Iron-Sulfur Cluster Signaling: The Common Thread in Fungal Iron Regulation

Abstract

Iron homeostasis in fungi involves balancing iron uptake and storage with iron utilization to achieve adequate, non-toxic levels of this essential nutrient. Extensive work in the non-pathogenic yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have uncovered unique iron regulation networks for each organism that control iron metabolism via distinct molecular mechanisms. However, common themes have emerged from these studies. The activities of all fungal iron-sensing transcription factors characterized to date are regulated via iron-sulfur cluster signaling. Furthermore, glutaredoxins often play a key role in relaying the intracellular iron status to these DNA-binding proteins. Recent work with fungal pathogens, including Candida and Aspergillus species and Cryptococcus neoformans, have revealed novel iron regulation mechanisms, yet similar roles for iron-sulfur clusters and glutaredoxins in iron signaling have been confirmed. This review will focus on these recent discoveries regarding iron regulation pathways in both pathogenic and non-pathogenic fungi.

Graphical Abstract

Introduction

Regulation of intracellular iron metabolism is crucial for the survival of almost all living organisms. As a cofactor for a wide variety of essential proteins and enzymes, the uptake and mobilization of iron is tightly controlled. However, excess iron must be minimized or safely stored to avoid the generation of damaging oxygen radicals by labile iron as well as the mismetallation of non-iron metalloproteins. The single-celled eukaryotes Saccharomyces cerevisiae and Schizosaccharomyces pombe have served as excellent model systems to tease out these intracellular regulation pathways and compare and contrast different strategies for adapting to both high iron and low iron environments (for recent reviews see [1–3]). In each case, iron homeostasis pathways employ a network of proteins working in concert to control the uptake, utilization, and storage of iron in response to changes in intracellular iron levels. A unifying theme in these regulation pathways is the central role of iron-sulfur (Fe-S) clusters as sensors of iron bioavailability. Even though the transcription factors that control iron metabolism are not conserved between these two divergent yeast species, they have all been shown to bind Fe-S clusters that regulate their protein-protein interactions and/or DNA binding activity. Furthermore, in vivo and in vitro studies have established that [2Fe-2S]-binding cytosolic glutaredoxins (Grxs) with a Cys-Gly-Phe-Ser (CGFS) active site post-translationally regulate the activity of these transcription factors via direct binding and/or Fe-S cluster transfer.

The same principle of Fe-S cluster-dependent signaling via CGFS Grxs to transcriptional regulators has recently been demonstrated in a variety of other fungi that act as human pathogens. The transcription factors that control iron metabolism in these organisms share homology with either S. cerevisiae or S. pombe transcription factors, providing the framework to decipher their molecular mechanisms (Table 1, Figure 1). Importantly, these regulation pathways are often essential for virulence, providing potential targets for the development of anti-fungal compounds. In this article, we will focus on recent insights into iron-responsive transcriptional regulation for both pathogenic and non-pathogenic fungi, highlighting how Fe-S clusters and CGFS Grxs play commons roles in these pathways and elucidating the importance of such regulation to survival in the environment or the human host. A deeper understanding of iron regulation pathways by connecting information gained across species may help in the development of novel strategies for treating iron-related diseases and combatting microbial invasions.

Table 1.

Fungal transcription factors with verified iron regulation activity. Iron-dependent activators are listed in green and repressors are shown in red. Transcription factors with both activator and repressor activities are listed in blue.

Fungal species	Aft1/2 regulator	Hap4-like regulator	GATA-type regulator	Sef1 regulatora	Refs. establishing roles for mitochondrial Fe-S cluster biogenesis, CGFS Grxs, and/or GSH in iron sensing
					Mito Fe-S biogenesis	CGFS Grx	GSH
Non-pathogenic
Saccharomyces cerevisiae (budding yeast)	Aft1/Aft2	Yap5	-	-	[29,60,61]	[62,63]	[61,64,65]
Schizosaccharomyces pombe (fission yeast)	-	Php4	Fep1	-	[39]	[38,49,51]	[40,51]
Pathogenic
Candida albicans (commensal-opportunistic pathogenic yeast)	-	Hap43	Sfu1	Sef1	[45,46,59]	[47]	NDb
Candida glabrata (commensal-opportunistic pathogenic yeast)	Aft1/Aft2	Yap5	-	Sef1	ND	ND	ND
Cryptococcus neoformans (opportunistic pathogenic yeast)	-	HapX	Cir1	-	[57]	[55]	ND
Aspergillus fumigatus (opportunistic pathogenic mold)	-	HapX	SreA	-	[41]	[27]	[41]

^aA Sef1 ortholog exists in S. cerevisiae but is not listed here because there is no evidence for its involvement in iron regulation.

^bND, Not determined

Figure 1. Schematic protein domain structure for the four families of fungal iron regulators.

DNA-binding domains (either Zn-finger or bZIP) are shaded in blue with specific Zn-finger binding residues shown in blue text. CBC-binding regions are shaded in purple. Coiled-coil regions are shown in gray. Confirmed Fe-S binding domains are shaded dark orange with specific Fe-S binding residues in red text. Putative Fe-S binding domains and other Cys-rich regions are shaded in light orange. A conserved C-terminal region in HapX/Hap43 regulators that is required for their response to iron starvation is shaded in green. Sc, S. cerevisiae; Cg, C. glabrata; Sp, S. pombe; An, A. nidulans; Af, A. fumigatus; Ca, C. albicans; Cn, C. neoformans.

Aft1 and Aft2 control the low iron response in S. cerevisiae

The iron regulon in the well-studied budding yeast S. cerevisiae comprises a set of ~30 genes that encode proteins involved in iron acquisition at the plasma membrane, iron import into the mitochondria, iron-sulfur cluster biosynthesis and vacuolar iron export [1,2]. Upregulation of these genes in the face of iron deficit is designed to scavenge iron from extracellular sources and mobilize intracellular stores. The molecular mechanism employed by the paralogous transcriptional activators Aft1 and Aft2 has been illuminated from numerous in vivo and in vitro studies stretching over the past three decades. Under iron-deficient conditions, Aft1/2 accumulates in the nucleus, binds DNA, and activates the iron regulon (Fig. 2A, top) [4,5]. Conversely, when iron is replete, Aft1/2 dissociates from DNA and is exported to the cytosol by the exportin Msn5, thereby deactivating the iron regulon [6,7]. Control of DNA binding activity and nuclear-cytosolic trafficking of Aft1 and Aft2 in response to iron is dependent on a conserved Cys-Asp-Cys (CDC) motif located just downstream of the DNA-binding, WRKY-GCM1 Zn finger domain (Fig. 1). Replacement of one or both Cys in this motif abolishes Aft1/2 inhibition in response to iron causing constitutive activation of the iron regulon. This motif was recently shown to be essential for binding a [2Fe-2S] cluster that regulates the DNA binding affinity of Aft2 (and presumably Aft1) [8,9]. This inhibitory Fe-S cluster is trafficked to Aft1/Aft2 by the cytosolic Grx paralogs Grx3 and Grx4 that form heterodimeric [2Fe-2S]-bridged complexes with Bol2 (formerly named Fra2) (Fig. 2A) [10,11]. The cluster ligands in these heterocomplexes are provided by the Cys in the Grx CGFS active site, a Cys from GSH, and two residues provided by Bol2 (His and possibly a Cys) [12]. Fe-S cluster assembly/delivery to these cytosolic trafficking factors, in turn, is dependent on the mitochondrial iron-sulfur cluster (ISC) assembly pathway, the redox-active tripeptide glutathione (GSH), and the mitochondrial ABC transporter Atm1 [2,13]. In contrast, the cytosolic Fe-S cluster assembly (CIA) pathway is not essential for signaling iron bioavailability to Aft1 or Aft2. Additional modes of iron-independent, post-translational regulation of Aft1/2 activity have also been described recently, including phosphorylation by Hog1 protein kinase in response to changes in sphingolipid metabolism [6,14]. Furthermore, Aft1/2 activity is linked to and complemented by the mRNA binding proteins Cth1 and Cth2, which are included in the Aft1/2 iron regulon. Their expression under low iron conditions facilitates the degradation of mRNA targets that encode proteins involved in non-essential, iron-consuming pathways (e.g. respiration, TCA cycle, heme biosynthesis), serving as a post-transcriptional mechanism to conserve iron for essential functions (see [15] for a recent review).

Figure 2. Proposed models for regulation by iron-dependent transcription factors in the non-pathogenic fungi S. cerevisiae (A) and S. pombe (B).

When iron levels are low in S. cerevisiae (A, top), Aft1 and Aft2 activate expression of iron acquisition genes as well as the mRNA binding proteins Cth1 and Cth2 that degrade transcripts encoding proteins in non-essential, iron-consuming metabolic pathways. Meanwhile, Yap5 binds the YRE in target genes via its bZIP domain and represses expression of iron storage and sequestration genes. Based on evidence from the C. glabrata Yap5 homolog, Yap5 may also interact with the CCAAT binding complex (CBC); however, direct evidence for this interaction in S. cerevisiae is lacking. During conditions of iron sufficiency (A, bottom), Grx3 and Grx4 form Fe-S-bridged heterodimers with Bol2 and deliver [2Fe-2S] clusters to Aft1/2, which promotes their DNA dissociation, dimerization, and export to the cytosol. This change, in turn, leads to deactivation of Aft1/2-regulated genes. Under these same conditions, Yap5 binds [2Fe-2S] clusters that likely promote conformational changes that lead to activation of its target genes involved in iron storage/sequestration. During iron deficiency in S. pombe (B, top), Php4 economizes iron usage by binding the CBC and repressing transcription of the iron-responsive repressor Fep1 as well as genes encoding proteins in non-essential, iron-utilizing pathways. Grx4 maintains an interaction with Php4 but does not interfere in its association with the CBC. Fep1 dissociates from its target genes when iron is low, leading to the expression of iron acquisition genes as well as PHP4. The dissociation of Fep1 from DNA is proposed to be triggered by reverse transfer of iron or [2Fe-2S] clusters from Fep1 to the Grx4-Fra2(Bol2) heterodimer. When iron levels increase (B, bottom), Grx4 and Php4 bind a [2Fe-2S] cluster that promotes dissociation of Php4 from the CBC and nuclear export, leading to derepression of the Php4 regulon. Meanwhile, Fep1 binds [2Fe-2S] clusters, presumably delivered by Fra2-Grx4 heterodimers, which facilities its DNA binding activity and repression of its target genes.

Regulation of iron homeostasis by Aft1/Aft2 is not unique to S. cerevisiae

Aft1 and Aft2 orthologs exist in other yeast species; however, in some cases, iron-responsive transcriptional activity is not conserved. For example, in the opportunistic pathogen Candida albicans and the methylotrophic yeast Pichia pastoris, Aft1/2 orthologs possess a Zn-finger DNA binding domain that is homologous to ScAft1, but lack the CDC iron-responsive motif. Consequently, these transcriptional regulators have little or no impact on iron metabolism in these organisms [16,17]. On the other hand, several recent reports have confirmed that Aft1 is the primary regulator of the iron starvation response in Candida glabrata, similar to S. cerevisiae ([18–20] and for a recent review see [21]). Consequently, a C. glabrata aft1Δ deletion strain exhibits a severe growth defect in iron-limited conditions and decreased virulence in an ex vivo infection model [19]. Although this yeast switches between commensal and pathogenic lifestyles similar to C. albicans, C. glabrata is evolutionarily closer to the nonpathogenic S. cerevisiae, which is reflected in its iron regulation systems. In addition to Aft1 and Aft2, genomic and transcriptomics studies have identified orthologs of the S. cerevisiae high iron sensing basic leucine zipper (bZIP) transcription factor Yap5 (see below), and the mRNA binding regulator Cth2, each performing largely similar functions in C. glabrata as previously demonstrated in S. cerevisiae (Fig. 3A) [18,19,22,23]. C. glabrata also possesses orthologs of the cytosolic [2Fe-2S] cluster trafficking proteins Grx3, Grx4 and Bol2; however, their ability to bind [2Fe-2S] clusters and their specific roles in post-translational control of CgAft1/2 activity have not yet been addressed. Furthermore, the impact of Fe-S cluster assembly pathways and the role of GSH in C. glabrata iron regulation are unknown, highlighting important future areas of study. Interestingly, the iron-dependent regulation of GRX3 and GRX4 genes in C. glabrata is somewhat different from S. cerevisiae. Expression of GRX3 is up-regulated by CgAft1 and CgAft2 during iron starvation [18,19], while GRX4 expression is repressed by CgYap5 under the same conditions, but activated by CgYap5 during iron overload [22]. In contrast, neither ScAft1 nor ScAft2 regulates GRX3 or GRX4 expression in S. cerevisiae, whereas ScYap5 activates GRX4 during iron excess. Nevertheless, this regulation pattern implicates functions for CgGrx3 and CgGrx4 in iron metabolism, although their specific molecular roles may differ from each other and from their S. cerevisiae orthologs.

Figure 3. Proposed models for regulation by iron-dependent transcription factors in Candida species C. glabrata (A) and C. albicans (B).

Iron regulation in C. glabrata parallels S. cerevisiae since CgAft1/2 activates iron uptake gene and expression of the mRNA binding protein Cth2 when iron is scarce (A, top), while CgYap5 activates iron storage and sequestration genes when iron is abundant via direct interaction with the CBC at the promoters of Yap5-regulated genes (A, bottom). However, in contrast to S. cerevisiae, the transcriptional activator Sef1 has a minor role in activating the expression of a handful of TCA cycle enzymes and ISC assembly factors during iron deficiency in C. glabrata. Nevertheless, in all cases, the post-translational mechanisms for regulating the activities of these transcription factors in response to iron have not been established. Presumably, CgAft1/2 and CgYap5 bind [2Fe-2S] clusters similar to their S. cerevisiae orthologs, whereas nothing is known about the post-translational regulation of CgSef1. C. albicans also employs a tripartite iron regulatory system. During iron deficit (B, top), CaSef1 is phosphorylated by the protein kinase Ssn3, triggering its nuclear localization and DNA binding activity, which in turn leads to activation of iron acquisition genes and expression of the CBC-binding repressor CaHap43. CaHap43, in turn, represses expression of the GATA-type repressor Sfu1 in addition to genes involved in non-essential iron utilization pathways in order to conserve iron, while also activating expression of several genes involved in siderophore and high affinity iron uptake. Meanwhile, CaSfu1 dissociates from its target DNA, promoting expression of iron acquisition genes and SEF1, completing the three-part regulatory circuit. Under conditions of iron sufficiency (B, bottom), CaSef1 is retained in the cytosol via interaction with CaSfu1, leading to its destabilization. CaSfu1 also binds and represses SEF1 and iron acquisition genes. The mechanisms for deactivation of CaHap43 and CaSfu1 repressor activity in response to iron are unclear, but may involve DNA dissociation and/or interaction with CaGrx3. Each of these post-translational control mechanisms are proposed to involve Fe-S cluster pathways in some manner, but direct evidence for Fe-S cluster binding by these transcription factors is lacking.

High iron response by Yap5 in S. cerevisiae

As mentioned earlier, the bZIP transcription factor Yap5 orchestrates the response to high iron conditions in S. cerevisiae. Yeast have no mechanism for exporting excess iron nor do their genomes encode a ferritin-like iron storage protein. Thus, excess iron is stored in the vacuole or sequestered in protein-bound pools to avoid toxicity. Yap5 mounts this defense against high iron by activating expression of the vacuolar iron importer Ccc1, the Cu-binding metallothionein Cup1, and the cytosolic Fe-S binding proteins Grx4 and Tyw1 [1,24,25]. Upregulation of CCC1 by Yap5 plays a major role in tackling high iron conditions by facilitating the clearance of excess iron from the cytosol to the vacuole [26]. Since Grx4 inhibits Aft1/2 activity during iron sufficiency, its increased expression in iron excess conditions may further limit Aft1/2 activation of iron uptake genes. Increased Cup1 protein may limit Cu availability for the multicopper oxidases that function in high-affinity iron uptake. Interestingly, the Grx4 ortholog in A. fumigatus (GrxD) was recently shown to physically interact with the Cup1 ortholog (CmtA) under iron excess conditions [27], hinting at a possible function for fungal metallothioneins in chelating iron or Fe-S clusters. Similarly, the upregulation of TYW1, which encodes a [4Fe-4S]-dependent enzyme that modifies tRNA, may provide an expanded pool of Fe-S binding sites to safely sequester excess iron [24]. The molecular mechanism used by Yap5 for responding to iron bioavailability show some parallels to Aft1/2 with some distinct differences. Notably, Yap5 forms dimers and binds [2Fe-2S] clusters in vitro via two cysteine rich domains [28]. Interestingly, the Fe-S binding motif in one of these Cys-rich domains (CGFCX₅CXC) is well conserved among fungal Yap5 orthologs and is reminiscent of the Fe-S binding site in the CGFS Grxs that regulate Aft1/Aft2 activity (Fig. 1). Yap5 activation in vivo is dependent on the mitochondrial Fe-S synthesis machinery (ISC pathway) but not the cytosolic/nuclear machinery (CIA pathway) [29]. As mentioned earlier, modulation of Aft1/2 activity in response to iron is also dependent on the ISC and not the CIA pathway, albeit with the opposite effect on Aft1/2 function (inhibition) compared with Yap5 (activation). Furthermore, Yap5 is constitutively bound to the promoter of its target genes [26], unlike Aft1 and Aft2 which undergo iron-dependent nucleocytoplasmic shuttling [5]. Instead, Yap5 is proposed to undergo a conformational change upon binding [2Fe-2S] clusters at the dimer interface that facilitates gene expression (Fig. 2A) [26,28]. The donor of these clusters remains unidentified since Yap5 activity is not impacted by the absence of the cluster trafficking proteins Grx3 and Grx4, in contrast to Aft1/2 [29].

Yap5 regulates iron overload in C. glabrata via interaction with the CCAAT-binding complex

The Yap5 transcription factor in C. glabrata performs a similar function to its S. cerevisiae ortholog by activating the expression of proteins that alleviate iron overload stress (GRX4, CCC1, TYW1), as well as additional Fe-S binding proteins (GLT1, RLI1, ACO1, SDH2, ISA1) and proteins involved in heme biosynthesis (HEM3) [21,22]. Interestingly, Thiébaut and coworkers recently demonstrated that CgYap5 functions with the CCAAT-binding complex (CBC) in regulating iron homeostasis (Fig. 3A) [23]. CBC proteins in yeasts are evolutionarily conserved transcription factors that form heterotrimeric DNA binding complexes (Hap2/3/5 in S. cerevisiae) that bind a CCAAT motif in the promoters of their target genes. In both S. cerevisiae and C. glabrata, the core DNA binding CBC is constitutively expressed, whereas a fourth regulatory subunit (Hap4) is induced in the absence of glucose, interacting with the CBC to activate transcription of genes required for respiration [23,30]. Surprisingly, the iron-responsive CgYap5 was shown to possess a Hap4-like (Hap4L) domain that mediates its interaction with Hap2/3/5 and is required for CgYap5 to bind DNA and activate gene transcription. Furthermore, the CgYap5 DNA binding motif (YRE, or Yap Response Element) is found within 10–14 base pairs of a CCAAT motif in CgYap5 target genes, supporting the physical interaction between these transcription factors and the dependence of CgYap5 on CBC for recruitment to DNA (Fig. 3A). Thus, in C. glabrata, the CBC controls both respiratory metabolism and iron homeostasis via interaction with Hap4 or Yap5, respectively [23]. Interestingly, the authors propose that a similar interaction between Yap5 and Hap2/3/5 may exist in S. cerevisiae based on the conservation of the Hap4L domain in ScYap5 and the location of CCAAT motifs in close proximity to the YREs in ScYap5 target genes (Fig. 2A) [21]. Additional studies are required to confirm this hypothesis. In addition, the role of Fe-S cluster binding in regulating CgYap5 activity has not yet been addressed, although the Cys-rich Fe-S binding domains in ScYap5 are well conserved in CgYap5 (Fig. 1) [28], hinting at a similar Fe-S cluster-dependent regulation mechanism.

Regulation of iron economy by Hap4-like transcriptional repressors (Php4/Hap43/HapX)

While CBC-binding Hap4-like regulators have a role in mitigating iron toxicity in C. glabrata, in the fission yeast Schizosaccharomyces pombe and some fungal pathogens, Hap4-like regulators serve as repressors in iron-limiting conditions to economize iron usage. In these organisms, iron deficiency induces expression of the Hap4-like subunits of the CBC, namely Php4 in S. pombe, Hap43 and HapX in the pathogenic yeasts C. albicans and C. neoformans, respectively, and HapX in the filamentous fungi A. nidulans and A. fumigatus (see Table 1) [3,17,31,32]. These iron-responsive regulatory components bind to the CBC, leading to repression of genes involved in iron-rich metabolic pathways, thus reserving dwindling iron stores for essential pathways. In C. albicans, C. neoformans, and A. fumigatus, Hap43 and HapX have also been shown to activate expression of siderophore, heme, and/or high affinity iron uptake systems during iron deficiency. Consequently, hapX/hap43 null mutants display decreased virulence in mouse infection models, underscoring their critical role for survival in the host [33–36]. The molecular mechanisms for controlling Hap4-like regulators in response to iron have been unveiled in several recent reports. This mechanism is probably best understood in S. pombe, in which low iron induces Php4 to bind to the CBC (Php2/3/5) through its Hap4L domain (Fig. 2B, top) [3]. When iron is sufficient, Php4 forms a [2Fe-2S] cluster binding complex with the cytosolic CGFS glutaredoxin Grx4 via two conserved Cys residues [37], which promotes its dissociation from the CBC and export from the nucleus (Fig. 2B, bottom) [38]. Similar to the iron-dependent regulation of Aft1/2 activity in S. cerevisiae, regulation of Php4 activity in vivo is dependent on mitochondrial Fe-S cluster biogenesis [39] and GSH [40], which likely reflects the requirement for the GSH-ligated [2Fe-2S]-Grx4 complex to regulate Php4 activity. A recent study confirms a similar situation in A. fumigatus, in which depletion of mitochondrial ISC components or GSH disrupts iron regulation by both HapX and the GATA-type repressor SreA (Table 1 and see below), while the cytosolic CIA pathway is dispensable for iron regulation [41]. Furthermore, the cytosolic CGFS glutaredoxin GrxD was recently shown to control activity of both AfHapX and AfSreA via physical interaction with these proteins in vivo (Fig. 4A) [27]. Genetic studies further confirmed that under iron starvation conditions, the Cys in the CGFS active site of AfGrxD is essential for signaling iron starvation to AfHapX, while inactivating repression of iron acquisition genes by AfSreA. These authors also demonstrate that recombinant AfHapX forms a complex with AfGrxD and binds an Fe-S cluster in vitro in the presence or absence of AfGrxD [27]. This observation contrasts SpPhp4, which did not purify with an Fe-S cluster in the absence of Grx4 [37]. Of note, the HapX/Hap43 orthologs in pathogenic fungi have additional Cys-rich domains that are absent in SpPhp4 (Fig. 1). Interestingly, two of these Cys-rich motifs (B and C in Fig. 1) share high sequence identity with the verified Fe-S binding sites in ScYap5 [28]. Accordingly, AfHapX was found to play a key role in regulation of iron excess in addition to iron starvation, thus combining traits from SpPhp4 and ScYap5 (Fig. 4A) [42]. In the opportunistic pathogen C. albicans, the specific domains in Hap43 (also known as Cap2) that dictate the response to iron starvation were recently delineated via functional analysis of truncation and deletion mutants in vivo. These studies revealed that deletion of individual Cys-rich regions in CaHap43 (A, B, or C in Fig. 1) did not significantly impact its activity under low iron conditions, whereas deletion or mutations in the N-terminal DNA-binding domain or the C-terminal conserved region (green shaded region of HapX/Hap43 in Fig. 1) abolished the CaHap43-dependent response to iron starvation and recruitment to target promoters (Fig. 3B) [43,44]. In addition, disruption of mitochondrial Fe-S cluster biogenesis or deletion of the cytosolic CGFS Grx (Grx3) has been shown to deregulate iron homeostasis in C. albicans [45–47], although the impact of the CIA system or GSH metabolism on Hap43 activity in this yeast has not yet been reported. Even less is known about the iron-dependent mechanism for regulating HapX activity in the fungal pathogen C. neoformans (Fig. 4B). This mechanism will likely be different for specific gene targets since C. neoformans HapX represses transcription of genes involved in iron-consuming pathways under low iron conditions similar to its Php4/Hap43/HapX orthologs, while also activating expression of siderophore transport genes [36].

Figure 4. Proposed models for regulation by iron-dependent transcription factors in the pathogenic fungi A. fumigatus (A) and C. neoformans (B).

Iron regulation in A. fumigatus resembles S. pombe since AfHapX represses iron utilization genes and sreA when iron is scarce (A, top), whereas AfSreA represses iron uptake genes and hapX when iron is abundant (A, bottom). However, in contrast to its ortholog in S. pombe (Php4), AfHapX also functions as an activator of siderophore biosynthesis and has a bZIP domain that directly interacts with target DNA in a constitutive manner. During iron deficiency, AfGrxD is required to promote both the repressor and activator functions of AfHapX, while inactivating the repressor function of AfSreA via physical interactions with both transcription factors, which may facilitate the removal of their [2Fe-2S] clusters. When iron is sufficient, AfGrxD forms [2Fe-2S]-binding complexes with AfHapX and presumably AfSreA, which may lead to conformational changes in each that alter their activity. However, the HapX-GrxD interaction is not essential for promoting the activator function of AfHapX under these conditions. [2Fe-2S] cluster binding has been confirmed for AfHapX in vitro in both the presence and absence of AfGrxD, but has not yet been addressed for AfSreA. During iron scarcity in C. neoformans (B, top), CnHapX represses expression of iron-rich metabolic pathways while also activating expression of CIR1 and Fe-siderophore uptake genes. CnGrx4 interacts with CnCir1 under these conditions, which is proposed to trigger derepression of genes encoding high affinity iron uptake systems and siderophore import pathways. Under iron replete conditions (B, bottom), CnGrx4 is partially localized to the cytosol, whereas CnCir1 remains in the nucleus repressing expression of iron acquisition genes. Meanwhile, CnHapX activates expression of iron utilization pathways and weakly activates CIR1 expression. Fe-S cluster binding by CnGrx4, CnHapX, and/or CnCir is proposed to trigger the functional changes in these regulatory proteins, but has not yet been confirmed.

Regulation of iron acquisition by GATA-type transcriptional repressors

In S. pombe, C. albicans, and Aspergillus sp., the CBC-binding Php4/Hap43/HapX proteins function in a negative feedback loop with GATA-type transcriptional repressors in order to maintain precise control of intracellular iron levels. The CBC-binding proteins repress expression of iron utilization pathways and the iron-responsive GATA regulators under iron deficiency, whereas the GATA regulators reciprocally repress expression of iron acquisition pathways and the CBC-binding proteins when iron levels rise. Interestingly, in C. albicans and C. glabrata, this feedback loop includes a third regulator, Sef1 [17,48], discussed in the next section. Iron-responsive GATA-type repressors bind a conserved 5′-(A/T)GATA(A/T)-3′ sequence to facilitate transcriptional repression of genes involved in siderophore, heme, and/or ionic iron uptake during iron sufficiency. Representatives in this family include Fep1 in S. pombe, Sfu1 in C. albicans, SreA in Aspergillus species, and Cir1 in C. neoformans (Table 1) [3]. A similar iron-sensing GATA factor was lost from the S. cerevisiae genome during its evolution and is functionally replaced with the Aft1/Aft2 activators. Most of these GATA-type regulators have two zinc fingers at the N-terminus (ZnF1 and ZnF2) required for high-affinity DNA binding and a highly conserved intervening Cys-rich region (Fig. 1). The molecular mechanism of iron sensing by the GATA-type regulators is an area of active research. Labbé and coworkers established that Grx4 post-translationally modulates Fep1 activity in S. pombe via specific interactions between the N- and C-terminal domains of both proteins, and furthermore Cys-172 in the CGFS motif of SpGrx4 is critical to the iron-dependent inhibition of SpFep1 (Fig. 2B) [49]. The S. pombe Bol2 ortholog (Fra2) was also shown to co-regulate the iron-dependent inhibition of SpFep1 activity in vivo, and coimmunoprecipitate in a complex with SpFep1 and SpGrx4 (Fig. 2B) [50]. Hidalgo et al. recently followed up this work by further examining the interplay between SpGrx4, SpFra2, and GSH in controlling SpFep1 activity [51]. Under GSH depletion conditions, they demonstrate that SpGrx4 remains bound to SpFep1 under low and high iron conditions allowing expression of SpFep1-repressed genes, presumably due to the lack of Fe-S clusters. However, in a grx4Δ strain, Fep1-regulated genes are constitutively repressed, with or without GSH present. This result suggests that apo-SpGrx4 functions to inhibit SpFep1 activity in the absence of a bound Fe-S cluster. These authors also purified recombinant SpGrx4, SpFra2, and SpFep1 and report UV-visible spectra that are reminiscent of [2Fe-2S] cluster-binding proteins, although they were unable to detect acid labile sulfide in the purified SpFep1 sample, concluding that SpFep1 only binds Fe via the four conserved Cys in the Cys-rich region [51]. Based on the genetic data and Fe transfer assays between SpGrx4-SpFra2 and SpFep1, they propose that iron starvation causes loss of the [2Fe-2S] cluster bridging SpGrx4 and SpFra2, triggering a reverse iron transfer from SpFep1 to SpGrx4-SpFra2, which in turn inactivates its repressor activity (Fig. 2B, top). However, using additional spectroscopic techniques (electron paramagnetic resonance (EPR), circular dichroism (CD), and resonance Raman), the Roe and di Patti groups established that recombinant Fep1 from S. pombe and its ortholog in Pichia pastoris are bona fide [2Fe-2S] cluster-binding proteins [52–54], thus giving a new perspective to this regulatory mechanism. The modified proposal suggests that Fe-S cluster transfer between SpFep1 and SpGrx4-SpFra2 serves as the on/off switch regulating SpFep1’s activity (Fig. 2B) [52]. Studies in A. fumigatus support this inhibition mechanism since the cytosolic CGFS Grx (GrxD) was shown to interact with the Fep1 ortholog SreA in vivo as mentioned earlier, and iron acquisition genes regulated by AfSreA are constitutively repressed in the absence of grxD (Fig. 4A) [27]. However, the genomes of Aspergillus spp. do not appear to encode a cytosolic Bol2-like protein, suggesting some key variances in the regulation mechanism of the iron-responsive GATA-type factors in these organisms. In C. neoformans, Kronstad and coworkers recently demonstrated that Grx4 interacts with the GATA-type regulator Cir1 and impacts its transcriptional activity (Fig. 4B) [55]. The Fe-S binding domain of CnGrx4 is essential for this regulatory function; however, whether or not the interaction between CnGrx4 and CnCir1 directly involves Fe-S cluster binding has not yet been addressed. In any case, both CnCir1 and CnGrx4 are critical for the virulence of this pathogen, highlighting their essential roles in regulating iron homeostasis [55,56]. Of note, CnCir1 is likely structurally different from the other fungal iron-responsive GATA-type regulators because it lacks one of the zinc fingers (Fig. 1); nevertheless, the Cys-rich Fe-S binding motif in SpFep1 is well conserved in CnCir1. Another clue pointing to Fe-S-dependent post-translational regulation of this protein is the fact that defects in the mitochondrial Atm1 transporter, which is required for cytosolic Fe-S cluster maturation, derepresses the expression of Cir1-regulated iron uptake genes in C. neoformans [57].

Sef1-mediated iron regulation in Candida species

Unexpectedly, the yeast C. albicans utilizes a third iron-responsive transcription factor that facilitates its adaptation to low iron bioavailability during bloodstream infection [17,48]. The Zn₂Cys₆ DNA-binding protein Sef1 serves as an intermediary regulator between the GATA factor Sfu1 and the CBC regulator Hap43. Under iron limitation, CaSef1 activates HAP43 and iron uptake genes, while CaHap43, in turn, represses SFU1 along with genes involved in iron-rich metabolic pathways (Fig. 3B, top). During iron sufficiency, CaSfu1 represses SEF1 as well as genes encoding iron uptake systems to avoid iron overload (Fig. 3B, bottom). This tripartite iron regulatory circuit thus allows this commensal opportunistic pathogen to easily adapt to the iron-rich environment of the gut as well as the iron-poor environment of the bloodstream [17,48]. As evidence, CaSef1 was shown to be essential for C. albicans virulence in a mouse model. On the other hand, CaSfu1 is dispensable for bloodstream infection, but instead promotes commensalism in the gut [17]. On the post-translational level, CaSef1 is phosphorylated by the protein kinase Ssn3 when intracellular iron levels are low, which triggers its import into the nucleus and DNA-binding activity (Fig. 3B, top). When iron levels rise, CaSef1 is retained in the cytosol by physical association with CaSfu1, where it is destabilized (Fig. 3B, bottom) [58]. The question arises as to how these transcription factors sense changes in iron bioavailability. Ror and Panwar provide a hypothesis in a recent study examining the impact of mitochondrial biogenesis on Sef1 activity in C. albicans [59]. They observe increased SEF1 expression in a mutant defective in mitochondrial biogenesis (fzo1Δ/Δ) that can be rescued with overexpression of mitochondrial ISC components. This result suggests that the iron sensing by the GATA-type factor CaSfu1 that represses SEF1 is dependent on mitochondrial Fe-S cluster biogenesis, as demonstrated for its orthologs in S. pombe, C. neoformans, and A. fumigatus [32,39,41]. Furthermore, they noted that nucleocytoplasmic shuttling of CaSef1 in response to iron was disrupted in the fzo1Δ/Δ strain, leading to constitutive nuclear localization [59]. These authors thus propose that CaSef1 either directly senses an Fe-S cluster or Fe-S cluster derived product, or interacts with an Fe-S cluster binding protein (possibly CaSfu1) that promotes its cytosolic localization during iron sufficiency. If this hypothesis is true, then this regulation mechanism mirrors that observed for S. cerevisiae Aft1 and Aft2, in which Fe-S cluster binding to these regulatory proteins promotes their localization to the cytosol when iron is replete, leading to deactivation of iron uptake genes (Fig. 2A, bottom) [7,9]. However, further studies are required to test this regulation mechanism and uncover the specific link between mitochondrial Fe-S cluster biogenesis and CaSef1 function. These are no clear Fe-S binding motifs in the Sef1 protein sequence (Fig 1), although there are >10 conserved Cys/His residues downstream of the Zn₂Cys₆ DNA-binding motif that could possibly perform this function.

Interestingly, Gerwien et al. recently demonstrated that Sef1 has a minor role in iron homeostasis in C. glabrata, even though the overall iron regulation network in this Candida pathogen is more similar to S. cerevisiae (Table 1). As described earlier, CgAft1 and the mRNA binding protein CgCth2 are the primary regulators of the iron starvation response in C. glabrata via transcriptional and post-transcriptional mechanisms, respectively [18–21]. CgSef1 was found to positively regulate expression of only a few TCA cycle and Fe-S cluster assembly genes (Fig. 3A). Consequently, the sef1Δ strain exhibited a moderate growth defect under iron limitation conditions and decreased survival in human blood [19]. Nevertheless, further work is required to nail down the specific role of CgSef1 in iron regulation and pathogenesis, and the molecular mechanism for regulating Sef1 activity in C. glabrata. We note here that a Sef1 ortholog is also present in S. cerevisiae but has no known iron regulation functions, unlike its Candida counterparts [17].

Conclusions and Future Directions

Much of the groundwork in our understanding of fungal iron metabolism has been established over the past few decades using the non-pathogenic model yeasts S. cerevisiae and S. pombe. Critical iron trafficking pathways and regulation factors have been identified, providing a foundation to tease out the elaborate control mechanisms required to regulate this essential metal. As outlined in this review, recent studies have focused on pinpointing the protein-protein and metal-protein interactions that govern activity of the iron-responsive transcription factors in these model organisms. This molecular-level information can then be reconciled with in vivo functional studies in order to develop a cohesive model for iron regulation at both the cellular and molecular level. Thus far, specific Fe-S binding sites in ScAft1/2, ScYap5, SpPhp4, and SpFep1 have been identified via site-directed mutagenesis and their respective Fe-S coordination chemistries have been analyzed via biophysical spectroscopy. Furthermore, the roles of CGFS Grxs and Bol2/Fra2 proteins in controlling the transcriptional activity for each of these proteins have been systematically analyzed, revealing that CGFS Grxs regulate activity of ScAft1/2, SpPhp4, and SpFep1 via Fe-S cluster binding and/or Fe-S transfer, whereas Bol2/Fra2 proteins co-regulate ScAft1/2 and SpFep1 activity in partnership with CGFS Grxs. However, additional studies are required to hammer out the details of the regulation mechanisms for each of these regulators. For example, the only crystal structure available for any of these regulators is a truncated form of ScAft2 that lacks the Fe-S-binding motif [9]. Additional atomic-level structural information will provide novel insights into the impact of Fe-S binding on the dynamic protein-protein interactions and protein-DNA interactions that govern the regulation mechanisms for each of these transcription factors.

Meanwhile, progress in understanding iron regulation mechanisms in pathogenic fungi is a step behind the non-pathogenic species but rapidly catching up. Unraveling the iron regulation pathways in these organisms has revealed some common themes with some unique twists that reflect adaption to specific niches in the human host. For example, most fungal species employ a pair of transcriptional regulators (either Aft1/2 and Yap5 or Hap4-like and GATA-type factors) to control iron acquisition and iron utilization, whereas both Candida species have integrated a third transcriptional regulator (Sef1) into this circuit to fine-tune the transcriptional response to rapidly changing iron conditions in the human host. After identification of the specific regulators that control the response to iron starvation vs. iron excess in pathogenic fungi, the most recent advancements have explored the molecular mechanisms for regulating these transcription factors in response to iron fluctuations. As previously demonstrated in S. cerevisiae and S. pombe, critical roles for both mitochondrial Fe-S cluster biogenesis and CGFS Grxs in iron regulation have now been confirmed in C. albicans, A. fumigatus, and C. neoformans, thus reflecting a ubiquitous function for Fe-S-binding CGFS Grxs as iron sensors that relay the status of mitochondrial Fe-S cluster biogenesis to the nuclear transcriptional machinery. However, distinct mechanistic differences have been teased out that likely reflect the specific adaption of these fungi to their unique environmental niches. Future research in this area will likely follow the path of the S. cerevisiae and S. pombe studies by exploring the molecular-level details of the regulation mechanisms for each of these iron-responsive transcription factors in pathogenic fungi.

Highlights.

• Fungi balance iron uptake/storage with iron utilization to maintain non-toxic iron levels.

• Regulation of iron metabolism is essential for pathogenic and non-pathogenic fungi.

• Iron-responsive transcription factors in diverse fungal species use iron-sulfur clusters to sense iron bioavailability.

• Glutaredoxins serve as iron-sulfur cluster sensing and trafficking factors in fungi.

Abbreviations

bZIP

basic leucine zipper

Af

Aspergillus fumigatus

An

Aspergillus nidulans

Ca

Candida albicans

CBC

CCAAT-binding complex

Cg

Candida glabrata

CGFS

Cys-Gly-Phe-Ser

CIA

cytosolic iron-sulfur assembly

Cn

Cryptococcus neoformans

Fe-S

iron-sulfur

GRX

glutaredoxin

GSH

glutathione

ISC

iron-sulfur cluster

Sc

Saccharomyces cerevisiae

Sp

Schizosaccharomyces pombe