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

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).


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 into C. gattii adaptation for arboreal growth and the production of infectious propagules by b-CA independent mechanism (s).

Plasmids and oligonucleotides
Plasmids and oligonucleotides used in this study are listed in Table 2. The fulllength 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 5 EU723699; CAN2 5 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.

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% CO 2 were washed with sterile water, inoculated at OD 600 5 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, 5ml of serial dilutions of yeast suspension from original stock of 10 7 /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% CO 2 ) or in 5% CO 2 .

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 CO 2 (5%) were washed twice with sterile distilled water, and were re-suspended in water at a concentration of 5 6 10 7 cells/ml. An equal number of cells of the opposite mating type cells was mixed, and 5ml of the mixture inoculated on buffered V8 medium, and incubated at 30˚C with or without CO 2 for up to 8 weeks. For monokaryotic fruiting, 5-10ml of individually washed cells (5 6 10 7 /ml) were inoculated on filament agar and buffered V8 agar media, and incubated with or without CO 2 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% CO 2 . Cells were washed with sterile distilled water, counted, and adjusted to 10 8 /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% CO 2 for respective assessments of capsule, urease, and phospholipase production. For determination of stress sensitivity, yeast cells grown as described above were serially diluted (10 3 -10 7 ), and spotted on YPD medium containing redox cycling agents menadione (3mg/ml), paraquat (1mM), sodium nitrite (0-10 mM), and sodium chloride (1-1.8 M), and incubated at 30˚C with 5% CO 2 .

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% CO 2 were washed, and then resuspended in sterile phosphate buffered saline (PBS), pH 7.4, at a concentration of 1 6 10 7 /ml. Group of five mice were injected intravenously with 10 6 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 CO 2 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 10 5 CFU of each strain in a volume of 30ml 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% CO 2 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 4mm and stained with mucicarmine (Richard-Allan Scientific, Kalamazoo, MI).

Results
CAN2 but not CAN1 is a major b-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 b-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 b-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% CO 2 ) or in a high-CO 2 (5%) environment. The can2 mutant in contrast, exhibited a severe growth defect in ambient air, but not in a high-CO 2 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 reintroduction 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-CO 2 environment (Fig. 1b).
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, a-ketoglutarate failed to complement the CAN2 is major b-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 CO 2 (0.036%; ambient air) or high CO 2 (5%) for 2-4 days. The can2 mutant did not grow on YPD agar at either 30˚C or 37˚C in low CO 2 , but grew well in high CO 2 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-CO 2 environment. Control plates were incubated directly in high CO 2 environment. Results were expressed as the percentage of C. gattii killed 5 [1-(CFU of experiment/CFU of control)] 6 100. The percentage of can2 mutant cell death was significantly higher than WT and reconstituted strains (p,0.05).
doi:10.1371/journal.pone.0113147.g001 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.
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-CO 2 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 CO 2 in both can2 mutant and in the WT strain and also CAC1 expression appeared to be marginally induced without CO 2 , 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 CO 2 environment. These results indicated that CAN2 gene expression is not regulated by CO 2 (Fig. 2c).

CO 2 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 CO 2 for photosynthesis, and they possess a CO 2 concentration mechanism through RubisCO, an enzyme specifically found in chloroplasts of bundle sheath cells [18], we asked whether high CO 2 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 CO 2 . To our surprise, we found that C. gattii WT strain undergoes hyphae development as part of monokaryotic fruiting more vigorously in high CO 2 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 CO 2 , compared to 4-weeks post-incubation under low CO 2 . 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 CO 2 but not at pH 5.0. However, filamentation was not as robust as on filament agar (data not shown).
In contrast, high CO 2 completely suppressed sexual mating (a-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 CO 2 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) 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 CO 2 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 CO 2 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 CO 2 . 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.
doi:10.1371/journal.pone.0113147.g002 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 CO 2 is a powerful inducer of monokaryotic hyphae differentiation but not in sexual mating providing important distinction in these two developmental programs in C. gattii. b-CA activity is not required for the expression of C. gattii virulence repertoire Since b-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-CO 2 environment. Similarly, mutants did not exhibit any altered sensitivity to oxidative, osmotic or nitrosative stress (Figure S2). These results indicated that b-CA activity is neither required for general stress response nor for the expression of virulence traits, at least when CO 2 is in abundance. Furthermore, b-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).
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].

Discussion
The present study revealed that high CO 2 strongly induced monokaryotic hyphae development in C. gattii while it completely repressed sexual (a-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 CO 2 through RuBisCO [32], the observed association between high CO 2 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 CO 2 (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 CO 2 compared to its appearance at 4 weeks in a low-CO 2 (ambient air) environment. Recently, CO 2 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 CO 2 while opposite was true for hyphae development where high CO 2 served as powerful inducer. These results indicate that C. gattii CAC1 may not be directly involved in CO 2 -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 CO 2 -HCO 3 2 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% CO 2 ). In this regard, C. gattii is similar to its closely related species C. neoformans where CAN2 was major b-CA for growth under ambient air [20][21][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 Figure 6. b-CA activity is dispensable for in vivo colonization. (a) Organ load determination: BALB/c mice (6-8 weeks) were infected intra-nasally with 10 5 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 CO 2 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, 6 100). (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.
doi:10.1371/journal.pone.0113147.g006 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 1-2mm in diameter, a size consistent with spores [1]. Also, all of the isolates from this outbreak belonged to MATa 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 MATa and MATa strains of C. gattii have been identified colonizing hollows in Eucalyptus trees in Australia [37][38][39][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 CO 2 conditions induced robust development of monokaryotic hyphae and spores in C. gattii. Conversely, high CO 2 completely repressed hyphae development in sexual mating. Both CAN1 and CAN2 were dispensable for CO 2 induced morphogenetic transitions and expression of pathogenic traits. Further investigations are warranted to dissect CO 2 -mediated signaling pathways to determine relevant sensor(s) required for monokaryotic fruiting. 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. doi:10.1371/journal.pone.0113147.s001 (TIF) Figure S2. b-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% CO 2 , washed, and adjusted to OD 600 51.0. The 10-fold serial dilutions were prepared and 4ml of each dilution was spotted on YPD alone, YPD containing NaNO 2 (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. doi:10.1371/journal.pone.0113147.s002 (TIF) 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.