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Med Mycol. 2016 August; 54(6): 605–615.
Published online 2016 April 26. doi:  10.1093/mmy/myw013
PMCID: PMC4943159

The ZIP family zinc transporters support the virulence of Cryptococcus neoformans

Abstract

Zinc is an essential element in living organisms and a cofactor for various metalloproteins. To disseminate and survive, a pathogenic microbe must obtain zinc from the host, which is an environment with extremely limited zinc availability. In this study, we investigated the roles of the ZIP family zinc transporters Zip1 and Zip2 in the human pathogenic fungus Cryptococcus neoformans. Zip1 and Zip2 are homologous to Zrt1 and Zrt2 of the model fungus, Saccharomyces cerevisiae, respectively. We found that the expression of ZIP1 was regulated by the zinc concentration in the environment. Furthermore, the mutant lacking ZIP1 displayed a severe growth defect under zinc-limited conditions, while the mutant lacking ZIP2 displayed normal growth. Inductively coupled plasma–atomic emission spectroscopy analysis showed that the absence of Zip1 expression significantly reduced total cellular zinc levels relative to that in the wild type, while overexpression of Zip1 was associated with increased cellular zinc levels. These findings suggested that Zip1 plays roles in zinc uptake in C. neoformans. We also constructed a Zip1-FLAG fusion protein and found, by immunofluorescence, not only that the protein was localized to the periphery implying it is a membrane transporter, but also that the protein was N-glycosylated. Furthermore, the mutant lacking ZIP1 showed attenuated virulence in a murine inhalation model of cryptococcosis and reduced survival within murine macrophages. Overall, our data suggest that Zip1 plays essential roles in zinc transport and the virulence of C. neoformans.

Keywords: Cryptococcus neoformans, Fungi, Metal, Virulence, Zinc

Introduction

Zinc plays multiple essential roles in biological systems. Enzymes that require zinc as a cofactor include alkaline phosphatases, Cu-Zn superoxide dismutases, and alcohol dehydrogenases. Regulatory proteins that contain zinc-binding domains, such as the C2H2-type zinc finger domain, the LIM domain, and the RING finger domain, also require zinc for structural stabilization.1,2 The importance of this cofactor is evident from the fact that zinc deficiency can cause severe problems. On the other hand, excess zinc can also have a detrimental effect on cells. Therefore, the cellular zinc concentration must be tightly regulated.3,4 The role of zinc in host-pathogen interactions has also been considered intensively. As a means of nutritional immunity during microbial infection, the vertebrate host sequesters intra- and extracellular zinc using a number of zinc importers and exporters as well as calprotectin, which is a Zn/Mn-chelating protein released by neutrophils.5 However, in general, pathogenic microbes including fungi possess efficient zinc acquisition machinery to counteract zinc sequestration by hosts, allowing their survival within the host environment and the progression of disease.6,7

Among fungi, proteins involved in zinc uptake and zinc storage have been best characterized in Saccharomyces cerevisiae. In this fungus, zinc uptake at the plasma membrane is mediated by two members of the Zrt- and Irt-related protein (ZIP) families of metal transporters designated Zrt1 and Zrt2.8,9 The high-affinity zinc transporter Zrt1 is expressed under conditions of zinc deficiency and delivers zinc into the cytoplasm, while the low-affinity zinc transporter Zrt2 is expressed in response to zinc repletion.10 The low-affinity iron transporter Fet4 and the high-affinity inorganic phosphate transporter Pho84 were also shown to facilitate zinc uptake.11,12 Excess zinc is stored in vacuoles during zinc repletion in S. cerevisiae, and the cation diffusion facilitator Cot1 and its paralog Zrc1 mediate zinc transport into vacuoles.1315 Stored vacuolar zinc is transported into the cytoplasm by the vacuole membrane transporter Zrt3, which is another member of the ZIP family of metal transporters in S. cerevisiae.15

Similar zinc transport systems have been identified in the pathogenic fungus Aspergillus fumigatus. Three plasma membrane zinc transporters, ZrfA, ZrfB, and ZrfC, have been identified, and among them, ZrfC was shown to be essential for virulence of A. fumigatus, but only in the absence of ZrfA and ZrfB.7,16 Zinc acquisition has also been studied for another major human fungal pathogen, Candida albicans, and Citiulo et al. identified and characterized a zinc acquisition locus, PRA1-ZRT1, in this fungus. PRA1 encodes an extracellular zinc scavenger called a ‘zincophore,’ and ZRT1 encodes a predicted high affinity zinc transporter.6 Citiulo et al. showed that Pra1 has zinc-binding properties and is required for zinc acquisition within the host, and they suggested that Zrt1 plays an essential role in binding of Pra1 to the fungal cell surface.6 This Pra1-Zrt1 system was shown to be required for zinc acquisition within the host environment.

There is no information available on zinc transporters in Cryptococcus neoformans, the pathogenic fungus that causes a life-threatening meningoencephalitis, primarily in immunocompromised people such as those with AIDS. However, a recent study by Schneider et al. identified the transcription factor Zap1 that regulates expression of putative zinc transporters in another Cryptococcus species, C. gattii, which was responsible for an outbreak of cryptococcosis in immunocompetent individuals in western North America.17,18 Zap1 is a C2H2-type zinc finger transcription factor that is homologous to S. cerevisiae Zap1. The mutant lacking ZAP1 displayed significantly reduced expression of putative zinc transporters, growth deficiency under zinc-depleted conditions, and reduced virulence in a murine model of cryptococcosis, suggesting that Zap1 plays an important role in zinc metabolism and virulence of C. gattii. Although the importance of zinc transport and metabolism is suggested in C. gattii, no study has been conducted on zinc uptake and homeostasis in C. neoformans, which is the major cause of cryptococcosis.

In this study, we sought to identity and characterize zinc transporter genes in the genome of C. neoformans to understand zinc uptake systems in this fungus. We identified genes that were homologous to ZRT1 and ZRT2 of S. cerevisiae and found that the expression of these homologs was regulated by the concentration of zinc in the environment. The functional characteristics of zinc transporters and their roles in the virulence of C. neoformans were also investigated.

Materials and Methods

Strains and growth conditions

The C. neoformans strains used in this study are listed in Table S1. The strains were routinely cultured in yeast extract-Bacto peptone medium with 2.0% glucose (YPD; Difco) or yeast nitrogen base (YNB; Difco) with 2.0% glucose. The low-zinc medium was prepared by adding N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) to the YPD at a final concentration of 100 μM (YPD + TPEN) or using YNB-ZnSO4 medium (low-Zn YNB; Sunrise Science Products). Zinc-replete media were prepared by adding ZnCl2 to the low-zinc medium at concentrations indicated in the text.

Construction of strains

The sequences of the genes encoding ZIP1 (CNAG_00895.2) and ZIP2 (CNAG_03398.2) were obtained from the C. neoformans var. grubii serotype A genome database (http://www.broad.mit.edu/annotation/genome/cryptococcus_neoformans). Gene deletion mutants were constructed by homologous recombination using a gene-specific knockout cassette amplified by overlapping PCR with primers listed in Table S2.19 To construct the zip1 mutant, the gene-specific knockout cassette was amplified by PCR using primers zip1-1, zip1-2, zip1-3, zip1-4, zip1-5, zip1-6, zip1-7, zip1-8, zip1-9PO, zip1-10PO, and zip1-Neo_PO with genomic DNA and the plasmid pJAF1 as templates. To construct the zip2 mutant, the gene-specific knockout cassette was prepared by PCR using primers zip2-1, zip2-2, zip2-3, zip2-4, zip2-5, zip2-6, zip2-7PO, and zip2-Nat_PO, with genomic DNA and the plasmid pCH233 as templates. The wild-type strain was biolistically transformed with the amplified knockout constructs, and 1,360- and 1,686-bp regions of the coding sequence of ZIP1 and ZIP2 were replaced with the neomycin-resistant gene (NEO) and nourseothricin acetyltransferase gene (NAT), respectively. The positive transformants were selected by PCR and Southern blot analysis. To construct the zip1 zip2 double mutant, the ZIP1 knock-out cassette was introduced into the zip2 mutant by biolistic transformation, and positive transformants were identified by PCR. To construct the ZIP1-reconstituted strain, the ZIP1 gene was amplified by PCR using primers zip1_Re.1, zip1_Re.5, zip1_fus, M13-R, M13-F, and NAT-up with wild-type genomic DNA and the plasmid pCH233 as templates. The amplified 4.9 Kb DNA fragment containing the wild-type ZIP1 gene was digested with BspHI and introduced into the zip1 mutant. Positive transformants were identified by PCR and confirmed by phenotypic assays. To construct the ZIP2-reconstituted strain, the ZIP2 gene was amplified by PCR using primers zip2_F_SacI and zip2_R_BamHI from wild-type genomic DNA, followed by digestion with SacI and BamHI. The digested fragments were cloned into the plasmid pJAF1. The resulting plasmid pWH147 was digested with BspHI and introduced into the zip2 mutant by biolistic transformation. Positive transformants were identified by PCR. To construct the Zip1-FLAG fusion protein, pWH109, which contained the 3×FLAG epitope, the Gal7 terminator, and NEO, was used.20 The ZIP1 open reading frame was amplified by PCR from wild-type genomic DNA, and primers Zip1_F and Zip1_R. The amplified DNA fragments were digested with HindIII and BglII, and ligated with pWH109. The resulting plasmid pWH164 was used as a template for PCR with primers M13-R and Gal7termR. The amplified DNA fragments were digested with HindIII and SpeI, and ligated with pCH233. The resulting plasmid pWH178 was digested with HindIII and introduced into the zip1 mutant by biolistic transformation. Positive transformants containing the ZIP1-FLAG fusion were identified by PCR.

Northern blot analysis

To analyze transcript levels of ZIP1 and ZIP2 in response to a range of zinc concentrations, the wild-type strain was grown in 5 ml of YPD overnight, harvested, and washed twice with metal-chelated water using Chelex 100 (Bio-rad), and then transferred to YPD medium containing 100 μM TPEN with various concentrations of ZnCl2 as indicated in the text. Cells were then grown at 30°C for 12 hours and harvested. For the time course analysis, the wild-type strain was grown in 50 ml of YPD medium for 12 hours. TPEN was added at a final concentration of 100 μM, and cells were harvested at the indicated times. Total RNA was extracted using Trizol (Invitrogen), and 15 μg from each sample was used for northern blot analysis as described elsewhere.21 The hybridization probes were amplified using genomic DNA from the wild-type strain as a template and with the primers listed in Table S2, and labeled with 32P-dCTP using a DNA labeling kit (Ambion). The hybridized membrane was exposed to a phosphor screen (PerkinElmer) and scanned using a Packard Cyclone Phosphor Imager (PerkinElmer).

Western blot analysis

The strain expressing the Zip1-FLAG fusion protein was grown in 50 ml of YPD medium for 12 hours. TPEN was added at a final concentration of 100 μM, cells were harvested at the times indicated in the text, and total protein was extracted as previously described.20 The quantity of total protein was measured by Bradford assay.22 To remove the N-linked glycans, 2.5 μg of total lysates were incubated with 250 U of PNGase F (New England Biolabs) at 37°C for 4 hours. Samples were denatured at 100°C for 5 min, separated in a polyacrylamide gel (SDS-PAGE), and transferred to a nitrocellulose membrane (Amersham). Western blot analysis was performed using an anti-DDDDK polyclonal rabbit antibody (Abcam) as the primary antibody and a goat anti-rabbit IgG horseradish peroxidase conjugate (Santa Cruz Biotechnology) as the secondary antibody, followed by visualization by chemiluminescence.

Immunofluorescence

Cells were cultured in 5 ml of YPD at 30°C overnight and washed twice with metal-chelated water. Cells were then resuspended in YPD medium containing 100 μM of TPEN, cultured at 30°C for 3 hours, and visualized by immunofluorescence microscopy as described previously.23 Briefly, cells were fixed with formaldehyde (Sigma), washed with PBS, and resuspended with SCB buffer (1 M sorbitol, 10 mM sodium citrate, pH 5.8). Cells were treated with 40 mg/mL of lysing enzymes from Trichoderma harzianum (Sigma) and 35 mM β-mercaptoethanol, and incubated at 30°C for 4 hours to generate spheroplasts. Cells were washed twice with SCB buffer, resuspended in PBS buffer (PBS, 1 M sorbitol, 0.2% BSA), spotted on poly-L-lysine treated slides, and incubated for 15 min until buffer was dried completely. The cell permeabilizing step with detergent was omitted because topology prediction of the Zip1-FLAG fusion protein indicated the C-terminal fused epitope tag faces extracellularly. The slides were then incubated with blocking buffer containing 10 μg/mL of anti-DDDDK polyclonal rabbit antibody (Abcam) at 4°C overnight in a moist chamber, followed by incubation with blocking buffer containing 4 μg/ml of goat anti-rabbit IgG with Alexa Fluor® 488 conjugate (Abcam) at room temperature for 1 hour. The samples were washed 7 times with blocking buffer and mounted with ProLong® Gold antifade reagent with DAPI (Molecular Probes). The immunofluorescence images were acquired using a BX53 microscope (Olympus).

Inductively coupled plasma–atomic emission spectroscopy (ICP-AES)

Total cellular zinc levels were determined by ICP-AES. Cells were grown in 5 ml of YPD overnight and washed twice with metal-chelated PBS (pH 7.4). Then, cells were transferred and incubated in 50 ml of low-Zn YNB medium overnight at 30°C. Cells were harvested and washed twice with metal-chelated PBS, resuspended in 200 ml of low-Zn YNB medium containing either glucose or galactose, and held overnight at 30°C. Cells were washed twice with metal-chelated PBS and then lyophilized. The lyophilized samples were digested by the microwave system (START D) and analyzed using an OPTIMA 5300 DV (PerkinElmer) system.24

Virulence assay

Virulence was assayed in an inhalation model of cryptococcosis using female BALB/c mice (4 to 6 weeks old) from Charles River Laboratories (Ontario, Canada) as previously described.25 Briefly, C. neoformans strains were grown in 5 mL of YPD at 30°C overnight, washed twice with PBS (Invitrogen Canada), and resuspended in PBS. BALB/c mice, in groups of 10, were intranasally inoculated with a suspension of 2 ×105 cells in 50 μl. The health status of the mice was monitored daily post-inoculation. Mice reaching the humane endpoint were euthanized by CO2 anoxia. Statistical analyses of survival differences were performed by log rank tests using GraphPad Prism 5 for Windows (GraphPad software). The protocols for the virulence assay (protocol A13-0093) were approved by the University of British Columbia Committee on Animal Care.

Macrophage survival assay

The murine macrophage cell line J774A.1 was maintained at 37°C in 10% CO2 in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FBS), 1% nonessential amino acids, 100 μg/ml penicillin-streptomycin, and 4 mM L-glutamine (Invitrogen). Cryptococcus cells were opsonized with monoclonal antibody 18B7 against the capsular polyscaccharide glucuronoxylomannan (1 μg/ml), and macrophages were incubated with 150 ng/ml phorbol 12-myristate 13-acetate (PMA) in serum-free DMEM medium for 1 hour for stimulation prior to co-incubation at a multiplicity of infection (MOI) of 10:1.26,27 Macrophages were inoculated with each strain indicated and then washed after 1 hour of incubation to remove unattached, extracellular fungal cells. After 1 hour and 24 hours of incubation, sterile, ice-cold distilled H2O was applied to each well to lyse the macrophages, and the fungal cells were harvested by centrifugation and plated on YPD agar media to determine colony-forming units (CFUs).

Results

Identification of the ZIP family zinc transporters in C. neoformans

Two genes, CNAG_00895 and CNAG_03398, encoding proteins that are homologous to S. cerevisiae Zrt1 and Zrt2, respectively, were identified from the genome of C. neoformans var. grubii. CNAG_00895 and CNAG_03398 encode predicted polypeptides of 369 and 394 amino acids that showed 39% identity and 54% similarity, and 31% identity and 51% similarity, to S. cerevisiae Zrt1 and Zrt2, respectively. Both CNAG_00895 and CNAG_03398 contain the ZIP family zinc transporter domains and therefore were named Zip1 and Zip2, respectively (Fig. 1).28 Computational prediction of the transmembrane protein topology revealed that C. neoformans Zip1 and Zip2 had 8 and 9 transmembrane domains, respectively.29

Figure 1.
Zip1 and Zip2 encode ZIP family zinc transporters. The alignments of the amino acid sequences of Zip1 and Zip2 to the ZIP family zinc transport proteins in other fungi are shown. The GenBank accession numbers for the protein sequences of the fungal ZIP ...

To test whether expression of ZIP1 and ZIP2 transcripts was regulated by zinc, we grew the wild-type cells in low-zinc YNB media with various concentrations of zinc and evaluated transcript levels by northern blot analysis. Transcripts of ZIP1 were abundant when cells were grown in low-zinc YNB media without any zinc supplementation, and less abundant transcripts were found from the cells grown in the same medium containing 0.25 μM of zinc. However, the addition of more than 2.5 μM of zinc in the medium significantly reduced ZIP1 transcript levels, with virtually no transcripts detected in these cells (Fig. 2A). These results suggested that zinc deprivation induces expression of ZIP1 and that ZIP1 is required for the high-affinity zinc transport in C. neoformans. While abundant ZIP1 transcripts in media with low-zinc concentrations were observed, no visible ZIP2 transcripts were detected from the cells grown in media with various concentrations of zinc, suggesting a minor contribution of Zip2 to zinc transport in C. neoformans. To confirm expression of ZIP1 upon zinc deprivation, transcript levels of ZIP1 were also monitored over time following the addition of TPEN, which has been used extensively to study zinc homeostasis in various organisms, to YPD medium.30 The results from the time-course expression analysis showed that ZIP1 transcripts became visible at 3 hours after the addition of TPEN to the medium, and transcript signals were highly induced after this time (Fig. 2B). These results confirmed zinc-dependent regulation of ZIP1 expression, which resembles the expression patterns of high-affinity zinc transporters in S. cerevisiae and A. fumigatus.8,16

Figure 2.
Analysis of ZIP1 and ZIP2 mRNA expression. A. Transcript levels of ZIP1 and ZIP2 were monitored by northern blot analysis after growing the wild-type strain in low-zinc YNB medium supplemented with different amounts of ZnCl2 as indicated. B. The wild-type ...

Zip1 is the major zinc transporter in C. neoformans

To characterize the functions of ZIP1 and ZIP2 in C. neoformans, zip1 and zip2 mutants were constructed using a targeted gene deletion method with biolistic transformation. ZIP1-reconstituted and ZIP2-reconstituted strains were also constructed by integration of the wild-type genes into their original loci in the zip1 mutant and the zip2 mutant, respectively. To account for potential redundant roles for the genes, a zip1 zip2 double mutant, in which both ZIP1 and ZIP2 were deleted, was constructed and included in this study (see Materials and Methods).

Loss of ZIP1 or ZIP2 function in each mutant was first confirmed by their growth in media containing various concentrations of zinc. While all strains grew well on the YPD control medium, the zip1 mutant showed a significant growth defect on low-zinc medium, suggesting that ZIP1 is required for high-affinity zinc transport. Growth of the zip1 mutant was restored with the addition of 250 μM zinc to the medium, indicating the existence of a Zip1-independent low-affinity zinc transport system in C. neoformans. The reintroduction of ZIP1 in the zip1 mutant strain also restored growth, confirming that the phenotype of the mutant was due to the deletion of ZIP1 (Fig. 3). The growth defect of the zip1 mutant in the low-zinc medium supported the results of our expression analysis, which showed highly abundant transcripts of ZIP1 in wild-type cells grown under the same conditions. These results suggested that Zip1 plays a role in high-affinity zinc transport and that an additional low-affinity zinc transport system is present to support the growth of the mutants under conditions with high concentration of zinc. In contrast to the zip1 mutant, the zip2 mutant showed no visible growth defect in media with different concentrations of zinc, suggesting that Zip2 does not play a critical role in zinc transport in vitro. Furthermore, the zip1 zip2 double mutants displayed identical phenotypes to the zip1 single mutant, indicating that the contribution of Zip2 in zinc transport is minimal.

Figure 3.
ZIP1 is required for growth on zinc-limited media. Ten-fold serial dilutions of cells (starting at 104 cells) were spotted onto solid, low-Zn YNB medium (control) and low-Zn YNB medium containing different amounts of ZnCl2 as indicated. The plates were ...

To verify the involvement of Zip1 in zinc transport, the intracellular zinc concentrations in the zip1 mutant were determined by inductively coupled plasma–atomic emission spectroscopy (ICP–AES) and compared with the wild-type strain. Significantly reduced cellular zinc levels were observed in the zip1 mutant compared to the wild-type strain, suggesting the loss of zinc transport activity in these cells (Table 1). The results of this analysis also showed slightly increased intracellular iron and manganese levels, suggesting that defects in zinc transport may influence iron and manganese uptake, although further experiments are required to verify this relationship. To support our hypothesis that Zip1 is a zinc transporter in C. neoformans, we constructed a strain expressing Zip1 under the control of the galactose-inducible GAL7 promoter (PGAL7-ZIP1), cultured it in the low-zinc medium containing either glucose or galactose, and then measured intracellular levels of zinc using ICP-AES. The strain containing PGAL7-ZIP1 grown in the presence of glucose (non-induction condition) displayed zinc levels similar to the zip1 mutant. However, increased zinc accumulation was observed from the same strain grown in the presence of galactose (induction condition) (Fig. 4). These data support the conclusion that Zip1 functions as a zinc importer in C. neoformans.

Table 1.
Metal contents of the strains lacking ZIP1 or ZIP2.
Figure 4.
Expression of ZIP1 is correlated with cellular zinc levels. Cellular zinc levels were determined by ICP analysis. Values indicate weight per dry weight of the cells and are averages from three independent experiments with standard deviations (p < ...

Zip1 is a glycoprotein that localizes to the plasma membrane

Different subcellular locations are possible for fungal ZIP family zinc transporters. For example, S. cerevisiae Zrt1 and Zrt2 localize to the plasma membrane, while another ZIP family member, Zrt3, localizes to vacuoles. To ascertain the localization of Zip1 in C. neoformans, we constructed the Zip1-FLAG epitope fusion and introduced it into the zip1 mutant. This strain showed restored growth in low-zinc medium, confirming functionality of the Zip1-FLAG fusion protein (Fig. 5A). We next tested whether the Zip1-FLAG fusion protein showed zinc-dependent regulation similar to the corresponding transcripts. Overall expression patterns of the Zip1-FLAG fusion protein were identical to that of the mRNA. However, the western blot analysis revealed multiple bands, including a smear in the region greater than 135 kDa, suggesting significant post-translational modification of Zip1. We suspected glycosylation of Zip1 because the corresponding S. cerevisiae homolog Zrt1 is significantly glycosylated.31 Protein extracts were treated with peptide N-glycosidase F, which cleaves between the asparagine and the innermost N-acetylglucosamine of glycoproteins.31 After treatment, higher molecular weight smear bands were shifted to less than 135 kDa and the Zip1-FLAG fusion protein band became more intense, indicating that Zip1 is indeed N-linked glycosylated. We also noted that a prominent species of lower mobility was present at ~80 kDa, which may reflect incomplete digestion or addition modification (e.g., O-linked glycosylation) (Fig. 5B). The subcellular localization of the fusion protein was investigated by immunofluorescence using an anti-FLAG antibody, and the results showed that the Zip1-FLAG protein was mainly present at the periphery (Fig. 6). Considering that Zip1 contains the transmembrane domains, we concluded that it is likely to be localized to the plasma membrane.

Figure 5.
Zip1 is a glycoprotein. A. The Zip1-FLAG fusion protein restored growth of the zip1 mutant, confirming that the fusion protein was functional. Ten-fold serial dilutions of cells (starting at 104 cells) were spotted onto solid low-Zn YNB medium (control) ...
Figure 6.
Zip1 is localized in the cell membrane. Localization of Zip1-FLAG was investigated by immunofluorescent microscopy. DAPI was used to stain nuclei. Two independently generated strains expressing Zip1-FLAG were investigated, and the isogenic reconstituted ...

Deletion of ZIP1 caused the attenuated virulence in a mouse inhalation model of cryptococcosis

We next evaluated the virulence of the mutant strains in comparison with the wild-type and the reconstituted strains using a mouse inhalation model of cryptococcosis. Groups of 10 BALB/c mice were infected with each strain, and their survival was monitored daily. The results in Figure Figure7A7A show that the virulence of the zip1 mutant was significantly attenuated compared to that of the wild-type strain and the ZIP1-reconstituted strain, suggesting the requirement of ZIP1 for fully virulent C. neoformans. The zip1 zip2 double mutant displayed slightly increased host survival, implying that ZIP2 contributes to virulence, although no phenotype characteristic of the zip2 mutant was observed in vitro throughout the study. Interestingly, the zip1 and zip1 zip2 mutants did eventually cause disease thus indicating that additional mechanisms for zinc acquisition are present in C. neoformans. Furthermore, we noticed that the ZIP1-reconstituted strain, although clearly more virulent than the mutant, was less virulent than the wild-type strain, perhaps due to poor expression of the gene or other changes that occurred during strain construction.

Figure 7.
The zip1 mutant exhibits the attenuated virulence. A. Ten female BALB/c mice were infected intranasally with each of the strains indicated, and the survival of the mice was monitored twice per day. The results from this assay indicate that ZIP1 is required ...

To support our conclusion that ZIP1 is required for the virulence of C. neoformans, we performed a macrophage survival assay. Stimulated murine macrophage J774A.1 cells were incubated with the wild-type strain, the zip1 mutant, the zip2 mutant, or the zip1 zip2 double mutant cells for 1 hour and 24 hours, and intracellular fungal survival was determined by comparing CFUs/ml. While no difference was found at 1 hour, the zip1 mutant and the zip1 zip2 double mutant showed significantly reduced CFUs compared to the wild-type strain after 24 hour of incubation, suggesting impaired intracellular survival of the mutants (Fig. 7B). These results were consistent with the attenuated virulence of the zip1 mutant and the zip1 zip2 double mutant and confirmed the requirement for a high-affinity zinc transporter for virulence of C. neoformans.

As mentioned, C. neoformans expresses virulence factors such as melanin synthesis and capsule formation that support the growth of fungal cells within the host environment.17,32 Although ZIP1 was shown to be required for growth of C. neoformans in a mammalian host, we found that the zip1 mutant displayed no change in melanin synthesis and capsule formation compared to the wild-type strain. Similarly the zip2 mutant and the zip1 zip2 double mutant showed no alteration in melanin synthesis and capsule formation (data not shown). These results suggested that the attenuated virulence of the zip1 mutant may have been caused solely by the deficiency in high-affinity zinc transport activity, and that ZIP1 plays an important role in the virulence of C. neoformans.

Discussion

The contribution of essential metals, such as iron and copper, to the pathogenesis of C. neoformans has been documented. Transport systems for these metals have been identified and their roles in virulence have been studied. However, a role of another essential metal, zinc, in the virulence C. neoformans has not been studied, and its transporter has not been identified and characterized. We therefore sought genes in the C. neoformans genome that could be responsible for zinc acquisition in the fungus and studied their contribution in virulence. Two genes, ZIP1 and ZIP2, which encode proteins homologous to S. cerevisiae Zrt1 and Zrt28,9 and A. fumigatus ZrfA and ZrfB, were identified.16 Our data demonstrated that Zip1 plays a major role in zinc uptake as a high-affinity zinc transporter in a zinc-limited environment. Evidence supporting a role for Zip1 in high-affinity zinc transport includes the fact that deletion of ZIP1 resulted in growth defects in zinc-depleted medium, with growth restored by addition of more than 25 μM zinc to the medium, and that expression of ZIP1 was increased with the depletion of zinc and abolished by the addition of zinc into the medium. Moreover, cellular zinc levels were markedly reduced in the zip1 mutant and overexpression of Zip1 increased zinc levels in C. neoformans.

In contrast to Zip1, we failed to identify a function for Zip2 in C. neoformans. In S. cerevisiae, Zrt2, a homolog of C. neoformans Zip2, is responsible for low-affinity zinc transport, with an estimated Km of 10 μM total zinc. Moreover, expression of ZRT2 in S. cerevisiae showed good correlation with low-affinity zinc transport activity, which was abolished upon deletion of the gene.9 However, unlike Zrt2 in S. cerevisiae, we were unable to detect expression of ZIP2 at any zinc concentration tested in our study. Furthermore, no specific phenotype associated with the deletion of ZIP2 was observed. In addition to Zip1 and Zip2, our data suggested the existence of an additional zinc transport system, since the zip1 zip2 double mutant was viable. The existence of such a system in S. cerevisiae has also been suggested, since additional zinc transport activity, presumably by a divalent cation transporter, was observed from a zrt1 zrt2 double mutant with an estimated Km of 500 μM total zinc.9,33

Glycosylation of the high-affinity membrane zinc transporter in the model yeast S. cerevisiae has been reported. Western blot results suggested that the HA-tagged Zrt1 forms a multimer and is highly glycosylated, a characteristic that was confirmed by deglycosylation using PNGaseF.31 In the present study, we showed that Zip1 in C. neoformans is also glycosylated. Our in silico analysis of the amino acid sequence of Zip1 resulted in the identification of two putative glycosylation sites, Asn129 and Asn170. Glycosylation might be a common feature of key membrane zinc transporters in eukaryotic cells. In humans, Zip5 possesses three potential N-glycosylation sites, Asn49, Asn91, and Asn158.34 Human Zip8 and Zip14 are also highly glycosylated and have at least three glycosylation sites in their N-terminal regions. However, in humans, it has been suggested that deglycosylation of Zip8 does not influence zinc uptake.35 In addition, in the case of Zip14, deglycosylation influences proteasomal degradation of the protein.36 Therefore, how glycosylation influences the function and stability of Zip1 in C. neoformans needs to be determined.

A number of studies have suggested that active zinc sequestration takes place upon microbial infection of a vertebrate host.37 An example includes the results of laser ablation with inductively coupled plasma mass spectrometry that revealed no detectable zinc in the region where the bacterial pathogen Staphylococcus aureus localized within the infected liver, while sufficient zinc levels were displayed in the non-infected tissue of the organ.5 To overcome this host zinc sequestration, microbial pathogens possess systems for zinc acquisition to aid in their survival and pathogenesis. In bacterial pathogens, the ZnuABC transport systems are mainly responsible for zinc acquisition within the host, and eliminating such systems causes significantly reduced virulence for Campylobacter jejuni, Salmonella enterica, Haemophilus ducreyi, Escherichia coli, Brucella abortus, and Streptococcus pyogenes.38 Failure of zinc acquisition also hampers virulence for pathogenic fungi. For example, in A. fumigatus, a mutant lacking the genes encoding the high-affinity zinc transporters had significantly reduced virulence.7 The absence of the transcriptional regulator Csr1/Zap1, which activates the expression of genes required for zinc uptake under zinc-limited conditions, and deletion of the gene encoding the extracellular zinc scavenger Pra1 resulted in attenuated virulence in C. albicans.6,39 Our findings revealed a significant reduction in virulence for the zip1 single mutant and the zip1 zip2 double mutant using a murine model of cryptococcosis and macrophages. These results agree with the previous observations in A. fumigatus and C. albicans and suggest that zinc transport via Zip1 acquisition plays a critical role in survival and virulence of C. neoformans within the host environment.

During the preparation of our manuscript, Schneider et al. reported an analysis of ZIP family zinc transporters in C. gattii, a Cryptococcus species that mainly infects immunocompetent individuals.40 A total of four ZIP family zinc transporters, ZIP1 (CNBG_6066), ZIP2 (CNBG_2209), ZIP3 (CNBG_5361), and ZIP4 (CNBG_3633), were identified in the genome of C. gattii, and among them, ZIP1 and ZIP2 were functionally characterized. ZIP1 and ZIP2 in C. gattii are orthologs of ZIP1 and ZIP2 in C. neoformans, respectively, and the C. gattii mutant lacking ZIP1 displayed a growth defect in zinc-limited conditions, while the C. gattii zip2 mutant showed a phenotype comparable to the wild-type strain. These results are in agreement with our data for C. neoformans. However, we note that the contribution of Zip1 to the virulence of C. neoformans might be different from that of C. gattii. Specifically, the C. gattii mutant lacking ZIP1 was as virulent as the wild-type strain, while we observed attenuated virulence of the C. neoformans zip1 mutant in the current study. Considering that both the Schneider et al. study and our own study used the same murine model of cryptococcosis with identical procedures, this discrepancy implies that the role of Zip1 is different between C. gattii and C. neoformans. Because the primary infection sites are different between C. neoformans and C. gattii, the brain for the former and the lung for the latter, free zinc concentrations in the sites of infection may also contribute to differences in virulence between the two species, although such differences seem to be marginal.4143

Overall, our study showed that Zip1 is the main player for zinc transport in C. neoformans, and that zinc acquisition is important to the virulence of the fungus. Given that the zip1 and zip1 zip2 mutants retained the ability to eventually cause disease, our result suggest the need for further investigations into zinc transport and homeostasis to understand the mechanisms of pathogenesis in C. neoformans.

Acknowledgments

We thank Jeongmi Kim and Il Ye Cho for their assistance with the northern blot analysis and for phenotypic observations of the mutants.

Funding Information

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 NRF-2013R1A1A1A05007037 (WJ), and by the National Institute of Allergy and Infectious Diseases (RO1 AI053721) (JWK).

Declaration of interest

The authors report no conflicts of interset. The author alone are responsible for the content and the writing of the papaer.

Supplementary material

Supplementary material is available at Medical Mycology online (http://www.mmy.oxfordjournals.org/).

SUPPLEMENTARY MATERIAL:

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