ABSTRACT
Iron acquisition is critical for the ability of the pathogenic yeast Cryptococcus neoformans to cause disease in vertebrate hosts. In particular, iron overload exacerbates cryptococcal disease in an animal model, defects in iron acquisition attenuate virulence, and iron availability influences the expression of major virulence factors. C. neoformans acquires iron by multiple mechanisms, including a ferroxidase-permease high-affinity system, siderophore uptake, and utilization of both heme and transferrin. In this study, we examined the expression of eight candidate ferric reductase genes and their contributions to iron acquisition as well as to ferric and cupric reductase activities. We found that loss of the FRE4 gene resulted in a defect in production of the virulence factor melanin and increased susceptibility to azole antifungal drugs. In addition, the FRE2 gene was important for growth on the iron sources heme and transferrin, which are relevant for proliferation in the host. Fre2 may participate with the ferroxidase Cfo1 of the high-affinity uptake system for growth on heme, because a mutant lacking both genes showed a more pronounced growth defect than the fre2 single mutant. A role for Fre2 in iron acquisition is consistent with the attenuation of virulence observed for the fre2 mutant. This mutant also was defective in accumulation in the brains of infected mice, a phenotype previously observed for mutants with defects in high-affinity iron uptake (e.g., the cfo1 mutant). Overall, this study provides a more detailed view of the iron acquisition components required for C. neoformans to cause cryptococcosis.
INTRODUCTION
Iron is an essential element for almost all organisms, due to its role in various metabolic processes. However, its bioavailability in the environment is extremely limited because it is rapidly oxidized in the presence of atmospheric oxygen to insoluble ferric hydroxides. In addition, available iron in mammals is also severely limited because it is in heme-containing proteins and bound to extracellular carrier proteins, such as transferrin and lactoferrin, as well as to the intracellular storage protein ferritin. To overcome this challenge of limited iron availability, pathogens have evolved multiple mechanisms to acquire iron from the environment and from within the mammalian host. These include a reductive high-affinity uptake system, the production and uptake of siderophores, which are low-molecular-weight iron chelators that have a high affinity specifically for ferric iron, and the use of both heme and transferrin.
Fungal pathogens of humans employ different iron acquisition mechanisms that are likely to be important virulence determinants. For example, siderophores play an important role both in iron acquisition and in virulence for the invasive mold Aspergillus fumigatus (1–3). Although the pathogenic yeasts Cryptococcus neoformans and Candida albicans are not known to produce their own siderophores, they have siderophore transporters that are employed to obtain iron from siderophores produced by other organisms (4, 5). However, siderophore iron uptake does not appear to be important in the virulence of these two pathogens. In addition, both C. neoformans and C. albicans are able to utilize heme as an iron source. The cell wall mannoproteins Cig1 (in C. neoformans) and Rbt5 and Rbt51 (in C. albicans), as well as the heme oxygenase Hmx1 (in C. albicans), are involved in acquiring iron from heme (6–11). The reductive high-affinity iron uptake system is an important virulence determinant in C. neoformans and C. albicans but not in A. fumigatus, and this mechanism is employed by these two pathogens to acquire iron from transferrin in mammalian hosts (7, 8, 12). This iron uptake system involves the reduction of ferric iron to ferrous iron by cell surface reductases, followed by ferrous iron oxidation by a ferroxidase coupled to an iron permease for transport. Since the iron must be reduced before it becomes a substrate for the ferroxidase-permease complex, enzymatic and/or nonenzymatic ferric reductase activity is critically required for iron acquisition through the reductive uptake system. Therefore, the candidate genes encoding these enzymes in C. neoformans are the focus of the study reported in this article.
Ferric reductases are integral membrane proteins that require NADPH, flavin mononucleotide (FMN), and heme for activity. They catalyze the oxidation of cytoplasmic NADPH and transfer the electron across the plasma membrane to reduce metals like iron and copper. For fungi, these reductases have been studied extensively in Saccharomyces cerevisiae and also in C. albicans, Schizosaccharomyces pombe, and A. fumigatus (12–17). The number of ferric reductases varies across the fungi, with 8 in S. cerevisiae, 2 in S. pombe, 16 in C. albicans, and 15 in A. fumigatus. In S. cerevisiae, Fre1 and Fre2 account for the majority of the cell surface ferric reductase activity. These reductases also have cupric reductase activity and are able to facilitate the use of siderophore-bound iron (18, 19). Fre3 and Fre4 from S. cerevisiae are also able to catalyze the reductive release of iron from siderophores (19). The expression of the genes encoding these reductases and also Fre5 and Fre6 is induced by iron depletion. The transcription of FRE1 and FRE7 is also induced by copper depletion (18, 20, 21). In C. albicans, Fre10 and Fre7 are the major ferric reductases, and they also have cupric reductase activity (12, 15, 22). The genes encoding these and other reductases respond to a wide range of environmental conditions, including iron depletion (23, 24). The S. pombe ferric reductase Frp1 is essential for reductive iron uptake and is transcriptionally repressed by iron (17). In A. fumigatus, a role for FreB in iron acquisition via the reductive uptake system has been demonstrated (13). Although all of these studies implicated their respective ferric reductases in iron acquisition, no role in virulence was observed in A. fumigatus and C. albicans.
The C. neoformans genome encodes eight putative metallo/ferric reductases (hence called “ferric reductases”). In this study, we examined the expression of these genes in response to iron limitation and constructed deletion mutants of all eight reductases to begin to analyze their roles in iron acquisition. We found that Fre2 is required for growth on hemin and transferrin as the sole iron sources and that Fre4 has a role in melanin production and resistance to azole drugs. The genes coding for these two proteins also had a shared pattern of regulation that was distinct from that of the other six genes. We therefore focused the bulk of our analyses on Fre2 and Fre4 and demonstrated that mutants lacking FRE2 alone or both FRE2 and FRE4 were attenuated for virulence in a mouse model of cryptococcosis.
MATERIALS AND METHODS
Strains and growth assays.The C. neoformans variety grubii strains used in this study are listed in Table 1. All strains were maintained on YPD medium (1% yeast extract, 2% peptone, 2% glucose). Yeast nitrogen base low-iron medium (YNB-LIM) (YNB [pH 7.2] plus 150 μM bathophenanthroline disulfonate [BPS]) and defined low-iron medium (LIM) were prepared as described previously (7, 25, 26) and used as iron-limiting media, supplemented as indicated, for phenotypic characterization. YNB low-copper medium (YNB-LCM) (YNB [pH 7.2] plus 100 μM bathocuproine disulfonate [BCS]) was prepared similarly to YNB-LIM. Defined low-copper medium (LCM) was also prepared similar to defined LIM, except that CuSO4 was omitted and FeCl3 was added. Cells were starved for iron or copper by growing in the respective limiting media at 30°C for 2 days. For growth assays in liquid media, a final concentration of 5 × 104 cells ml−1 was inoculated in the medium with or without supplemented iron sources. Cells were incubated at 30°C, and growth was monitored by measuring the optical density at 600 nm. For growth assays on solid media, 10-fold serial dilutions of cells were spotted on agar plates with or without supplemented iron sources. Plates were incubated at 30°C for 2 days before being photographed. Growth of the strains was also assessed by a high-throughput 96-well microplate assay using the Infinite M200 PRO plate reader (Tecan, Austria). For this assay, iron-starved cells were inoculated in 200 μl of YNB-LIM with or without supplemented iron sources for 3 to 4 days at 30°C (final inoculum of 104 cells). Growth was monitored by measuring optical density at 600 nm, and the resulting data were analyzed using the Magellan software (Tecan, Austria).
Strains used in this study
Construction of deletion and complemented strains.All deletion mutants were constructed by homologous recombination using gene-specific knockout cassettes, which were amplified by three-step overlapping PCR (27, 28) with the primers listed in Table S1 in the supplemental material. All three antibiotic resistance markers, namely the resistance markers for nourseothricin (NAT), neomycin (NEO), and hygromycin (HYG), were amplified by PCR using the primers M13-F and M13-R and the plasmids pCH233, pJAF1, and pJAF15, respectively, as the templates. In general, the gene-specific knockout primers 1 and 2 and 3 and 4 were used to amplify the flanking sequences of their respective genes; and the nested knockout primers 5 and 6 were used to amplify the gene-specific deletion construct containing the resistance marker. Primers 2 and 3 were chimeric primers containing a 30-bp sequence at their 5′ end that was complementary to the sequence from resistance markers to facilitate overlap during PCR. All constructs for deletions or complementation were introduced into the H99 wild-type (WT) strain or into the fre2Δ or fre4Δ mutants, by biolistic transformation, as described previously (29).
To construct the fre1Δ mutant, a deletion cassette was constructed by PCR using primers H07770-ko-1, H07770-ko-2, H07770-ko-3, H07770-ko-4, H07770-ko-5, and H07770-ko-6, with H99 genomic DNA and the plasmid pJAF15 as the templates. The 3.31-kb open reading frame of FRE1 was replaced with the 2.09-kb HYG cassette using 5′ and 3′ flanking sequences of FRE1. Positive transformants were identified by PCR and confirmed by Southern blotting.
To construct the fre2Δ mutant, a deletion cassette was constructed by PCR using primers H06821-ko-1, H06821-ko-2, H06821-ko-3, H06821-ko-4, H06821-ko-5, and H06821-ko-6, with H99 genomic DNA and the plasmid pCH233 as the templates. The 2.38-kb open reading frame of FRE2 was replaced with the 1.65-kb NAT cassette using 5′ and 3′ flanking sequences of FRE2. Positive transformants were identified by PCR and confirmed by Southern blotting. To construct the fre2Δ + FRE2 complemented strain, a complementation construct containing the FRE2 gene and HYG resistance marker was generated by cloning. A 0.84-kb DNA fragment downstream of the FRE2 translation stop site was PCR amplified using primers H06821C-Sp and H06821C-Hi with the introduction of the SpeI restriction enzyme site, with genomic DNA as the template. The amplified 0.84-kb DNA fragment was digested with HindIII/SpeI and ligated with pJAF15 to construct pHYGFRE2L. A 3.24-kb DNA fragment containing the FRE2 gene, along with the promoter and terminator regions, was PCR amplified using primers H06821C-Xb and H06821C-No with the introduction of XbaI and NotI restriction enzyme sites, with genomic DNA as the template. The amplified 3.24-kb DNA fragment was digested with XbaI/NotI and ligated with pHYGFRE2L to construct pSS117. This plasmid was linearized with XbaI and introduced into the fre2Δ mutant.
To construct the fre3Δ mutant, a deletion cassette was constructed by PCR using primers H06524-ko-1, H06524-ko-2, H06524-ko-3, H06524-ko-4, H06524-ko-5, and H06524-ko-6, with H99 genomic DNA and the plasmid pJAF1 as the templates. The 2.50-kb open reading frame of FRE3 was replaced with the 1.86-kb NEO cassette using 5′ and 3′ flanking sequences of FRE3. The resulting transformants were screened and confirmed by PCR.
To construct the fre4Δ mutant, a deletion cassette was constructed by PCR using primers H07334-ko-1, H07334-ko-2, H07334-ko-3, H07334-ko-4, H07334-ko-5, and H07334-ko-6, with H99 genomic DNA and plasmid pJAF1 as the templates. The 2.35-kb open reading frame of FRE4 was replaced with the 1.86-kb NEO cassette using 5′ and 3′ flanking sequences of FRE4. Positive transformants were identified by PCR and confirmed by Southern blotting. To construct the fre4Δ + FRE4 complemented strain, a complementation construct containing the FRE4 gene and HYG resistance marker was generated by cloning. A 0.87-kb DNA fragment downstream of the FRE4 translation stop site was PCR amplified using primers H07334C-Sp and H07334C-Hi with the introduction of SpeI restriction enzyme site, with genomic DNA as the template. The amplified 0.87-kb DNA fragment was digested with HindIII/SpeI and ligated with pJAF15 to construct pHYGFRE4L. A 3.65-kb DNA fragment containing the FRE4 gene, along with the promoter and terminator regions, was PCR amplified using primers H07334C-Xb and H07334C-No with the introduction of XbaI and NotI restriction enzyme sites, with genomic DNA as the template. The amplified 3.65-kb DNA fragment was digested with XbaI/NotI and ligated with pHYGFRE4L to construct pSS118. This plasmid was linearized with XbaI and introduced into the fre4Δ mutant.
To construct the fre5Δ mutant, a deletion cassette was constructed by PCR using primers H07604-ko-1, H07604-ko-2, H07604-ko-3, H07604-ko-4, H07604-ko-5, and H07604-ko-6, with H99 genomic DNA and the plasmid pJAF1 as the templates. The 2.29-kb open reading frame of FRE5 was replaced with the 1.86-kb NEO cassette using 5′ and 3′ flanking sequences of FRE5. The resulting transformants were screened and confirmed by PCR.
To construct the fre6Δ mutant, a deletion cassette was constructed by PCR using primers H06976-ko-1, H06976-ko-2, H06976-ko-3, H06976-ko-4, H06976-ko-5, and H06976-ko-6, with H99 genomic DNA and the plasmid pCH233 as the templates. The 1.71-kb open reading frame of FRE6 was replaced with the 1.65-kb NAT cassette using 5′ and 3′ flanking sequences of FRE6. The resulting transformants were screened and confirmed by PCR.
To construct the fre7Δ mutant, a deletion cassette was constructed by PCR using primers H00876-ko-1, H00876-ko-2, H00876-ko-3, H00876-ko-4, H00876-ko-5, and H00876-ko-6, with H99 genomic DNA and the plasmid pJAF1 as the templates. The 2.24-kb open reading frame of FRE7 was replaced with the 1.86-kb NEO cassette using 5′ and 3′ flanking sequences of FRE7. The resulting transformants were screened and confirmed by PCR.
To construct the fre201Δ mutant, a deletion cassette was constructed by PCR using primers H03498-ko-1, H03498-ko-2, H03498-ko-3, H03498-ko-4, H03498-ko-5, and H03498-ko-6, with H99 genomic DNA and the plasmid pCH233 as the templates. The 2.36-kb open reading frame of FRE201 was replaced with the 1.65-kb NAT cassette using 5′ and 3′ flanking sequences of FRE201. The resulting transformants were screened and confirmed by PCR.
To construct the fre2Δ fre4Δ double mutant, the FRE2-specific deletion cassette (described above) was introduced into the fre4Δ strain (described above) by biolistic transformation. Positive transformants were identified by PCR and confirmed by Southern blotting. To construct the fre2Δ fre4Δ + FRE2 and fre2Δ fre4Δ + FRE4 complemented strains, the linearized plasmids pSS17 (FRE2) and pSS118 (FRE4) (described above), respectively, were introduced into the fre2Δ fre4Δ double mutant. The resulting transformants were screened and confirmed by PCR.
Real-time qRT-PCR.Real-time quantitative reverse transcription-PCR (qRT-PCR) was performed as follows. To examine gene expression, three biological replicates of the WT and fre4Δ mutant strains were grown in 5 ml of YPD medium overnight at 30°C. Cells were washed twice in Chelex-treated water followed by growth in YNB-LIM or YNB-LCM for 2 days to starve the cells for iron or copper, respectively. Cells were then washed and grown either in 5 ml YNB-LIM (with or without 100 μM FeCl3 and 10 μM hemin) or in 5 ml YNB-LCM (with or without 100 μM CuSO4) for 6 h at 30°C (final density of 107 cells ml−1). Total RNA was extracted and cDNA was synthesized as described previously (30). Relative gene expression was quantified based on the threshold cycle (2−ΔΔCT) method (31). 18S rRNA was used as an internal control for normalization.
Ferric and cupric reductase assays.Ferric and cupric reductase activities were measured as described previously (32). Briefly, cells grown overnight in YPD medium were starved for iron or copper in defined LIM or defined LCM, respectively. The iron- or copper-starved cells were then grown in defined LIM or defined LCM with or without 100 μM FeCl3 or 100 μM CuSO4, respectively, for 6 h. For the ferric reductase assay (in a total volume of 2 ml), 1 ml of cells was mixed with BPS and Fe(III)-Na EDTA (1 mM each), and the mixture was incubated for 1 h in the dark before the A535 (ε = 22,140 M−1 cm−1) of the colored Fe(II)-BPS complex was read. Similarly for the cupric reductase assay, 1 ml of cells was mixed with BCS and CuSO4 (1 mM each), and the Cu reductase activity was measured by reading the A478 (ε = 9,058 M−1 cm−1) of the colored Cu(I)-BCS complex.
Noniron metalloporphyrin uptake assay.Cells grown overnight in YPD medium were starved for iron in YNB-LIM. YPD or YNB-LIM plates with or without 10 μM hemin or 100 μM FeCl3 were spread with 200 μl of 10 μM Ga-PPIX (Frontier Scientific), 10 μM Mn-PPIX (Frontier Scientific), or 10 μM GaCl3 (Sigma-Aldrich) and then were spotted with 10-fold serial dilutions of the cells. The plates were incubated for 2 days at 30°C before being photographed.
Melanin assay.Melanin production was examined on l-3,4-dihydroxyphenylalanine (l-DOPA) agar plates containing 0.1% glucose.
Virulence assay.The virulence of cryptococcal strains was examined in an inhalation model of cryptococcosis using female BALB/c mice (4 to 6 weeks old) from Charles River Laboratories (Senneville, Ontario, Canada), as described previously (30). Briefly, cells grown overnight in YPD medium were washed twice and resuspended in phosphate-buffered saline (PBS) (Invitrogen, Canada). The BALB/c mice were intranasally inoculated with a suspension of 104 cells in 50 μl. The health status of the mice was monitored daily after inoculation. Mice that showed disease symptoms and reached 15% weight loss were euthanized by CO2 anoxia. Statistical analyses of survival differences were performed by log rank tests using GraphPad Prism software (San Diego, CA). The fungal loads in the organs of three mice from each group were determined as described previously (30). The protocol for the virulence assay (protocol A13-0093) was approved by the University of British Columbia Committee on Animal Care. For histopathology studies, the lungs from three euthanized mice from each group were fixed in 10% neutral buffered formalin. The tissue was then embedded in paraffin and cut into 5-μm-thick sections, stained with hematoxylin and eosin (H&E) or Mayer's mucicarmine (MM), and then fixed on slides. Slides were examined by light microscopy under 5× objective magnification.
RESULTS
The C. neoformans genome encodes eight putative ferric reductases.We previously found that two iron regulators, the GATA factor Cir1 and the regulatory subunit HapX of the CCAAT-binding complex, both influenced the expression of a set of genes encoding predicted ferric reductases under iron-limiting conditions (30). This regulation suggested a role for these putative ferric reductases in iron acquisition. Therefore, we searched the C. neoformans genome by BLAST to identify genes corresponding to homologues of the S. cerevisiae ferric reductases as part of the present study, and this analysis identified eight candidates. The proteins encoded by these genes contained characteristic features of ferric reductases, including a flavin adenine dinucleotide (FAD)-binding domain (IPR013112/PF08022), a ferric reductase NAD-binding domain (IPR013121/PF08030), and a ferric reductase transmembrane component-like domain (IPR013130/PF01794) (Fig. 1). However, the ferric reductase transmembrane domain was not present in the predicted polypeptide encoded by CNAG_06976.7. We named the corresponding genes based on their closest similar orthologues in the S. cerevisiae genome database viz. FRE1 (CNAG_07770.7), FRE2 (CNAG_06821.7), FRE3 (CNAG_06524.7), FRE4 (CNAG_07334.7), FRE5 (CNAG_07604.7), FRE6 (CNAG_06976.7), and FRE7 (CNAG_00876.7). Although the top hit for CNAG_03498.7 in S. cerevisiae (Fre2) was more similar to the product encoded by CNAG_06821.7, we named the gene corresponding to CNAG_03498.7 “FRE201” to reflect the relationship to FRE2. These putative ferric reductases contained four to nine transmembrane regions, except that Fre6 contained only one transmembrane region. In addition, four conserved histidine residues that have a role in heme coordination were present in all of the candidate ferric reductases except Fre5. The first canonical heme-spanning histidine in this reductase was replaced by an arginine residue that is highly conserved in bacterial ferric reductases (33). Comparisons of the identities and similarities of the amino acid sequences of the eight ferric reductases are shown in Table S2 in the supplemental material.
Domain structure of C. neoformans ferric reductases. Colored boxes indicate the ferric reductase transmembrane component-like domain (red box), the flavin adenine dinucleotide (FAD)-binding domain (blue box), and the ferric reductase NAD-binding domain (green box). An asterisk indicates the bis-heme motif comprising four conserved histidine residues. The length in number of amino acids is indicated adjacent to the sequences. Numbers in parentheses indicate the number of transmembrane regions.
Expression of the FRE genes is iron and copper responsive.In S. cerevisiae and other microorganisms, the expression of FRE genes is transcriptionally regulated by iron and/or copper availability. In a previous microarray study, we found that a subset of the putative cryptococcal ferric reductases was transcriptionally regulated by iron and that this regulation was mediated, at least in part, by the transcription factors Cir1 and HapX (30). To validate and extend this analysis, we studied the expression of all eight putative ferric reductases in response to iron or copper availability during growth. The wild-type (WT) strain was grown either in YNB low-iron medium (YNB-LIM) without or with 100 μM FeCl3 or 10 μM hemin or in YNB low-copper medium (YNB-LCM) without or with 100 μM CuSO4 for 6 h, and the expression of FRE genes was analyzed by real-time qRT-PCR. The transcript levels of the FRE2 and FRE4 genes were reduced in the presence of both FeCl3 and hemin (Fig. 2A). This result suggests that the proteins encoded by these genes have a role in iron acquisition under iron-limited conditions, likely through a contribution to the high-affinity iron uptake system. However, all of the other FRE genes (FRE1, FRE3, FRE5, FRE6, FRE7, and FRE201) showed differential regulation depending upon the iron source. In particular, the transcripts of most of these genes, particularly FRE3, FRE5, and FRE7, were increased in the presence of hemin. However, the presence of FeCl3 had only a moderate repressive effect on the expression of these FRE genes. In the presence of copper, the transcript levels of the FRE2 to -5, FRE7, and FRE201 genes were reduced, with FRE4 showing the greatest reduction (Fig. 2B). The results for these genes may suggest roles in copper metabolism. In contrast, the expression of the FRE1 and FRE6 genes was elevated under high-copper conditions, with a particularly robust response observed for FRE6.
Expression analysis of C. neoformans FRE transcript levels. (A) Relative expression of FRE genes in the WT strain in the presence of iron, as examined by qRT-PCR. RNA was extracted from cells incubated in YNB-LIM with no iron or with either 100 μM FeCl3 or 10 μM hemin for 6 h. (B) Relative expression of FRE genes in the WT strain in the presence of copper, as examined by qRT-PCR. RNA was extracted from cells incubated in YNB-LCM with no copper or with 100 μM CuSO4 for 6 h. The data were normalized using 18S rRNA as an internal control. The data are from three biological replicates, and the bars represent standard errors.
Ferric and cupric reductase activities of FRE deletion strains.We next constructed deletion mutants in the WT background to characterize the functions of each FRE gene, including their contribution in reducing ferric and/or cupric ion to the ferrous and/or cuprous forms. Two independent mutants for each gene were subsequently used in all phenotypic analyses, and the data are shown here for only one representative independent mutant for each gene. In addition, the fre2Δ and fre4Δ mutations were complemented with the WT genes as described below. For ferric ion reduction, the WT and freΔ deletion strains were grown in defined LIM without or with 100 μM FeCl3. In the absence of iron, the ferric reductase activity in all of the freΔ mutants, except fre2Δ, was significantly lower than that in the WT strain (P < 0.05) (Fig. 3A). The fre2Δ mutant produced significantly higher ferric reductase activity than the WT (P < 0.05). When grown in the presence of iron, the ferric reductase activity was downregulated in the WT strain and in most of the freΔ mutants, including the fre1Δ to fre4Δ and fre7Δ strains (Fig. 3A). Compared to the WT, the ferric reductase activities in the fre1Δ, fre3Δ, fre4Δ, fre7Δ, and fre201Δ mutants were significantly lower under high-iron conditions (P < 0.05). However, under these conditions, fre2Δ and fre6Δ mutants exhibited significantly higher ferric reductase activities than the WT (P < 0.05), while the fre5Δ mutant had a WT level of ferric reductase activity. Interestingly, the ferric reductase activity in the fre201Δ mutant was significantly lower than that in the WT strain (P < 0.05), irrespective of the iron levels in the growth medium. This result indicates that Fre201 makes the greatest contribution to overall activity, although in general, ferric reductase activity was detected in all freΔ mutants, suggesting some functional redundancy.
Reductase activities of freΔ mutants. (A) Ferric reductase activity of WT and freΔ mutant strains grown in defined LIM with no iron (−Fe) or with 100 μM FeCl3 (+Fe). (B) Cupric reductase activity of WT and freΔ mutant strains grown in defined LCM with no copper (−Cu) or with 100 μM CuSO4 (+Cu). The data are from three replicates, and the bars represent standard errors.
Given that some ferric reductases also have cupric reductase activity (18, 22, 34), we also tested each freΔ deletion strain for this activity. For cupric ion reduction, the WT and freΔ deletion strains were grown in defined LCM without or with 100 μM CuSO4. Under low-copper conditions, the fre3Δ to fre6Δ mutants produced a WT level of cupric reductase activity (Fig. 3B). However, cupric reductase activity in the fre1Δ and fre7Δ mutants was lower than that in the WT strain. Under high-copper conditions, the cupric reductase activity was upregulated in all strains, except in the fre4Δ, fre5Δ, and fre7Δ mutants (Fig. 3B). Additionally, in this medium, the fre4Δ and fre7Δ mutants had significantly lower cupric reductase activity than that in the WT (P < 0.05). However, the cupric reductase activity in the fre2Δ and fre201Δ mutants was significantly higher than that in the WT (P < 0.05), irrespective of the copper concentration in the media (Fig. 3B).
Deletion of FRE4 impaired melanin production.Next, we examined all of the fre deletion mutants for virulence-related phenotypes, including their ability to grow at 37°C (host body temperature) and elaboration of the major virulence factors capsule and melanin. These analyses showed that all of the fre mutants grew at 37°C and produced capsule similar to the WT strain (data not shown). However, when the freΔ deletion strains were tested on l-DOPA medium for melanin formation, the fre4Δ strain showed a defect in melanin biosynthesis (Fig. 4A). Because exogenous copper induces melanin production and laccase activity (a copper-requiring enzyme that catalyzes the conversion of l-DOPA to melanin) and Fre4 could also have cupric reductase activity, we reasoned that the melanin-negative phenotype of the fre4Δ strain was due to poor copper availability. To test this hypothesis, the WT and fre4Δ strains were grown on l-DOPA plates with 50 μM CuSO4 and also on control plates without CuSO4. Addition of copper restored melanin biosynthesis in the fre4Δ strain to a level similar to that in the WT (Fig. 4B). Furthermore, qRT-PCR analysis for LAC1 (which encodes laccase) expression showed no differences in mRNA expression levels between the mutant and the WT strains (see Fig. S1 in the supplemental material), ruling out a transcriptional effect of Fre4 on LAC1.
The fre4Δ mutant is defective in melanin production. (A) Melanin production in WT and freΔ mutant strains was compared by growing cells on l-DOPA plates for 2 days. (B) Melanin production in the fre4Δ mutant was restored to the WT level by addition of exogenous copper. Cells were grown on l-DOPA plates with no CuSO4 (−Cu) or with 50 μM CuSO4 (+Cu) for 2 days.
Fre2 is required for robust growth on hemin and transferrin.To further characterize the functions of the FRE genes, we tested the growth of the freΔ mutants on various iron sources in vitro. All strains were first grown for 2 days in YNB-LIM to exhaust intracellular iron stores. After iron starvation, we tested growth of all the strains by agar spotting assays on YNB-LIM without or with FeCl3 or hemin at different concentrations. These spot assays showed that the fre201Δ and fre2Δ strains had a subtle growth defect in the presence of FeCl3 (at 100 μM) and hemin (at 10 μM), respectively (Fig. 5A). We also carried out YNB-LIM liquid assays for these two deletion strains and confirmed these growth defects, although the phenotypes were more pronounced under these growth conditions (Fig. 5B). This difference may be the result of free iron contaminating the agar in solid medium. Importantly, we also observed that a mutant with a transfer DNA (T-DNA) insertion in FRE2 had a severe growth defect in the presence of 10 μM hemin (Fig. 5C). This insertion mutant was obtained in a previously reported screen for mutants with reduced growth on hemin, and it had a more obvious growth defect than with the fre2Δ strain (35). Interestingly, the fre2 insertion mutant was generated in the background of a cfo1Δ mutant that lacks the ferroxidase of the high-affinity iron uptake system (35). Thus, the difference in the growth defects between the fre2 T-DNA insertion and deletion mutants indicates that the high-affinity iron uptake system, along with Fre2, makes a contribution to iron acquisition from hemin. The cfo1Δ mutant does not have a growth defect on hemin, and this finding therefore highlights the specific contribution of Fre2 (7). The growth defect of the fre2Δ strain was overcome by excess hemin (at 100 μM), suggesting the presence of other systems to acquire iron from hemin at higher concentrations.
Fre2 is required for robust growth on hemin. Strains were first iron starved in YNB-LIM for 2 days, washed, and then transferred to either solid agar plates or liquid medium. (A) Tenfold serial dilutions of iron-starved strains were spotted on YPD, YNB-LIM, and YNB-LIM with different concentrations of FeCl3 or hemin as indicated. Plates were incubated at 30°C for 2 days. (B) Iron-starved strains were inoculated into YNB-LIM with no iron or with different concentrations of FeCl3 or hemin as indicated. Cultures were incubated at 30°C with shaking, and turbidity was measured every 12 h. Reduced growth of fre2Δ and fre201Δ mutants was consistently observed in several repeats of the experiments. (C) Tenfold serial dilutions of iron-starved strains were spotted on YNB-LIM and YNB-LIM with different concentrations of hemin as indicated. Plates were incubated at 30°C for 2 days.
We also tested the growth of the freΔ mutants on other iron sources using a high-throughput assay in liquid media (see Materials and Methods). The strains were grown in YNB-LIM liquid media without or with various iron sources, including the mammalian host iron source transferrin (10 μM) or the siderophores ferrichrome (10 μM) and enterobactin (5 μM). All of the freΔ mutants grew to a similar level as the WT strain in the presence of the siderophores (data not shown). However, the fre2Δ and fre201Δ strains showed a subtle growth defect in the presence of transferrin as the sole iron source (Fig. 6).
Fre2 and Fre201 are required for robust growth on transferrin. Strains were first iron starved in YNB-LIM for 2 days, washed, and then inoculated into YNB-LIM with no transferrin or with 10 μM transferrin. Cultures were incubated at 30°C with shaking, and turbidity was measured every 12 h. Reduced growth of fre2Δ and fre201Δ mutants was consistently observed in several repeats of the experiments.
We selected FRE2, along with FRE4, for more detailed subsequent analyses because of the hemin phenotype of the fre2Δ strain and the melanin-negative phenotype of the fre4Δ strain. In addition, these two genes were of interest because they shared a pattern of expression that was distinct from all of the other genes, and they were the only two genes with lower transcript levels in response to hemin. To address potential redundant contributions to reductase activity, we generated double mutants lacking both FRE2 and FRE4. As mentioned above, we also constructed complemented strains wherein a WT copy of FRE2 or FRE4 was reintroduced into the single mutants. The fre2Δ fre4Δ double mutant was also complemented with a WT copy of either FRE2 (ectopic) or FRE4 (at the original locus). Although we observed interesting phenotypes for the fre201Δ strain, a more detailed analysis of FRE201 is currently under further investigation. We repeated the growth assays for the fre2Δ strain and the complemented strain in the presence of hemin and transferrin. As observed earlier, the fre2Δ strain showed less robust growth in the presence of hemin (see Fig. S2 in the supplemental material) and transferrin (see Fig. S3 in the supplemental material). This growth defect in both hemin and transferrin was overcome by the introduction of a WT copy of FRE2 at the original locus (see Fig. S2 and S3), thus further strengthening the conclusion that Fre2 is required for robust growth on these iron sources. In addition, the melanization defect of the fre4Δ strain was reversed by the introduction of a WT copy of FRE4 at the original locus (see Fig. S4 in the supplemental material).
Fre2 is required for hemin iron acquisition and not for heme uptake.Given that Fre2 had a role in growth on hemin as the sole iron source, we tested whether Fre2 acted as a component of a heme uptake system. That is, it was possible that Fre2 played a role in hemin internalization. To test this hypothesis, we used the noniron metalloporphyrin (MP) Ga-PPIX, which is a toxic heme analog (36). Owing to their structural similarity to heme, MPs enter the cells by a “Trojan horse” mechanism via a heme uptake pathway and act intracellularly to cause toxicity. The WT, fre2Δ, and complemented strains were iron starved before comparison of their growth on hemin in the presence or absence of Ga-PPIX. All strains, including the WT, grew well on YPD in the presence or absence of Ga-PPIX suggesting that the heme uptake pathway is not essential under these iron-replete conditions (Fig. 7). However, all of the strains showed weak or no growth on FeCl3 and hemin in the presence of Ga-PPIX, indicating that heme uptake leads to toxicity under these conditions. In addition, all of the strains grew well in the presence of GaCl3, thus attributing the toxic effect to Ga-PPIX and not to gallium. Together, these results demonstrate that loss of Fre2 does not block Ga-PPIX uptake and toxicity and that this candidate reductase therefore probably has no role in heme uptake.
Fre2 is not required for hemin uptake. Strains were first iron starved in YNB-LIM for 2 days, washed, and then transferred to solid agar plates. Tenfold serial dilutions of iron-starved strains were spotted on YPD without or with 10 μM Ga-PPIX, YNB-LIM plus 100 μM FeCl3, or YNB-LIM plus 10 μM hemin without or with 10 μM Ga-PPIX or 10 μM GaCl3 as indicated. Plates were incubated at 30°C for 2 days.
Loss of FRE4 caused increased susceptibility to antifungal drugs.Previously, a connection between iron availability and antifungal drug susceptibility was observed in C. neoformans, S. cerevisiae, and Candida spp. (7, 8, 37, 38, 39). In C. neoformans, loss of CFO1 and CFT1 (high-affinity iron uptake components) resulted in increased susceptibility to azole as well as polyene drugs. This phenotype was reversed by exogenous hemin, thus supporting the idea that heme biosynthesis was affected in these mutants, leading to drug susceptibility (7, 8). In this context, we tested the freΔ strains for susceptibility to the antifungal drugs fluconazole, miconazole, and amphotericin B. These drugs are commonly used to treat or reduce the symptoms of cryptococcal meningitis. None of the deletion strains showed altered susceptibility to the polyene antifungal drug amphotericin B (Fig. 8A). However, the fre4Δ strain showed increased susceptibility to the azole antifungal drugs fluconazole and miconazole (Fig. 8A). As reported earlier (7, 8), we tested whether this phenotype was related to iron deficiency by studying the response to the drugs in the presence of different iron sources. We found that addition of hemin again reversed the susceptibility to fluconazole (Fig. 8B). Even the addition of inorganic salts FeCl3 and CuSO4 could overcome the susceptibility to the azole drug. These findings are similar to those observed for C. albicans iron acquisition mutants (39). Overall, our data suggest that the deletion of FRE4 resulted in iron deficiency, thereby affecting heme biosynthesis and ultimately affecting ergosterol biosynthesis.
Deletion of FRE4 increases susceptibility to antifungal drugs. (A) Tenfold serial dilutions of strains were spotted on YPD without or with the antifungal drugs as indicated. Plates were incubated at 30°C for 2 days. (B) Addition of exogenous hemin, FeCl3, or CuSO4 suppressed the antifungal drug susceptibility of the fre4Δ mutant. Tenfold serial dilutions of strains were spotted on YPD with fluconazole without or with an iron or copper source as indicated. Plates were incubated at 30°C for 2 days.
Fre2 contributes to virulence in mice.Finally, we tested the virulence of the fre2Δ and fre4Δ single mutants, the fre2Δ fre4Δ double mutant and the complemented strains in a mouse inhalation model of cryptococcosis. We tested 13 BALB/c mice per strain for the WT, the fre2Δ and fre4Δ single mutants and their respective complemented strains, and two independent fre2Δ fre4Δ double mutants. Of the 13 mice, 3 mice for each strain were used for fungal load analysis at the time that the mice succumbed to infection with the WT strain; the other 10 mice were monitored for the development of disease symptoms. We observed that mice infected with the WT strain, the fre4Δ mutant, and the FRE2 or the FRE4 complemented strains showed 100% mortality by day 21. However, mice infected with the fre2Δ mutant and fre2Δ fre4Δ double mutants survived to days 26 and 27, respectively (Fig. 9). These results indicate that the deletion of FRE2, and FRE2 in combination with FRE4, caused a significant attenuation of virulence, suggesting that Fre2 has a role during infection. Since the fre2Δ mutant showed a growth defect in the presence of hemin, we hypothesize that heme iron is important for the growth of C. neoformans in mammalian hosts.
The fre2Δ mutant and the fre2Δ fre4Δ double mutants are attenuated in virulence. Thirteen BALB/c mice were infected intranasally with each of the strains indicated, and the survival of the mice was monitored over the time course indicated on the x axis. The difference in survival rates between the fre2Δ mutant and the WT strain was significant (P < 0.0001), as was the difference between the survival rates of the fre2Δ fre4Δ double mutants and the WT strain (P < 0.0001).
We also analyzed the distribution of fungal cells in the lungs and brains of infected mice. Three infected mice for each strain were sacrificed at the same time point (day 20) as the WT-infected mice from the virulence assay (Fig. 9). The numbers of fungal cells in the lungs of mice infected with each strain, except the fre2Δ mutant (1 order of magnitude lower than the WT), were comparable. However, the numbers of fungal cells in the brains of mice infected with the fre2Δ mutant and the fre2Δ fre4Δ double mutant were 2 to 4 orders of magnitude lower than those in the brains of mice infected with the WT strain (Fig. 10) suggesting a defect in dissemination and/or colonization of organs. We also noted that the burden for the fre2Δ mutant in lungs and brains was lower than the burden for the fre2Δ fre4Δ double mutants in both tissues. This result suggests that loss of Fre4 improves the ability of the fre2Δ mutant to accumulate or persist in the host. Consistent with the fungal load data, the histopathology analysis of the lungs at day 20 of infection showed less abundance of fre2Δ mutant cells compared to the WT and FRE2 complemented strains (see Fig. S5 in the supplemental material).
The fre2Δ mutant and the fre2Δ fre4Δ double mutants showed reduced fungal load in the brain in a mouse model of cryptococcosis. The distribution of fungal cells in the lungs and brains of mice was analyzed with three mice for each strain sacrificed at day 20 when WT-infected mice reached the endpoint. The dashed horizontal line indicates the detection limit of the assay, and the error bars indicate standard errors.
DISCUSSION
Iron acquisition is critical for the pathogenesis of C. neoformans, and there is a well-established role for the reductive, high-affinity uptake system in overall disease and colonization of the central nervous system (5–8, 25, 30, 40, 41). However, the molecular genetics of the enzymatic reduction of ferric to ferrous iron have not been investigated for this pathogen. Ferric reductases are present in all fungal species, with variable numbers predicted across fungal genomes; these enzymes have been studied extensively in S. cerevisiae and, to some extent, in C. albicans, S. pombe, and A. fumigatus (13–24, 42). In our study, we identified eight candidate ferric reductase (FRE) genes in C. neoformans, characterized their expression and contributions to reductase activity, and investigated their roles in virulence factor production. In some fungi, specific reductases account for the majority of the cell-surface reductase activity, and these include ScFre1p and ScFre2p in S. cerevisiae and CaFre10 and CaFre7 in C. albicans. For C. neoformans, we found that the deletion of individual FRE genes did not completely eliminate reductase activity under the conditions tested, although one gene (FRE201) made a major contribution. In addition, loss of Fre2 resulted in elevated ferric reductase activity, perhaps due to an influence on iron uptake and intracellular iron levels that resulted in elevated expression of other reductase genes. This result, along with the discoveries of a role for Fre2 in iron acquisition from heme and a contribution of Fre4 to production of the virulence factor melanin, served to focus our attention on these two reductases.
We found that the transcript levels of both FRE2 and FRE4 were reduced in the presence of iron and that this reduction was independent of the iron source. Unlike the expression of these two genes, the expression pattern of all the other FRE genes was either induced or repressed, depending upon the iron source. Additionally, the transcript level of FRE4 was strongly repressed in the presence of copper. Based on this observation and the finding that the fre4Δ mutant showed reduced cupric reductase activity, we hypothesize that Fre4 may also be a cupric reductase. We also note that a previous study reported upregulation of FRE7 in C. neoformans under low-copper conditions (43). Besides metal regulation, transcription of ferric reductase genes in fungal pathogens is also affected by pH, macrophage engulfment, antifungal drugs, and infection, suggesting distinct regulation of different ferric reductases in response to specific environmental conditions (23, 44–46). These factors remain to be examined in C. neoformans, and more detailed interpretation of expression patterns and reductase activities will also require knowledge of the localization of each enzyme.
Our discovery of a role for Fre4 in melanization further supports the possible involvement of this reductase in copper homeostasis. The melanin defect for the fre4Δ mutant was not due to the decreased transcription of the LAC1 gene that encodes the multicopper oxidase (laccase) for melanin production, as similar levels of LAC1 expression were observed between the mutant and the WT strain. In contrast, addition of copper restored melanin production in the fre4Δ mutant, as seen in other melanin-negative or -deficient mutants of C. neoformans: e.g., mel1, mel5, mel7, vph1, and clc-a mutations (47–50). Ferric reductases are also known to reduce copper at the cell surface, as reported in S. cerevisiae and C. albicans (18, 22, 34). It is therefore possible that Fre4 in C. neoformans is a multifunctional enzyme with the ability to reduce copper, and thus its loss resulted in a defect in copper uptake and subsequently affected laccase activity. A defect in copper uptake could also affect the function of additional copper-dependent enzymes in C. neoformans. For example, a mutant lacking Sod1 (Cu/Zn superoxide dismutase) is deficient in melanin and also exhibits reduced laccase activity (51). A decrease in ferric/cupric reductase and laccase activities was also observed in the melanin-deficient oxy2 (copper-sensing transcription factor) mutant and was attributed to the lack of copper (52). Similar observations have been reported in Podospora anserina, wherein the loss of the GRISEA gene caused a melanin-negative phenotype that was repaired by copper supplementation (53, 54).
We also found that deletion of FRE4 increased susceptibility to azole drugs, perhaps due to an influence of altered metal homeostasis on iron-dependent ergosterol biosynthesis. Previous studies have also suggested increased membrane fluidity as well as iron deficiency as contributing factors in the increased susceptibility of iron uptake mutants to antifungal drugs (7, 8, 39). For example, the ftr1 and ftr2 iron uptake mutants and the ccc2 copper transporter mutant showed increased susceptibility to the antifungal drug fluconazole in C. albicans (39). This susceptibility was overcome by addition of iron. Similarly, the drug susceptibility of cfo1Δ and cft1Δ mutants in C. neoformans was suppressed by addition of hemin, as well as the siderophore deferoxamine (for the cfo1Δ mutant) (7, 8). We tested the influence of iron with the fre4Δ mutant and found that addition of hemin suppressed the drug susceptibility. We also found that addition of FeCl3 and CuSO4 suppressed the susceptibility of the fre4Δ mutant, as observed for the C. albicans mutants. It was interesting that the addition of CuSO4 reduced the drug susceptibility of the mutant. We speculate that loss of Fre4 resulted in defective copper acquisition, thereby affecting iron availability. This is because the high-affinity iron uptake component Cfo1 is a copper-requiring enzyme and Fre4 may also be a cupric reductase. A similar role for copper chelation in increasing susceptibility to drugs was observed in C. albicans (39). More recently, the heme-binding protein Dap1 in Candida glabrata was shown to be required for growth under low-iron conditions and also for azole drug tolerance (38).
Heme and transferrin are both important sources of iron for pathogens during infection of vertebrate hosts. Mechanisms of heme uptake have been well characterized in pathogenic bacteria and involve the recognition of heme or heme-loaded hemophores by surface receptors before delivery to membrane transporters and subsequent internalization (55). Although less is known about the use of heme in parasites and pathogenic fungi, Leishmania amazonensis was recently found to have a heme receptor that was required for heme utilization, and the pathogenic fungi C. albicans and Histoplasma capsulatum utilize heme and/or hemoglobin via cell surface receptors (9, 10, 56, 57). C. neoformans also uses heme and hemoglobin as iron sources, and the mannoprotein Cig1 was recently implicated in heme uptake (6). Cig1 shows heme-binding properties, and its loss delayed growth in hemin and reduced susceptibility to noniron metalloporphyrins. In our study, we discovered that Fre2 contributes to heme use by C. neoformans. Loss of Fre2 resulted in a subtle growth defect in the presence of hemin, but unlike Cig1, Fre2 was involved in acquisition of iron from hemin rather than uptake. The fre2Δ mutant had a less severe growth defect in hemin than a cig1Δ mutant, and this suggests (i) a minor role for the enzyme, (ii) a contribution as part of a cell surface activity involving other components, and (iii) the presence of additional heme utilization systems like the one involving Cig1. Support for Fre2's acting together with other components at the cell surface comes from the observation that loss of both Fre2 and the ferroxidase Cfo1 for high-affinity uptake resulted in a more severe growth defect on hemin than the loss of Fre2 alone. Both ferric reductase and the high-affinity iron uptake system appear to contribute to the use of iron from hemin. Furthermore, the growth defect of the fre2Δ mutant was overcome by supplementing the growth medium with excess hemin. This suggests the presence of other systems for acquisition of iron from hemin at higher concentrations. A similar conclusion was recently reported during the analysis of Cig1 (6).
We also observed a role for Fre2 in growth on transferrin, and we note that a role for reductive iron uptake from transferrin has previously been observed in C. albicans (12). In that study, CaFre10 facilitated the reduction and release of iron from transferrin for subsequent uptake by the high-affinity permease-multicopper oxidase system. The contribution of CaFre10 activity was greater under acidic conditions, and another cell surface reductase, CaFre1, contributed to reductive transferrin-iron uptake under alkaline conditions (12). Our earlier studies revealed that the reductive, high-affinity iron uptake system (involving Cfo1 and Cft1) was required for iron utilization from transferrin and FeCl3 in C. neoformans (7, 8). In the present study, we extended our understanding of this process by identifying the ferric reductases (Fre2 and additionally Fre201) that release transferrin iron for subsequent utilization via the high-affinity reductive system. The contribution of Fre201 in transferrin utilization was comparatively larger than that of Fre2, and the role of Fre201 is currently under further investigation. Analogous to C. albicans, we speculate that Fre2 and Fre201 may contribute to the use of iron from transferrin under conditions that are specific for each reductase.
Finally, we analyzed the virulence of the fre2Δ, fre4Δ, and fre2Δ fre4Δ mutants using a mouse inhalation model of cryptococcosis and found that Fre2 but not Fre4 contributed to virulence. This result revealed Fre2 as a new component of the iron acquisition mechanisms that impact virulence in C. neoformans and, interestingly, indicated that the melanin defect of the fre4Δ mutant was not relevant for virulence. An analysis of the fungal burden in infected mice also showed that deletion of FRE2 impaired the dissemination of the mutant from lungs to the brain and/or colonization of the brain. This defect is reminiscent of the reduced fungal burden observed for mutants lacking the Cft1 and Cfo1 components of the high-affinity uptake system in C. neoformans (6, 7, 8). Since the fre2Δ mutant showed a defect in the use of hemin as an iron source, it is likely that hemin may be important for the growth of C. neoformans during infection. It was also interesting that deletion of FRE4 improved the ability of the fre2Δ mutant to accumulate or persist in the brains and lungs of infected mice. This result suggests the potential for complex interactions between reductases and possible competing contributions to metal homeostasis. In general, the identification of a role of ferric reductases in cryptococcal virulence is a novel finding for fungal pathogens of humans. Ferric reductases have been characterized in C. albicans and A. fumigatus, but none of these enzymes have so far been implicated in virulence (13, 22, 23, 46). For C. neoformans, additional work is needed to understand the interactions between reductases, the contributions of each of the enzymes to virulence, and the mechanisms by which Fre2 participates in iron use from heme and transferrin.
ACKNOWLEDGMENTS
This research was supported by a grant from the National Institute of Allergy and Infectious Diseases (RO1 AI053721). J.K. gratefully acknowledges a Scholar Award in Molecular Pathogenic Mycology from the Burroughs Wellcome Fund. D.O. was supported by a grant from CNPq, Brazil.
We acknowledge Wax-it Histology Services, Inc., for participating in the histopathology experiment.
FOOTNOTES
- Received 22 October 2013.
- Returned for modification 13 November 2013.
- Accepted 2 December 2013.
- Accepted manuscript posted online 9 December 2013.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.01357-13.
- Copyright © 2014, American Society for Microbiology. All Rights Reserved.
