Elucidation of the calcineurin-Crz1 stress response transcriptional network in the human fungal pathogen Cryptococcus neoformans

Abstract

Calcineurin is a highly conserved Ca2+/calmodulin-dependent serine/threonine-specific protein phosphatase that orchestrates cellular Ca2+ signaling responses. In Cryptococcus neoformans, calcineurin is activated by multiple stresses including high temperature, and is essential for stress adaptation and virulence. The transcription factor Crz1 is a major calcineurin effector in Saccharomyces cerevisiae and other fungi. Calcineurin dephosphorylates Crz1, thereby enabling Crz1 nuclear translocation and transcription of target genes. Here we show that loss of Crz1 confers phenotypes intermediate between wild-type and calcineurin mutants, and demonstrate that deletion of the calcineurin docking domain results in the inability of Crz1 to translocate into the nucleus under thermal stress. RNA-sequencing revealed 102 genes that are regulated in a calcineurin-Crz1-dependent manner at 37°C. The majority of genes were down-regulated in cna1Δ and crz1Δ mutants, indicating these genes are normally activated by the calcineurin-Crz1 pathway at high temperature. About 58% of calcineurin-Crz1 target genes have unknown functions, while genes with known or predicted functions are involved in cell wall remodeling, calcium transport, and pheromone production. We identified three calcineurin-dependent response element motifs within the promoter regions of calcineurin-Crz1 target genes, and show that Crz1 binding to target gene promoters is increased upon thermal stress in a calcineurin-dependent fashion. Additionally, we found a large set of genes independently regulated by calcineurin, and Crz1 regulates 59 genes independently of calcineurin. Given the intermediate crz1Δ mutant phenotype, and our recent evidence for a calcineurin regulatory network impacting mRNA in P-bodies and stress granules independently of Crz1, calcineurin likely acts on factors beyond Crz1 that govern mRNA expression/stability to operate a branched transcriptional/post-transcriptional stress response network necessary for fungal virulence. Taken together, our findings reveal the core calcineurin-Crz1 stress response cascade is maintained from ascomycetes to a pathogenic basidiomycete fungus, but its output in C. neoformans appears to be adapted to promote fungal virulence.

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.

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)

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).

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)

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.

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).

Fig 7. Model of calcineurin-Crz1 signaling pathway in C. neoformans.

Under stress conditions such as high Ca²⁺ concentration or thermal stress, calcineurin is activated and dephosphorylates Crz1, resulting in its translocation to the nucleus. In the nucleus, Crz1 upregulates the expression of stress response genes, such as those involved in cell wall maintenance or remodeling and small molecule transport. Calcineurin also translocates to P-bodies and stress granules [27] where it may act upon other factors that influence mRNA expression or stability to operate a bifurcated transcriptional/post-transcriptional stress response network necessary for fungal virulence.