siderophore native to Streptomyces pilosus biosynthesised by the DesABCD enzyme cluster as a high affinity Fe(III) chelator. Although DFOB has a long clinical history for the treatment of chronic iron overload, limitations encourage the development of new analogues. This review describes a recent body of work that has used precursor-directed biosynthesis (PDB) to access new DFOB analogues. PDB exploits the native biosynthetic machinery of a producing organism in culture medium augmented with non-native substrates that compete against native substrates during metabolite assembly. The method allows access to analogues of natural products using benign methods, compared to multistep organic synthesis. The disadvantages of PDB are the production of metabolites in low yield and the need to purify complex mixtures. Streptomyces pilosus medium was supplemented with different types of non-native diamine substrates to compete against native 1,5-diaminopentane to generate DFOB analogues containing alkene bonds, fluorine atoms, ether or thioether functional groups, or a disulfide bond. All analogues retained function as Fe(III) chelators and have properties that could broaden the utility of DFOB. These PDB studies have also added knowledge to the understanding of DFOB biosynthesis.

The evolution of three siderophore biosynthetic clusters in environmental and host-associating strains of Pantoea.

For many pathogenic members of the Enterobacterales, siderophores play an important role in virulence, yet the siderophores of the host-associating members of the genus Pantoea remain unexplored. We conducted a genome-wide survey of environmental and host-associating strains of Pantoea to identify known and candidate siderophore biosynthetic clusters. Our analysis identified three clusters homologous to those of enterobactin, desferrioxamine, and aerobactin that were prevalent among Pantoea species. Using both phylogenetic and comparative genomic approaches, we demonstrate that the enterobactin-like cluster was present in the common ancestor of all Pantoea, with evidence for three independent losses of the cluster in P. eucalypti, P. eucrina, and the P. ananatis-P. stewartii lineage. The desferrioxamine biosynthetic cluster, previously described and characterized in Pantoea, was horizontally acquired from its close relative Erwinia, with phylogenetic evidence that these transfer events were ancient and occurred between ancestral lineages. The aerobactin cluster was identified in three host-associating species groups, P. septica, P. ananatis, and P. stewartii, with strong evidence for horizontal acquisition from human-pathogenic members of the Enterobacterales. Our work identifies and describes the key siderophore clusters in Pantoea, shows three distinct evolutionary processes driving their diversification, and provides a foundation for exploring the roles that these siderophores may play in human opportunistic infections.

Noninvasive Trafficking of Brentuximab Vedotin and PET Imaging of CD30 in Lung Cancer Murine Models.

CD30 has been considered a unique diagnostic and therapeutic target for CD30-positive lymphomas and some lung diseases. Additionally, CD30 has shown high expression in clinical lung cancer samples. In this study, 89Zr-radiolabeled brentuximab vedotin (BV) was developed for in vivo tracking of BV and imaging CD30 expression in lung cancer models via conjugation with desferrioxamine (Df). CD30 expression in three lung cancer cell lines (H460, H358, and A549) was quantified by Western blot. Flow cytometry and saturation binding assays were used to evaluate the binding capabilities of the tracer in vitro. After longitudinal positron emission tomography (PET) imaging and quantitative analysis were performed, ex vivo biodistribution and histological studies were used to verify PET results. Finally, dosimetric extrapolation of murine data to humans was performed. At the cellular level, CD30 was found to be expressed on H460 and A549 cells with the highest and lowest levels of expression, respectively. Both Df-BV and 89Zr-Df-BV displayed high binding affinity to H460 cells. PET images and their quantification verified that BV accumulated in H460 tumor models (9.93 ± 2.70% ID/g at 24 h after injection; n = 4) at the highest level, followed by H358 and A549 tumors (8.05 ± 2.43 and 5.00 ± 1.56% ID/g; n = 4). The nonspecific 89Zr-labeled IgG showed a low tumor uptake of 5.2 ± 1.0% ID/g for H460 models. Ex vivo biodistribution and fluorescence immunohistochemistry also corroborated these findings. Dosimetric results displayed safe dose estimations. Therefore, 89Zr-Df-BV provides a potential agent for evaluating CD30 expression noninvasively in lung cancer, and also for imaging of brentuximab vedotin for better understanding of its pharmacokinetics.