IAI Accepts, published online ahead of print on 7 April 2014 Infect. Immun. doi:10.1128/IAI.01607-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Defects in phosphate acquisition and storage influence the virulence of Cryptococcus neoformans

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Melissa Caza1, Ju Hun Yeon3, Jeongmi Kim2, Christian J. Kastrup3, Won Hee Jung2 and

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James W. Kronstad1#

Matthias Kretschmer1, Ethan Reiner1, Guanggan Hu1, Nicola Tam1, Debora L. Oliveira1,

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Michael Smith Laboratories, Department of Microbiology and Immunology, and Faculty of

Land and Food Systems, University of British Columbia, Vancouver, B.C., Canada V6T 1Z4 2

Department of Systems Biotechnology, Chung-Ang University, Anseong-Si, Gyeonggi-Do,

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Republic of Korea

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University of British Columbia, Vancouver, BC, Canada V6T 1Z4

Michael Smith Laboratories, Department of Biochemistry and Molecular Biology,

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# To whom correspondence should be addressed:

Michael Smith Laboratories, The

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University of British Columbia, 301-2185 East Mall, Vancouver, BC, Canada, V6T 1Z4

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E-mail: [email protected]; Tel.: (+1) 604 822 4732; Fax.: (+1) 604 822 2114

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Running title: Phosphate acquisition in C. neoformans

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Key words: fungal pathogenesis, polyphosphate, PKA, calcineurin, Cir1, Vtc4

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ABSTRACT

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transcriptional profiling indicated that the fungal pathogen Cryptococcus neoformans, which

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causes meningoencephalitis in immunocompromised individuals, encounters phosphate

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limitation during proliferation in phagocytic cells. We therefore tested the hypothesis that

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phosphate acquisition and polyphosphate metabolism are important for cryptococcal

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virulence.

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phosphate medium, perturbed the formation of virulence factors (capsule and melanin),

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reduced survival in macrophages and attenuated virulence in a mouse model of

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cryptococcosis.

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revealed regulatory connections between phosphate acquisition and storage, and the iron

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regulator Cir1, cAMP-dependent protein kinase A (PKA) and the calcium-calmodulin-

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activated protein phosphatase calcineurin.

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polyphosphate polymerase blocked the ability of C. neoformans to produce polyphosphate.

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The vtc4 mutant behaved liked the wild-type strain in interactions with macrophages, and in

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the mouse infection model. However, the fungal load in the lung was significantly increased

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in mice infected with vtc4 deletion mutants. In addition, the mutant was impaired in the

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ability to trigger blood coagulation in vitro, a trait associated with polyphosphate. Overall,

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this study reveals that phosphate uptake in C. neoformans is critical for virulence and that its

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regulation is integrated with key signalling pathways for nutrient sensing.

Nutrient acquisition and sensing are critical aspects of microbial pathogenesis. Previous

Deletion of the high affinity uptake system interfered with growth on low

Additionally, analysis of nutrient sensing functions for C. neoformans

Deletion of the VTC4 gene encoding a

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INTRODUCTION

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Cryptococcus neoformans (1). Approximately one million new cases of this disease and

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>600,000 fatalities are estimated to occur every year, and immunocompromised people are

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particularly at risk (2). The sister species C. gattii recently emerged as a primary pathogen

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causing infections in otherwise healthy individuals in North America (3, 4). C. neoformans

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and C. gattii are acquired by inhalation of spores or desiccated yeast cells and, in the absence

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of immune containment, fungal cells spread systemically and cause meningoencephalitis.

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The most prominent virulence factors are the polysaccharide capsule, the deposition of

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melanin in the cell wall and growth at host temperature (reviewed in 5).

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macrophages in the lungs are the first line of immune defense after infection. However, C.

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neoformans is known to persist in the intracellular environment and resist the unfavourable

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conditions of the phagolysosome (e.g., acidic pH, degradative enzymes and nutrient

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limitation) (6, 7). C. neoformans is also known to modulate the immune response with the

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production of capsule and prostaglandins (8).

Cryptococcosis is a fungal disease of humans caused by the basidiomycete yeast

Alveolar

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We are interested in identifying the mechanisms by which fungal pathogens sense and

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acquire nutrients during disease. Two observations indicate a role for phosphate uptake and

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storage in the virulence of C. neoformans. First, candidate components of the high affinity

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phosphate uptake system are upregulated during the interaction of C. neoformans with

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macrophages (9, 10). Second, the transcription of these components is controlled by key

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regulators of virulence including the master iron regulator Cir1, the pH-response regulator

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Rim101 and the cAMP-dependent protein kinase PKA (11-13). For example, Cir1 positively

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regulates the expression of three genes encoding putative high affinity phosphate transporters

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(PHO840 (+3.9x); PHO84 (+12.4x); PHO89 (+13.3x)) as well as VTC4, (+2.1x) a putative

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polyphosphate polymerase, upon iron limitation (13). These findings focused our attention on

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phosphate because of its essential role in many biomolecules and biochemical processes, and 3

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because the ability to sense and adapt to the environmental availability of phosphate is critical

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for fungal proliferation.

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Phosphate acquisition, storage and metabolism in fungi have been best studied in

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Saccharomyces cerevisiae and Neurospora crassa (14-17).

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uptake system in S. cerevisiae consists of Pho84, which mediates proton-coupled co-transport,

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and Pho89, which performs sodium-coupled transport upon phosphate limitation (18-20).

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The transcription factors Pho4 and Pho2 regulate the expression of the PHO genes in response

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to activation by the cyclin dependent kinase Pho85, the cyclin Pho80 and the cyclin

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dependent kinase inhibitor Pho81 (with similar components in N. crassa) (15, 21-26). A

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mitogen-activated kinase (Mak2) in N. crassa, and cAMP/PKA in yeast also regulate the

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PHO pathway (15, 17, 27).

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The high affinity phosphate

Inorganic phosphate (Pi) is stored as polyphosphate, a linear chain of phosphate

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groups with high-energy bonds similar to ATP (28, 29).

In bacteria, polyphosphate is

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synthesized by polyphosphate kinases that reversibly transfer Pi from ATP or GTP to

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polyphosphate. Polyphosphate is thought to be a phosphate and energy storage molecule, a

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scavenger of toxic cations and a regulator of gene expression (29). Furthermore, it has blood

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coagulating and immune modulatory activities, and it contributes to the virulence of several

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bacteria (e.g., Salmonella ssp., Neisseria ssp. or Mycobacterium ssp.) and parasites (e.g.,

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Trypanosoma ssp.) (30-32).

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component of the polyphosphate polymerase in the vacuolar transport chaperone complex that

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synthesizes polyphosphate at the vacuolar membrane (33, 34). In this context, we previously

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showed that cAMP/PKA signalling influenced the expression of genes for phosphate uptake

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and storage in the fungal pathogen of maize, Ustilago maydis (35). One of the genes encoded

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Vtc4 and deletion of this gene reduced polyphosphate formation, influenced the transition

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between yeast and filamentous growth, and attenuated virulence (36).

In S. cerevisiae, Vtc4 was previously identified as a key

4

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In this study, we identified and genetically characterized candidate genes encoding

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functions for high affinity phosphate uptake, polyphosphate storage and reactivation, and

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mitochondrial phosphate uptake in C. neoformans. We also investigated connections between

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phosphate acquisition and storage, and nutrient sensing functions including PKA, calcineurin

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and the transcription factors Cir1 and Rim101. Furthermore, we investigated the influence of

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phosphate uptake and polyphosphate storage on the virulence of C. neoformans in

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macrophages and in a murine model of infection. These studies revealed an important role for

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phosphate uptake in cryptococcal virulence and indicated that polyphosphate production

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influences fungal colonization of pulmonary tissue.

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MATERIALS AND METHODS Mutant construction and growth conditions.

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C. neoformans mutants were constructed in the serotype A strain H99 (Table S1) and

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strains were grown in yeast peptone dextrose (YPD, broth or agar, pH 7.0) or yeast nitrogen

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base (YNB, broth or agar, pH 5.4) with different concentrations of inorganic phosphate

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provided as KH2PO4 (0, 7.34 or 100 mM). Defined low iron medium (LIM) was employed to

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induce capsule (37).

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All genes were identified from the genome sequence of the serotype A strain H99

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(Broad Institute; http://www.broadinstitute.org//scientific-community/data) with a BLASTp

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search for orthologs from the S. cerevisiae genome database (http://www.yeastgenome.org/).

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Gene designations and similarities to the S. cerevisiae orthologs are listed in Table 1. An

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overlap PCR strategy was used to delete the genes for the candidate orthologs designated

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MIP1, MIP2, EPP1, XPP1, VTC4, PHO84, PHO840 and PHO89. All genotypes of the

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resulting mutants were confirmed by PCR, and genomic hybridization was additionally

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performed for the mutants lacking the PHO phosphate transporters (Fig. S1). The constructs

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to delete the complete coding sequence of each gene were designed as described previously

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(38).

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designated 1 and 2, 5 and 6 or 3 and 4 for each gene, respectively. The fragments were fused

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by a second round of PCR and reamplified in a third PCR reaction using nested primers 7 and

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8. All primers and plasmids used to construct the fragments are listed in Tables S2 and S3.

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Deletion of the genes was achieved via biolistic transformation as described by Toffaletti et

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al. (39). Transformants were grown overnight on YPD with 1 M Sorbitol and then transferred

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to YPD with 100 μg ml-1 nourseothricin, 200 μg ml-1 neomycin or 200 μg ml-1 hygromycin B.

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PCR, gel electrophoresis, restriction enzyme digestion and genomic hybridization were

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performed using standard procedures (40).

Briefly, left arm, right arm and marker sequences were amplified using primers

6

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To delete the genes individually, the final deletion constructs for PHO84 (3.9kb,

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NEO), PHO840 (3.9kb, HYG), PHO89 (3.7kb, NAT), VTC4 (3.5kb, NEO), MIP1 (2.9kb,

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NAT), MIP2 (3.3kb, NEO), XPP1 (3.8kb, NAT) and EPP1 (2.8kb, NEO) were biolistically

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transformed into H99. Single mutants of pho84¨ were retransformed with the PHO89 or

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PHO840 constructs to generate the double mutants pho84¨ pho89¨ or pho84¨ pho840¨,

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respectively. To generate triple mutants, the pho84¨ pho89¨ mutant was retransformed with

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the PHO840 construct; these mutants are henceforth designated as pho¨¨¨. Independent

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single mutants and double mutants were used to generate the double and triple mutants,

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respectively. For the polyphosphatase double mutants, the epp1¨ mutant was retransformed

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with the XPP1 construct or vice versa. For the mitochondrial phosphate uptake transporter

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double mutants, the MIP2 construct was retransformed into the mip1¨ mutant or vice versa.

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Because only three resistance markers are available for transformation of C. neoformans, the

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triple mutant could not be complemented and therefore two independent mutants were

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employed for the analysis.

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changes. Independent mutants were therefore analyzed instead of constructing complemented

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strains.

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Quantification of gene expression under phosphate starvation.

Single and double mutants only showed minor phenotypic

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Wild-type (wt) cells were pre-grown in YPD overnight, washed once in YNB without

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phosphate, and transferred to YNB without phosphate and with arabinose as carbon source

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(2%). Arabinose was used to avoid potential problems with catabolite repression of nutrient

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acquisition genes caused by glucose. After 24h of starvation, the culture was divided and

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transferred into YNB with arabinose and with (250 mM) or without phosphate. RNA was

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extracted at the 0h, 0.5h, 1h and 5h time points as described previously (38). DNAse

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treatment, cDNA synthesis and qPCR were performed as described previously with the

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endogenous control genes for glyceraldehyde phosphate dehydrogenase (GAPDH) and actin

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(38). The 0h time point was set as the reference expression point for the analyzed genes 7

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(MIP1, MIP2, EPP1, XPP1, VTC4, PHO84, PHO840, PHO89, the cyclin dependent kinase

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inhibitor PHO81 and a putative low affinity vacuolar Pi transporter (PHO91;

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CNAG_02180)). The fold change was calculated at the 0.5h, 1h and the 5h time points

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between the no phosphate and high phosphate samples. Values from the 24h starvation

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samples and the 5h high phosphate (250 mM) samples were used to compare the transcript

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levels of the high affinity phosphate transporters relative to the transcript level of PHO84. To

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investigate the regulation of phosphate metabolism genes by Cir1, wt and cir1¨ cells were

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grown as described above and the transcript levels were determined for the EPP1, XPP1,

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VTC4, PHO84, PHO840, and PHO89 genes at 1h after the transfer from starvation medium to

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starvation or phosphate-replete medium.

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biological replicates.

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Drug and metal resistance assays.

The analysis was repeated three times with

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YPD was amended with the following concentrations of agents to impose stress or

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metals: 5 mM ZnCl2, 1 mM CdCl2, 0.2 mM HAsNa2O4*7H2O, 3 mM NiSO4, 200 mM CsCl,

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3 mM K2CrO7, 100 mM SrCl2, 0.75 mM CoCl2, 25 mM FeCl3, 3 mM SnCl2, 7.5 mM MnCl2,

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5 mM PbNH3, 1 M NaCl, 7.5 mM AlCl3, 1 mM VCl3, 0.3 mM CuCl2, 100 mM LiCl, 0.5 M

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CaCl2, 100 μg ml-1 cyclosporine A or 50 mM CaCl2 plus 100 μg ml-1 cyclosporine A (75 μg

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ml-1 in some cases for 37°C), and 1 M Sorbitol. All media were adjusted to pH 5.4 and YPD

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was also buffered to pH 4.0 or 9.0. Additional media tested included 20% citrate buffered

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sheep’s blood in low iron water at pH 5.5, and YNB with 1% glycerol or sodium acetate as

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carbon source at pH 5.4, as described by Kretschmer et al. (38). Cells were pregrown in

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YPD, washed once, counted and adjusted to 2x106 cells ml-1 in YPD. Serial dilutions were

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performed and 5 μl of each dilution were spotted onto the agar medium.

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incubated at 30°C or 37°C for two days unless stated otherwise. Assays were repeated three

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times.

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Polyphosphate detection.

Plates were

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Polyphosphate was assayed as described by Boyce et al. (36). Briefly, RNA was

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extracted with a citrate buffer and bead beating in a bead mill to break open the cells and

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release RNA and polyphosphate. Total RNA (5 μg to 10 μg) was loaded onto a native DNA

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polyacrylamide gel, with subsequent electrophoresis in 1x TBE buffer.

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polyphosphate were fixed with acetate, stained with toluidine blue O and destained in acetate,

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as described (33, 36). Gels were scanned and Adobe photoshop (Adobe photoshop 7.0;

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Adobe Systems Software Ireland Ltd., Dublin, Ireland) was used to visualize polyphosphate

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and to determine the amount of polyphosphate in relationship to a polyphosphate (10 μg)

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loading control (type 45; Sigma-Aldrich, St. Louis, MO, USA) and the amount of loaded

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RNA. To examine the influence of cAMP for the wt, cir1¨ and pho84¨ pho840¨ strains,

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cells were pregrown overnight in YPD at pH 7.0. The cultures were divided into control and

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cAMP samples, 1 mM CPT-cAMP was added for 3h and 5h, and RNA was extracted.

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Cyclospsorin A (CsA) was added to wt cells at 100 μg ml-1 followed by incubation at 30°C

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for 5h and RNA extraction.

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Virulence factor assays.

RNA and

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Capsule formation was examined by differential interference microscopy (DIC) after

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incubation for 24h and 48h at 30°C in liquid low-iron medium (LIM), and staining with India

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ink. DMEM medium was also used to induce capsule formation. Melanin production was

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examined on L-3,4-dihydroxyphenylalanine (L-DOPA) agar containing 0.1% glucose. Cells

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were pregrown in YPD, washed once, counted and adjusted to 2x106 cells ml-1 in YPD. Serial

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dilutions were performed and 5 μl of each dilution were spotted onto agar medium. Plates

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were incubated at 30°C or 37°C for two days unless stated otherwise. To test if melanin

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defects could be remediated with external copper, 0.5 M copper chloride was added at the

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time that cells were spotted.

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Macrophage uptake and survival assay.

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Macrophage infections were performed as described (10).

Briefly, cells of the

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J774.A1 macrophage-like cell line were grown to 80% confluence in DMEM supplemented

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with 10% fetal bovine serum and 2 mM L-glutamine at 37oC with 5% CO2. Macrophages

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were stimulated 2h prior to infection with 150 ng ml-1 phorbol myristate acetate (PMA).

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Fungal cells were grown in YPD overnight, washed with PBS and opsonized in DMEM with

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0.5 ȝg ml-1 of the monoclonal antibody 18B7 for 30min at 37oC. Stimulated macrophages

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were incubated with 2x105 opsonized fungal cells (MOI 1:1) for 2h and 24h at 37°C with 5%

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CO2. Macrophages containing internalized cryptococci were washed four times with PBS and

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then lysed with sterile water for 30min at room temperature. Lysate dilutions were plated on

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YPD agar and incubated at 30oC for 48h, at which time the resulting colony forming units

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(CFUs) were counted. To measure the uptake of cryptococcal cells by macrophages, the

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number of macrophages with internalized cryptococcal cells was determined after 2h of co-

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culture.

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Virulence assays.

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The virulence of C. neoformans strains was evaluated in BALB/c mice by intranasal

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inoculation with 2x105 cells in a total volume of 50 μl PBS. Cells were pregrown in YPD,

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washed three times in PBS, and counted. Mice were sacrificed at 15% weight loss or at three

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or seven days after inoculation to examine a time course of infection. The fungal load was

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determined in the lungs, brains, kidney, spleen and liver of infected animals by plating

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homogenized tissue on YPD and counting CFUs. All experiments with vertebrate animals

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were conducted in compliance with the guidelines of the Canadian Council on Animal Care

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and the University of British Columbia’s Committee on Animal Care. The studies involving

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mice were approved by the University of British Columbia’s Committee on Animal Care

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(protocol A13-0093).

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Assay for blood coagulation.

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Cells were grown in YPD, washed once in PBS, counted and 2x107 cells were pelleted

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in a 0.65 ml reaction tube. Citrated blood plasma (10 μl) was placed on top of the cells, and

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calcium-rich saline solution (3.3 μl of water containing 40 mM CaCl2 and 90 mM NaCl) was

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added. Recalcified blood plasma without cells served as a negative control and recalcified

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blood plasma containing fragments of glass, a known activator of clotting, served as a positive

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control. The time to reach complete gelation of the blood plasma was determined visually by

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monitoring the movement of a small air bubble, which was placed in the blood plasma. The

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time point at which the air bubble was fixed in the forming clot was used as the blood

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coagulation time for the sample. The assay was performed at least three times.

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Histopathology.

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Lungs of infected mice were isolated, fixed and stained with mucicarmine or

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hematoxylin and eosin (H&E) staining to investigate the progression of infection for the wt or

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the vtc4¨ strains at three and seven days post infection.

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Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES).

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Cellular levels of P, Na, Fe and Zn were determined by ICP-AES as described

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elsewhere (41). Briefly, cells were grown in YPD at pH 7.0, washed twice with water, frozen

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and lyophilized. A total of 0.15 g of cell biomass was digested with 3 ml of H2O2 and 5 ml of

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HNO3 using a microwave digestion system (START D). ICP-AES analysis was performed

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using the OPTIMA 5300 DV (PerkinElmer) system. The scaling and normalization process

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were based on the total cell number.

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Statistical Analysis.

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The data are representative of at least three independent experiments. Values are

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given as the mean of triplicates ± SD. The virulence data were analyzed with the log rank test

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for statistical differences. A two-tailed unpaired Students t-test was used for all other tests for

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statistical difference.

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RESULTS

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Identification of phosphate metabolism genes in C. neoformans.

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To investigate the relevance of phosphate uptake and polyphosphate metabolism for

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virulence in C. neoformans, we initially searched the genome for genes with high sequence

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similarity to phosphate transport, regulation and storage functions in S. cerevisiae. We

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identified two orthologs of the yeast inorganic phosphate (Pi) transporter Pho84, designated

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PHO84 and PHO840, and one ortholog of the yeast Pho89 transporter (Table 1, Table S1).

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The phosphate uptake system is regulated by the transcription factors Pho4, Pho2 and Spl2 in

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S. cerevisiae, but candidate orthologs could not be definitively identified in C. neoformans.

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Candidate regulatory components with similarity to the cyclin-dependent kinase Pho85

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(CNAG_07871), the cyclin Pho80 (CNAG_01922) or the cyclin-dependent kinase inhibitor

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Pho81 (CNAG_02541) were found in C. neoformans. We also identified one gene encoding a

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protein with similarity to the yeast vacuolar transport chaperone Vtc4 (CNAG_01263) for

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polyphosphate synthesis, as well as candidate orthologs for endo- (EPP1) and

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exopolyphosphatases (XPP1) that reactivate phosphate from polyphosphate (Table 1; Table

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S1). Phosphate is also important for mitochondrial functions in S. cerevisiae, which possesses

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the mitochondrial Pi transporters Mir1 and Pic2. We found two putative mitochondrial Pi

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transporters, designated Mip1 and Mip2, in C. neoformans, which have higher similarity to

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Mir1 than to Pic2, respectively (Table 1; Table S1). Taken together, the use of the well-

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characterized set of phosphate-related genes from S. cerevisiae identified a core set of similar

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genes in C. neoformans. Although additional phosphate functions unique to this fungus

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would not be detected by this approach, the core set provided a starting point to assess their

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contributions to cryptococcal virulence.

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Expression of phosphate genes during phosphate starvation.

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We first examined the transcript levels of the genes for phosphate metabolism under

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conditions of high and low phosphate (Fig. 1A). Starvation for phosphate is predicted to

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induce the expression of functions for high affinity uptake and reactivation, as well as repress

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components for storage. We therefore starved wt cells for phosphate (for 24h) to maximally

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de-repress high affinity phosphate uptake and then transferred the cells in media with or

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without phosphate. Transcript levels were examined under the two phosphate conditions at

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0h, 0.5h, 1h and 5h for the genes MIP1, MIP2, EPP1, XPP1, VTC4, PHO84, PHO840, and

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PHO89, as well as the cyclin dependent kinase inhibitor PHO81, and a putative low affinity

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vacuolar phosphate transporter PHO91. Addition of phosphate resulted in lower transcript

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levels for PHO84 (i.e., 37.8X at 0.5h and 115.2X at 5h), PHO89 (i.e., 20.3X at 0.5h and

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38.7X at 5h), VTC4 (i.e., 4.1X at 0.5h to 7.9X at 5h) and PHO840 (i.e., 3.1X at 0.5h and 2.7X

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at 5h) at each time point. PHO81 showed a transient reduction in transcript levels at 0.5h

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(3.0X) and 3h (5.0X), and unchanged expression at 5h (0.8X) compared to the initial

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starvation condition (0h). The MIP1, XPP1 and PHO91 genes showed no or very minor

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changes in transcript levels between starved and phosphate-replete conditions. EPP1 showed

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a transient induction of gene expression after addition of phosphate (i.e., 5.3X at 0.5h versus

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1.4X at 5h), while MIP2 showed an induction of gene expression at all time points after

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addition of phosphate (i.e., 5.7X at 0.5h and 3.8X at 5h).

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The experiment shown in Fig. 1A revealed that PHO84 was the most responsive to

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phosphate addition, followed by PHO89 and then PHO840.

To assess the potential

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contribution of each transporter to phosphate acquisition under no or high phosphate

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conditions, we compared the transcript levels of the PHO840 and PHO89 genes relative to

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PHO84. PHO84 is highly expressed under phosphate deplete conditions and PHO840 showed

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an almost identical transcript level under this condition (74.5% of PHO84), while PHO89

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showed 346.7X lower expression than PHO84 (Fig. 1B). This result suggests that Pho84 and

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Pho840 may be the main transporters for phosphate under starvation conditions. In contrast, 13

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PHO84 is highly repressed under high phosphate conditions, while PHO840 showed 16.1X

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higher expression and PHO89 showed 6.4X lower expression versus PHO84 (Fig.1B). Thus

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Pho840 might also contribute to phosphate acquisition during high phosphate conditions.

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As described earlier, some genes for phosphate uptake are regulated by Cir1 upon iron

319

limitation (13).

To determine whether Cir1 is also a positive regulator of phosphate

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acquisition upon phosphate starvation, the expression of the EPP1, XPP1, VTC4, PHO84,

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PHO840 and PHO89 genes was examined for wt and the cir1¨ mutant at the 1h time point

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(Fig. 1C). The expression of the EPP1, XPP1, VTC4, PHO84 and PHO840 genes was

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unchanged between mutant and wt for the low or high phosphate conditions (Fig. 1C; data not

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shown). However, PHO89 showed an increase in transcript level of 7.8X in the mutant under

325

the low phosphate condition, with a considerably lower (0.2X) transcript level in the mutant

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compared with the wt strain under high phosphate conditions. Apart from PHO89, these

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results suggest only a minor direct regulation of the phosphate genes by Cir1 in the context of

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phosphate availability.

329 330

Phosphate uptake and nutrient sensing functions are important for growth in

331

phosphate-limited media.

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All of the identified genes for the high affinity transporters (PHO84, PHO840,

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PHO89), polyphosphate metabolism (VTC4, EPP1, XPP1) and the putative mitochondrial Pi

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transporters (MIP1, MIP2) were deleted singly and in combinations (Table S1), and the

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resulting mutants were phenotypically characterized. We initially tested the mutants for their

336

ability to grow in phosphate-replete and limited media at 30°C or 37°C. We found that the

337

pho¨¨¨ triple mutant, but none of the other mutants, showed reduced growth on low

338

phosphate medium, and that growth was rescued by the addition of 100 mM KH2PO4 (Fig. 2).

339

The pho¨¨¨ mutant showed the most severe growth defect at 30°C, while growth was

340

slightly improved at 37°C (Fig. 2 and data not shown). This could be caused by increased 14

341

membrane permeability or activation of other phosphate acquisition pathways at the elevated

342

temperature. Growth defects upon phosphate limitation were not observed for the vtc4, epp1,

343

xpp1, mip1 and mip2 mutants (data not shown).

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In S. cerevisiae, Pho84 is thought to mediate phosphate uptake with a pH optimum of

345

4.5, while Pho89 is proposed to have an optimum of pH 9.5 (18-20). We therefore analyzed

346

the growth of all of the C. neoformans mutants at pH 4.0, 5.4 and 9.0. Deletion of the genes

347

encoding the three candidate phosphate high affinity transporters to generate the pho¨¨¨

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triple mutant reduced growth on YPD medium at pH 4.0 and 9.0, while a growth defect was

349

not observed at pH 5.4. Incubation on YPD medium at pH 4.0, 5.4 or 9.0 at 37°C did not

350

further exacerbate the growth defect of the pho¨¨¨ triple mutants in relation to the wt (Fig. 2

351

and data not shown). For all of the other mutants, no differences were seen on YPD for any

352

tested pH or temperature (data not shown). Taken together, these experiments revealed that

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the loss of all three candidate transporters was needed to impair growth on low phosphate

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medium, and that extremes of pH also influenced the growth of the triple mutant.

355

As indicated in the Introduction, previous transcription profiling experiments revealed

356

that defects in PKA, the pH regulator Rim101 and in the iron regulator Cir1 (under iron

357

starvation) influenced the expression of the phosphate uptake genes PHO84, PHO840, and

358

PHO89 (11-13; Fig. 1C). PKA is an important sensor of nutrient availability and regulates

359

virulence factor expression in C. neoformans (42). We therefore tested mutants defective in

360

Cir1, Rim101 and in the catalytic (Pka1) and regulatory (Pkr1) subunits of PKA, for their

361

growth upon phosphate limitation. This analysis revealed reduced growth for the pkr1¨ and

362

the cir1¨ mutants, with rescue by addition of 100 mM KH2PO4 (Fig. 2). The cir1¨ mutant

363

generally displayed slower growth on YPD relative to wt, and also showed a rescue of the

364

phenotype at lower pH, while growth was severely reduced at high temperature (as previously

365

reported 43). In contrast, the rim101 mutant did not show reduced growth upon phosphate

366

limitation, but showed reduced growth at high pH, as described by O’Meara et al. (12). 15

367

Overall, these results established a connection between the iron regulator Cir1, PKA, and

368

phosphate acquisition in C. neoformans.

369 370

Phosphate uptake, polyphosphate metabolism and nutrient sensing influence

371

susceptibility to zinc and cyclosporine A.

372

We next tested the mutants for changes in susceptibility to zinc, calcium and

373

cyclosporine A (CsA). The assay with zinc was predicated on observations in S. cerevisiae

374

that the high affinity phosphate uptake system also has a low affinity for bivalent metals such

375

as arsenate, zinc, cobalt and nickel (44-46).

376

polyphosphate in the vacuole and/or in acidocalcisomes is involved in detoxification of toxic

377

metals and the storage of calcium (47). CsA is an immune modulating and antimicrobial

378

compound which binds cyclophilin A and inhibits calcineurin, a Ca2+/calmodulin-dependent

379

serine/threonine protein phosphatase (48). We examined the susceptibility to CsA because of

380

observations that calcineurin is involved in the regulation of the PHO pathway in Aspergillus

381

fumigatus (49). In particular, a mutant defective in the ΔphoBPho80 cyclin that regulates the

382

PHO pathway shows enhanced susceptibility to CsA. In C. neoformans, calcineurin is known

383

to regulate growth at high temperature and cell wall integrity (50).

In addition, the storage of phosphate as

384

Upon testing our mutants, we found that zinc susceptibility was affected by defects in

385

the phosphate uptake system, the polyphosphate-synthesizing enzyme Vtc4, the

386

exopolyphosphatase and regulators of nutrient sensing. Specifically, deletion of PHO840 or

387

the deletion of both PHO84-related transporters increased zinc susceptibility; this finding may

388

indicate a compensatory increase in uptake mediated by other components such as Pho89. In

389

support of this idea, deletion of all three transporters led to reduced susceptibility indicating a

390

transport activity for the Pho84, Pho840 and Pho89 proteins (Fig. 3). Deletion of VTC4 and

391

loss of polyphosphate formation (see below) led to a dramatic zinc susceptibility, thus

392

suggesting a scavenging effect of polyphosphate. Also the deletion of the gene for the 16

393

putative

cytoplasmic

exopolyphosphatase

Xpp1,

but

not

the

predicted

vacuolar

394

endopolyphosphatase Epp1, led to a slightly increased susceptibility towards zinc.

395

Interestingly, the cir1¨ and the pkr1¨ mutants also showed increased zinc susceptibility,

396

while the pka1¨ and rim101¨ mutants did not.

397

pronounced at higher temperature, except for the xpp1¨ mutant (Fig. 3).

In general, the phenotypes were more

398

Increased CsA susceptibility was observed for the pho840¨, pho84¨ pho840¨,

399

pho¨¨¨, cir1¨ and pkr1¨ mutants, while increased resistance was seen for the pka1¨ and

400

xpp1¨ mutants (Fig 3). No difference in CsA susceptibility was observed upon deletion of

401

RIM101, VTC4 or EPP1. Calcium susceptibility was unaltered for all mutants, except for the

402

pho¨¨¨ and cir1¨ mutants, which were slightly more sensitive in the presence of 0.5 M

403

calcium. A combination of calcium and CsA gave similar results to CsA alone. As with zinc

404

susceptibility, the influence of CsA was more marked at the higher temperature (Fig. 3).

405

Taken together, these results establish links between phosphate uptake and storage, nutrient

406

sensing functions, and the susceptibility to zinc, calcium and treatment with CsA.

407 408

Phosphate uptake and nutrient sensing functions influence susceptibility to bivalent

409

ions.

410

As mentioned, phosphate uptake and storage are thought to influence metal

411

susceptibility because of low affinity uptake by the Pho transporters and the ability of

412

polyphosphate to scavenge positively charged ions. We tested growth on an additional set of

413

metals (Material and Methods) for all of the phosphate metabolism- and nutrient-sensing

414

mutants to further examine the extent of metal susceptibility.

415

PHO840 slightly increased the resistance of C. neoformans to arsenate, nickel and cobalt, and

416

deletion of all three PHO genes further increased the resistance to these metals. In contrast,

417

deletion of CIR1 or PKR1 increased susceptibility to the metals, while deletion of PKA1

418

increased the resistance or showed unchanged susceptibility compared to wt. The phenotypes

Deletion of PHO84 and

17

419

could be observed at both 30°C and 37°C, and were generally more pronounced at the higher

420

temperature (Fig. 4A). Because Pho89 in S. cerevisiae uses sodium as co-substrate, we also

421

tested the susceptibility of the mutants against this metal and did not find differences relative

422

to the wt strain. For manganese, the pkr1¨ mutant was more susceptible and the pka1¨

423

mutant was more resistant thus revealing a reciprocal effect (Fig. 4B). All other mutants did

424

not show a difference in susceptibility. We should also note that none of the mutants showed

425

differences in the use of a variety of carbon sources or the response to osmotic (sorbitol)

426

stress (data not shown).

427 428

Levels of phosphate, sodium, iron and zinc are altered in mutants defective in high

429

affinity phosphate uptake.

430

The growth defects under low phosphate conditions and the differences in metal

431

susceptibility (Fig. 2 and 4), prompted an examination of the total phosphate, sodium, iron

432

and zinc ion content per cell for the wt and the following mutants: pho840¨, pho84¨

433

pho840¨, pho84¨ pho89¨, pho¨¨¨, vtc4¨ and epp1¨ xpp1¨ (Table 2). Markedly reduced

434

phosphate concentrations were found for the pho84¨ pho840¨ mutant (76.7% of wt), the

435

pho84¨ pho89¨ mutant (78.7%) and especially the pho¨¨¨ mutant (26.4% or 4-fold lower

436

phosphate ions per cell compared to wt). The amount of sodium per cell was increased for the

437

pho840¨ mutant (+150.7%), the pho84¨ pho840¨ mutant (+155.7%) and the pho¨¨¨ mutant

438

(+312.2%) compared to wt. This result may reflect the upregulation of other transporters,

439

including PHO89 in the single and double mutants, in an attempt to increase phosphate

440

uptake that results in a concommitant increase in sodium. The total amount of iron in the

441

pho84¨ pho840¨ (77.9%), pho84¨ pho89¨ (74.2%) and vtc4¨ (78.2%) mutants was

442

decreased compared to wt. However, the pho¨¨¨ triple mutant showed an increase in iron

443

ions compared to wt (129.8%). Zinc ions were reduced in the pho84¨ pho89¨ (79.6%)

444

mutant and increased in the pho¨¨¨ triple mutant (152.8%) compared to wt. All other 18

445

mutants showed changes of less than 20% compared to wt for the ions tested (Table 2). In

446

general, the triple mutant showed increased amounts of all ions except for phosphate and, as

447

mentioned, this may reflect increased expression of other transporters to compensate for the

448

loss of high affinity phosphate uptake.

449 450

Defects in phosphate metabolism and nutrient sensing perturb polyphosphate

451

accumulation.

452

Cells store inorganic phosphate as linear chains of polyphosphate when phosphate is

453

abundant. It is known that polyphosphate in yeasts and filamentous fungi is synthesized at the

454

vacuolar membrane by a vacuolar transport chaperone complex with Vtc4 as the

455

polyphosphate polymerase (33, 34, 36). However, little is known about the synthesis and

456

function of polyphosphate in fungal pathogens of humans and we therefore examined

457

polyphosphate accumulation in our mutants. As in S. cerevisiae and U. maydis, deletion of

458

VTC4 in C. neoformans abolished the formation of polyphosphate (1.45% of the wt level)

459

(Fig. 5A and B). However, mutants with defects in high affinity phosphate uptake also had

460

impaired polyphosphate accumulation as follows: pho840¨ (73.0%), pho84¨ pho840¨

461

(31.8%), pho84¨ pho89¨ (81.8%) and pho¨¨¨ (14.3%). In contrast to the phosphate uptake

462

system and the polyphosphate polymerase, deletion of the enzymes responsible for

463

reactivation of phosphate from polyphosphate led to increased polyphosphate in the mutants.

464

Deletion of XPP1 alone led only to a slight increase in polyphosphate (111.7%), while

465

deletion of EPP1 and especially the deletion of both polyphosphatases led to a greater

466

increase in polyphosphate (122.7% and 155.7%, respectively) (Fig. 5A and B). Furthermore,

467

the mobility of the polyphosphate from the epp1Δ xpp1Δ double mutant was slightly reduced,

468

suggesting the accumulation of chains of greater length (Fig. 5A).

469

Mutants lacking nutrient sensing and regulatory functions also interfered with

470

polyphosphate formation. Thus, levels were reduced in mutants for Pka1 (60.3%), Pkr1 19

471

(32.5%) and Cir1 (34.9%), but not for Rim101. Interestingly, even though loss of the pH

472

response regulator Rim101 did not have an effect, the pH of the growth medium was found to

473

influence polyphosphate formation. Specifically, wt cells grown overnight at pH 5.4 versus

474

pH 9.0 accumulated 73.7% more polyphosphate at the higher pH. This was also seen for all

475

of the nutrient-sensing and regulatory mutants except cir1¨ (Fig. 5C).

476

Because the pho840¨ and pho84¨ pho840¨ mutants have similar total Pi amounts

477

compared to the pho84¨ pho89¨ mutant, but lower polyphosphate levels, the Pho84 proteins

478

(and especially Pho840) may act as sensors for the phosphate state of the cell as well as

479

transporters. A transport and receptor (transceptor) function for Pho84 has been described in

480

S. cerevisiae (17, 51). In addition, Pho84 is involved in the activation of PKA in S. cerevisiae

481

(52). PKA is a key nutrient sensing function in C. neoformans (42) and the cAMP signalling

482

pathway has regulatory interactions with Cir1 (43). To investigate these sensing functions

483

further, we attempted to rescue polyphosphate formation by the cir1¨ and the pho84¨

484

pho840¨ mutants with extracellular cAMP. Specifically, we tested an analogue of cAMP,

485

CPTcAMP, that is membrane permeable and that has the same PKA activating properties as

486

cAMP. Wt cells showed an increase in polyphosphate of 17.2% and 11.0% after 3h and 5h,

487

respectively, upon treatment with 1 mM CPTcAMP (Fig. 5D; data not shown). However,

488

rescue of polyphosphate accumulation was not seen for the pho84¨ pho840¨ and the cir1¨

489

mutants (Fig. 5D and data not shown). These results further support the involvement of the

490

transcription factor Cir1 in polyphosphate metabolism as well as connections between PKA

491

and a possible sensing function of the transporters for phosphate availability.

492

We also tested whether CsA treatment influenced polyphosphate accumulation

493

because some phosphate acquisition and nutrient-sensing mutants showed differences in both

494

polyphosphate formation and CsA susceptibility. The wt strain showed a 30.5% increase in

495

polyphosphate after 5h of treatment with 100 μg ml-1 CsA at 30°C (Fig. 5E). This result

20

496

further supports a connection between calcineurin and phosphate metabolism in C.

497

neoformans.

498 499

A defect in high affinity phosphate uptake influences capsule, melanin formation and

500

cell size.

501

The major virulence factors of C. neoformans include formation of an extracellular

502

polysaccharide capsule and the pigment melanin in the cell wall (reviewed in 5). In addition,

503

the ability to grow at human body temperature and the formation of enlarged (giant/titan) cells

504

are important for the infection process (53). In preparation for the examination of virulence in

505

an animal model of cryptococcosis, we first tested all of our phosphate mutants for capsule

506

and melanin formation. Only the pho¨¨¨ triple mutant showed poor formation of a capsule

507

in inducing conditions (low iron or DMEM media).

508

containing L-DOPA was only disturbed for the pho¨¨¨ mutant (Fig. 6B). The mutant

509

showed reduced growth on different media including L-DOPA, but a longer incubation period

510

to allow further growth or addition of copper did not increase melanin formation (data not

511

shown).

Melanin formation on medium

512

Further examination of the mutant cells also revealed additional phenotypes. For

513

example, granular particles were observed in the cytosol of the pho¨¨¨ triple mutant and in

514

the pho84¨ pho89¨ mutant during growth in LIM (Fig. 6A, indicated by arrows).

515

Interestingly, the size and shape of the cells for the pho¨¨¨ mutant were altered upon growth

516

in YPD (Fig. 6C, D, E). This phenotype could be seen after growth for 12h and 36h at 30°C

517

where 5-10% of the cells were enlarged (2-4 fold in diameter) and had an irregular shape (Fig.

518

6E). The frequency was twice as high when cells were grown at 37°C (up to 23%), and was

519

not dramatically altered at 12h vs. 36h of growth (Fig. 6D). Irregular cell shape and increased

520

cell size of the pho¨¨¨ triple mutant could also be seen with cells grown on solid YPD

521

medium (data not shown).

21

522 523

Loss of phosphate high affinity uptake attenuates virulence.

524

Functions for phosphate acquisition, including components of the high affinity uptake system,

525

were upregulated during macrophage infection with C. neoformans (9, 10). We therefore

526

employed our mutants to test the requirement for phosphate uptake and storage for

527

intracellular survival and proliferation. Specifically, we tested the uptake and survival of the

528

vtc4¨, epp1¨ xpp1¨ and pho¨¨¨ mutants during macrophage interaction. Upon opsonisation

529

of the strains, similar levels of initial uptake of the fungal cells by the macrophage cell line

530

were seen after 2h for the deletion strains compared to wt (data not shown). C. neoformans is

531

able to survive and to replicate within the phagolysosome upon uptake by macrophages. We

532

therefore examined survival after 24h infection of the macrophages and found that the wt

533

reached a level of 272.1% compared to the starting cell numbers. The vtc4¨ and epp1¨

534

xpp1¨ mutants did not show a significant difference compared to the wt in terms of survival.

535

However, the pho¨¨¨ mutants did not replicate in the macrophages and showed a survival

536

rate of ~67% after 24h of interaction (Fig. 7A). The triple mutants were unable to proliferate

537

in DMEM but did not lose viability when tested for 24h in parallel conditions without

538

macrophages (data not shown). Together, these results indicate that the compromised growth

539

of mutants with a defect in phosphate acquisition extends to intracellular replication and,

540

additionally, that the mutants have reduced survival in macrophages relative to the wt strain.

541

We hypothesized that the pho¨¨¨ mutants would have reduced virulence during a

542

murine infection assay because of the reduced survival in macrophages, the inability to grow

543

upon phosphate limitation, and the reduced capsule and melanin production.

544

inoculation of mice, we found that the two independent mutants allowed an extended period

545

of survival by 12d and 17d, compared to wt, but the mice all eventually succumbed to the

546

disease (Fig. 7B). Both mutants showed similar but slightly decreased fungal loads relative to

547

the wt strain in the lung and brain at the time that the mice succumbed to disease (Fig. 7C).

Upon

22

548

Overall, we conclude that deletion of the three phosphate transporters reduced virulence, but

549

that the mutants were still able to disseminate to the brain.

550 551

Loss of polyphosphate influences blood coagulation and proliferation in the lung, but

552

not overall virulence.

553 554

Polyphosphate is known to influence blood coagulation and to have immune

555

modulating activity (30-32).

It is also implicated in the virulence of some bacterial

556

pathogens. However, nothing is known about these potential contributions of polyphosphate

557

to the virulence of fungal pathogens of humans. We found that cells of C. neoformans were

558

able to increase the rate of blood clotting (Fig. 8A). In contrast, deletion of VTC4 led to a

559

significant reduction in the rate of blood clotting for cells grown in liquid culture (Fig. 8A).

560

This suggests that the reduced polyphosphate production altered the surface properties of the

561

cells or components released from the cells. During a murine infection assay, no differences

562

in survival were seen between the two independent vtc4¨ mutants and the wt strain (Fig. 8B).

563

However, the fungal load in the lung, but not in brain, kidney, liver or spleen, was markedly

564

increased by a factor of 13.5 and 29.4 fold for the two mutants at the endpoint of the

565

experiment, compared to the wt strain (Fig. 8C). This increased fungal burden in the lung of

566

vtc4¨-infected mice could also be seen during a time course of infection. The vtc4¨ mutant

567

showed 1.4 times and 5.2 times more cells in the lung than the wt strain after 3 and 7 days,

568

respectively (Fig. 8D). Histopathology of the lung tissue at 7dpi showed no differences in

569

inflammatory response and tissue damage for mice infected with the wt strain versus the

570

vtc4¨ mutant (Fig. 7E). An examination of the sections also supported the conclusion from

571

the CFU counts that the cells of the vtc4¨ mutant were more abundant than those of the wt

572

strain in the lungs of mice (Fig. 7E).

573

23

574 575

DISCUSSION

576

Phosphate uptake and virulence.

577

The identification of specific nutritional requirements for fungal proliferation in

578

mammalian hosts and an understanding of the relationship between nutrient sensing and

579

virulence factor expression may provide opportunities for antifungal therapy. In this study,

580

we constructed deletion mutants to test the hypothesis that high affinity phosphate uptake and

581

polyphosphate storage are important for the virulence of C. neoformans. We found that it was

582

necessary to delete all three genes for candidate transporters (PHO84, PHO840 and PHO89)

583

to obtain a mutant with poor growth upon phosphate limitation. This pho¨¨¨ triple mutant

584

also displayed resistance to bivalent cations, reduced formation of capsule and melanin, and

585

an increased proportion of enlarged cells. Cell enlargement may indicate a problem with cell

586

cycle control and cell division because phosphate regulation is linked with the cell cycle in

587

yeast (54, 55). We anticipated that phosphate uptake was important for virulence because

588

candidate components of the phosphate high affinity uptake system were upregulated during

589

macrophage interaction (9, 10). As predicted, the pho¨¨¨ mutants were compromised for

590

survival in macrophages and showed attenuated virulence in a murine model of

591

cryptococcosis, although the fungal load in lung and brain was comparable to wt at time of

592

decease.

593

susceptibility to phosphate starvation, and attenuated expression of the key virulence factors,

594

capsule and melanin. Overall, we conclude that phosphate uptake functions are important but

595

not essential for virulence or for proliferation/dissemination of the pathogen. It is interesting

596

that the uptake mutants still caused diseased and this may result from the compensatory

597

expression of alternative uptake functions during colonization of host tissue.

These virulence defects likely arise because of poor survival in macrophages,

598

We know very little about the relationship between phosphate acquisition and

599

virulence for fungal pathogens of humans. Phosphate uptake has been best characterized in

24

600

saprophytic fungi such as S. cerevisiae and N. crassa, and some components have been

601

examined in the pathogens C. glabrata, C. albicans, and Aspergillus fumigatus (49, 56-58).

602

In A. fumigatus, mutants lacking orthologs of the transporter Pho84 or the regulator Pho80 did

603

not show reduced virulence in mice (49). For C. albicans, phosphate starvation is thought to

604

trigger a more virulent state and to promote the transition to filament formation (58). This

605

study also revealed that deletion of the gene for the candidate regulator, Pho4, resulted in

606

increased induction of filamentation in response to phosphate limitation.

607

There is considerable evidence that the sensing and transport of phosphate is important

608

for the virulence of several bacterial pathogens, in contrast to the paucity of information for

609

fungi. The emerging theme in bacteria is that phosphate sensing and uptake are part of a

610

complex network that links nutritional adaptation, virulence factor expression and the

611

response to stress (59). For example, expression of the Pho regulon in E. coli is controlled by

612

the PhoR/PhoB two-component regulatory system and the phosphate specific transport system

613

(Pst) is part of the regulon.

614

phosphorylates PhoB to control the expression of the regulon (59-62). The Pho regulon has

615

functions related to virulence in extra-intestinal pathogenic E. coli and intestinal pathogenic

616

E. coli. In the former, mutations in the phosphate specific transport system led to reduced

617

serum survival, reduced capsular antigen at the cell surface, changes in lipid A composition of

618

the outer membrane and an imbalance of cyclopropane and unsaturated fatty acids (59-61, 63-

619

65). These changes contribute to virulence defects in a newborn pig infection model.

620

Similarly, deletion of components of the Pst in Edwardsiella tarda also led to reduced

621

replication in serum and virulence, and a lower capacity to replicate in phagocytes (66, 67).

622

Loss of the Pst results in constitutive activation of the Pho regulon probably via the

623

PhoR/PhoB two component system, and inactivation of the regulator also influences

624

virulence.

625

Corynebacterium glutamicum, while hemolysin expression is PhoB dependent in Vibrio

Phosphate is sensed by PhoR and, upon limitation, PhoR

For example, PhoB/PhoR is important for the expression of siderophores in

25

626

cholerae (68, 69).

Interestingly, defects in PhoR/B likely influence virulence through

627

changes in cell surface components such as lipids and exopolysaccharides (59). In this

628

context, it is interesting that defects in phosphate uptake functions also influenced surface

629

properties including elaboration of the polysaccharide capsule and deposition of melanin in

630

the cell wall for C. neoformans.

631

Phosphate storage and virulence.

632

Deletion of VTC4 in C. neoformans abolished polyphosphate formation thus indicating

633

functional as well as sequence similarity to VTC4 in S. cerevisiae and U. maydis (34, 36).

634

Polyphosphate is found in the vacuole, acidocalciomes, cytosol, mitochondria, nucleus and

635

cell wall in fungi (47, 70, 71). The vacuole and the acidocalciomes are the major storage sites

636

for polyphosphate, which is negatively charged and thus able to chelate calcium, magnesium,

637

zinc, iron, sodium and potassium (28). Our results indicate that zinc is probably stored as a

638

polyphosphate/zinc aggregate in the vacuole in C. neoformans because deletion of the Pho

639

transporters and reduced polyphosphate formation lead to altered zinc susceptibility. As in S.

640

cerevisiae, the endo- and exopolyphosphatases in C. neoformans influence the level of

641

polyphosphate, most likely through a role in reactivation of inorganic phosphate from

642

polyphosphate.

643

The virulence defect observed for a mutant lacking the three phosphate transporter

644

genes prompted us to also consider the role of phosphate storage in virulence. No differences

645

in uptake or survival in macrophages were seen for the vtc4¨ and epp1¨ xpp1¨ mutants of C.

646

neoformans.

647

compared to wt suggesting that polyphosphate might normally be exposed on the surface. An

648

impact on coagulation could potentially influence the ability of the fungus to enter or exit the

649

blood stream, survive in blood and in organs, and cross the blood brain barrier.

650

differences were seen for the vtc4 mutant and the wt strain in host survival or colonisation of

651

brain, kidney, liver and spleen in a mouse model of cryptococcosis. However, the fungal load

However, the ability of the vtc4 mutant to coagulate blood was reduced

No

26

652

in the lung was increased at the time that the mice were sacrificed and during a time course of

653

infection experiment. This suggests that polyphosphate has a function during proliferation of

654

C. neoformans in the lung or contributes to the migration of fungal cells from the lung into the

655

blood stream. Given the influence on blood coagulation, we also tested the growth of all of

656

the mutants on blood agar and did not find any haemolytic activity for C. neoformans or any

657

differences between wt and mutants (data not shown). In general, the interaction of the vtc4

658

mutant with the host provides the impetus for additional work to better understand the

659

contribution of polyphosphate to cryptococcal disease.

660

The subtle influence of the vtc4 mutation on cryptococcal virulence was unexpected

661

because we previously found that the comparable mutation in the basidiomycete plant

662

pathogen, Ustilago maydis, reduced both the filamentous growth that is a virulence trait as

663

well as symptom formation in the corn host (36). Similarly, polyphosphate is important for

664

the virulence of bacterial pathogens. In particular, polyphosphate is involved in survival

665

during starvation (e.g., in low phosphate) for Campylobacter jejuni and Salmonella enterica

666

(72, 73). The polyphosphate kinase in bacteria is also essential for biofilm formation in

667

Pseudomonas aeruginosa, Vibrio cholerae and E. coli (74-76), while deletion of the kinase

668

gene in Campylobacter jejuni increased biofilm formation (72).

669

macrophage interaction or during infection of the host was also reduced for polyphosphate

670

metabolism mutants in P. aeruginosa, Helicobacter pylori, C. jejuni, Salmonella enterica,

671

Mycobacterium tuberculosis and E. coli (72-74, 76-83).

672

Connections between phosphate, PKA and iron.

Virulence during

673

Pho84 functions as both as a transporter and receptor (a transceptor) for phosphate in

674

S. cerevisiae (84, 85). The transport activity is not essential for the sensor function of Pho84

675

and targets of the PKA pathway are known to be regulated by the sensing activity of Pho84,

676

thus indicating an influence on PKA activity (51, 52, 86, 87). Our previous work also

677

revealed that deletions in genes encoding components of PKA perturbed phosphate 27

678

metabolism and polyphosphate formation in U maydis (35, 36). Pho84 (and particularly

679

Pho840) may also have a sensing function and influence PKA activity in C. neoformans. In

680

particular, we noted that the pho84¨ pho840¨ mutant only produced 31.8% of the wt level of

681

polyphosphate versus 81.8% for the pho84¨ pho89¨ mutant.

682

phosphate available for polyphosphate biosynthesis was similar between these mutants:

683

pho84¨ pho840¨ (76.7% of wt) and pho84¨ pho89¨ (78.7% of wt). Also, deletion of

684

PHO840 causes a reduction of polyphosphate to 73% compared to 81.8% for the pho84¨

685

pho89¨ mutant.

686

polyphosphate production in the wt strain, but not the pho84¨ pho840¨ mutant, was increased

687

with addition of exogenous cAMP. This indicates that PKA, as a target of cAMP activation,

688

is involved in sensing, but also that Pho84 and/or Pho840 may contribute sensor activity.

689

Further evidence for PKA involvement comes from differences in growth for the pka1¨ and

690

pkr1¨ mutants in low phosphate medium, in resistance to zinc, arsenate, nickel, cobalt, and in

691

the impaired formation of polyphosphate in the pka1¨ and pkr1¨ mutants. These phenotypes

692

all resemble those of mutants with defects in phosphate metabolism.

However, the amount of

Pho840 may therefore be a sensor of phosphate levels.

Furthermore,

693

The formation of titan/giant cells by C. neoformans is known to be regulated by PKA

694

and Rim101, and to play an important role in virulence (53, 88). Although we did not find a

695

connection between Rim101 and phosphate uptake, there is a relationship with PKA and we

696

did observe a higher percentage of enlarged cells for mutants defective in phosphate uptake in

697

vitro. Whether these enlarged cells are related to titan/giant cells and influence virulence will

698

require further investigation. However, these results suggest a connection between phosphate

699

as a potentially limiting nutrient in the host and changes in cell size for C. neoformans.

700

We previously identified and characterized Cir1 as the master regulator of iron

701

homeostasis in C. neoformans (43). This protein regulates a large number of functions

702

including nutrient uptake, cell wall and sterol biosynthesis, and signalling pathways; it is also

703

required for elaboration of the major virulence factors including capsule, melanin and growth 28

704

at 37°C.

During iron limitation, Cir1 induces the expression of all three high affinity

705

transporters for phosphate and the polyphosphate kinase VTC4 (13). In contrast to iron

706

limitation, this regulation of the high affinity uptake system by Cir1 was not seen upon

707

phosphate starvation, with the exception of modest regulation of PHO89 by Cir1. However,

708

the cir1¨ mutant shared phenotypes with the phosphate mutants including reduced growth

709

under phosphate limitation with rescue of the phenotype in replete conditions, and increased

710

susceptibility to both divalent cations and CsA. These phenotypes were also shared by the

711

pkr1 deletion mutant lacking the regulatory subunit of PKA, and this suggests that they

712

resulted from activation of PKA.

713

polyphosphate formation, which was not rescued with exogenous cAMP, and that total iron

714

was reduced in the pho84¨ pho840¨, pho84¨ pho89¨ and vtc4¨ mutants compared to the wt

715

strain (Table 2). Taken together, these results indicate an indirect interconnection between

716

phosphate, PKA and iron sensing and regulation in C. neoformans. Similar connections have

717

previously been observed in bacterial pathogens and S. cerevisiae (46, 89). For example, the

718

PHO regulator PhoB-PhoR and the ferric uptake regulator (Fur) sense phosphate and iron to

719

control virulence in Edwardsiella tarda (89).

720

between the phosphate and the iron regulators, as well as a physical interaction between

721

components of the two systems (Fur and PhoU; Fur and EsrC). In addition, the iron response

722

regulator Aft1 is activated in a pho80¨ mutant in S. cerevisiae (46).

723

Phosphate and calcineurin.

We also found that the cir1¨ mutant had reduced

Chakraborty et al. (89) showed crosstalk

724

Our analysis also revealed a connection between phosphate uptake and calcineurin in

725

C. neoformans. Deletion of pho840¨, pho84¨ pho840¨, pho¨¨¨¸ pkr1¨ and cir1¨ all lead to

726

increased CsA sensitivity, while the deletion of xpp1¨ and the catalytic subunit of PKA

727

(pka1¨) lead to resistance to CsA at 37°C. Furthermore, the production of polyphosphate by

728

wt cells was increased by CsA treatment. These observations are consistent with the finding

729

in S. cerevisiae that the cyclin dependent kinase complex Pho80-Pho85 and PKA negatively 29

730

regulate the transcription factor Crz1, the major target of calcineurin (90, 91). The Crz1

731

ortholog in C. neoformans influences growth at high temperature, the response to oxygen

732

limitation, cell wall biosynthesis, fluconazole sensitivity and biofilm formation in a calcium-

733

dependent manner (92, 93). In this context, it is interesting that recent work in Candida

734

glabrata revealed that the aspartyl protease CgYps1 (yapsin 1) influences vacuolar and ion

735

homeostasis, cell wall remodelling, calcineurin sensitivity and polyphosphate levels (94).

736

When combined with our results and the connections between the PHO pathway, calcineurin

737

and cAMP signalling in A. fumigatus (49), these observation suggest that a conserved

738

regulatory network exists in fungi that involves cross talk between phosphate metabolism,

739

PKA and calcineurin.

740

In summary, our results reveal that phosphate is sensed by C. neoformans to influence

741

virulence factor expression and that its acquisition is important for virulence. There is a

742

growing body of evidence that virulence and nutrition are coordinated in C. neoformans (7).

743

For example, changes such as β-oxidation and central carbon metabolism have a pleiotropic

744

influence on the elaboration of virulence factors such as capsule and melanin (10, 38).

745

Therefore, the connection between nutrient availability and the expression of virulence factors

746

that is well established for bacterial pathogens also applies to the fungal pathogen C.

747

neoformans (95).

748 749 750

ACKNOWLEDGEMENTS

751

This work was supported by a grant from the Canadian Institutes of Health Research (to

752

J.W.K.) and the Basic Science Research Program through the National Research Foundation

753

of Korea funded by the Ministry of Science, ICT and Future Planning NRF-

754

2013R1A1A1A05007037 (to W.H.J.). D.L.O. was supported by a grant from Conselho

755

Nacional de Desenvolvimento Cientifico e Tecnologico (CsF-CNPq, Brazil). J.W.K. is a 30

756

Burroughs Wellcome Fund Scholar in Molecular Pathogenic Mycology. The authors thank

757

Dr. Arturo Casadevall for monoclonal antibody 18B7 and Wax-It services for assistance with

758

the histopathology study.

759

31

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TABLES AND FIGURES

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Table 1: Identification of C. neoformans genes/proteins for phosphate uptake and storage. Yeast EIdentity Similarity Putative Cryptococcus Designation in C. proteina value [%] [%] localizationc neoformans geneb neoformans High affinity phosphate transporter Pho84 CNAG_02777 PHO84 e-140 50 65 plasma membrane CNAG_05459 PHO840 2e-27 26 41 plasma membrane Pho89 CNAG_05075 PHO89 1e-95 37 52 plasma membrane Mitochondrial phosphate transporters Mir1 CNAG_06377 MIP1 1e-96 60 71 mitochondrial (7e-58) (Pic2) (40) (59) membrane CNAG_03824 MIP2 2e-67 43 58 mitochondrial (3e-45) (34) (55) membrane Polyphosphate storage and processing Vtc4 CNAG_01263 VTC4 e-155 64 80 vacuolar membrane Ppx1 CNAG_04354 XPP1 2e-24 26 44 cytosol Ppn1 CNAG_07629 EPP1 1e-76 43 61 vacuole a Yeast proteins where identified in the S. cerevisiae genome data base (http://www.yeastgenome.org). b Cryptococcus orthologs from the Broad Institute database (http://www.broadinstitute.org). c Putative protein location based on the S. cerevisiae proteins.

1129 1130 1131 1132 1133 1134 1135 1136 1137 1138

Table 2: Analysis of ion content by inductively coupled plasma mass spectrometry for mutants lacking high affinity phosphate uptake, polyphosphate polymerase or polyphosphate phosphatases. P Na Fe Zn Strain 1010 ions per cell 109 ions per cell 107 ions per cell 107 ions per cell 108 111 170 69 H99 ±6.5 ±6.9 ±4.4 ±1.1 Mutant values as percentage of wt 89.8% 82.2% 101.2% 150.7% pho840¨ ±3.8 ±2.2 ±2.1 ±8.9 pho84¨ pho840¨

76.7% ±4.5

155.7% ±10.4

77.9% ±2.7

89.9% ±0.8

pho84¨ pho89¨

78.7% ±2.8

96.5% ±2.5

74.2% ±2.3

79.6% ±2.7

pho¨¨¨

26.4% ±1.6

312.2% ±23.7

129.8% ±6.9

152.8% ±5.0 40

vtc4¨

1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 1172 1173 1174 1175 1176 1177 1178 1179 1180 1181 1182 1183

87.5% ±3.9

114.2% ±5.6

78.2% ±0.7

93.3% ±2.7

82.1% 92.5% 86.8% 81.2% epp1¨ ±7.0 ±9.7 ±7.7 ±2.0 xpp1¨ All values except Na for pho84¨ pho89¨ and epp1¨ xpp1¨, and Zn for pho840¨, are significantly different from wt (by Students t-test). All values that differ by at least 20% of the values for wt are marked in bold.

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 at the time points 0h, 0.5h, 1h and 5h after transfer from phosphate starvation conditions (for 24h) to high and low phosphate media (Materials and Methods). B) The transcript levels of the high affinity uptake transporters PHO840 and PHO89, relative to PHO84, were determined for cells from 24h phosphate-starved cultures (-Pi) and in 5h high phosphate (250 mM; +Pi) cultures. C) The transcript levels for phosphate-regulated genes were determined in wt and cir1¨ strains (cir1¨/wt) grown under no phosphate or high phosphate conditions at the one-hour time point. The experimental conditions from (A) were used. Each experiment was performed in triplicate. GAPDH and actin were used as endogenous controls. The dashed lines indicate a two-fold change in transcript levels. 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 2d at 30°C or 37°C. FIG 3 Defects in phosphate acquisition and storage, or regulation, influence growth in the presence of zinc, calcium and cyclosporine A. Strains were spotted in serial dilution on YPD plates at pH 5.4 with 5 mM Zn, 100 μg ml-1 CsA, 0.5 M CaCl2 or 50 mM CaCl2 plus 100 μg ml-1 CsA, and incubated for 2d at 30°C or 37°C. Note that 75 μg ml-1 CsA was used for some plates at 37°C to reveal the increased sensitivity of the deletion strains compared to wt. Two independent high affinity phosphate uptake system triple mutants (pho¨¨¨) and two independent double mutants for endo- and exopolyphosphatases (xpp1ǻ epp1ǻ) were examined. 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 2d at 30°C or 37°C. FIG 5 Defects in phosphate uptake and storage, or regulation, interfere with polyphosphate formation. 41

1184 1185 1186 1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 1200 1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216

A) Visualization of polyphosphate on a native polyacrylamide gel stained with toluidine blue O. Polyphosphate type 45 (10 μg) was loaded as 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 wt at p

Defects in phosphate acquisition and storage influence virulence of Cryptococcus neoformans.

Nutrient acquisition and sensing are critical aspects of microbial pathogenesis. Previous transcriptional profiling indicated that the fungal pathogen...
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