Fungi use various, partially redundant iron acquisition systems, depending on their current host niche. To find common key components of iron acquisition and trafficking in C. glabrata, S. cerevisiae, and C. albicans, we searched their genome databases (Candida Genome Database, CGD, and Saccharomyces Genome Database, SGD) for orthologs with known or postulated iron-related functions based on previously published data or iron-related mutant phenotypes (Table 1 and Supplementary Table 1). Genes involved in intracellular iron trafficking were found highly conserved between the species (Supplementary Table 1). These code for transporters in membranes of the vacuole, a major iron storage site (Ccc1, Smf1, Smf3, and Fth1), and of the mitochondria, where a constant supply of iron is needed to maintain respiration and iron-sulfur cluster biosynthesis (Mmt1, Atm1, Mrs3, and Mrs4). Similarly, all three species possess the main components of the reductive HA iron uptake (the permease Ftr1, the associated ferroxidases Fet3/Fet34, and ferric reductases; Table 1). Notably, the ferric reductase family is considerably smaller in C. glabrata (3 Fre orthologs) than in C. albicans (17 Fre orthologs) and S. cerevisiae (9 Fre orthologs). With regard to host iron source-related acquisition systems, however, major differences between the species were observed (Table 1). While all three species encode orthologs of transporters for xenosiderophore uptake (Sit1/Arn1-4), both C. glabrata and S. cerevisiae lack orthologs of C. albicans proteins associated with iron acquisition from host ferritin (Als3) or heme and hemoglobin (Rbt5, Rbt51, Csa1, Csa2, and Pga7).

Thus, although C. glabrata is a successful pathogen with a large overlap in host niches to C. albicans, it seems to lack the components needed for the utilization of the most abundant host iron sources.

The in silico data indicated a limited set of host iron sources available for utilization by C. glabrata. We thus tested different iron sources commonly found in the host. To this end we grew C. glabrata, S. cerevisiae, and C. albicans without free iron, but in presence of ferritin, transferrin, hemin, or hemoglobin. Addition of 100 μM or 1 mM FeCl₃ served as a control for moderate or high iron levels. The uptake of xenosiderophores via C. glabrata Sit1 has been described elsewhere (Nevitt and Thiele, 2011). To account for possible effects of the pH on host iron source stability and solubility (Almeida et al., 2008), we performed these tests under conditions of different pH (Figure 1). Moderate FeCl₃ levels and ferritin supported robust growth by all three species under acidic pH conditions (pH 4.5 and 5.8). However, under slightly alkaline conditions (pH 7.3) C. glabrata and S. cerevisiae, but not C. albicans, were unable to grow (Figures 1B,D). An increase in iron concentration to high levels, however, allowed growth of all species (Figure 1C). In agreement with our in silico analysis, C. glabrata and S. cerevisiae were not able to use hemoglobin, hemin, or transferrin over the whole tested range of media pH. In contrast, hemoglobin, hemin, and transferrin were all exploited as iron sources by C. albicans in a pH-dependent manner, in which alkaline pH conditions allowed the most robust growth (Figures 1E–G).

Taken together, C. glabrata is much less versatile than C. albicans in the utilization of host iron sources, and more similar to S. cerevisiae in that respect. C. glabrata thus seems to rely on ferritin and non-protein-bound ferric iron, in combination with acidic pH, for growth under iron-limiting conditions in the host.

To determine which pathways allow C. glabrata iron acquisition and growth with ferritin or FeCl₃ as iron sources, we investigated deletion mutants lacking genes for two major components of the HA iron uptake system – the HA permease Ftr1 and the associated ferroxidase Fet3. Both mutants displayed severe growth defects with either ferritin as sole iron source or in presence of only moderate FeCl₃ levels. Only high-level iron supplementation (1 mM FeCl₃) partially restored growth of these mutants, predominantly at acidic pH (Figure 2). Hence, the reductive HA iron uptake, particularly the permease-ferroxidase complex, is needed for ferritin- and FeCl₃-dependent growth of C. glabrata.

The initial step of HA iron uptake, the extracellular reduction of ferric iron, is typically mediated by membrane-integral NAD(P)H-dependent ferric reductases (Fre). The C. glabrata genome, in contrast to C. albicans and S. cerevisiae contains only few (three) potential FRE gene orthologs (Table 1), none of them with proven Fre function. We scanned the genomes of C. glabrata, C. albicans, and S. cerevisiae for additional putative ferric reductases. This in silico analysis included a BLAST search for the ferric reductase transmembrane component-like domain (Pfam family PF01794). We identified 17 of the 18 known C. albicans ferric reductases (Jeeves et al., 2011), and the nine ferric reductases already described in S. cerevisiae. Both families show only marginal overlap, which points to different evolutionary origins in both species. Notably, CaFre10 and ScFre1/ScFre2 (green dot, Figure 3A) are known to account for 75% and 90–98% of the whole cell surface reductase activity, respectively (Dancis et al., 1990; Anderson et al., 1992; Georgatsou and Alexandraki, 1994; Knight et al., 2002, 2005), but are non-orthologous. Overall, the gene families of the two species contain ferric as well as cupric reductases, with different cellular localizations depicted as intracellular (mitochondria, ER, vacuole), or extracellular/surface-associated (Figure 3A and Supplementary Table 2). Our analysis furthermore revealed that most (potential) Fres additionally contained a FAD-binding domain, a NAD-binding domain, multiple transmembrane domains and a signal peptide (Figure 3A).

A BLAST search in the predicted protein sequences of C. glabrata using the ferric reductase transmembrane component-like domain of major C. albicans and S. cerevisiae surface ferric reductases (CaFre10 and ScFre1) as the query revealed only the three aforementioned putative C. glabrata ferric reductases CgFre6, CgFre8, and CgAim14. No additional, thus far unknown ferric reductases were found. CgFre6 showed 37% protein identity to ScFre6 and contained the characteristic ferric reductase domains – an N-terminal signal peptide, the ferric reductase transmembrane component-like domain, a FAD- and NAD-binding domain, and a transmembrane domain (Figure 3). CgFre8 showed 41% protein identity to ScFre8 and 29.9% identity to its presumed ortholog in C. albicans, C1_09780C. Like its S. cerevisiae and C. albicans orthologs, CgFre8 lacks both, the FAD-/NAD-binding domains and a signal peptide. CgAim14 showed 38 and 28.3% protein identity to its S. cerevisiae and C. albicans orthologs, respectively. In contrast to its cross-species counterparts, however, CgAim14 does not contain any signal peptide. Importantly, the closest S. cerevisiae and C. albicans orthologs of CgFre6, CgFre8, and CgAim14 have all not been described as surface-localized ferric reductases. In addition, and in contrast to CgFre6, the lack of a predicted signal peptide in CgFre8 and CgAim14 suggests that these proteins may not enter the secretory pathway and thus do not have access to the cell surface or the extracellular space.

In conclusion, our in silico analysis confirmed the comparatively small number of three ferric reductase family members and the lack of orthologs of known surface ferric reductases of S. cerevisiae and C. albicans in C. glabrata. The presence of ferric reductase protein domains and a signal peptide in CgFre6, which is absent in the other putative C. glabrata ferric reductases, suggests CgFre6 to be the potential major surface reductase of this species.

Expression of uptake-associated ferric reductases is typically induced under iron deprivation (Georgatsou and Alexandraki, 1999; Jeeves et al., 2011). Similarly to S. cerevisiae (Puig et al., 2005), iron acquisition in C. glabrata is largely activated by the transcription factor Aft1, whereas iron consuming processes are inhibited by Cth2-dependent degradation of their mRNA transcripts (Gerwien et al., 2016).

To analyze the iron-dependent regulation of the three potential C. glabrata ferric reductases, we tested for Aft1 and Cth2 dependency of CgFRE6, CgFRE8, and CgAIM14 transcription under iron limitation. We found a moderate induction of CgFRE6 and CgFRE8 transcript levels in the wild-type (WT), in line with their presumed roles as ferric reductases (Figure 3B). CgAFT1 deletion almost completely abolished CgFRE6 expression, but led to an overexpression of CgFRE8. In contrast, both CgFRE6 and CgFRE8 were not significantly affected by CTH2 deletion. Finally, transcript levels of CgAIM14 were largely unaffected by iron deprivation and by deletion of CgAFT1 or CgCTH2.

Taken together, the CgAft1-dependent suppression indicates that there is no apparent role of CgFre8 in iron uptake, while the CgAft1-mediated up-regulation of CgFRE6 transcription supports our in silico prediction of CgFre6 as the main C. glabrata uptake-associated surface ferric reductase.

We wanted to measure the contribution of these proteins to the surface ferric reductase activity of C. glabrata by a 2,3,5-triphenyltetrazolium chloride (TTC)- and ferrozine-based assay (Figure 4; see Materials and Methods section). Surprisingly, while C. albicans and S. cerevisiae exhibited surface ferric reductase activity, indicated by red coloration in the TTC assay (supplemented with antimycin A) (Figure 4A) or the formation of a purple halo around colonies in the ferrozine assay (Figure 4B), C. glabrata showed no such activity. This was the case for our standard WT strain and also for a set of clinical strains isolated from different anatomical sites. Iron and copper starvation is known to increase surface reductase activity (Georgatsou and Alexandraki, 1999; Santos et al., 2003). Enhanced activity was indeed observed for the C. albicans WT with the TTC assay, but not using ferrozine (Figures 4C,D and Supplementary Figure 1). In contrast C. glabrata lacked any detectable surface ferric reductase activity even under these inducing conditions (Supplementary Figure 1). We continued to elucidate the function of the C. glabrata ferric reductases by creating Cgfre6Δ and Cgfre8Δ single and double deletion mutants; we were, however, not able to delete CgAIM14. In accordance with the lack of surface ferric reductase activity and in contrast to mutants lacking other major components of the HA iron uptake system (Figure 2), C. glabrata ferric reductase mutants showed no defects in growth with ferritin, under moderate FeCl₃ levels (Figures 5A,B) or under copper and iron starvation (Supplementary Figure 1). Thus, neither CgFre6 nor CgFre8 are required for HA iron uptake of C. glabrata.

Taken together, these results point to major differences between the surface ferric reduction properties of C. albicans and S. cerevisiae in comparison to C. glabrata. The absence of measurable ferric reductase activity in C. glabrata argues against surface reductase functions of CgFre6 or CgFre8, and indicates a loss of surface ferric reduction ability in C. glabrata.

The importance of surface ferric reduction has been demonstrated in various bacteria and fungi (Schröder et al., 2003). However, some fungi such as Histoplasma capsulatum are also able to secrete either ferric reductases (Zarnowski and Woods, 2005; Zarnowski et al., 2008) or low molecular weight ferric reductants into the environment (Timmerman and Woods, 1999). We therefore postulated that C. glabrata might engage in a similar strategy to compensate for its lack of surface ferric reductase activity. To measure extracellular ferric reduction activity in culture supernatants of C. glabrata, C. albicans, and S. cerevisiae, we monitored conversion of Fe³⁺ to Fe²⁺ by spectrophotometric detection of Fe²⁺-ferrozine complexes, with the reductant 1,4-Dithiothreitol (DTT) as a positive control. For all analyzed species, ferric reduction activities were detected (Figure 6A).

Microbial ferric reductases typically act as oxido-reductases and require a cofactor as electron donor. This cofactor is frequently NAD(P)H (Schröder et al., 2003), and more rarely glutathione (Zarnowski and Woods, 2005; Zarnowski et al., 2008). In addition, certain bacteria additionally require flavins (FAD and FMN) as intermediate electron acceptor (Schröder et al., 2003). Accordingly, we tested the potential cofactor function of glutathione, FAD, FMN, NADH, or NADPH in extracellular ferric reduction (Figure 6A). Only addition of NADH or NADPH led to a significant increase in ferric reduction in all species. A slight increase upon FMN addition was additionally detected in S. cerevisiae supernatants. NADH was therefore chosen as a cofactor for further experiments.

Our data thus show that all three species are able to reduce ferric iron extracellularly.

To test whether the putative C. glabrata ferric reductases contribute to extracellular ferric reduction, we analyzed culture supernatants of the CgFRE6 and CgFRE8 single and double deletion mutants in presence of NADH. The deletion mutants showed similar extracellular ferric reduction capabilities as the WT (Figure 6B), indicating that CgFre6 and CgFre8 are not only dispensable for surface activity, but also for extracellular ferric reduction by C. glabrata.

To elucidate whether the observed extracellular ferric reduction is dependent on another protein or a smaller molecule, we separated the supernatant into a low molecular weight fraction (<10 kDa) and a high molecular weight fraction (>10 kDa), and optionally treated both with proteinase K (Figure 7A). The majority of extracellular ferric reduction activity was found in the low molecular weight fraction for all species. As this activity was not (C. glabrata and C. albicans) or only slightly (S. cerevisiae) affected by proteinase K treatment, these data point to a small non-protein molecule as the main reducing agent. The high molecular weight fraction contributed only a very small reductase activity in all species, which was almost completely abolished by addition of proteinase K. This points to a minor contribution of a potentially secreted protein. In support of these findings, extracellular ferric reduction was not affected by heat or ultraviolet light (UV) treatment to denature proteins (Figure 7B), or by growth in presence of brefeldin A, an inhibitor of protein secretion in eukaryotes (Kossaczka et al., 1995) (Figure 7C).

In summary, major extracellular ferric reduction activity in the three analyzed species is likely carried out by a low molecular weight non-protein.

The C. glabrata deletion mutant Cgfre6Δ was generated in an ATCC 2001 background by replacing the CgFRE6 with a TEF1 promoter-driven nourseothricin resistance (NAT1) cassette (Schwarzmüller et al., 2014). The cassette was amplified with 500–1,000 bp gene-specific flanks by PCR (primer: Fre6 na fwd × Fre6 na rev) from a previously existing mutant (Schwarzmüller et al., 2014). The PCR-amplified insert (primer Fre6 fwd × Fre6 rev) was used for C. glabrata transformation using a modified heat shock method (Sanglard et al., 1996) with 45°C heat shock for 15 min. For the double mutant Cgfre6Δfre8Δ, CgFRE6 was deleted in an ATCC 2001 his3Δ background strain (Schwarzmüller et al., 2014) as described above, followed by CgFRE8 deletion using CgHIS3 as an auxotrophy selection marker (including 861 bp promoter sequence and 232 bp terminator sequence): A HIS3 fragment (primer HIS3 gene fwd × HIS3 gene rev) was fused with 1,000 bp FRE8-specific 5′- and 3′-flanks (primer Fre8-fwd-pUC19 × Fre8-HIS3 5′flank-BC, Fre8-HIS3 3′flank-BC × Fre8-rev-pUC19) and integrated into a XbaI-linearized pUC19 vector using the Infusion HD Cloning Kit (Clontech). The PCR-amplified insert (primer Fre8 fwd × Fre8 rev) was used for transformation. The transformants were plated onto YPD agar (2% agar, 1% yeast extract, 2% peptone, 2% glucose) containing 250 μg/ml nourseothricin, or SD agar supplemented with 0.079% complete supplement mix (CSM) without histidine (Formedium), 2.5 μM Cu₂O, 2.5 μM CuSO₄, 25 μM FeCl₃, and 25 μM FeSO₄. Knockout strains were verified by PCR (primer gene naCoP1 × gene naCoP4) and Sanger sequencing. Strains used in this study are shown in Table 2 and all primers are listed in Supplementary Table 3.

Strains were cultivated over-night (oN) at 37°C (C. glabrata) or 30°C (C. albicans and S. cerevisiae) in liquid synthetic defined media (SD: 0.67% YNB, 2% Glucose, 0.079% CSM [Formedium]) with 180°rpm shaking. They were then transferred to citrate-buffered SD (pH 5.8) containing 500 μM (for C. albicans) or 200 μM (for C. glabrata and S. cerevisiae) of the extracellular iron chelator bathophenanthrolinedisulfonic acid (BPS) to induce iron starvation. BPS precultures were incubated for 22 h, washed three times by centrifugation and re-elution in iron-free water, and then resuspended in iron-free water to on OD₆₀₀ of 0.1. The yeast suspension (20 μl) was added to 180 μl citrate-buffered SD (pH 4.5, 5.8, or 7.3) containing 200 μM BPS and supplemented with either 100 μM FeCl₃ (stock in 1% HCl), 100 μg/ml horse ferritin (stock in iron-free 5 mM HEPES, 0.15 M NaCl, 4× filtered through 50 KDa molecular weight cut-off columns [Amicon Ultra 0.5 ml]), 100 μg/ml transferrin (Calbiochem, stock in 0.15 M iron-free Na₂CO₃, 2× filtered through 50 KDa columns [Amicon Ultra 0.5 ml]), 0.1 mg/ml bovine hemoglobin (Sigma, stock in H₂O), or 1 μM hemin (Sigma, stock in DMSO). Growth was recorded over 2 days in biological triplicates by OD₆₀₀ measurement every 30 min (with intermittent shaking) at 37°C in a Tecan Infinite 200 ELISA reader.

SD oN cultures were washed twice with iron-free water, and adjusted to 2 × 10⁷ cells/ml in iron-free water for spotting of 1:10 serial dilutions on unbuffered SD agar containing the metal chelator ethylenediaminetetraacetic acid – (EDTA) (3 μM), the iron chelator BPS (10 μM), or the copper chelator ammonium tetrathiomolybdate – (ATTM) (7 μM). For more alkaline conditions, SD agar was phosphate-buffered to pH 6.4. Plates were incubated oN at 37°C. For the negative control, one plate was inactivated after oN cultivation by heat treatment (1 h, 72°C). Subsequently, 1.5% agarose was melted in 1 × TAE and supplemented with 0.1% TTC (Roth), plus 20 μg/ml antimycin A (Sigma) to inhibit mitochondrial reduction activity (Hsu et al., 2011) where indicated. The plates were overlaid with this TTC agarose, and formation of red formazan, indicating cell surface reduction, was detected in biological triplicates after 1 h incubation.

Serial dilution growth plates were prepared as described before, but with an overlay mixture of 0.5% agarose, 10 mM MgCl₂, 0.1 mg/ml NADH, 1 mg/ml ferrozine, 5 mg/ml ferric ammonium citrate instead. Formation of a purple halo around colonies, indicating surface ferric reductase activity, was documented in biological triplicates after 5 min incubation.

Ferric reduction activity was measured for all three species from stationary oN cultures. Preliminary experiments revealed that low pH medium [such as unbuffered rich medium (YPD) and minimal medium (SD)] or a low pH due to secretion of fungal metabolic products lead to increased background signals (not shown). We therefore chose buffered nutrient-limited medium for our assays, which resulted in the lowest background noise and satisfactory growth for all tested species – SD BR medium [0.67% YNB, 2% Glucose, 50% Britton Robinson buffer (40 mM H₃BO₄, 40 mM Acetic acid, 40 mM H₃PO₄, 3 g/L KCl, pH 5.8)]. Precultures were prepared in SD BR, and cultures were treated with an inhibitor of Golgi/ER-dependent protein secretion, brefeldin A (10 μg/ml) (Kossaczka et al., 1995) where indicated. The average wet cell volume per 200 μl oN culture was determined by cell volume tubes (TPP) for normalization. The cell-free supernatant was obtained by centrifugation for 4 min at 4,000 ×g and filtering through Minisart syringe filters (Sigma, 0.2 μm). Supernatant and media control were treated identically: Heat-treatment (70°C, 20 min), UV-treatment (120 mJ, 360 s), fractionation on molecular weight (Ultra 0.5 ml Amicon centrifugal protein filters, 10 kDa molecular weight cut-off with restoration of original volume of the high molecular weight fraction in adequate medium), or proteinase K-treatment (100 μg/ml, 30 min, 37°C). Culture supernatant or control (180 μl) were combined with 20 μl detection mix consisting of five volumes Britton-Robinson buffer, 1 volume 1 M MgCl₂, 0.5 volumes 15 mg/ml ammonium ferric citrate, 1 volume 20 mM ferrozine, and 2.5 volumes of potential cofactor (stock either 10 mg/ml NADH, 10 mg/ml NADPH, 6.5 mM glutathione, 20 μM FMN, or 20 μM FAD). As positive control, 5 mM of the reductant DTT was added. Iron reduction (as ferrous iron binding to ferrozine) was detected by OD₅₆₈ measurement every 5 min for 4 h at 37°C in a Tecan Infinite 200 ELISA reader in biological triplicates. Data of the 2 h time point was normalized to the initial wet cell volume and adjusted to a scale from 0% (medium control) to 100% (DTT).

Sample preparation and RNA isolation was conducted as described previously (Gerwien et al., 2016). Briefly, an SD oN culture was harvested, washed, and 1 × 10⁷ cells/ml were inoculated into iron-free citrate-buffered SD (pH 5.8) supplemented with 5 μM FeCl₃ and grown for 4 h at 37°C and 180 rpm. The 0 h sample was immediately frozen in liquid nitrogen and stored at -80°C. The remaining culture was washed four times with iron-free SD (start time set as first contact with the medium) and incubated in the same medium for the 1 and 4 h samples. RNA was isolated with the RNAeasy Mini Kit (Qiagen), and quality was verified using the Agilent 2100 Bioanalyzer Nanochip system according to the manufacturer’s protocol. The concentration was determined using a NanoDrop 1000 instrument.

Quantitative real-time PCR was conducted as described previously (Gerwien et al., 2016). Briefly, 600 ng high-quality RNA was treated with DNAse (Epicentre) and reversely transcribed into cDNA using oligo-dT primers and Superscript III (Invitrogen). 1 μl diluted cDNA (1:20) was used for gene expression analysis in a C1000 thermocycler (Bio-Rad, CFX96 Realtime system) with the EvaGreen system (Bio & Sell). The expression rates were determined in biological triplicates and normalized to the house keeping genes EFB1 and EFT2 using Bio-Rad CFX Manager 3.1.

Information about gene orthologs, protein structure, and BLAST results were obtained from the Candida Genome Database (CGD)¹ and the Saccharomyces Genome Database (SGD)². BLAST search for proteins with a predicted ferric reductase domain in C. glabrata (pfam family PF01794, ferric reductase transmembrane component-like domain) was performed using the domain sequence of ScFre1 and CaFre10 as query. Additionally, the presence of additional conserved protein domains were predicted by CGD and SGD-associated prediction tools (SignalP, TMHMM, Pfam) and verified by SMART³. Subcellular localization was predicted by TargetP 1.1⁴. GraphPad Prism 5 (GraphPad Inc.) was used for statistics. All data are reported as the mean ± SEM or standard deviation where appropriate, and two-tailed, unpaired Student’s t-test was performed, if not stated otherwise. Statistically significant results were marked as ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001.