Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Dec 4;9(6):e02377-18.
doi: 10.1128/mBio.02377-18.

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

Affiliations
Free PMC article

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

Rodgoun Attarian et al. mBio. .
Free PMC article

Erratum in

Abstract

The acquisition of iron and the maintenance of iron homeostasis are important aspects of virulence for the pathogenic fungus Cryptococcus neoformans In this study, we characterized the role of the monothiol glutaredoxin Grx4 in iron homeostasis and virulence in C. neoformans Monothiol glutaredoxins are important regulators of iron homeostasis because of their conserved roles in [2Fe-2S] cluster sensing and trafficking. We initially identified Grx4 as a binding partner of Cir1, a master regulator of iron-responsive genes and virulence factor elaboration in C. neoformans We confirmed that Grx4 binds Cir1 and demonstrated that iron repletion promotes the relocalization of Grx4 from the nucleus to the cytoplasm. We also found that a grx4 mutant lacking the GRX domain displayed iron-related phenotypes similar to those of a cir1Δ mutant, including poor growth upon iron deprivation. Importantly, the grx4 mutant was avirulent in mice, a phenotype consistent with observed defects in the key virulence determinants, capsule and melanin, and poor growth at 37°C. A comparative transcriptome analysis of the grx4 mutant and the WT strain under low-iron and iron-replete conditions confirmed a central role for Grx4 in iron homeostasis. Dysregulation of iron-related metabolism was consistent with grx4 mutant phenotypes related to oxidative stress, mitochondrial function, and DNA repair. Overall, the phenotypes of the grx4 mutant lacking the GRX domain and the transcriptome sequencing (RNA-Seq) analysis of the mutant support the hypothesis that Grx4 functions as an iron sensor, in part through an interaction with Cir1, to extensively regulate iron homeostasis.IMPORTANCE Fungal pathogens cause life-threatening diseases in humans, particularly in immunocompromised people, and there is a tremendous need for a greater understanding of pathogenesis to support new therapies. One prominent fungal pathogen, Cryptococcus neoformans, causes meningitis in people suffering from HIV/AIDS. In the present study, we focused on characterizing mechanisms by which C. neoformans senses iron availability because iron is both a signal and a key nutrient for proliferation of the pathogen in vertebrate hosts. Specifically, we characterized a monothiol glutaredoxin protein, Grx4, that functions as a sensor of iron availability and interacts with regulatory factors to control the ability of C. neoformans to cause disease. Grx4 regulates key virulence factors, and a mutant is unable to cause disease in a mouse model of cryptococcosis. Overall, our study provides new insights into nutrient sensing and the role of iron in the pathogenesis of fungal diseases.

Keywords: capsule; cryptococcosis; melanin; nuclear localization; transcriptome.

Figures

FIG 1
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
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
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
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
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
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
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
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
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
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
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.

Similar articles

See all similar articles

Cited by 5 articles

References

    1. Park BJ, Wannemuehler KA, Marston BJ, Govender N, Pappas PG, Chiller TM. 2009. Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS. AIDS 23:525–530. doi:10.1097/QAD.0b013e328322ffac. - DOI - PubMed
    1. May RC, Stone NR, Wiesner DL, Bicanic T, Nielsen K. 2016. Cryptococcus: from environmental saprophyte to global pathogen. Nat Rev Microbiol 14:106–117. doi:10.1038/nrmicro.2015.6. - DOI - PMC - PubMed
    1. Ballou ER, Johnston SA. 2017. The cause and effect of Cryptococcus interactions with the host. Curr Opin Microbiol 40:88–94. doi:10.1016/j.mib.2017.10.012. - DOI - PubMed
    1. Rajasingham R, Smith RM, Park BJ, Jarvis JN, Govender NP, Chiller TM, Denning DW, Loyse A, Boulware DR. 2017. Global burden of disease of HIV-associated cryptococcal meningitis: an updated analysis. Lancet Infect Dis 17:873–881. doi:10.1016/S1473-3099(17)30243-8. - DOI - PMC - PubMed
    1. Jung WH, Kronstad JW. 2008. Iron and fungal pathogenesis: a case study with Cryptococcus neoformans. Cell Microbiol 10:277–284. doi:10.1111/j.1462-5822.2007.01077.x. - DOI - PubMed

Publication types

Feedback