Regulatory and pathogenic mechanisms in response to iron deficiency and excess in fungi

Abstract

Iron is an essential element for all eukaryote organisms because of its redox properties, which are important for many biological processes such as DNA synthesis, mitochondrial respiration, oxygen transport, lipid, and carbon metabolism. For this reason, living organisms have developed different strategies and mechanisms to optimally regulate iron acquisition, transport, storage, and uptake in different environmental responses. Moreover, iron plays an essential role during microbial infections. Saccharomyces cerevisiae has been of key importance for decrypting iron homeostasis and regulation mechanisms in eukaryotes. Specifically, the transcription factors Aft1/Aft2 and Yap5 regulate the expression of genes to control iron metabolism in response to its deficiency or excess, adapting to the cell's iron requirements and its availability in the environment. We also review which iron‐related virulence factors have the most common fungal human pathogens (Aspergillus fumigatus, Cryptococcus neoformans, and Candida albicans). These factors are essential for adaptation in different host niches during pathogenesis, including different fungal‐specific iron‐uptake mechanisms. While being necessary for virulence, they provide hope for developing novel antifungal treatments, which are currently scarce and usually toxic for patients. In this review, we provide a compilation of the current knowledge about the metabolic response to iron deficiency and excess in fungi.

Regulatory and pathogenic mechanisms in response to iron deficiency and excess. Iron‐related virulence factors in fungal human pathogens. Metabolic response to iron deficiency and excess in fungi.

INTRODUCTION

Iron (Fe) is the fourth most abundant element in the Earth's crust and is essential for most prokaryotic and all eukaryotic organisms (Frey & Reed, 2012). Iron is a vital micronutrient that functions mainly as a redox cofactor of various kinds of enzymes in the form of haeme, iron‐sulphur clusters (ISC) and mononuclear iron or oxo‐diiron centres. These iron‐dependent enzymes are required for multiple cellular processes, including the tricarboxylic acid cycle, mitochondrial respiration, oxygen transport, lipid and carbon metabolism, ribosome biogenesis and recycling, DNA replication and repair, chromatin remodelling, translation and amino acid biogenesis, photosynthesis, and nitrogen fixation (Berman‐Frank et al., 2003; Neilands, 1974; Terry & Low, 1982). Furthermore, iron has the capacity to be reversibly oxidized and reduced, having a crucial impact on the cellular redox status, and contributing to the formation of reactive oxygen species (ROS). This term describes the chemical reactive molecules that are formed upon incomplete reduction of oxygen, including superoxide (O₂ ^·−), peroxides (H₂O₂ and ROOH), and free radicals (^·OH and RO^·) (Dixon & Stockwell, 2014; Lambeth & Neish, 2014). Iron is involved in the production of hydroxyl radicals (^·OH), the most damaging chemical species in biological systems, through Fenton's reaction (Fenton, 1894), where H₂O₂ reacts with a ferrous iron (Fe²⁺) to produce a ferric iron (Fe³⁺), hydroxide (OH⁻), and a hydroxyl radical. Subsequently, the hydroxyl radical reacts with H₂O₂ to produce a superoxide radical (O^2·−), promoting the reduction of Fe³⁺ to Fe²⁺ and O₂, completing the catalytic electron transport cycle of iron known as the Haber‐Weiss reaction (Crielaard et al., 2017; Haber & Weiss, 1932).

Certain amounts of iron and ROS are needed in order to correctly sustain and develop cellular and organismal life (Dixon & Stockwell, 2014; Frey & Reed, 2012). However, a large excess of iron, ROS, or both is related to several inflammatory disorders and chronic degenerative conditions (Kell, 2009; Lambeth & Neish, 2014; Mittal et al., 2014). At high concentrations, iron is extremely toxic to cell or organism growth and viability, leading to nonspecific oxidation and damage to nucleic acids, lipids, and proteins (Stadtman, 1992). On the other hand, an insufficient level of iron causes iron deficiency, anaemia, and adverse effects on the immune system as well as cognitive development (Camaschella, 2015; Philpott & Protchenko, 2008). In fact, iron deficiency is the most common human nutritional disorder in the world, affecting more than 2 billion people (Zimmermann & Hurrell, 2007). Therefore, all organisms have evolved sophisticated strategies to regulate intracellular iron levels in order to maintain essential iron‐dependent processes (Ehrensberger & Bird, 2011).

The complexity of iron homeostasis has been studied in detail in different eukaryotic model organisms such as the yeast Saccharomyces cerevisiae as well as in some human fungal pathogens such as Aspergillus fumigatus, Cryptococcus neoformans, and Candida albicans, as iron plays a key role during microbial infections (Barluzzi et al., 2002; Gerwien et al., 2017; Matthaiou et al., 2018). For example, the human pathogenic fungi C. albicans is able to adapt to extremes of iron availability in different host niches, such as the bloodstream (iron‐poor) or the gastrointestinal tract (iron‐rich). In these cases, C. albicans has developed a complex and efficient regulatory system for iron acquisition and storage to circumvent iron limitation within the human host (Ramanan & Wang, 2000; Knight, Vilaire, et al., 2005; Gerwien et al., 2017).

Because of the importance of understanding the role of iron metabolism regulation due to its influence on human physiological alterations and diseases, in this review we provide a compilation of the current knowledge about the metabolic response to iron deficiency and excess, both in model organisms such as S. cerevisiae and fungal human pathogens.

REGULATORY MECHANISMS OF IRON HOMEOSTASIS IN S. CEREVISIAE

The iron bioavailability can fluctuate from high scarcity conditions to overly abundance (Philpott et al., 2012; Sanvisens et al., 2011); in each situation S. cerevisiae cells exhibit different strategies to respond and adapt, mainly by triggering a complex rearrangement of gene expression.

Under iron‐scarcity conditions, the transcription factors Aft1 and its paralogue Aft2 (standing for Activator of Ferrous Transport), which sense intracellular iron levels, are activated and play a central role by regulating the expression of a subset of genes known as ‘the iron regulon’ (Kaplan & Kaplan, 2009; Philpott et al., 2012). The iron regulon includes genes involved in intracellular iron transport, iron uptake, metabolic remodelling, or iron scavenging (Ehrensberger & Bird, 2011; Rutherford & Bird, 2004). In addition, Aft1/2 up‐regulates CTH1 and CTH2, encoding for mRNA‐binding proteins that destabilize certain mRNAs through binding to specific AU‐rich elements (ARE) located in their 3′‐UTR (Pedro‐Segura et al., 2008; Puig et al., 2005, 2008). Thus, Cth2 contributes to remodelling metabolism by destabilizing a broad number of mRNAs encoding proteins involved in iron‐dependent metabolic pathways, such as mitochondrial respiration, the tricarboxylic acid cycle, haeme biosynthesis, ISC formation, and biotin synthesis (Puig et al., 2005), moreover Cth2 also inhibits the expression of target mRNAs by limiting their translation in response to iron deficiency (Ramos‐Alonso et al., 2018).

By contrast, under iron‐overload conditions, the transcription factor Yap5 (Yeast AP‐1) regulates the expression of a subset of genes whose promoter contain the AP‐1 binding site. One of the main Yap5 targets is CCC1, which encodes an iron vacuolar transporter that mediates iron detoxification by sequestering the iron excess into the vacuole (Li et al., 2001, 2008; Pimentel et al., 2012) in the form of ferric polyphosphate, which in S. cerevisiae can be later reused (Nguyen et al., 2019). As in plants, the yeast cell vacuole functions as a major iron storage and detoxifying compartment (Li et al., 2008). However, mammal cells do not have such storage organelle, and iron is stored within a specialized hollow protein called ferritin (Finazzi & Arosio, 2014; Pantopoulos et al., 2012), mostly in the liver. If iron uptake overloads the hepatic storage capacity, it may form aberrant iron depositions in the tissues, promoting cell damage, oxidative stress, and a wide range of diseases depending on the affected organ(s) (Sousa et al., 2020). Most fungi do not have iron‐rich proteins such as ferritin, except for some species belonging to the Mucoromycota phylum (Matzanke, 2020).

The role of Aft1 and Aft2 transcription factors and its linkage with Fe–S clusters in response to iron deficiency

Under conditions of iron depletion in S. cerevisiae, the transcription factors Aft1 and Aft2 are essential regulators of cellular iron homeostasis, which promote the transcriptional activation of the iron regulon (Ehrensberger & Bird, 2011; Ramos‐Alonso et al., 2020; Shakoury‐Elizeh et al., 2010; Waldron et al., 2009). It includes ~30–40 genes that encode proteins involved in the following processes (Figure 1):

• reductive iron uptake system: metalloreductases (Fre1‐4), high‐affinity iron uptake complex (Fet3/Ftr1), copper‐delivery proteins (Atx1 and Ccc2), and oxygen‐independent low‐affinity plasma membrane iron and copper transporter (Fet4) (Stearman et al., 1996; Yun et al., 2001).

• non‐reductive iron import machinery: cell wall mannoproteins (Fit1‐3) and iron‐xenosiderophore transporters (Arn1‐4) (Heymann et al., 2000; Protchenko et al., 2001).

• haeme degradation: haeme oxygenase (Hmx1) (Protchenko & Philpott, 2003).

• intracellular iron transport: iron importer into mitochondria (Mrs4), vacuolar metalloreducatse (Fre6), and iron exporters from vacuole (Fet5/Fth1, Smf3) (Mühlenhoff et al., 2003; Singh et al., 2007; Urbanowski & Piper, 1999).

• metabolic remodelling: Cth1/Cth2 mRNA‐binding proteins and biotin transporters (Puig et al., 2008).

• iron‐independent alternatives to use iron (biotin and 7‐keto‐8‐aminopelargonic acid (KAPA) importers Vht1 and Bio5) (Weider et al., 2006).

FIGURE 1.

Schematic representation of iron regulon members in Saccharomyces cerevisiae. It includes the main genes that encode proteins involved in acquisition of extracellular iron, acquisition of intracellular iron, iron scavenging, intracellular iron transport, and metabolic remodelling response of iron‐dependent processes.

Recent studies have identified novel genes regulated by Aft1 in iron scarcity conditions such as MMT1 and MMT2 (mitochondrial metal exporter genes) (Li et al., 2020), KHA1 (trans‐Golgi network K⁺/H⁺ exchanger) (Wu et al., 2016), and RNR1 (ribonucleotide reductase enzyme) (Ros‐Carrero et al., 2020). Aft1 DNA‐binding activity and subcellular localization are influenced by intracellular iron status. When the iron concentration is low, Aft1 accumulates in the nucleus, bound to the IREs (iron responsive element) consensus sequence (YRCACCCR) present in the promoter region of iron regulon genes and activating transcription (Kaplan & Kaplan, 2009; Yamaguchi‐Iwai et al., 1996) (Figure 2A). Aft2 responds to intracellular iron status changes in a similar way to Aft1. In spite of the overlapping roles of Aft1 and Aft2, both factors may have independent functions in accordance with the slight variations in the nucleotide sequence flanking these recognized DNA motifs. Aft2 promotes the transcription of genes involved in intracellular iron response (Courel et al., 2005; Rutherford et al., 2001). In addition, several studies revealed that Aft1 and Aft2 could additionally participate in other functions different from iron homeostasis regulation, such as chromosome maintenance and benomyl resistance, by a transcription‐independent mechanism (Berthelet et al., 2010).

FIGURE 2.

Schematic representation of the response mechanisms to iron deficiency (A) and excess (B) in Saccharomyces cerevisiae.

Several conserved cysteine desulfurases, scaffold proteins, iron chaperones, and thioredoxins located in the mitochondria are required for the assembly of iron‐sulphur clusters (ISC), which is the previous step to their insertion into enzymes that require these cofactors. The mitochondrial ISC machinery is also necessary for the iron‐dependent nuclear export of Aft1. Under iron‐sufficient conditions, the Aft1/2 transcription factors detect a mitochondrial iron signal, diminishing the transcription of iron starvation response genes. However, under iron scarcity conditions, ISC synthesis decreases and Aft1/2 accumulates in the nucleus, bound to the IREs, activating the iron regulon (Kaplan & Kaplan, 2009; Martínez‐Pastor et al., 2013; Stehling & Lill, 2013; Ueta et al., 2012). The ability of Aft1/2 to sense the iron signal is regulated by a complex formed by the two‐monothiol glutaredoxins Grx3 and Grx4, as well as Fra1 and Fra2 (the aminopeptidase P‐like protein and the BolA‐like protein iron repressors of activation, respectively; Fra stands for Fe Repressor of Activation) (Kumánovics et al., 2008; Ojeda et al., 2006; Pujol‐Carrion et al., 2006) (Figure 2A,B).

ISC are synthetized in the mitochondria. In the first place, iron is imported by inner membrane carriers Mrs3‐Mrs4 into the mitochondria (Mühlenhoff et al., 2010; Schulz et al., 2022; Ueta et al., 2012). Then, a [2Fe–2S] cluster is synthesized on the scaffold protein Isu1. The reaction requires the cysteine desulfurase complex Nfs1‐Isd11 as a sulphur donor, frataxin (Yfh1) as an iron donor or as a regulator of desulfurase activity, and the electron donor ferredoxin Yah1. The [2Fe–2S] cluster is transferred from Isu1 to the apoproteins with the help of the monothiol glutaredoxin Grx5 and a dedicated chaperone system composed of Ssq1, Jac1, and Mge1. The mentioned components are essential in the biogenesis and maturation of all ISC proteins and are thus termed the core ISC proteins. Specialized components catalyse the generation of [4Fe–4S] clusters and assist the insertion into client apoproteins (Schulz et al., 2022). Finally, Grx5, the ABC transporter Atm1, and the sulfhydryl oxidase Erv1 are needed for the generation and export of an unknown sulphur‐ and possibly iron‐ and glutathione (GSH)‐containing precursor termed X‐S to the cytosol, constituting the founding stone for the extramitochondrial ISC protein biogenesis, executed by the cytoplasmic ISC assembly (CIA) machinery (Lill et al., 2012, 2015; Mühlenhoff et al., 2015; Stehling & Lill, 2013). Moreover, the X‐S precursor is required to generate a [2Fe–2S] cluster that is inserted into the Grx3‐Grx4. These proteins function as an iron sensor, forming a dimer bound to a [2Fe–2S] cluster coordinated by two GSH molecules (Mühlenhoff et al., 2010; Ojeda et al., 2006; Pujol‐Carrion et al., 2006; Ueta et al., 2012) and interacting with Fra2, forming a [2Fe–2S]‐Fra2‐Grx3‐Grx4 complex (Li, Mapolelo, et al., 2011; Martínez‐Pastor et al., 2017) (Figure 2A,B). Under iron‐sufficient conditions, the [2Fe–2S] cluster is transferred from the Fra2‐Grx3‐Grx4 complex to the Aft1/2 transcription factors, which homodimerize, decreasing their DNA affinity and being exported to the cytosol (Li et al., 2012; Li & Outten, 2019; Poor et al., 2014) (Figure 2B). Recent studies have demonstrated that the mitogen‐activated protein (MAP) kinase Hog1 regulates Aft1 function by promoting its nuclear export. This is due to the interaction between Hog1 and Aft1, which directly phosphorylates this transcription factor at residues S210 and S224 (Martins et al., 2018).

The role of Cth2 in the remodelling of iron metabolism

In iron starvation conditions, Aft1/2 transcript factors activate the expression of two mRNA binding proteins, called Cth1 and Cth2. They are essential for an adequate adaptation to iron deficiency because they coordinate the metabolic reprogramming response (Martínez‐Pastor & Puig, 2020; Philpott et al., 2012). Cth1/Cth2 proteins belong to the tristetraprolin family, characterized by the presence of two CCCH tandem zing fingers (TZF) that specially interact with adenosine and uridine AU‐rich elements (AREs) in the 3′UTR regions of mRNAs to post‐transcriptionally inhibit their expression (Martínez‐Pastor & Puig, 2020; Puig et al., 2005; Wells et al., 2017). CTH1 expression is low, whereas CTH2 is highly induced upon iron deficiency; for this reason Cth2, a nucleocytoplasmic shuttling protein, plays a more relevant role in the adaptation to iron deficiency conditions and it is responsible for the downregulation of more than 200 mRNAs, whereas Cth1 is only implicated in the regulation of approximately 60 transcripts (Martínez‐Pastor et al., 2013).

The CTH‐driven degradation mechanism, known as ARE‐mediated mRNA decay (AMD), implicates the recruitment of ARE‐containing mRNAs in the nucleus (mediated by Cth2), followed by the maturation and export of a messenger ribonucleoprotein complex to the cytosol (Ramos‐Alonso et al., 2019). Once in the cytosol, the targeted mRNAs are degraded from 5′ to 3′ by an iron‐independent process that involves a conserved N‐terminal region of Cth2 and the RNA helicase Dhh1, the Ccr4‐Pop2 deadenylase complex and the exonuclease Xrn1 (Pedro‐Segura et al., 2008; Perea‐García et al., 2020; Prouteau et al., 2008; Vergara et al., 2011). After this process, Cth2 is released from the decay machinery and can re‐enter to the nucleus to initiate a new AMD cycle.

In response to iron deficiency conditions, Cth2 post‐transcriptionally inhibits the expression of ARE‐containing mRNAs encoding proteins involved in the following cellular functions:

• Vacuolar iron storage, by down‐regulating CCC1 mRNA (Puig et al., 2005).

• Enzymes of the tricarboxylic acid (TCA) cycle and respiratory chain, such as succinate dehydrogenase, aconitase, isocitrate dehydrogenase, and alpha‐ketoglutarate dehydrogenase (Puig et al., 2005, 2008).

• Haeme biosynthesis (Puig et al., 2005, 2008).

• Biosynthesis of several amino acids (leucine, lysine, glutamate, or methionine) (Puig et al., 2005, 2008).

• Synthesis of ergosterol, sphingolipids, and fatty acids (Jordá et al., 2022).

• Synthesis of enzymatic cofactors (lipoic acid and biotin) (Puig et al., 2008).

• Promoting the degradation of the ARE‐containing WTM1 mRNA, mediating the regulation of the ribonucleotide reductase enzyme (Sanvisens et al., 2011).

The role of Yap5 in response to iron excess

In iron overload conditions, the transcription factor Yap5 coordinates a crucial response in the activation of the expression of a few target genes. Yap5 is a member of the basic‐region leucine zipper (bZIP) transcription factor family, which in yeast is composed by eight members. They include the major oxidative stress regulator Yap1 (Rietzschel et al., 2015; Rodrigues‐Pousada et al., 2010) which associates to Yap response elements (YREs) within the promoter of its target genes (with TTASTAA consensus sequence).

In response to high iron levels, Yap5 specifically activates the transcription of the following genes:

• CCC1, a vacuolar transporter that mediates the import of excess cytosolic iron into the vacuole (Li et al., 2001).

• GRX4, monothiol glutaredoxin, which binds and transfers the iron‐derived mitochondrial signal of Aft1/2 transcription factors (Pujol‐Carrion et al., 2006).

• CUP1, the cytosolic copper‐binding metallothionein to protect cells from oxidative stress and limit copper availability (Li, Jia, et al., 2011; Rodrigues‐Pousada et al., 2010).

• TYW1, which encodes a [4Fe–4S]‐containing enzyme implicated in iron buffering and synthesis of wybutosine‐modified tRNA (Li, Jia, et al., 2011; Noma et al., 2006).

Moreover, in iron rich‐conditions, Aft1 is dissociated from DNA and is shuttled to the cytosol by the nuclear exportin Msn5, halting transcription of the iron regulon (Ueta et al., 2012; Yamaguchi‐Iwai et al., 2002) (Figure 2B).

Yap5‐independent transcriptional activation of CCC1

As ccc1 mutants are more sensitive to iron than yap5 mutants (Rietzschel et al., 2015), this indicates that other factors than Yap5 activate the expression of CCC1 to protect against high cytosolic iron toxicity. The Snf1 complex regulates the transcriptional changes associated with glucose derepression and the general stress transcription factors Msn2 and Msn4 contribute to the regulation of CCC1 transcription in an Yap5‐independent manner (Li & Ward, 2018).

IRON TRANSPORT MECHANISMS IN PATHOGENIC FUNGI

While being largely abundant on the Earth's crust, iron is present mostly in the oxidized ferric form (Fe³⁺) in aerobic conditions, being hardly soluble at neutral pH (Ratledge & Dover, 2000). This problem in iron bioavailability is solved by microorganisms in three ways: (i) environment acidification, (ii) reduction of the iron to a more soluble ferrous form (Fe²⁺), or (iii) secretion of soluble iron‐chelating molecules; fungi employ mostly the last two strategies, often simultaneously (Philpott, 2006).

One of the few examples of iron mobilization and uptake upon acidification of the environment has been described in Neurospora crassa (Winkelmann, 1979). The reductive iron assimilation pathway (RIA) usually consists of a sequential process that first reduces the insoluble iron present in the cell environment via the membrane‐bound ferric reductase Fre5, then iron is re‐oxidized by the ferroxidase Fet3, and finally ferric ions are internalized by the high‐affinity iron permease Ftr1 (Askwith et al., 1994; Lesuisse & Labbe, 1994; Stearman et al., 1996; Wang et al., 2003). The reason why fungi need to re‐oxidize iron after reducing it is unknown, but it is proposed to be a specific mechanism for iron since other divalent ions do not change their oxidation state as often as iron, being excluded from the RIA pathway (Haas et al., 2008; Kosman, 2003) (Figure 3). The siderophore‐mediated iron assimilation pathway (SIA) is based on the use of siderophores (Greek for ‘iron carrier’), small molecules of 100–500 Da with very high affinity to the ferric ion (Hider, 2007), which are synthetized and excreted by fungi and other microorganisms. About 500 different compounds are identified as siderophores (Boukhalfa & Crumbliss, 2002), which can be classified into three main categories: catecholates, hydroxamates, and carboxylates. Most of the fungal siderophores belong to the hydroxamate category, which is derived from the amino acid l‐ornithine (Haas et al., 2008). Fungal siderophores can be further divided into four structural families: the rhodotorulic acid, the fusarinine, the coprogen, and the ferrichrome family (Haas et al., 2008) (Figure 3). Siderophore biosynthesis starts with the hydroxylation of l‐ornithine (Mei et al., 1993), followed by the addition of acyl and amino acid groups to the growing molecule, providing the main backbone of each of the families; final modifications such as methylation or acetylation produce further variants within each family. After synthesis and secretion, siderophores bind with high affinity to insoluble iron sources in the environment, which can be later internalized via specific siderophore‐iron transporters (SITs), which are proton symporters powered by the plasma membrane potential (Haas et al., 2003; Philpott & Protchenko, 2008) (Figure 4). Some fungi species like S. cerevisiae lack the siderophore biogenesis machinery; nonetheless, they are capable to absorb iron bound by siderophores produced by other organisms (xeno‐siderophores) by expressing siderophore transporters (Froissard et al., 2007).

FIGURE 3.

Chemical structures of the major fungal siderophore types bound to ferric ion (except for rhodotorulic acid). Compared to TAFC, in FsC, the acetyl group (circled yellow) is replaced by H atom.

FIGURE 4.

Schematic representation of the reductive (RIA) and siderophore mediated (SIA) iron acquisition mechanisms in fungi. SIT stands for ‘siderophore‐iron transporter’.

It has been shown that calcium supplementation has a synergistic effect with iron deprivation in slowing growth on some pathogenic fungi, by further inhibiting iron uptake mechanisms transcriptionally and causing oxidative stress triggered by mitochondrial calcium overload. In the case of A. fumigatus, this synergistic effect of calcium has been observed in murine models, besides in the in vitro conditions where the other two organisms have been tested for this effect (Ye et al., 2022). Moreover, in C. neoformans, it has been shown that increase on calcium levels disrupts the calcineurin pathway, affecting cell wall integrity and capsule formation, thus reducing virulence (Stempinski et al., 2022).

Iron homeostasis, specifically reductive iron uptake, has also been associated with morphological development in dimorphic or pleomorphic fungal pathogens that can switch between yeast‐like growth and pseudo or hyphal growth (Bairwa et al., 2017). For instance, in Candida albicans, the reductive iron uptake system is coordinated with the adhesion protein Als3 (agglutinin‐like sequence 3) to facilitate iron acquisition from ferritin during hyphal growth (Almeida et al., 2008). In C. albicans, the ability to switch between yeast and hyphal morphologies contributes to virulence, and there is a link between the expression of adhesion proteins (MP65 and PGA62) and chromatin remodelling in response to iron availability (Puri et al., 2014). Additionally, the connections between cell morphology, reductive iron uptake, and siderophore‐mediated uptake in the dimorphic pathogen Talaromyces marneffei have been studied (Pasricha et al., 2016).

In a pathogenic environment (i.e., inside a mammal), free iron is a scarce resource since most of the iron is already bound with high affinity to transferrin, haemoglobin, or other iron‐containing molecules. Consequently, pathogenic fungi have developed virulence factors improving their iron uptake from the host organisms. Note that the host environment is usually harsh for the fungal pathogen, as they rarely cause infections in healthy individuals; however, those infections can be lethal in immunocompromised patients (Philpott, 2006). Below, we will detail the most relevant iron‐related virulence factors used by the most common fungal pathogens in humans.

Iron homeostasis and uptake mechanisms in Aspergillus fumigatus virulence

Aspergillus fumigatus is a saprophytic fungus and the most common air‐borne fungal pathogen of humans (Dagenais & Keller, 2009; Tekaia & Latgé, 2005). Clinical manifestations range from a local allergic disease manifesting as asthma or sinusitis (the most extreme condition of which is called Allergic bronchopulmonary aspergillosis or ABPA) to a life‐threatening invasive disease in immunocompromised patients, termed ‘invasive aspergillosis’ (IA) (Barnes & Marr, 2006). All its virulence‐associated genes are transcriptionally upregulated during iron starvation, encoding important products for survival in iron‐scarce conditions (Haas, 2012). A. fumigatus and its less virulent relative A. nidulans lack systems for direct uptake of iron sources from the host, such as haeme, transferrin, or ferritin (Eisendle et al., 2003; Schrettl et al., 2004); they obtain iron via low affinity ferrous permeases that also can transport copper and zinc ions, the SIA pathway, and the RIA pathway (Figure 5).

FIGURE 5.

Iron homeostasis in Aspergillus fumigatus. Gene regulatory network controlling iron homeostasis in iron deficiency (up) and iron excess (down) conditions (separated by the thick dotted line) as well as iron‐related metabolic pathways activated in each condition. Inside the nucleus, solid lines represent direct transcriptional activation or repression while dashed lines connecting the master regulators represent transcriptional repression as well. Question marks indicate processes in which the effector protein is unknown or the process itself is not proved to exist. Grey and red arrows represent intracellular iron trafficking for storage and use, respectively. ETC, electron transport chain; ISC, iron–sulphur cluster; LAA, low affinity acquisition; Orn, l‐ornithine; RIA, reductive iron acquisition; SIA, siderophore iron acquisition; TCA, tricarboxylic acid cycle.

Aspergillus spp. excretes fusarinine C (FsC) and triacetylfusarinine C (TAFC) to sequester iron and then internalizes the siderophore‐bound ferric ions through SITs. A. fumigatus possesses five potential SITs (MirB, MirC, MirD, Sit1, and Sit2), which are transcriptionally repressed by iron. Besides extracellular siderophores, A. fumigatus produces two intracellular ferrichrome‐type siderophores named ferricrocin (FC) and hydroxyferricrocin (HFC) for iron storage, distribution, intracellular handling of iron, hyphal ferricrocin, and conidial hydroxyferricrocin (Haas, 2014; Schrettl et al., 2007; Wallner et al., 2009); iron can be detoxified and stored into the vacuole via the CccA vacuolar transporter; however, it is not clear if this iron form can be reused (Gsaller et al., 2012), or internalized to the mitochondria for its use in biosynthetic pathways via the importer MrsA (Long et al., 2016). Siderophore production is crucial for A. fumigatus virulence, as blockage of siderophore biosynthesis (ΔsidA mutant) results in absolute avirulence in murine models of IA (Hissen et al., 2005; Schrettl et al., 2004), while depletion of intracellular or extracellular siderophores results in partial infection attenuation (Schrettl et al., 2007; Yasmin et al., 2012). Iron bound to extracellular siderophores is internalized by the SITs; MirB and MirD are specific for TAFC and FC recognition and transport (Aguiar et al., 2022; Raymond‐Bouchard et al., 2012), Sit1 and Sit2 have been found to be involved in the transport of xenosiderophores ferrichrome and ferrioxamine B (Park et al., 2016), whereas the substrate specificities of MirC remains elusive (Aguiar et al., 2021). In a murine aspergillosis model, MirB was found to be crucial for virulence (Aguiar et al., 2022); this transporter is absent in the mammalian host, so they may constitute a therapeutic target for specific antifungals (Wencewicz et al., 2013). The siderophore system is also important for intracellular survival of the pathogen after phagocytosis by alveolar macrophages in murine models, and siderophore biosynthesis defects change the immune response of the host (Schrettl et al., 2010; Seifert et al., 2008). After cellular uptake of TAFC‐ and FsC‐iron complexes by SITs, hydrolysis takes place in the cytosol, two esterases are involved in this process have been identified: EstB and SidJ, which belong to different protein families (Gründlinger et al., 2013; Kragl et al., 2007). EstB is homologous to E. coli siderophore degrading enzyme Fes, which is responsible for the release of iron from the catecholate‐type siderophore enterobactin (Haas, 2014). EstB specifically acts on TAFC, while SidJ is specific to FsC families (Gründlinger et al., 2013; Kragl et al., 2007).

Interestingly, EstB and SidJ are not essential for siderophore hydrolysis, but rather optimize the process and facilitate the release of iron, since inactivation of EstB (ΔestB mutant) leads to a decrease in the growth rate during iron starvation, reduces the transfer of iron from TAFC to metabolism, and intracellular siderophore FC, and consequently delays iron sensing (Kragl et al., 2007). The lack of FC and vacuolar storage, particularly in the absence of both enzymes, results in an accumulation of siderophore (in the iron chelated form) breakdown products, indicating that the transfer of iron from extracellular siderophores to metabolism, FC, or the vacuole occurs prior to the recycling of siderophore breakdown products (Gsaller et al., 2012).

As a preclinical application, SITs present a promising opportunity to enhance the diagnosis of fungal infections. In animal models, the use of ⁶⁸Gallium (⁶⁸Ga) chelated with TAFC or the bacterial siderophore ferrioxamine E has demonstrated the ability to enable in vivo imaging of infections caused by A. fumigatus and specific visualization of the infection site through Positron Emission Tomography (PET). This imaging technique relies on the selective accumulation of the radiolabelled compounds in fungal cells, allowing for improved detection and diagnosis of invasive fungal infections (Haas, 2014; Kriegl et al., 2022; Petrik et al., 2020).

Siderophore biosynthesis and iron homeostasis are transcriptionally regulated by the SreA and HapX transcription factors, via a negative feedback loop (Haas, 2012; Hortschansky et al., 2007; Schrettl et al., 2010). HapX represses iron utilization pathways in low iron conditions; the ΔhapX mutant is avirulent in the murine model of IA and TAFC production levels are reduced (Schrettl et al., 2010). SreA is a transcriptional repressor of iron acquisition genes, and a ΔsreA mutant retains virulence towards the murine model of IA (Schrettl et al., 2008) (Figure 5). Other transcription factors regulating iron homeostasis and uptake apart from the main SreA/HapX control system are SrbA, AcuM, PrtT, HacA/IreA, and MpkA (Moore, 2013).

The leucine biosynthetic pathway has emerged as a critical factor for the adaptation of A. fumigatus to iron starvation and for its virulence (Orasch et al., 2019), where the multifaceted role of the transcription factor LeuB in coordinating the expression of key genes involved in the regulation of branched chain amino acids (BCAA) biosynthesis, nitrogen metabolism, and iron homeostasis in A. fumigatus has been demonstrated in vitro and in vivo (Long et al., 2018). LeuB not only directly activates genes involved in iron acquisition but also influences the expression of iron‐related genes through its interaction with HapX. Moreover, activation of LeuB is influenced by α‐isopropylmalate (α‐IPM), an intermediate that accumulates during iron starvation due to the dependence of BCAA enzymes dihydroxyacid dehydratase (Ilv3A) and α‐IPM isomerase (LeuA) on iron–sulphur (Fe–S) clusters (Misslinger et al., 2021).

Aspergillus fumigatus possess a single glutaredoxin named GrxD which is involved in iron sensing. During iron excess, HapX is believed to coordinate [2Fe–2S] clusters independently of GrxD, transforming HapX into an activator of iron‐consuming pathways and iron detoxification (Misslinger et al., 2019). This activation relies on specific conserved motifs, such as the CGFCX₅CXC motif found in S. cerevisiae Yap5 (Gsaller et al., 2014). On the other hand, the repression of iron acquisition by SreA during iron availability is assumed to involve [2Fe–2S] cluster coordination through a conserved motif in a GRX‐independent manner. During iron starvation, GrxD appears to remove [2Fe–2S] clusters from both SreA and HapX, inactivating SreA and converting HapX from its ‘iron excess’ to its ‘iron starvation’ function. Overall, the absence of GrxD leads to the constitutive derepression of iron consumption and constitutive repression of iron acquisition, locking HapX and SreA in the ‘iron form’. Consequently, the lack of Grx4 results in iron‐regulatory defects specifically during iron starvation (Misslinger et al., 2021).

On the other hand, A. fumigatus genome encodes 15 putative metalloreductases (with FreB among them (Blatzer et al., 2011)), but blockage of the RIA pathway (ΔftrA mutant) does not affect A. fumigatus virulence (Schrettl et al., 2004). However, several lines of evidence indicate a putative role of the RIA pathway in virulence (McDonagh et al., 2008; Schrettl et al., 2004), as mutants lacking either extracellular siderophore biosynthesis still retain some virulence. Moreover, the RIA pathway is crucial for virulence of siderophore biogenesis‐lacking pathogens such as Candida spp. and Cryptococcus spp.

Iron acquisition virulence factors in Candida albicans

Candida albicans and other Candida species are common commensal fungi that live perpetually associated with their mammalian host (Odds, 1988), and they are present in the oral cavity of up to 75% of the population (Ruhnke, 2006) without any clinical manifestation. However, immunocompromised individuals can develop superficial infections in the oral cavity termed ‘oral candidiasis’ (OC) (Ruhnke, 2006). Moreover, Candida spp. can also infect the vulvovaginal area, causing ‘vulvovaginal candidiasis’ (VVC), which 75% of women have suffered at least once in their life (Sobel, 2007) even without an explicit immunocompromised precondition. While these superficial infections are bothersome and quite common in the population, they are usually non‐lethal; in contrast, systemic (or disseminated) infections of C. albicans are associated with a high mortality rate (Pfaller & Diekema, 2007). Risk factors for systemic candidiasis include neutropenia, damage to the gastrointestinal mucosa, the use of central venous catheters, the application of broad‐spectrum antibacterials, and trauma or gastrointestinal surgery, as all those processes that disrupt physical and biological barriers and that can facilitate the transfer of Candida spp. from its natural skin niche to the bloodstream and other internal organs (Koh et al., 2008; Spellberg et al., 2011).

Candida spp. can switch between yeast and hyphae phenotypic morphologies. Although hyphae forms are more invasive, both morphologies have a relevant role in pathogenesis and virulence (Jacobsen et al., 2012). C. albicans possess lots of virulence mechanisms, including the use of specialized proteins termed adhesins and invasins, biofilm formation on both biotic and abiotic surfaces regulated by contact sensing, hydrolase secretion, pH sensing and regulation, the use of heat shock proteins, transcriptional programmes for morphology switching, and metal acquisition mechanisms (Mayer et al., 2013). The main iron use and storage subcellular localization are the vacuole and the mitochondria, and intracellular iron homeostasis is controlled by the Mrs4–Ccc1–Smf3 pathway; Mrs4 is the mitochondrial iron importer, while Ccc1 and Smf3 are the vacuole iron importer and exporter, respectively (Figure 6). Altering the balance of these transporters either genetically cause an imbalance in iron homeostasis, deriving in mitochondrial disfunction, cell wall integrity, and reduced virulence in murine models (Xu et al., 2014). Thus, the Mrs4‐Ccc1‐Smf3 pathway is an interesting target for development of novel drugs against C. albicans infections.

FIGURE 6.

Iron homeostasis in Candida albicans. Gene regulatory network controlling iron homeostasis in iron deficiency (up) and iron excess (down) conditions (separated by the thick dotted line), as well as iron‐related metabolic pathways activated in each condition. Inside the nucleus, solid lines represent direct transcriptional activation or repression, while dashed lines connecting the master regulators represent transcriptional repression as well; the dotted line connecting Sfu1 and Hap43 represents that the transcriptional repression exerted on Hap43 is indirect. Question marks indicate processes in which the effector protein is unknown. Grey arrows represent intracellular iron trafficking for storage and use. LAA, low affinity acquisition; RIA, reductive iron acquisition; SIT, siderophore iron transport.

The most widely investigated metal regarding pathogenesis is iron, which can be acquired by C. albicans by the RIA pathway, a siderophore uptake system and a haeme‐iron uptake system (Almeida et al., 2009). Depending on the iron concentration of the environmental niche, C. albicans regulates iron homeostasis using a complex regulatory network controlled by a transcriptional activator named Sef1 and two iron‐responsive transcriptional repressors named Sfu1 and Hap43. In an iron scarce niche such as the bloodstream, Sef1 activates iron uptake genes and Hap43 represses iron utilization genes and SFU1; while in a high iron concentration niche such as the gastrointestinal tract, Sfu1 represses iron uptake genes and SEF1 as well as HAP43 (indirectly), forcing a high utilization of the available iron and limiting its toxicity (Blankenship & Mitchell, 2011; Chen et al., 2011) (Figure 6).

Candida albicans do not produce its own siderophores, but it can scavenge iron bound to xeno‐siderophores; mutation in the only siderophore‐iron transporter described in C. albicans (sit1Δ/Δ) results in unimpaired virulence in murine models of systemic candidiasis, but it reduces the capacity to damage ex vivo human keratinocyte tissue (Heymann et al., 2002). The haeme‐iron uptake system in C. albicans is regulated by Hap1 (Andrawes et al., 2022) and mediated by the expression of the CFEM hemophore system (Common in Fungal Extracellular Membrane) RBT5, RBT51, CSA1, CSA2, and PGA7, which internalize and transport haeme bound to haeme‐binding proteins such as haemoglobin and human serum albumin from the external medium to the plasma membrane through CFEM hemophore cascade (Kornitzer & Roy, 2020). To introduce haeme into the cell for haeme‐iron utilization, recent studies demonstrated a strict requirement for the vacuolar ATPase activity, and the involvement of components from endocytosis and as well as components of ESCRT (Endosomal Sorting Complex Required for Transport) complexes (Kornitzer & Roy, 2020). Later iron is released after haeme digestion by Hmx1. localized to the vacuole (Hmx1 is anchored to the ER in S. cerevisiae) (Kornitzer & Roy, 2020; Santos et al., 2003); a rbt5Δ/Δ mutant exhibits normal virulence in mice, and the role of the other five haeme‐binding proteins in virulence remains untested (Almeida et al., 2009; Weissman & Kornitzer, 2004).

Candida albicans use the RIA pathway to steal iron from the host's ferritin and transferrin deposits, as well as acquiring free iron (Knight, Lively, et al., 2005; Almeida et al., 2008); C. albicans bounds the host's ferritin via the surface and adhesion protein Als3, especially in the hyphae form (Almeida et al., 2008); Als3 also play a role in biofilm formation and adhesion to host's cells (Nobile et al., 2006; Zhao et al., 2004, 2006), and ALS3 deletion suppresses virulence in surface infection models, but not in systemic infection models (Almeida et al., 2008; Cleary et al., 2011; Zhao et al., 2004). C. albicans has two characterized ferric reductases, called Cfl1 and Cfl95 and localized in the plasma membrane (Hammacott et al., 2000; Knight et al., 2002; Yamada‐Okabe et al., 1996), of a total pool of 17 ferric reductase putative genes. Ferrous iron is reconverted to the ferric form after solubilization by the multicopper oxidase CaFet3, which requires copper supplied by the intracellular copper transporter Ccc2 (Weissman et al., 2002); a fet3Δ/Δ mutant has impaired growth in iron‐limiting medium, but its virulence is just slightly lower than the wild‐type strain (Eck et al., 1999). Ferroxidized iron is internalized via the CaFtr1 high affinity iron permease, encoded by a gene essential for survival in limiting iron media as well as for virulence (Ramanan & Wang, 2000) (Figure 6); C. albicans has another putative high affinity iron permease (CaFtr2), but evidence shows that this second permease is not essential for survival or pathogenesis and it is transcriptionally regulated differently than CaFtr1, while being a functional high affinity iron permease (Ramanan & Wang, 2000).

Candida albicans is becoming increasingly resistant to existing treatments, and drug repurposing is a promising strategy to find new therapeutic approaches. One of them is related to iron metabolism and consists of limiting the pathogen's iron availability by using the FDA‐approved iron chelator ‘deferasirox’. This treatment has been tested in murine models of OC, which resulted in a significant reduction on C. albicans CFU per gram of tongue tissue, as well as reduced salivary iron levels and neutrophil‐mediated inflammation (Puri et al., 2019). Another potential therapeutic strategy consists on the administration of a monoclonal antibody named C7, which has fungicidal properties and it is known to bind Als3, which may block the iron uptake from the host's ferritin (Brena et al., 2007, 2011). Contrarily, iron overload in the host results in more susceptibility towards Candida spp. infections, both superficial and systemic (Mencacci et al., 1997; Tripathi et al., 2022).

Iron‐regulated virulence factors of Cryptococcus neoformans

Cryptococcus neoformans is a human fungal pathogen that can cause meningoencephalitis in immunocompromised individuals, especially on those affected by HIV/AIDS in developing countries (Park et al., 2009) with a very high mortality rate. It also causes a pulmonary disease, both in immunocompetent and immunocompromised patients; the most common symptom among them is cough, following a benign clinical course in immunocompetent patients, but deriving into a more aggressive disease causing cavitary lesions in immunocompromised patients (Chang et al., 2006). Cryptococcosis is exacerbated by experimental iron overload in murine models, highlighting the importance of iron homeostasis in virulence (Barluzzi et al., 2002).

There are several transcription factors regulating iron homeostasis in adaptation to the host environment. The main driver of transcriptional response to iron limitation is Cir1, through both positive and negative regulatory influences in a wide variety of virulence‐relevant functions, such as cAMP/PKA, calmodulin and MAP kinase signalling pathways (Jung et al., 2006), some of them affecting iron homeostasis. Moreover, Cir1 is needed for capsule formation, growth at 37°C and virulence, as well as to influence processes that require iron‐dependent enzymes like ergosterol biosynthesis and glycolysis (Choi et al., 2012), but curiously it has an inhibitory role on the RIA pathway (Jung et al., 2006). A second layer of control is exerted by HapX and Rim101. HapX induces siderophore transporter expression and represses electron transport in response to low‐iron conditions, and it also induces Cir1 and Rim101 expression (Jung et al., 2010); loss of HapX has a small but measurable decrease in virulence. Rim101 nuclear localization is regulated by the Rim/Pal signalling pathway upon changes in pH (Ost et al., 2015), and it induces genes related to different pathways of iron uptake and capsule formation (O'Meara et al., 2010, 2014). Surprisingly, a rim101 mutant triggered a more severe pulmonary inflammation than the wildtype, but less severe neurological symptoms, likely because of changes in the host's immune response upon aberrant capsule formation (O'Meara et al., 2013). As said before, Cir1 boosts cAMP/PKA signalling, which in turn its activating Rim101, as well as promoting the expression of the genes of the RIA pathway (O'Meara et al., 2010); another important iron uptake and virulence regulator is Sre1 (a homologue of SrbA from A. fumigatus), which is induced upon hypoxia (as happens during infection) and promotes the expression of the RIA genes, as well as the formation of melanin (Chang et al., 2007) (Figure 7).

FIGURE 7.

Iron homeostasis in Cryptococcus neoformans. Gene regulatory network controlling iron homeostasis during host infection, as well as iron‐related metabolic pathways activated in this situation. Inside the nucleus, solid lines represent direct transcriptional activation or repression of the indicated gene set; for clarity, the arrows have the same colour as their corresponding regulator. Solid lines among the regulators represent protein–protein interactions, while dashed lines connecting the master regulators represent transcriptional activation; the dotted line between nuclear and cytoplasmic Rim101 represents nuclear import. Question marks indicate processes in which the effector protein is doubtful or unknown. Grey arrows represent intracellular iron trafficking for storage and use. 3‐HAA, 3‐Hydroxyanthranilic acid; ESCRT, endosomal sorting complex required for transport; ETC, electron transport chain; HemeA, Heme Acquisition; ISC, iron–sulphur cluster; LAA, low affinity acquisition; RIA, reductive iron acquisition; SIT, siderophore iron transport.

Iron regulates the synthesis of key virulence factors of C. neoformans including the pigment melanin and the polysaccharide capsule (Jung et al., 2006; Liu & Nizet, 2009). Iron uptake mechanisms include high and low affinity iron permeases (Jacobson et al., 1998), iron reduction activities performed by melanin and laccase, iron reductases and secretion of the reductant 3‐hydroxyanthranilic acid (Nyhus et al., 1997), internalization of xeno‐siderophores and haeme‐iron uptake (Kronstad et al., 2013). C. neoformans has six putative xeno‐siderophore transporters (Sit1‐6), but it does not produce its own siderophores and available evidence indicates that siderophore uptake is not required for virulence in murine models (Jacobson & Petro, 1987; Tangen et al., 2007). Haeme‐iron uptake is mediated by the extracellular mannoprotein Cig1, which contributes to virulence if the high affinity uptake system is disabled (Cadieux et al., 2013). Another important player in haeme‐iron acquisition is the ESCRT‐I (endosomal sorting complex required for transport) protein Vps23; which is needed for proper polysaccharide capsule and melanin formation (major virulence factors of C. neoformans). In fact, the vps23 mutant exhibited a reduced virulence in a murine model of cryptococcosis, while Vps23 overexpression displayed hypervirulence (Hu et al., 2013). The best characterized iron acquisition mechanism in C. neoformans is the RIA pathway, exploiting the host's iron storage proteins like transferrin. C. neoformans genome has eight putative ferric reductases, being Fre201 the one with the greatest contribution to the overall reductase activity. However, all of them display activity, suggesting functional redundancy, as Fre4 is necessary for proper melanin production and its loss increased susceptibility to antifungal drugs, while Fre2 has the major contribution to virulence in a murine model of cryptococcosis (Saikia et al., 2014). The Cfo1 ferroxidase must be localized to the plasma membrane for proper function, mediated by cAMP‐signalling. Cfo1 is necessary for virulence, as well as for resistance to the antifungal drugs fluconazole and amphotericin B, but these phenotypes can be counterbalanced if exogenous haeme or siderophores are present, suggesting that high enough iron intracellular levels are required for antifungal resistance (Jung et al., 2009); an homologous protein is also encoded in C. neoformans genome (CFO2), but evidence suggests that it is not relevant for pathogenesis. The two high affinity iron transporters Cft1 and Cft2 are important for virulence, an apparently Cft2 was not essential for growth in low iron environments; one hypothesis for the Cft2 function is that it may serve as a vacuolar iron transporter, mobilizing to the cytoplasm the iron reserves stored inside the vacuole (Jung et al., 2008). Cft1 was required for virulent growth in the brain of infected murine models of cryptococcosis, while dissemination in the lungs was unaffected, suggesting that transferrin may be an especially important iron source in brain tissue (Figure 7). The expression of both CFT1 and CFT2 genes is regulated by a cAMP‐dependent protein kinase, and Cir1 has opposite roles in regulation of each of those genes.

CONCLUSIONS AND FUTURE DIRECTIONS

Iron is a key element in a plethora of biological functions, and a well‐balanced homeostasis is required. Saccharomyces cerevisiae has been a great model to unravel the transcriptional response to varying iron concentrations and iron uptake strategies, and both processes have been key for understanding the conserved mechanisms shared by other eukaryotes as well as for modelling human fungal pathogens, which are a severe threat to a significant part of the population.

Understanding the differences and similarities between the host and the fungal pathogen regarding iron homeostasis and iron uptake strategies is of great importance in order to find novel therapeutic targets against pathogenic fungi. The available antifungals are very limited both in spectrum and toxicity towards the host, and some pathogenic strains are becoming resistant to the available treatments. Disrupting the pathogen's ability to access the host's iron sources is a promising strategy for enlarging our therapeutic toolset, by repurposing already existing drugs that may interact with those targets or by directed drug development against fungal‐specific mechanisms for iron uptake. It has been reported that statins (HGM‐CoA reductase inhibitors, used for hypercholesterolemia treatment) have some antifungal activity, since the isoprenoid biosynthesis pathway converges with the siderophore biosynthesis pathway leading to FsC and TAFC production (Balhara et al., 2016; Macreadie et al., 2006); the required dose if used alone is beyond the safe therapeutic dose of statins, nonetheless combining statins and antifungal azoles has synergistic effects against various pathogenic fungi while being used in clinically achievable concentrations (Nyilasi et al., 2010), but its shared hepatic metabolism would discourage its use for invasive infections, limiting its application to therapy for patients with oropharyngeal candidiasis or other mucocutaneous infections. A screening with the Prestwick Chemical library (including 1200 FDA‐approved compounds) using fluorescent reporters to sense disruption of metal homeostasis in C. albicans has identified two promising candidates named artemisinin and pyrvinium pamoate; they both show fungistatic rather than fungicidal properties, thus its therapeutic application would require a combination of these compounds with other antifungal drugs (Simm & May, 2019). A recent preprint has shown the therapeutic potential of a compound analogous to Streptomyces‐produced Collismycin A (ColA), which induces an iron starvation response. While ColA possesses substantial cytotoxic activities against mammalian cells besides its antifungal and antimicrobial activities, its analogous NR‐6226C has little toxicity towards mammalian cells while keeping its antifungal activity on azole‐ and echinocandin‐resistant Candida species, and its effectiveness has been proven in vivo in Galleria mellonella larvae infected with Candida glabrata (Corrales et al., 2023). Indeed, further research is needed to develop all those promising therapeutic targets into specific clinical applications against fungal infections.

AUTHOR CONTRIBUTIONS

Jordi Pijuan: Conceptualization (lead); writing – original draft (lead); writing – review and editing (lead). David F. Moreno: Conceptualization (lead); writing – original draft (lead); writing – review and editing (lead). Galal Yahya: Conceptualization (lead); writing – original draft (lead); writing – review and editing (lead). Mihaela Moisa: Funding acquisition (equal); writing – original draft (equal); writing – review and editing (equal). Ihtisham Ul Haq: Writing – original draft (equal); writing – review and editing (equal). Katarzyna Krukiewicz: Writing – original draft (equal); writing – review and editing (equal). Rasha Mosbah: Writing – original draft (equal); writing – review and editing (equal). Kamel Metwally: Writing – original draft (equal); writing – review and editing (equal). Simona Cavalu: Funding acquisition (equal); writing – original draft (equal); writing – review and editing (equal).

FUNDING INFORMATION

None.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

Pijuan, J. , Moreno, D.F. , Yahya, G. , Moisa, M. , Ul Haq, I. , Krukiewicz, K. et al. (2023) Regulatory and pathogenic mechanisms in response to iron deficiency and excess in fungi. Microbial Biotechnology, 16, 2053–2071. Available from: 10.1111/1751-7915.14346