Iron Starvation Induces Ferricrocin Production and the Reductive Iron Acquisition System in the Chromoblastomycosis Agent Cladophialophora carrionii

Iron is a micronutrient required by almost all living organisms. Despite being essential, the availability of this metal is low in aerobic environments. Additionally, mammalian hosts evolved strategies to restrict iron from invading microorganisms. In this scenario, the survival of pathogenic fungi depends on high-affinity iron uptake mechanisms. Here, we show that the production of siderophores and the reductive iron acquisition system (RIA) are employed by Cladophialophora carrionii under iron restriction. This black fungus is one of the causative agents of chromoblastomycosis, a neglected subcutaneous tropical disease. Siderophore biosynthesis genes are arranged in clusters and, interestingly, two RIA systems are present in the genome. Orthologs of putative siderophore transporters were identified as well. Iron starvation regulates the expression of genes related to both siderophore production and RIA systems, as well as of two transcription factors that regulate iron homeostasis in fungi. A chrome azurol S assay demonstrated the secretion of hydroxamate-type siderophores, which were further identified via RP-HPLC and mass spectrometry as ferricrocin. An analysis of cell extracts also revealed ferricrocin as an intracellular siderophore. The presence of active high-affinity iron acquisition systems may surely contribute to fungal survival during infection.


Introduction
Iron is necessary for essentially all living organisms. Due to its oxidation states, Fe 2+ and Fe 3+ , this metal has the ability to gain or lose electrons, an important feature in many cellular processes, including cellular respiration [1]. Regardless of its essentiality, the Fe 3+ resulting from the oxidation of Fe 2+ is insoluble in water and with a neutral pH [2,3]. In addition, iron in excess triggers, via a Fenton/Haber Weiss reaction [4], the formation of hydroxyl radicals that are highly reactive and cause cell damage. Therefore, iron acquisition and homeostasis are based on ferric iron solubilization, transport across the plasma membrane into the cytosol and regulation of the uptake and utilization processes in order to avoid toxicity.
Pathogenic fungi face an iron-deprived scenario inside their hosts, which employ diverse strategies of nutritional immunity to decrease plasma and phagosome iron contents [5,6]. The relevance of this metal in the host-pathogen relationship is demonstrated by the increase in the frequency and severity of infections in people with iron overload [7]. induces both systems in this fungus. Considering the low iron availability imposed by the nutritional immunity, those high-affinity iron acquisition strategies may be relevant for pathogenesis.

Strain and Growth Conditions
All experiments were conducted with the C. carrionii KSF strain. The fungus was maintained in Potato Dextrose Agar (PDA) at 22 • C for seven days. Conidia were produced by inoculating mycelium fragments into Potato Dextrose Broth (PDB; 20% potato infusion, 2% glucose), under agitation at 150 rpm, at 28 • C, for seven days. Then, the conidia enrichment was achieved through the glass wool filtering of cultures [34]. The filtrate was centrifuged at 3000× g for 5 min, and conidia were resuspended in saline (0.9% NaCl). The cell suspensions were quantified in a Neubauer chamber.

In Silico Analysis of Siderophore and RIA-Related Sequences
The search for genes related to siderophore acquisition, the RIA system and transcriptional regulation was based on sequence similarity defined using the protein BLAST tool at FungiDB (https://fungidb.org/fungidb/app/workspace/blast/new accessed on 15 January 2021). Amino acid sequences of A. fumigatus, Aspergillus nidulans, S. cerevisiae and C. neoformans were used as input in the analysis (sequences IDs are found in Table  S1). Expectation values less than or equal to 10 −10 were considered the cut-off. The definition of orthologs was also considered based on the orthology analysis at FungiDB. Transmembrane segments of Ftr and Fet orthologs were identified using Protter (https: //wlab.ethz.ch/protter/start/ accessed on 15 January 2021) [36]. Conserved motifs and amino acid residues in protein sequences were inspected manually.
The DNA Pattern Find tool from Sequence Manipulation Suite (http://www.bioinformatics. org/sms2/dna_pattern.html accessed on 15 January 2021) was employed for the identification of consensus sequences in the promoters of genes. For that, 1500 bp regions upstream the start codon of hapX, sreA, sidI, sidA1, ftrA1 and ftrA2 were used. Protein sequence alignments were obtained with MAFFT (https://mafft.cbrc.jp/alignment/server/ accessed on 15 January 2021), and the IQ-TREE web service (http://iqtree.cibiv.univie.ac.at/ accessed on 15 January 2021) was used for construction of the siderophore transporter phylogenetic tree [37].

RNA Isolation and Quantitative Real-Time PCR (qRT-PCR) Analysis
Cells grown in iron-deprived (50 µM BPS) and control [100 µM (NH 4 ) 2 Fe(SO 4 ) 2 ] conditions were submitted to total RNA extraction. The lysis was carried out with trizol (TRI Reagent ® , Sigma-Aldrich, St. Louis, MO, USA) and 0.5 µm glass beads in a mechanical beater (Mini-Beadbeater, Biospec Products Inc., Bartlesville, OK, USA) according to the manufacturer's instructions. To avoid amplification due to genomic DNA, RNA was treated with DNAse I (Promega Corporation, Madison, WI, USA) and then used for cDNA synthesis (High-Capacity cDNA Reverse Transcription Kits, AppliedBiosystems Inc., Vilnius, Lithuania). The cDNA was used in qRT-PCR assays using SYBR Green PCR Master Mix (Life Technologies, Foster City, CA, USA) in the StepOnePlus TM system (Applied Biosystems Carlsbad, CA, USA). The reaction conditions were as follows: 40 cycles (95 • C for 15 s/60 • C for 1 min), after initial denaturation at 95 • C for 10 min. The mRNA quantification was conducted in triplicate. The relative expression levels of transcripts were defined using the relative standard curve method [38]. The expression of the genes of interest were normalized with transcript levels of the actin gene (CLCR_02925). A melting curve analysis confirmed the primer specificity. Table S2 lists the oligonucleotides used. A student's t-test was conducted to compare the expression levels of two conditions (p ≤ 0.05).

Overlay-Chrome Azurol S (O-CAS) Assay
The O-CAS assay [39] was used to detect siderophores secreted by C. carrioni. Solid 10-day-old MMcM mycelium cultures under iron-deprivation (without iron supplementation) and in the presence of iron [100 µM (NH 4 ) 2 Fe(SO 4 ) 2 ] were prepared. The cultures were covered with 15 mL of O-CAS solution, monitored for changes in color and then photographed. All the reagents necessary for the CAS solution were purchased from Sigma-Aldrich, St. Louis, MO, USA. Residual traces of iron were removed from glassware with acid treatment [40].

Ferric Perchlorate Assay
Supernatants from C. carrioni 30-day-old cultures under iron deprivation (without iron supplementation) and control conditions [100 µM (NH 4 ) 2 Fe(SO 4 ) 2 ] were submitted to the ferric perchlorate colorimetric assay [41]. The samples were filtered (0.22 µM pore membrane) and lyophilized. Dried supernatants were resuspended in water (MilliQ-water) using one-tenth of the original volume. For the assay, 250 µL of samples was added to 1.250 mL of 5 mM Fe(ClO 4 ) 3 . The same volumes of sterile MMcM with and without iron were used as controls. The samples were incubated at room temperature for 5 min and monitored for a change in color.

Siderophore Identification
To induce siderophore production, C. carrionii mycelia were cultivated in an iron-free MMcM medium for 30 days. Culture supernatants were collected and filtered in 0.22 µM membranes in order to remove any fungal cells. Samples were freeze-dried and then resuspended in one-tenth of the initial volume. These samples were used for extracellular siderophore identification. For intracellular siderophore identification, cells were collected via centrifugation at 3000× g and 4 • C for 15 min and washed 5 times with PBS to remove extracellular siderophores. Fungal cells were lysed through mechanical disruption in a mortar, and the samples were kept frozen by adding liquid nitrogen. The lysate containing the intracellular siderophores was resuspended in sterile water (1 mL/4 mL of culture). Cell debris was removed via centrifugation, and the obtained supernatant was filtered in 0.22 µM and lyophilized. For identification assays, the lyophilized material was resuspended in one-tenth of the original volume in MilliQ water.
For siderophore identification, 100 mM FeSO 4 was firstly added to the samples. Then, the siderophores were purified with an Amberlite XAD-16 column (Rohom and Haas, Philadelphia, PA, USA). Elution was performed with methanol, and the eluates were dried under a vacuum. Then, 100 µL of water was used for pellet solubilization; a 10 µL aliquot was loaded onto the HPLC containing a reversed-phase column (Nucleosil C18, 5 mm, 4 × 250 mm, Macherey-Nagel, Duren, Germany). The chromatography was performed as described [42]. The collected fractions were vacuum-dried, resuspended in a 50 methanol/0.1% formic acid solution, and analyzed in the LTQ Velos ion trap mass spectrometer (Thermo Fisher Scientific Waltham, MA, USA) equipped with an electrospray source (ESI-MS, Electrospray Ionization Mass Spectrometry). The electrospray source parameters were 4.0 kV and 270 • C.

Genes for High-Affinity Iron Acquisition Are Conserved in C. carrionii
As a first step in the investigation of iron acquisition mechanisms in C. carrionii, an orthology analysis was conducted. Genes of A. fumigatus, S. cerevisiae, A. nidulans and C. neoformans were used in the search (Table S1). All genes required for the synthesis of hydroxamate siderophores were identified in the C. carrionii genome (Table 1), with the exception of sidG, involved in TAFC biosynthesis in A. fumigatus. A genome location inspection indicated that genes are localized near each other ( Figure 1A). Two ornithine monooxygenase orthologs were identified in C. carrionii. One of them, described here as sidA1, is located in and shares the promoter region with NRPS sidC. A second gene cluster includes five orthologs involved in siderophore biosynthesis: NRPS sidD, transacylase sidF, enoyl-CoA hydratase sidH, acyl-CoA synthetase sidI and ornithine monooxygenase (described here as sidA2). Interestingly, this cluster also harbors a putative ABC multidrug transporter (CLCR_02231). Table 1. Ortholog genes putatively involved in high-affinity iron acquisition in C. carrionii # .

Iron Related Pathway
Cladophialophora carrionii Genes * Siderophore biosynthesis Reductive iron acquisition (RIA) Regulation of iron metabolism CLCR_05818 (SreA) CLCR_01554 (HapX) Considering that C. carrionii can synthesize siderophores, according to the genom analysis, we next searched for the presence of siderophore transporters. A. fumigatus, nidulans, S. cerevisiae and C. neoformans amino acid sequences were used in the similari analysis (Table S1). C. carrionii presents six putative siderophore transporters (Table Considering that C. carrionii can synthesize siderophores, according to the genome analysis, we next searched for the presence of siderophore transporters. A. fumigatus, A. nidulans, S. cerevisiae and C. neoformans amino acid sequences were used in the similarity analysis (Table S1). C. carrionii presents six putative siderophore transporters ( Table 1), one of which is localized near the sidA2 in the siderophore biosynthetic cluster (CLCR_02321, Figure 1A). The number of transmembrane domains (TMs) varies from 13 to 14, which is in accordance with the predicted TMs (12)(13)(14) in MFS members [9]. Phylogeny analysis suggested that C. carrionii carries orthologs to MirB (CLCR_03365), MirD (CLCR_02321), Sit1 (CLCR_01135) and Sit2 (CLCR_06890 and CLCR_03414) ( Figure 2). The C. carrionii transporter CLCR_04614 did not cluster with any of the analyzed sequences. A deeper investigation revealed that CLCR_04614 shares a higher sequence similarity (E-value 3 × 10 −156 ) with Afu3g01360, an A. fumigatus MFS transporter for which the function is not well defined [43]. In addition to siderophore production and uptake, the RIA system allows for highaffinity iron acquisition in fungi. This system comprises three groups of proteins: ferric reductases (Fres), multicopper ferroxidases (Fets) and high-affinity iron permeases (Ftrs). Fres are characterized by the presence of three conserved domains: a ferric reductase domain (FRD) in the transmembrane regions, a FAD-binding and NADPH-binding domains. The last two are localized in the C-terminal cytoplasmic region of Fres [44]. The FRD domain, predicted using the Pfam database (PF01794), was searched against the C. carrionii amino acid sequence database in FungiDB (h ps://fungidb.org/fungidb/app/, accessed on 15 January 2021). Twelve sequences were retrieved, eleven of which contain both the FAD-binding (PF08022/IRP017927) and the NADPH-binding domains (PF08030) ( Table 2). The number of transmembrane domains varies from five to eight, and three sequences have predicted signal peptides.  In addition to siderophore production and uptake, the RIA system allows for highaffinity iron acquisition in fungi. This system comprises three groups of proteins: ferric reductases (Fres), multicopper ferroxidases (Fets) and high-affinity iron permeases (Ftrs). Fres are characterized by the presence of three conserved domains: a ferric reductase domain (FRD) in the transmembrane regions, a FAD-binding and NADPH-binding domains. The last two are localized in the C-terminal cytoplasmic region of Fres [44]. The FRD domain, predicted using the Pfam database (PF01794), was searched against the C. carrionii amino acid sequence database in FungiDB (https://fungidb.org/fungidb/app/, accessed on 15 January 2021). Twelve sequences were retrieved, eleven of which contain both the FAD-binding (PF08022/IRP017927) and the NADPH-binding domains (PF08030) ( Table 2). The number of transmembrane domains varies from five to eight, and three sequences have predicted signal peptides.  Based on similarity analysis with S. cerevisiae and A. fumigatus sequences (Table S1), we found that C. carrionii carries two orthologs of the iron permease Ftr1/FtrA and two orthologs of the ferroxidase Fet3/FetC (Table 1). C. carrionii sequences were defined as FtrA1, FtrA2, FetC1 and FetC2. Each member of the permease-ferroxidase pairs, FtrA1-FetC1 and FtrA2-FetC2, is localized next to each other in the genome and shares the same promoter ( Figure 1B). Iron permeation by the Ftr1 depends on specific motifs, including the elements REXLE and DXXE [54,55]. Both were identified in the amino acid sequences of the two putative C. carrionii permeases (CLCR_07299 and CLCR_03583) ( Figure 3A). The REXLE element is located in the transmembrane domains 1 and 4, while the DXXE domain is found in the extracellular loop between transmembrane domains 6 and 7. Additionally, the topology of putative C. carrionii Ftrs is similar to that found in S. cerevisiae and C. albicans [54,55], presenting seven transmembrane domains with an extracellular N-terminal and intracellular C-terminal ( Figure S1A,B).

NAD-Binding
Ferric iron transported by Ftr1 results from the action of Fet3. This is a type 1 membrane protein that contains only one transmembrane domain. The N-terminus is extracellularly localized while the C-terminus is cytoplasmic [56,57]. As an MCO, Fet3 presents three types of copper-binding sites that allow for its ferroxidation activity [58]. The two putative C. carrionii ferroxidases (CLCR_03382 and CLCR_06747) present the same topology as S. cerevisiae Fet3, with three MCO domains localized in the extracellular N-terminus ( Figure S1C,D). In addition to the copper-binding sites, Fet3 has some specific amino acid residues involved in (I) Fe 2+ binding, (II) the coupling of electron transfer and/or (III) Fe 3+ transfer to Ftr1. Those residues include E185, D278, D283, Y354 and D409. E185 and D409 have been demonstrated to play a role in all three processes [58]. Y354 and D278 is necessary for the ferroxidase activity of Fet3 [59,60], and D283 is related to the trafficking of Fe 3+ to Ftr1 [58]. Sequence alignment of S. cerevisiae Fet3, A. fumigatus FetC and the predicted Fets of C. carrionii demonstrated that all the above-mentioned amino acid residues are conserved in this black fungus ( Figure 3B). Altogether, these findings strongly suggest that C. carrionii may acquire iron via the RIA system.
Siderophore-based iron acquisition, as well as the RIA system, is regulated by iron availability via SreA and HapX transcription factors, for which orthologs were also identified in the C. carrionii genome (Table 1 and Table S1). SreA is a GATA-type regulator that represses high-affinity iron uptake under iron sufficiency, while the bZIP factor HapX induces RIA and siderophore uptake in low iron availability [61,62]. Taken together, our in silico approach demonstrated that the machinery responsible for high-affinity iron uptake is conserved in this black fungus.
the elements REXLE and DXXE [54,55]. Both were identified in the amino acid sequences of the two putative C. carrionii permeases (CLCR_07299 and CLCR_03583) ( Figure 3A). The REXLE element is located in the transmembrane domains 1 and 4, while the DXXE domain is found in the extracellular loop between transmembrane domains 6 and 7. Additionally, the topology of putative C. carrionii Ftrs is similar to that found in S. cerevisiae and C. albicans [54,55], presenting seven transmembrane domains with an extracellular N-terminal and intracellular C-terminal ( Figure S1A,B).

Iron Availability Regulates the Expression of Iron Acquisition Genes
Genes related to iron acquisition are usually regulated by the availability of this metal in the extracellular environment. To test this hypothesis in C. carrionii, the expression of selected genes was determined under iron-deprived conditions. As shown in Figure 4A, sidA1 and sidI, representative of siderophore biosynthesis, as well as ftrA1 and ftrA2, involved in RIA, were induced in the presence of the iron chelator BPS. The transcription factors sreA and hapX were regulated by iron as well. sreA, known to repress the iron acquisition system in iron-sufficient conditions, was repressed under iron starvation, while hapX, an inducer of iron acquisition, was positively regulated in BPS, as expected.
An inspection of the promoter regions of the regulated genes ( Figure 4B) revealed the presence of consensus sequences recognized by SreA (HGATAR, and its extended version ATCWGATAA) [42,61,63] and HapX (CCAAT) [64], indicating that both transcription factors may regulate iron acquisition in C. carrionii. sidA1 shares the 5 upstream region with the putative NRPS SidC (CLCR_04490), while the promoters of the permeases ftrA1 and ftrA2 are shared with fetC1 and fetC2, respectively ( Figure 1). Therefore, we can infer that sidC, fetC1 and fetC2 are regulated by iron availability as well. An inspection of the promoter regions of the regulated genes ( Figure 4B) revealed the presence of consensus sequences recognized by SreA (HGATAR, and its extended version ATCWGATAA) [42,61,63] and HapX (CCAAT) [64], indicating that both transcription factors may regulate iron acquisition in C. carrionii. sidA1 shares the 5′ upstream region with the putative NRPS SidC (CLCR_04490), while the promoters of the permeases ftrA1 and ftrA2 are shared with fetC1 and fetC2, respectively ( Figure 1). Therefore, we can infer that sidC, fetC1 and fetC2 are regulated by iron availability as well.

C. carrionii Produces and Secretes Hydroxamate Siderophores under Low-Iron Conditions
Based on the genomic information that C. carrionii is capable of synthesizing siderophores, this phenotype was investigated. Cells were incubated for 10 days in solid MMcM medium supplemented (100 µM) or not with iron. Next, the plates were overlaid with chrome azul S (O-CAS), a blue solution for which color is changed in the presence of siderophores since these molecules "steal" the Fe ion from CAS. As shown in Figure 5A, in 15-day-old cultures, CAS changed from blue to orange in low-iron medium. This indicates that siderophores were produced and secreted by C. carrionii. As expected, the presence of iron blocked siderophore biosynthesis.

C. carrionii Produces and Secretes Hydroxamate Siderophores under Low-Iron Conditions
Based on the genomic information that C. carrionii is capable of synthesizing siderophores, this phenotype was investigated. Cells were incubated for 10 days in solid MMcM medium supplemented (100 µM) or not with iron. Next, the plates were overlaid with chrome azul S (O-CAS), a blue solution for which color is changed in the presence of siderophores since these molecules "steal" the Fe ion from CAS. As shown in Figure 5A, in 15-day-old cultures, CAS changed from blue to orange in low-iron medium. This indicates that siderophores were produced and secreted by C. carrionii. As expected, the presence of iron blocked siderophore biosynthesis.
The development of an orange color from CAS solution indicates the presence of hydroxamate-type siderophores [39]. To confirm the nature of siderophores produced by C. carrionii, the ferric perchlorate assay was performed. A Fe(ClO 4 ) 3 solution was mixed with concentrated supernatants of 30-day cultures in low-iron medium. The appearance of an orange-red color in the −Fe supernatant revealed the presence of hydroxamates ( Figure 5B). As expected, the +Fe condition inhibited the production of siderophores. Non-inoculated MMcM mixed with Fe(ClO 4 ) 3 is shown as an additional control.

Ferricrocin Is Produced by C. carrionii as an Intra-and Extracellular Siderophore
Aiming for the identification of the hydroxamate siderophores secreted by C. carrionii, culture supernatants were submitted to RP-HPLC and mass spectrometry analysis. Following incubation in iron-deprived MMcM for 30 days, the culture supernatant was filter-sterilized and concentrated ten times with ultrapure water. FeSO 4 was then added to the sample in order to saturate the secreted siderophores. An analysis via RP-HPLC demonstrated the presence of a compound displaying absorption at 430 nm, typical of ironsaturated siderophores. The retention time at which the peak was observed matched that of ferricrocin, a hydroxamate siderophore usually utilized for iron storage ( Figure 6). To confirm the siderophore identity, the RP-HPLC peak was subjected to high-resolution mass spectrometry. The presence of ferricrocin (C 28 H 47 N 9 O 13 Fe) was reinforced through the visualization of four different ionizing adducts: H + (m/z = 771.2471), NH 4 + (m/z = 788.2733), Na + (m/z = 793.2279) and K + (m/z = 809.2021) ( Figure S2).
Based on the genomic information that C. carrionii is capable of synthesizing siderophores, this phenotype was investigated. Cells were incubated for 10 days in solid MMcM medium supplemented (100 µM) or not with iron. Next, the plates were overlaid with chrome azul S (O-CAS), a blue solution for which color is changed in the presence of siderophores since these molecules "steal" the Fe ion from CAS. As shown in Figure 5A, in 15-day-old cultures, CAS changed from blue to orange in low-iron medium. This indicates that siderophores were produced and secreted by C. carrionii. As expected, the presence of iron blocked siderophore biosynthesis.  The development of an orange color from CAS solution indicates the presence of hydroxamate-type siderophores [39]. To confirm the nature of siderophores produced by C. carrionii, the ferric perchlorate assay was performed. A Fe(ClO4)3 solution was mixed with concentrated supernatants of 30-day cultures in low-iron medium. The appearance of an orange-red color in the −Fe supernatant revealed the presence of hydroxamates ( Figure 5B). As expected, the +Fe condition inhibited the production of siderophores. Noninoculated MMcM mixed with Fe(ClO4)3 is shown as an additional control.

Ferricrocin Is Produced by C. carrionii as an Intra-and Extracellular Siderophore
Aiming for the identification of the hydroxamate siderophores secreted by C. carrionii, culture supernatants were submi ed to RP-HPLC and mass spectrometry analysis. Following incubation in iron-deprived MMcM for 30 days, the culture supernatant was filter-sterilized and concentrated ten times with ultrapure water. FeSO4 was then added to the sample in order to saturate the secreted siderophores. An analysis via RP-HPLC demonstrated the presence of a compound displaying absorption at 430 nm, typical of iron-saturated siderophores. The retention time at which the peak was observed matched that of ferricrocin, a hydroxamate siderophore usually utilized for iron storage ( Figure 6). To confirm the siderophore identity, the RP-HPLC peak was subjected to highresolution mass spectrometry. The presence of ferricrocin (C28H47N9O13Fe) was reinforced through the visualization of four different ionizing adducts: H + (m/z = 771.2471), NH4 + (m/z = 788.2733), Na + (m/z = 793.2279) and K + (m/z = 809.2021) ( Figure S2). Next, the presence of intracellular siderophores was evaluated in cell extracts obtained after incubation for 30 days in iron-free MMcM. Following centrifugation, cells were washed five times with PBS to eliminate extracellular siderophores and processed as described in the Materials and Methods. RP-HPLC analysis was conducted after saturation of the sample with FeSO 4 , and a peak compatible with ferricrocin was again identified (Figure 6, third panel) and confirmed via high-resolution mass spectrometry.

Discussion
Although the importance of siderophore production by black yeasts in the adaptation to the human host was cited a long time ago [33], the molecular strategies used by those fungi for iron acquisition are still poorly explored. Here, we showed that C. carrionii KSF expresses two high-affinity iron-acquisition systems, siderophore production and RIA, under iron limiting conditions. Firstly, a homology analysis was carried out to search for gene orthologs to those involved in iron assimilation in other fungi. The machinery for the biosynthesis and uptake of siderophores was identified. Most of the genes are clustered, a characteristic hallmark of secondary metabolite production, found in various fungal classes belonging to basidiomycetes and the Ascomycota phylum [42,65,66]. This arrangement enables the genes to be co-regulated. C. carrionii presents two sidA orthologs, a common feature in some plant and human pathogens [9], including the closely related Cladophialophora bantiana [66]. Genomic analyses of C. bantiana isolated from a brain abscess revealed the presence of two clusters of siderophore biosynthesis genes. One includes sidA and sidC orthologs and the other is represented by sidD, sidF, sidI, sidA and a siderophore transporter [66]. In C. carrionii, we found the same pattern of gene distribution into two clusters, with the addition of sidH, which shares the 5 region with sidF. Two siderophore biosynthetic clusters were also identified in the black yeast-like Aureobasidium melanogenum, one including sidA and sidC orthologs and the other composed by sidD, sidF and sidI [21].
An orthology survey indicated six putative siderophore transporters in the C. carrionii genome. Phylogenetic analysis, however, showed that CLCR_04614 is not closely related to a siderophore transporter with a proven function in other fungi. In contrast, it seems to be more similar to an uncharacterized A. fumigatus MFS transporter [43]. The number of siderophore transporter paralogs in C. carrionii is equivalent to that in A. fumigatus, which encodes five transporters [61], and the basidiomycete C. neoformans, which presents six [9]. CLCR_02321 is localized within the larger cluster found in C. carrionii and shares its promoter region with the ornithine monooxygenase-encoding gene sidA2. The presence of a siderophore transporter in a siderophore biosynthesis cluster is a feature conserved in other ascomycetes, including C. bantiana [66], Blastomyces dermatitidis [67], A. fumigatus [61], Histoplasma capsulatum [63], A. melanogenum [21] and the Paracoccidioides genus [42].
The recognition of siderophores by the plasma membrane transporters is highly stereospecific [68]. C. carrionii presents six putative siderophore transporters, five of which (MirB, MirD, Sit1 and Sit2) are orthologs to A. fumigatus proteins for which substrate specificity has been elucidated. In A. fumigatus, MirB and MirD transport TAFC and fusarinine C, respectively [43], while Sit1 and Sit2 have overlapping and unique substrate specificities. Both Sit1 and Sit2 mediate the transport of ferrichrome, ferricrocin and coprogen-type siderophores, whereas only Sit1 transports the bacterial ferroxiamines [69]. The last are considered xenosiderophores, that is, those produced by other microorganisms. Considering the diversity of siderophore transporters and putative specificities in C. carrionii, we may infer that this fungus is able to take up a variety of molecules, including its native and the xenosiderophores. In fact, various fungal species that lack the siderophore synthesis machinery, like C. albicans, Candida glabrata, S. cerevisiae and C. neoformans, have siderophores transporters for the uptake of xenosiderophores [70].
In the environment, C. carrionii can be found in soil, where ferrioxamines, ferricrocin and ferrichromes are abundantly found [71], decaying matter, wood and spines and plants, mainly in Cactaceae [72,73]. In an analysis of cactus microbiomes, Fonseca-Garcia and Coleman-Derr [74] identified bacteria able to produce siderophores. These facts reinforce the hypothesis that C. carrionii could be able to use xenosiderophores in the environment. Additionally, secondary bacterial infections are the most frequent complications of CBM [75] and siderophores produced by these prokaryotes may be a source of iron for C. carrionii in the mammalian host.
Beyond siderophore synthesis and uptake, the RIA system is also conserved in C. carrionii. Twelve sequences containing the FRD domains, a hallmark of ferric reductases, were identified in the C. carrionii genome. Most of them belong to ortholog groups that harbor characterized Fres in other fungi. S. cerevisiae has eight FRE orthologs, FRE1-FRE8. FRE1 and FRE2 are involved in both ferric and cupric ion reductions [45,46,76], FRE3-FRE6 are regulated specifically by iron, while FRE7 is specifically regulated by copper [77]. FRE8 is related to both iron and copper homeostasis [53]. Additionally, FRE3 and FRE4 are required for the uptake of Fe 3+ bound to siderophores via the reduction of this ion to Fe 2+ [78]. The several Fre orthologs in C. carrionii may correlate with diversity in function, i.e., the reduction of free iron and copper, as well as siderophore-bound iron. Further investigations are necessary to test this hypothesis.
Two orthologs of Ftr1/FtrA and Fet3/FetC were identified in C. carrionii. FtrA and FetC are divergently transcribed from the same intergenic region, a characteristic conserved in many fungal divisions [79], including the black yeast-like A. melanogenum [21]. Three ferroxidases and two iron permeases were found in the genome of C. bantiana isolated from the brain abscess of a patient. Two of the Fets are located next to the two Ftrs [66]. C. carrionii ftrA1 and ftrA2 are up-regulated upon iron deprivation. As both share an intergenic region with fetC1 and fetC2, respectively, we assume that the oxidases are also regulated by iron availability. The co-expression of Ftr-Fet occurs in many fungal species, including A. fumigatus [14] and the mucormycosis agent Lichtheimia corymbifera [79]. Indeed, in S. cerevisiae and C. albicans, the co-expression of Ftr1 is required for the trafficking of Fet3 to the plasma membrane and the efficient localization of Frt1 at the cell surface depends on the co-expression of Fet [55,80].
The adaptation to iron availability in A. fumigatus is regulated by SreA and HapX [61,62]. This transcriptional control is conserved in fungi, and the functions of SreA and/or HapX orthologs have been described in model fungal species, as well as in human and plant pathogens [81][82][83][84][85][86][87]. In most pathogenic species, these transcription factors are virulence determinants. Here, we demonstrated that iron availability regulates sreA and hapx expression in an opposite way. While sreA is repressed, hapX is induced by iron starvation. This agrees with the postulated negative feed-back loop that connects these two proteins in A. fumigatus: SreA is induced under iron sufficiency and represses hapX, whereas HapX is positively regulated by iron limitation and represses sreA. The functional connection of SreA and HapX in C. carrionii is reinforced by the presence of regulatory sequences in their promoter regions. Siderophore biosynthesis (sidA1 and sidI)and RIA (ftrA1 and ftrA2)-related genes were up-regulated by iron starvation, probably via the action of HapX. A. fumigatus sidA mutants are unable to grow upon iron deprivation and are avirulent in a mouse model of invasive aspergillosis [14,88], and sidI deletion also impacts growth under low iron in this mold [11]. Aureobasidium species present SreA and HapX orthologs that are regulated by iron, as in A. fumigatus. Additionally, siderophore biosynthesis genes harbor SreA and HapX consensus sequences in their promoter regions, confirming the role of these two transcription factors in the regulation of iron homeostasis in these black yeast-like strains [21,89]. Indeed, sidA expression was derepressed under iron sufficiency in an Sre1-knockout A. pullulans strain [89].
The induction of siderophore and RIA acquisition systems was also reported in the black yeast Exophiala dermatitidis recovered from an ex vivo skin model of infection [90]. This fact confirms that iron limitation is faced by implantation pathogens, like C. carrionii, when in contact with the host. A C. albicans strain defective in a siderophore transporter is less efficient in the invasion of keratinocyte layers, which demonstrates the fundamental role of siderophore uptake in invasion and the penetration of epithelial tissue [91]. In addition, the lack of a functional RIA system makes C. albicans and C. neoformans attenuated for virulence [15,17,18] A comparative genomic survey of pathogenic and environmental siblings of Cladophialophora and the Fonsecaea genus identified an enrichment of genes related to siderophore biosynthesis and uptake in the clinical species. This suggests a role of these cellular processes in virulence since no enrichment was observed in the environmental species [92].
Considering that the siderophore biosynthesis pathway is up-regulated upon iron deficiency, the production of hydroxamates was evaluated in vitro. The O-CAS and ferric perchlorate assays confirmed that C. carrionii produces and secretes hydroxamate-type siderophores. This class includes four families with distinct structures: fusarinines, coprogens, ferrichromes and rhodotorulic acid [9]. An analysis of supernatants of iron-deprived cultures revealed the presence of ferricrocin, a typical intracellular siderophore that belongs to the ferrichrome family, of which the synthesis depends on SidC (CLCR_04490 in C. carrionii) [93]. Ferricrocin is used as an iron-storage compound in Aspergillus species and Neurospora crassa [93,94] and is involved in intra-and transcellular iron distribution during conidiogenesis in A. fumigatus [95]. By contrast, in the plant pathogen Magnaporthe grisea, ferricrocin is dispensable for conidiation but is essential for pathogenesis [96]. A lack of ferricrocin also causes attenuation of virulence in a murine aspergillosis model [10]. Indeed, it was recently demonstrated that in A. fumigatus, ferricrocin contributes to iron acquisition as an extracellular siderophore, in addition to the intracellular role described previously [97]. The black yeast Aureobasidium melanogenum HN6.2 strain produces ferricrocin as an intraand extracellular siderophore as well [21]. Sit1 and Sit2 of A. fumigatus [69] and Sit1 of C. albicans [91] are the membrane transporters responsible that mediate ferricrocin uptake. The presence of orthologs to those proteins in C. carrionii suggests that secreted ferricrocin is further utilized by the fungus. As expected, the presence of ferricrocin was confirmed in C. carrionii cellular extracts as well.
In summary, our data demonstrate that C. carrionii employs two high-affinity iron uptake systems (Figure 7). The siderophore-mediated acquisition comprises enzymes involved in hydroxamate biosynthesis and plasma membrane siderophore transporters. The RIA pathway includes ferric reductases, multicopper ferroxidases and high-affinity iron permeases. Representative genes for each pathway are induced by iron starvation, and this regulation is probably accomplished by the activities of HapX and SreA transcription factors.

Conclusions
To the best of our knowledge, this is the first report of iron acquisition systems in the Cladophialophora genus. This black fungus encodes the genomic machinery for siderophore production and RIA systems, which are induced by iron starvation in vitro. In such

Conclusions
To the best of our knowledge, this is the first report of iron acquisition systems in the Cladophialophora genus. This black fungus encodes the genomic machinery for siderophore production and RIA systems, which are induced by iron starvation in vitro. In such conditions, the hydroxamate siderophore ferricrocin is found in cell extracts and is also secreted, as recently demonstrated in A. fumigatus. C. carrionii codifies six putative siderophore transporters suggesting a versatility in the uptake of different types of siderophores. In addition, the RIA system may be a contributor in the acquisition of siderophore-bound iron. Siderophores are essential for virulence in various plant and human fungal pathogens. As such, these molecules have been investigated as potential biomarkers of fungal infections and as Trojan horses for the oriented delivery of antimicrobials, since SITs are fungal-specific transporters. Furthermore, siderophore biosynthesis is not found in humans, making this pathway a target for drugs. Finally, the scarce number of investigations focusing on molecular mechanisms of virulence and pathogenicity of CBM agents confirms the neglected nature of this disease. Thus, efforts must be made in order to gather information on such matters to open new possibilities of more effective treatments. This meets some objectives of the 2030 Agenda of the United Nations, in which the relevance of neglected tropical diseases is highlighted.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/jof9070727/s1, Figure S1: Schematic representation of characteristic domains and amino acid residues in putative iron oxidase/permease complex components in C. carrionii. A and B. Representation of the seven transmembrane domains and REXLE (yellow) and DXXE (orange) motifs in C. carrioni Ftr1 orthologs (CLCR_03583 and CLCR_07299). C and D. Structural representation of the transmembrane domain and essential amino acids residues (orange) in C. carrioni Fet3 orthologs (CLCR_03382 and CLCR_06747). The three multicopper oxidase domains are represented by shades of blue; Figure S2: High-resolution mass spectrometry of C. carrionii ferricrocin. The peaks identified in the RP-HPLC inspection ( Figure 6) were submitted to mass spectrometry analysis for definition of ferricrocin molecular mass. The four different ionizing adducts are: H+ (m/z = 771.24), NH4+ (m/z = 788.27), Na+ (m/z = 828 793.23) and K+ (m/z = 809.20); Table S1: Orthology analysis of genes involved in high affinity iron acquisition in C. carrionii; Table S2: Oligonucleotides used in qRT-PCR experiments.