Fungal and Parasitic Infections

Defects in Phosphate Acquisition and Storage Influence Virulence of Cryptococcus neoformans

Matthias Kretschmer, Ethan Reiner, Guanggan Hu, Nicola Tam, Debora L. Oliveira, Melissa Caza, Ju Hun Yeon, Jeongmi Kim, Christian J. Kastrup, Won Hee Jung, James W. Kronstad
G. S. Deepe Jr., Editor
DOI: 10.1128/IAI.01607-14

ABSTRACT

Nutrient acquisition and sensing are critical aspects of microbial pathogenesis. Previous transcriptional profiling indicated that the fungal pathogen Cryptococcus neoformans, which causes meningoencephalitis in immunocompromised individuals, encounters phosphate limitation during proliferation in phagocytic cells. We therefore tested the hypothesis that phosphate acquisition and polyphosphate metabolism are important for cryptococcal virulence. Deletion of the high-affinity uptake system interfered with growth on low-phosphate medium, perturbed the formation of virulence factors (capsule and melanin), reduced survival in macrophages, and attenuated virulence in a mouse model of cryptococcosis. Additionally, analysis of nutrient sensing functions for C. neoformans revealed regulatory connections between phosphate acquisition and storage and the iron regulator Cir1, cyclic AMP (cAMP)-dependent protein kinase A (PKA), and the calcium-calmodulin-activated protein phosphatase calcineurin. Deletion of the VTC4 gene encoding a polyphosphate polymerase blocked the ability of C. neoformans to produce polyphosphate. The vtc4 mutant behaved like the wild-type strain in interactions with macrophages and in the mouse infection model. However, the fungal load in the lungs was significantly increased in mice infected with vtc4 deletion mutants. In addition, the mutant was impaired in the ability to trigger blood coagulation in vitro, a trait associated with polyphosphate. Overall, this study reveals that phosphate uptake in C. neoformans is critical for virulence and that its regulation is integrated with key signaling pathways for nutrient sensing.

INTRODUCTION

Cryptococcosis is a fungal disease of humans caused by the basidiomycete yeast Cryptococcus neoformans (1). Approximately one million new cases of this disease and >600,000 fatalities are estimated to occur every year, and immunocompromised people are particularly at risk (2). The sister species Cryptococcus gattii recently emerged as a primary pathogen causing infections in otherwise healthy individuals in North America (3, 4). C. neoformans and C. gattii are acquired by inhalation of spores or desiccated yeast cells, and in the absence of immune containment, fungal cells spread systemically and cause meningoencephalitis. The most prominent virulence factors are the polysaccharide capsule, the deposition of melanin in the cell wall, and growth at host temperature (reviewed in reference 5). Alveolar macrophages in the lungs are the first line of immune defense after infection. However, C. neoformans is known to persist in the intracellular environment and resist the unfavorable conditions of the phagolysosome (e.g., acidic pH, degradative enzymes, and nutrient limitation) (6, 7). C. neoformans is also known to modulate the immune response with the production of capsule and prostaglandins (8).

We are interested in identifying the mechanisms by which fungal pathogens sense and acquire nutrients during disease. Two observations indicate a role for phosphate uptake and storage in the virulence of C. neoformans. First, candidate components of the high-affinity phosphate uptake system are upregulated during the interaction of C. neoformans with macrophages (9, 10). Second, the transcription of these components is controlled by key regulators of virulence, including the master iron regulator Cir1, the pH response regulator Rim101, and cyclic AMP (cAMP)-dependent protein kinase A (PKA) (11–13). For example, Cir1 positively regulates the expression of three genes encoding putative high-affinity phosphate transporters (PHO840 [3.9-fold {3.9×}], PHO84 [12.4×], and PHO89 [13.3×]) as well as VTC4 (2.1×), the gene for a putative polyphosphate polymerase, upon iron limitation (13). These findings focused our attention on phosphate because of its essential role in many biomolecules and biochemical processes and because the ability to sense and adapt to the environmental availability of phosphate is critical for fungal proliferation.

Phosphate acquisition, storage, and metabolism in fungi have been best studied with Saccharomyces cerevisiae and Neurospora crassa (14–17). The high-affinity phosphate uptake system in S. cerevisiae consists of Pho84, which mediates proton-coupled cotransport, and Pho89, which performs sodium-coupled transport upon phosphate limitation (18–20). The transcription factors Pho4 and Pho2 regulate the expression of the PHO genes in response to activation by the cyclin-dependent kinase Pho85, the cyclin Pho80, and the cyclin-dependent kinase inhibitor Pho81 (with similar components in N. crassa) (15, 21–26). A mitogen-activated kinase (Mak2) in N. crassa and cAMP/PKA in yeast also regulate the PHO pathway (15, 17, 27).

Inorganic phosphate (Pi) is stored as polyphosphate, a linear chain of phosphate groups with high-energy bonds similar to ATP (28, 29). In bacteria, polyphosphate is synthesized by polyphosphate kinases that reversibly transfer Pi from ATP or GTP to polyphosphate. Polyphosphate is thought to be a phosphate and energy storage molecule, a scavenger of toxic cations, and a regulator of gene expression (29). Furthermore, it has blood-coagulating and immunomodulatory activities, and it contributes to the virulence of several bacteria (e.g., Salmonella spp., Neisseria spp., and Mycobacterium spp.) and parasites (e.g., Trypanosoma spp.) (30–32). In S. cerevisiae, Vtc4 was previously identified as a key component of the polyphosphate polymerase in the vacuolar transport chaperone complex that synthesizes polyphosphate at the vacuolar membrane (33, 34). In this context, we previously showed that cAMP/PKA signaling influenced the expression of genes for phosphate uptake and storage in the fungal pathogen of maize, Ustilago maydis (35). One of the genes encoded Vtc4, and deletion of this gene reduced polyphosphate formation, influenced the transition between yeast and filamentous growth, and attenuated virulence (36).

In this study, we identified and genetically characterized candidate genes encoding functions for high-affinity phosphate uptake, polyphosphate storage and reactivation, and mitochondrial phosphate uptake in C. neoformans. We also investigated connections between phosphate acquisition and storage and nutrient sensing functions, including PKA, calcineurin, and the transcription factors Cir1 and Rim101. Furthermore, we investigated the influence of phosphate uptake and polyphosphate storage on the virulence of C. neoformans in macrophages and in a murine model of infection. These studies revealed an important role for phosphate uptake in cryptococcal virulence and indicated that polyphosphate production influences fungal colonization of pulmonary tissue.

MATERIALS AND METHODS

Mutant construction and growth conditions.C. neoformans mutants were constructed in the serotype A strain H99 (see Table S1 in the supplemental material), and strains were grown in yeast extract-peptone-dextrose (YPD) (broth or agar; pH 7.0) or yeast nitrogen base (YNB) (broth or agar; pH 5.4) with different concentrations of inorganic phosphate provided as KH2PO4 (0, 7.34, or 100 mM). Defined low-iron medium (LIM) was employed to induce capsule (37).

All genes were identified from the genome sequence of the serotype A strain H99 (Broad Institute; http://www.broadinstitute.org//scientific-community/data) with a BLASTp search for orthologs from the Saccharomyces Genome Database (http://www.yeastgenome.org/). Gene designations and similarities to the S. cerevisiae orthologs are listed in Table 1. An overlap PCR strategy was used to delete the genes for the candidate orthologs designated MIP1, MIP2, EPP1, XPP1, VTC4, PHO84, PHO840, and PHO89. All genotypes of the resulting mutants were confirmed by PCR, and genomic hybridization was additionally performed for the mutants lacking the PHO phosphate transporters (see Fig. S1 in the supplemental material). The constructs to delete the complete coding sequence of each gene were designed as described previously (38). Briefly, left arm, right arm, and marker sequences were amplified using primers designated 1 and 2, 5 and 6, and 3 and 4 for each gene, respectively. The fragments were fused by a second round of PCR and reamplified in a third PCR using nested primers 7 and 8. All primers and plasmids used to construct the fragments are listed in Tables S2 and S3 in the supplemental material. Deletion of the genes was achieved via biolistic transformation as described by Toffaletti et al. (39). Transformants were grown overnight on YPD with 1 M sorbitol and then transferred to YPD with 100 μg ml−1 of nourseothricin, 200 μg ml−1 of neomycin, or 200 μg ml−1 of hygromycin B. PCR, gel electrophoresis, restriction enzyme digestion, and genomic hybridization were performed using standard procedures (40).

View this table:
TABLE 1

Identification of C. neoformans genes and proteins for phosphate uptake and storage

To delete the genes individually, the final deletion constructs for PHO84 (3.9 kb; NEO; neomycin resistance gene in place of the open reading frame), PHO840 (3.9 kb; HYG; hygromycin resistance gene), PHO89 (3.7 kb; NAT; nourseothricin resistance gene), VTC4 (3.5 kb; NEO), MIP1 (2.9 kb; NAT), MIP2 (3.3 kb; NEO), XPP1 (3.8 kb; NAT), and EPP1 (2.8 kb; NEO) were biolistically transformed into H99. pho84Δ single mutants were retransformed with the PHO89 or PHO840 constructs to generate the pho84Δ pho89Δ or pho84Δ pho840Δ double mutants, respectively. To generate triple mutants, the pho84Δ pho89Δ mutant was retransformed with the PHO840 construct; these mutants are henceforth designated phoΔΔΔ mutants. Independent single mutants and double mutants were used to generate the double and triple mutants, respectively. For the polyphosphatase double mutants, the epp1Δ mutant was retransformed with the XPP1 construct or vice versa. For the mitochondrial phosphate uptake transporter double mutants, the MIP2 construct was retransformed into the mip1Δ mutant or vice versa. Because only three resistance markers are available for transformation of C. neoformans, the triple mutant could not be complemented; therefore, two independent mutants were employed for the analysis. Single and double mutants showed only minor phenotypic changes. Independent mutants were therefore analyzed instead of constructing complemented strains.

Quantification of gene expression under phosphate starvation.Wild-type (wt) cells were pregrown in YPD overnight, washed once in YNB without phosphate, and transferred to YNB without phosphate and with arabinose as a carbon source (2%). Arabinose was used to avoid potential problems with catabolite repression of nutrient acquisition genes caused by glucose. After 24 h of starvation, the culture was divided and transferred into YNB with arabinose and with (250 mM) or without phosphate. RNA was extracted at the 0-, 0.5-, 1-, and 5-h time points as described previously (38). DNase treatment, cDNA synthesis, and quantitative PCR (qPCR) were performed as described previously with the endogenous control genes for glyceraldehyde phosphate dehydrogenase (GAPDH) and actin (38). The 0-h time point was set as the reference expression point for the analyzed genes (MIP1, MIP2, EPP1, XPP1, VTC4, PHO84, PHO840, PHO89, a gene for acyclin-dependent kinase inhibitor [PHO81], and a gene for a putative low-affinity vacuolar Pi transporter [PHO91; CNAG_02180]). The fold change was calculated at the 0.5-, 1-, and 5-h time points between the no-phosphate and high-phosphate samples. Values from the 24-h starvation samples and the 5-h high-phosphate (250 mM) samples were used to compare the transcript levels of the high-affinity phosphate transporters relative to the transcript level of PHO84. To investigate the regulation of phosphate metabolism genes by Cir1, wt and cir1Δ cells were grown as described above and the transcript levels were determined for the EPP1, XPP1, VTC4, PHO84, PHO840, and PHO89 genes at 1 h after the transfer from starvation medium to starvation or phosphate-replete medium. The analysis was repeated three times with biological replicates.

Drug and metal resistance assays.YPD was amended with the following concentrations of agents to impose stress or metals: 5 mM ZnCl2, 1 mM CdCl2, 0.2 mM HAsNa2O4·7H2O, 3 mM NiSO4, 200 mM CsCl, 3 mM K2CrO7, 100 mM SrCl2, 0.75 mM CoCl2, 25 mM FeCl3, 3 mM SnCl2, 7.5 mM MnCl2, 5 mM PbNH3, 1 M NaCl, 7.5 mM AlCl3, 1 mM VCl3, 0.3 mM CuCl2, 100 mM LiCl, 0.5 M CaCl2, 100 μg ml−1 of cyclosporine (CsA) or 50 mM CaCl2 plus 100 μg ml−1 of cyclosporine (75 μg ml−1 in some cases for 37°C), and 1 M sorbitol. All media were adjusted to pH 5.4, and YPD was also buffered to pH 4.0 or 9.0. Additional media tested included 20% citrate buffered sheep's blood in low-iron water at pH 5.5 and YNB with 1% glycerol or sodium acetate as a carbon source at pH 5.4, as described by Kretschmer et al. (38). Cells were pregrown in YPD, washed once, counted, and adjusted to 2 × 106 cells ml−1 in YPD. Serial dilutions were performed, and 5 μl of each dilution was spotted onto the agar medium. Plates were incubated at 30°C or 37°C for 2 days unless stated otherwise. Assays were repeated three times.

Polyphosphate detection.Polyphosphate was assayed as described by Boyce et al. (36). Briefly, RNA was extracted with a citrate buffer and bead beating in a bead mill to break open the cells and release RNA and polyphosphate. Total RNA (5 μg to 10 μg) was loaded onto a native DNA polyacrylamide gel, with subsequent electrophoresis in 1× Tris-borate-EDTA (TBE) buffer. RNA and polyphosphate were fixed with acetate, stained with toluidine blue O, and destained in acetate, as described previously (33, 36). Gels were scanned, and Adobe Photoshop 7.0 (Adobe Systems Software Ireland Ltd., Dublin, Ireland) was used to visualize polyphosphate and to determine the amount of polyphosphate in relationship to a polyphosphate (10 μg) loading control (type 45; Sigma-Aldrich, St. Louis, MO) and the amount of loaded RNA. To examine the influence of cAMP for the wt, cir1Δ, and pho84Δ pho840Δ strains, cells were pregrown overnight in YPD at pH 7.0. The cultures were divided into control and cAMP samples, 1 mM 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate (CPT)-cAMP was added for 3 h and 5 h, and RNA was extracted. CsA was added to wt cells at 100 μg ml−1, followed by incubation at 30°C for 5 h and RNA extraction.

Virulence factor assays.Capsule formation was examined by differential interference microscopy (DIC) after incubation for 24 h and 48 h at 30°C in liquid low-iron medium (LIM) and staining with India ink. Dulbecco modified Eagle medium (DMEM) was also used to induce capsule formation. Melanin production was examined on l-3,4-dihydroxyphenylalanine (l-DOPA) agar containing 0.1% glucose. Cells were pregrown in YPD, washed once, counted, and adjusted to 2 × 106 cells ml−1 in YPD. Serial dilutions were performed, and 5 μl of each dilution was spotted onto agar medium. Plates were incubated at 30°C or 37°C for 2 days unless stated otherwise. To test if melanin defects could be remediated with external copper, 0.5 M copper chloride was added at the time that cells were spotted.

Macrophage uptake and survival assay.Macrophage infections were performed as described previously (10). Briefly, cells of the J774.A1 macrophage-like cell line were grown to 80% confluence in DMEM supplemented with 10% fetal bovine serum and 2 mM l-glutamine at 37°C with 5% CO2. Macrophages were stimulated 2 h prior to infection with 150 ng ml−1 of phorbol myristate acetate (PMA). Fungal cells were grown in YPD overnight, washed with phosphate-buffered saline (PBS), and opsonized in DMEM with 0.5 μg ml−1 of monoclonal antibody 18B7 for 30 min at 37°C. Stimulated macrophages were incubated with 2 × 105 opsonized fungal cells (multiplicity of infection [MOI], 1:1) for 2 h and 24 h at 37°C with 5% CO2. Macrophages containing internalized cryptococci were washed four times with PBS and then lysed with sterile water for 30 min at room temperature. Lysate dilutions were plated on YPD agar and incubated at 30°C for 48 h, at which time the resulting CFU were counted. To measure the uptake of cryptococcal cells by macrophages, the number of macrophages with internalized cryptococcal cells was determined after 2 h of coculture.

Virulence assays.The virulence of C. neoformans strains was evaluated in BALB/c mice by intranasal inoculation with 2 × 105 cells in a total volume of 50 μl of PBS. Cells were pregrown in YPD, washed three times in PBS, and counted. Mice were sacrificed at 15% weight loss or at 3 or 7 days after inoculation to examine the time course of infection. The fungal load was determined in the lungs, brain, kidneys, spleen, and liver of infected animals by plating homogenized tissue on YPD and counting CFU. All experiments with vertebrate animals were conducted in compliance with the guidelines of the Canadian Council on Animal Care and the University of British Columbia's Committee on Animal Care. The studies involving mice were approved by the University of British Columbia's Committee on Animal Care (protocol A13-0093).

Assay for blood coagulation.Cells were grown in YPD, washed once in PBS, and counted, and 2 × 107 cells were pelleted in a 0.65-ml reaction tube. Citrated blood plasma (10 μl) was placed on top of the cells, and calcium-rich saline solution (3.3 μl of water containing 40 mM CaCl2 and 90 mM NaCl) was added. Recalcified blood plasma without cells served as a negative control, and recalcified blood plasma containing fragments of glass, a known activator of clotting, served as a positive control. The time to reach complete gelation of the blood plasma was determined visually by monitoring the movement of a small air bubble, which was placed in the blood plasma. The time point at which the air bubble was fixed in the forming clot was used as the blood coagulation time for the sample. The assay was performed at least three times.

Histopathology.Lungs of infected mice were isolated, fixed, and stained with mucicarmine or hematoxylin and eosin (H&E) to investigate the progression of infection for the wt or the vtc4Δ strain at 3 and 7 days postinfection.

ICP-AES.Cellular levels of P, Na, Fe, and Zn were determined by inductively coupled plasma-atomic emission spectroscopy (ICP-AES) as described elsewhere (41). Briefly, cells were grown in YPD at pH 7.0, washed twice with water, frozen, and lyophilized. A total of 0.15 g of cell biomass was digested with 3 ml of H2O2 and 5 ml of HNO3 using a microwave digestion system (START D). ICP-AES analysis was performed using the OPTIMA 5300 DV (PerkinElmer) system. The scaling and normalization processes were based on the total cell number.

Statistical analysis.The data are representative of at least three independent experiments. Values are given as the means of triplicates ± standard deviations (SD). The virulence data were analyzed with the log rank test for statistical differences. A two-tailed unpaired Student t test was used for all other tests for statistical difference.

RESULTS

Identification of phosphate metabolism genes in C. neoformans.To investigate the relevance of phosphate uptake and polyphosphate metabolism for virulence in C. neoformans, we initially searched the genome for genes with high sequence similarity to phosphate transport, regulation, and storage functions in S. cerevisiae. We identified two orthologs of the yeast inorganic phosphate (Pi) transporter Pho84, whose genes are designated PHO84 and PHO840, and one ortholog of the yeast Pho89 transporter (Table 1; see also Table S1 in the supplemental material). The phosphate uptake system is regulated by the transcription factors Pho4, Pho2, and Spl2 in S. cerevisiae, but candidate orthologs could not be definitively identified in C. neoformans. Candidate regulatory components with similarity to the cyclin-dependent kinase Pho85 (CNAG_07871), the cyclin Pho80 (CNAG_01922), or the cyclin-dependent kinase inhibitor Pho81 (CNAG_02541) were found in C. neoformans. We also identified one gene encoding a protein with similarity to the yeast vacuolar transport chaperone Vtc4 (CNAG_01263) for polyphosphate synthesis, as well as candidate orthologs for endopolyphosphatases (EPP1) and exopolyphosphatases (XPP1) that reactivate phosphate from polyphosphate (Table 1; see also Table S1). Phosphate is also important for mitochondrial functions in S. cerevisiae, which possesses the mitochondrial Pi transporters Mir1 and Pic2. We found two putative mitochondrial Pi transporters, designated Mip1 and Mip2, in C. neoformans, which have higher similarity to Mir1 than to Pic2 (Table 1; see also Table S1). Taken together, the results show that the use of the well-characterized set of phosphate-related genes from S. cerevisiae identified a core set of similar genes in C. neoformans. Although additional phosphate functions unique to this fungus would not be detected by this approach, the core set provided a starting point to assess their contributions to cryptococcal virulence.

Expression of phosphate genes during phosphate starvation.We first examined the transcript levels of the genes for phosphate metabolism under conditions of high and low phosphate (Fig. 1A). Starvation for phosphate is predicted to induce the expression of functions for high-affinity uptake and reactivation, as well as repress components for storage. We therefore starved wt cells for phosphate (for 24 h) to maximally derepress high-affinity phosphate uptake and then transferred the cells in media with or without phosphate. Transcript levels were examined under the two phosphate conditions at 0 h, 0.5 h, 1 h, and 5 h for the genes MIP1, MIP2, EPP1, XPP1, VTC4, PHO84, PHO840, and PHO89, as well as the gene for a cyclin-dependent kinase inhibitor, PHO81, and the gene for a putative low-affinity vacuolar phosphate transporter, PHO91. Addition of phosphate resulted in lower transcript levels for PHO84 (i.e., lower by 37.8× at 0.5 h and 115.2× at 5 h), PHO89 (i.e., lower by 20.3× at 0.5 h and 38.7× at 5 h), VTC4 (i.e., lower by 4.1× at 0.5 h to 7.9× at 5 h), and PHO840 (i.e., lower by 3.1× at 0.5 h and 2.7× at 5 h) at each time point. PHO81 showed a transient reduction in transcript levels at 0.5 h (lower by 3.0×) and 3 h (reduced by 5.0×) and unchanged expression at 5 h (0.8×) compared to the initial starvation condition (0 h). The MIP1, XPP1, and PHO91 genes showed no or very minor changes in transcript levels between starvation and phosphate-replete conditions. EPP1 showed a transient induction of gene expression after addition of phosphate (i.e., 5.3× at 0.5 h versus 1.4× at 5 h), while MIP2 showed an induction of gene expression at all time points after addition of phosphate (i.e., 5.7× at 0.5 h and 3.8× at 5 h).

FIG 1
FIG 1

Phosphate starvation influences the transcript levels for genes encoding candidate phosphate uptake and storage functions. (A) The transcript levels for the indicated genes were determined by qPCR. RNA from wt cells was prepared 0 h, 0.5 h, 1 h, and 5 h after transfer from phosphate starvation conditions (for 24 h) to high- and low-phosphate media (see Materials and Methods). (B) The transcript levels of the high-affinity uptake transporters PHO840 and PHO89, relative to PHO84, were determined for cells from 24-h phosphate-starved cultures (− Pi) and in 5-h high-phosphate (250 mM; + Pi) cultures. (C) The transcript levels for phosphate-regulated genes were determined in wt and cir1Δ strains grown under no-phosphate or high-phosphate conditions at the 1-h time point. The graph shows the transcript levels for each gene in the cir1Δ mutant relative to the wild-type strain. The experimental conditions for panel A were used. Each experiment was performed in triplicate. GAPDH and actin were used as endogenous controls. The dashed lines indicate a 2-fold change in transcript levels.

The experiment shown in Fig. 1A revealed that PHO84 was the most responsive to phosphate addition, followed by PHO89 and then PHO840. To assess the potential contribution of each transporter to phosphate acquisition under no- or high-phosphate conditions, we compared the transcript levels of the PHO840 and PHO89 genes relative to PHO84. PHO84 is highly expressed under phosphate-depleted conditions, and PHO840 showed an almost identical transcript level under this condition (74.5% of PHO84), while PHO89 showed 346.7× lower expression than PHO84 (Fig. 1B). This result suggests that Pho84 and Pho840 may be the main transporters for phosphate under starvation conditions. In contrast, PHO84 is highly repressed under high-phosphate conditions, while PHO840 showed 16.1× higher expression and PHO89 showed 6.4× lower expression than PHO84 (Fig. 1B). Thus, Pho840 might also contribute to phosphate acquisition under high-phosphate conditions.

As described earlier, some genes for phosphate uptake are regulated by Cir1 upon iron limitation (13). To determine whether Cir1 is also a positive regulator of phosphate acquisition upon phosphate starvation, the expression of the EPP1, XPP1, VTC4, PHO84, PHO840, and PHO89 genes was examined for the wt and the cir1Δ mutant at the 1-h time point (Fig. 1C). The expression of the EPP1, XPP1, VTC4, PHO84, and PHO840 genes was unchanged between the mutant and wt for the low- or high-phosphate condition (Fig. 1C and data not shown). However, PHO89 showed an increase in transcript level of 7.8× in the mutant under the low-phosphate condition, with a considerably lower (0.2×) transcript level in the mutant than in the wt strain under the high-phosphate condition. Apart from PHO89, these results suggest only a minor direct regulation of the phosphate genes by Cir1 in the context of phosphate availability.

Phosphate uptake and nutrient sensing functions are important for growth in phosphate-limited media.All of the identified genes for the high-affinity transporters (PHO84, PHO840, and PHO89), polyphosphate metabolism (VTC4, EPP1, and XPP1), and the putative mitochondrial Pi transporters (MIP1 and MIP2) were deleted singly and in combinations (see Table S1 in the supplemental material), and the resulting mutants were phenotypically characterized. We initially tested the mutants for their ability to grow in phosphate-replete and limited media at 30°C or 37°C. We found that the phoΔΔΔ triple mutant, but none of the other mutants, showed reduced growth on low-phosphate medium and that growth was rescued by the addition of 100 mM KH2PO4 (Fig. 2). The phoΔΔΔ mutant showed the most severe growth defect at 30°C, while growth was slightly improved at 37°C (Fig. 2 and data not shown). This could be caused by increased membrane permeability or activation of other phosphate acquisition pathways at the elevated temperature. Growth defects upon phosphate limitation were not observed for the vtc4, epp1, xpp1, mip1, and mip2 mutants (data not shown).

FIG 2
FIG 2

Candidate high-affinity phosphate transporters and regulatory proteins influence growth at extreme pH and under low-phosphate conditions. Cells of the mutants listed on the left were spotted on the indicated media in serial dilutions starting from 104 cells, and the plates were incubated for 2 days at 30°C or 37°C.

In S. cerevisiae, Pho84 is thought to mediate phosphate uptake with a pH optimum of 4.5, while Pho89 is proposed to have an optimum of pH 9.5 (18–20). We therefore analyzed the growth of all of the C. neoformans mutants at pHs 4.0, 5.4, and 9.0. Deletion of the genes encoding the three candidate phosphate high-affinity transporters to generate the phoΔΔΔ triple mutant reduced growth on YPD medium at pHs 4.0 and 9.0, while a growth defect was not observed at pH 5.4. Incubation on YPD medium at pH 4.0, 5.4, or 9.0 at 37°C did not further exacerbate the growth defect of the phoΔΔΔ triple mutants in relation to the wt (Fig. 2 and data not shown). For all of the other mutants, no differences were seen on YPD for any tested pH or temperature (data not shown). Taken together, these experiments revealed that the loss of all three candidate transporters was needed to impair growth on low-phosphate medium and that extremes of pH also influenced the growth of the triple mutant.

As indicated in the introduction, previous transcription profiling experiments revealed that defects in PKA, the pH regulator Rim101, and the iron regulator Cir1 (under iron starvation) influenced the expression of the phosphate uptake genes PHO84, PHO840, and PHO89 (11–13) (Fig. 1C). PKA is an important sensor of nutrient availability and regulates virulence factor expression in C. neoformans (42). We therefore tested mutants defective in Cir1, Rim101, and the catalytic (Pka1) and regulatory (Pkr1) subunits of PKA for their growth upon phosphate limitation. This analysis revealed reduced growth for the pkr1Δ and cir1Δ mutants, with rescue by addition of 100 mM KH2PO4 (Fig. 2). The cir1Δ mutant generally displayed slower growth on YPD relative to the wt and also showed a rescue of the phenotype at lower pH, while growth was severely reduced at a high temperature (as previously reported [43]). In contrast, the rim101 mutant did not show reduced growth upon phosphate limitation but showed reduced growth at high pH, as described by O'Meara et al. (12). Overall, these results established a connection between the iron regulator Cir1, PKA, and phosphate acquisition in C. neoformans.

Phosphate uptake, polyphosphate metabolism, and nutrient sensing influence susceptibility to zinc and cyclosporine.We next tested the mutants for changes in susceptibility to zinc, calcium, and cyclosporine (CsA). The assay with zinc was predicated on observations in S. cerevisiae that the high-affinity phosphate uptake system also has a low affinity for bivalent metals such as arsenate, zinc, cobalt, and nickel (44–46). In addition, the storage of phosphate as polyphosphate in the vacuole and/or in acidocalcisomes is involved in detoxification of toxic metals and the storage of calcium (47). CsA is an immunomodulating and antimicrobial compound which binds cyclophilin A and inhibits calcineurin, a Ca2+/calmodulin-dependent serine/threonine protein phosphatase (48). We examined the susceptibility to CsA because of observations that calcineurin is involved in the regulation of the PHO pathway in Aspergillus fumigatus (49). In particular, a mutant defective in the ΔphoBPho80 cyclin that regulates the PHO pathway shows enhanced susceptibility to CsA. In C. neoformans, calcineurin is known to regulate growth at high temperatures and cell wall integrity (50).

Upon testing our mutants, we found that zinc susceptibility was affected by defects in the phosphate uptake system, the polyphosphate-synthesizing enzyme Vtc4, the exopolyphosphatase, and regulators of nutrient sensing. Specifically, deletion of PHO840 or the deletion of both PHO84-related transporters increased zinc susceptibility; this finding may indicate a compensatory increase in uptake mediated by other components such as Pho89. In support of this idea, deletion of all three transporters led to reduced susceptibility, indicating a transport activity for the Pho84, Pho840, and Pho89 proteins (Fig. 3). Deletion of VTC4 and loss of polyphosphate formation (see below) led to dramatic zinc susceptibility, thus suggesting a scavenging effect of polyphosphate. Also, the deletion of the gene for the putative cytoplasmic exopolyphosphatase Xpp1, but not the predicted vacuolar endopolyphosphatase Epp1, led to a slightly increased susceptibility toward zinc. Interestingly, the cir1Δ and pkr1Δ mutants also showed increased zinc susceptibility, while the pka1Δ and rim101Δ mutants did not. In general, the phenotypes were more pronounced at higher temperature, except for the xpp1Δ mutant (Fig. 3).

FIG 3
FIG 3

Defects in phosphate acquisition and storage or regulation influence growth in the presence of zinc, calcium, and cyclosporine. Strains were spotted in serial dilution on YPD plates at pH 5.4 with 5 mM Zn, 100 μg ml−1 of CsA, 0.5 M CaCl2, or 50 mM CaCl2 plus 100 μg ml−1 of CsA and incubated for 2 days at 30°C or 37°C. Note that 75 μg ml−1 of CsA was used for some plates at 37°C to reveal the increased sensitivity of the deletion strains compared to the wt. Two independent high-affinity phosphate uptake system triple mutants (phoΔΔΔ) and two independent double mutants for endo- and exopolyphosphatases (xpp1Δ epp1Δ) were examined.

Increased CsA susceptibility was observed for the pho840Δ, pho84Δ pho840Δ, phoΔΔΔ, cir1Δ, and pkr1Δ mutants, while increased resistance was seen for the pka1Δ and xpp1Δ mutants (Fig. 3). No difference in CsA susceptibility was observed upon deletion of RIM101, VTC4, or EPP1. Calcium susceptibility was unaltered for all mutants except for the phoΔΔΔ and cir1Δ mutants, which were slightly more sensitive in the presence of 0.5 M calcium. A combination of calcium and CsA gave results similar to those obtained with CsA alone. As with zinc susceptibility, the influence of CsA was more marked at the higher temperature (Fig. 3). Taken together, these results establish links between phosphate uptake and storage, nutrient sensing functions, and the susceptibility to zinc, calcium, and treatment with CsA.

Phosphate uptake and nutrient sensing functions influence susceptibility to bivalent ions.As mentioned, phosphate uptake and storage are thought to influence metal susceptibility because of low-affinity uptake by the Pho transporters and the ability of polyphosphate to scavenge positively charged ions. We tested growth on an additional set of metals (see Materials and Methods) for all of the phosphate metabolism and nutrient-sensing mutants to further examine the extent of metal susceptibility. Deletion of PHO84 and PHO840 slightly increased the resistance of C. neoformans to arsenate, nickel, and cobalt, and deletion of all three PHO genes further increased the resistance to these metals. In contrast, deletion of CIR1 or PKR1 increased susceptibility to the metals, while deletion of PKA1 increased the resistance or showed unchanged susceptibility compared to that of the wt. The phenotypes could be observed at both 30°C and 37°C and were generally more pronounced at the higher temperature (Fig. 4A). Because Pho89 in S. cerevisiae uses sodium as a cosubstrate, we also tested the susceptibility of the mutants against this metal and did not find differences relative to the wt strain. For manganese, the pkr1Δ mutant was more susceptible and the pka1Δ mutant was more resistant, thus revealing a reciprocal effect (Fig. 4B). All other mutants did not show a difference in susceptibility. We should also note that none of the mutants showed differences in the use of a variety of carbon sources or the response to osmotic (sorbitol) stress (data not shown).

FIG 4
FIG 4

Defects in phosphate acquisition or regulation influence growth in the presence of metals. Cells were spotted in serial dilutions on YPD plates at pH 5.4 with or without addition of 0.2 mM HAsNa2O4·7H2O (As), 3 mM NiSO4 (Ni), or 0.75 mM CoCl2 (Co) (A) or 1 M NaCl (Na) or 7.5 mM MnCl2 (Mn) (B). The plates were incubated for 2 days at 30°C or 37°C.

Levels of phosphate, sodium, iron, and zinc are altered in mutants defective in high-affinity phosphate uptake.The growth defects under low-phosphate conditions and the differences in metal susceptibility (Fig. 2 and 4) prompted an examination of the total phosphate, sodium, iron, and zinc ion content per cell for the wt and the following mutants: the pho840Δ, pho84Δ pho840Δ, pho84Δ pho89Δ, phoΔΔΔ, vtc4Δ, and epp1Δ xpp1Δ mutants (Table 2). Markedly reduced phosphate concentrations were found for the pho84Δ pho840Δ mutant (76.7% of wt), the pho84Δ pho89Δ mutant (78.7%), and especially the phoΔΔΔ mutant (26.4% or 4-fold-lower phosphate ions per cell than for the wt). The amount of sodium per cell was increased for the pho840Δ mutant (150.7%), the pho84Δ pho840Δ mutant (155.7%), and the phoΔΔΔ mutant (312.2%) compared to the wt. This result may reflect the upregulation of other transporters, including PHO89 in the single and double mutants, in an attempt to increase phosphate uptake that results in a concomitant increase in sodium. The total amount of iron in the pho84Δ pho840Δ (77.9%), pho84Δ pho89Δ (74.2%), and vtc4Δ (78.2%) mutants was decreased compared to that in the wt. However, the phoΔΔΔ triple mutant showed an increase in iron ions compared to the wt (129.8%). Zinc ions were reduced in the pho84Δ pho89Δ (79.6%) mutant and increased in the phoΔΔΔ triple mutant (152.8%) compared to the wt. All other mutants showed changes of less than 20% compared to the wt for the ions tested (Table 2). In general, the triple mutant showed increased amounts of all ions except for phosphate, and as mentioned above, this may reflect increased expression of other transporters to compensate for the loss of high-affinity phosphate uptake.

View this table:
TABLE 2

Analysis of ion content by inductively coupled plasma mass spectrometry for mutants lacking high-affinity phosphate uptake, polyphosphate polymerase, or polyphosphate phosphatasesa

Defects in phosphate metabolism and nutrient sensing perturb polyphosphate accumulation.Cells store inorganic phosphate as linear chains of polyphosphate when phosphate is abundant. It is known that polyphosphate in yeasts and filamentous fungi is synthesized at the vacuolar membrane by a vacuolar transport chaperone complex with Vtc4 as the polyphosphate polymerase (33, 34, 36). However, little is known about the synthesis and function of polyphosphate in fungal pathogens of humans, and we therefore examined polyphosphate accumulation in our mutants. As in S. cerevisiae and U. maydis, deletion of VTC4 in C. neoformans abolished the formation of polyphosphate (1.45% of the wt level) (Fig. 5A and B). However, mutants with defects in high-affinity phosphate uptake also had impaired polyphosphate accumulation as follows: pho840Δ (73.0%), pho84Δ pho840Δ (31.8%), pho84Δ pho89Δ (81.8%), and phoΔΔΔ (14.3%). In contrast to the phosphate uptake system and the polyphosphate polymerase, deletion of the enzymes responsible for reactivation of phosphate from polyphosphate led to increased polyphosphate in the mutants. Deletion of XPP1 alone led to only a slight increase in polyphosphate (111.7%), while deletion of EPP1 and especially the deletion of both polyphosphatases led to greater increases in polyphosphate (122.7% and 155.7%, respectively) (Fig. 5A and B). Furthermore, the mobility of the polyphosphate from the epp1Δ xpp1Δ double mutant was slightly reduced, suggesting the accumulation of chains of greater length (Fig. 5A).

FIG 5
FIG 5

Defects in phosphate uptake and storage or regulation interfere with polyphosphate formation. (A) Visualization of polyphosphate on a native polyacrylamide gel stained with toluidine blue O. Polyphosphate type 45 (10 μg) was loaded as a marker together with 5 μg of RNA from each strain (grown overnight in YPD at pH 7.0). The experiment was repeated several times, and a representative gel is shown. (B) The amount of polyphosphate from each strain was normalized to the polyphosphate loading control and the amount of RNA loaded on the gel. The results of three independent experiments are shown. An asterisk indicates values that are significantly different from the wt at a P value of <0.05. (C) The amount of polyphosphate from each mutant with a defect in signaling or regulatory functions was normalized to the polyphosphate loading control and the amount of RNA loaded on the gel. Cells were grown overnight in YPD at pH 5.4 or pH 8.0. An asterisk indicates values that are significantly different from the wt at a P value of <0.05 or between the same strain at pH 5.4 or pH 8.0. (D) Rescue of polyphosphate synthesis in the wt, pho84Δ pho840Δ, and cir1Δ strains upon incubation with 1 mM CPT-cAMP for 3 h. Cells were pregrown in YPD pH 7.0 overnight. The culture was divided and 1 mM CPT-cAMP added. An asterisk indicates a significant difference from strains without cAMP (P < 0.05). (E) Amount of polyphosphate for the wt strain in YPD pH 7.0 with or without addition of 100 μg ml−1 CsA for 5 h. An asterisk indicates a significant difference for cells cultured with or without CsA (P < 0.05).

Mutants lacking nutrient sensing and regulatory functions also interfered with polyphosphate formation. Thus, levels were reduced in mutants for Pka1 (60.3%), Pkr1 (32.5%), and Cir1 (34.9%) but not for Rim101. Interestingly, even though loss of the pH response regulator Rim101 did not have an effect, the pH of the growth medium was found to influence polyphosphate formation. Specifically, wt cells grown overnight at pH 5.4 versus pH 9.0 accumulated 73.7% more polyphosphate at the higher pH. This was also seen for all of the nutrient-sensing and regulatory mutants except the cir1Δ mutant (Fig. 5C).

Because the pho840Δ and pho84Δ pho840Δ mutants have total Pi amounts similar to that of the pho84Δ pho89Δ mutant but lower polyphosphate levels, the Pho84 proteins (and especially Pho840) may act as sensors for the phosphate state of the cell as well as transporters. A transport and receptor (transceptor) function for Pho84 has been described to occur in S. cerevisiae (17, 51). In addition, Pho84 is involved in the activation of PKA in S. cerevisiae (52). PKA is a key nutrient sensing function in C. neoformans (42), and the cAMP signaling pathway has regulatory interactions with Cir1 (43). To investigate these sensing functions further, we attempted to rescue polyphosphate formation by the cir1Δ and pho84Δ pho840Δ mutants with extracellular cAMP. Specifically, we tested an analog of cAMP, CPT-cAMP, that is membrane permeative and that has the same PKA activating properties as cAMP. wt cells showed increases in polyphosphate of 17.2% and 11.0% after 3 h and 5 h, respectively, upon treatment with 1 mM CPT-cAMP (Fig. 5D and data not shown). However, rescue of polyphosphate accumulation was not seen for the pho84Δ pho840Δ and cir1Δ mutants (Fig. 5D and data not shown). These results further support the involvement of the transcription factor Cir1 in polyphosphate metabolism as well as connections between PKA and a possible sensing function of the transporters for phosphate availability.

We also tested whether CsA treatment influenced polyphosphate accumulation because some phosphate acquisition and nutrient-sensing mutants showed differences in both polyphosphate formation and CsA susceptibility. The wt strain showed a 30.5% increase in polyphosphate after 5 h of treatment with 100 μg ml−1 of CsA at 30°C (Fig. 5E). This result further supports a connection between calcineurin and phosphate metabolism in C. neoformans.

A defect in high-affinity phosphate uptake influences capsule, melanin formation, and cell size.The major virulence factors of C. neoformans include formation of an extracellular polysaccharide capsule and the pigment melanin in the cell wall (reviewed in reference 5). In addition, the ability to grow at human body temperature and the formation of enlarged (giant/titan) cells are important for the infection process (53). In preparation for the examination of virulence in an animal model of cryptococcosis, we first tested all of our phosphate mutants for capsule and melanin formation. Only the phoΔΔΔ triple mutant showed poor formation of a capsule under inducing conditions (low iron or DMEM). Melanin formation on medium containing l-DOPA was disturbed only for the phoΔΔΔ mutant (Fig. 6B). The mutant showed reduced growth on different media including l-DOPA, but a longer incubation period to allow further growth or addition of copper did not increase melanin formation (data not shown).

FIG 6
FIG 6

A mutant lacking high-affinity phosphate uptake shows reduced capsule and melanin formation and increased cell size. (A) Cells were pregrown in YPD and transferred to LIM to induce capsule formation. Capsule was visualized after 24 h and 48 h with India ink staining. Representative pictures at 48 h are shown. The phoΔΔΔ triple mutant lacking high-affinity phosphate uptake and the pho84Δ pho89Δ double mutant showed dense granules in the cytoplasm, as indicated by arrows. (B) Melanin formation was evaluated following growth on l-DOPA medium for 48 h. Two independent phoΔΔΔ mutants were tested. (C) Comparison of cell size for the wt strain and the phoΔΔΔ mutant following growth in YPD for 12 h at 37°C. (D) The percentage of cells exhibiting an enlarged cell phenotype is shown for two independent phoΔΔΔ mutants following growth at 30°C or 37°C in YPD for 12 h and 36 h. Standard deviations are indicated. (E) Comparison of cell shape between the wt and the phoΔΔΔ mutants at 30°C or 37°C in YPD for 12 h.

Further examination of the mutant cells also revealed additional phenotypes. For example, granular particles were observed in the cytosol of the phoΔΔΔ triple mutant and in the pho84Δ pho89Δ mutant during growth in LIM (Fig. 6A, arrows). Interestingly, the size and shape of the cells for the phoΔΔΔ mutant were altered upon growth in YPD (Fig. 6C to E). This phenotype could be seen after growth for 12 h and 36 h at 30°C, where 5 to 10% of the cells were enlarged (2- to 4-fold in diameter) and had an irregular shape (Fig. 6E). The frequency was twice as high when cells were grown at 37°C (up to 23%) and was not dramatically altered at 12 h versus 36 h of growth (Fig. 6D). Irregular cell shape and increased cell size of the phoΔΔΔ triple mutant could also be seen with cells grown on solid YPD medium (data not shown).

Loss of phosphate high-affinity uptake attenuates virulence.Functions for phosphate acquisition, including components of the high-affinity uptake system, were upregulated during macrophage infection with C. neoformans (9, 10). We therefore employed our mutants to test the requirement for phosphate uptake and storage for intracellular survival and proliferation. Specifically, we tested the uptake and survival of the vtc4Δ, epp1Δ xpp1Δ, and phoΔΔΔ mutants during macrophage interaction. Upon opsonization of the strains, similar levels of initial uptake of the fungal cells by the macrophage cell line were seen after 2 h for the deletion strains and the wt (data not shown). C. neoformans is able to survive and to replicate within the phagolysosome upon uptake by macrophages. We therefore examined survival after 24 h infection of the macrophages and found that the wt reached a level of 272.1% compared to the starting cell numbers. The vtc4Δ and epp1Δ xpp1Δ mutants did not show a significant difference from the wt in terms of survival. However, the phoΔΔΔ mutants did not replicate in the macrophages and showed a survival rate of ∼67% after 24 h of interaction (Fig. 7A). The triple mutants were unable to proliferate in DMEM but did not lose viability as determined by testing for 24 h in parallel conditions without macrophages (data not shown). Together, these results indicate that the compromised growth of mutants with a defect in phosphate acquisition extends to intracellular replication and, additionally, that the mutants have reduced survival in macrophages relative to the wt strain.

FIG 7
FIG 7

Phosphate uptake is important for survival in macrophages and virulence in mice. (A) The survival of opsonized wt, vtc4Δ, epp1Δ xpp1Δ, and phoΔΔΔ strains within the macrophage-like cell line J774.A1 is shown after 24 h of interaction. The results were statistically different, as indicated by the double asterisk (P < 0.01). (B) Virulence of the wt strain and two independent phoΔΔΔ triple mutants in a mouse model of cryptococcosis. BALB/c mice were inoculated with 2 × 105 cells in 50 μl of PBS and sacrificed at 15% weight loss. The results were statistically different between the wt strain and each mutant, according to a log rank test (P < 0.001). (C) Fungal load in the lungs and brains of three mice at the time that the mice inoculated with each strain succumbed to disease. There were no statistically significant differences between the wt and the mutants.

We hypothesized that the phoΔΔΔ mutants would have reduced virulence during a murine infection assay because of the reduced survival in macrophages, the inability to grow upon phosphate limitation, and the reduced capsule and melanin production. Upon inoculation of mice, we found that the two independent mutants allowed an extended period of survival, by 12 days and 17 days, compared to the wt, but the mice all eventually succumbed to the disease (Fig. 7B). Both mutants showed similar but slightly decreased fungal loads relative to the wt strain in the lungs and brain at the time that the mice succumbed to disease (Fig. 7C). Overall, we conclude that deletion of the three phosphate transporters reduced virulence but that the mutants were still able to disseminate to the brain.

Loss of polyphosphate influences blood coagulation and proliferation in the lungs but not overall virulence.Polyphosphate is known to influence blood coagulation and to have immunomodulating activity (30–32). It is also implicated in the virulence of some bacterial pathogens. However, nothing is known about these potential contributions of polyphosphate to the virulence of fungal pathogens of humans. We found that cells of C. neoformans were able to increase the rate of blood clotting (Fig. 8A). In contrast, deletion of VTC4 led to a significant reduction in the rate of blood clotting for cells grown in liquid culture (Fig. 8A). This suggests that the reduced polyphosphate production altered the surface properties of the cells or components released from the cells. During a murine infection assay, no differences in survival were seen between the two independent vtc4Δ mutants and the wt strain (Fig. 8B). However, the fungal load in the lungs, but not in brain, kidneys, liver, or spleen, was markedly increased, by a factor of 13.5 and 29.4 for the two mutants at the endpoint of the experiment, compared to that in the wt strain (Fig. 8C). This increased fungal burden in the lungs of vtc4Δ mutant-infected mice could also be seen during a time course of infection. The vtc4Δ mutant showed 1.4 times and 5.2 times more cells in the lung than the wt strain after 3 and 7 days, respectively (Fig. 8D). Histopathology of the lung tissue at 7 days postinfection (dpi) showed no differences in inflammatory response and tissue damage for mice infected with the wt strain versus the vtc4Δ mutant (Fig. 7E). An examination of the sections also supported the conclusion from the CFU counts that the cells of the vtc4Δ mutant were more abundant than those of the wt strain in the lungs of mice (Fig. 7E).

FIG 8
FIG 8

Loss of polyphosphate influences blood coagulation in vitro and lung colonization but not virulence in mice. (A) Blood coagulation time was evaluated as described in Materials and Methods for the wt and the vtc4Δ strains. Cells were pregrown in YPD and washed once in PBS prior to the assay. The experiment was repeated at least three times. (B) A virulence assay was performed with the wt strain and two independent vtc4Δ mutants in BALB/c mice under the same conditions as the experiment whose results are shown in Fig. 7. No statistically significant differences in virulence were observed according to a log rank test. (C) Determination of fungal load at the end of the virulence assay shown in panel B. CFU were measured in lungs, brain, liver, kidneys, and spleen. A statistical significant difference (**, P < 0.01) was observed only for the lungs of mice infected with the vtc4Δ mutant compared to mice infected with the wt strain. (D) Analysis of the fungal load in lungs during a time course of infection. Three mice were employed for each strain (wt and vtc4Δ) at each time point (3 dpi and 7 dpi). No significant differences between wt and mutant were seen at 3 dpi, and the differences observed for the mice with the vtc4Δ mutant after 7 dpi were significantly different from findings with the wt (**, P < 0.01). ns, not significant. (E) Histopathology of the lungs from the experiment shown in panel D. Lungs were fixed and stained with mucicarmine. Bar equals 100 μm.

DISCUSSION

Phosphate uptake and virulence.The identification of specific nutritional requirements for fungal proliferation in mammalian hosts and an understanding of the relationship between nutrient sensing and virulence factor expression may provide opportunities for antifungal therapy. In this study, we constructed deletion mutants to test the hypothesis that high-affinity phosphate uptake and polyphosphate storage are important for the virulence of C. neoformans. We found that it was necessary to delete all three genes for candidate transporters (PHO84, PHO840, and PHO89) to obtain a mutant with poor growth upon phosphate limitation. This phoΔΔΔ triple mutant also displayed resistance to bivalent cations, reduced formation of capsule and melanin, and an increased proportion of enlarged cells. Cell enlargement may indicate a problem with cell cycle control and cell division because phosphate regulation is linked with the cell cycle in yeast (54, 55). We anticipated that phosphate uptake was important for virulence because candidate components of the phosphate high-affinity uptake system were upregulated during macrophage interaction (9, 10). As predicted, the phoΔΔΔ mutants were compromised for survival in macrophages and showed attenuated virulence in a murine model of cryptococcosis, although the fungal load in lungs and brain was comparable to that of the wt at the time of death. These virulence defects likely arise because of poor survival in macrophages, susceptibility to phosphate starvation, and attenuated expression of the key virulence factors, capsule and melanin. Overall, we conclude that phosphate uptake functions are important but not essential for virulence or for proliferation and dissemination of the pathogen. It is interesting that the uptake mutants still caused disease, and this may result from the compensatory expression of alternative uptake functions during colonization of host tissue.

We know very little about the relationship between phosphate acquisition and virulence for fungal pathogens of humans. Phosphate uptake has been best characterized in saprophytic fungi such as S. cerevisiae and N. crassa, and some components have been examined in the pathogens Candida glabrata, Candida albicans, and Aspergillus fumigatus (49, 56–58). For A. fumigatus, mutants lacking orthologs of the transporter Pho84 or the regulator Pho80 did not show reduced virulence in mice (49). For C. albicans, phosphate starvation is thought to trigger a more virulent state and to promote the transition to filament formation (58). This study also revealed that deletion of the gene for the candidate regulator, Pho4, resulted in increased induction of filamentation in response to phosphate limitation.

There is considerable evidence that the sensing and transport of phosphate are important for the virulence of several bacterial pathogens, in contrast to the paucity of information for fungi. The emerging theme in bacteria is that phosphate sensing and uptake are part of a complex network that links nutritional adaptation, virulence factor expression, and the response to stress (59). For example, expression of the Pho regulon in E. coli is controlled by the PhoR/PhoB two-component regulatory system, and the phosphate-specific transport system (Pst) is part of the regulon. Phosphate is sensed by PhoR, and upon limitation, PhoR phosphorylates PhoB to control the expression of the regulon (59–62). The Pho regulon has functions related to virulence in extraintestinal pathogenic E. coli and intestinal pathogenic E. coli. In the former, mutations in the phosphate-specific transport system led to reduced serum survival, reduced capsular antigen at the cell surface, changes in lipid A composition of the outer membrane, and an imbalance of cyclopropane and unsaturated fatty acids (59–61, 63–65). These changes contribute to virulence defects in a newborn pig infection model. Similarly, deletion of components of the Pst in Edwardsiella tarda also led to reduced replication in serum and virulence and a lower capacity to replicate in phagocytes (66, 67). Loss of the Pst results in constitutive activation of the Pho regulon probably via the PhoR/PhoB two-component system, and inactivation of the regulator also influences virulence. For example, PhoB/PhoR is important for the expression of siderophores in Corynebacterium glutamicum, while hemolysin expression is PhoB dependent in Vibrio cholerae (68, 69). Interestingly, defects in PhoR/B likely influence virulence through changes in cell surface components such as lipids and exopolysaccharides (59). In this context, it is interesting that defects in phosphate uptake functions also influenced surface properties, including elaboration of the polysaccharide capsule and deposition of melanin in the cell wall for C. neoformans.

Phosphate storage and virulence.Deletion of VTC4 in C. neoformans abolished polyphosphate formation, thus indicating functional as well as sequence similarity to VTC4 in S. cerevisiae and U. maydis (34, 36). Polyphosphate is found in the vacuole, acidocalcisomes, cytosol, mitochondria, nucleus, and cell wall in fungi (47, 70, 71). The vacuole and the acidocalcisomes are the major storage sites for polyphosphate, which is negatively charged and thus able to chelate calcium, magnesium, zinc, iron, sodium, and potassium (28). Our results indicate that zinc is probably stored as a polyphosphate/zinc aggregate in the vacuole in C. neoformans, because deletion of the Pho transporters and reduced polyphosphate formation lead to altered zinc susceptibility. As in S. cerevisiae, the endo- and exopolyphosphatases in C. neoformans influence the level of polyphosphate, most likely through a role in reactivation of inorganic phosphate from polyphosphate.

The virulence defect observed for a mutant lacking the three phosphate transporter genes prompted us to also consider the role of phosphate storage in virulence. No differences in uptake or survival in macrophages were seen for the vtc4Δ and epp1Δ xpp1Δ mutants of C. neoformans. However, the ability of the vtc4 mutant to coagulate blood was reduced compared to that of the wt, suggesting that polyphosphate might normally be exposed on the surface. An impact on coagulation could potentially influence the ability of the fungus to enter or exit the bloodstream, survive in blood and in organs, and cross the blood-brain barrier. No differences were seen for the vtc4 mutant and the wt strain in host survival or colonization of brain, kidneys, liver, and spleen in a mouse model of cryptococcosis. However, the fungal load in the lung was increased at the time that the mice were sacrificed and during an experiment investigating the time course of infection. This suggests that polyphosphate has a function during proliferation of C. neoformans in the lungs or contributes to the migration of fungal cells from the lungs into the bloodstream. Given the influence on blood coagulation, we also tested the growth of all of the mutants on blood agar and did not find any hemolytic activity for C. neoformans or any differences between the wt and mutants (data not shown). In general, the interaction of the vtc4 mutant with the host provides the impetus for additional work to better understand the contribution of polyphosphate to cryptococcal disease.

The subtle influence of the vtc4 mutation on cryptococcal virulence was unexpected because we previously found that the comparable mutation in the basidiomycete plant pathogen, Ustilago maydis, reduced both the filamentous growth that is a virulence trait and symptom formation in the corn host (36). Similarly, polyphosphate is important for the virulence of bacterial pathogens. In particular, polyphosphate is involved in survival during starvation (e.g., in low phosphate) for Campylobacter jejuni and Salmonella enterica (72, 73). The polyphosphate kinase in bacteria is also essential for biofilm formation in Pseudomonas aeruginosa, Vibrio cholerae, and Escherichia coli (74–76), while deletion of the kinase gene in Campylobacter jejuni increased biofilm formation (72). Virulence during macrophage interaction or during infection of the host was also reduced for polyphosphate metabolism mutants of P. aeruginosa, Helicobacter pylori, C. jejuni, Salmonella enterica, Mycobacterium tuberculosis, and E. coli (72–74, 76–83).

Connections between phosphate, PKA, and iron.Pho84 functions as both as a transporter and receptor (a transceptor) for phosphate in S. cerevisiae (84, 85). The transport activity is not essential for the sensor function of Pho84, and targets of the PKA pathway are known to be regulated by the sensing activity of Pho84, thus indicating an influence on PKA activity (51, 52, 86, 87). Our previous work also revealed that deletions in genes encoding components of PKA perturbed phosphate metabolism and polyphosphate formation in U maydis (35, 36). Pho84 (and particularly Pho840) may also have a sensing function and influence PKA activity in C. neoformans. In particular, we noted that the pho84Δ pho840Δ mutant produced only 31.8% of the wt level of polyphosphate, versus 81.8% for the pho84Δ pho89Δ mutant. However, the amounts of phosphate available for polyphosphate biosynthesis were similar for the pho84Δ pho840Δ (76.7% of wt) and pho84Δ pho89Δ (78.7% of wt) mutants. Also, deletion of PHO840 causes a reduction of polyphosphate to 73%, compared to 81.8% for the pho84Δ pho89Δ mutant. Pho840 may therefore be a sensor of phosphate levels. Furthermore, polyphosphate production in the wt strain, but not the pho84Δ pho840Δ mutant, was increased with addition of exogenous cAMP. This indicates that PKA, as a target of cAMP activation, is involved in sensing but also that Pho84 and/or Pho840 may contribute sensor activity. Further evidence for PKA involvement comes from differences in growth for the pka1Δ and pkr1Δ mutants in low-phosphate medium, in resistance to zinc, arsenate, nickel, and cobalt, and in the impaired formation of polyphosphate in the pka1Δ and pkr1Δ mutants. These phenotypes all resemble those of mutants with defects in phosphate metabolism.

The formation of titan/giant cells by C. neoformans is known to be regulated by PKA and Rim101 and to play an important role in virulence (53, 88). Although we did not find a connection between Rim101 and phosphate uptake, there is a relationship with PKA, and we did observe a higher percentage of enlarged cells for mutants defective in phosphate uptake in vitro. Whether these enlarged cells are related to titan/giant cells and influence virulence will require further investigation. However, these results suggest a connection between phosphate as a potentially limiting nutrient in the host and changes in cell size for C. neoformans.

We previously identified and characterized Cir1 as the master regulator of iron homeostasis in C. neoformans (43). This protein regulates a large number of functions, including nutrient uptake, cell wall and sterol biosynthesis, and signaling pathways; it is also required for elaboration of the major virulence factors, including capsule, melanin, and growth at 37°C. During iron limitation, Cir1 induces the expression of all three high-affinity transporters for phosphate and the polyphosphate kinase VTC4 (13). In contrast to iron limitation, this regulation of the high-affinity uptake system by Cir1 was not seen upon phosphate starvation, with the exception of modest regulation of PHO89 by Cir1. However, the cir1Δ mutant shared phenotypes with the phosphate mutants, including reduced growth under phosphate limitation with rescue of the phenotype in replete conditions and increased susceptibility to both divalent cations and CsA. These phenotypes were also shared by the pkr1 deletion mutant lacking the regulatory subunit of PKA, and this suggests that they resulted from activation of PKA. We also found that the cir1Δ mutant had reduced polyphosphate formation, which was not rescued with exogenous cAMP, and that total iron was reduced in the pho84Δ pho840Δ, pho84Δ pho89Δ, and vtc4Δ mutants compared to the wt strain (Table 2). Taken together, these results indicate an indirect interconnection between phosphate, PKA, and iron sensing and regulation in C. neoformans. Similar connections have previously been observed in bacterial pathogens and S. cerevisiae (46, 89). For example, the PHO regulator PhoB-PhoR and the ferric uptake regulator (Fur) sense phosphate and iron to control virulence in Edwardsiella tarda (89). Chakraborty et al. (89) showed cross talk between the phosphate and the iron regulators, as well as a physical interaction between components of the two systems (Fur and PhoU as well as Fur and EsrC). In addition, the iron response regulator Aft1 is activated in a pho80Δ mutant in S. cerevisiae (46).

Phosphate and calcineurin.Our analysis also revealed a connection between phosphate uptake and calcineurin in C. neoformans. The pho840Δ, pho84Δ pho840Δ, phoΔΔΔ, pkr1Δ, and cir1Δ mutations all led to increased CsA sensitivity, while the xpp1Δ mutation and the catalytic subunit of PKA (pka1Δ) led to resistance to CsA at 37°C. Furthermore, the production of polyphosphate by wt cells was increased by CsA treatment. These observations are consistent with the finding in S. cerevisiae that the cyclin-dependent kinase complex Pho80-Pho85 and PKA negatively regulate the transcription factor Crz1, the major target of calcineurin (90, 91). The Crz1 ortholog in C. neoformans influences growth at high temperature, the response to oxygen limitation, cell wall biosynthesis, fluconazole sensitivity, and biofilm formation in a calcium-dependent manner (92, 93). In this context, it is interesting that recent work with Candida glabrata revealed that the aspartyl protease CgYps1 (yapsin 1) influences vacuolar and ion homeostasis, cell wall remodelling, calcineurin sensitivity, and polyphosphate levels (94). When combined with our results and the connections between the PHO pathway, calcineurin, and cAMP signaling in A. fumigatus (49), these observations suggest that a conserved regulatory network exists in fungi that involves cross talk between phosphate metabolism, PKA, and calcineurin.

In summary, our results reveal that phosphate is sensed by C. neoformans to influence virulence factor expression and that its acquisition is important for virulence. There is a growing body of evidence that virulence and nutrition are coordinated in C. neoformans (7). For example, changes such as β-oxidation and central carbon metabolism have a pleiotropic influence on the elaboration of virulence factors such as capsule and melanin (10, 38). Therefore, the connection between nutrient availability and the expression of virulence factors that is well established for bacterial pathogens also applies to the fungal pathogen C. neoformans (95).

ACKNOWLEDGMENTS

This work was supported by a grant from the Canadian Institutes of Health Research (to J.W.K.) and the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT and Future Planning (grant NRF-2013R1A1A1A05007037 to W.H.J.). D.L.O. was supported by a grant from Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CsF-CNPq, Brazil). J.W.K. is a Burroughs Wellcome Fund Scholar in Molecular Pathogenic Mycology.

We thank Arturo Casadevall for monoclonal antibody 18B7 and Wax-It services for assistance with the histopathology study.

FOOTNOTES

    • Received 13 February 2014.
    • Returned for modification 3 March 2014.
    • Accepted 2 April 2014.
    • Accepted manuscript posted online 7 April 2014.

  • Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.01607-14.

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