Zap1 regulates zinc homeostasis and modulates virulence in Cryptococcus gattii

Abstract

Zinc homeostasis is essential for fungal growth, as this metal is a critical structural component of several proteins, including transcription factors. The fungal pathogen Cryptococcus gattii obtains zinc from the stringent zinc-limiting milieu of the host during the infection process. To characterize the zinc metabolism in C. gattii and its relationship to fungal virulence, the zinc finger protein Zap1 was functionally characterized. The C. gattii ZAP1 gene is an ortholog of the master regulatory genes zafA and ZAP1 that are found in Aspergillus fumigatus and Saccharomyces cerevisiae, respectively. There is some evidence to support an association between Zap1 and zinc metabolism in C. gattii: (i) ZAP1 expression is highly induced during zinc deprivation, (ii) ZAP1 knockouts demonstrate impaired growth in zinc-limiting conditions, (iii) Zap1 regulates the expression of ZIP zinc transporters and distinct zinc-binding proteins and (iv) Zap1 regulates the labile pool of intracellular zinc. In addition, the deletion of ZAP1 reduces C. gattii virulence in a murine model of cryptococcosis infection. Based on these observations, we postulate that proper zinc metabolism plays a crucial role in cryptococcal virulence.

Figure 1. Identification of the ZAP1 zinc regulator in C. gattii.

A. The zinc finger domain architecture was evaluated using the ScanProsite tool employing the consensus sequence C-X(2,4)-C-X(12)-H-X(3,5)-H in the ZAP1 homologs of different fungi: S. cerevisiae ZAP1 (Sacce_Zap1 – Genbank NP_012479.1), A. fumigatus ZafA (Aspfu_ZafA – Genbank ABJ98717.1), C. albicans ZAP1 (Canal_Zap1 – Genbank XP_717199.1), C. neoformans serotype D ZAP1 (CryneoD_Zap1 – Genbank XP_572252), C. neoformans serotype A ZAP1 (CryneoA_Zap1 – Broad Institute CNAG_05392) and C. gattii ZAP1 (Cryga_Zap1 – Broad Institute CNBG_4460). The zinc finger domain is represented by black bars, and the length of each protein sequence (in amino acids) is indicated to the right. B. Phylogenetic analysis applying the Neighbor-Joining method and including Zap1 sequences from distinct fungi. The bar marker indicates the genetic distance, which is proportional to the number of amino acid substitutions. C. Quantitative real time RT-PCR of ZAP1 gene transcripts after growth of C. gattii in YNB with or without TPEN. D. Quantitative real time RT-PCR of ZIP gene transcripts after growth of C. gattii in YNB with or without TPEN. The measured quantity of the mRNA in each of these samples was normalized using the Ct values obtained for the actin gene. Data are shown as the mean ± SD from three experimental replicates of three biological replicates. ^* P<0.05. ^** P<0.01. ^*** P<0.001.

Figure 2. C. gattii ZAP1 null mutants are defective in zinc metabolism.

(A) Growth of the WT, zap1Δ mutant and zap1Δ::ZAP1 complemented strains in low-zinc or control media was evaluated spectrophotometrically. The ratio between growth in low-zinc and control conditions is show as the mean ± SD from three biological replicates. (B) Fluorometric determination of intracellular zinc was accomplished using the probe Fluozin-1-AM. The relative zinc concentration was determined based on the fluorescence in WT or zap1Δ mutant cells cultured in YNB or YNB +10 μM TPEN as a control to determine the level of background fluorescence. Bars represent the mean of the cell count of normalized fluorescence levels. C. Quantitative real time RT-PCR of ZIP gene transcripts after growth of the C. gattii WT or zap1Δ mutant in YNB + TPEN. The measured quantity of the mRNA in each of the samples was normalized using the Ct values obtained for the actin gene. Data are shown as the mean ± SD from three experimental replicates of three biological replicates. ^** P<0.01. ^*** P<0.001. NS, not significant.

Figure 3. Disruption of ZAP1 generates an imbalance in ROS metabolism.

(A) Fluorometric determination of intracellular ROS levels employing the probe CM-H2DCFDA. The relative ROS levels were determined based on the fluorescence in WT, zap1Δ mutant or zap1Δ::ZAP1 complemented cells cultured in YNB + TPEN. Bars represent the mean of the cell count with normalized fluorescence levels. (B) Quantitative real time RT-PCR of CAT gene transcripts after growth of C. gattii WT or zap1Δ mutant cells in YNB + TPEN. (C) Quantitative real time RT-PCR of Mn-SOD or Cu/Zn-SOD gene transcripts after growth of the C. gattii WT or zap1Δ mutant cells in YNB + TPEN. The measured quantity of the mRNA in each of the samples was normalized using the Ct values obtained for the actin gene. Data are shown as the mean ± SD from three experimental replicates of three biological replicates. ^* P<0.05. ^** P<0.01. NS, not significant.

Figure 4. Lack of ZAP1 leads to alterations in glutathione metabolism.

(A) The WT, zap1Δ mutant and zap1Δ::ZAP1 complemented strains were incubated in YNB or YNB +0.5 mM DEM. After 24 h of incubation, the cell density was spectrophotometrically determined. The ratio between growth in DEM and control conditions is shown as the mean ± SD from three biological replicates. (B) Quantitative real time RT-PCR of GPX gene transcripts after growth of C. gattii WT or zap1Δ mutant cells in YNB + TPEN. The measured quantity of the mRNA in each of the samples was normalized using the Ct values obtained for the actin gene. Data are shown as the mean ± SD from three experimental replicates of three biological replicates. ^* P<0.05. ^** P<0.01. ^*** P<0.001. NS, not significant.

Figure 5. C. gattii ZAP1 gene null mutant shows defects in the response to RNS.

The WT, zap1Δ mutant and zap1Δ::ZAP1 complemented strains were incubated in YNB or YNB +1 mM DETA-NONOate. After 24 h of incubation, the cell density was spectrophotometrically determined. The ratio between growth in DEM and control conditions is shown as the mean ± SD from three biological replicates. ^*** P<0.001.

Figure 6. Zap1 is required for full C. gattii virulence in mice and influences phagocytosis by macrophages.

(A) Virulence assay of WT, zap1Δ mutant and zap1Δ::ZAP1 complemented strains in an intranasal inhalation infection model with BALB/c mice. (B) CFU counts after macrophage infection with WT, zap1Δ mutant and zap1Δ::ZAP1 complemented strains. ^*** P<0.001.