Factors Influencing the Nitrogen-Source Dependent Flucytosine Resistance in Cryptococcus Species

ABSTRACT Flucytosine (5-FC) is an antifungal agent commonly used for treatment of cryptococcosis and several other systemic mycoses. In fungi, cytosine permease and cytosine deaminase are known major players in flucytosine resistance by regulating uptake and deamination of 5-FC, respectively. Cryptococcus species have three paralogs each of cytosine permease (FCY2, FCY3, and FCY4) and cytosine deaminase (FCY1, FCY5 and FCY6). As in other fungi, we found FCY1 and FCY2 to be the primary cytosine deaminase and permease gene, respectively, in C. neoformans H99 (VNI), C. gattii R265 (VGIIa) and WM276 (VGI). However, when various amino acids were used as the sole nitrogen source, C. neoformans and C. gattii diverged in the function of FCY3 and FCY6. Though there was some lineage-dependent variability, the two genes functioned as the secondary permease and deaminase, respectively, only in C. gattii when the nitrogen source was arginine, asparagine, or proline. Additionally, the expression of FCY genes, excluding FCY1, was under nitrogen catabolic repression in the presence of NH4. Functional analysis of GAT1 and CIR1 gene deletion constructs demonstrated that these two genes regulate the expression of each permease and deaminase genes individually. Furthermore, the expression levels of FCY3 and FCY6 under different amino acids corroborated the 5-FC susceptibility in fcy2Δ or fcy1Δ background. Thus, the mechanism of 5-FC resistance in C. gattii under diverse nitrogen conditions is orchestrated by two transcription factors of GATA family, cytosine permease and deaminase genes.

(2). C. neoformans and C. gattii species share 85 to 90% genomic similarity, and infections caused by the two species complexes are treated by the same therapeutic measures (3). However, the fundamental biological differences between the two species have been increasingly revealed as the two species complexes have been closely examined. C. neoformans is distributed worldwide while the C. gattii was known to be concentrated in subtropical to tropical regions (4)(5)(6) until the outbreak of C. gattii infection occurred in Vancouver Island, Canada, during early 2000 (7). The major environmental niche of C. neoformans is pigeon droppings and trees, whereas C. gattii has not been isolated from pigeon droppings (4). The morphology of yeast cells in both species is indistinguishable, but the morphology of the sexual spores is distinct (8,9). The utilization of amino acids as nitrogen source is different between the two species complexes (10,11). For example, L-phenylalanine and L-tryptophan can be utilized by C. neoformans but not by C. gattii. D-alanine and D-proline can be utilized by C. gattii but not by C. neoformans. Most isolates of C. neoformans are susceptible to canavanine, a structural analog of arginine while isolates of C. gattii are resistant to the compound (12,13). The nitrogen utilization in both species complexes, however, is regulated by the GATA family transcription factor Gat1 (10).
Flucytosine (5-FC) is a fluorinated cytosine currently used in combination with amphotericin B or azoles for the treatment of cryptococcosis (14)(15)(16). 5-FC itself is a prodrug that does not have any antifungal property but its metabolized product prevents fungal growth. 5-FC is transported into fungal cells via Fcy2, a purine-cytosine permease. 5-FC is then converted to 5-fluorouridine (5-FUrd) by Fcy1, a cytosine deaminase. An uracil phosphoribosyltransferase, Fur1, then mediates the conversion of 5-FUrd to 5-fluorouridine monophosphate (5-FUMP). The 5-FUMP is further metabolized to 5-fluorouridine triphosphate, which is integrated into RNA and inhibits protein synthesis, or to 5-fluorodeoxyuridine monophosphate (5-FdUMP), which inhibits DNA synthesis and cell division (17,18). The major drawbacks of 5-FC treatment are that the fungal cells rapidly develop resistance to the drug and thus cannot be used for monotherapy (19). The combination therapy of 5-FC with amphotericin B renders the reduction of polyene toxicity and increases the therapeutic efficiency (20,21).
Although 5-FC is used as a gold standard for the treatment of cryptococcosis, the 5-FC resistance mechanism in cryptococci is less than clear. There have been several studies elucidating the mechanism of 5-FC resistance in Cryptococcus species. By transcriptome analysis, Song et al. revealed 59 novel genes that are associated with the C. neoformans response to 5-FC. These genes are known to be involved in various aspects of cellular functions, including signal transduction, translation, translational modification and protein turnover, structure and biogenesis of ribosome, or control of cell cycle, cell division, and chromosome partitioning (22). The key components responsible for fungal resistance to 5-FC are known to be the FCY2-FCY1-FUR1 pathway, but based on comparative sequence analysis of 5-FC resistant C. gattii clinical isolates Vu et al. suggested the presence of another protein or pathway that may affect 5-FC susceptibility in C. gattii (23). In addition, a classical type I myosin, Myo5, has been reported to be involved in 5-FC accumulation in the yeast cells of C. gattii (24), and the UXS1 that encodes UDP-glucuronic acid decarboxylase involved in capsule biosynthesis is associated with 5-FC resistance in Cryptococcus (25). A recent report demonstrated that mutations in UXS1 cause accumulation of UDP-glucuronic acid in the cell which downregulates FCY2 and reduce cellular uptake of the drug (19).
Even with these efforts to elucidate the 5-FC resistance mechanism in Cryptococcus species, no work has been attempted to compare the 5-FC resistance between C. neoformans and C. gattii in the light of nitrogen metabolism, one of the major biochemical differences between complexes of C. neoformans and C. gattii species. There is one study that showed the relationship between nitrogen source and 5-FC resistance in the model yeast Saccharomyces cerevisiae. Costa et al. showed by transcriptome analysis that nitrogen, arginine, cell wall, and lipid metabolism are associated with 5-FC resistance in the yeast (26). In addition, they showed supplement of arginine in growth media increases the 5-FC resistance of Candida glabrata (26). These reports suggest how considerable aspects of 5-FC metabolism remain unexplored in cryptococci.
In this study, we aimed to elucidate the nitrogen-source dependent 5-FC resistance mechanism in Cryptococcus species. Although the cytosine permease Fcy2 and cytosine deaminase Fcy1 play the major role in uptake and deamination of the drug as previously reported (19), we found the secondary cytosine permease gene, FCY3, and cytosine deaminase gene, FCY6, to function in the nitrogen-source dependent 5-FC resistance only in C. gattii. We identified that two GATA family transcription factors, GAT1 and CIR1, control the gene expression of FCY3 and FCY6, thereby regulating 5-FC susceptibility in C. gattii. Furthermore, we demonstrated that the expression of most of these genes was repressed in the presence of NH 4 . Our study provides further understanding on the mechanism of 5-FC resistance, which is coordinated by Gat1 and Cir1 transcription factors, cytosine permeases, and cytosine deaminases.

RESULTS AND DISCUSSIONS
C. neoformans and C. gattii behave differently in nitrogen-source dependent 5-FC resistance. Nitrogen metabolism, especially that of arginine, is known to be involved in the susceptibility to 5-FC in S. cerevisiae, and supplementation of arginine in the growth media increases the resistance of C. glabrata to 5-FC (26). Interestingly, we found that 5-FC uptake was affected by different nitrogen sources in the growth media in C. gattii R265 strain (Fig. S1). We suspected that 5-FC susceptibility of Cryptococcus species may be influenced by different nitrogen sources in growth media. We tested 11 different amino acids as the sole nitrogen source and used 5-FC Etest strips to determine the MIC among three representative genome sequenced strains, C. neoformans H99 (VNI), C. gattii R265 (VGIIa), and WM276 (VGI) ( Fig. 1 and Fig. S2). Among the 11 tested amino acids, several amino acids affected the growth of the wildtype strains. We focused our studies on 3 amino acids, arginine (Arg), asparagine (Asn), and proline (Pro), for the following reasons. These three amino acids did not affect growth of tested strains and produced markedly different susceptibility to 5-FC compared to NH 4 . Furthermore, Asn affected the 5-FC uptake of C. gattii (Fig. S1), Arg is known to affect 5-FC susceptibility of C. glabrata (26), and proline metabolism gene, PRO2, was differentially expressed by 5-FC treatment in S. cerevisiae (26). No clear difference of 5-FC MIC was observed in C. neoformans H99 on different nitrogen sources except for slightly higher MIC on Pro media ( Fig. 1 and Table S1). It is known that many biological processes are influenced by the presence of NH 4 in the culture media which is termed nitrogen catabolic repression (NCR) (27)(28)(29). We supplemented 10 mM NH 4 to the media containing different amino acid and measured the 5-FC MIC. Addition of NH 4 to Pro media slightly reduced the 5-FC MIC in H99 (0.094 versus 0.032 to 0.094 mg/mL) while addition of NH 4 to other media had no effect. The difference of 5-FC MIC among different medium was clearly higher in C. gattii R265 than in H99. The 5-FC MIC was highest on Pro media for R265 (0.125 to 0.25 mg/mL, Fig. 1, and Table S1). R265 showed lower 5-FC MIC on Arg and Asn than on NH 4 . Interestingly, addition of NH 4 increased the 5-FC MIC of R265 on Arg and Asn but decreased the 5-FC MIC on Pro. The 5-FC MIC was lowest in C. gattii WM276 in most of the tested nitrogen sources compared to H99 and R265. WM276 showed similar 5-FC MIC on all media with or without supplement of NH 4 ( Fig. 1 and Table S1). These results suggested that the nitrogen source in growth media affects the 5-FC resistance differently among different lineages of cryptococcal strains.
It is known that FCY2-FCY1-FUR1 pathway is important for 5-FC resistance in fungal species. To determine the importance of this pathway in cryptococcal 5-FC susceptibility, we deleted FCY2, FCY1, and FUR1 in both C. neoformans (H99) and C. gattii (R265 and WM276). As expected, all the fcy1D, fcy2D, and fur1D mutants were resistant to 5-FC when ammonium sulfate was the sole nitrogen source (Fig. 1, Fig. S2, and Table S1), suggesting that in the presence of ammonium sulfate FCY2, FCY1, and FUR1 are the major genes contributing to the 5-FC susceptibility in Cryptococcus species. However, differences were observed in 5-FC susceptibility among the three strains when they were tested on YNB media containing Arg, Asn, or Pro as the sole nitrogen source ( Fig. 1 and Table S1). Both the fcy1D and fcy2D mutants of H99 were resistant to 5-FC on all three tested amino acids, whereas the fcy2D mutant of R265 was not resistant to 5-FC and showed the similar 5-FC MIC as the wild type on the media containing Arg and Asn. In addition, the fcy2D mutant of R265 had higher MIC and exhibited high background growth in the 5-FC inhibitory zone on the Pro media. The fcy1D mutant of WM276 was resistant to 5-FC on these nitrogen sources except for Asn, which had a reduced 5-FC susceptibility with higher MIC compared to the wild type (0.25 to 0.38 versus 0.008 to 0.023 mg/mL). In contrast, the WM276 fcy2D mutant was still susceptible to 5-FC and displayed the similar 5-FC MIC as the wild type on all three amino acids.
These results suggested the possible existence of secondary permeases and deaminases that function under different nitrogen sources in C. gattii species complex. Strikingly, all the fcy1D and fcy2D mutants of R265 and WM276 became resistant to 5-FC when ammonium sulfate was supplemented in the culture media, indicating that ammonium sulfate can suppress all the effects exerted by different amino acids on 5-FC susceptibility in C. gattii species complex. Unlike the fcy1D and fcy2D mutants, the fur1D mutants from both C. neoformans and C. gattii lineages were resistant to 5-FC on all tested conditions (Fig. 1, Fig. S2, and Table S1). Taken together, C. neoformans H99

FIG 1
The 5-FC susceptibility is different on different nitrogen sources in Cryptococcus species. Each indicated strain was spread on YNB without ammonium sulfate without amino acid agar media supplemented with 2% glucose and 10 mM indicated nitrogen source. "1NH 4 " indicates additional 10 mM ammonium sulfate was supplemented to the indicated amino acid containing media. After application of 5-FC MIC test strip, plates were incubated for 3 days at 30°C and photographed. The experiments were repeated at least two times.
Flucytosine Resistance in Cryptococcus Species mBio and two C. gattii strains, R265 and WM276, have different mechanisms for nitrogen-source dependent 5-FC resistance governed by cytosine permeases and cytosine deaminases. FCY3 and FCY6 are the secondary cytosine permease and cytosine deaminase genes, respectively, in C. gattii. Based on the results of 5-FC Etest for the fcy1D and fcy2D mutants, we hypothesized that there are other cytosine deaminase(s) and cytosine permease(s) involved in nitrogen source dependent 5-FC susceptibility of C. gattii. In S. cerevisiae, there is only one cytosine deaminase gene, FCY1, but three cytosine permease genes, FCY2, FCY21, and FCY22. The amino acid sequence of FCY21 and FCY22 are very similar to FCY2, but they cannot substitute the function of FCY2 (30). Furthermore, S. cerevisiae contains a URK1 gene showing high similarity to uracil phosphoribosyltransferase gene (FUR1). BLAST searches indicated that two genes showing high similarity to FCY2 exist in the genome of H99, R265 and WM276, which we designated FCY3 and FCY4 (Table S2). Additionally, two genes showing low similarity to FCY1 were found in the three strains, which we designated FCY5 and FCY6 (Table S2). We also found that there are two genes showing similarity to FUR1, which we named FUR2 and URK1/FUR3 (Table S2).
To elucidate if other permease gene(s) were involved in the 5-FC resistance under different nitrogen source on which the fcy2D mutant exhibited susceptibility to 5-FC, we constructed double knockout as well as triple knockout mutants with all possible combinations of FCY2, FCY3, and FCY4 genes. We focused our studies on WM276 (VGI) strain because this strain not only showed the greatest variations in 5-FC susceptibility ( Fig. 1) but also is the most frequent lineage isolated from global clinical cases (2). The fcy3D, fcy4D, and fcy3D fcy4D mutants exhibited the similar 5-FC MIC as the WT strain in all tested media ( Fig. 2A). However, the fcy2D fcy3D and fcy2D fcy3D fcy4D mutants were both resistant to 5-FC on all nitrogen sources, indicating that FCY3 is the secondary cytosine permease gene in WM276. FIG 2 FCY3 and FCY6 are the secondary cytosine permease and cytosine deaminase genes important for 5-FC resistance. Cytosine permease deletion mutants (A) and cytpsome deaminase deletion mutants (B) derived from WM276 strain were spread on YNB without ammonium sulfate without amino acid agar media supplemented with 2% glucose and 10 mM indicated nitrogen source. After application of 5-FC MIC test strip, plates were incubated for 3 days at 30°C and photographed. The experiments were repeated at least two times.

Flucytosine Resistance in Cryptococcus Species mBio
To identify a secondary cytosine deaminase gene, we constructed double and triple knockout mutants in all possible combinations of FCY1, FCY5, and FCY6 genes in WM276 strain and tested their susceptibility to 5-FC. 5-FC susceptibility of the fcy5D, fcy6D, and fcy5D fcy6D mutants was similar to the WT strain in all media (Fig. 2B). The fcy1D fcy5D mutant showed a 5-FC MIC similar to the fcy1D mutant on Asn media. In contrast, the fcy1D fcy6D and fcy1D fcy5D fcy6D mutants were resistant to 5-FC in all media. Furthermore, since we found the fcy1D mutant like that on Asn was also susceptible to 5-FC on media containing alanine (Ala), leucine (Leu), methionine (Met), and serine (Ser) (Fig. S2), we also examined the effects of FCY6 deletions in those media. Similarly, only the fcy1D fcy6D and fcy1D fcy5D fcy6D mutants were clearly resistant to 5-FC on all four media (Fig. S3). Taken together, these results suggested that FCY6 is the secondary cytosine deaminase gene involved in the 5-FC metabolism of WM276.
RNA-seq analysis indicates FCY2-FCY1-FUR1 pathway related genes are differentially expressed in different nitrogen source. Since nitrogen source had great impacts on 5-FC susceptibility in WM276, we performed RNA-seq from cells grown in media containing NH 4 , Asn, and Pro as the sole source of nitrogen to explore the transcription network functioning under these conditions. We found 741 genes upregulated and 471 genes downregulated in Asn compared to NH 4 and 763 genes upregulated and 707 genes downregulated in Pro. Among the differentially expressed genes, the number of genes commonly up-or downregulated between Asn and Pro conditions was 302 and 272, respectively. We found that the expression levels of three putative cytosine permeases FCY2, FCY3, and FCY4, and three putative cytosine deaminases FCY1, FCY5, and FCY6 genes were differentially expressed (Table 1). For the uracil phosphoribosyltransferase genes, only two of the three paralogue genes, FUR1 and FUR2, showed expression changes greater than 2-fold under different nitrogen source (Table 1). More importantly, the RNA levels of FCY3 significantly increased in Asn (115-fold) and Pro (174-fold) compared to NH 4 as determined by quantitative RT-PCR, which corroborated the observed a Only the genes showed false discovery rate (FDR) ,0.05 in at least one set of comparison are displayed. Fold change is the ratio of the relative expression levels.
Flucytosine Resistance in Cryptococcus Species mBio phenotype of the fcy3D mutants described above ( Fig. 2A). FCY6 RNA levels were significantly increased in the presence of Asn (14-fold) compared to NH 4, which also corroborated the observed phenotypes of the fcy6D mutants (Fig. 2B). GAT1 and CIR1 are involved in the 5-FC resistance on different nitrogen sources. Because supplementation of ammonium sulfate in different amino acids media altered the 5-FC susceptibility in the FCY1 and FCY2 deletion mutants (Fig. 1), it was possible that the expression of the genes encoding cytosine permease and deaminase was under NCR. Transcription factors of GATA family are known to be involved in the regulating NCR sensitive genes under the absence or limitation of preferred nitrogen source in Cryptococcus (10). We found four of the 10 GATA family transcription factors, GAT1, GLN3, GAT201 and CIR1, were differentially expressed in different amino acid media (Table 1). To investigate whether these transcription factors play a role in the nitrogen source-dependent 5-FC resistance in C. gattii, deletion mutant of each gene was constructed in various WM276 background and tested for their 5-FC susceptibility.
First, the gat1D mutant grew poorly and showed lower MIC compared to WM276 in all tested media ( Fig. 3A; photographs of 3-day incubation and Fig. S4A; photographs of 6-day incubation). Addition of NH 4 to the three amino acid media slightly improved Flucytosine Resistance in Cryptococcus Species mBio the growth and marginally increased the MIC of the gat1D mutant. Interestingly, deletion of GAT1 in the strains of fcy1D background became resistant to 5-FC in the Asn media ( Fig. 3A and Fig. S4A). Since the fcy1D fcy6D mutant was resistant to 5-FC in the Asn media (Fig. 2B), it was possible that GAT1 positively regulates FCY6 and, hence, deletion of GAT1 in the fcy1D background altered its 5-FC susceptibility. Also, deletion of GAT1 in the fcy2D strain background resulted in the 5-FC resistance in all media ( Fig. 3A and Fig. S4A). Since the fcy2D fcy3D strain was resistant to 5-FC in all media ( Fig. 2A), it was possible that GAT1 positively regulates FCY3 and, therefore, deletion of GAT1 in the fcy2D background resulted in the changes of 5-FC susceptibility. Next, the cir1D mutant showed a similar growth rate and level of 5-FC resistance on NH 4 medium but it was more susceptible to 5-FC on Arg, Asn, and Pro media compared to the wild type (WM276 MIC 0.012 mg/mL versus cir1D MIC 0.008 mg/mL in Arg, 0.012 mg/mL versus 0.006 mg/mL in Asn, and 0.012 mg/mL versus 0.008 mg/mL in Pro) (Fig. 3B). Supplementation of NH 4 to each of the three amino acid media restored the cir1D levels of 5-FC MIC similar to the wild type. Deletion of CIR1 in the fcy1D background markedly reduced the 5-FC MIC on Arg and Asn media compared to that of the fcy1D mutant. In addition, we observed a clear halo of the fcy1D cir1D mutant on Pro media after 2 days incubation (Fig. S4B), but this halo became very faint on the 3rd day (Fig. 3B). Since the fcy1D fcy6D and fcy1D fcy6D cir1D mutants were resistant to 5-FC on those media ( Fig. 2B and Fig. 3B), it was possible that CIR1 negatively regulates the expression of FCY6 and, consequently, deletion of CIR1 in the fcy1D background decreases the 5-FC resistance. Deletion of CIR1 in the fcy2D mutant markedly reduced the MIC on all media supplemented with NH 4 compared to that of the fcy2D mutant (Fig. 3B). We further constructed the fcy2D fcy3D cir1D mutant and measured its 5-FC MIC. Interestingly, as the fcy2D fcy3D mutant, the fcy2D fcy3D cir1D mutant was completely resistant to 5-FC on all tested condition (Fig. 3B). These results indicated that CIR1 negatively regulates the 5-FC resistance of C. gattii in FCY3 dependent manner.
We also constructed deletion mutants of the other two GATA family transcription factors, GLN3 and GAT201, whose expressions were influenced by nitrogen source ( Table 1). The 5-FC susceptibility of all GLN3 and GAT201 deletion mutants constructed either in the wild-type background or in the fcy2D background was similar to the control strains in all tested conditions ( Fig. S4C and D). These results indicated that the GLN3 and GAT201 genes were not involved in the regulation of 5-FC susceptibility.
Expression of FCY genes is influenced by nitrogen source and is under the NCR regulation. To examine and confirm the RNA-seq results that the expression of FCY genes was affected by different nitrogen source in growth media, qRT-PCR was performed using RNA isolated from WM276 grown under various nitrogen conditions. First, we determined the expression levels of the two cytosine deaminase genes, FCY1 and FCY6, which was found to play a role in 5-FC resistance in the study of deletion mutants. Although the expression of the major cytosine deaminase FCY1 was significantly increased on Arg and Pro compared to NH 4 media, the increase was less than 2fold (Fig. 4A). On Asn, no significant change was observed in the expression of FCY1. We noted in the RNA-seq data that the expression levels of FCY1 on Asn and Pro had small decrease (,3-fold) compared to that of the NH 4 media ( Table 1). Since it is known that RNA-seq data are prone to have large variations, we concluded that the expression of FCY1 was not markedly affected by different nitrogen source based on the qRT-PCR result. The secondary cytosine deaminase FCY6 showed significantly increased expression on Asn (more than 41-fold) and Pro (more than 68-fold) (Fig. 4B). Furthermore, the increases in FCY6 expression were abolished when NH 4 was added to the amino acid containing media indicating that FCY6 expression was under the NCR regulation. However, the expression levels of FCY6 showed no statistically significant changes on Arg compared to NH 4 .
Next, we measured the expression levels of the two cytosine permeases, FCY2 and FCY3. Both genes displayed significantly higher levels of expression on Arg, Asn, and Pro compared to NH 4 . FCY2 expression was induced about 90-fold on Arg, 76-fold on Asn, and 49-fold on Pro compared to that of NH 4 (Fig. 4C). Similarly, FCY3 expression was induced about 53-fold on Arg, 197-fold on Asn, and 163-fold on Pro compared to that of NH 4 (Fig. 4D). The induction of cytosine permease gene expression disappeared when NH 4 was added to Arg, Asn, or Pro media. These results indicate that similarly to FCY6, both FCY2 and FCY3 expression were also under the NCR regulation.
The expression of GAT1 and CIR1 is influenced by nitrogen sources and is under the regulation of NCR. Based on 5-FC MIC data of deletion mutants and qRT-PCR results of the FCY genes, we hypothesized that GAT1 and CIR1 are involved in the nitrogen-source dependent 5-FC resistance via the transcriptional regulation of the FCY genes. To investigate the function of GAT1 and CIR1 in regulating the expression of FCY genes, we performed the qRT-PCR using RNA isolated from the wild-type strain, gat1D, and cir1D strains. First, we found the expression levels of GAT1 were significantly induced on Arg (3.1-fold) and Pro (4.8-fold) but not on Asn (1.8-fold) compared to NH 4 in the wild-type WM276 strain (Fig. 5A). In contrast, the expression levels of CIR1 were significantly induced on Arg (2.7-fold), Asn (5.1-fold) and Pro (3.6-fold) compared to NH 4 in the wild-type strain (Fig. 5B). We observed the similar changes of CIR1 expression in RNA-seq results (Table 1). However, GAT1 expression was induced on both Asn (2.2-fold) and Pro (3.5-fold) in RNA-seq results. Interestingly, the increased expression of GAT1 and CIR1 all disappeared when NH 4 was supplemented in media containing different amino acids indicating that the expression of GAT1 and CIR1 is also under NCR. Furthermore, when CIR1 was deleted, the GAT1 expressions was not affected in all tested nitrogen sources compared to the wild type (Fig. 5A) indicating that CIR1 does not regulate the expression of GAT1 in the tested media. Nonetheless, the CIR1 expression was significantly increased on NH 4 and decreased on Asn in the gat1D mutant compared to the wild-type strain (Fig. 5B) indicating that GAT1 regulates the expression of CIR1 on NH 4 and Asn media. GAT1 and CIR1 regulate the expression of FCY genes. We then examined the roles of GAT1 and CIR1 in the expression of FCY genes. For the cytosine deaminases, deletion of GAT1 significantly affected the expression of FCY1 on Pro (2.5-fold) and deletion of CIR1 significantly affected the expression of FCY1 on Asn (2.8-fold) (Fig. 6A). Since the fluctuation of the FCY1 expression levels was small in all tested conditions even in the wild-type background, it is likely that FCY1 expression is not impacted by nitrogen-source dependent regulation. In contrast, the expression of FCY6 was significantly decreased in the gat1D mutant on all media which supports our previous notion that GAT1 positively regulates the expression of FCY6. Furthermore, the decrease of FCY6 expression might result in smaller amount of cytosine deaminase and contributes to the increased 5-FC resistance in the fcy1D gat1D mutant compared to the fcy1D mutant on Asn media (Fig. 3A). In contrast, FCY6 expression in the cir1D mutant was significantly increased on media containing NH 4 or Arg compared to the wild-type strain while the FCY6 expression levels on Asn media were significantly lower in the cir1D than in the wild-type strain. Regardless of the differences, the expression levels of FCY6 in cir1D mutant were still higher on Arg (3.5-fold), Asn (4-fold), and Pro (7.9fold) compared to NH 4 (Fig. 6B). Thus, when CIR1 was deleted in the fcy1D mutant, the increased FCY6 expression might result in the larger amount of cytosine deaminase and contributes to the higher 5-FC susceptibility on Arg, Asn, and Pro media compared to the fcy1D mutant (Fig. 3B).
For the cytosine permease, the FCY2 expression was significantly increased in NH 4 (39-fold) in the gat1D mutant (Fig. 6C) indicating that GAT1 negatively regulates the expression of FCY2 under preferred nitrogen source. In addition, FCY2 expression was significantly decreased in Arg (1.7-fold), Asn (2.4-fold), and Pro (1.4-fold) in the gat1D mutant compared to the wild type. The FCY2 expression in the cir1D mutant was significantly increased on NH 4 (3.3-fold) compared to the wild type. Deletion of CIR1 caused significant decrease of FCY2 expression only in Asn (1.4-fold). For FCY3, the observed expression increases in the wild-type strain grown on three amino acids all vanished when GAT1 was deleted (Fig. 6D), indicating that GAT1 positively regulates the FCY3 expression under poor nitrogen sources. The low expression levels of FCY3 in the gat1D mutant corroborated with the 5-FC resistance phenotype of fcy2D gat1D mutant on all tested nitrogen conditions ( Fig. 3A and 6D). In contrast, the expression of FCY3 was significantly increased in the cir1D mutant on NH 4 and Arg media. This suggests that CIR1 negatively regulates the expression of FCY3 on NH 4 and Arg media and contributes to the increased 5-FC susceptibility in the fcy2D cir1D compared to fcy2D mutant on NH 4 and Arg media (Fig. 3B and 6D). In comparison, the expression levels of FCY3 in the cir1D mutant were similar to the levels of the wild type on Asn and Pro media. However, the fcy2D cir1D mutant was more susceptible to 5-FC than the fcy2D mutant on Asn and Pro (Fig. 3B). Since the expression of FCY1 was significantly upregulated in the cir1D mutant (Fig. 6A), it is likely that increased expression of FCY1 may have resulted in the larger amount of cytosine deaminase and contributed to the 5-FC susceptibility in the fcy2D cir1D mutant on Asn and Pro.
Taken together, we show that the two GATA transcription factors, GAT1 and CIR1, either positively or negatively regulate the expression of the paralogs in FCY2-FCY1-FUR1 pathway under different nitrogen conditions in C. gattii WM276. Fig. 7 summarizes the results of these interactions.
Our study reveals that nitrogen source in growth media affects the 5-FC susceptibility differently in Cryptococcus species. Importantly, we show that several genes related to 5-FC susceptibility are under the influence of NCR. NCR is a global transcription mechanism, including downregulation of genes involved in the utilization of poor nitrogen when the preferred nitrogen sources are available (for review see 28,31). Generally, ammonium and glutamate are the preferred nitrogen sources for protein synthesis in eukaryotic microorganisms. In S. cerevisiae, when favorable nitrogen sources are available, NCR inhibits the expression of genes encoding permeases as well as catabolic enzymes which are required for cellular entry and utilization of poor nitrogen sources such as arginine, urea, allantoine, GABA (g -aminobutyrate) and proline (28). Here, we show that not only the FCY genes are under NCR, but also GAT1 and CIR1 themselves are under NCR in nitrogen sources such as Arg, Asn and Pro (Fig. 7). In S. cerevisiae, it is known that GATA family transcription factors can regulate the expression of each other via the GATA motif. For instance, DAL80 expression is Gln3 and Gat1dependent and regulated by Dal80 itself (32). Moreover, Gln3 and Dal80 bind to promoter of DAL80. In turn, GAT1 expression is Gln3 dependent and Dal80 regulated but is not autogenously regulated like DAL80 (32). It is possible that similar regulation of GAT1 and CIR1 also exists in Cryptococcus. Because the environmental niches are different between C. neoformans and C. gattii, it is possible that NCR may contribute to the different growth behavior in these species.
In this study, we identified the paralogs of the cytosine permease and deaminase genes in Cryptococcus species and revealed their roles in 5-FC resistance by deleting the genes in C. gattii WM276. We identified and deleted three paralogs of cytosine deaminase and permease genes in C. gattii WM276. The function of secondary cytosine permease FCY3 and deaminase FCY6 in 5-FC resistance is manifested only under the poor nitrogen source conditions. Although FCY4 and FCY5 were differentially expressed under different nitrogen sources and under NCR ( Fig. S5A and B), they were not important for 5-FC resistance under our tested conditions. Three paralogue genes encoding uracil phosphoribosyltransferase, FUR1, FUR2, and URK1/FUR3 were found in WM276. FUR1 expression was induced on Arg, Asn, and Pro compared to NH 4 and was under the regulation of NCR (Fig. S5C). Additionally, FUR1 expression was negatively regulated by GAT1 in all tested nitrogen sources but was not regulated by CIR1 (Fig. S5D and Fig. 7D). FUR2 was also under the regulation of NCR (Fig. S5E and Fig. 7E). FUR2 expression was positively regulated by GAT1 on Arg, Asn, and Pro. FUR2 expression was also positively regulated by CIR1 on Asn and Pro ( Fig. S5F and Fig. 7D). Since the fur1D mutants were resistant to 5-FC in all tested conditions, we did not pursue the function of FUR2 and URK1/FUR3 in 5-FC resistance. It is not clear if the paralogs of FCY2-FCY1-FUR1 pathway have additional roles in other biological or experimental environments. Nevertheless, several studies suggest that cryptococcal cells encounter nutritional limitations under infection conditions (33)(34)(35)(36). Furthermore, RNA-seq analysis of C. gattii R265 has revealed that intense modulation of gene expression in the pathways of amino acid metabolism and transporters takes place during infection (37). It would be interesting to learn if the paralogs of cytosine permease and deaminase play any role in the cryptococcal response to 5-FC during treatment of infection.
We found four GATA family transcription factors, GAT1, GLN3, GAT201 and CIR1, which were differentially expressed under different nitrogen source but only GAT1 and CIR1 were found important for 5-FC susceptibility. It has been shown that CIR1 regulates nitrogen acquisition by direct regulation of amino acid permease genes, including AAP2, AAP4, AAP5, and AAP7 and ammonium transporter genes, AMT1 and AMT2 in C. neoformans (38). We also found that the iron related genes, CFT2, CFO2, FRE6, and FRE7, which are known to be regulated by CIR1 were also differentially regulated by nitrogen source ( Table 1) which indicates that iron metabolism pathway may be involved in the nitrogen response in C. gattii. Nonetheless, the importance of those genes in 5-FC resistance remains to be elucidated. In conclusion, present study extends our understanding of 5-FC resistance mechanisms in Cryptococcus species and further reveals the biological differences between the two species complex, C. neoformans and C. gattii.

MATERIALS AND METHODS
Strains and culture conditions. The strains used in this study are listed in Table S3. Strains were cultured in yeast extract-peptone-dextrose (YPD) medium. Yeast nitrogen base (YNB) without amino acids and without ammonium sulfate containing 2% glucose and 10 mM indicated nitrogen source (ammonium sulfate [NH 4 ], arginine [Arg], asparagine [Asn], and proline [Pro]) was used to test the nitrogensource dependent 5-FC resistance.
Construction of deletion mutant strains. The strains were constructed by either biolistic transformation (39) or CRISPR-Cas9 coupled electroporation system (40). Briefly, the Cas9 construct was amplified from pXL1-Cas9 by using GPD1 promoter L and GPD1 terminator R primer pair. The single guide RNA (sgRNA) construct was amplified from pRH003 plasmid by overlap PCR. To generate the gene deletion cassette, the 59 and 39 flanking region of target gene were amplified from genomic DNA. Then, 59 flanking region, selection marker gene (HYG r , NEO r , or NAT r gene), and 39 flanking region were assembled by second round PCR for the complete gene deletion cassette. The primers used in this study were listed in Table S4. DNA was delivered by electroporation using Bio-Rad electroporator with 0.7 kV and 25 X. Drug resistant transformants were screened by colony PCR and confirmed by Southern blotting analysis.
5-FC susceptibility test. The 5-FC susceptibility of each strain was determined by plating approximately 2 Â 10 6 cells on YNB plates without amino acids and without ammonium sulfate supplemented with 2% glucose and 10 mM amino acids (ammonium sulfate [NH 4 ], arginine [Arg], asparagine [Asp], or proline [Pro]) as a nitrogen source followed by application of the Liofilchem MTSTM (MIC Test Strips) (Liofilchem, Waltham, MA). The plates were incubated for indicated days at 30°C and photographed.
RNA extraction, sequencing, and differential expression analysis. Cells were grown in 20 mL YPD medium at 30°C for 16 h. Then, about 5 mL of the overnight culture was inoculated into 100 mL of fresh YPD medium to make the OD 600 of culture as 0.4 and further incubated for 4 to 5 h at 30°C until OD 600 of culture medium reached approximately 1.0. The cultures were divided into four 20 mL each in 50 mL tubes. The cells were pelleted by centrifugation at 3,500 rpm for 5 min and washed twice with 10 mL of YNB medium without ammonium sulfate and amino acid. The cells were resuspended in 10 mL of YNB with NH 4 , Arg, Asn, or Pro media and were further incubated for 2 h at 30°C and pelleted by centrifugation at 3,500 rpm for 5 min. Then, the cells were frozen in dry ice with ethanol and lyophilized overnight. Three independent cultures for each strain were prepared for RNA isolation as biological replicate. Total RNAs were isolated by the TRIzol reagent (Invitrogen, Carlsbad, CA) and RNAeasy minikit (Qiagen). RNAs were treated with Turbo RNase-free DNase (Ambion) to remove genomic DNA. RNA was checked by agarose gel electrophoresis and Agilent 2100 for integrity and quantitation. polyA-RNA was purified from total RNA, processed, and paired-end directional libraries (75 bp) were prepared and sequenced using an Illumina HiSeq 4000 platform (Novogene, Davis, CA). The RNA-seq data were analyzed by Partek Flow software, version 7.0, 2017 (Partek Inc., St. Louis, MO). Illumina reads were aligned to the reference genome of WM276 (ASM18594v1) using STAR. After trimming and mapping, approximately 15 to 38 million total paired aligned reads were recovered from libraries of each RNA-Seq. DESeq2 was used to calculate the differentially expressed genes. Differential expression deemed significant if the changes are at least 2-fold and p-adj is , 0.05.
Real-time quantitative reverse transcription-PCR (qRT-PCR). Total RNA was converted to cDNAs using a high-capacity cDNA reverse transcription kit (Applied Biosystems). qRT-PCR was performed with primers listed in Table S4 by using the SYBR green PCR master mix (Applied Biosystems) and a Quantudio 3 real-time PCR system (Thermofisher). The PCR efficiency and threshold cycle (CT) determination were performed using an algorithm as described previously (41). Data were normalized to actin gene levels and expressed as the amount relative to actin RNA levels. Each reaction was run in triplicate for technical repeats and three independent biological replicates were performed for each experimental condition. Statistical analyses were performed using Graph Pad Prism (version 8.4.3). Student's t test and ANOVA followed by multiple comparison test were used to determine whether there were significant differences between indicated samples.
Data availability. All RNA-Seq data for this study are available in the Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/) database under accession number GSE215311.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.

ACKNOWLEDGMENTS
This work was supported by the Division of Intramural Research (DIR), NIAID, NIH.