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Open Access

Peer-reviewed

Research Article

Carbon Dioxide is a Powerful Inducer of Monokaryotic Hyphae and Spore Development in Cryptococcus gattii and Carbonic Anhydrase Activity is Dispensable in This Dimorphic Transition

  • Ping Ren,

    Affiliation Mycology Laboratory, Wadsworth Center, New York State Department of Health, 120 New Scotland Avenue, Albany, New York, United States of America

    ⨯
  • Vishnu Chaturvedi,

    Affiliations Mycology Laboratory, Wadsworth Center, New York State Department of Health, 120 New Scotland Avenue, Albany, New York, United States of America, Department of Biomedical Sciences, School of Public Health, State University at New York, Albany, New York, United States of America

    ⨯
  • Sudha Chaturvedi

    Sudha.Chaturvedi@health.ny.gov

    Affiliations Mycology Laboratory, Wadsworth Center, New York State Department of Health, 120 New Scotland Avenue, Albany, New York, United States of America, Department of Biomedical Sciences, School of Public Health, State University at New York, Albany, New York, United States of America

    ⨯

Abstract

Cryptococcus gattii is unique among human pathogenic fungi with specialized ecological niche on trees. Since leaves concentrate CO2, we investigated the role of this gaseous molecule in C. gattii biology and virulence. We focused on the genetic analyses of β-carbonic anhydrase (β-CA) encoded by C. gattii CAN1 and CAN2 as later is critical for CO2 sensing in a closely related pathogen C. neoformans. High CO2 conditions induced robust development of monokaryotic hyphae and spores in C. gattii. Conversely, high CO2 completely repressed hyphae development in sexual mating. Both CAN1 and CAN2 were dispensable for CO2 induced morphogenetic transitions. However, C. gattii CAN2 was essential for growth in ambient air similar to its reported role in C. neoformans. Both can1 and can2 mutants retained full pathogenic potential in vitro and in vivo. These results provide insight into C. gattii adaptation for arboreal growth and production of infectious propagules by β-CA independent mechanism(s).

Citation: Ren P, Chaturvedi V, Chaturvedi S (2014) Carbon Dioxide is a Powerful Inducer of Monokaryotic Hyphae and Spore Development in Cryptococcus gattii and Carbonic Anhydrase Activity is Dispensable in This Dimorphic Transition. PLoS ONE 9(12): e113147. https://doi.org/10.1371/journal.pone.0113147

Editor: Yong-Sun Bahn, Yonsei University, Republic Of Korea

Received: March 7, 2014; Accepted: October 22, 2014; Published: December 5, 2014

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Funding: This study was supported by NIAID AI05887702. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Cryptococcus gattii, a basidiomycetous yeast, is an emerging pathogen in North America causing fatal disease in both healthy and immunocompromised humans as well in a wide range of animals including birds, domestic and wild mammals [1], [2]. A large outbreak of C. gattii infection among humans and animals in Vancouver Island, British Columbia, Canada, and the isolation of C. gattii from several genera of trees other than Eucalyptus, have indicated that this fungus must have broader geographic distribution including Pacific Northwest in the United States, and around the world [1], [3]–[8]. In extensive ecological investigations, C. gattii was isolated readily from soil, air, and water surrounding trees, in regions in the vicinity of Vancouver Island; evidently, C. gattii dispersal in the environment has been occurring through distribution of tree byproducts, aerosolization, water flow, and arthropogenic factors [9], [10].

Given the numerous possibilities for C. gattii dispersal, the organism’s de novo colonization mechanisms on trees and regions surrounding these trees are far from clear. Xue et al [11] have demonstrated that the young Arabidopsis thaliana plant surfaces represent a permissible environment, in which C. gattii and its closely related species C. neoformans can complete their sexual cycle (α-a mating). This intriguing finding raised the possibility that plants might serve as a critical host in the production of infectious propagules in the form of sexual spores (basidiospores). However, the predominance of α mating type both clinically and environmentally indicated that sexual mating in nature might be a limited and rare event. A number of studies raised the possibility that monokaryotic fruiting (α-α mating or same sex mating) might be a widespread phenomenon in C. neoformans var. neoformans, C. neoformans var. grubii and C. gattii [12]–[14]. Studies examining strains from outbreak investigations on Vancouver Island have found diploid isolates of α mating type with heterozygous alleles at their α- mating locus suggesting that the hypervirulent C. gattii VGII outbreak strains arose as a result of α-α mating [15]. Interestingly, the fruiting body (basidium) containing basidiospores as a result of α-α mating were not observed in C. gattii in the laboratory setting [16]. Therefore, it is possible that monokaryotic fruiting results from mating-dependent and mating-independent developmental pathways. A recent study from C. neoformans var. neoformans found cell cycle arrest induced mating-independent monokaryotic fruiting[17].

C. gattii is unique among human pathogenic fungi in its ecological niche; it predominantly inhabits trees by mechanisms not yet clearly understood. Since plants concentrate CO2 through the action of Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCo), it is conceivable that C. gattii is sensing CO2 for its survival and propagation in the environment [18]. A number of reports provide insight into how pathogenic fungi sense environmental CO2 via carbonic anhydrase (CA) and fungal adenyl cyclase [19]–[22]. CO2 diffusion into or out of the cells is facilitated by its conversion to biocarbonate ions (HCO3−), which are utilized for several cellular processes in the cell. CO2-HCO3− inter-conversion is catalyzed by CAs, which are zinc metalloenzymes and are grouped into five evolutionarily unrelated families, α, β, γ, δ, and ε-CA [23]–[25]. Of these, β-CA is unique to fungi and reported to be essential for fungal growth in ambient air (CO2 ∼ 0.036%) but not in a high CO2 (5%) environment [19]–[22].

In the present study, we focused on the genetic analyses of β-carbonic anhydrase (β-CA) encoded by C. gattii CAN1 and CAN2 as later is critical for CO2 sensing in a closely related pathogen C. neoformans. Our results provide insight into C. gattii adaptation for arboreal growth and the production of infectious propagules by β-CA independent mechanism (s).

Methods

Strains and media

The C. gattii strains used in this study are listed in Table 1. These strains were routinely maintained on yeast extract peptone dextrose agar (YPD) slants, and were stored in 15% glycerol at −70°C. YPD containing nourseothricin (100 µg/ml) or hygromycin B (200 µg/ml) was used to screen can1, and can2 single mutant and can1can2 double mutant strains [26]. The preparation of the various media -V8 medium for sexual (α-a) mating, filament agar for monokaryotic fruiting, Niger seed agar for melanin production, urea agar for urease production, and agar based Dulbecco’s modified Eagle’s (DME) medium for capsule production were used as described [27]. YPD containing menadione (3 µg/ml), or paraquat (1 mM) was used for oxidative stress, NaCl (1.4 M, and 1.8 M) for osmotic stress, and NaNO2 (1 mM) for nitrosative stress were prepared as described [26]. Yeast nitrogen base (YNB) broth containing various sugars was prepared as described [28]. For determination of amino acid requirements, synthetic dextrose (SD) medium containing 0.17% YNB and 1% glucose was supplemented with adenine (20 mg/l), uracil (30 mg/l), L-arginine (20 mg/l), leucine (60 mg/l), histidine (20 mg/l), tryptophane (30 mg/l). For determination of fatty acid requirements, YPD agar supplemented with palmitate (1–10 mM) or myristate (1–10 mM) and with 1% Tween 80 as surfactant was prepared as described previously [20].

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Table 1. Cryptococcus gattii strains used in this study.

https://doi.org/10.1371/journal.pone.0113147.t001

Plasmids and oligonucleotides

Plasmids and oligonucleotides used in this study are listed in Table 2. The full-length CAN1 and CAN2 gene sequences from C. neoformans were BLAST searched in the NCBI database for C. gattii (R265) (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi), which yielded R265 cont1.355, and R265 cont1.479, for CAN1 and CAN2, respectively. Primers were designed to amplify approximately 1500-bp fragments of the CAN1 and CAN2 genes from genomic DNA of NIH 444 strain of C. gattii. The nucleotide sequences for the CAN1 and CAN2 genes from NIH 444 have been submitted to the GenBank database (CAN1  =  EU723699; CAN2  =  EU723700). The C. neoformans cDNA sequences from CAN1 and CAN2 were aligned with the C. gattii CAN1 and CAN2 genomic sequences, using the GAP function of the GCG Wisconsin package to obtain exon/intron boundaries. cDNA sequences for C. gattii retrieved through this analysis were used in multiple alignments for comparison with C. neoformans.

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Table 2. Plasmids and oligonucleotides used in this study.

https://doi.org/10.1371/journal.pone.0113147.t002

Disruption of C. gattii CAN1 and CAN2 genes

Gene disruption was carried out as described previously [26], [29]. Disruption cassettes for CAN1 and CAN2 were constructed by PCR fusion [30]. In brief, upstream and downstream regions flanking the CAN1 and CAN2 genes (approximately 1 kb on either side) and the full-length NAT marker gene from pCH333 plasmid were PCR-amplified. PCR amplicons were gel-purified, added in a molar ratio of 1∶3∶1, as 5’-flanking (CAN1 or CAN2):marker (NAT):3’-flanking (CAN1 or CAN2) amplicons, followed by reaction at 94°C for 2 min, and 15 cycles at 94°C for 30 s and 58°C for 10 min to allow fusion to occur. The fusion product was used as template in conventional PCR, to obtain can1::NAT and can2::NAT alleles. The constructs were directly used to transform C. gattii NIH 444 wild type (WT) strain by biolistic delivery, and transformants were selected on YPD containing nourseothricin. The potential can1 and can2 mutants were screened by diagnostic PCR using primer pair V1609/V1610 designed from the CAN1 flanking NAT gene and V1496/1497 from the CAN2 flanking NAT gene. The can1 and can2 mutants were further confirmed for gene deletion and single integration events by reverse transcriptase (RT)-PCR and Southern blot analyses, respectively. The can1can2 double knockout mutants were created by disruption of the CAN2 gene in the can1 mutant using the can2::HYG allele, followed by diagnostic PCR and Southern blot analyses, as described for the can2 single mutant. Two of these clones termed can1can2-1, and can1can2-2 were used for further studies.

For construction of the CAN2 reconstituted strain, a 2.9-kb fragment containing full-length CAN2 ORF was PCR-amplified from genomic DNA of C. gattii WT strain using primers V1467/V1470. The PCR fragment was cloned into pCR2.1-TOPO (Invitrogen) to yield pCR2.1-CAN2, and then sequenced for confirmation. The plasmid was digested with EcoRI, and the CAN2 full-length fragment was biolistically transformed into the can2 mutant, and transformants were selected on YPD medium in ambient air. Since the can2 mutant did not grow in ambient air, clones recovered under these conditions were potential CAN2 reconstituted strains. These transformants were patched on YPD-nourseothricin plates. Inability to grow on this medium was considered as an indication of can2+CAN2 reconstituted strains with CAN2 integration in the native locus, resulting in the removal of the can2::NAT allele. These reconstituted strains were further confirmed for single CAN2 homologous integration event by Southern blot. One of these clones termed as can2+CAN2 was used for further investigations.

Analysis of nutritional requirements of can2 mutant

Cultures grown overnight in YPD broth at 30°C with 5% CO2 were washed with sterile water, inoculated at OD600  =  0.1 in YNB containing various sugars, and incubated in ambient air with shaking for 1 week. To determine amino acid and fatty acid requirements, 5µl of serial dilutions of yeast suspension from original stock of 107/ml were spotted on an appropriate medium supplemented with various amino acids or fatty acids. Cultures were incubated for 2-5 days at 30°C in ambient air (0.036% CO2) or in 5% CO2.

Mating assays

V8 medium, buffered either with 100 mM MOPS for pH 7.0 or with sodium citrate for pH 5.0 and filament agar (pH 5.0) was used for mating and monokaryotic fruiting assays [20]. Cultures grown overnight in YPD broth at 30°C with high CO2 (5%) were washed twice with sterile distilled water, and were re-suspended in water at a concentration of 5 × 107 cells/ml. An equal number of cells of the opposite mating type cells was mixed, and 5µl of the mixture inoculated on buffered V8 medium, and incubated at 30°C with or without CO2 for up to 8 weeks. For monokaryotic fruiting, 5-10µl of individually washed cells (5 × 107/ml) were inoculated on filament agar and buffered V8 agar media, and incubated with or without CO2 for 8 weeks. Images of hyphal growth were captured with an Olympus AX70 microscope equipped with a digital camera as described previously [27].

Assays for virulence factor expression and stress sensitivity

The C. gattii WT, can1, can2 single mutants, can1can2 double mutant, and can2+CAN2 reconstituted strains were incubated for 16–18 hours in YPD broth at 30°C with 5% CO2. Cells were washed with sterile distilled water, counted, and adjusted to 108/ml. Five microliters of yeast suspension were spotted on DME agar, on Christensen’s agar, and on egg yolk agar and incubated for 24–72 hours at 30°C with 5% CO2 for respective assessments of capsule, urease, and phospholipase production. For determination of stress sensitivity, yeast cells grown as described above were serially diluted (103–107), and spotted on YPD medium containing redox cycling agents menadione (3µg/ml), paraquat (1µM), sodium nitrite (0–10 mM), and sodium chloride (1–1.8 M), and incubated at 30°C with 5% CO2.

Virulence assays

The pathogenic potentials of the C. gattii WT, can1, can2 single mutants, can1can2 double mutant, and can2+CAN2-reconstituted strains were assessed in a mouse model of pulmonary and systemic cryptococcosis [26], [29]. BALB/c mice (6–8 weeks) were procured from Charles River Laboratories, Inc., and procedures for safe and pain-free handling of animals were followed as per the protocol approved by the Institutional Animal Care and Use Committee (IACUC), Wadsworth Center, New York State Department of Health, Albany, NY, USA. Cultures grown overnight in YPD broth at 30°C with 5% CO2 were washed, and then re-suspended in sterile phosphate buffered saline (PBS), pH 7.4, at a concentration of 1 × 107/ml. Group of five mice were injected intravenously with 106 CFU of each strain. The animals were given food and water ad libitum, and were observed twice daily for any sign of distress. Mice that appeared moribund or in pain were sacrificed using CO2 inhalation and cervical dislocation as per the protocol approved by the Institutional Animal Care and Use Committee (IACUC), Wadsworth Center, New York State Department of Health, Albany, NY, USA. Survival data were analyzed by Kaplan-Meyer survival curve using the SAS software (SAS Institute Inc., Cary, NC, USA).

To determine the pathogenic potential of test strains in pulmonary infection, we inoculated a group of three mice with 105 CFU of each strain in a volume of 30µl via nasal inhalation as previously described [26]. Animal care procedures were as per approved IACUC protocol. Animals were sacrificed after 14 days of infection, lungs and brains were removed aseptically, homogenized, serially diluted, and plated on YPD agar, and incubated at 30°C with 5% CO2 for CFU enumeration.

For histopathology, the left lung lobe was dissected and immersion-fixed in formalin; it was embedded and processed into paraffin blocks, sectioned at 4µm and stained with mucicarmine (Richard-Allan Scientific, Kalamazoo, MI).

Results

CAN2 but not CAN1 is a major β-CA required for C. gattii growth at ambient air

We identified two CA encoding genes, CAN1 and CAN2 in the C. gattii genome database for related strain R265 (www.broad.mit.edu/annotation/genome/cryptococcus_neoformans_b/Blast.html). The pair-wise comparison revealed 58% and 42% identity at nucleotide and amino acid levels. Both deduced Can1p and Can2p sequences exhibited β-CA signature motif comprising one histidine, two cysteins, and one aspartate residue critical for zinc-binding and enzyme activity. Comparison of deduced amino acid sequences of C. gattii Can1p and Can2p with that of C. neoformans Can1p and Can2p revealed them to be 89% and 97% identical, respectively, indicating that the two genes are highly conserved in C. neoformans and C. gattii [20].

To assess the role of β-CA in C. gattii biology, we created can1, and can2 single knockout mutants and a can1can2 double knockout mutant through homologous integration (see method and Figure S1). The can1 mutant did not exhibit any growth defects in either ambient air (0.036% CO2) or in a high-CO2 (5%) environment. The can2 mutant in contrast, exhibited a severe growth defect in ambient air, but not in a high-CO2 environment. The can1can2 double knockout mutants exhibited a growth phenotype similar to that of can2 single mutant. The severe growth defect of the can2 mutant in ambient air was rescued by re-introduction of wild-type CAN2 allele (Fig. 1a). These results indicated that CAN2 but not CAN1 is essential for C. gattii growth in ambient air. Prolonged incubation of the can2 mutant in ambient air was irreversibly lethal; majority of the cells could not be rescued by a shift to a high-CO2 environment (Fig. 1b).

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Figure 1. Role of β-CA in C. gattii growth in ambient air and in high CO2 environment.

(a). CAN2 is major β-CA essential for C. gattii growth in ambient air. C. gattii WT and various can mutant strains were spotted on YPD agar and incubated at 30°C and 37°C in low CO2 (0.036%; ambient air) or high CO2 (5%) for 2–4 days. The can2 mutant did not grow on YPD agar at either 30°C or 37°C in low CO2, but grew well in high CO2 condition. The severe growth defect phenotype of can2 was rescued by re-introduction of WT CAN2 allele. (b). CAN2-mediated C. gattii growth inhibition in ambient air is fungicidal. Approximately 100 colony forming unit (CFU) each of the C. gattii WT, the can2 mutant, and the can2+CAN2 reconstituted strain were inoculated on YPD agar, and plates were incubated for 2, 3, and 7 days at 30°C in ambient air, and then transferred to a high-CO2 environment. Control plates were incubated directly in high CO2 environment. Results were expressed as the percentage of C. gattii killed  =  [1- (CFU of experiment/CFU of control)] × 100. The percentage of can2 mutant cell death was significantly higher than WT and reconstituted strains (p<0.05).

https://doi.org/10.1371/journal.pone.0113147.g001

CAN2 is critical for fatty acid biosynthesis but not required for adenyl cyclase (CAC1) gene expression

We reasoned that the inability of the can2 mutant to grow in air could be due to limiting amounts of bicarbonate, a critical substrate required for the synthesis of several cellular carboxylases important in metabolism [31]. Bicarbonate is also a critical substrate for CAC1 gene activation, and that in turn leads to the synthesis of cAMP, a ubiquitous second messenger that regulates a large variety of essential physiological processes [21], [22]. Interestingly, addition of exogenous cAMP (2–10 mM) or sodium bicarbonate (1–10 mM) either singly or in combination, failed to complement the growth defect of the can2 mutant in ambient air. Similarly, addition of various cellular metabolites and carbon sources, including citrate, succinate, oxalaacetate, malate, α-ketoglutarate failed to complement the growth defect of the can2 mutant (data not shown). In contrast to the report published for C. neoformans, the growth defect of the can2 mutant was barely rescued by addition of exogenous fatty acids, 0.1 mM and 1 mM palmitate (Fig. 2a), indicating that CAN2 is essential for fatty-acid biosynthetic processes in ambient air in C. gattii. We observed a clear zone surrounding the colonies of C. gattii WT and can2+CAN2 reconstituted strains (2 mM and 5 mM palmitate) (Fig. 2a). This might be due to the fact that WT and reconstitute strains were able to utilize fatty acids from media resulting in clear zone surrounding the growth.

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Figure 2. CAN2 is critical for fatty acid biosynthesis but not essential for adenyl cycalse (CAC1) gene expression.

(a). Palmitic acid barely restored can2 mutant growth in ambient air. C. gattii strains grown overnight in high CO2 were collected, washed, serially diluted, and spotted on YPD medium containing palmitate with 1% Tween 80 as surfactant. Cells were incubated at 30°C in ambient air for 5 days. The growth defect of can2 mutant was barely rescued in the presence of low but not high concentration of palmitate in ambient air. The halo surrounding the growth patches of WT and reconstituted strains reflects efficient utilization of fatty acids from media. (b) Semi-quantitative RT-PCR confirmed CAC1 gene expression is independent of CAN2. C. gattii WT, can2 mutant, and can2+CAN2 reconstituted strain were grown in YPD broth in high CO2 or in YPD broth containing 1 mM sodium palmitate in ambient air, for 3 days at 30°C. Total RNA was isolated and reverse transcribed (cDNA) with 100-ng aliquots in 1∶10 serial dilutions. SOD1 was used as a loading control. (c) Semi-quantitative RT-PCR confirmed CAN2 gene expression is not regulated by CO2. CAN2 transcript in total RNA was determined from C. gattii WT strain grown in various conditions as indicated. SOD1 was used as a loading control.

https://doi.org/10.1371/journal.pone.0113147.g002

To explore the link between CAN2 and CAC1, RNA was extracted from WT, can2 mutant, and can2+CAN2 reconstitute strain grown for 3 days in ambient air in the presence of 1mM sodium palmitate or in a high-CO2 environment. We found that the can2 mutant remains viable (100%) but do not multiply in the presence of 1 mM sodium palmitate in ambient air for up to 4 days (data not shown). Semi-quantitative RT-PCR revealed that CAC1 transcript was expressed with or without CO2 in both can2 mutant and in the WT strain and also CAC1 expression appeared to be marginally induced without CO2, which was consistent for WT, can2 mutant and can2+CAN2 reconstituted strains (Fig. 2b). These results indicated that CAC1 expression is independent of CAN2, in other words, CAN2 is not required for CAC1 expression. Also, semi-quantitative RT-PCR analysis of CAN2 transcript in C. gattii WT revealed similar expression pattern in both ambient air or in high CO2 environment. These results indicated that CAN2 gene expression is not regulated by CO2 (Fig. 2c).

CO2 is a powerful inducer of monokaryotic hyphae development in C. gattii

Mating is an important process by which Cryptococcus generates filaments and spores that might be important in its ecological fitness. It is clear that C. gattii associates with various plant species in nature [1]. However, it is not clear how this fungus survives and propagates on plant substrates. Since most of the plants utilize CO2 for photosynthesis, and they possess a CO2 concentration mechanism through RubisCO, an enzyme specifically found in chloroplasts of bundle sheath cells [18], we asked whether high CO2 induces mating and hyphae development in C. gattii. The C. gattii WT, can1 and can2 single mutants, can1can2 double mutant, and can2+CAN2 reconstitute strains were inoculated on filament agar and V8 agar for monokaryotic and sexual mating. The inoculated plates were incubated in ambient air or in high CO2. To our surprise, we found that C. gattii WT strain undergoes hyphae development as part of monokaryotic fruiting more vigorously in high CO2 than in ambient air (Fig. 3a). Filaments on the edges of C. gattii WT growth appeared as early as 1-week post-incubation under high CO2, compared to 4-weeks post-incubation under low CO2. The can2 but not can1 mutation caused further enhancement of filamentation as judged by long and dense filaments on the colony edges (Fig. 3a; lower panel). Light microscopic mounts of these filamentous projections from the WT as well from the can2 mutant revealed hyphae and blastospores but not basidiospores. The can2 mutant hyphae harbored several blastospores, whereas the WT and can2+CAN2 reconstituted strains harbored few blastospores (Fig. 3b; lower panel). The topology of hyphae harboring blastospores was consistent with our earlier report where these structures were analyzed by scanning electron microscope [27]. The monokaryotic filamentation was also observed on V8 agar at pH 7.0 with or without CO2 but not at pH 5.0. However, filamentation was not as robust as on filament agar (data not shown).

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Figure 3. CO2 is a powerful inducer of monokaryotic hyphae development in C. gattii.

C. gattii strains were individually cultured on filament agar and hyphae development was assessed macroscopically at 8 weeks-post incubation. (a) Upper panel- Few filamentous projections (arrow) seen at the edge of the colonies of WT and can2+CAN2 reconstituted strains. No growth of can2 mutant in ambient air (low CO2). Lower panel-Robust filamentation (arrow) in the presence of high CO2 with dense and long hyphal extension in can2 mutant. (b) Upper panel - Light microscopic analyses of hyphae development (magnification, × 100) in WT, can2, and can2+CAN2 strains in the presence of high CO2. Lower panel - Filamentous growth on the edge of the colony were carefully removed, mounted on lactophenol cotton blue and gently pressed and photographed (magnification, × 200). Filaments bearing blastospores (arrows) seen in all the strains except that can2 mutant revealed more blastospores compared to the WT and can2+CAN2 reconstituted strains.

https://doi.org/10.1371/journal.pone.0113147.g003

In contrast, high CO2 completely suppressed sexual mating (α-a) in C. gattii WT, can2 mutant, and can2+CAN2 reconstituted strains, as no filamentation on the edges of the colonies was observed even after 8 weeks of incubation on V8 agar medium adjusted to either pH 5 or 7 (Fig. 4a). C. gattii WT and can2+CAN2 reconstituted strains showed robust sexual mating under low CO2 in V8 agar medium adjusted to pH 7.0 (Fig. 4b), but not to pH 5.0 (data not shown). Hyphae cells produced during sexual mating contained two nuclei (single arrow) that were linked by fused clamp (double arrow) connections (Fig. 4c & 4d). It should be pointed out here that we used only unilateral crossing in which WT MATa strain (NIH 198) was used as the opposite mating partner in the sexual mating assay. Overall, these results indicated that high CO2 is a powerful inducer of monokaryotic hyphae differentiation but not in sexual mating providing important distinction in these two developmental programs in C. gattii.

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Figure 4. High CO2 inhibits sexual mating in C. gattii and CAN2 does not rescue this inhibition.

An equal number of C. gattii WT, can2 mutant, and can2+CAN2 reconstituted strains were mixed with the MATa strain (NIH 198), inoculated on V8 agar and incubated in high (5%) or low (0.036%) CO2 and mating was assessed at 8 weeks post-incubation. (a) Light microscopic analyses (magnification, × 100) of the representative edges of the mating patches showing no filamentation in high CO2. (b) Light microscopic analyses (magnification, × 100) of the representative edges of the mating patches with extensive filamentation in low CO2 (ambient air). No growth of can2 mutant in ambient air. (c) Filamentous growth on the edge of the colony was carefully removed, and stained with SYTOX Green and assessed under fluorescent microscope (magnification, × 400). Filaments showing characteristic fused clamp connection (single arrow) and pairs of nuclei (double arrows) upon mating of WT (α) × WT (a), and can2+CAN2 (α) × WT (a). No growth of can2 mutant was evident in ambient air. (d) Light microscopy of same structures as shown in c (magnification, × 400).

https://doi.org/10.1371/journal.pone.0113147.g004

β-CA activity is not required for the expression of C. gattii virulence repertoire

Since β-CA activity was found to be essential for C. gattii growth in ambient air, we asked whether this enzyme is required for C. gattii virulence factor expression, and furthermore, for disease development in mammalian hosts. The can1 and can2 single mutants, as well can1can2 double mutant strains expressed major virulence factors (melanin, capsule, phospholipase, urease) at levels comparable to those for the WT strain, in a high-CO2 environment. Similarly, mutants did not exhibit any altered sensitivity to oxidative, osmotic or nitrosative stress (Figure S2). These results indicated that β-CA activity is neither required for general stress response nor for the expression of virulence traits, at least when CO2 is in abundance. Furthermore, β-CA activity is not essential for C. gattii to induce disease in mammalian host. Mice infected intravenously with the can1 or can2 single mutant, or can1can2 double mutant strains manifested severe disease similarly to mice infected with the WT strain (Fig. 5a & 5b).

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Figure 5. β-CA activity is dispensable for C. gattii pathogenesis.

(a-b) Systemic cryptococcosis model: The WT, can1, can2 single mutants, can1can2 double mutants and can2+CAN2 reconstituted strains grown overnight in YPD broth in high CO2 were washed with PBS, and counted, and a 100µl suspension containing 106 cells was injected intravenously into 5–6 weeks old BALB/c mice (5 mice/group). Mice were monitored twice daily and sacrificed if any symptoms of distress were apparent. No significant difference on survival rate of mice infected with WT or mutant strains observed (p>0.05).

https://doi.org/10.1371/journal.pone.0113147.g005

Given the importance of gaseous exchange in the lungs, with the high oxygen content in the terminal alveoli, as well the lungs’ vigorous defense mechanisms against pathogens, we probed if can2 mutant is able to colonize the lungs as efficiently as the WT strain. The organ load experiment revealed that the fungal burden imposed by the can2 mutant was almost as high as the burden imposed by the WT strain (Fig. 6a). Also, the can2 mutant was able to produce capsule in the lungs as large as those produced by the WT strain (Fig. 6b). Furthermore, histopathological examinations of lungs infected with can2 mutant or the WT strain revealed similar tissue responses, including severe and diffuse interstitial pneumonia, and the presence of numerous organisms in the alveoli and airways (Fig. 6c). Altogether, these results confirmed that CAN2 deletion has no influence on C. gattii virulence traits and pathogenesis, in agreement with previous findings for C. neoformance and Candida albicans [20], [21].

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Figure 6. β-CA activity is dispensable for in vivo colonization.

(a) Organ load determination: BALB/c mice (6–8 weeks) were infected intra-nasally with 105 yeast cells of the WT, can2, or can2+CAN2 strain (3 mice/strain). After 14 days, mice were sacrificed, and their lungs were removed aseptically, weighed, homogenized, and diluted in PBS, and cultured on YPD agar in high CO2 for CFU enumeration. Results were expressed as CFU per gram of tissue. No significant difference in organ load of WT or can2 mutant was observed (p>0.05). (b) In vivo capsule production: WT, can2, and can2+CAN2 strains recovered from mice lungs were visualized with India ink (magnification, × 100). (c) Histopathological examination of lungs: Left lung lobes from mice infected with the WT, the can2 mutant, or the can2+CAN2 reconstituted strain for 14-days were fixed, sectioned, and stained with Mayer’s mucicarmine. Alveoli (single arrow) and airways (double arrow) showed the presence of numerous organisms for each of the infecting strain and similar tissue response was noted for WT, can2 and can2+CAN2.

https://doi.org/10.1371/journal.pone.0113147.g006

Discussion

The present study revealed that high CO2 strongly induced monokaryotic hyphae development in C. gattii while it completely repressed sexual (α-a) hyphae development, indicating an important distinction in environmental responses by theses two developmental programs. Considering the fact that C. gattii grows on plants known to concentrate CO2 through RuBisCO [32], the observed association between high CO2 and morphological transition in C. gattii indicates an ecological adaptation for survival and propagation in nature.

Nitrogen starvation, water deprivation and high temperature have been linked to monokaryotic fruiting in C. neoformans [16], [17]. Also, darkness is an additional factor associated with hyphae production and fruiting structures in C. neoformans [33]. We have now added high CO2 (5%) to this list as it strongly induced hyphae development in C. gattii; filamentation was discernable as early as 1-week post incubation in high CO2 compared to its appearance at 4 weeks in a low-CO2 (ambient air) environment. Recently, CO2 has also been shown to be powerful inducer of filamentation in C. albicans that requires CAC1 but bypasses Ras [21]. CAC1 activation requires both bicarbonate and G proteins in C. albicans as well as in C. neoformans [21], [22], [34]. Interestingly, we did not find any link between CAN2 and CAC1 as can2 mutant produced equivalent amount of CAC1 transcript as the WT strain. Additionally, CAC1 transcript was induced more in ambient air than in high CO2 while opposite was true for hyphae development where high CO2 served as powerful inducer. These results indicate that C. gattii CAC1 may not be directly involved in CO2-induced monokaryotic hyphae development as opposed to its critical role assessed in sexual mating [34]. These results support the hypothesis that there are probably different signaling pathways in the development of hyphal projection, a prerequisite for spore formation in monokaryotic fruiting and sexual mating. The search of C. gattii database for a related strain R265 (http://www.broad.mit.edu) revealed single copy of CAC1 gene as reported earlier for C. neoformans [34]. Interestingly, we found that can2 mutant undergoes robust monokaryotic filamentation with blastospore formation indicating that either CAN2 serves as a repressor, or certain threshold levels of CO2-HCO3− interconversion is critical in this developmental pathway.

We also found that CAN2, but not CAN1, was essential for C. gattii growth under ambient air (0.035% CO2). In this regard, C. gattii is similar to its closely related species C. neoformans where CAN2 was major β-CA for growth under ambient air [20]–[22]. The precise mechanism for observed growth defects of C. gattii can2 mutant in ambient air is not clear at present but defective fatty acid biosynthesis might be partially responsible, consistent with earlier report for C. neoformans [20]. Since CAN2 was dispensable for survival, proliferation, and lethality during intravenous and intranasal infection, its role in C. gattii pathogenesis appeared to be redundant.

Although very little is known about morphological forms of C. gattii in nature, a hyphal phase appears to be an integral part of C. gattii biology. The recent outbreak of C. gattii on Vancouver Island revealed that the fungus inhabits several tree species (Douglas fir, alder, maple, and Garry oak) [1], [4], [9]. The Vancouver Island air samples contain particles of 1–2µm in diameter, a size consistent with spores [1]. Also, all of the isolates from this outbreak belonged to MATα mating type, further bearing out the predominant mode of reproduction possibly through monokaryotic fruiting. Additionally, the endemic nature of C. gattii in Australia, majority of Australian isolates being sterile, and their well-known association with Eucalyptus trees strongly suggest that the monokaryotic fruiting might be the driving force for the survival and propagation of C. gattii in nature [35], [36]. Although, mixed populations of MATα and MATa strains of C. gattii have been identified colonizing hollows in Eucalyptus trees in Australia [37]–[40], no meiotic recombination has been detected in isolates recovered from these hollows; thus monokaryotic fruiting could still be the main mode of propagation of C. gattii in nature.

In summary, we have demonstrated that high CO2 conditions induced robust development of monokaryotic hyphae and spores in C. gattii. Conversely, high CO2 completely repressed hyphae development in sexual mating. Both CAN1 and CAN2 were dispensable for CO2 induced morphogenetic transitions and expression of pathogenic traits. Further investigations are warranted to dissect CO2-mediated signaling pathways to determine relevant sensor(s) required for monokaryotic fruiting.

Supporting Information

Figure S1.

Characterization of can1, and can2 single knockout mutants, can1can2 double knockout mutant, and can2+CAN2 reconstituted strains. (a-b) Diagnostic PCR and Southern hybridization analysis for can1 mutants: (a) Primers (V1609/v1610) designed from the CAN1 flanking NAT gene amplified 1.7-kb PCR product from the genomic DNA of C. gattii WT and 3.0-kb amplicon from the genomic DNA of can1-1 and can1–2 mutants obtained through two independent transformation events. (b) Genomic DNA was digested with Sac I (cuts once within CAN1 gene) and probed with 612-bp PCR product amplified from CAN1 ORF. The C. gattii WT produced 1.4-kb band, while both can1-1 and can1-2 mutants produced 3.3-kb bands. (c-e) Diagnostic PCR, RT-PCR, and Southern hybridization analyses of can2 mutant and can2+CAN2 reconstituted strains. (c) Primers (V1496/V1497) designed from the CAN2 flanking NAT gene amplified 1.4-kb PCR product from the genomic DNA of C. gattii WT and can2+CAN2 reconstituted strains while same primer set produced 2.9-kb PCR product from the genomic DNA of can2 mutant. (d) Total RNA was isolated, reverse transcribed to cDNA and amplified with primers (V1600/V1532) directed against CAN2 or primers (V548/V549) directed against SOD1. RT-PCR products were fractionated by electrophoresis in a 1% agarose gel and stained with ethidium bromide. C. gattii WT and can2+CAN2 reconstituted strains yielded 515-bp CAN2 transcript while can2 mutant did not. SOD1 transcript served as a loading control. (e) Genomic DNA from C. gattii WT, can2 mutant, and can2+CAN2 reconstituted strains were cut with Hind III (non-cutter within CAN2 gene), and probed with 372-bp PCR product amplified from the CAN2 gene. The C. gattii WT and can2+CAN2 reconstituted strains produced 3.0-kb band while can2 mutant produced 4.5-kb band. (f-g) Diagnostic PCR and Southern hybridization analyses of can1can2 double knockout strains: For creation of can1can2 double knockout strain, CAN2 gene was disrupted in can1 mutant using can2:HYG allele. (f) Primers (V1496/V1497) yielded 1.4-kb amplicon from the genomic DNA of C. gattii WT as shown in figure C while same primer pair yielded 3.2-kb amplicon from the genomic DNA of can1can2 double knockout strains. (g) Genomic DNA from C. gatti WT, can1can2-1, and can1-can2-2 double knockout mutants were cut with Hind III and probed with CAN2 PCR product. The C. gattii WT produced 3.0-kb band, while both can1can2 double knockout mutants produced 4.9-kb bands.

https://doi.org/10.1371/journal.pone.0113147.s001

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Figure S2.

β-CA activity is dispensable for virulence factor production and for various stresses in C. gattii. WT and various can mutant strains were grown overnight at 30°C in 5% CO2, washed, and adjusted to OD600  = 1.0. The 10-fold serial dilutions were prepared and 4µl of each dilution was spotted on YPD alone, YPD containing NaNO2 (nitrossative), NaCl (osmotic), menadione and paraquat (oxidative) and incubated at 30°C for 72 h. Also assessed were the production of melanin (Niger seed agar), urease (Christensen agar), phospholipase (egg-yolk agar) and capsule (DME agar). Mutant strains neither exhibited any altered sensitivity to stress nor were defective in the production of major virulence factors.

https://doi.org/10.1371/journal.pone.0113147.s002

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Acknowledgments

We thank Adriana Verschoor for editorial comments. We also thank Wadsworth Center Histopathology Core (Dr. Melissa Behr) for reading slides, Molecular Genetics Core for nucleotide sequencing and Media & Tissue Culture Core for the preparation of media and reagents. This study was supported in part with funds from NIAID (SC, VC).

Author Contributions

Conceived and designed the experiments: SC VC. Performed the experiments: PR SC. Analyzed the data: PR SC VC. Contributed reagents/materials/analysis tools: PR SC VC. Wrote the paper: SC VC. Acquisition of data and interpretation of data: PR SC VC. Revised the manuscript critically for important intellectual content: SC VC. Final approval of the version to be published: PR SC VC.

References

  1. 1. Kidd SE, Hagen F, Tscharke RL, Huynh M, Bartlett KH, et al. (2004) A rare genotype of Cryptococcus gattii caused the cryptococcosis outbreak on Vancouver Island (British Columbia, Canada). Proc Natl Acad Sci U S A 101:17258–17263.
  2. 2. Chaturvedi S, Dyavaiah M, Larsen RA, Chaturvedi V (2005) Cryptococcus gattii in AIDS patients, Southern California. Emer Infect Dis 11:1686–1692.
  3. 3. Stephen C, Lester S, Black W, Fyfe M, Raverty S (2002) Multispecies outbreak of cryptococcosis on southern Vancouver Island, British Columbia. Can Vet J 43:792–794.
  4. 4. Kidd SE, Chow Y, Mak S, Bach PJ, Chen H, et al. (2007) Characterization of environmental sources of the human and animal pathogen Cryptococcus gattii in British Columbia, Canada, and the Pacific Northwest of the United States. Appl Environ Microbiol 73:1433–1443.
  5. 5. Callejas A, Ordonez N, Rodriguez MC, Castaneda E (1998) First isolation of Cryptococcus neoformans var. gattii, serotype C, from the environment in Colombia. Med Mycol 36:341–344.
  6. 6. Lazera MS, Salmito Cavalcanti MA, Londero AT, Trilles L, Nishikawa MM, et al. (2000) Possible primary ecological niche of Cryptococcus neoformans. Med Mycol 38:379–383.
  7. 7. Fortes ST, Lazera MS, Nishikawa MM, Macedo RC, Wanke B (2001) First isolation of Cryptococcus neoformans var. gattii from a native jungle tree in the Brazilian Amazon rainforest. Mycoses 44:137–140.
  8. 8. Granados DP, Castaneda E (2005) Isolation and characterization of Cryptococcus neoformans varieties recovered from natural sources in Bogota, Colombia, and study of ecological conditions in the area. Microb Ecol 49:282–290.
  9. 9. Kidd SE, Bach PJ, Hingston AO, Mak S, Chow Y, et al. (2007) Cryptococcus gattii dispersal mechanisms, British Columbia, Canada. Emerg Infect Dis 13:51–57.
  10. 10. MacDougall L, Kidd SE, Galanis E, Mak S, Leslie MJ, et al. (2007) Spread of Cryptococcus gattii in British Columbia, Canada, and detection in the Pacific Northwest, USA. Emerg Infect Dis 13:42–50.
  11. 11. Xue C, Tada Y, Dong X, Heitman J (2007) The human fungal pathogen Cryptococcus can complete its sexual cycle during a pathogenic association with plants. Cell Host Microbe 1:263–273.
  12. 12. Lin X, Hull CM, Heitman J (2005) Sexual reproduction between partners of the same mating type in Cryptococcus neoformans. Nature 434:1017–1021.
  13. 13. Hiremath SS, Chowdhary A, Kowshik T, Randhawa HS, Sun S, et al. (2008) Long-distance dispersal and recombination in environmental populations of Cryptococcus neoformans var. grubii from India. Microbiology 154:1513–1524.
  14. 14. Saul N, Krockenberger M, Carter D (2008) Evidence of recombination in mixed-mating-type and alpha-only populations of Cryptococcus gattii sourced from single Eucalyptus tree hollows. Eukaryot Cell 7:727–734.
  15. 15. Fraser JA, Giles SS, Wenink EC, Geunes-Boyer SG, Wright JR, et al. (2005) Same-sex mating and the origin of the Vancouver Island Cryptococcus gattii outbreak. Nature 437:1360–1364.
  16. 16. Wickes BL, Mayorga ME, Edman U, Edman JC (1996) Dimorphism and haploid fruiting in Cryptococcus neoformans: Association with the α-mating type. Proc Natl Acad Sci 93:7327–7331.
  17. 17. Fu J, Morris RM, Wickes BL (2013) The production of monokaryotic hyphae by Cryptococcus neoformans can be induced by high temerature arrest of the cell cycle and is independent of same-sex mating. PLoS Pathog 9:e1003335.
  18. 18. Hatch MD (1992) C4 photosynthesis: An unlikely process full of surprises. Plant Cell Physiol 33:333–342.
  19. 19. Gotz R, Gnann A, Zimmermann FK (1999) Deletion of the carbonic anhydrase-like gene NCE103 of the yeast Saccharomyces cerevisiae causes an oxygen-sensitive growth defect. Yeast 15:855–864.
  20. 20. Bahn YS, Cox GM, Perfect JR, Heitman J (2005) Carbonic anhydrase and CO2 sensing during Cryptococcus neoformans growth, differentiation, and virulence. Curr Biol 15:2013–2020.
  21. 21. Klengel T, Liang WJ, Chaloupka J, Ruoff C, Schroppel K, et al. (2005) Fungal adenylyl cyclase integrates CO2 sensing with cAMP signaling and virulence. Curr Biol 15:2021–2026.
  22. 22. Mogensen EG, Janbon G, Chaloupka J, Steegborn C, Fu MS, et al. (2006) Cryptococcus neoformans senses CO2 through the carbonic anhydrase Can2 and the adenylyl cyclase Cac1. Eukaryot Cell 5:103–111.
  23. 23. Hewett-Emmett D, Tashian RE (1996) Functional diversity, conservation, and convergence in the evolution of the alpha-, beta-, and gamma-carbonic anhydrase gene families. Mol Phylogenet Evol 5:50–77.
  24. 24. Smith KS, Ferry JG (2000) Prokaryotic carbonic anhydrases. FEMS Microbiol Rev 24:335–366.
  25. 25. So AK, Espie GS, Williams EB, Shively JM, Heinhorst S, et al. (2004) A novel evolutionary lineage of carbonic anhydrase (epsilon class) is a component of the carboxysome shell. J Bacteriol 186:623–630.
  26. 26. Narasipura SD, Chaturvedi V, Chaturvedi S (2005) Characterization of Cryptococcus neoformans var. gattii SOD2 reveals distinct roles of the two superoxide dismutases in fungal biology and virulence. Mol Microbiol 55:1782–1800.
  27. 27. Ren P, Springer DJ, Behr MJ, Samsonoff WA, Chaturvedi S, et al. (2006) Transcription factor STE12alpha has distinct roles in morphogenesis, virulence, and ecological fitness of the primary pathogenic yeast Cryptococcus gattii. Eukaryot Cell 5:1065–1080.
  28. 28. Kurtzman CP (2007) Blastobotrys americana sp. nov., Blastobotrys illinoisensis sp. nov., Blastobotrys malaysiensis sp. nov., Blastobotrys muscicola sp. nov., Blastobotrys peoriensis sp. nov. and Blastobotrys raffinosifermentans sp. nov., novel anamorphic yeast species. Int J Syst Evol Microbiol 57:1154–1162.
  29. 29. Narasipura SD, Ault JG, Behr MJ, Chaturvedi V, Chaturvedi S (2003) Characterization of Cu,Zn superoxide dismutase (SOD1) gene knockout mutant of Cryptococcus neoformans var. gattii:role in biology and virulence. Mol Microbiol 47:1681–1694.
  30. 30. Yu JH, Hamari Z, Han KH, Seo JA, Dominguez YR, et al. (2004) Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi. Fungal Gen Biol 41:973–981.
  31. 31. Cronan JE Jr, Waldrop GL (2002) Multi-subunit acetyl-CoA carboxylases. Prog Lipid Res 41:407–435.
  32. 32. Tabita FR, Hanson TE, Li H, Satagopan S, Singh J, et al. (2007) Function, structure, and evolution of the RubisCO-like proteins and their RubisCO homologs. Microbiol Mol Biol Rev 71:576–599.
  33. 33. Idnurm A, Bahn YS, Nielsen K, Lin X, Fraser JA, et al. (2005) Deciphering the model pathogenic fungus Cryptococcus neoformans. Nat Rev Microbiol 3:753–764.
  34. 34. Alspaugh JA, Pukkila-Worley R, Harashima T, Cavallo LM, Funnell D, et al. (2002) Adenylyl cyclase functions downstream of the Galpha protein Gpa1 and controls mating and pathogenicity of Cryptococcus neoformans. Eukaryot Cell 1:75–84.
  35. 35. Sorrell TC (2001) Cryptococcus neoformans variety gattii. Med Mycol 39:155–168.
  36. 36. Fraser JA, Subaran RL, Nichols CB, Heitman J (2003) Recapitulation of the sexual cycle of the primary fungal pathogen Cryptococcus neoformans var. gattii: implications for an outbreak on Vancouver Island, Canada. Eukaryot Cell 2:1036–1045.
  37. 37. Fisher D, Burrow J, Lo D, Currie B (1993) Cryptococcus neoformans in tropical northern Australia: predominantly variant gattii with good outcomes. Aust N Z J Med 23:678–682.
  38. 38. Halliday CL, Carter DA (2003) Clonal reproduction and limited dispersal in an envronmentl population of Cryptococcus neoformans var. gattii isolate from Australia. J Clin Microbiol 41:703–711.
  39. 39. Halliday CL, Bui T, Krockenberger M, Malik R, Ellis DH, et al. (1999) Presence of alpha and a mating types in environmental and clinical collections of Cryptococcus neoformans var. gattii strains from Australia. J Clin Microbiol 37:2920–2926.
  40. 40. Chen S, Sorrell T, Nimmo G, Speed B, Currie B, et al. (2000) Epidemiology and host- and variety-dependent characteristics of infection due to Cryptococcus neoformans in Australia and New Zealand. Australasian Cryptococcal Study Group. Clin Infect Dis 31:499–508.