Article Figures & Data
Figures
- FIG 1
Grx4 interacts with Cir1. A yeast two-hybrid assay was used to examine the interaction of Grx4 and Cir1. DBD and AD indicate the Gal4 DNA binding and activation domains fused to Grx4 and Cir1, respectively. The vector designation indicates the empty vector control. All combinations of transformants grew in the absence of leucine (Leu) and tryptophan (Trp), confirming plasmid retention in the strains. Only yeast cells transformed with plasmids containing GRX4 and CIR1 grew in the absence of histidine (His), confirming an interaction to allow expression of HIS3. 3-Amino-1,2,4-triazole (3AT) was included at different concentrations to enhance the stringency of the HIS3 selection. Qualitative and quantitative analyses of β-galactosidase activity were performed using X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) or chlorophenol red-β-d-galactopyranoside as a substrate, respectively, and the quantitative numbers represent the mean values of three assays with the standard error of the mean provided in parentheses.
- FIG 2
Grx4 is localized in nuclei upon iron limitation. (A) Grx4-mCherry and Cir1-GFP were colocalized in nuclei under low-iron conditions (defined low-iron medium [LIM]), but Grx4-mCherry shifted to the cytosol with addition of iron (FeCl3 or heme) and Cir1-GFP remained in the nucleus. Size bar = 5 μm. (B) Colocalization of Cir1-GFP with DAPI in nuclei under all conditions. (C) Quantitation of the Grx4-mCherry signal in the nucleus and cytoplasm in response to different levels of iron, as described in Materials and Methods. The graph shows the average of each treatment with 95% confidence intervals (CI; n ≥ 20). One-way ANOVA and Tukey statistical tests were performed to analyze the signal intensity, and *** indicates P < 0.0001. (D) Deletion of CIR1 caused mislocalization of Grx4-mCherry to the cytosol under the low-iron condition (YNB + BPS medium). Grx4-mCherry was partially retained in nuclei under the high-iron condition upon treatment with bortezomib (BTZ), a proteasome inhibitor. The same results were obtained with cells grown in defined LIM. Size bar = 5 μm.
- FIG 3
Grx4 influences the elaboration of three major virulence factors. (A) The sensitivity of the WT strain of C. neoformans, two independent grx4 mutants, and the GRX4 reconstituted strain to temperature (30 and 37°C) was examined using spot assays on YPD medium. (B) Spot assays were performed with each strain on l-DOPA plates with incubation at 30°C to examine melanin production. (C) Cells were grown in defined low-iron medium at 30°C for 48 h, and capsule formation was assessed by India ink staining for the indicated strains. Size bar = 10 μm. (D) Fifty cells of each strain from panel C were measured for cell diameter and capsule size. Each bar represents the average of the 50 measurements with standard deviations. Statistical significance relative to the WT capsule size is indicated by ** (Student's t test, P < 0.01).
- FIG 4
Grx4 is required for virulence in a mouse inhalation model. Ten female BALB/c mice were challenged by intranasal inoculation with 105 cells of the WT strain (H99), the grx4-JL mutant, or the GRX4 reconstituted strain. Survival differences between groups of mice were evaluated by the log rank Mantel-Cox test. The P values for the mice infected with the WT and mutant strains were statistically different (*, P <0.001). Also shown is the distribution of fungal cells in the organs (brain, lung, and spleen) of infected mice. Organs infected with the WT, the grx4-JL mutant, or the GRX4 reconstituted strain were collected at the humane endpoint of the experiment, and fungal burdens were monitored in organs by determining CFU upon plating on YPD medium. Three mice for each strain were used for the experiments, and horizontal bars in each graph represent the average CFU. In all organs, differences in fungal burdens between the grx4 mutant and the WT strain and between the grx4 mutant and the reconstituted strain, were statistically significant by the nonparametric Mann-Whitney two-tailed U test (*, P < 0.05).
- FIG 5
Grx4 is required for robust growth on inorganic iron or heme as the sole iron source. (A) Spot assays with each strain were performed on YNB-BPS medium with different concentrations of FeSO4 or FeCl3. (B) Cells of the WT, the grx4 mutant, and the GRX4 reconstituted strain were inoculated into liquid YNB medium plus 150 μM BPS without and with supplementation with FeCl3 as the iron source. The cultures were incubated at 30°C, and OD600 values were measured. The cir1 mutant strain was included for comparison with the grx4 strains. (C) The indicated strains were also tested for growth without and with supplementation with heme by the same method as in panel B. (D) Spot assays of each strain on YNB-BPS medium with different concentrations of heme.
- FIG 6
Grx4 is involved in iron homeostasis. (A and B) Disruption of GRX4 leads to increased sensitivity to the iron-chelating drugs curcumin and ferrozine. (A) Spot assays with the WT, two independent grx4 mutants, and the GRX4 complemented strains (without iron starvation) on YPD plates with or without curcumin (CCM) at a concentration of either 150 or 300 μM, supplemented with 0, 10, or 150 μM heme as the iron source. (B) Spot assays with each strain without iron starvation on YPD plates with or without 75 μM or 750 μM ferrozine supplemented with 0 or the indicated amount of FeEDTA (15). (C) Spot assays with each strain grown in YPD medium overnight and spotted onto YPD supplemented with either 1, 5, 20, or 40 mM FeCl3.
- FIG 7
Impact of loss of the GRX domain of Grx4 on the transcripts for specific Gene Ontology categories. (A and B) Gene Ontology (GO) enrichment analysis of the differentially expressed genes between WT and grx4 strains under low-iron (A) and high-iron (B) conditions (with total gene numbers within each functional category shown as the percentage of genes showing differential expression).
- FIG 8
Grx4 regulates genes involved in metal ion transport, heme biosynthesis, and iron-sulfur cluster binding in response to iron availability. (A) Changes in transcript abundance of the genes encoding functions in iron-sulfur cluster binding between the WT and grx4 mutant strains grown under low- and high-iron conditions, with the results represented by the heat map. (B) Changes in transcript abundance of the genes encoding functions in heme biosynthesis and binding between the WT and grx4 mutant strains grown under low- and high-iron conditions, with the results represented by the heat map. (C) Spot assays of the indicated strains on YPD medium with or without the antimalarial drug chloroquine (6 mM), CoCl2 (a hypoxia-mimicking agent [600 μM]), or phleomycin (an iron-dependent inhibitor [8 μg/ml]).
- FIG 9
Grx4 is implicated in the regulation of functions for electron transport and the response to oxidative stress. (A) Heat map representation of changes in transcript abundance of the genes encoding functions in electron carrier activity between the WT and grx4 mutant strains grown under low- and high-iron conditions. (B) Spot assays on YPD medium indicate that the grx4 mutation leads to sensitivity to inhibitors of electron transport chain complexes I to IV and the alternative oxidase (75 μg/ml rotenone, 2 mM malonic acid, 5 μg/ml antimycin A, 10 mM potassium cyanide [KCN], 10 mM salicylic hydroxamate [SHAM], and 50 μM diphenyleneiodonium [DPI]). (C) Spot assays on YPD medium indicate that the grx4 mutation of GRX4 leads to the sensitivity to agents that provoke oxidative stress (2 mM t-BOOH, 0.01% H2O2, and 5 µg/ml 1-chloro-2,4-dinitrobenzene [CDNB]). (D) Spot assays on YPD medium indicate that the grx4 mutants have altered susceptibilities to inhibitors of reactive oxygen species (50 µM plumbagin, 5 µg/ml menadione, and 500 µM paraquat).
- FIG 10
Grx4 regulates function for DNA repair and confers resistance to DNA-damaging agents. (A) Heat map representation of changes in transcript abundance of the genes encoding functions in DNA repair between the WT strain and grx4 mutant grown under low- and high-iron conditions. (B) Spot assays on YPD medium with exposure to UV light (400 J/m2), the DNA repair inhibitor hydroxyurea (HU [25 mM]), and the DNA-damaging agent methyl methanesulfonate (MMS [0.03%]).
- FIG 11
Proposed model for the interaction of Grx4 with Cir1 in C. neoformans and comparisons with Grx4 interactions in S. pombe. During iron repletion, Grx4 in C. neoformans partially relocalizes to the cytoplasm, potentially influencing the extent of its interaction with Cir1 and the repression of genes for iron uptake. Under this condition in S. pombe, Grx4 is known to interact with Fep1 but does not inhibit its ability to repress iron uptake genes (24, 26, 27, 29). Upon iron depletion, Grx4 in S. pombe inhibits Fep1, leading to depression via dissociation from the promoters of genes for iron uptake. We hypothesize that Grx4 similarly influences the activity of Cir1 upon iron limitation. Grx4 also regulates the activity of Php4 in S. pombe, and the proposed model for this yeast indicates that Grx4 promotes the exit of Php4 from the nucleus through interaction with the nuclear exportin Crm1 upon iron repletion, thereby allowing Php2, -3, and -5 to activate genes for iron utilization (32, 33). The expression of these genes is repressed by Grx4 in a complex with Php2, -3, and -5 and Php4 upon iron limitation (35). These interactions in S. pombe provide a framework for future studies of the interaction of Grx4 with HapX, the Php4 ortholog in C. neoformans.
Tables
- TABLE 1
Number of genes differentially regulated by iron availability and/or Grx4
Comparison No. of genesa: Upregulated Downregulated 2-fold 5-fold 10-fold Total 2-fold 5-fold 10-fold Total WT: low vs high iron 525 16 0 541 328 53 27 408 grx4 vs WT Low iron 498 99 50 647 317 62 25 404 High iron 580 100 56 736 290 52 16 358 grx4: low vs high iron 100 2 0 102 237 11 0 248 ↵a The gene numbers were calculated using P value-based statistics (P < 0.01).
- TABLE 2
Grx4-regulated genes encoding iron transport and homeostasis and mitochondrial functions
Result for iron level comparisona: Gene ID Gene
nameFunction WT low
vs highgrx4 low
vs WT lowgrx4 high
vs WT highgrx4 low
vs highIron transport CNAG_00815 SIT1 Siderochrome-iron uptake transporter 1.86 1.06 2.09 0.94 CNAG_06761 SIT3 Siderophore-iron transporter Str1 2.29 0.37 0.89 0.96 CNAG_07387 SIT4 Siderophore-iron transporter 2.81 3.92 10.55 1.04 CNAG_07519 SIT5 Siderophore-iron transporter 2.23 0.64 2.07 0.68 CNAG_07751 SIT6 Siderophore iron transporter MirB 3.63 2.32 8.35 1.00 CNAG_03498 FRE201 Metalloreductase 1.45 2.06 3.39 0.88 CNAG_06524 FRE3 Ferric reductase 2.50 5.07 16.25 0.77 CNAG_06976 FRE6 Ferric reductase 2.40 0.66 1.16 1.35 CNAG_00876 FRE7 Ferric reductase 0.40 0.89 0.49 0.72 CNAG_03498 FRE8 Ferric reductase 1.45 2.06 3.39 0.88 CNAG_04864 CIR1 Iron regulator 1 0.81 0.02 0.01 1.23 CNAG_01242 HAPX Conserved hypothetical protein 1.93 1.21 2.14 1.08 CNAG_02950 GRX4 Grx4 family monothiol glutaredoxin 1.72 0.11 0.15 1.21 CNAG_00727 MMT2 Mitochondrial protein with role in iron
accumulation0.50 0.89 0.43 1.04 CNAG_03465 LAC1 Laccase 1 0.99 10.22 7.55 1.32 CNAG_05154 CCC1 Membrane fraction protein 0.57 5.20 5.50 0.54 CNAG_07519 SIT1/ARN1 Conserved hypothetical protein 2.23 0.64 2.07 0.68 CNAG_01653 CIG1 Cytokine inducing-glycoprotein, putative
a hemophore2.29 0.64 1.32 1.10 CNAG_07865 Ferro-O2-oxidoreductase 2.55 0.94 1.99 1.20 Heme biosynthesis CNAG_01721 HEM3 Porphobilinogen deaminase 0.15 2.62 1.71 0.22 CNAG_02460 HEM13 Coproporphyrinogen III oxidase 0.39 0.82 0.27 1.16 CNAG_03939 HEM1 5-Aminolevulinic acid synthase 0.19 2.52 1.10 0.42 CNAG_03187 HEM14 Protoporphyrinogen oxidase 0.92 0.45 0.45 0.91 CNAG_01908 HEM4 Uroporphyrinogen-III synthase 0.04 10.60 2.72 0.14 Mitochondrial ISC
assemblyCNAG_03395 NFU1 NifU-like protein C 0.10 3.78 1.59 0.25 CNAG_03589 YAH1 Adrenodoxin-type ferredoxin 0.54 2.24 2.22 0.54 CNAG_02522 MRS3/4 Carrier 1.25 0.85 1.11 0.95 CNAG_03985 GRX5 Monothiol glutaredoxin-5 0.45 1.95 2.04 0.43 CNAG_05199 SSQ1 Heat shock protein 0.41 0.81 0.52 0.64 CNAG_04288 JAC1 Conserved hypothetical protein 1.78 0.34 0.60 1.01 CNAG_02131 ISA1 Iron sulfur assembly protein 1 0.32 2.20 1.58 0.44 CNAG_00491 ISA2 Iron sulfur assembly protein 2 0.92 0.70 0.82 0.77 CNAG_00389 IBA57 Mitochondrial protein 0.25 2.42 1.17 0.52 Cytosolic ISC
assemblyCNAG_04202 NAR1 Iron hydrogenase 0.15 3.04 1.61 0.28 CNAG_01802 DRE2 Cytoplasmic protein 0.50 3.10 2.41 0.63 CNAG_01137 ACO1 Aconitase 0.21 1.47 1.35 0.23 ISC-containing proteins CNAG_07908 ACO2 Aconitate hydratase 0.20 5.64 2.13 0.53 CNAG_06621 BIO2 Biotin synthase 0.35 1.28 0.98 0.45 CNAG_00462 CIR2 Oxidoreductase 0.05 8.51 1.35 0.29 CNAG_01914 COQ11 Mitochondrial protein 0.98 0.58 0.56 1.00 CNAG_07572 ELP3 Pol II transcription elongation factor 0.27 2.90 1.76 0.44 CNAG_04862 GLT1 Glutamate synthase 0.22 7.36 7.15 0.22 CNAG_07491 GRX6 Conserved hypothetical protein 1.06 0.71 0.92 0.82 CNAG_00237 LEU1 3-Isopropylmalate dehydratase 0.22 9.54 3.01 0.70 CNAG_05194 LIP5 Lipoic acid synthetase 0.47 1.89 2.16 0.41 CNAG_02565 LYS4 Homoaconitate hydratase 0.07 8.81 1.64 0.40 CNAG_05070 MET5 Sulfite reductase beta subunit 0.14 3.60 2.13 0.23 CNAG_03206 NTG2 DNA-(apurinic or apyrimidinic site) lyase 0.23 3.50 1.89 0.42 CNAG_02315 RIP1 Ubiquinol-cytochrome c reductase iron-sulfur
subunit0.22 2.47 1.30 0.42 CNAG_07667 SAT4 Other/HAL protein kinase 0.89 0.92 0.83 0.98 CNAG_07356 SHH3 Succinate dehydrogenase 0.16 2.19 1.13 0.32 CNAG_06558 TAH18 NADPH-ferrihemoprotein reductase 0.81 3.20 1.82 1.41 Electron transport CNAG_05318 CYB2 l-Mandelate dehydrogenase 0.41 2.78 1.84 0.61 CNAG_05169 CYB2 Cytochrome b2 0.21 4.54 1.86 0.50 CNAG_07480 MCR1 Cytochrome b5 reductase 2.24 0.95 1.67 1.27 CNAG_00716 CYC7 Electron carrier 0.22 2.14 1.96 0.23 CNAG_03666 Acyl-CoA dehydrogenase 0.06 2.83 1.16 0.14 CNAG_03629 YHB1 NADH-ubiquinone oxidoreductase 0.05 4.30 1.15 0.20 CNAG_01229 CYB2 l-Mandelate dehydrogenase 0.13 3.91 2.05 0.25 CNAG_05631 NADH-ubiquinone oxidoreductase 0.07 5.40 1.29 0.30 CNAG_01078 ALD5 Aldehyde dehydrogenase 1.51 2.39 3.00 1.20 CNAG_04189 SDH1 Succinate dehydrogenase flavoprotein subunit 0.17 1.52 1.03 0.24 CNAG_04521 Oxidoreductase 1.40 1.83 2.54 1.00 CNAG_03663 CYB2 l-Lactate dehydrogenase 0.10 3.02 1.31 0.24 CNAG_03226 SDH2 Succinate dehydrogenase iron-sulfur subunit 0.09 1.48 0.87 0.15 CNAG_05179 QCR2 Ubiquinol-cytochrome c reductase complex core 0.17 2.12 0.95 0.37 CNAG_05258 Glucose-methanol-choline oxidoreductase 5.68 4.72 17.36 1.53 CNAG_01323 QCR7 Ubiquinol-cytochrome c reductase 0.27 3.20 1.96 0.43 CNAG_05909 CYT1 Electron transporter 0.12 3.18 1.18 0.32 Mitochondrial functions CNAG_00162 Alternative oxidase 0.02 52.56 1.80 0.68 CNAG_03824 MIR1 Phosphate transport protein MIR1 1.19 0.26 0.31 1.00 CNAG_00499 Carnitine/acyl carnitine carrier 0.36 1.70 1.04 0.59 CNAG_05909 Electron transporter 0.12 3.18 1.18 0.32 CNAG_02315 Ubiquinol-cytochrome c reductase 0.22 2.47 1.30 0.42 CNAG_05132 Cytochrome c oxidase 0.48 1.14 0.95 0.56 CNAG_03225 Malate dehydrogenase 0.91 0.56 0.44 1.14 CNAG_00237 3-Isopropylmalate dehydratase 0.22 9.54 3.01 0.70 CNAG_03596 Dihydrolipoamide succinyltransferase 0.46 0.87 0.74 0.54 CNAG_07908 Aconitate hydratase 0.20 5.64 2.13 0.53 CNAG_05031 Succinyl-CoA:3-ketoacid-coenzyme A transferase 0.96 0.22 0.65 0.32 CNAG_06138 NADH dehydrogenase (ubiquinone) Fe-S 6 0.22 2.05 1.01 0.45 DNA repair CNAG_07552 RAD8 DNA repair Rad8 0.87 6.75 4.97 1.17 CNAG_00178 REV1 DNA repair REV1 1.40 6.12 7.12 1.19 CNAG_05198 RAD7 DNA repair RAD7 1.27 5.49 7.27 0.95 CNAG_02544 SWI5 DNA repair Swi5/Sae3 1.56 5.06 7.04 1.10 CNAG_02512 RAD16 DNA repair RAD16 1.97 4.84 8.86 1.06 CNAG_01163 RAD54 DNA repair and recombination RAD54 1.24 3.42 3.98 1.06 CNAG_00299 RAD5 DNA repair RAD5 0.75 2.82 2.19 0.96 CNAG_02771 RAD54b DNA repair and recombination RAD54B 1.10 7.91 8.79 0.98 CNAG_00720 RAD51 DNA repair RAD51 1.51 7.11 10.72 0.99 CNAG_03637 Double-strand break repair factor and silencing regulator 1.09 3.61 3.71 1.05 Ergosterol metabolism CNAG_00519 ERG3 2.18 0.24 0.39 1.30 CNAG_01129 ERG7 1.66 0.43 0.54 1.32 CNAG_00854 ERG2 2.46 0.25 0.57 1.08 CNAG_02830 ERG4 1.82 0.46 0.94 0.88 CNAG_01737 ERG25 2.19 0.17 0.35 1.06 Oxidative stress CNAG_00575 CAT3 Catalase 3 (CAT3) 0.48 5.59 3.31 0.81 CNAG_00654 SRX1 Conserved hypothetical protein (SRX1, sulfiredoxin) 4.34 4.85 14.41 1.45 CNAG_01138 CCP1 Cytochrome c peroxidase (CCP1) 0.07 181.00 27.35 0.44 CNAG_03482 TSA1 Thioredoxin-dependent peroxide reductase (TSA1) 1.31 5.10 4.62 1.44 CNAG_03985 GRX5 Monothiol glutaredoxin-5 (GRX5) 0.45 1.95 2.04 0.43 CNAG_04388 SOD2 Mitochondrial superoxide dismutase Sod2 0.98 0.58 0.51 1.12 CNAG_05847 TRR1 Thioredoxin-disulfide reductase (TRR1) 1.24 12.12 10.13 1.47 ↵a The numbers in boldface are the measurements where the log2 value is ≥2; the numbers in italic are log2 values of <0.5.
FIG S1
Grx4 from C. neoformans has a conserved C-terminal GRX domain with a signature “CGFS” motif. (A) Alignment of the amino acid sequence of the C-terminal Grx domain of C. neoformans Grx4 (XP_012047837.1) with selected Grx sequences from other organisms. UhGrx4, Ustilago hordei, CCF50760.1; UmGrx4, Ustilago maydis, XP_011390702.1; SpGrx4, Schizosaccharomyces pombe, NP_596647.1; ScGrx3, Saccharomyces cerevisiae, AJV06961.1; ScGrx4, S. cerevisiae, NP_011101.3; CaGrx4, Candida albicans, KHC50815.1; HcGrx4, Histoplasma capsulatum, EEH02725.1; HsGrx4, Homo sapiens, AAF28841.1. (B) Phylogeny tree generated using neighbor-joining tree analysis in MEGA (v.7.0.26). Download FIG S1, PDF file, 0.8 MB.
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FIG S2
The phenotypes of tagged strains (Grx4-mCherry, Cir1-GFP, and Grx4-mCherry Cir1-GFP) are the same as the WT strain. Ten-fold serial dilutions of each strain grown in YPD medium overnight were spotted onto YPD or l-DOPA medium. The plates were incubated at either 30 or 37°C for 2 days before being photographed. Download FIG S2, PDF file, 0.6 MB.
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FIG S3
Abundance of Cir1-GFP or Cir1-mCherry assessed by Western blot analysis. (A) Proteins from cells expressing Grx4-mCherry and WT cells grown under the low-iron condition were analyzed by Western blot analysis using anti-mCherry antibody. Intact Grx4-mCherry (58 kDa) was detected in cells expressing Grx4-mCherry but not in WT cells. (B) Proteins from cells expressing Grx4-mCherry in either the WT strain or the cir1 mutant grown under conditions in low- versus high-iron medium for 5 h were analyzed using anti-mCherry antibody. (C) The proteins from cells expressing Cir1-GFP in either the WT or grx4 mutant background grown under the indicated conditions (low versus high iron) for 5 h were analyzed using anti-GFP antibody. Download FIG S3, PDF file, 0.9 MB.
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TABLE S1
List of transcripts upregulated in the grx4 mutant under both low- and high-iron conditions. Download Table S1, DOCX file, 0.1 MB.
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TABLE S2
List of transcripts downregulated in the grx4 mutant under both low- and high-iron conditions. Download Table S2, DOCX file, 0.08 MB.
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FIG S4
STRING analysis of upregulated transcripts in the grx4 cells under either the low- or high-iron condition. STRING was used to visualize predicted protein-protein interactions for the identified 639 Grx4-regulated proteins (http://string-db.org) using the corresponding proteins from C. neoformans strain JEC21 in the database. Notably, functions relevant to mitochondria (cellular respiration, electron transport, and metal binding) and oxidative phosphorylation were identified. Download FIG S4, PDF file, 2.2 MB.
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FIG S5
Verification of RNA-Seq data by qPCR. qPCR was used to verify the transcript levels of CIR1, LAC1, and FRE3 in the WT and grx4 mutant cells, grown under either the low-iron (A) or high-iron (B) condition. The expression pattern of these three genes is consistent with the data revealed by RNA-Seq analysis. Download FIG S5, PDF file, 0.6 MB.
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FIG S6
Grx4 influences susceptibility to various inhibitors and DNA-damaging agents on minimal medium supplemented with iron. Ten-fold serial dilutions of each strain were spotted onto YNB medium plus BPS and FeCl3 (100 μM) supplemented with the agents indicated, and the plates were incubated at 30°C for 2 days before being photographed. The concentrations of the inhibitors were as follows: chloroquine, 6 mM; CoCl2, 600 μM; phleomycin, 8 μg/ml; SHAM, 10 mM; and paraquat, 500 µM. DNA-damaging agents included UV light (100 J/m2) and 0.03% methyl methanesulfonate (MMS). Download FIG S6, PDF file, 0.5 MB.
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TABLE S3
Primers for strain construction and qRT-PCR. Download Table S3, DOCX file, 0.02 MB.
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TABLE S4
BioProject and BioSample accession numbers for the RNA-Seq data. Download Table S4, DOCX file, 0.07 MB.
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