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. 2022 Aug 9;60(8):myac056. doi: 10.1093/mmy/myac056

Cryptococcus neoformans can utilize ferritin as an iron source

Moonyong Song 1, Eun Jung Thak 2, Hyun Ah Kang 3, James W Kronstad 4, Won Hee Jung 5,✉
PMCID: PMC9387142  PMID: 35943215

Abstract

Ferritin, a major iron storage protein in vertebrates, supplies iron upon iron deficiency. Ferritin is also found extracellularly, and acts as an iron carrier and a contributor to the immune response to invading microbes. Some microbial pathogens take advantage of ferritin as an iron source upon infection. However, no information is currently available on whether the human fungal pathogen Cryptococcus neoformans can acquire iron from ferritin. Here, we found that C. neoformans grew well in the presence of ferritin as a sole iron source. We showed that the binding of ferritin to the surface of C. neoformans is necessary and that acidification may contribute to ferritin-iron utilization by the fungus. Our data also revealed that the high-affinity reductive iron uptake system in C. neoformans is required for ferritin-iron acquisition. Furthermore, phagocytosis of C. neoformans by macrophages led to increased intracellular ferritin levels, suggesting that iron is sequestered by ferritin in infected macrophages. The increase in intracellular ferritin levels was reversed upon infection with a C. neoformans mutant deficient in the high-affinity reductive iron uptake system, indicating that this system plays a major role in iron acquisition in the phagocytosed C. neoformans in macrophages.

Lay Summary

Cryptococcus neoformans is an opportunistic fungal pathogen causing life-threatening pulmonary disease and cryptococcal meningitis, mainly in immunocompromised patients. In this study, we found that C. neoformans can use ferritin, a major iron storage protein in vertebrates, as a sole iron source.

Keywords: Cryptococcus neoformans, ferritin, iron, macrophage

Introduction

Iron is an essential metal for almost all organisms because of its role in important cellular processes such as oxygen transport, DNA synthesis, and ATP production. Iron also plays a critical role in the interaction of pathogenic microbes with hosts because iron is required for the survival and proliferation of microorganisms in host tissue. At the same time, the vertebrate host sequesters iron using several mechanisms to prevent iron acquisition by microbes, through a process termed nutritional immunity.1 A primary mechanism of iron sequestration in the vertebrate host involves the iron-storage protein ferritin.

Ferritin is the second major iron-storage protein (30%) in the human body after hemoglobin (65%), and, in an iron-depleted state, the protein releases iron mainly by lysosomal degradation.2,3 Iron oxidation and mineral core formation in ferritin have been well-studied. Human ferritin consists of 24 subunits of heavy and light chains, which are differentially expressed in a tissue-specific manner.4 The heavy chain possesses ferroxidase activity that oxidizes ferrous to ferric iron, and the light chain facilitates mineralization and iron core formation.4 Most of the ferritin is located in the cytoplasm of cells. However, some iron-loaded ferritin is also secreted via the multivesicular body–exosome pathway under the control of CD63 and is found extracellularly in the serum, cerebrospinal fluid, lung tissue, and bronchoalveolar lavage fluid.5–7

The primary roles of intracellular ferritin are iron storage and antioxidant activity. In addition, intracellular ferritin contributes to nutritional immunity by limiting iron availability against intracellular and extracellular pathogens. For example, ferritin levels increased dramatically when macrophages were infected with Mycobacterium avium, leading to the inhibition of growth of the bacterial cells.8 The roles of extracellular ferritin include H-kininogen cleavage, inhibition of lymphoid and myeloid cell proliferation, development of erythroid progenitor cells, and iron transport to other organs, such as the brain.9–12 The iron stored in ferritin can also serve as an iron source for pathogenic microorganisms. For example, Neisseria meningitidis, which causes bacterial meningitis, induces an iron-depleted condition in epithelial cells, resulting in ferritin aggregation and lysosomal degradation, and the bacterium uses released iron for its replication in the epithelial cells.13Burkholderia cenocepacia, an opportunistic pathogen often found in cystic fibrosis patients, can also acquire iron from ferritin by proteolysis of the protein in a serine protease-dependent manner.14 Additionally, it has been reported that Bacillus cereus, which causes food-related illness, can utilize ferritin directly using the ferritin receptor, IlsA. Once the IlsA protein of B. cereus binds to ferritin, iron is released, and the bacterial cell subsequently obtains iron using its siderophore, bacillibactin.15 Pathogenic fungi, like Candida albicans, can also utilize ferritin as an iron source. Similar to B. cereus, C. albicans binds to ferritin using a cell adhesin protein, Als3, and subsequently induces acidification of the surroundings, resulting in iron release, which is then imported by the high-affinity reductive iron uptake system.16

Cryptococcus neoformans is an opportunistic fungal pathogen causing life-threatening pulmonary disease and cryptococcal meningitis, mainly in immunocompromised patients. Infection by C. neoformans is initiated by inhalation of the fungal spores or desiccated cells. After infection, C. neoformans is phagocytosed by alveolar macrophages, but the fungus can survive in a phagolysosome within the macrophage.17 Subsequently, the fungus can disseminate from the lungs to reach the central nervous system. Iron plays a critical role in the survival of C. neoformans in the host but also in the expression of major virulence factors, such as capsule production and melanin deposition.18–20 Within the host environment, C. neoformans is challenged by mechanisms of nutritional immunity to limit iron availability. To overcome iron sequestration by the host, C. neoformans has developed at least three major iron-uptake systems: the high-affinity reductive iron-uptake system, siderophore transport system, and heme-uptake system. High-affinity reductive iron uptake involves cell-surface ferric reductases (Fre2, Fre4), a ferroxidase (Cfo1), and an iron permease (Cft1). Ferric iron is first reduced to the ferrous form by Fre2 and Fre4. Ferrous iron is then oxidized by Cfo1 and finally transported by Cft1.21–23 A previous study showed that Cft1 is required for transferrin utilization, suggesting C. neoformans utilizes transferrin as an iron source via high-affinity reductive iron uptake.23 A siderophore is a secondary metabolite with a high affinity for ferric iron and is produced by several microorganisms. Although C. neoformans cannot synthesize siderophores, the fungus can use iron from siderophores produced by other microorganisms.24 Components of the endosomal sorting complex required for transport (ESCRT) and a hemophore are also involved in the use of heme as an iron source by C. neoformans.25–27

We have found that C. neoformans can utilize iron from multiple sources in the host, such as transferrin and heme. However, no information is currently available on whether ferritin could be used as an iron source by the fungus. Therefore, in this study, we investigated the growth of C. neoformans in a medium containing ferritin as a sole iron source. Mutants lacking the genes responsible for high-affinity reductive iron uptake were also tested for their ability to use ferritin to elucidate the mechanism of ferritin-iron acquisition. Furthermore, we studied the influence of infection by C. neoformans on intracellular ferritin in macrophages.

Materials and methods

Strains and growth conditions

Cryptococcus neoformans H99 (serotype A) was used as the wild-type strain. The mutant strains lacking CFO1, CFT1, CAP59, ALG3, or KTR3 were described in previous studies.21–23,28–30Candida albicans SC5314 was used as a control.31 All fungal cells were cultured in a yeast extract-Bacto peptone medium supplemented with 2% glucose (YPD) or defined yeast nitrogen base medium containing 2% glucose (YNB). Defined low-iron medium (LIM) was prepared as described previously.22 Ferritin, FeCl3, ferrioxamine, and hemin were added to LIM as iron sources throughout the study. The iron-starved fungal cells were prepared by preculturing in LIM at 30°C for 2 days and used for experiments as described previously.23

Growth analysis

For growth analysis in liquid culture, iron-starved C. neoformans cells were grown in liquid LIM with or without 0.1, 0.5, 1, 2, and 3 μm ferritin (Sigma, St Louis, MO, USA) at 30°C, and the cell density was monitored using a spectrophotometer. For growth analysis in solid media, 10-fold serially diluted C. neoformans and C. albicans cells were spotted onto LIM plates and incubated at 30°C for 3 days.

An MWCO 6–8 kDa dialysis tube (Spectrum, New Brunswick, NJ, USA) was used to investigate whether physical contact with ferritin is necessary for the growth of C. neoformans. First, 100 μl of ferritin (13 μm) was placed inside the dialysis tube, and both ends of the tube were tied tightly with thread. In parallel, another dialysis tube containing the same amount of ferritin was prepared but with only one end of the tube open. The dialysis tubes were added to LIM, C. neoformans cells were inoculated and cultured at 30°C, and the cell growth was monitored visually and spectrophotometrically.

Immunofluorescence analysis of ferritin binding to surface of fungal cells

Iron-starved C. neoformans and C. albicans cells (5 × 105 cells for each strain) were attached to a poly-l-lysine-coated coverslip and washed with iron-chelated phosphate-buffered saline (PBS) that had been treated with Chelex-100 resin (Bio-Rad, Richmond, CA, USA). Ferritin (1 μm) was added to the fungal cells and incubated at 30°C (for C. neoformans) and 37°C (for C. albicans) for 1 h. Cells were washed three times with iron-chelated PBS to remove non-bound ferritin. The cells were fixed with 4% formaldehyde at 4°C for 1 h. After washing three times with iron-chelated PBS, cells were blocked for 15 min at room temperature (RT) in iron-chelated PBS with 1% bovine serum albumin (BSA). Next, the anti-ferritin rabbit polyclonal antibody against the light chain polypeptide (MyBioSource, San Diego, CA, USA; Cloud-Clone, Houston, TX, USA) was added as the primary antibody and incubated at 4°C overnight. The cells were washed seven times with blocking buffer, and goat anti-rabbit conjugated DyLight-649 (MyBioSource, San Diego, CA, USA) was subsequently added as the secondary antibody. Cells were incubated at RT for 1 h and washed seven times with blocking buffer. The coverslip was mounted on a glass slide with ProLong™ Gold Antifade Reagent (Invitrogen, Waltham, MA, USA), and the fluorescence signal of ferritin was observed using a confocal fluorescence microscope (LSM800Airy, Zeiss, Oberkochen, Germany). The zen 3.4 (blue edition) software (Zeiss) was used to process the images. To prepare the cells in the iron-replete conditions, iron-starved C. neoformans cells were preincubated in LIM containing 10 μm ferrioxamine and 100 μm hemin at 30°C for 2 h before ferritin supplement.

Immunofluorescence analysis to evaluate intracellular ferritin levels in macrophages

Cells of the J774a.1 murine macrophage-like cell line (5 × 105 cells) were seeded on coverslips in 24-well plates and incubated at 37°C in 5% CO2 overnight. The C. neoformans wild-type strain was cultured in YPD medium at 30°C overnight. Macrophages were activated and co-incubated with C. neoformans cells, which were opsonized with 10 μg/ml mouse immunoglobulin G (IgG) 18B7 antibody (kindly provided by Arturo Casadevall, Johns Hopkins School of Public Health) at 37°C for 1 h. The cells were washed three times with PBS and incubated in fresh Dulbecco's modified Eagle medium (DMEM, GenDEPOT, Katy, TX, USA) at 37°C in 5% CO2 for 24 h, and nonphagocytosed C. neoformans cells were removed by washing. Next, cells were fixed using 4% formaldehyde at 4°C for 1 h and washed three times with PBS. Macrophages were permeabilized with 0.5% Triton X-100 at RT for 15 min and washed three times with PBS. Cells were blocked in PBS with 1% BSA (RMBio, Missoula, MT, USA) at RT for 10 min. Anti-ferritin heavy chain rabbit polyclonal antibody (GeneTex, Irvine, CA, USA) was added as the primary antibody, and donkey anti-rabbit IgG conjugated DyLight-649 (BioLegend, San Diego, CA, USA) was added as the secondary antibody. The coverslips were mounted onto the glass slide using ProLong™ Gold Antifade Reagent with DAPI (Invitrogen), and the fluorescence signal was observed using a confocal fluorescence microscope (LSM800Airy, Zeiss).

Macrophage infection and Western blot analysis

The J774a.1 murine macrophage cell line was maintained in DMEM with 10% fetal bovine serum and 100 μg/ml penicillin-streptomycin at 37°C in 5% CO2. Macrophages (1.5 × 106 cells) were seeded in 6-well plates and incubated at 37°C in 5% CO2 overnight. To prepare infected macrophages, C. neoformans wild-type cells were incubated in YPD medium at 30°C overnight and opsonized with 9.33 μg/ml monoclonal 18B7 antibody (kindly gifted by Arturo Casadevall, Johns Hopkins School of Public Health) at 37°C for 1 h. Macrophages were activated using 10 n m phorbol 12-myristate 13-acetate (PMA; Sigma) for 1 h. Opsonized fungal cells were added and incubated with activated macrophages at 37°C in 5% CO2 for 1 h. Non-phagocytosed fungal cells were removed by washing the wells three times with PBS, and the plate was incubated at 37°C in 5% CO2 for an additional 24 h. After 24 h, all samples were washed with PBS and harvested. To extract total protein, macrophages were washed twice with ice-cold PBS, and cell pellets were resuspended in 100 μl RIPA buffer (25 m m Tris-HCl pH 7.5, 150 m m NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) containing 1 m m PMSF and lysed at 4°C overnight. Supernatants containing total protein were obtained after centrifugation. The total protein concentration was estimated by the Bradford assay.32 Total protein (15 μg) was separated on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and subsequently transferred to the nitrocellulose membrane. To detect macrophage ferritin, anti-ferritin heavy chain rabbit polyclonal antibody (GeneTex) as the primary antibody and mouse anti-rabbit IgG horseradish peroxidase conjugate (Santa Cruz Biotechnology, Dallas, TX, USA) as the secondary antibody were used. Finally, the ferritin proteins on the membrane were visualized using chemiluminescence.

To evaluate ferritin levels in the culture supernatant during the fungal culture, iron-starved C. neoformans cells were incubated in 2 μm ferritin at 30°C, and supernatants were collected at different time points (0, 9, 12, 18, 24, and 36 h). The samples were denatured for 5 min, and the levels of the ferritin proteins were evaluated as described above. The total proteins from the same culture supernatants were mixed with 5X native protein-loading dye and separated on a 12% native protein PAGE gel. The levels of intact ferritin remaining in the medium were detected by Coomassie blue staining.

Results

Cryptococcus neoformans can utilize ferritin as an iron source via the high-affinity reductive iron uptake system

Ferritin is normally found in the cytoplasm of mammalian cells, as well as extracellularly in serum and cerebrospinal fluid. Therefore, we hypothesized that C. neoformans encounters and utilizes ferritin as an iron source in infected host tissue. We initially performed growth assays to investigate whether C. neoformans can utilize ferritin as a sole iron source in culture. As shown in Figure 1A, C. neoformans grew well in the medium containing different concentrations of ferritin as the sole iron source. Similarly, C. neoformans grew in solid medium containing ferritin, and the growth was comparable to that of C. albicans, a fungal pathogen known to use ferritin (Fig. 1B).16 These results suggest that C. neoformans can utilize ferritin as an iron source and that extracellular ferritin could support the growth of fungi in host tissue.

Figure 1.

Figure 1.

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Cryptococcus neoformans can use ferritin as a sole iron source. (A) Iron-starved C. neoformans cells were cultured in LIM containing different concentrations of ferritin at 30°C, and the growth of fungal cells was monitored. (B) Ten-fold serial dilutions of fungal cells (starting at 106 cells) were spotted onto the solid media indicated in the figure. The plates were incubated at 30°C for 3 days. Candidaa albicans was used as a positive control. (C) Iron-starved C. neoformans wild-type cells, the cfo1 mutant, and the cft1 mutant were inoculated in LIM containing ferritin as a sole iron source and incubated at 30°C. The growth of cells was monitored spectrophotometrically.

The high-affinity reductive iron uptake system is known to play a role in ferritin utilization by C. albicans.16 Therefore, we examined whether the high-affinity reductive iron uptake system in C. neoformans contributes to the use of ferritin by testing the growth of mutants lacking the CFO1 (ferroxidase) and CFT1 (iron permease) genes that we previously characterized.22,23 Growth was monitored in the presence of ferritin, and we found that the proliferation of both cfo1△ and cft1△ was significantly reduced, while the wild-type cells grew normally (Fig. 1C). These data indicate that the high-affinity reductive iron uptake system is required for ferritin utilization in C. neoformans.

Ferritin binds directly to the surface of C. neoformans

In C. albicans and B. cereus, Als3 and IlsA are cell-surface proteins responsible for ferritin binding and iron utilization, respectively.15,16 In this context, we verified that physical contact is required for the use of ferritin iron by C. neoformans. Ferritin was placed inside a dialysis tube to prevent physical contact with fungal cells. The dialysis tube with one side open was also included as a control. Iron-starved C. neoformans cells were inoculated in the media containing the dialysis tubing, and turbidity was evaluated visually. After 12 h, we observed a significantly higher turbidity in the sample that allowed direct contact between the C. neoformans cells and the ferritin protein than the one that blocked the physical contact (Fig. 2). These results suggest that physical contact is necessary for C. neoformans to use ferritin iron.

Figure 2.

Figure 2.

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Physical contact is required for C. neoformans to utilize ferritin. The necessity of physical contact with ferritin for utilization as an iron source was examined using dialysis tubing. Iron-starved C. neoformans cells were cultured in LIMin the presence of a completely closed or a one-side open dialysis tube containing ferritin. Photographs were taken at 0 and 12 h after starting the culture. The experiments were repeated three times, and the same results were obtained.

Next, we performed immunofluorescence microscopy using an anti-ferritin antibody labeled with a red fluorophore. It is known that ferritin binds to the surface of C. albicans hyphal cells.16 Therefore, C. albicans was included as a positive control. Iron-starved C. albicans and C. neoformans cells were cultured with ferritin, and the cell surface fluorescence was observed. As expected, fluorescence was detected from the C. albicans hyphal cells cultured in the presence of ferritin, while no signal was detected from the cells grown in medium without ferritin (Fig. 3A). Like C. albicans, fluorescence signals were observed from the C. neoformans cells grown in the medium containing ferritin (Fig. 3B). These results suggest that ferritin binds directly to the surface of C. neoformans cells.

Figure 3.

Figure 3.

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Ferritin binds to the surface of C. neoformans. Iron-starved C. albicans (A) and C. neoformans (B) were cultured with ferritin, and the fluorescence signal was detected using confocal fluorescence microscopy. The fungal cells cultured without ferritin served as a negative control. C. neoformans cells precultured in LIM-containing ferrioxamine (C) or hemin (D) as an iron source were cultured with ferritin, and the fluorescence signal was detected using confocal fluorescence microscopy. The images were processed as described in the ‘Materials and methods’ section. Ferritin is shown in red. DIC, differential interference contrast.

To test if the binding of ferritin is dependent on the intracellular iron levels, C. neoformans cells were first precultured in the medium containing the siderophore ferrioxamine or hemin as an iron source, washed, and transferred to the medium containing ferritin, and the fluorescence signals were examined. We found that the signals were significantly reduced in the cells precultured with ferrioxamine or hemin, suggesting that ferritin binding on the surface of C. neoformans is influenced by the iron levels within the cells (Fig. 3C, D).

Additionally, we investigated whether cell wall or capsular components of C. neoformans are responsible for ferritin binding. For this, mutants lacking ALG3, KTR3, or CAP59 were cultured in the medium containing ferritin as an iron source, and ferritin binding was analyzed by immunofluorescence. ALG3 and KTR3 encode α-1,3- and α-1,2-mannosyltransferases involved in the biosynthesis of N-glycan and O-glycan, respectively, and CAP59 contributes to glucuronoxylomannan synthesis and export for capsule synthesis.28,33–35 Immunofluorescence analysis showed that fluorescence signals were still detected in the mutants lacking ALG3, KTR3, or CAP59. Furthermore, we observed that the growth of the same mutant strains in the medium containing ferritin as a sole iron source was similar to that of the wild-type strain (Fig. 4). These results suggest that cell wall or capsular components are not required for ferritin binding but rather a yet unknown component might play a role in ferritin binding.

Figure 4.

Figure 4.

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Capsule and cell wall components are not required for ferritin binding in C. neoformans. (A) The iron-starved C. neoformans wild-type cells and the mutant cells lacking CAP59, ALG3, or KTR3 were incubated with ferritin and fixed by formaldehyde. Ferritin binding was confirmed by immunofluorescence microscopy using anti-ferritin light chain antibody and anti-rabbit antibody conjugated Dylight-649. (B) The growth of the wild-type cells and the same mutant cells was monitored spectrophotometrically.

Acidification by C. neoformans is required for ferritin utilization

As mentioned earlier, C. albicans binds to ferritin and induces subsequent acidification in the surroundings, resulting in the release of iron molecules from the protein.16 In this context, we cultured C. neoformans cells in LIM containing ferritin as a sole iron source and measured the pH of the medium and cell density to investigate whether the fungus acidifies the medium for iron release from the ferritin protein. We recorded a gradual decrease in the pH of the culture medium during fungal proliferation in the presence of ferritin (Fig. 5A).

Figure 5.

Figure 5.

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Acidification contributes to ferritin utilization in C. neoformans. (A) Iron-starved C. neoformans cells were grown in LIM with or without ferritin. The cell density and pH of the medium were monitored. (B) The fungal cells were incubated in LIM containing ferritin as a sole iron source, and the culture supernatants were obtained at the different time points indicated in the figure. Levels of ferritin remaining in the medium were evaluated by Western blot analysis using anti-ferritin light chain antibody. The supernatant from the C. neoformans culture without ferritin was used as a negative control (right panel). The ferritin levels in the medium containing ferritin without the C. neoformans cells remained constant, indicating that the ferritin protein was stable in the medium (right panel). (C) The fungal cells were incubated in LIM containing ferritin as a sole iron source, and the culture supernatants were obtained at the different time points indicated in the figure. Levels of ferritin remaining in the medium were evaluated in a 12% native PAGE gel to detect the intact ferritin complex. The protein levels were detected by Coomassie blue staining. (D) Additional MOPS buffer was added to the medium to enhance the buffering capacity, and the growth of C. neoformans was observed.

Previously, it was demonstrated that the structure of intact ferritin is unstable and that the ferritin shell disassembles at acidic pH levels below 3.4.36 To confirm that ferritin was intact during the C. neoformans culture, we examined the protein content in the culture supernatant for up to 45 h by Western blot analysis. The results showed that the bands representing intact ferritin were visible until 30 h but gradually disappeared afterward, suggesting disassembly of the protein (Fig. 5B). We also evaluated intact ferritin protein levels in the culture supernatant by subjecting the samples to electrophoresis in the native gel and found similar results (Fig. 5C). We should note that the pH of the medium started to drop below 3.7 after 30 h, implying that the disassembly of ferritin after 30 h may have been caused by the reduction in the pH. However, in the medium containing ferritin, C. neoformans started to grow exponentially after 24 h, at which time ferritin was still intact, and the pH was above 6.0. Therefore, we hypothesized that localized acidification by C. neoformans at the interface between intact ferritin and the fungal cell surface may occur, and the acidification solubilizes or releases ferric iron molecules from the intact protein. Released iron molecules might become readily available to the fungus for its growth. To further support our hypothesis, we increased the buffering capacity of the media by adding 200 m m 3-morpholinopropanesulfonic acid (MOPS) and monitored the growth of C. neoformans in the presence of ferritin. The results showed that the growth of C. neoformans was significantly decreased in the medium containing 200 m m MOPS compared to that in the same medium without buffering (Fig. 5D). These results further support the conclusion that acidification, in addition to physical contact, is required for ferritin utilization in C. neoformans. However, the mechanism of acidification in the interaction between ferritin and the cell surface of C. neoformans remains to be identified.

Cryptococcus neoformans infection increases intracellular ferritin levels in macrophages

In addition to the utilization of extracellular ferritin as an iron source, changes in intracellular ferritin levels in macrophages upon infection by C. neoformans were investigated. For this, we performed Western blot analysis to evaluate the change in the intracellular ferritin levels. Our analysis revealed that the protein levels were significantly increased in the macrophages infected with wild-type C. neoformans, suggesting that iron sequestration by a ferritin-associated mechanism may have occurred within the host cells containing C. neoformans (Fig. 6A). We should note that, as observed in another study,37 activation of macrophages by PMA also caused a slight increase in ferritin expression, but the levels were markedly lower than those of the macrophages infected with wild-type C. neoformans. In addition to the Western blot experiment, immunofluorescence analysis using the fluorophore-conjugated ferritin-specific antibody was performed to confirm increased intracellular ferritin levels in the macrophages upon infection by C. neoformans. The macrophages containing C. neoformans showed a significantly higher fluorescence than those without fungal cells (Fig. 6B). This analysis, consistent with our Western blot results, confirms that intracellular ferritin was highly expressed upon phagocytosis of C. neoformans by macrophages, a strong indication of iron sequestration to decrease the available iron that is necessary for pathogen survival and to enhance its role in the antimicrobial immune response.

Figure 6.

Figure 6.

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Phagocytosed C. neoformans cells increased the intracellular ferritin levels. (A) The murine macrophages were activated by PMA, followed by co-incubating with opsonized C. neoformans cells for 24 h. Total proteins from the macrophages phagocytosed C. neoformans cells were extracted, and Western blot analysis was performed using rabbit anti-ferritin heavy chain antibody. The experiments were repeated three times, and the similar results were obtained. (B) The murine macrophages (J774a.1) alone or containing phagocytosed C. neoformans cells were fixed, and intracellular ferritin was stained by anti-rabbit antibody conjugated DyLight-649. The fluorescence signals were detected by confocal fluorescence microscopy. (C) The murine macrophages were activated by PMA and co-incubated with the C. neoformans wild-type strain, the cft1 mutant, the cft2 mutant, and the cft1 cft2 double mutant. Total proteins from the macrophages phagocytosed C. neoformans cells were extracted, and Western blot analysis was performed using rabbit anti-ferritin heavy chain antibody. The experiments were repeated three times, and the similar results were obtained.

Intracellular ferritin levels were also investigated in the macrophages infected with the mutants lacking CFT1, CFT2, or both. As mentioned above, CFT1 encodes the iron permease in the high-affinity reductive iron uptake system, and its paralog, CFT2, presumably plays a role in low-affinity iron uptake in C. neoformans. The results showed that the level of intracellular ferritin was reduced in the macrophages infected with the cft1Δ mutant compared to the cells infected with the wild type. By contrast, the macrophages infected with the cft2Δ mutant displayed intracellular ferritin levels similar to those of the cells containing the wild type, suggesting that the high-affinity reductive iron uptake mediated by Cft1 in C. neoformans was mainly responsible for triggering iron acquisition by the fungus in the phagolysosome of the infected macrophages (Fig. 6C). In the same analysis, we noted that the level of intracellular ferritin was reduced even more in the macrophages infected with the cft1Δ cft2Δ double mutant than those infected with the cft1Δ single mutant. These results suggest that the macrophages no longer responded with a change in ferritin level in the presence of the cft1Δ cft2Δ double mutant and that the deletion of CFT2 in the absence of CFT1 caused complete abolition of iron uptake in C. neoformans within the phagolysosome.

Discussion

In this study, we investigated whether C. neoformans utilizes intracellular and extracellular ferritin as an iron source. We showed that C. neoformans could utilize ferritin as a sole iron source. Furthermore, our data demonstrated that ferritin-iron utilization requires the direct binding of ferritin to the cell surface of C. neoformans. As mentioned earlier, Als3 plays an important role in ferritin binding in C. albicans.16 Als3, a multifunctional adhesin protein, is involved in attachment to epithelial cells, endothelial cells, and extracellular matrix proteins in the mammalian host.38 The role of Als3 in ferritin binding in C. albicans led us to search for its homolog in the genome of C. neoformans, but no homologous protein was identified. One candidate is Cfl1, which is the first identified adhesin protein in C. neoformans.39 However, we excluded a possible contribution of Cfl1 to ferritin binding and utilization because the protein is mainly responsible for hyphal morphogenesis.39 Furthermore, our previous study showed that the expression of CFL1 is not affected by iron levels and that the gene is not regulated by the major iron regulatory protein Cir1 in C. neoformans.40 However, the same study showed that one of the Cfl1 homologs, Cfl105 (CNAG_03 454), is a direct target of Cir1 in the low-iron condition, suggesting a possible contribution of the Cir1 protein to ferritin binding and utilization in C. neoformans.40,41

Our study showed that the acidification of the medium contributed to ferritin utilization by C. neoformans. We hypothesized that localized acidification caused iron release from ferritin because the increased buffering capacity of the medium reduced the growth of the fungus and the protein initially remained intact while C. neoformans grew exponentially. The mechanism by which C. neoformans acidifies the medium and induces iron release from ferritin is unknown. However, we speculate that a reducing agent secreted from C. neoformans is responsible for releasing iron, at least partly, because previous studies showed that several reducing agents, such as thioglycolic acid, can remove iron from ferritin to generate apoferritin.42,43 In C. neoformans, a role for the secreted reductant 3-hydroxyanthranilic acid (3-HAA) in reducing ferric iron to ferrous iron has been suggested.44 In addition to reducing agents, ferric iron-chelating agents, such as aceto- and benzohydroxamic acids, can penetrate the ferritin shell and remove ferric iron from its iron core.45 However, we ruled out a possible contribution of a ferric iron chelator to ferritin-iron release by C. neoformans because of the absence of the biosynthesis pathway for siderophores, microbial ferric iron-chelating agents, in the fungus.46 We should note that acidification and iron reduction by a yet unknown reducing agent are not sufficient for ferritin-iron utilization by C. neoformans because our data also demonstrated that physical interaction between ferritin and the fungus is necessary. A cell surface protein responsible for ferritin binding to C. neoformans appears to be essential for ferritin-iron utilization and remains to be identified.

In this study, we found significantly reduced growth of the cfo1 and cft1 mutants in the medium containing ferritin as an iron source, indicating that the high-affinity reductive iron uptake system mainly comprised Cfo1 and Cft1 plays an essential role in iron uptake from ferritin. The Fet3 and Ftr1 proteins in the model yeast S. cerevisiae are homologous to Cfo1 and Cft1, respectively, and studies suggest that the substrate of Fet3 is ferrous iron, not ferric iron. Therefore, we hypothesize that Cfo1 oxidizes ferrous iron released from ferritin at the cell surface and that Cft1 transports ferrous iron in C. neoformans.

In addition to our study on the utilization of ferritin iron by C. neoformans in culture, we observed that intracellular ferritin levels were increased in response to C. neoformans infection in murine macrophages. We hypothesized that the expression of ferritin was increased to prevent iron acquisition by phagocytosed C. neoformans cells. Support was found for this hypothesis in that the ferritin levels remained unaltered in macrophages infected with the mutant lacking CFT1, which would block the high-affinity reductive iron uptake system in C. neoformans. This result is consistent with the possibility that the increase in ferritin sequesters iron from phagocytosed C. neoformans and that the high-affinity reductive iron uptake is the major iron acquisition system in the fungus within the phagolysosome of the macrophage.

In mammalian cells, multiple factors are involved in the regulation of ferritin expression, and these could potentially be responsive to the activity of phagocytosed cryptococcal cells. Cytoplasmic trans-acting mRNA-binding IRPs (IRP1 and IRP2) are the main regulatory proteins that control intracellular ferritin levels. IRPs bind to the iron-responsive element (IRE) of the 5′-UTR of ferritin mRNA and inhibit translation in iron-depleted cells. In the iron-replete condition, IRPs are released from ferritin mRNA, but a distinct mechanism governs the dissociation of each protein from the 5′-UTR of ferritin mRNA. The iron–sulfur cluster (4Fe–4S) binds to IRP1, and the protein subsequently dissociates from the IRE of the 5′-UTR. By contrast, IRP2 is degraded via the proteasome pathway in iron-replete cells.47,48 In addition to intracellular iron levels, nitric oxide (NO) and oxidative stress induce an increase in the IRP1 and IRP2 binding activities, resulting in reduced ferritin synthesis.49,50 Clues to explain how ferritin levels were increased upon C. neoformans infection may come from studies with other pathogens. For example, a study using mouse bone marrow-derived macrophages (BMMs) infected with the pathogenic bacteria M. avium described the possible underlying mechanisms of ferritin regulation, at least partly. The increase in ferritin expression upon infection by M. avium was mediated transcriptionally, rather than post-transcriptionally, in an IRP-independent manner. Moreover, NO did not influence the increase in ferritin expression upon the infection. However, the study demonstrated that Toll-like receptor 2 (TLR2) was mainly responsible for the regulation of ferritin in BMM-infected M. avium.8 IRP-independent regulation of ferritin expression was also observed in an in vitro study using the murine macrophage cell line infected with Salmonella enterica serotype Typhimurium.51 Taken together, we hypothesize that, similarly to the infection with bacterial pathogens, transcriptional and IRP-independent regulation controls ferritin expression in macrophages infected with C. neoformans to sequester iron from the fungus, and further study is required to verify this hypothesis.

In summary, our study showed that extracellular ferritin is a potential iron source for C. neoformans in the vertebrate host upon infection. Both acidification and the physical interaction between ferritin and the cell surface of C. neoformans are required for ferritin-iron utilization. Moreover, we found that, once released from ferritin, iron is transported via the high-affinity reductive iron system in C. neoformans (Fig. 7). Our study also demonstrated that macrophages increase cytoplasmic ferritin levels in response to infection by C. neoformans to sequester iron from the phagocytosed fungus and that the high-affinity reductive iron system in the fungus is the main iron-uptake pathway for iron acquisition in the phagolysosome that induces the up-regulation of ferritin in the host macrophage.

Figure 7.

Figure 7.

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Model of ferritin utilization in C. neoformans. Ferritin initially binds to the surface of C. neoformans, and iron, most likely ferric iron, is released from ferritin by acidification by the fungus. Released iron becomes reduced by an unknown mechanism, including a cell surface reductase. Reduced iron is then transported via the high-affinity reductive iron uptake system in C. neoformans.

Acknowledgments

We thank Maggie P. Wear and Arturo Casadevall for providing 18B7 antibody.

Contributor Information

Moonyong Song, Department of Systems Biotechnology, Chung-Ang University, Anseong 17546, Korea.

Eun Jung Thak, Department of Life Science, Chung-Ang University, Seoul 06974, Korea.

Hyun Ah Kang, Department of Life Science, Chung-Ang University, Seoul 06974, Korea.

James W Kronstad, Michael Smith Laboratories, Department of Microbiology & Immunology, University of British Columbia, Vancouver, B, C V6T 1Z4, Canada.

Won Hee Jung, Department of Systems Biotechnology, Chung-Ang University, Anseong 17546, Korea.

Funding

This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning 2021M3A9I4021431 (W.J.), and 2022R1F1A1065306 (W.J.); and the National Institute of Allergy and Infectious Diseases RO1 AI053721 (J.K.). J.K. is a Burroughs Wellcome Fund Scholar in Molecular Pathogenic Mycology and a Canadian Institute for Advanced Research (CIFAR) Fellow in the Fungal Kingdom: Threats & Opportunities Program.

Institutional Review Board statement

Not applicable.

Informed Consent Statement

Not applicable.

Data availability

Not applicable.

Declaration of interest

The authors declare no conflict of interest.

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