Reductive Iron Assimilation Research Articles

Excess airway iron during severe SARS-CoV-2 infection impairs neutrophil antifungal activity enabling 2° mold infection

Abstract COVID-19-Associated Pulmonary Aspergillosis (CAPA) is an invasive 2° mold infection caused by Aspergillus species affecting ~5% of patients with severe SARS-CoV-2 (SARS-2) infection. In this study, we hypothesized that excess airway iron released during SARS-2 infection promotes 2° infection via enhancement of fungal growth and impairment of neutrophil antifungal activity. We show that SARS-2 bronchoaveolar lavage samples (n=48) have higher levels of total iron, ferric iron, and oxyhemoglobin relative to bacterial pneumonia (n=40). Likewise, FFPE lung tissues from SARS-2 infection exhibited increased extracellular ferric iron, ferrous iron, ferritin, and hemoglobin. We show that both iron and hemoglobin enhance the growth rate of Aspergillus fumigatus via siderophore and reductive iron assimilation dependent mechanism. Utilizing lung epithelial cell transmigration model, we show that iron enhances neutrophil intracellular ROS production resulting in enhanced conidiacidal activity. In contrast, transmigrated neutrophils exposed to both heme and hemoglobin exhibit decreased ROS production and NET formation with significant impairment in hyphal killing. When both neutrophils and fungi are co-cultured with iron, heme, or hemoglobin, fungi consistently win the battle characterized by uncontrolled hyphal proliferation. These data identify both microbial and host mechanisms which may be targeted pharmacologically to mitigate the iron-dependent growth of 2° mold infection.

The Transcription Factors AcuK and AcuM Influence Siderophore Biosynthesis of Aspergillus fumigatus.

The mold Aspergillus fumigatus employs two high-affinity uptake systems, reductive iron assimilation (RIA) and siderophore-mediated iron acquisition (SIA), for the acquisition of the essential trace element iron. SIA has previously been shown to be crucial for virulence in mammalian hosts. Here, we show that a lack of AcuK or AcuM, transcription factors required for the activation of gluconeogenesis, decreases the production of both extra- and intracellular siderophores in A. fumigatus. The lack of AcuM or AcuK did not affect the expression of genes involved in RIA and SIA, suggesting that these regulators do not directly regulate iron homeostasis genes, but indirectly affect siderophore production through their influence on metabolism. Consistent with this, acetate supplementation reversed the intracellular siderophore production defect of ΔacuM and ΔacuK. Moreover, ΔacuM and ΔacuK displayed a similar growth defect under iron limitation and iron sufficiency, which suggests they have a general role in carbon metabolism apart from gluconeogenesis. In agreement with a potential role of the glyoxylate cycle in adaptation to iron starvation, transcript levels of the malate synthase-encoding acuE were found to be upregulated by iron limitation that is partially dependent on AcuK and AcuM. Together, these data demonstrate the influence of iron availability on carbon metabolism.

Essential roles of ferric reductase-like proteins in growth, development, stress response, and virulence of the filamentous entomopathogenic fungus Beauveria bassiana

In yeasts, ferric reductase catalyzes reduction of ferric ion to ferrous form, which is essential for the reductive iron assimilation system. However, the physiological roles of ferric reductases remain largely unknown in the filamentous fungi. In this study, genome-wide annotation revealed thirteen ferric reductase-like (Fre) proteins in the filamentous insect pathogenic fungus Beauveria bassiana, and all their functions were genetically characterized. Ferric reductase family proteins exhibit different sub-cellular distributions (e.g., cell periphery and vacuole), which was due to divergent domain architectures. Fre proteins had a synergistic effect on fungal virulence, which was ascribed to their distinct functions in different physiologies. Ten Fre proteins were not involved in reduction of ferric ion in submerged mycelia, but most proteins contributed to blastospore development. Only two Fre proteins significantly contributed to B. bassiana vegetative growth under the chemical-induced iron starvation, but most Fre proteins were involved in resistance to osmotic and oxidative stresses. Notably, a bZIP-type transcription factor HapX bound to the promoter regions of all FRE genes in B. bassiana, and displayed varying roles in the transcription activation of these genes. This study reveals the important role of BbFre family proteins in development, stress response, and insect pathogenicity, as well as their distinctive role in the absorption of ferric iron from the environment.

High-affinity iron uptake is required for optimal Epichloë festucae colonization of Lolium perenne and seed transmission.

Epichloë festucae uses a siderophore-mediated system to acquire iron, which is important to maintain endophyte-grass symbioses. Here we investigate the roles of the alternative iron acquisition system, reductive iron assimilation (RIA), via disruption of the fetC gene, which encodes a multicopper ferroxidase, either alone (i.e., ΔfetC) or in combination with disruption of the gene sidA, which encodes a siderophore biosynthesis enzyme (i.e., ΔfetC/ΔsidA). The phenotypic characteristics of these mutants were compared to ΔsidA and wild-type (WT) strains during growth under axenic culture conditions (in culture) and in symbiosis with the host grass, perennial ryegrass (in planta). Under iron deficiency, the colony growth rate of ΔfetC was slightly slower than that of WT, while the growth of ΔsidA and ΔfetC/ΔsidA mutants was severely suppressed. Siderophore analyses indicated that ΔfetC mutants hyperaccumulate ferriepichloënin A (FEA) at low iron concentrations and ferricrocin and FEA at higher iron concentrations. When compared to WT, all mutant strains displayed hyperbranching hyphal structures and a reduced ratio of Epichloë DNA to total DNA in planta. Furthermore, host colonization and vertical transmission through infection of the host seed were significantly reduced in the ΔfetC/ΔsidA mutants, confirming that high-affinity iron uptake is a critical process for Epichloë transmission. Thus, RIA and siderophore iron uptake are complementary systems required for the maintenance of iron metabolism, fungal growth, and symbiosis between E. festucae and perennial ryegrass.

The Siderophore Ferricrocin Mediates Iron Acquisition in Aspergillus fumigatus.

The opportunistic fungal pathogen Aspergillus fumigatus utilizes two high-affinity iron uptake mechanisms, termed reductive iron assimilation (RIA) and siderophore-mediated iron acquisition (SIA). The latter has been shown to be crucial for virulence of this fungus and is a target for development of novel strategies for diagnosis and treatment of fungal infections. So far, research on SIA in this mold focused mainly on the hyphal stage, revealing the importance of extracellular fusarinine-type siderophores in iron acquisition as well as of the siderophore ferricrocin in intracellular iron handling. The current study aimed to characterize iron acquisition during germination. High expression of genes involved in biosynthesis and uptake of ferricrocin in conidia and during germination, independent of iron availability, suggested a role of ferricrocin in iron acquisition during germination. In agreement, (i) bioassays indicated secretion of ferricrocin during growth on solid media during both iron sufficiency and limitation, (ii) ferricrocin was identified in the supernatant of conidia germinating in liquid media during both iron sufficiency and limitation, (iii) in contrast to mutants lacking all siderophores, mutants synthesizing ferricrocin but lacking fusarinine-type siderophores were able to grow under iron limitation in the absence of RIA, and (iv) genetic inactivation of the ferricrocin transporter Sit1 decreased germination in the absence of RIA. Taken together, this study revealed that ferricrocin has not only an intracellular role but also functions as an extracellular siderophore to support iron acquisition. The iron availability-independent ferricrocin secretion and uptake during early germination indicate developmental, rather than iron regulation. IMPORTANCE Aspergillus fumigatus is one of the most common airborne fungal pathogens for humans. Low-molecular-mass iron chelators, termed siderophores, have been shown to play a central role in iron homeostasis and, consequently, virulence of this mold. Previous studies demonstrated the crucial role of secreted fusarinine-type siderophores, such as triacetylfusarinine C, in iron acquisition, as well as of the ferrichrome-type siderophore ferricrocin in intracellular iron storage and transport. Here, we demonstrate that ferricrocin is also secreted to mediate iron acquisition during germination together with reductive iron assimilation. During early germination, ferricrocin secretion and uptake were not repressed by iron availability, indicating developmental regulation of this iron acquisition system in this growth phase.

Regulation of High-Affinity Iron Acquisition, Including Acquisition Mediated by the Iron Permease FtrA, Is Coordinated by AtrR, SrbA, and SreA in Aspergillus fumigatus.

Iron acquisition is crucial for virulence of the human pathogen Aspergillus fumigatus. Previous studies indicated that this mold regulates iron uptake via both siderophores and reductive iron assimilation by the GATA factor SreA and the SREBP regulator SrbA. Here, characterization of loss of function as well as hyperactive alleles revealed that transcriptional activation of iron uptake depends additionally on the Zn2Cys6 regulator AtrR, most likely via cooperation with SrbA. Mutational analysis of the promoter of the iron permease-encoding ftrA gene identified a 210-bp sequence, which is both essential and sufficient to impart iron regulation. Further studies located functional sequences, densely packed within 75 bp, that largely resemble binding motifs for SrbA, SreA, and AtrR. The latter, confirmed by chromatin immunoprecipitation (ChIP) analysis, is the first one not fully matching the 5'-CGGN12CCG-3' consensus sequence. The results presented here emphasize for the first time the direct involvement of SrbA, AtrR, and SreA in iron regulation. The essential role of both AtrR and SrbA in activation of iron acquisition underlines the coordination of iron homeostasis with biosynthesis of ergosterol and heme as well as adaptation to hypoxia. The rationale is most likely the iron dependence of these pathways along with the enzymatic link of biosynthesis of ergosterol and siderophores. IMPORTANCE Aspergillus fumigatus is the most common filamentous fungal pathogen infecting humans. Iron acquisition via siderophores has previously been shown to be essential for virulence of this mold species. Here, we demonstrate that AtrR, a transcription factor previously shown to control ergosterol biosynthesis, azole resistance, and adaptation to hypoxia, is essential for activation of iron acquisition, including siderophore biosynthesis and uptake. Dissection of an iron-regulated promoter identified binding motifs for AtrR and the two previously identified regulators of iron acquisition, SrbA and SreA. Altogether, this study identified a new regulator required for maintenance of iron homeostasis, revealed insights into promoter architecture for iron regulation, and emphasized the coordinated regulation of iron homeostasis ergosterol biosynthesis and adaptation to hypoxia.

Molecular Regulatory Mechanism of the Iron-Ion-Promoted Asexual Sporulation of Antrodia cinnamomea in Submerged Fermentation Revealed by Comparative Transcriptomics.

Antrodia cinnamomea is a precious edible and medicinal fungus with activities of antitumor, antivirus, and immunoregulation. Fe2+ was found to promote the asexual sporulation of A. cinnamomea markedly, but the molecular regulatory mechanism of the effect is unclear. In the present study, comparative transcriptomics analysis using RNA sequencing (RNA-seq) and real time quantitative PCR (RT-qPCR) were conducted on A. cinnamomea mycelia cultured in the presence or absence of Fe2+ to reveal the molecular regulatory mechanisms underlying iron-ion-promoted asexual sporulation. The obtained mechanism is as follows: A. cinnamomea acquires iron ions through reductive iron assimilation (RIA) and siderophore-mediated iron assimilation (SIA). In RIA, ferrous iron ions are directly transported into cells by the high-affinity protein complex formed by a ferroxidase (FetC) and an Fe transporter permease (FtrA). In SIA, siderophores are secreted externally to chelate the iron in the extracellular environment. Then, the chelates are transported into cells through the siderophore channels (Sit1/MirB) on the cell membrane and hydrolyzed by a hydrolase (EstB) in the cell to release iron ions. The O-methyltransferase TpcA and the regulatory protein URBS1 promote the synthesis of siderophores. HapX and SreA respond to and maintain the balance of the intercellular concentration of iron ions. Furthermore, HapX and SreA promote the expression of flbD and abaA, respectively. In addition, iron ions promote the expression of relevant genes in the cell wall integrity signaling pathway, thereby accelerating the cell wall synthesis and maturation of spores. This study contributes to the rational adjustment and control of the sporulation of A. cinnamomea and thereby improves the efficiency of the preparation of inoculum for submerged fermentation.

Expression Patterns in Reductive Iron Assimilation and Functional Consequences during Phagocytosis of Lichtheimia corymbifera, an Emerging Cause of Mucormycosis.

Iron is an essential micronutrient for most organisms and fungi are no exception. Iron uptake by fungi is facilitated by receptor-mediated internalization of siderophores, heme and reductive iron assimilation (RIA). The RIA employs three protein groups: (i) the ferric reductases (Fre5 proteins), (ii) the multicopper ferroxidases (Fet3) and (iii) the high-affinity iron permeases (Ftr1). Phenotyping under different iron concentrations revealed detrimental effects on spore swelling and hyphal formation under iron depletion, but yeast-like morphology under iron excess. Since access to iron is limited during pathogenesis, pathogens are placed under stress due to nutrient limitations. To combat this, gene duplication and differential gene expression of key iron uptake genes are utilized to acquire iron against the deleterious effects of iron depletion. In the genome of the human pathogenic fungus L. corymbifera, three, four and three copies were identified for FRE5, FTR1 and FET3 genes, respectively. As in other fungi, FET3 and FTR1 are syntenic and co-expressed in L. corymbifera. Expression of FRE5, FTR1 and FET3 genes is highly up-regulated during iron limitation (Fe-), but lower during iron excess (Fe+). Fe- dependent upregulation of gene expression takes place in LcFRE5 II and III, LcFTR1 I and II, as well as LcFET3 I and II suggesting a functional role in pathogenesis. The syntenic LcFTR1 I–LcFET3 I gene pair is co-expressed during germination, whereas LcFTR1 II- LcFET3 II is co-expressed during hyphal proliferation. LcFTR1 I, II and IV were overexpressed in Saccharomyces cerevisiae to represent high and moderate expression of intracellular transport of Fe3+, respectively. Challenge of macrophages with the yeast mutants revealed no obvious role for LcFTR1 I, but possible functions of LcFTR1 II and IVs in recognition by macrophages. RIA expression pattern was used for a new model of interaction between L. corymbifera and macrophages.

Defects in the Ferroxidase That Participates in the Reductive Iron Assimilation System Results in Hypervirulence in Botrytis Cinerea.

The plant pathogen Botrytis cinerea is responsible for gray-mold disease, which infects a wide variety of species. The outcome of this host-pathogen interaction, a result of the interplay between plant defense and fungal virulence pathways, can be modulated by various environmental factors. Among these, iron availability and acquisition play a crucial role in diverse biological functions. How B. cinerea obtains iron, an essential micronutrient, during infection is unknown. We set out to determine the role of the reductive iron assimilation (RIA) system during B. cinerea infection. This system comprises the BcFET1 ferroxidase, which belongs to the multicopper oxidase (MCO) family of proteins, and the BcFTR1 membrane-bound iron permease. Gene knockout and complementation studies revealed that, compared to the wild type, the bcfet1 mutant displays delayed conidiation, iron-dependent sclerotium production, and significantly reduced whole-cell iron content. Remarkably, this mutant exhibited a hypervirulence phenotype, whereas the bcftr1 mutant presents normal virulence and unaffected whole-cell iron levels and developmental programs. Interestingly, while in iron-starved plants wild-type B. cinerea produced slightly reduced necrotic lesions, the hypervirulence phenotype of the bcfet1 mutant is no longer observed in iron-deprived plants. This suggests that B. cinerea bcfet1 knockout mutants require plant-derived iron to achieve larger necrotic lesions, whereas in planta analyses of reactive oxygen species (ROS) revealed increased ROS levels only for infections caused by the bcfet1 mutant. These results suggest that increased ROS production, under an iron sufficiency environment, at least partly underlie the observed infection phenotype in this mutant.IMPORTANCE The plant-pathogenic fungus B. cinerea causes enormous economic losses, estimated at anywhere between $10 billion and $100 billion worldwide, under both pre- and postharvest conditions. Here, we present the characterization of a loss-of-function mutant in a component involved in iron acquisition that displays hypervirulence. While in different microbial systems iron uptake mechanisms appear to be critical to achieve full pathogenic potential, we found that the absence of the ferroxidase that is part of the reductive iron assimilation system leads to hypervirulence in this fungus. This is an unusual and rather underrepresented phenotype, which can be modulated by iron levels in the plant and provides an unexpected link between iron acquisition, reactive oxygen species (ROS) production, and pathogenesis in the Botrytis-plant interaction.

Genome mining and biosynthesis of a polyketide from a biofertilizer fungus that can facilitate reductive iron assimilation in plant

Fungi have the potential to produce a large repertoire of bioactive molecules, many of which can affect the growth and development of plants. Genomic survey of sequenced biofertilizer fungi showed many secondary metabolite gene clusters are anchored by iterative polyketide synthases (IPKSs), which are multidomain enzymes noted for generating diverse small molecules. Focusing on the biofertilizer Trichoderma harzianum t-22, we identified and characterized a cryptic IPKS-containing cluster that synthesizes tricholignan A, a redox-active ortho-hydroquinone. Tricholignan A is shown to reduce Fe(III) and may play a role in promoting plant growth under iron-deficient conditions. The construction of tricholignan by a pair of collaborating IPKSs was investigated using heterologous reconstitution and biochemical studies. A regioselective methylation step is shown to be a key step in formation of the ortho-hydroquinone. The responsible methyltransferase (MT) is fused with an N-terminal pseudo-acyl carrier protein (ψACP), in which the apo state of the ACP is essential for methylation of the growing polyketide chain. The ψACP is proposed to bind to the IPKS and enable the trans MT to access the growing polyketide. Our studies show that a genome-driven approach to discovering bioactive natural products from biofertilizer fungi can lead to unique compounds and biosynthetic knowledge.

An insight into the iron acquisition and homeostasis in Aureobasidium melanogenum HN6.2 strain through genome mining and transcriptome analysis.

Aureobasidium melanogenum HN6.2 is a unique yeast strain who can produce the siderophore of fusigen under iron starvation to guarantee its survival. However, a comprehensive understanding of mechanisms involved in iron acquisition and homeostasis for it is still vacant. In this study, genome sequencing and mining revealed that A. melanogenum HN6.2 strain was the first yeast species that exclusively possessed all the four known mechanisms for the iron acquisition: (i) the siderophore-mediated iron uptake; (ii) reductive iron assimilation; (iii) low-affinity ferrous uptake; and (iv) heme utilization, which suggested its stronger adaptability than Aspergillus fumigatus and Saccharomyces cerevisiae. This HN6.2 strain also employed the vacuolar iron storage for immobilizing the excessive iron to avoid its cellular toxicity. Specially, genome mining indicated that A. melanogenum HN6.2 strain could also synthesize ferricrocin siderophore. Further HPLC and Q-Tof-MS analysis confirmed that the siderophores synthesized by this strain consisted of cyclic fusigen, linear fusigen, ferricrocin, and hydroxyferricrocin and they played parallel roles as both intracellular and extracellular siderophores. Also, the heme utilization for this strain was experimentally verified by the knock-out of heme oxygenase gene. For iron homeostasis, the transcriptome analysis revealed that this strain mainly employed two central regulators of SreA/HapX to tune iron uptake and storage at the transcriptional level. It was also noted that mitogen-activated protein kinase C gene (MpkC) exhibited a transcriptional up-regulation under iron sufficiency, suggesting that it may serve as another factor involved in the repression of siderophore biosynthesis. This is the first genetic blueprint of iron acquisition and homeostasis for A. melanogenum.

Genomic Organization and Expression of Iron Metabolism Genes in the Emerging Pathogenic Mold Scedosporium apiospermum.

The ubiquitous mold Scedosporium apiospermum is increasingly recognized as an emerging pathogen, especially among patients with underlying disorders such as immunodeficiency or cystic fibrosis (CF). Indeed, it ranks the second among the filamentous fungi colonizing the respiratory tract of CF patients. However, our knowledge about virulence factors of this fungus is still limited. The role of iron-uptake systems may be critical for establishment of Scedosporium infections, notably in the iron-rich environment of the CF lung. Two main strategies are employed by fungi to efficiently acquire iron from their host or from their ecological niche: siderophore production and reductive iron assimilation (RIA) systems. The aim of this study was to assess the existence of orthologous genes involved in iron metabolism in the recently sequenced genome of S. apiospermum. At first, a tBLASTn analysis using A. fumigatus iron-related proteins as query revealed orthologs of almost all relevant loci in the S. apiospermum genome. Whereas the genes putatively involved in RIA were randomly distributed, siderophore biosynthesis and transport genes were organized in two clusters, each containing a non-ribosomal peptide synthetase (NRPS) whose orthologs in A. fumigatus have been described to catalyze hydroxamate siderophore synthesis. Nevertheless, comparative genomic analysis of siderophore-related clusters showed greater similarity between S. apiospermum and phylogenetically close molds than with Aspergillus species. The expression level of these genes was then evaluated by exposing conidia to iron starvation and iron excess. The expression of several orthologs of A. fumigatus genes involved in siderophore-based iron uptake or RIA was significantly induced during iron starvation, and conversely repressed in iron excess conditions. Altogether, these results indicate that S. apiospermum possesses the genetic information required for efficient and competitive iron uptake. They also suggest an important role of the siderophore production system in iron uptake by S. apiospermum.

Aspergillusfumigatus protein phosphatase PpzA is involved in iron assimilation, secondary metabolite production, and virulence.

Metal restriction imposed by mammalian hosts during an infection is a common mechanism of defence to reduce or avoid the pathogen infection. Metals are essential for organism survival due to its involvement in several biological processes. Aspergillus fumigatus causes invasive aspergillosis, a disease that typically manifests in immunocompromised patients. A.fumigatus PpzA, the catalytic subunit of protein phosphatase Z (PPZ), has been recently identified as associated with iron assimilation. A.fumigatus has 2 high-affinity mechanisms of iron acquisition during infection: reductive iron assimilation and siderophore-mediated iron uptake. It has been shown that siderophore production is important for A.fumigatus virulence, differently to the reductive iron uptake system. Transcriptomic and proteomic comparisons between ∆ppzA and wild-type strains under iron starvation showed that PpzA has a broad influence on genes involved in secondary metabolism. Liquid chromatography-mass spectrometry under standard and iron starvation conditions confirmed that the ΔppzA mutant had reduced production of pyripyropene A, fumagillin, fumiquinazoline A, triacetyl-fusarinine C, and helvolic acid. The ΔppzA was shown to be avirulent in a neutropenic murine model of invasive pulmonary aspergillosis. PpzA plays an important role at the interface between iron starvation, regulation of SM production, and pathogenicity in A.fumigatus.

Differentially regulated high-affinity iron assimilation systems support growth of the various cell types in the dimorphic pathogen Talaromyces marneffei.

Iron is a key trace element important for many biochemical processes and its availability varies with the environment. For human pathogenic fungi iron acquisition can be particularly problematical because host cells sequester free iron as part of the acute-phase response to infection. Fungi rely on high-affinity iron uptake systems, such as reductive iron assimilation (RIA) and siderophore-mediated iron uptake (non-RIA). These have been extensively studied in pathogenic fungi that exist outside of host cells, but much less is known for intracellular fungal pathogens. Talaromyces marneffei is a dimorphic fungal pathogen endemic to Southeast Asia. In the host T. marneffei resides within macrophages where it grows as a fission yeast. T. marneffei has genes of both iron assimilation systems as well as a paralogue of the siderophore biosynthetic gene sidA, designated sidX. Unlike other fungi, deletion of sidA or sidX resulted in cell type-specific effects. Mutant analysis showed that T. marneffei yeast cells also employ RIA for iron acquisition, providing an additional system in this cell type that differs substantially from hyphal cells. These data illustrate the specialized iron acquisition systems used by the different cell types of a dimorphic fungal pathogen and highlight the complexity in siderophore-biosynthetic pathways amongst fungi.

Siderophore Biosynthesis but Not Reductive Iron Assimilation Is Essential for the Dimorphic Fungus Nomuraea rileyi Conidiation, Dimorphism Transition, Resistance to Oxidative Stress, Pigmented Microsclerotium Formation, and Virulence

Iron is an indispensable factor for the dimorphic insect pathogenic Nomuraea rileyi to form persistent microsclerotia which can replace conidia or blastospores for commercial mass production. There are two high affinity iron acquisition pathways in N. rileyi, siderophore-assisted iron mobilization and reductive iron assimilation systems. Transcription of the two iron uptake pathways related genes is induced under iron-limiting conditions. Stage-specific iron uptake-related genes expression during microsclerotia development shows siderophore-mediated iron acquisition genes are rigorously upregulated specifically during the formation and mature period while reductive iron assimilation related genes just display a higher expression at the late maturation period. Abrogation of reductive iron assimilation, by the deletion of the high affinity iron permease (NrFtrA), has no visible effect on microsclerotia biogenesis in N. rileyi. In sharp contrast, N. rileyi L-ornithine-N5-monooxygenase (NrSidA), required for synthesis of all siderophores, is absolutely necessary for the development of pigmented microsclerotia. In agreement with the lower intracellular iron contents of microsclerotia in ΔNrSidA strains, not only the pigments, but both the number and the biomass are also noticeably reduced. Certain concentration of ROS is required for promoting microsclerotia biogenesis. Combined with expression pattern analysis of related genes and quantitative of intracellular iron or extracellular siderophore in WT and mutants, these data demonstrate the lack of adequate intracellular iron caused by the loss of the siderophore results in the deficiency of ROS detoxication. Furthermore, ΔNrSidA strains show significantly increased sensitivity to hydrogen peroxide. Besides, NrSidA, but not NrFtrA, play a crucial role in vegetative growth under iron-limiting conditions, conidiation, and dimorphic switching. Remarkably, the slower growth of the ΔNrSidA strains in vivo due to a reduced capacity for iron acquisition leads to the loss of virulence in Spodoptera litura while the ΔNrFtrA mutants behaved as WT during infection. Together, these results prove siderophore-assisted iron mobilization is the major pathway of cellular iron uptake and essential for conidiation, dimorphism transition, oxidative stress resistance, pigmented microsclerotium formation and full virulence.

A Putative Mitochondrial Iron Transporter MrsA in Aspergillus fumigatus Plays Important Roles in Azole-, Oxidative Stress Responses and Virulence.

Iron is an essential nutrient and enzyme co-factor required for a wide range of cellular processes, especially for the function of mitochondria. For the opportunistic fungal pathogen Aspergillus fumigatus, the ability to obtain iron is required for growth and virulence during the infection process. However, knowledge of how mitochondria are involved in iron regulation is still limited. Here, we show that a mitochondrial iron transporter, MrsA, a homolog of yeast Mrs4p, is critical for adaptation to iron-limited or iron-excess conditions in A. fumigatus. Deletion of mrsA leads to disruption of iron homeostasis with a decreased sreA expression, resulted in activated reductive iron assimilation (RIA) and siderophore-mediated iron acquisition (SIA). Furthermore, deletion of mrsA induces hypersusceptibility to azole and oxidative stresses. An assay for cellular ROS content in ΔmrsA combined with rescue from the mrsA-defective phenotype by the antioxidant reagent L-ascorbic acid indicates that the increased sensitivity of ΔmrsA to the azole itraconazole and to oxidative stress is mainly the result of abnormal ROS accumulation. Moreover, site-directed mutation experiments verified that three conserved histidine residues related to iron transport in MrsA are required for responses to oxidative and azole stresses. Importantly, ΔmrsA causes significant attenuation of virulence in an immunocompromised murine model of aspergillosis. Collectively, our results show that the putative mitochondrial iron transporter MrsA plays important roles in azole- and oxidative-stress responses and virulence by regulating the balance of cellular iron in A. fumigatus.

Paracoccidioides spp. ferrous and ferric iron assimilation pathways.

Iron is an essential micronutrient for almost all organisms, including fungi. Usually, fungi can uptake iron through receptor-mediated internalization of a siderophore or heme, and/or reductive iron assimilation (RIA). Traditionally, the RIA pathway consists of ferric reductases (Fres), ferroxidase (Fet3) and a high-affinity iron permease (Ftr1). Paracoccidioides spp. genomes do not present an Ftr1 homolog. However, this fungus expresses zinc regulated transporter homologs (Zrts), members of the ZIP family of membrane transporters that are able in some organisms to transport zinc and iron. A 2,3,5-triphenyltetrazolium chloride (TTC)-overlay assay indicates that both Pb01 and Pb18 express a ferric reductase activity; however, 59Fe uptake assays indicate that only in Pb18 is this activity coupled to a reductase-dependent iron uptake pathway. In addition, Zrts are up-regulated in iron deprivation, as indicated by RNAseq and qRT-PCR using Pb01 transcripts. RNAseq strategy also demonstrated that transcripts related to siderophore uptake and biosynthesis are up-regulated in iron-deprived condition. The data suggest that the fungus could use both a non-classical RIA, comprising ferric reductases and Fe/Zn permeases (Zrts), and siderophore uptake pathways under iron-limited conditions. The study of iron metabolism reveals novel surface molecules that could function as accessible targets for drugs to block iron uptake and, consequently, inhibit pathogen's proliferation.

Reductive iron assimilation and intracellular siderophores assist extracellular siderophore-driven iron homeostasis and virulence.

Iron is an essential nutrient and prudent iron acquisition and management are key traits of a successful pathogen. Fungi use nonribosomally synthesized secreted iron chelators (siderophores) or reductive iron assimilation (RIA) mechanisms to acquire iron in a high affinity manner. Previous studies with the maize pathogen Cochliobolus heterostrophus identified two genes, NPS2 and NPS6, encoding different nonribosomal peptide synthetases responsible for biosynthesis of intra- and extracellular siderophores, respectively. Deletion of NPS6 results in loss of extracellular siderophore biosynthesis, attenuated virulence, hypersensitivity to oxidative and iron-depletion stress, and reduced asexual sporulation, while nps2 mutants are phenotypically wild type in all of these traits but defective in sexual spore development when NPS2 is missing from both mating partners. Here, it is reported that nps2nps6 mutants have more severe phenotypes than both nps2 and nps6 single mutants. In contrast, mutants lacking the FTR1 or FET3 genes encoding the permease and ferroxidase components, respectively, of the alternate RIA system, are like wild type in all of the above phenotypes. However, without supplemental iron, combinatorial nps6ftr1 and nps2nps6ftr1 mutants are less virulent, are reduced in growth, and are less able to combat oxidative stress and to sporulate asexually, compared with nps6 mutants alone. These findings demonstrate that, while the role of RIA in metabolism and virulence is overshadowed by that of extracellular siderophores as a high-affinity iron acquisition mechanism in C. heterostrophus, it functions as a critical backup for the fungus.

Genome mining and functional genomics for siderophore production in Aspergillus niger.

Iron is an essential metal for many organisms, but the biologically relevant form of iron is scarce because of rapid oxidation resulting in low solubility. Simultaneously, excessive accumulation of iron is toxic. Consequently, iron uptake is a highly controlled process. In most fungal species, siderophores play a central role in iron handling. Siderophores are small iron-specific chelators that can be secreted to scavenge environmental iron or bind intracellular iron with high affinity. A second high-affinity iron uptake mechanism is reductive iron assimilation (RIA). As shown in Aspergillus fumigatus and Aspergillus nidulans, synthesis of siderophores in Aspergilli is predominantly under control of the transcription factors SreA and HapX, which are connected by a negative transcriptional feedback loop. Abolishing this fine-tuned regulation corroborates iron homeostasis, including heme biosynthesis, which could be biotechnologically of interest, e.g. the heterologous production of heme-dependent peroxidases. Aspergillus niger genome inspection identified orthologues of several genes relevant for RIA and siderophore metabolism, as well as sreA and hapX. Interestingly, genes related to synthesis of the common fungal extracellular siderophore triacetylfusarinine C were absent. Reverse-phase high-performance liquid chromatography (HPLC) confirmed the absence of triacetylfusarinine C, and demonstrated that the major secreted siderophores of A. niger are coprogen B and ferrichrome, which is also the dominant intracellular siderophore. In A. niger wild type grown under iron-replete conditions, the expression of genes involved in coprogen biosynthesis and RIA was low in the exponential growth phase but significantly induced during ascospore germination. Deletion of sreA in A. niger resulted in elevated iron uptake and increased cellular ferrichrome accumulation. Increased sensitivity toward phleomycin and high iron concentration reflected the toxic effects of excessive iron uptake. Moreover, SreA-deficiency resulted in increased accumulation of heme intermediates, but no significant increase in heme content. Together with the upregulation of several heme biosynthesis genes, these results reveal a complex heme regulatory mechanism.

Infection Structure-Specific Reductive Iron Assimilation Is Required for Cell Wall Integrity and Full Virulence of the Maize Pathogen Colletotrichum graminicola

Ferroxidases are essential components of the high-affinity reductive iron assimilation pathway in fungi. Two ferroxidase genes, FET3-1 and FET3-2, have been identified in the genome of the maize anthracnose fungus Colletotrichum graminicola. Complementation of growth defects of the ferroxidase-deficient Saccharomyces cerevisiae strain Δfet3fet4 showed that both Fet3-1 and Fet3-2 of C. graminicola represent functional ferroxidases. Expression of enhanced green fluorescent protein fusions in yeast and C. graminicola indicated that both ferroxidase proteins localize to the plasma membrane. Transcript abundance of FET3-1 increased dramatically under iron-limiting conditions but those of FET3-2 were hardly detectable. Δfet3-1 and Δfet3-2 single as well as Δfet3-1/2 double-deletion strains were generated. Under iron-sufficient or deficient conditions, vegetative growth rates of these strains did not significantly differ from that of the wild type but Δfet3-1 and Δfet3-1/2 strains showed increased sensitivity to reactive oxygen species. Furthermore, under iron-limiting conditions, appressoria of Δfet3-1 and Δfet3-1/2 strains showed significantly reduced transcript abundance of a class V chitin synthase and exhibited severe cell wall defects. Infection assays on intact and wounded maize leaves, quantitative data of infection structure differentiation, and infection stage-specific expression of FET3-1 showed that reductive iron assimilation is required for appressorial penetration, biotrophic development, and full virulence.