Deletion of the calcineurin docking motif affects Crz1 function in C. neoformans

The transcription factor Crz1 is a major downstream effector of calcineurin in various model and pathogenic fungi. Recent studies have identified CNAG_00156 as the putative C. neoformans CRZ1 ortholog, but drew conflicting conclusions that suggested this gene is 1) the ortholog of Crz1, 2) not the ortholog of Crz1, or 3) might be the ortholog of Crz1 [28–30]. In our recent study, we identified CNAG_00156 as a substrate of calcineurin [31], and in this study reciprocal BLAST searches revealed that CNAG_00156 is an ortholog of S. cerevisiae CRZ1 (S1 Fig). Similar results were obtained with Crz1 from C. albicans and A. fumigatus CrzA (S1 Fig). Phylogenetic analysis performed using the protein sequences of Cryptococcus Crz1, including the outgroup species Cryptococcus amylolentus and Tsuchiyaea wingfieldii, demonstrated that the Crz1 protein is highly divergent between different fungal species, but all feature at least two zinc finger domains at the C-terminus (S1 Fig).

In S. cerevisiae and C. neoformans, activated calcineurin dephosphorylates Crz1 leading to translocation of Crz1 into the nucleus and subsequent activation of stress genes [13]. In S. cerevisiae, the PIISIQ motif has been characterized as the calcineurin docking domain (CDD) in Crz1 [32]. In order to predict the CDD in C. neoformans Crz1, the PxIxIT consensus (P[^PG][IVFL][^PG][IVFL][TSHEDQNKR]) was queried against the CNAG_00156 ORF [31, 33]. We identified two candidate motifs in Crz1: ⁴⁵¹PMICIQ⁴⁵⁶ and ⁸⁶⁸PALSIS⁸⁷³ (Figs 1A and S3). Comparison of the Crz1 protein sequences in the pathogenic Cryptococcus species complex, and two outgroup species, C. amylolentus and T. wingfieldii, revealed that the PALSIS motif is conserved across all of these species. However, the PMICIQ motif was conserved among the pathogenic species, while in the outgroup species, the cysteine has been substituted with arginine (Fig 1A).

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Fig 1. Deletion of the ⁴⁵¹PMICIQ⁴⁵⁶ motif prevents translocation of Crz1 into the nucleus under thermal stress.

(A) Schematic diagram showing the location of the candidate ⁴⁵¹PMICIQ⁴⁵⁶ motif. The PMICIQ motif is conserved in the pathogenic species complex. The green box represents the PolyQ domain; red boxes represent the zinc finger domains. (B) Strains ECt172 (Crz1^WT) and ECt386 (Crz1^PMICIQΔ) were grown overnight at 24°C, washed, and total cell lysates were extracted. Samples were resolved by SDS-PAGE and Western blotting was performed to verify expression of the Crz1^WT and the mutant Crz1^PMICIQΔ constructs. (C) The CRZ1^WT-mCherry strain (ECt172) (Crz1-mCH) was grown at 24°C and then shifted to 37°C for 15 min. To verify that Crz1 translocation was due to calcineurin activity, FK506 (1 μg/ml) was added 30 min prior to the 37°C temperature shift. Strains ECt172 and ECt386 were grown at 24°C and then shifted to 38°C for 20 min. Cells were fixed with 4% formaldehyde for 15 min and washed, prior to imaging by direct fluorescence microscopy using the Zeiss Axioskop 2 Plus microscope. Scale bar = 5 μm. Note that GFP-Nop1 served as a nucleolar marker. (D) Wild-type (H99), cna1Δ (KK1), crz1Δ (AFA3-3) mutants, and strains ECt172 (crz1Δ + Crz1^WT) and ECt375 (crz1Δ + Crz1^PMICIQΔ) were grown in YPD media, washed, and resuspended in PBS. Five 10-fold serial dilutions of each strain were spotted on YPD solid media, with the various additives as described and incubated at 30°C for 48 h, unless otherwise stated. Strains were incubated at 39°C for 72 h before imaging. CFW: calcofluor white.

https://doi.org/10.1371/journal.pgen.1006667.g001

To test if ⁴⁵¹PMICIQ⁴⁵⁶ is a calcineurin docking domain, the motif was deleted from the wild-type Crz1, and both the Crz1^WT and the Crz1^PMICIQΔ mutant were fused with the mCherry protein and expressed in the crz1Δ mutant strain; expression of the Crz1-mutant construct was confirmed by Western blot (Fig 1B). We first verified if the calcineurin-dependent dephosphorylation of Crz1^WT-mCherry expressed in the crz1Δ mutant strain results in its nuclear translocation. The strain expressing the Crz1^WT-mCherry tagged protein (ECt172) was grown at 24°C, shifted to 37°C for 15 min, and then examined by direct fluorescence microscopy. At 24°C, the Crz1-mCherry signal was diffuse and evenly distributed throughout the cytosol (Fig 1C). Following incubation at 37°C for 15 min, Crz1-mCherry fluorescence was localized to the nucleus as evidenced by co-localization with the nucleolar marker protein GFP-Nop1 (Fig 1C). Furthermore, addition of the calcineurin inhibitor FK506 to the culture 30 min prior to the temperature shift prevented the translocation of Crz1 into the nucleus, consistent with Crz1 acting downstream of calcineurin (S2 Fig). Next, the strain expressing the Crz1^PMICIQΔ-mCherry tagged protein (ECt375) was grown at 24°C, shifted to 38°C for 20 min, and then examined by direct fluorescence microscopy. At 24°C, the mCherry signal was diffuse and evenly distributed throughout the cytosol (Fig 1C). Following incubation at 38°C for 20 min, mCherry fluorescence was still localized in the cytosol (Fig 1C).

Earlier studies have shown that C. neoformans calcineurin mutant strains are sensitive to high temperature and cell wall stresses [25, 26]. First, we determined if the deletion of CRZ1 results in calcineurin-related phenotypes by assessing growth of the crz1Δ mutant at 37°C and 39°C and with a panel of different cell wall stresses. As previously reported, the cna1Δ mutant exhibits dramatic temperature sensitivity at 37°C and 39°C (Fig 2A). In contrast, the crz1Δ mutant displayed a modest temperature defect at 37°C compared to wild-type, and this was more pronounced at 39°C (Fig 2A). Similar to the cna1Δ mutant, the crz1Δ mutant displayed a growth defect on media containing 0.03% SDS. However, the crz1Δ mutant displayed an intermediate phenotype compared to the wild-type and cna1Δ mutants on media containing 0.35 M CaCl₂ or 1% Congo red, and a very slight growth defect on media containing 5 mg/ml calcofluor white (Fig 2A). Complementation of the crz1Δ mutant with the wild-type CRZ1 gene restored a wild-type phenotype under these stress conditions.

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Fig 2. Deletion of CRZ1 confers partial phenotypes in comparison to wild-type and cna1Δ mutant.

(A) Wild-type (H99), cna1Δ (KK1), crz1Δ (AFA3-3) mutants and their respective complemented strains (KK5 and AFA3-3-17), and the cna1Δ crz1Δ double mutant strain (XW245) were grown in YPD media, washed, and resuspended in PBS. Five 10-fold serial dilutions of each strain were spotted on YPD solid media, with the various additives as listed and incubated at 30°C for 48 h, unless otherwise stated. Strains were incubated at 39°C for 72 h before imaging. CFW: calcofluor white. (B) Wild-type (H99 and KN99a), cna1Δ (KK1 and KK8), and crz1Δ (AFA3-3 and AFA1-4) strains of opposite mating type were grown in YPD media, washed, and resuspended in PBS. Equal cell numbers of opposite mating types were mixed and spotted on MS media. Mating assays were incubated at room temperature in the dark for 12 days and imaged. (C) 12 G. mellonella larvae per group were infected by injection with 4x10³ cells of wild-type (H99), crz1Δ (AFA3-3), or the crz1Δ + CRZ1 complemented strains (AFA3-3-17 and ECt3). The larvae were incubated at 37°C and monitored daily. (H99 vs PBS, p-value <0.0001; AFA3-3 vs PBS, p-value = 0.0006; AFA3-3-17 vs PBS, p-value <0.0001; ECt3 vs PBS, p-value <0.0001; H99 vs AFA3-3, p-value = 0.0142; AFA3-3 vs AFA3-3-17, p-value = 0.0022; AFA3-3 vs ECt3, p-value = 0.0035; H99 vs AFA3-3-17, p-value = 0.2804; H99 vs ECt3, p-value = 0.5209) (D) Ten female BALB/c mice per group were infected through intranasal instillation with 1x10⁶ cells of wild-type (H99), crz1Δ (AFA3-3), and the crz1Δ + CRZ1 complemented (AFA3-3-17) strains. The mice were monitored daily and sacrificed at predetermined clinical end points that are predictive of imminent mortality. (H99 vs AFA3-3, p-value <0.0001; AFA3-3 vs AFA3-3-17, p-value = 0.0002; H99 vs AFA3-3-17, p-value <0.0001)

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To determine if the deletion of PMICIQ motif confers any phenotypes, the crz1Δ + Crz1^WT and crz1Δ + Crz1^PMICIQΔ strains were assayed at 37°C and 39°C, in addition to a panel of different cell wall stresses (Fig 1D). We found that compared to the crz1Δ + Crz1^WT strain, the crz1Δ + Crz1^PMICIQΔ was growth impaired at 39°C, or in the presence of 0.02% SDS. The crz1Δ + Crz1^PMICIQΔ strain presented an intermediate phenotype between the wild-type and crz1Δ on media containing 1% Congo red (Fig 1D). In summary, our results show that deletion of the PMICIQ domain results in the inability of Crz1 to translocate to the nucleus and sensitivity to growth at high temperature, strongly suggesting that this domain is functional.

The crz1Δ mutant displays an intermediate phenotype in comparison to wild-type and the calcineurin cna1Δ mutant

Calcineurin mutations impair hyphal elongation during sexual reproduction [25]. To determine if crz1 mutations affect mating, unilateral and bilateral mating crosses of crz1Δ mutants of opposite mating types were conducted. Unlike the cna1Δ mutants, hyphal formation was not affected in the crz1Δ mutants in either unilateral or bilateral mating crosses, suggesting that either another target is involved or Crz1 and a second target of calcineurin are together redundant for the control of mating (Fig 2B). These phenotypic results support roles for Crz1 in cell wall integrity and calcium sequestration in the calcineurin signaling pathway, and suggest that calcineurin acts on factors other than Crz1 to govern thermotolerance and mating.

Calcineurin has been previously shown to be required for virulence and deletion of CNA1 resulted in an avirulent phenotype [26]. In the Galleria infection model, we observed that loss of Crz1 resulted in reduced virulence in comparison to wild-type (crz1Δ mutant median survival = 6 days, WT median survival = 5 days; WT vs crz1Δ mutant p-value = 0.0142), which was restored by complementation of the crz1Δ mutant strain with the wild-type CRZ1 gene (crz1Δ mutant vs crz1Δ + CRZ1 complemented strain (ECt3 median survival = 4 days), p-value = 0.0035; crz1Δ mutant vs crz1Δ + CRZ1 complemented strain (AFA3-3-17 median survival = 5 days), p-value = 0.0022) (Fig 2C). To determine if Crz1 is also required for the virulence of C. neoformans in a vertebrate model, animals were infected with the wild-type strain (H99), the crz1Δ mutant strain (AFA3-3), and the crz1Δ + CRZ1 complemented strain (AFA3-3-17) (intranasal infection, 10⁵ CFUs). The crz1Δ mutant (median survival = 39 days) displayed attenuated virulence compared to wild-type (median survival = 23 days; p-value <0.0001) (Fig 2D). Complementation of the crz1Δ mutant with the wild-type CRZ1 gene largely restored virulence (median survival = 32 days; crz1Δ + CRZ1 vs wild-type p-value <0.0001; crz1Δ mutant vs crz1Δ + CRZ1 p-value = 0.0002) (Fig 2D).

We observed that the loss of CRZ1 resulted in attenuated virulence in both infection models, which is in contrast to the fully avirulent phenotype displayed by the cna1 calcineurin mutation (Fig 2C and 2D). The crz1Δ virulence defect was largely remediated and restored to wild-type when the crz1Δ mutant was complemented with the native CRZ1 gene.

Identification of calcineurin-Crz1 target genes by RNA-sequencing

A goal of this study was to identify the genomic targets of the calcineurin-Crz1-signaling pathway under high temperature stress. To this end, we performed RNA-sequencing of the wild-type strain, cna1Δ and crz1Δ mutant strains, and their respective complemented strains. In brief, strains were grown at 24°C and then shifted to 37°C for 1 hour, with three biological replicates. Direct polyA RNA sequencing was conducted and pairwise analyses of wild-type against the cna1Δ mutant and wild-type against the crz1Δ mutant were performed. Genes were regarded as differentially expressed if the expression ratio was altered ≥2-fold at a false discovery rate < 0.2.

Principle component analysis (PCA) and hierarchical clustering were used to explore the relationship in gene expression between samples (Fig 3A and 3B). PCA demonstrated, unsurprisingly, that high-temperature stress has a larger effect on gene expression than either of the mutations. PC1 clearly distinguishes the samples grown at 37°C from the 24°C samples, which clustered to the right or left side of the graph respectively (Fig 3A). Moreover, the 24°C samples tightly clustered on PC1 and PC2, indicating much lower variability in gene expression under this growth condition, as is expected. In contrast, we observed greater variability in gene expression under temperature stress at 37°C for each of the strains analyzed (Fig 3A). A hierarchical cluster analysis was also preformed to verify the observations from the PCA. We observed the same clustering pattern, whereby gene expression patterns of strains grown at 24°C were more similar compared to each other than when compared to the gene expression of strains grown at 37°C (Fig 3B). The variability of gene expression at 37°C was again greater than that at 24°C, and the effect of the temperature shift is greater than the effect of the mutations (Fig 3B).

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Fig 3. Expression dynamics of calcineurin and Crz1 target genes.

(A) Principal component analysis (PCA) was performed to compare gene expression levels of the WT (H99), cna1Δ (KK1), cna1Δ + CNA1 (KK5), crz1Δ (AFA3-3), and crz1Δ+ CRZ1 (AFA3-3-17) strains grown at 24°C and 37°C. Lighter colors indicate the 24°C samples and the bright colors indicate the 37°C samples. Note that results for one cna1Δ 37°C biological replicate were not included in the analysis, due to sample contamination. (B) Hierarchical cluster analysis recapitulates the observation from the PCA. The heatmap indicates the pairwise distance between samples. The x-axis of the color key indicates the distances between the samples, with the darker blue signifying a higher similarity (i.e. small distance) between the samples, while the lighter blue signifies a lower similarity (i.e larger distance) between the samples. The color bar above the heatmap indicates the sample type. Gene expression levels at 24°C were different from the gene expression levels at 37°C. In general, gene expression of samples grown at 24°C showed less variability (dark blue). However, gene expression of samples grown at 37°C showed more variability (light blue).

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Quantitative real-time PCR was performed to validate the results obtained from RNA sequencing. Of the 22 genes analyzed, 20 genes showed very similar fold changes in expression compared to RNA-sequencing (S3 Fig). The magnitude of fold change in two genes (CNAG_02415 and CNAG_00025 (VCX1)) observed in the real-time PCR was higher than in the RNA-sequencing but still supported the relationships of the gene expression patterns among the isolates at 37°C. Only in one case (CNAG_07725 (Rox1)) were we not able to validate the RNA-sequencing results as the magnitude of fold change in wild-type at 37°C was lower by real-time PCR.

Pairwise analyses of wild-type against the cna1Δ mutant, and wild-type against the crz1Δ mutant at 24°C, showed that under non-stress inducing conditions, loss of CNA1 and CRZ1 did not have a significant impact on gene expression at a whole genome level. From the pairwise analysis of wild-type against the cna1Δ mutant, 32 genes were identified as being differentially expressed, and 50% of the genes were downregulated in the cna1Δ mutant, and the other half were up-regulated (S1 Table). Comparing the gene expression of the crz1Δ mutant against the wild-type, we found that only five genes were differentially expressed; of the five genes, four genes were also differentially expressed in the cna1Δ mutant (S1 Table).

At 37°C, 495 genes were identified as being differentially expressed and regulated in a calcineurin-dependent manner in the comparison between wild-type and the cna1Δ mutant. In contrast, only 161 genes were differentially expressed in the pairwise analysis of wild-type against the crz1Δ mutant at 37°C (Fig 4A).

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Fig 4. Gene suite regulated by the calcineurin-Crz1 pathway in C. neoformans.

(A) Pairwise analyses of WT vs. cna1Δ and WT vs. crz1Δ gene sets were compared to determine the regulated genes. 102 genes from both gene sets have differential gene expression compared with the wild-type at 37°C and thus are controlled by both calcineurin and Crz1. 393 genes were differentially regulated by calcineurin, independently of Crz1, while 59 genes were regulated by Crz1 in a calcineurin-independent manner. (B) Comparison of the 102 genes against the known S. cerevisiae (144 genes) and A. fumigatus (137 genes) gene sets revealed that only two genes were shared in common between the three species (*Afu3g10690 and Afu7g01303 are both calcineurin dependent homologs of CNAG_0123 and YGL006W –refer to S5 Table). (C) Calcineurin-Crz1-dependent genes of known functions. Gene names and descriptions listed were identified using the FungiDB search portal; gene orthology was determined using the GO function. (Log2FC = Log2 Fold change)

https://doi.org/10.1371/journal.pgen.1006667.g004

To elucidate gene networks that are regulated by the calcineurin-Crz1 signaling pathway, the gene sets obtained from the pairwise analyses were compared. From the comparison of the 37°C gene sets, 102 genes were regulated in a calcineurin-Crz1 dependent manner (Fig 4A). 99 genes (97.1%) were down regulated in both the cna1Δ and crz1Δ mutants, indicating that these genes are normally positively activated by the calcineurin-Crz1-signaling pathway under thermal stress.

A majority of the genes (393 genes, 80%) identified as being calcineurin-dependent were directly or indirectly regulated independently of Crz1 (Fig 4A and S2 Table). 261 genes (66%) were down-regulated and 132 genes (34%) were up-regulated in the cna1Δ mutant, suggesting that calcineurin exerts a positive regulation of genes under thermal stress. Just over half of the genes (199 genes, 51%) encoded proteins of unknown function. 194 genes belonged to ascribed functional categories including: oxidation-reduction processes (35 genes, 18%), membrane transport including the ammonia permeases Amt1 and Amt2 and the inositol transporters Itr4 and Itr6 (32 genes, 8%), carbohydrate metabolism (20 genes, 5%), signaling and transcriptional regulation (14 genes, 3.5%), DNA replication (11 genes, 2.8%), sterol biosynthesis (5 genes, 1.3%), stress response (4 genes, 1%), and the remaining 73 gene products (18.6%) were involved in other metabolic and biological processes (S2 Table).

We identified a smaller subset of genes (59 genes) as being regulated by Crz1, independently of calcineurin, under thermal stress (S3 Table). 43 genes (73%) were down-regulated in the crz1Δ mutant, indicating that these genes are normally activated by Crz1 under thermal stress, and 16 genes (27%) were up-regulated. A majority of the genes (43 genes; 73%) encoded proteins of unknown functions. The remaining 16 genes had ascribed functional categories including: cell wall integrity (FKS1 and CHS8), membrane transport (6 genes, 10%), oxidation-reduction processes (4 genes, 6.8%), carbohydrate metabolism (4 genes, 6.8%), protein kinases (3 genes, 5%), encoded vesicle-mediated transport (2 genes, 3.4%), and other metabolic and biological processes (5 genes, 8.5%) (S3 Table).

Of the 102 genes that are controlled by the calcineurin-Crz1 pathway, 64 (62.75%) are genes of unknown functions (S4 Table). Consistent with the hypothesis that Crz1 acts downstream of calcineurin to regulate cell wall integrity based on the phenotypic assays, 11% of the genes encode proteins that contribute to cell wall integrity/maintenance; examples of these include: CHS7, CHS6, CHS5, KRE6, CDA2, CTS2, EXG1, and BGL2 (Fig 4C). We also identified genes involved in membrane transport (VCX1, PMC1, and RIM1), melanin production (LAC2), signaling and transcription (ROX1), and amino acid and carbohydrate metabolism (Fig 4C).

Calcineurin-Crz1-signaling pathway has been adapted in C. neoformans to enable growth at 37°C

In S. cerevisiae, 163 calcineurin-Crz1-dependent genes were identified in a genome wide-profiling study under Ca²⁺ and Na⁺ stresses, including genes involved in ion homeostasis and cell wall integrity [11]. Here we focused on those that were calcium regulated (153 genes), and also excluded nine that were hypothetical genes, and from the remaining 144 genes we found that 79 have an ortholog in C. neoformans. Interestingly, when we compared the 102 calcineurin-Crz1-dependent genes identified in our study with the S. cerevisiae calcineurin-Crz1-dependent genes identified upon calcineurin stimulation by high Ca²⁺ stress, we found that while 30 genes have an ortholog in S. cerevisiae, only two genes (CNAG_01232 PMC1 and CNAG_02217 CHS7) have a yeast ortholog that was also regulated by the calcineurin-Crz1 pathway (Fig 4B and S5 Table). Both genes were downregulated in the crz1Δ mutant, indicating that the genes are upregulated in response to thermal stress. The two S. cerevisiae orthologs (PMC1 and CHS1) are similarly upregulated in response to Ca²⁺ stress. To determine the significance of this overlap between the two gene sets, we performed a resampling test to evaluate the probability that the overlap occurred by chance (as described in Materials and Methods). The estimated probability for the C. neoformans and S. cerevisiae gene sets was 0.250, indicating that two-gene overlap between the two gene sets is likely due to chance, not conservation of a regulated gene.

In the pathogenic fungus A. fumigatus, 141 calcineurin-dependent genes were identified upon high Ca²⁺ stress [23]. From these we excluded four hypothetical proteins and found that from the remaining 137 there were 78 genes that have an ortholog in C. neoformans. When we compared the 102 genes identified from our study to the A. fumigatus gene set, we found that 56 genes have an ortholog in A. fumigatus, and from this subset, five genes (CNAG_00025 VCX1, CNAG_01232 PMC1, CNAG_02217 CHS7, CNAG_03412 CTS1, and CNAG_04737) have six orthologs that were regulated by the calcineurin pathway in A. fumigatus (Fig 4B and S5 Table). The five genes are downregulated in the crz1Δ mutant, indicating that the genes are upregulated in response to thermal stress. Of the five genes, two genes (CNAG_04737 and CTS1) have A. fumigatus orthologs that were differentially regulated. In the A. fumigatus crzAΔ mutant, chiB1 (Afu7g08490) and Afu6g03450 are upregulated under Ca²⁺ stress. We repeated the resampling test, and found that the estimated probability for the overlap between the C. neoformans and A. fumigatus gene sets was 0.002. The low probability of an overlap of at least six genes by chance suggests some conservation of calcineurin/Crz1 regulation between C. neoformans and A. fumigatus.

We further applied this analysis to compare the S. cerevisiae gene set with the gene set from A. fumigatus. From the OrthoMCL analysis, we found that 116 out of 144 S. cerevisiae calcineurin-dependent genes have orthologs in A. fumigatus, and 63 out of 137 A. fumigatus calcineurin-dependent genes have orthologs in S. cerevisiae. When we compared the two subsets, we found three S. cerevisiae genes (YGL006W PMC1, YLR350W ORM2, and YNL192W CHS1) overlapping with four A. fumigatus orthologs (S5 Table). The resampling analysis found a probability of 0.268 suggesting that this number of overlapping genes could occur by chance, as noted in the comparison between C. neoformans and S. cerevisiae.

This analysis shows that the genes regulated by the calcineurin-Crz1 pathway differ significantly between C. neoformans and S. cerevisiae, C. neoformans and A. fumigatus, and A. fumigatus and S. cerevisiae. Given that the gene sets of S. cerevisiae and A. fumigatus were obtained using different stress-inducing conditions, our results could suggest that under thermal stress, the C. neoformans calcineurin-Crz1 pathway may regulate a subset of genes that is different from the genes it may regulate under ionic stresses. But given that the genes regulated in S. cerevisiae and A. fumigatus differ significantly in response to the same stress condition (calcium ion stress) lends support to the conclusion that the output of the pathway differs between these species.

We found that the C. neoformans calcineurin-Crz1 pathway positively regulated both PMC1 and the calcium ion transporter gene VCX1, unlike in S. cerevisiae, where the Vcx1 protein is negatively regulated by calcineurin post-transcriptionally and independently of Crz1 [12, 34–36]. In addition to PMC1, a small number of genes have been previously identified as calcineurin-dependent gene targets in S. cerevisiae: FKS2, which encodes β-1,3-glucan synthase; the P-type ATPases PMR1 and ENA1; and the calcineurin regulator, RCN1 [34, 36–38]. We found that FKS1, encoding the sole β-1,3-glucan synthase, and MPK1, encoding the downstream protein kinase in the PKC pathway, were regulated by Crz1 independently of calcineurin under thermal stress. Adler et al. previously concluded that Pkc1 positively regulates Crz1 under glucose starvation, through phosphorylation. From our RNA-sequencing analysis results, we propose that Crz1 is regulated by calcineurin under thermal stress, and that the PKC pathway may act antagonistically to calcineurin.

Motif enrichment analyses

Crz1 target genes have been shown to contain a Crz1 binding motif in their promoter regions, also known as the calcineurin-dependent response element (CDRE). To identify the CDRE motif in C. neoformans, we performed a motif search employing MEME (Multiple Em for Motif Elucidation) with the S. cerevisiae, C. albicans, M. oryzae, and A. fumigatus CDRE motif sequences serving as consensus sequences [12, 22, 23]. However, we were unable to identify any unique motif sequences. We next attempted to identify the putative CDRE motifs with MEME by using 1 kb of upstream promoter sequences of the 102 calcineurin-Crz1 regulated genes and restricting the maximum size of the motif to 10 bp. Two putative motifs were identified: motif 1 (5’-[G/A]CACAGC[C/A]AC-3’) was found in 48 genes, and motif 2 (5’-GAAGATG[A/G]T[G/A]-3’) was present in 52 genes (Fig 5A and S6 Table).

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Fig 5. Genes regulated by the calcineurin-Crz1 pathway feature three motifs.

(A) DNA motifs generated by MEME from promoter analysis of the 102 target genes. (B) DNA motif generated by DREME from promoter analysis of CHS6 (CNAG_00546), and three other genes (CNAG_00588, CNAG_04891, and CNAG_00407). (C) Comparison of the presence or absence of the motifs against the phenotypic analyses of the target gene deletion mutants. CaCl₂ = 0.5 M calcium chloride; CR = 1% Congo red; CFW = 5 mg/mL calcofluor white.

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A second attempt was performed using the motif discovery algorithm DREME (Discriminative Regular Expression Motif Elicitation), using 1 kb of the upstream promoter sequences of the CHS6 gene (CNAG_00546), which has been found to be a target of Crz1, and three other genes (CNAG_00588, CNAG_04891, and CNAG_00407) that displayed the highest fold-change in expression levels in the RNA-seq analysis. Corresponding promoter sequences from the orthologs in C. deneoformans, C. gattii, and C. amylolentus were also used in order to increase specificity of the motif search. From this search, a third motif that is 7 bp in length (5’-GCACA[G/A]C-3’) was identified with an e-value of 1.3e^-4 (Fig 5B). This motif is a shorter variant of motif 1 identified in the MEME analysis. We used Analysis of Motif Enrichment (AME) to check for motif enrichment among the promoter regions of the 102 calcineurin-Crz1 regulated genes, and found that the motif was enriched (p-value = 1.95e^-4), and is present in the promoter regions of 29 genes. We found that 21 genes did not contain any of the three putative motifs, suggesting that these genes may be indirect targets of the calcineurin-Crz1 pathway (S6 Table).

To verify if genes containing the motifs were direct targets of Crz1, we performed ChIP-PCR using the crz1Δ mutant complemented with the Crz1-4xFLAG construct described in [31]. Cultures were grown at 24°C and then shifted to 37°C for 1 hour, with and without FK506, with biological duplicates. Three genes with the highest fold-changes (CNAG_00588, CNAG_00407, CNAG_04819) were selected, and primers were designed to amplify 150–280 bp segments of the promoter regions containing the motifs (S4 Fig). We observed that in general, Crz1 binding intensity was increased upon temperature shift to 37°C compared to 24°C and this increase was inhibited by exposure to FK506 (Fig 6).

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Fig 6. Crz1 binding of promoter regions increases under thermal stress.

Cultures of the Crz1-4xFLAG strain were grown to exponential phase at 24°C, shifted to 37°C with and without FK506 for 30 min, and crosslinked with formaldehyde. Whole cell lysates (WCE) were prepared and ChIP-PCR was performed employing anti-FLAG nickel beads and specific DNA primers for three genes (CNAG_00588, CNAG_00407, and CNAG_04861) as described in material and methods. PCR DNA products for the DNA obtained from the anti-FLAG CHIP-PCR or from WCE-PCR were fractionated in agarose gels. Primers for each gene tested were designed to produce a product 150–280 bp long. Quantification of band intensity was analyzed using ImageJ, and values were normalized to the 24°C control. Results shown are representative of two biological replicates (labelled as Rep 3 gel and Rep 4 gel above).

https://doi.org/10.1371/journal.pgen.1006667.g006

Deletion mutants of genes regulated by the calcineurin-Crz1 pathway display differential phenotypes

Of the 102 genes regulated in a calcineurin-Crz1 dependent manner, we were able to phenotypically assay 70 gene deletion mutants from the C. neoformans gene deletion collection deposited at the FGSC by the Madhani lab [39]. From the group of 70 mutants tested, 10 mutants displayed similar phenotypes to wild-type when assayed on media containing 0.5 M CaCl₂, 1% Congo red, and 5 mg/mL calcofluor white, or growth at 37°C and 39°C. 14 mutants presented with an intermediate phenotype compared to the cna1Δ and crz1Δ mutants at 37°C, while 55 mutants presented with similar temperature sensitive growth phenotypes to the crz1Δ mutant (Fig 5C, S5 and S6 Figs). One mutant (cnag_03463Δ) encoding a protein of unknown function displayed slower growth across all conditions assayed (S5 Fig). Interestingly, two deletion mutants (cnag_02864Δ and cnag_03227Δ) displayed a slightly increased resistance at 39°C (S6 Fig). In summary, 60 out of 70 genes regulated by the calcineurin-Crz1 pathway, when mutated, conferred stress sensitivity to different degrees. This represents a significant enrichment of this phenotype, reinforcing the model that genes regulated by the calcineurin-Crz1 pathway have roles in stress resistance.

Strains and plasmids

Fungal strains and plasmids constructed in this study are listed in S8 Table. All fungal strains listed in S8 Table were derived from C. neoformans var. grubii laboratory reference strain H99 (#4413), unless otherwise stated. Fungal transformation was carried out using the biolistic method [44]. Fungal strains were maintained on YPD agar (1% yeast extract, 2% bactopeptone, and 2% dextrose) supplemented with the relevant antibiotics and grown at 30°C.

The crz1Δ::NAT deletion mutant was generated using overlap PCR and biolistic transformation. To construct the Crz1-mCherry epitope-tagged strains for the site-directed mutagenesis, the Crz1 ORF was PCR amplified and fused with the mCherry fusion protein by splice overlap PCR, cloned into pCR2.1-TOPO (Invitrogen) to yield plasmid pXW15 which was verified by sequencing. The 7.96 kb BamHI Crz1-mCherry fusion fragment was subcloned into the BamHI site of the safe haven plasmid pSDMA25 [45], to generate plasmid pEC13 which was verified by sequencing. The recombinant Crz1-mCherry plasmid was linearized using the restriction enzyme AscI, and the crz1Δ::NAT deletion mutant was biolistically transformed. Transformants were screened for proper integration into the safe haven locus as previously described [45]. Targeted integration of each construct was confirmed by PCR amplification (5’ junction: JOHE40956/JOHE40957; 3’ junction: JOHE40958/JOHE41562). Tandem array integration was ruled out by PCR using inverse primers (JOHE41450/JOHE41451) that would only yield a product if two or more copies of the construct were tandemly integrated at the safe haven site. The NAT selectable marker in pSL04, which contains the nucleolar fluorescence marker GFP-Nop1, was replaced with the HYG selectable marker from pJAF15. The GFP-Nop1 construct was introduced into the recombinant Crz1-mCherry mutant strains by ectopic integration. All primers that were used in this study are listed in S9 Table. The Crz1^PMICIQΔ –mCherry plasmid was constructed by Gibson cloning, using the plasmid pEC13 as the template.

Phenotypic assays

Strains were grown in liquid YPD media at 30°C overnight with shaking, and washed with PBS. Five 10-fold serial dilutions of each strain were made, and spotted out onto solid media. Unless otherwise stated, strains were incubated for 2 days at 30°C. YPD was supplemented with 2.5 M CaCl₂ solution, mixed to achieve a final concentration of 0.35 M, 0.4 M, or 0.5 M of CaCl₂. YPD was supplemented with 20% SDS, mixed to achieve a final concentration of 0.02% or 0.03% SDS. Congo red was added to YPD to a final concentration of 1% (w/v). Calcofluor white was added to final concentrations of 4 mg/mL or 5 mg/mL. MS mating medium was prepared as previously described [25]. Strains of opposite mating types were grown in liquid YPD media at 30°C overnight with shaking, and washed with PBS. Equal volumes of cell suspensions of each mating type were mixed, spotted onto MS mating media, and incubated at room temperature in the dark for 12 days before visualization using a Nikon Eclipse E400 microscope with an attached DXM200F digital camera.

Microscopy

Strains were grown overnight in liquid YPD medium at room temperature with shaking. Cells were then diluted to OD₆₀₀ = 0.1–0.2 with fresh YPD medium (20 mL), and grown for a further five hours, until OD₆₀₀ = 0.5–0.6 was achieved. A 5 mL aliquot of the cell culture was incubated in a 37°C water bath for 15 min. Cells incubated at 24°C and at 37°C were then fixed with 4% formaldehyde for 15 min, and washed with KPO₄/sorbitol buffer. To verify that translocation of Crz1 is due to calcineurin activity, FK506 (final concentration = 1 μg/mL) was added to the cultures, and cells were incubated for a further 15 min before being shifted to 37°C. To observe the localization of the Crz1^PMICIQΔ construct, cells were shifted to 38°C for 20 min prior to formaldehyde fixing. Cell suspensions were spotted onto slides with a layer of 1.5% YNB-agarose and covered with a coverslip. Cells were visualized by direct fluorescence microscopy using a Zeiss Axioskop 2 Plus microscope and AxioVision 4.6 image acquisition software. Nuclear fluorescence was quantified by ImageJ software.

RNA isolation and real-time PCR

Strains were grown overnight at 24°C with shaking, until an OD₆₀₀ = 0.5 was reached. Cell cultures were divided in two. One half was incubated at 24°C while the other half was transferred to 37°C, for a further 2 hours. Cells were collected by centrifugation, frozen at -80°C, and then lyophilized overnight. Total RNA was isolated from lyophilized cells using Trizol solution (Invitrogen Life Technologies, Carlsbad, CA) and the Qiagen RNeasy Mini kit (Qiagen, Valencia, CA). All RNA samples were collected in biological triplicates.

For quantitative PCR, total RNA was reversed transcribed into first strand cDNA by oligo dT priming using the AffinityScript cDNA synthesis kit according to the manufacturer’s instructions (Ailgent Technologies, Santa Clara, CA). The resulting cDNA was diluted with ultra-pure water. Real-time PCR was performed using the Brilliant III Ultra-Fast SYBR Green QPCR master mix (Agilent Technologies) and the Applied Sciences StepOnePlus Real Time-PCR system. Primers are listed in S9 Table.

RNA-sequencing data analysis

Direct polyA RNA sequencing was performed by the High Throughput Sequencing Facility at the University of North Carolina, Chapel Hill. Preliminary quality analysis of the raw FASTQ files was conducted using FastQC [46], quality filtering was then performed using fastq-mcf [47] to remove sequencing adapters (adapter sequences supplied by Illumina, Inc) and trim low quality bases. Tophat2 [48] was used to map the processed reads to the H99 genome and transcriptome as determined from the genome annotation (version 2, downloaded from the Broad Institute) [49], using the following parameters: "—transcriptome-max-hits 1—max-multihits 1—max-intron-length 4000—library-type fr-unstranded—no-coverage-search". The program htseq-count [50] was used to determine read counts per gene from the BAM file output by Tophat; the htseq-count parameters used were "—stranded = no—type = exon—idattr = gene_id". A custom script was developed in the R programming language [51] to analyse the count data in order to do quality testing, identify differentially expressed genes, and generate figures. This script used the R packages DESeq2 [52], gplots [53], RColorBrewer [54], and optparse [55].

Preliminary data analysis led us to excluded from analysis one of the cna1Δ 37°C replicates, which was found to contain reads mapping to the deleted portion of CNA1. This preliminary analysis also indicated that the CRZ1 complemented crz1Δ strains have much higher expression of CRZ1 than the wild-type strain.

The RNA-Seq data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus (GEO) [56] and are accessible through GEO Series accession number GSE93005 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE93005). The custom programs developed for processing and analyzing the RNA-Seq data are available in a GitHub software repository (https://github.com/granek/crne_cna1crz1_rnaseq). This repository includes all programs, support files, and instructions for automatically replicating all analyses presented here using the data available from GEO.

Orthology search and resampling test analysis

Orthologs were as determined by FungiDB [57], using OrthoMCL. The list of genes regulated by the calcineurin-Crz1 pathway in S. cerevisiae was taken from [11]; the study identified 163 regulated genes, of which 153 genes are regulated in response to Ca²⁺ stress. Nine genes (YNL043C, YMR007W, YPR197C, YMR304C-A, YDL011C, YDL172C, YPR170C, YDR535C, YGL165C) listed as “hypothetical genes” [11] were omitted from consideration, as the gene annotations were not found in FungiDB, to result in 144 genes that were further analyzed. The list of genes regulated by the calcineurin-Crz1 pathway in A. fumigatus was taken from [23]; 4 genes (Afu6g12810, Afu6g11860, Afu1g00190, Afu2g07390) listed as “hypothetical genes” were omitted from consideration as the gene annotations were not found in FungiDB. Further, FungiDB gives Afu2g05330 as the current name for the gene listed as Afu2g05325 in [23], so Afu2g05330 was used in this analysis. In total, 144 genes from S. cerevisiae, 137 genes from A. fumigatus and 102 genes from C. neoformans were analyzed.

The resampling test was performed to examine the likelihood that the overlap in regulated gene sets were to occur by chance. Briefly, sets of genes equivalent in size to the number of regulated genes with orthologs were randomly selected: 30 from all C. neoformans genes with an ortholog in S. cerevisiae, and 79 from all S. cerevisiae genes with an ortholog in C. neoformans. These two sets were compared to determine the number of orthologs shared between these two sets. This resampling procedure was repeated 1000 times, and the proportion of times that a simulated overlap was equal to or larger than the true overlap was tallied to give a probability estimate. The same test was carried out between C. neoformans (56 genes) and A. fumigatus (78 genes), as well as S. cerevisiae (116 genes) and A. fumigatus (63 genes). Scripts and data for replicating the resampling analysis are available in the GitHub software repository (https://github.com/granek/crne_cna1crz1_rnaseq).

Motif discovery

To perform motif discovery, we employed the CDRE fungal motif sequences as indicated in the results section and the MEME suite was employed through use of the MEME webserver. In order to identify candidate motifs, both MEME [58] and DREME [59] were employed, with the input sequences randomly shuffled to provide a negative data set for DREME. Candidate motifs were tested for enrichment in the upstream 1 kb promoter sequences of candidate calcineurin-Crz1 regulated genes using AME [60].

Chromatin immunoprecipitation

The ChiP analysis was conducted as previously described with some modifications [61]. Wild-type and crz1Δ + Crz1-4xFLAG strains were grown in liquid YPD media overnight at 30°C with shaking. Overnight cultures were diluted to an OD₆₀₀ = 0.2–0.3 with fresh media, and further incubated at room temperature for 4 h. FK506 (final concentration 10 mM) was then added to two sets of cultures; one set of cultures was incubated at 37°C and the other at room temperature for a further 1 h. The same was performed for two sets of cultures without FK506. Formaldehyde (1% final concentration) was added to each culture, and incubated at room temperature for 20 min with shaking. Glycine (final concentration 125 mM) was added to quench the reaction, with 5 min incubation at room temperature with shaking. Fixed cells were pelleted and washed with PBS + 125 mM glycine twice. Cells were suspended with 1 mL ChIP lysis buffer (50 mM HEPES pH 7.4, 140 mM NaCl, 1% Triton X100, 1 mM EDTA), and 100 μL volume of acid washed glass beads were added. Cells were bead-beat three times for 1 min at 4°C with 1 min rest intervals and cell lysates were collected. Cell lysates were sonicated (12 cycles of 10 s pulses, followed with 5 min incubation on ice between each interval) and cleared by centrifugation. An aliquot of each lysate sample was kept as the lysate input controls. Lysates were immunoprecipitated using anti-FLAG nickel beads (Invitrogen/Life technologies) according to manufacturer’s protocol. Immunocomplexes were eluted by adding 250 μL of elution buffer (TE buffer, 1% SDS, 0.1M NaHCO₃) to the beads, followed by a 5 min incubation at 65°C, prior to 15 min incubation at room temperature with constant rotation. The beads were then pelleted and the supernatant was collected. This step was repeated again to get a total volume of 500 μL. The eluates were de-crosslinked by adding 10 μL of 5M NaCl, and overnight incubation at 65°C. Following incubation, 10 μL of 0.5M EDTA, 20 μL of 1M Tris-HCl pH 6.8, and 2 μL of 20 mg/mL Proteinase K were added, and the eluates were further incubated for 1–2 hr at 45°C. Following extraction with phenol-chloroform-isoamyl alcohol, DNA was precipitated overnight at –20°C by adding 1 μL glycogen, 60 μL 3M NaOAc, and 1 mL 100% ethanol. DNA was pelleted by centrifugation at 13,000 rpm for 30 min at 4°C, and resolved in Ultra-Pure water and treated with 20 μg/mL RNaseA, before storage at -20°C.

DNA concentration was measured by Nanodrop, and diluted to an equimolar concentration for all samples. ChIP PCRs were done under typical laboratory PCR conditions with 1x ExTaq Buffer, 400nM dNTPs, 200nM forward primer, 200nM reverse primer, 0.625 units of ExTaq (Clonetech) and 1.2ul ChIP DNA template. Primers for each gene used can be found in S9 Table. PCR products were run on a 2% agarose gel containing 500μg/mL of ethidium bromide and imaged using the BioRad Gel Doc System.

Virulence assays

Galleria mellonella were infected as previously described [62]. Cells were grown overnight in liquid YPD medium at 30°C with shaking, washed with PBS and suspended in the same buffer. Cell density was calculated using a hemocytometer and cell suspensions of 10⁶ cells/mL were prepared. Groups of 12 larvae were injected with 4 μL of the cell suspension (4 x 10³ cells) through the proleg; one group of larvae was injected with PBS as a control. The larvae were incubated at 37°C, and death was monitored daily for 15 days. Larvae that pupated during the 15-day period were censored. G. mellonella infections were performed in triplicate.

5–6 week old BALB/c female mice were used for the murine infection model. Groups of 10 mice were infected with wild-type, crz1Δ mutant, and crz1Δ + CRZ1 complemented strains (10⁵ cells) using the intranasal inhalation model previously described [63]. Mice were monitored and weighed daily; mice that appeared moribund or weighed less than 20% of their initial starting weight were euthanized.

Survival curves for both infection models were adjusted using the Kaplan-Meier method and estimation of differences in survival were analyzed using the log rank and Wilcoxon tests with GraphPad Prism software (GraphPad, San Diego, CA). A p-value below 0.05 was considered significant.

Ethics statement

All experiments and animal care were conducted in accordance with the ethical guidelines of the Institutional Animal Care and Use Committee (IACUC) of Duke University Medical Center (DUMC). The DUMC IACUC approved all of the vertebrate studies under protocol number A245-13-09. Mice studies were conducted in the Division of Laboratory Animal Resources (DLAR) facilities that are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).