Iron Acquisition: A Novel Prospective on Mucormycosis Pathogenesis and Treatment

Abstract

Purpose of review

Mucormycosis is an increasingly common fungal infection with an unacceptably high mortality despite first-line antifungal therapy. Iron acquisition is a critical step in the causative organsisms’ pathogenetic mechanism. Therefore, abrogation of fungal iron acquisition is a promising therapeutic strategy to impact clinical outcomes for this deadly disease.

Recent findings

The increased risk of mucormycosis in patients in renal failure receiving deferoxamine iron chelation therapy is explained by the fact that deferoxamine actually acts as a siderophore for the agents of mucormycosis, supplying previously unavailable iron to the fungi. The iron liberated from deferoxamine is likely transported into the fungus by the high affinity iron permease. In contrast, two other iron chelators, deferiprone and deferasirox, do not supply iron to the fungus and were shown to be cidal against Zygomycetes in vitro. Further, both iron chelators were shown to effectively treat mucormycosis in animal models, and one has been successfully used as salvage therapy for a patient with rhinocerebral mucormycosis.

Summary

Further investigation and development of iron chelators is warranted as adjunctive therapy for mucormycosis.

I. Introduction

Mucormycosis is a life-threatening infection caused by fungi of the class Zygomycetes, order Mucorales. Fungi belonging to the family Mucoraceae, and specifically the species Rhizopus oryzae (Rhizopus arrhizus), are by far the most common cause of infection [1]. Strong clinical evidence has implicated iron availability as a major regulator of Zygomycetes virulence. This review will focus on the mechanism of iron uptake in R. oryzae and describe recent data suggesting the potential for iron chelators to be potential novel agents in the treatment of mucormycosis.

II. Overview of Mucormycosis

The agents of mucormycosis are opportunistic pathogens that almost uniformly affect immunocompromised hosts [2]. Patients with diabetic ketoacidosis are particularly susceptible to mucormycosis. Patients immunocompromised by cytotoxic chemotherapy or corticosteroid therapy are also susceptible to mucormycosis. A marked increase in the incidence of mucormycosis has occurred over the past two decades (Fig. 1). Similar increases have been reported by major stem cell transplant centers [3,4]. Given the increasing prevalence of diabetes, cancer, and organ transplantation in the aging United States population, the rise in incidence of mucormycosis is anticipated to continue unabated for the foreseeable future.

Figure 1. Increasing frequency of mucormycosis.

Data adapted from [10].

III. Available Antifungal Therapies for Mucormycosis

The standard therapy for invasive mucormycosis includes reversal of the underlying predisposing factors (if possible), emergent, wide-spread surgical debridement of the infected area (Fig. 2), and adjunctive antifungal therapy [5-8]. Amphotericin B (AmB) remains the only antifungal agent approved for the treatment of invasive mucormycosis [5-8]. Because the fungus is relatively resistant to AmB, high doses are required, which frequently results in nephrotoxicity [8]. Also, in the absence of surgical removal of the infected focus (such as excision of the eye in patients with rhinocerebral mucormycosis), antifungal therapy alone is rarely curative [5,6]. Even when surgical debridement is combined with high-dose AmB, the mortality associated with mucormycosis exceeds 50% [8], and in disseminated disease approaches 100% [9]. Because of this unacceptably high mortality rate, and the extreme morbidity of highly disfiguring surgical therapy (Fig. 2), it has been imperative to develop new strategies to treat and prevent invasive mucormycosis.

Figure 2.

Photograph of a teen patient who recovered from rhinocerebral mucormycosis but was left with a facial defect.

The nephrotoxicity of AmB has prompted clinicians in practice to adopt the use of lipid formulations of AmB, which are less nephrotoxic than AmB and can be administered at higher doses for a longer period of time [6,10]. Most recently, a retrospective review of outcomes in patients with rhino-orbital-cerebral mucormycosis suggested that combination therapy with lipid polyene plus caspofungin was superior to monotherapy with lipid polyenes [11]. Nevertheless, there is a great need for additional therapeutic strategies to improve outcomes in patients with these deadly infections.

IV. The Role of Iron in Mucormycosis Pathogenesis

A. Increased available serum iron is a risk factor for mucormycosis

Iron is required by virtually all microbial pathogens for growth and virulence [12]. In mammalian hosts, very little serum iron is available to microorganisms because it is highly bound to carrier proteins such as transferrin [13]. Sequestration of iron by serum is a major host defense mechanism against R. oryzae in particular [13]. The organism grows poorly in serum and this growth inhibition is reversed when exogenous iron is added [13,14].

Importantly, patients with elevated levels of available serum iron are uniquely susceptible to infection by R. oryzae and other Zygomycetes, but not to other pathogenic fungi, such as Candida or Aspergillus [6,8]. For example, patients treated with the iron chelator, deferoxamine, have a markedly increased incidence of invasive mucormycosis, which is associated with a mortality of >80% in these patients [15]. While deferoxamine acts as an iron chelator with respect to the human host, its effect on R. oryzae is just the opposite. Deferoxamine predisposes patients to Rhizopus infection by acting as a siderophore, which supplies previously unavailable iron to the fungus [14]. Rhizopus obtains iron from the iron-deferoxamine complex by intracellular transport of the reduced iron without deferoxamine internalization [16]. This transport is likely mediated by high-affinity iron permeases (Fig. 3).

Figure 3. Proposed mechanisms of iron assimilation by Zygomycetes in conditions of elevated available serum iron.

In patients in diabetic ketoacidosis (DKA), low pH conditions cause proton-mediated displacement of ferric iron (Fe³⁺) from serum carrier molecules, including transferrin (T). Ferric iron is then reduced at the cell surface to ferrous iron (Fe²⁺). In contrast, deferoxamine (D) directly chelates iron from transferrin, resulting in ferroxamine (iron-deferoxamine complex). Ferroxamine then binds to unidentified receptor(s) on the surface of Zygomycetes. The fungus then liberates the iron from ferroxamine by reduction at the cell surface, solubilizing ferrous iron from ferroxamine. In both cases, ferrous iron is then reoxidized back to ferric iron by copper oxidase (Cu-oxidase). High affinity iron permease (rFTR1), which physically complexes with copper oxidase in yeast, transports ferric iron nearly simultaneously to the oxidation step. Note that the oxidation to ferric iron prior to transport introduces specificity to iron transport by electrochemically separating the trivalent ferric iron from other divalent cations.

Patients with diabetic ketoacidosis have elevated levels of available serum iron, likely due to release of iron from binding proteins in the presence of acidosis [13]. Artis et al. showed that sera collected from patients with diabetic ketoacidosis supported growth of R. oryzae in the presence of acidic pH (7.3-6.88) but not in the presence of alkaline pH (7.78-8.38) [13]. Furthermore, adding exogenous iron to sera allowed R. oryzae to grow profusely at acidic conditions but not at pH ≥ 7.4. Finally, simulated acidotic conditions decreased the iron-binding capacity of sera collected from normal volunteers, suggesting that acidosis temporarily disrupts the capacity of transferrin to bind iron. Therefore, the increased susceptibility to mucormycosis of patients with diabetic ketoacidosis is likely due at least in part to an elevation in available serum iron during diabetic ketoacidosis, due to proton-mediated dissociation of iron from transferrin (Fig. 3).

B. Mechanisms and chemistry of iron assimilation in fungi

General concepts

Iron is present in two readily available ionization states, Fe²⁺ (ferrous) and Fe³⁺ (ferric). Because of its ability to exist in either of these two states, iron has the ability to donate and accept electrons, and therefore can participate in a wide variety of cellular oxidation-reduction reactions. However, the chemical properties of iron place two limitations on its cellular accumulation and utilization by microorganisms. First, the metal is mainly found in nature in an insoluble state, typically comprised of Fe³⁺ hydroxides [12]. The insolubility of Fe³⁺ hydroxides limits the ability of microorganisms to transport the iron intracellularly. Therefore, fungi have devised a variety of strategies to overcome this problem, as discussed below.

The second problem limiting iron utilization by fungi is that iron is potentially toxic because of its ability to catalyze the production of oxygen free radicals via the Fenton reaction [17] or the Haber-Weiss reaction [17]. Iron catalyzed production of oxygen free radicals leads to cellular injury by causing oxidative damage to a wide variety of cellular substrates [12]. Therefore, proper storage of excess iron is essential to prevent toxicity. For instance, soon after uptake, iron can be found in the ferrous form bound to polyphosphates in vacuoles of S. cerevisiae [18]. Alternatively, iron can be stored as part of iron-rich proteins (ferritins). To date, the only fungi identified that store iron in ferritins are members of the class Zygomycetes [19]. Three types of iron-rich proteins have been identified in Zygomycetes: 1) mycoferritin, which is closely related to the mammalians ferritins [12]; 2) bacterioferritin [20]; and 3) zygoferritin, which is unique to Zygomycetes [12]. Also, fungi can store iron as part of small proteins called siderophores, which specialize in obtaining iron from the environment [19]. This mechanism of storage is common among fungi belonging to the ascomycetes and basidiomycetes classes.

Iron uptake mechanisms

Three general mechanisms of iron uptake have been identified in fungi. These include: 1) a reductive iron uptake that involves reduction of the ferric form into the ferrous and subsequent transport by a permease [21-24]; 2) a siderophore permease that facilitates the uptake of siderophore-sequestered iron [25-27]; and 3) an uptake system for acquiring iron from haemin [28]. In the reductive system, fungi can use any of the following three methods to reduce ferric iron into the more soluble ferrous form: i) a low affinity iron reductase (Km, 40 μM) functions in iron-rich environments to reduce Fe³⁺ to Fe²⁺. Subsequently, Fe²⁺ is likely transported into the cell by the action of the low affinity iron permease. This iron permease also transports other bivalents elements, such as calcium and magnesium [12]; ii) an iron regulated high-affinity ferric reductase (Km, 0.15 μM) that reduces Fe³⁺ into Fe²⁺ and operates in iron-depleted environments, such as those present in the host. Even in hosts predicted to have elevated available serum iron, such as patients with diabetic ketoacidosis, most iron remains bound to carrier molecules, and free serum iron would still be present in submicromolar concentrations that induce the high-affinity rather than the low-affinity uptake system. The produced Fe²⁺ is further oxidized into Fe³⁺ by the action of a membrane copper oxidase before being transported across the cell membrane by a high-affinity iron permease [12,29]. The oxidation of ferrous iron back into ferric form is considered necessary to introduce specificity to transporting only iron into the cell. The copper oxidase and high-affinity iron permease exists as a complex enzyme [29] and their expression, as well as the expression of the high-affinity reductases, is controlled by the transcriptional regulator AFT1, which functions in low concentrations of iron [30]; and iii) non-enzymatic reduction of Fe³⁺ and transport of Fe²⁺. Phenolic compounds such as anthranilate and 3-hydroxyanthranilate are known to maintain a reduced environment to release and prolong the existence of Fe²⁺ at the fungal membrane until transport occurs [18]. However, the role of these compounds in solubilizing iron is considered to be limited compared to the enzymatic reduction processes.

The role of reductase/permease systems in Zygomycetes iron metabolism

We have cloned the high-affinity iron permease of R. oryzae and found the putative rFtr1p to have significant homology to known fungal high affinity iron permeases from C. albicans (46% identity) and S. cerevisiae (44% identity) [31]. As well, multiple regions of the predicted rFtr1p showed significant homology with putative transmembrane domains from S. cerevisiae [29] and C. albicans FTR1 [24]. Importantly, the putative REGLE motif, in which the glutamic acid residue is believed to interact directly with iron [29], was conserved in the predicted protein sequences of FTR1 from the three organisms and was embedded in a hydrophobic region. The rFTR1 was expressed in iron-depleted and not in iron-rich media. This iron-regulated expression of rFTR1 was also accompanied by an iron-regulated activity of ferric reductase that was induced or suppressed in low or high concentrations of iron, respectively (unpublished data). These data indicate that rFTR1 is likely to act in concert with ferric reductase to supply the cell with iron under iron-depleted conditions. Finally, rFTR1 restored the ability of an ftr1 null mutant of S. cerevisiae to grow on iron-limited medium and to take up radiolabeled iron, whereas S. cerevisiae transformed with the empty vector did not [31]. Recently, we have established the in vivo expression of rFtr1p in R. oryzae hyphae (unpublished data) and inhibition of rFtr1p expression by RNA-interference reduced the virulence of R. oryzae in diabetic ketoacidosis mouse model [32].

The role of siderophores in Zygomycetes iron metabolism

Fungi can produce siderophores, which provide the cell with much needed iron by chelating ferric iron [27,33]. To acquire iron, fungi can utilize their own secreted siderophores, siderophores secreted by other organisms (xenosiderophores), or both [12,14,16]. Siderophores supply iron to the host cell by one of the following four mechanisms: 1) Direct transfer of iron across the plasma membrane without entrance of the siderophore into the cell. In this case the transfer of iron is not an enzymatic membrane-reductive event, but rather an exchange between the gathering siderophore and an internal storage compound [34]; 2) Direct transfer of iron without entrance of the siderophore into the cell after reducing the chelated Fe³⁺ into Fe²⁺ [16]. This method is common among fungi utilizing iron from xenosiderophores [16]; 3) A shuttle mechanism encompassing the uptake of the entire siderophore-iron complex into the cell. Once internalized, iron is released by a reductase or by direct ligand exchange in which the recipient siderophore becomes the storage compound and the gathering siderophore is released into the environment to capture more iron [19]; and 4) An esterase-reductase mechanism by which Fe³⁺ is released from ferric triacetylfusarinine C (a siderophore belonging to the hydroxamate family, the most common fungal siderophores) by breaking the ester bond following internalization of the iron-siderophore complex [35]. The released Fe³⁺ is reduced and stored while the siderophore excreted to capture another iron molecule.

Zygomycetes are known for secreting rhizoferrin, a siderophore that belongs to the polycarboxylate family [36]. This siderophore supplies Rhizopus with iron through a receptor-mediated, energy dependant process [16,36]. However, it is not currently known by which mechanism of uptake this siderophore supplies the organism with iron. What is known is that rhizoferrin is inefficient in obtaining iron from the serum [14,16], and therefore the contribution of the organism’s endogenous siderophores to its virulence is likely minimal. Additionally, because of their antigenic properties, siderophores may not be effective iron scavengers in the host since they elicit an immune response [37].

Rhizopus can also utilize siderophores secreted by other organisms as xenosiderophores in their quest for iron. A prime example of the use of xenosiderophores by Rhizopus is provided by the clinical experience with the bacterial siderophore, deferoxamine [14]. In contrast to rhizoferrin, in vitro studies utilizing radiolabeled deferoxamine in serum demonstrated that R. oryzae efficiently liberates ferric iron from deferoxamine extracellularly before taking up the iron. This step is an energy-dependent, and requires the reduction of Fe³⁺ to Fe²⁺ prior to transporting the iron intracellularly, suggesting the involvement of the reductase/permease system [16].

The role of haemin utilization in Zygomycetes iron metabolism

C. albicans [28] and Histoplasma capsulatum [38] can utilize haemin as a source of iron. Haemin uptake kinetics in C. albicans have demonstrated two phases: a rapid phase of haemin binding followed by a slower uptake phase, both of which were induced in iron-depleted conditions [28]. The putative C. albicans haem oxygenase gene (CaHMX1) was required for iron assimilation from haemin and its expression was induced in iron deprived conditions, by haemin, and by a shift from 30 to 37°C. However, CaHMX1 was not involved in the uptake of haemin since a Cahmx1 null mutant was able to take up haemin similar to the wild-type. Finally, the three different iron uptake systems in C. albicans (reductive, siderophore and haemin) are regulated independently from each other, emphasizing the independence of the haemin uptake system. The Rhizopus genome project revealed two homologues (RO3G_07326 and RO3G_13316) of the CaHMX1. These two R. oryzae homologues may provide a means for invasive R. oryzae to obtain iron from host hemoglobin. Since iron is usually present in abundance in the human blood, the presence of these homologues might explain the angioinvasive nature of R. oryzae.

A potentially novel mode of iron acquisition by Zygomycetes

Another method of iron acquisition in fungi includes the acidification of the environment when the fungi are grown under anaerobic conditions. For example, under acidic conditions, S. cerevisiae [18] and Neurospora crassa [12] can accumulate iron at the cell surface and mobilize the iron by excreted hydroxy acids, such as citric acid, to transport iron intracellularly. This method might have in vivo relevance for Zygomycetes, especially in diabetic ketoacidotic patients, however, to date, no exploration of the role of such a mechanism in Zygomycetes iron acquisition has been undertaken.

V. Preliminary evidence of the benefit of iron chelation therapy for mucormycosis

The central role of iron metabolism in the pathogenesis of mucormycosis suggests the possibility of utilizing effective iron chelators as adjunctive antifungal therapy. In fact, in addition to deferoxamine, other experimental iron chelators have been studied in vitro against R. oryzae [15]. In contrast to deferoxamine, these other iron chelators did not allow the organism to take up iron, and did not support its growth in vitro in the presence of iron (Table 1) [15].

Table 1.

Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of iron chelators against zygomycetes (adapted from [39,40]).

Iron Chelator	24 hours		48 hours
	MIC (μg/ml)	MFC (μg/ml)	MIC (μg/ml)	MFC (μg/ml)
Deferiprone	3.12	100	6.25	6.25
Deferasirox	3.12-12.5	3.12-12.5	3.12-6.25	3.12-6.25
Deferoxamine	>100	>100	>100	>100

Furthermore, while deferoxamine significantly worsened disseminated R. oryzae infection in guinea pigs, one of the other chelators had no impact on in vivo infection and the other chelator more than doubled the mean survival time of infected guinea pigs [15]. This latter agent, deferiprone, is approved for clinical use as an iron chelator in Europe and India, and is available on a compassionate use basis for iron overload in the United States. We have confirmed the ability of deferiprone to inhibit growth of Zygomycetes in vitro (Table 1) and confirmed its efficacy in our diabetic-ketoacidotic murine model of R. oryzae infection [39]. Further, in 2005, deferasirox became the first orally bioavailable iron chelator approved for use by the United States (US) Food and Drug Administration (FDA), with an indication for treatment of transfusion-dependent iron overload. We found deferasirox to be effective at chelating iron from R. oryzae and demonstrated cidal activity in vitro against Zygomycetes at concentrations well below clinically achievable serum levels (Table 1). Additionally, deferasirox significantly improved survival of diabetic ketoacidotic or neutropenic mice with mucormycosis, with efficacy comparable to that of liposomal amphotericin B. Most importantly, deferasirox synergistically improved survival and reduced tissue fungal burden when combined with liposomal amphotericin B [40]. A recent study using Drosophila melanogaster as a model host also demonstrated that deferasirox significantly protected wild-type flies infected with R. oryzae when compared with placebo-treated flies [41]. Finally, deferasirox was recently successfully used as a salvage therapy to treat a patient with rhinocerebral mucormycosis who was failing months of polyene treatment [42]. A phase II clinical trail to determine the safety and efficacy of using deferasirox in combination with liposomal amphotericin B is currently underway.

VI. Conclusion

Mucormycosis is an increasingly common infection in immunocompromised patients, and the morality with standard therapy remains unacceptably high. The agents of mucormycosis are uniquely susceptible to variations in environmental iron concentrations. Therefore, abrogation of iron acquisition by the agents of mucormycosis is a promising therapeutic strategy to impact clinical outcomes. Iron chelators that cannot be utilized to supply Zygomycetes with iron have efficacy in treating experimental mucormycosis and at least one of them (i.e. deferasirox) has been successfully used in a salvage therapy for a case of rhinocerebral mucormycosis. Given the fact that deferasirox is FDA approved and deferiprone is approved for clinical use in Europe and India, further development of these agents, as well as other novel iron chelators, is warranted as adjunctive therapy for mucormycosis.