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

<div><p>Calcineurin is a highly conserved Ca²⁺/calmodulin-dependent serine/threonine-specific protein phosphatase that orchestrates cellular Ca²⁺ signaling responses. In <i>Cryptococcus neoformans</i>, 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 <i>Saccharomyces cerevisiae</i> 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 <i>cna1</i>Δ and <i>crz1</i>Δ 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 <i>crz1</i>Δ 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 <i>C</i>. <i>neoformans</i> appears to be adapted to promote fungal virulence.</p></div

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

<p><b>(A)</b> 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>(B)</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. <b>(C)</b> 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. <b>(D)</b> Wild-type (H99), <i>cna1</i>Δ (KK1), <i>crz1</i>Δ (AFA3-3) mutants, and strains ECt172 (<i>crz1</i>Δ + Crz1^WT) and ECt375 (<i>crz1</i>Δ + 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.</p

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

<p><b>(A)</b> DNA motifs generated by MEME from promoter analysis of the 102 target genes. <b>(B)</b> DNA motif generated by DREME from promoter analysis of <i>CHS6</i> (CNAG_00546), and three other genes (CNAG_00588, CNAG_04891, and CNAG_00407). <b>(C)</b> 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.</p

Deletion of <i>CRZ1</i> confers partial phenotypes in comparison to wild-type and <i>cna1</i>Δ mutant.

<p><b>(A)</b> Wild-type (H99), <i>cna1</i>Δ (KK1), <i>crz1</i>Δ (AFA3-3) mutants and their respective complemented strains (KK5 and AFA3-3-17), and the <i>cna1</i>Δ <i>crz1</i>Δ 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>(B)</b> Wild-type (H99 and KN99<b>a</b>), <i>cna1</i>Δ (KK1 and KK8), and <i>crz1</i>Δ (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. <b>(C)</b> 12 <i>G</i>. <i>mellonella</i> larvae per group were infected by injection with 4x10³ cells of wild-type (H99), <i>crz1</i>Δ (AFA3-3), or the <i>crz1</i>Δ + <i>CRZ1</i> complemented strains (AFA3-3-17 and ECt3). The larvae were incubated at 37°C and monitored daily. (H99 vs PBS, <i>p</i>-value <0.0001; AFA3-3 vs PBS, <i>p</i>-value = 0.0006; AFA3-3-17 vs PBS, <i>p</i>-value <0.0001; ECt3 vs PBS, <i>p</i>-value <0.0001; H99 vs AFA3-3, <i>p</i>-value = 0.0142; AFA3-3 vs AFA3-3-17, <i>p</i>-value = 0.0022; AFA3-3 vs ECt3, <i>p</i>-value = 0.0035; H99 vs AFA3-3-17, <i>p</i>-value = 0.2804; H99 vs ECt3, <i>p</i>-value = 0.5209) <b>(D)</b> Ten female BALB/c mice per group were infected through intranasal instillation with 1x10⁶ cells of wild-type (H99), <i>crz1</i>Δ (AFA3-3), and the <i>crz1</i>Δ + <i>CRZ1</i> 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, <i>p</i>-value <0.0001; AFA3-3 vs AFA3-3-17, <i>p</i>-value = 0.0002; H99 vs AFA3-3-17, <i>p</i>-value <0.0001)</p

Expression dynamics of calcineurin and Crz1 target genes.

<p><b>(A)</b> Principal component analysis (PCA) was performed to compare gene expression levels of the WT (H99), <i>cna1</i>Δ (KK1), <i>cna1</i>Δ + <i>CNA1</i> (KK5), <i>crz1</i>Δ (AFA3-3), and <i>crz1</i>Δ+ <i>CRZ1</i> (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 <i>cna1</i>Δ 37°C biological replicate were not included in the analysis, due to sample contamination. <b>(B)</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).</p

Model of calcineurin-Crz1 signaling pathway in <i>C</i>. <i>neoformans</i>.

<p>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 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006667#pgen.1006667.ref027" target="_blank">27</a>] 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.</p

Crz1 binding of promoter regions increases under thermal stress.

<p>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).</p