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Research Article | Host-Microbe Biology

The Monothiol Glutaredoxin Grx4 Regulates Iron Homeostasis and Virulence in Cryptococcus neoformans

Rodgoun Attarian, Guanggan Hu, Eddy Sánchez-León, Mélissa Caza, Daniel Croll, Eunsoo Do, Horacio Bach, Tricia Missall, Jennifer Lodge, Won Hee Jung, James W. Kronstad
Joseph Heitman, Editor
Rodgoun Attarian
Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, CanadaDepartment of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada
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Guanggan Hu
Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada
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Eddy Sánchez-León
Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada
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Mélissa Caza
Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada
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Daniel Croll
Laboratory of Evolutionary Genetics, Institute of Biology, University of Neuchâtel, Neuchâtel, Switzerland
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Eunsoo Do
Department of Systems Biotechnology, Chung-Ang University, Anseong, South Korea
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Horacio Bach
Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada
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Tricia Missall
Department of Biochemistry, Saint Louis University School of Medicine, St. Louis, Missouri, USA
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Jennifer Lodge
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA
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Won Hee Jung
Department of Systems Biotechnology, Chung-Ang University, Anseong, South Korea
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James W. Kronstad
Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, CanadaDepartment of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada
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  • ORCID record for James W. Kronstad
Joseph Heitman
Duke University
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Alexander Idnurm
University of Melbourne
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Bernhard Hube
Friedrich Schiller University Jena
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DOI: 10.1128/mBio.02377-18
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  • FIG 1
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    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
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    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
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    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
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    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
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    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
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    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
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    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
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    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
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    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
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    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
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    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

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  • TABLE 1

    Number of genes differentially regulated by iron availability and/or Grx4

    ComparisonNo. of genesa:
    UpregulatedDownregulated
    2-fold5-fold10-foldTotal2-fold5-fold10-foldTotal
    WT: low vs high iron5251605413285327408
    grx4 vs WT
        Low iron49899506473176225404
        High iron580100567362905216358
    grx4: low vs high iron10020102237110248
    • ↵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 IDGene
    name
    FunctionWT low
    vs high
    grx4 low
    vs WT low
    grx4 high
    vs WT high
    grx4 low
    vs high
    Iron transport
        CNAG_00815SIT1Siderochrome-iron uptake transporter1.861.062.090.94
        CNAG_06761SIT3Siderophore-iron transporter Str12.290.370.890.96
        CNAG_07387SIT4Siderophore-iron transporter2.813.9210.551.04
        CNAG_07519SIT5Siderophore-iron transporter2.230.642.070.68
        CNAG_07751SIT6Siderophore iron transporter MirB3.632.328.351.00
        CNAG_03498FRE201Metalloreductase1.452.063.390.88
        CNAG_06524FRE3Ferric reductase2.505.0716.250.77
        CNAG_06976FRE6Ferric reductase2.400.661.161.35
        CNAG_00876FRE7Ferric reductase0.400.890.490.72
        CNAG_03498FRE8Ferric reductase1.452.063.390.88
        CNAG_04864CIR1Iron regulator 10.810.020.011.23
        CNAG_01242HAPXConserved hypothetical protein1.931.212.141.08
        CNAG_02950GRX4Grx4 family monothiol glutaredoxin1.720.110.151.21
        CNAG_00727MMT2Mitochondrial protein with role in iron
    accumulation
    0.500.890.431.04
        CNAG_03465LAC1Laccase 10.9910.227.551.32
        CNAG_05154CCC1Membrane fraction protein0.575.205.500.54
        CNAG_07519SIT1/ARN1Conserved hypothetical protein2.230.642.070.68
        CNAG_01653CIG1Cytokine inducing-glycoprotein, putative
    a hemophore
    2.290.641.321.10
        CNAG_07865Ferro-O2-oxidoreductase2.550.941.991.20
    Heme biosynthesis
        CNAG_01721HEM3Porphobilinogen deaminase0.152.621.710.22
        CNAG_02460HEM13Coproporphyrinogen III oxidase0.390.820.271.16
        CNAG_03939HEM15-Aminolevulinic acid synthase0.192.521.100.42
        CNAG_03187HEM14Protoporphyrinogen oxidase0.920.450.450.91
        CNAG_01908HEM4Uroporphyrinogen-III synthase0.0410.602.720.14
    Mitochondrial ISC
    assembly
        CNAG_03395NFU1NifU-like protein C0.103.781.590.25
        CNAG_03589YAH1Adrenodoxin-type ferredoxin0.542.242.220.54
        CNAG_02522MRS3/4Carrier1.250.851.110.95
        CNAG_03985GRX5Monothiol glutaredoxin-50.451.952.040.43
        CNAG_05199SSQ1Heat shock protein0.410.810.520.64
        CNAG_04288JAC1Conserved hypothetical protein1.780.340.601.01
        CNAG_02131ISA1Iron sulfur assembly protein 10.322.201.580.44
        CNAG_00491ISA2Iron sulfur assembly protein 20.920.700.820.77
        CNAG_00389IBA57Mitochondrial protein0.252.421.170.52
    Cytosolic ISC
    assembly
        CNAG_04202NAR1Iron hydrogenase0.153.041.610.28
        CNAG_01802DRE2Cytoplasmic protein0.503.102.410.63
        CNAG_01137ACO1Aconitase0.211.471.350.23
    ISC-containing proteins
        CNAG_07908ACO2Aconitate hydratase0.205.642.130.53
        CNAG_06621BIO2Biotin synthase0.351.280.980.45
        CNAG_00462CIR2Oxidoreductase0.058.511.350.29
        CNAG_01914COQ11Mitochondrial protein0.980.580.561.00
        CNAG_07572ELP3Pol II transcription elongation factor0.272.901.760.44
        CNAG_04862GLT1Glutamate synthase0.227.367.150.22
        CNAG_07491GRX6Conserved hypothetical protein1.060.710.920.82
        CNAG_00237LEU13-Isopropylmalate dehydratase0.229.543.010.70
        CNAG_05194LIP5Lipoic acid synthetase0.471.892.160.41
        CNAG_02565LYS4Homoaconitate hydratase0.078.811.640.40
        CNAG_05070MET5Sulfite reductase beta subunit0.143.602.130.23
        CNAG_03206NTG2DNA-(apurinic or apyrimidinic site) lyase0.233.501.890.42
        CNAG_02315RIP1Ubiquinol-cytochrome c reductase iron-sulfur
    subunit
    0.222.471.300.42
        CNAG_07667SAT4Other/HAL protein kinase0.890.920.830.98
        CNAG_07356SHH3Succinate dehydrogenase0.162.191.130.32
        CNAG_06558TAH18NADPH-ferrihemoprotein reductase0.813.201.821.41
    Electron transport
        CNAG_05318CYB2l-Mandelate dehydrogenase0.412.781.840.61
        CNAG_05169CYB2Cytochrome b20.214.541.860.50
        CNAG_07480MCR1Cytochrome b5 reductase2.240.951.671.27
        CNAG_00716CYC7Electron carrier0.222.141.960.23
        CNAG_03666Acyl-CoA dehydrogenase0.062.831.160.14
        CNAG_03629YHB1NADH-ubiquinone oxidoreductase0.054.301.150.20
        CNAG_01229CYB2l-Mandelate dehydrogenase0.133.912.050.25
        CNAG_05631NADH-ubiquinone oxidoreductase0.075.401.290.30
        CNAG_01078ALD5Aldehyde dehydrogenase1.512.393.001.20
        CNAG_04189SDH1Succinate dehydrogenase flavoprotein subunit0.171.521.030.24
        CNAG_04521Oxidoreductase1.401.832.541.00
        CNAG_03663CYB2l-Lactate dehydrogenase0.103.021.310.24
        CNAG_03226SDH2Succinate dehydrogenase iron-sulfur subunit0.091.480.870.15
        CNAG_05179QCR2Ubiquinol-cytochrome c reductase complex core0.172.120.950.37
        CNAG_05258Glucose-methanol-choline oxidoreductase5.684.7217.361.53
        CNAG_01323QCR7Ubiquinol-cytochrome c reductase0.273.201.960.43
        CNAG_05909CYT1Electron transporter0.123.181.180.32
    Mitochondrial functions
        CNAG_00162Alternative oxidase0.0252.561.800.68
        CNAG_03824MIR1Phosphate transport protein MIR11.190.260.311.00
        CNAG_00499Carnitine/acyl carnitine carrier0.361.701.040.59
        CNAG_05909Electron transporter0.123.181.180.32
        CNAG_02315Ubiquinol-cytochrome c reductase0.222.471.300.42
        CNAG_05132Cytochrome c oxidase0.481.140.950.56
        CNAG_03225Malate dehydrogenase0.910.560.441.14
        CNAG_002373-Isopropylmalate dehydratase0.229.543.010.70
        CNAG_03596Dihydrolipoamide succinyltransferase0.460.870.740.54
        CNAG_07908Aconitate hydratase0.205.642.130.53
        CNAG_05031Succinyl-CoA:3-ketoacid-coenzyme A transferase0.960.220.650.32
        CNAG_06138NADH dehydrogenase (ubiquinone) Fe-S 60.222.051.010.45
    DNA repair
        CNAG_07552RAD8DNA repair Rad80.876.754.971.17
        CNAG_00178REV1DNA repair REV11.406.127.121.19
        CNAG_05198RAD7DNA repair RAD71.275.497.270.95
        CNAG_02544SWI5DNA repair Swi5/Sae31.565.067.041.10
        CNAG_02512RAD16DNA repair RAD161.974.848.861.06
        CNAG_01163RAD54DNA repair and recombination RAD541.243.423.981.06
        CNAG_00299RAD5DNA repair RAD50.752.822.190.96
        CNAG_02771RAD54bDNA repair and recombination RAD54B1.107.918.790.98
        CNAG_00720RAD51DNA repair RAD511.517.1110.720.99
        CNAG_03637Double-strand break repair factor and silencing regulator1.093.613.711.05
    Ergosterol metabolism
        CNAG_00519ERG32.180.240.391.30
        CNAG_01129ERG71.660.430.541.32
        CNAG_00854ERG22.460.250.571.08
        CNAG_02830ERG41.820.460.940.88
        CNAG_01737ERG252.190.170.351.06
    Oxidative stress
        CNAG_00575CAT3Catalase 3 (CAT3)0.485.593.310.81
        CNAG_00654SRX1Conserved hypothetical protein (SRX1, sulfiredoxin)4.344.8514.411.45
        CNAG_01138CCP1Cytochrome c peroxidase (CCP1)0.07181.0027.350.44
        CNAG_03482TSA1Thioredoxin-dependent peroxide reductase (TSA1)1.315.104.621.44
        CNAG_03985GRX5Monothiol glutaredoxin-5 (GRX5)0.451.952.040.43
        CNAG_04388SOD2Mitochondrial superoxide dismutase Sod20.980.580.511.12
        CNAG_05847TRR1Thioredoxin-disulfide reductase (TRR1)1.2412.1210.131.47
    • ↵a The numbers in boldface are the measurements where the log2 value is ≥2; the numbers in italic are log2 values of <0.5.

Supplemental Material

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  • Tables
  • 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.

    Copyright © 2018 Attarian et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • 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.

    Copyright © 2018 Attarian et al.

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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.

    Copyright © 2018 Attarian et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • 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.

    Copyright © 2018 Attarian et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • 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.

    Copyright © 2018 Attarian et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • 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.

    Copyright © 2018 Attarian et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • 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.

    Copyright © 2018 Attarian et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • 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.

    Copyright © 2018 Attarian et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S3

    Primers for strain construction and qRT-PCR. Download Table S3, DOCX file, 0.02 MB.

    Copyright © 2018 Attarian et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S4

    BioProject and BioSample accession numbers for the RNA-Seq data. Download Table S4, DOCX file, 0.07 MB.

    Copyright © 2018 Attarian et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

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The Monothiol Glutaredoxin Grx4 Regulates Iron Homeostasis and Virulence in Cryptococcus neoformans
Rodgoun Attarian, Guanggan Hu, Eddy Sánchez-León, Mélissa Caza, Daniel Croll, Eunsoo Do, Horacio Bach, Tricia Missall, Jennifer Lodge, Won Hee Jung, James W. Kronstad
mBio Dec 2018, 9 (6) e02377-18; DOI: 10.1128/mBio.02377-18

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The Monothiol Glutaredoxin Grx4 Regulates Iron Homeostasis and Virulence in Cryptococcus neoformans
Rodgoun Attarian, Guanggan Hu, Eddy Sánchez-León, Mélissa Caza, Daniel Croll, Eunsoo Do, Horacio Bach, Tricia Missall, Jennifer Lodge, Won Hee Jung, James W. Kronstad
mBio Dec 2018, 9 (6) e02377-18; DOI: 10.1128/mBio.02377-18
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KEYWORDS

cryptococcosis
capsule
melanin
nuclear localization
transcriptome

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