n-n--n---triacetylfusarinine-c and ferricrocin

Siderophores are a driver of Pinus sylvestris root responses to metabolites secreted by pathogenic and mycorrhizal fungi. Structurally different siderophores regulate the uptake of Fe by microorganisms and may play a key role in the colonization of plants by beneficial or pathogenic fungi. Siderophore action, however, may be dependent on the distribution of Fe within cells. Here, the involvement of siderophores in determining the changes of organelle morphology and element composition of some cellular fractions of root cells in Pinus sylvestris to trophically diverse fungi was investigated. Changes in the morphology and concentrations of different elements within organelles of root cells in response to three structurally different siderophores were examined by transmission electron microscopy combined with energy-dispersive X-ray spectroscopy. Weak development of mitochondrial cristae and the deposition of backup materials in plastids occurred in the absence of Fe in the structures of triacetylfusarinine C and ferricrocin. In response to metabolites of both pathogenic and mycorrhizal fungi, Fe accumulated mainly in the cell walls and cytoplasm. Fe counts increased in all of the analyzed organelles in response to applications of ferricrocin and triacetylfusarinine C. Chelation of Fe within the structure of siderophores prevents the binding of exogenous Fe, decreasing the abundance of Fe in the cell wall and cytoplasm. The concentrations of N, P, K, Ca, Mn, Cu, Mg, and Zn also increased in cells after applications of ferricrocin and triacetylfusarinine C, while the levels of these elements decreased in the cell wall and cytoplasm when Fe was present within the structure of the siderophores. These results provide insight into the siderophore-driven response of plants to various symbionts.

Topics: Cell Nucleus; Cell Wall; Cytoplasm; Deferoxamine; Ferric Compounds; Ferrichrome; Fungi; Hydroxamic Acids; Iron; Microscopy, Electron, Transmission; Mitochondria; Mycorrhizae; Organelles; Pinus sylvestris; Plant Roots; Siderophores

Siderophores represent important microbial virulence factors and infection biomarkers. Their monitoring in fermentation broths, bodily fluids, and tissues should be reproducible. Similar isolation, characterization, and quantitation studies can often have conflicting results, and without proper documentation of sample collection, data processing, and analysis methods, it is difficult to reexamine the data and reconcile these differences. In this Springer Nature Protocol, we present the procedure optimized for ferricrocin/triacetylfusarinine C extraction from biological material as well as for tissue fixation and cryosectioning for optical microscopy and for both elemental and molecular mass spectrometry imaging. Special attention is paid to siderophore data mining from conventional and product ion mass spectra, liquid chromatography, and mass spectrometry imaging datasets, performed here by our free software called CycloBranch.

Topics: Animals; Aspergillus fumigatus; Biomarkers; Chromatography, Liquid; Cryoultramicrotomy; Data Mining; Datasets as Topic; Disease Models, Animal; Ferric Compounds; Ferrichrome; Humans; Hydroxamic Acids; Invasive Pulmonary Aspergillosis; Mass Spectrometry; Rats; Siderophores; Software; Tissue Fixation

Ergothioneine (EGT; 2-mercaptohistidine trimethylbetaine) is a trimethylated and sulphurised histidine derivative which exhibits antioxidant properties. Here we report that deletion of Aspergillus fumigatus egtA (AFUA_2G15650), which encodes a trimodular enzyme, abrogated EGT biosynthesis in this opportunistic pathogen. EGT biosynthetic deficiency in A. fumigatus significantly reduced resistance to elevated H

Topics: Antioxidants; Aspergillus fumigatus; Carbon-Oxygen Lyases; Ergothioneine; Ferric Compounds; Ferrichrome; Fungal Proteins; Gene Deletion; Genetic Complementation Test; Gliotoxin; Glutathione; Histidine; Hydrogen Peroxide; Hydroxamic Acids; Lyases; Metals, Heavy; Oxidation-Reduction; Oxidative Stress; Proteomics; Reactive Oxygen Species; Siderophores; Vitamin K 3

Iron is an essential element for all microorganisms. Bacteria and fungi produce versatile siderophores for binding and storing this essential transition metal when its availability is limited in the environment. The aim of the study was to optimize the fermentation medium of Aspergillus fumigatus for siderophore production. Triacetyl-fusarinine C and ferricrocin yields were dependent on glucose and glycine supplementations as well as the initial pH of the culture media. The optimal fermentation medium for triacetylfusarinine C production contained 8% glucose, 0.4% glycine and the initial pH was set to 5.9. Meanwhile, maximal ferricrocin yields were recorded in the presence of 10% glucose, 0.5% glycine and at an initial pH of 7.4. Under optimized fermentation conditions, the yields for triacetylfusarinine C and ferricrocin increased up to 2.9 g/l culture medium and 18.9 mg/g mycelium, respectively.

Topics: Aspergillus fumigatus; Culture Media; Factor Analysis, Statistical; Fermentation; Ferric Compounds; Ferrichrome; Glucose; Glycine; Hydrogen-Ion Concentration; Hydroxamic Acids; Iron; Siderophores

A screening for siderophores produced by the ectomycorrhizal fungi Laccaria laccata and Laccaria bicolor in synthetic low iron medium revealed the release of several different hydroxamate siderophores of which four major siderophores could be identified by high resolution mass spectrometry. While ferricrocin, coprogen and triacetylfusarinine C were assigned as well as other known fungal siderophores, a major peak of the siderophore mixture revealed an average molecular mass of 797 for the iron-loaded compound. High resolution mass spectrometry indicated an absolute mass of m/z = 798.30973 ([M + H](+)). With a relative error of Δ = 0.56 ppm this corresponds to linear fusigen (C33H52N6O13Fe; MW = 797.3). The production of large amounts of linear fusigen by these basidiomycetous mycorrhizal fungi may possibly explain the observed suppression of plant pathogenic Fusarium species. For comparative purposes Fusarium roseum was included in this study as a well known producer of cyclic and linear fusigen.

Topics: Antibiosis; Chromatography, High Pressure Liquid; Culture Media; Ferric Compounds; Ferrichrome; Fusarium; Hydroxamic Acids; Iron; Laccaria; Mass Spectrometry; Molecular Weight; Plant Roots; Siderophores; Tracheophyta

Siderophores have been identified as virulence factors in the opportunistic fungal pathogen Aspergillus fumigatus. The 14-pass transmembrane protein MirB is postulated to function as a siderophore transporter, responsible for uptake of the hydroxamate siderophore N,N',N″-triacetylfusarinine C (TAFC). Our aim was to identify amino acids of A. fumigatus MirB that are crucial for uptake of TAFC. Site-directed mutagenesis was used to create MirB mutants. Expression of wild-type and mutant proteins in the Saccharomyces cerevisiae strain PHY14, which lacks endogenous siderophore transporters, was confirmed by Western blotting. TAFC transport assays using (55)Fe-labeled TAFC and growth assays with Fe-TAFC as the sole iron source identified alanine 125, tyrosine 577, loop 3, and the second half of loop 7 (Loop7Del2) as crucial for function, since their substitution or deletion abrogated uptake completely. Wild-type MirB transported ferricrocin and coprogen as well as TAFC but not ferrichrysin. MirB was localized by fluorescence microscopy using antisera raised against a MirB extracellular loop peptide. Immunofluorescence microscopy showed that in yeast, wild-type MirB had a punctate distribution under the plasma membrane, as did the A125D and Y577A strains, indicating that the defect in transport of these mutants was unlikely to be due to mislocalization or degradation. MirB immunolocalization in A. fumigatus showed that the transporter was found in vesicles which cycled between the cytoplasm and the plasma membrane and was concentrated at the hyphal tips. The location of MirB was not influenced by the presence of the siderophore TAFC but was sensitive to internal iron stores.

Topics: Amino Acids; Aspergillus fumigatus; Biological Transport; Blotting, Western; Cell Membrane; Computational Biology; Cytoplasm; Ferric Compounds; Ferrichrome; Fungal Proteins; Hydroxamic Acids; Hyphae; Iron; Membrane Transport Proteins; Microscopy, Fluorescence; Mutagenesis, Site-Directed; Proteolysis; Saccharomyces cerevisiae; Siderophores

The opportunistic fungal pathogen Aspergillus fumigatus produces four types of siderophores, low-molecular-mass iron chelators: it excretes fusarinine C (FsC) and triacetylfusarinine C (TAFC) for iron uptake and accumulates ferricrocin (FC) for hyphal and hydroxyferricrocin (HFC) for conidial iron distribution and storage. Siderophore biosynthesis has recently been shown to be crucial for fungal virulence. Here we identified a new component of the fungal siderophore biosynthetic machinery: AFUA_1G04450, termed SidL. SidL is conserved only in siderophore-producing ascomycetes and shows similarity to transacylases involved in bacterial siderophore biosynthesis and the N(5)-hydroxyornithine:anhydromevalonyl coenzyme A-N(5)-transacylase SidF, which is essential for TAFC biosynthesis. Inactivation of SidL in A. fumigatus decreased FC biosynthesis during iron starvation and completely blocked FC biosynthesis during iron-replete growth. In agreement with these findings, SidL deficiency blocked conidial accumulation of FC-derived HFC under iron-replete conditions, which delayed germination and decreased the size of conidia and their resistance to oxidative stress. Remarkably, the sidL gene is not clustered with other siderophore-biosynthetic genes, and its expression is not affected by iron availability. Tagging of SidL with enhanced green fluorescent protein suggested a cytosolic localization of the FC-biosynthetic machinery. Taken together, these data suggest that SidL is a constitutively active N(5)-hydroxyornithine-acetylase required for FC biosynthesis, in particular under iron-replete conditions. Moreover, this study revealed the unexpected complexity of siderophore biosynthesis, indicating the existence of an additional, iron-repressed N(5)-hydroxyornithine-acetylase.

Topics: Acetyl Coenzyme A; Acetyltransferases; Ascomycota; Aspergillus fumigatus; Cytoplasm; Ferric Compounds; Ferrichrome; Green Fluorescent Proteins; Hydroxamic Acids; Iron; Oxidative Stress; Phylogeny; Siderophores; Virulence Factors

Zinc plays a critical role in a diverse array of biochemical processes. However, excess of zinc is deleterious to cells. Therefore, cells require finely tuned homeostatic mechanisms to balance uptake and storage of zinc. Here we show that iron starvation affects zinc metabolism by downregulating expression of the plasma membrane zinc importer encoding zrfB and upregulating the putative vacuolar zinc transporter-encoding zrcA in Aspergillus fumigatus. Nevertheless, the zinc content of iron-starved mycelia exceeded that of iron replete mycelia, possibly due to unspecific metal uptake induced by iron starvation. In agreement with increased zinc excess and zinc toxicity during iron starvation, deficiency in siderophore-mediated high-affinity iron uptake caused hypersensitivity to zinc. Moreover, an increase of zinc uptake by conditional overexpression of zrfB was more toxic under iron depleted compared to iron replete conditions. This deregulated zinc uptake under iron starvation caused a decrease in heme production and an increase in protoporphyrin IX accumulation. Furthermore, zinc excess impaired production of the extracellular siderophore triacetylfusarinine C but not the intracellular siderophore ferricrocin. Taken together, these data demonstrate a fine tuned coordination of zinc and iron metabolism in A. fumigatus.

Topics: Aspergillus fumigatus; Carrier Proteins; Ferric Compounds; Ferrichrome; Fungal Proteins; Gene Expression Regulation, Fungal; Heme; Homeostasis; Hydroxamic Acids; Iron; Mycelium; Protoporphyrins; RNA, Fungal; Zinc

Aspergillus fumigatus, the most common airborne fungal pathogen of humans, employs two high-affinity iron uptake systems: iron uptake mediated by the extracellular siderophore triacetylfusarinine C and reductive iron assimilation. Furthermore, A. fumigatus utilizes two intracellular siderophores, ferricrocin and hydroxyferricrocin, to store iron. Siderophore biosynthesis, which is essential for virulence, is repressed by iron. Here we show that this control is mediated by the GATA factor SreA. During iron-replete conditions, SreA deficiency partially derepressed synthesis of triacetylfusarinine C and uptake of iron resulting in increased cellular accumulation of both iron and ferricrocin. Genome-wide DNA microarray analysis identified 49 genes that are repressed by iron in an SreA-dependent manner. This gene set, termed SreA regulon, includes all known genes involved in iron acquisition, putative novel siderophore biosynthetic genes, and also genes not directly linked to iron metabolism. SreA deficiency also caused upregulation of iron-dependent and antioxidative pathways, probably due to the increased iron content and iron-mediated oxidative stress. Consistently, the sreA disruption mutant displayed increased sensitivity to iron, menadion and phleomycin but retained wild-type virulence in a mouse model. As all detrimental effects of sreA disruption are restricted to iron-replete conditions these data underscore that A. fumigatus faces iron-depleted conditions during infection.

Topics: Aspergillus fumigatus; DNA, Fungal; Ferric Compounds; Ferrichrome; Fungal Proteins; GATA Transcription Factors; Gene Expression Profiling; Gene Expression Regulation, Fungal; Genes, Fungal; Genetic Complementation Test; Hydroxamic Acids; Iron; Molecular Sequence Data; Mutation; Oligonucleotide Array Sequence Analysis; Oxidative Stress; Promoter Regions, Genetic; Regulon; Repressor Proteins; RNA, Fungal; Siderophores; Virulence

The saprophytic fungus Aspergillus fumigatus is the most prevalent airborne fungal pathogen, which is responsible for invasive aspergillosis in immunocompromised patients. Iron plays an essential role for the growth and proliferation of A. fumigatus. This fungus synthesizes three major siderophores. It excretes triacetylfusarinine C to capture iron, while it accumulates ferricrocin and hydroxyferricrocin for hyphal and conidial iron storage, respectively. Herein, we investigated the role of the siderophore system of A. fumigatus in the modulation of immune effector pathways and iron homeostasis in macrophages. We set up a co-culture system consisting of the murine macrophage cell line RAW264.7 and either A. fumigatus wild type or a siderophore-deficient mutant (DeltasidA). We used real-time quantitative RT-PCR and Western blot analyses to study the expression of macrophage iron metabolism and innate immune response genes in response to pathogen challenge. Infection of macrophages with A. fumigatus wild type, but not with the DeltasidA mutant, induced expression of TNF and phagocyte oxidase subunit 47 at the transcriptional level. Moreover, infection with A. fumigatus wild type, but not with the DeltasidA mutant, compromised macrophage iron homeostasis. Infection with wild-type A. fumigatus decreased expression of the two cellular iron importers, the divalent metal transporter-1 and the transferrin receptor, and the only known iron exporter ferroportin. At the same time, it increased macrophage iron retention and ferritin synthesis. These data indicate that A. fumigatus affects the regulation of macrophage iron homeostasis and innate immune effector pathways via its siderophore system. The changes in immune response may be a consequence of macrophage iron restriction.

Topics: Animals; Aspergillosis; Aspergillus fumigatus; Cell Line; Coculture Techniques; Ferric Compounds; Ferrichrome; Hydroxamic Acids; Immunity, Innate; Iron; Macrophages; Mice; Phagocytosis; Receptors, Transferrin; Siderophores

Aspergillus fumigatus is an opportunistic fungal pathogen that causes life-threatening infections in immunocompromised patients. Despite low levels of free iron, A. fumigatus grows in the presence of human serum in part because it produces high concentrations of siderophores. The most abundant siderophores produced by A. fumigatus are N',N'',N'''-triacetylfusarinine C (TAF) and ferricrocin, both of which have thermodynamic iron binding constants that theoretically allow them to remove transferrin (Tf)-bound iron. Urea-polyacrylamide gel electrophoresis was used to measure the change in concentration of Tf species incubated with TAF or ferricrocin. The rate of removal of iron from diferric Tf by both siderophores was measured, as were the individual microscopic rates of iron removal from each Tf species (diferric Tf, N-terminal monoferric Tf and C-terminal monoferric Tf). TAF removed iron from all Tf species at a faster rate than ferricrocin. Both siderophores showed a preference for removing C-terminal iron, evidenced by the fact that k(1C) and k(2C) were much larger than k(1N) and k(2N). Cooperativity in iron binding was observed with TAF, as the C-terminal iron was removed by TAF much faster from monoferric than from diferric Tf. With both siderophores, C-terminal monoferric Tf concentrations remained below measurable levels during incubations. This indicates that k(2C) and k(1C) are much larger than k(1N). TAF and ferricrocin both removed Tf-bound iron with second-order rate constants that were comparable to those of the siderophores of several bacterial pathogens, indicating they may play a role in iron uptake in vivo and thereby contribute to the virulence of A. fumigatus.

Topics: Aspergillus fumigatus; Chelating Agents; Ferric Compounds; Ferrichrome; Hydroxamic Acids; Iron; Kinetics; Ligands; Molecular Structure; Thermodynamics; Transferrin