Connecting iron regulation and mitochondrial function in Cryptococcus neoformans

Abstract

Iron acquisition is essential for the proliferation of microorganisms, and human pathogens such as the fungus Cryptococcus neoformans must use sophisticated uptake mechanisms to overcome host iron sequestration. Iron is of particular interest for C. neoformans because its availability is an important cue for the elaboration of virulence factors. In fungi, extracellular iron is taken up through high affinity, low affinity, siderophore-mediated, and heme uptake pathways, and the details of these mechanisms are under active investigation in C. neoformans. Following uptake, iron is transported to intracellular organelles including mitochondria where it is used in heme biosynthesis and the synthesis of iron-sulfur (Fe-S) cluster precursors. One Fe-S cluster binding protein of note is the monothiol glutaredoxin Grx4 which has emerged as a master regulator of iron sensing in C. neoformans and other fungi through its influence on the expression of proteins for iron uptake or use. The activity of Grx4 likely occurs through interactions with Fe-S clusters and transcription factors known to control expression of the iron-related functions. Although the extent to which Grx4 controls the iron regulatory network is still being investigated in C. neoformans, it is remarkable that it also influences the expression of many genes encoding mitochondrial functions. Coupled with recent studies linking mitochondrial morphology and electron transport to virulence factor elaboration, there is an emerging appreciation of mitochondria as central players in cryptococcal disease.

Graphical abstract

Introduction

Invasive fungal infections are an underappreciated threat to human health [1–3]. Fungal diseases are increasing in frequency and there is a clear need for a detailed, mechanistic understanding of fungal pathogenesis to support improved diagnostics, the discovery and deployment of additional antifungal drugs, and the development of effective vaccines [1–3]. One of the most prevalent fungal diseases is caused by the basidiomycete yeast Cryptococcus neoformans [3–5**]. The bulk of cryptococcal disease occurs in immunocompromised individuals suffering from HIV/AIDS, and it is estimated that this fungus causes ~300,000 cases of meningoencephalitis per year, resulting in ~200,000 deaths globally [3–5**]. Cryptococcal meningitis is thought to be responsible for ~15% of all AIDS related deaths, with the greatest occurrence in Sub-Saharan Africa [3–5**].

Iron acquisition is a critical aspect of microbial pathogenesis and, as such, represents a potentially fruitful target for antifungal therapy. This is because fungi and other pathogens must overcome nutritional immunity (e.g., iron withholding) to proliferate and cause disease in mammalian hosts [6, 7]. Additionally, pathogens interpret the availability of iron and other nutrients to regulate the deployment of virulence factors. In the case of C. neoformans, iron levels control elaboration of the polysaccharide capsule that makes a major contribution to the virulence of the fungus [8, 9]. The characterization of iron acquisition functions and their potential as drug targets are being actively investigated for C. neoformans and many other pathogens [10–21]. For example, siderophores conjugated to antibiotics and other drugs show promise for the treatment of microbial infections [10–14]. Additionally, non-iron metallo-protoporphyrins, which are toxic analogs of heme, are inhibitory for bacterial and fungal pathogens, as are chelators and other molecules that interfere with iron acquisition [15–19]. Extracellular proteins for iron acquisition are also vaccine candidates as demonstrated for bacterial and fungal pathogens [20, 21].

In this review, we summarize aspects of iron acquisition in C. neoformans to set the stage for a discussion of recent studies on connections between iron and mitochondria, as well as iron-related regulatory networks that control the expression of mitochondrial processes. We focus on mitochondria because they play a central role in iron homeostasis by containing abundant iron-dependent proteins (e.g., for respiration) and the machinery for two key iron-related processes: heme biosynthesis and the biogenesis of iron-sulfur (Fe-S) proteins [22, 23]. We also highlight the emerging role of mitochondria in fungal virulence and note their importance as potential targets for antifungal therapy [24–27**]. We limit our discussion to C. neoformans, but refer readers to a number of recent reviews that more generally consider metal uptake and regulation for fungal pathogens [9, 28–32**].

An overview of mechanisms of iron acquisition in C. neoformans and connections to virulence

Fungi potentially acquire iron by four mechanisms: 1) low affinity transport of ferrous iron; 2) use of a ferroxidase-iron permease complex for high affinity uptake; 3) production and uptake of siderophores and; 4) acquisition of iron from heme and hemoglobin [9, 28–32**]. The components for these processes in C. neoformans have been reviewed recently and are summarized in Figure 1 [9, 28–30*]. In brief, physiological evidence indicates the presence of a low-affinity iron uptake system, but the underlying mechanisms and components have not yet been characterized [33]. High-affinity iron uptake involves the ferroxidase Cfo1 and the permease Cft1, and these proteins are required for acquisition from inorganic and host iron (e.g., transferrin) iron sources, as well as for virulence and proliferation in the central nervous system of mice [reviewed in 9, 28–30*]. Interestingly, mice infected with cfo1 or cft1 mutants eventually succumb to disease suggesting the presence of additional uptake mechanisms [28–30*]. Ferric reductases believed to reduce iron at the cell surface also support subsequent iron uptake by the Cft1-Cfo1 high affinity system [28–30*].

Figure 1. Mechanisms of iron acquisition and mitochondrial iron trafficking for C. neoformans.

Iron is taken up by a high affinity uptake system consisting of ferric reductases (e.g., Fre2 and Fre4), the iron permease Cft1 and the ferroxidase Cfo1 [9, 28–30]. C. neoformans also possesses six potential siderophore transporters (Sit1–6) and at least one system for heme uptake involving the candidate hemophore Cig1 and trafficking functions such as clathrin-mediated endocytosis (CME), endosomal sorting complex required for transport (ESCRT) and the regulator of vesicle fusion Vps45 [9, 28–30, 34*, 35*]. Iron transport and processing at the mitochondrion is illustrated with known components shown for import (Mrs3/4) and export (Atm1) [41, 44*, 45**]. The monothiol glutaredoxin Grx4 is also shown as a putative sensor of iron-sulfur (Fe-S) clusters and an interacting partner with transcription factors in the nucleus (Cir1 and HapX) [51**].

C. neoformans is able to take up siderophores produced by other microbes, although the fungus is not known to synthesize its own siderophores. One highly conserved siderophore transporter, Sit1, participates in the uptake of ferrioxamine B, but does not contribute to virulence in a mouse model of cryptococcosis [28–30*]. Heme is also an important iron source for C. neoformans and a number of functions for uptake have been identified. Earlier work identified a contribution of the ESCRT pathway as well as a putative hemophore, Cig1, and studies in the last year revealed contributions to heme trafficking for clathrin mediated endocytosis and the Sec1/Munc18 protein Vps45 that regulates vesicle trafficking and fusion [28–30*, 34*, 35*]. In general, loss of these functions results in attenuated virulence in mice [34*, 35*].

As mentioned, iron is not only important for C. neoformans as a limiting nutrient during proliferation in the host, but its availability also influences capsule size [8, 9]. This observation prompted a number of studies to identify regulatory factors that sense iron availability and ultimately control uptake, iron use, and virulence. The first factor identified was Cir1, a GATA transcription factor that not only regulates the expression of iron acquisition functions (e.g., CFT1 and CFO1) but also the major virulence-associated traits of growth at 37°C, capsule formation, and production of the melanin pigment that contributes to defense against the host immune system [36]. Subsequently, components of the CCAAT complex including HapX were identified as regulators of iron acquisition and iron use [37]. Interestingly, HapX has regulatory interactions with Cir1 and with other transcription factors including Rim101, a regulator of the pH response, and Mig1, a regulator of mitochondrial functions [38–40*]. For example, HapX positively influences the expression of RIM101, and Rim101 is important for growth in low iron medium and the expression of iron uptake genes such as CFO1, CFT1, and SIT1 [37–39]. A rim101 mutant also has increased HAPX expression in vivo [39].

Mitochondrial processing of iron

Mitochondria are a major site of iron processing and use in cells [22, 23]. Once in the cell, iron enters mitochondria through a conserved inner mitochondrial membrane transporter identified as Mrs3/4 in Saccharomyces cerevisiae [22, 23], and also characterized in C. neoformans [41]. Within mitochondria, iron serves as a cofactor for a number of conserved proteins with critical functions in the electron transport chain, the TCA cycle, fatty acid oxidation, and lipoate and biotin biosynthesis [22, 23]. However, before it can participate as a cofactor, imported ferric iron must be processed and incorporated into iron-containing compounds including heme and Fe-S clusters. The biogenesis of Fe-S clusters has been characterized in some detail in S. cerevisiae, and a subset of the core proteins is listed in Table 1 [22, 23]. The Frr3 and Frr4 proteins thought to participate in Fe-S cluster biogenesis in C. neoformans were identified through complementation of mutants that displayed constitutive iron reduction [42]. FRR3 turned out to encode an ortholog of the Isu1 and Isu2 scaffold proteins for Fe-S cluster assembly in S. cerevisiae, and FRR4 encodes an ortholog of the iron binding protein, frataxin that is also required for Fe-S cluster biogenesis in yeast [22, 23, 42]. Another Fe-S scaffold protein in yeast, Nfu1, has an ortholog in C. neoformans that is required for iron, copper, and manganese homeostasis [43]. There are several other proteins involved in Fe-S cluster biogenesis in S. cerevisiae including the cysteine desulfurase (Nfs1), scaffold proteins (Isa1, Isa2), and chaperones (Ssq1, Jac1, Mge1; reviewed in [22, 23]). Orthologs are predicted for many of these proteins in C. neoformans, although only a few have been studied to date (Table 1).

Table 1.

Genes encoding mitochondrial iron uptake and Fe-S cluster biogenesis proteins in S. cerevisiae, and candidate orthologs in C. neoformans.

S. cerevisiae	C. neoformans gene ID	Description	Reference
Nfs1	CNAG_01442#	Cysteine desulferase	-
lsu1/2	Frr3 (CNAG_04039)	Scaffold protein	[42]
Isa1	CNAG_02131#	Scaffold protein	-
Isa2	CNAG_00491#	Scaffold protein	-
Nfu1	Nfu1 (CNAG_3395)	Scaffold protein	[43]
Ssq1	CNAG_05199#	Hsp70-type chaperone	-
Jac1	CNAG_04288#	J-domain co-chaperone	-
Mge1	CNAG_01881#	Nucleotide exchange factor co-chaperone	-
Atm1	Atm1 (CNAG_04358)	Mitochondrial ABC-type transporter	[44*, 45**]
Yfh1	Frr4(CNAG 05011)	Frataxin	[42]
Mrs3/4	Mrs3 (CNAG_02522#	Mitochondrial carrier family	[41]

^#Predicted C. neoformans ortholog from strain H99 based on sequence similarity.

Fe-S cluster precursors can be exported via the mitochondrial ABC-transporter Atm1 and incorporated into cytosolic and nuclear proteins [22, 23, 44*, 45**]. For C. neoformans, a common theme emerges when mutants lacking the machinery for mitochondrial iron import and Fe-S cluster precursor export are examined. The total iron content in the mutants is higher than levels in wild-type cells in both the atm1Δ and the mrs3/4Δ mutants [41, 44*] suggesting that the inability to process iron in the mitochondria and return it to the cytosol in usable forms causes a dysregulation in iron uptake. This phenomenon is likely related to the role of Fe-S clusters in coordinating iron regulatory networks (see below). Importantly, recent work also implicated Atm1 in the response of C. neoformans to copper toxicity [45**]. Specifically, copper induces the expression of ATM1 by a mechanism that is dependent on the copper regulator Cuf1, and depletion of Atm1 results in enhanced sensitivity to copper stress and decreased fitness upon phagocytosis by macrophages [45**]. This work highlights the importance of Fe-S cluster-containing proteins and Fe-S protein biogenesis machinery in fungal susceptibility to the copper toxicity that is a component of mammalian defense [45**].

Proteins regulating iron homeostasis and mitochondrial function

Sensing Fe-S clusters is also a key aspect of the regulation of iron homeostasis in fungi, and detailed information on the participation of monothiol glutaredoxins is available for the model yeasts S. cerevisiae and Schizosaccharomyces pombe (reviewed in [46]). Monothiol glutaredoxins with CGFS active sites are typically small Fe-S coordinating proteins which function in Fe-S cluster biogenesis and the regulation of transcription factor activity [46]. For example, monothiol glutaredoxins influence the transcriptional activators Aft1/Aft2 that normally induce transcription of the iron regulon in response to low iron conditions in S. cerevisiae. Aft1 and Aft2 are known to respond to Fe-S clusters [47], and specifically Fe-S clusters of mitochondrial origin [48], and the glutaredoxins Grx3 and Grx4 attenuate the activity of Aft1 and Aft2 [49]. Similarly, a monothiol glutaredoxin, Grx4, is a key regulator of transcription factors involved in iron uptake and homeostasis in S. pombe [46, 50**]. In particular, Grx4 binds to and influences the activity of Fep1, a transcriptional repressor of iron-uptake functions, and Php4, a repressor of iron-utilizing functions [46, 50**].

Recent work also revealed that the monothiol glutaredoxin Grx4 is a central player in iron homeostasis and regulation in C. neoformans [51**]. The iron regulatory network in C. neoformans has similarities with that of S. pombe, although many of the details are still being uncovered and the extent of Grx4’s influence has only recently emerged [51**]. In C. neoformans, one of the major iron-responsive transcription factors, Cir1, shares some sequence similarity to Fep1. However, Cir1 is a larger polypeptide (952 amino acids versus 564 for Fep1) and it appears to function both as a transcriptional repressor and activator based on transcript abundance of genes encoding iron uptake and other functions [36]. As mentioned above, Cir1 is also a major regulator of virulence factor elaboration including growth at host temperature, capsule formation and the deposition of melanin in the cell wall [36]. Interestingly, HapX in C. neoformans has partial sequence similarity to the iron-responsive transcription factor Php4 from S. pombe; again, however HapX is a much larger polypeptide (718 amino acids versus 295 for Php4) [37]. A microarray study to compare the hapXΔ deletion mutant to the wild-type strain under low iron conditions also revealed HapX to be a transcriptional repressor of iron-uptake functions [37].

Not surprisingly, a grx4 deletion mutant of C. neoformans showed similar influences on transcript abundance when compared with the cir1Δ and hapxΔ mutants [36, 37, 51**]. For example, there was increased expression of genes encoding iron transporters, heme biosynthesis proteins, Fe-S cluster containing proteins, and Fe-S cluster biogenesis proteins in the absence of Grx4 [51**]. Grx4 also physically interacts with Cir1 as detected by a yeast two-hybrid analysis, and it is speculated that the protein also interacts with HapX through Fe-S clusters [51**]. Interestingly, the grx4Δ deletion mutant is also hypersensitive to many agents that impact mitochondrial function including inducers of oxidative stress and inhibitors of electron transport chain complexes (many components of which contain Fe-S clusters) [51**]. The connections between mitochondria and iron through Grx4 (and Cir1 and HapX) are consistent with observations in S. pombe. However, Grx4 in C. neoformans appears to have an influence on the expression of a greater number of functions, perhaps through interactions with other transcription factors that may connect iron and mitochondrial function [51**]. For example, the transcript levels for the transcription factors Mig1 and Rim101 are both influenced by HapX and it is tempting to speculate that Grx4 contributes to the activity of HapX by providing a sensing mechanism for Fe-S clusters (and iron availability) [37, 40*, 51**]. HapX has a negative influence on MIG1 expression while Mig1 has a positive influence on HAPX expression under low iron conditions [40*]. Furthermore, deletion of MIG1 altered the transcript levels for proteins involved in the electron transport chain and TCA cycle, and increased sensitivity to oxidative stress, the antifungal drug fluconazole, and inhibitors of the electron transport chain [40*]. Interestingly, deletion of MIG1 or HAPX individually did not impair survival in macrophages, but a double mutant had decreased survival [40*]. Similarly, the double mutant was only slightly less virulent that the wild-type strain upon inoculation in mice (and this was mostly due to the hapXΔ mutation), but the burden of the mutant was reduced in the kidney, lungs, liver, and spleen (but not the brain). Notably, HapX orthologs in other human fungal pathogens, including C. albicans and A. fumigatus, are also iron responsive, associated with mitochondrial function, and important for virulence [52, 53]. Overall, these results reinforce connections between iron-related transcription factors and mitochondrial function, but also suggest only minor roles in virulence for these factors.

Mitochondria and cryptococcal virulence

As highlighted by recent reviews, mitochondria are emerging as important contributors to the virulence of fungal pathogens, and as promising targets for antifungal therapy [24–27]. Recent studies emphasize these connections for C. neoformans [35**, 54*, 55*]. For example, dynamin-related proteins (DRPs) that mediate mitochondrial fusion and fission have been characterized with regard to roles in morphology, resistance to oxidative and nitrosative stress, and virulence [54*]. In particular, mutants lacking the fusion DRP Fzo1 had a virulence defect in a mouse inhalation model in that cells were cleared from the lungs within a week of infection (in contrast to the proliferation observed for wild-type cells). In contrast, deletion of genes from mitochondrial fission (MDV1, DNM1, and FIS1) did not influence virulence in the assay for lung proliferation. The fzo1Δ mutants were able to proliferate in culture at 37°C and produce a wild-type level of capsule, but they had defects in producing the virulence factor melanin, and they were sensitive to oxidative and nitrosative stresss and inhibitors of electron transport (rotenone and potassium cyanide). Interestingly, the mutants were also impaired in their ability to survive intracellularly in macrophages, and this phenotype could be rescued by scavengers of reactive oxygen species. Inhibitors for electron transport were also recently used to demonstrate a connection between mitochondrial function and elaboration of the polysaccharide capsule [55*]. Specifically, treatment with antimycin A, salicylhydroxamic acid, or rotenone impaired capsule growth, and enlargement of the capsule also correlated with an increase in mitochondrial membrane potential. These observations were used to suggest that capsule growth is a stress response and that additional studies are needed on the energetics of capsule formation. Finally, we note that recent work on the Sec1/Munc18 protein Vps45, as mentioned above, also plays an extensive role in iron/heme acquisition, is associated with mitochondria, and influences mitochondrial membrane permeability [35**]. A vps45Δ mutant also shows sensitivity to oxidative stress and inhibitors of the electron transport chain, and is impaired for virulence in mice and survival in macrophages thus providing another connection between iron, mitochondria, and virulence. The importance of mitochondria to virulence is underscored by the characterisation of hypervirulent isolates of a related species Cryptococcus gattii which were found to have increased expression of mitochondrial-associated genes resulting in enhanced proliferation in phagocytes [56]. Furthermore, a subset of these displayed a tubular mitochondrial morphology after phagocytosis which helped promote the growth of neighbouring cells [57]. Overall, these findings highlight mitochondria as key central players in virulence through studies on organellar morphology and on specific proteins contributing to trafficking and mitochondrial function.

Conclusions

Our understanding of the components and regulation of iron uptake pathways in C. neoformans is expanding and emerging themes from recent work highlight the importance of mitochondria as a target organelle for iron delivery and processing, and as contributors to the virulence of the fungus. In particular, mitochondria appear to be crucial for sensing that iron requirements have been met through the assembly and availability of Fe-S clusters, ultimately influencing the expression of iron uptake functions, and linking cellular energetics to virulence-related traits such as resistance to oxidative stress. We have focused on iron uptake and regulation, and connections with mitochondria in this review. However, it is important to highlight, as indicated is a number of excellent recent reviews, that mitochondrial functions are also relevant as potential targets for antifungal drugs and as contributors to drug resistance [24–27**]. Of course, mitochondria are complex organelles with a multitude of connections with key cellular processes. It will therefore be challenging but informative to further characterize the molecular connections between iron sensing, mitochondria and the regulation of virulence factors in C. neoformans. The potential pay off is that new opportunities may arise to target mitochondria for novel antifungal therapies.