Role of ferric reductases in iron acquisition and virulence in the fungal pathogen Cryptococcus neoformans

Abstract

Iron acquisition is critical for the ability of the pathogenic yeast Cryptococcus neoformans to cause disease in vertebrate hosts. In particular, iron overload exacerbates cryptococcal disease in an animal model, defects in iron acquisition attenuate virulence, and iron availability influences the expression of major virulence factors. C. neoformans acquires iron by multiple mechanisms, including a ferroxidase-permease high-affinity system, siderophore uptake, and utilization of both heme and transferrin. In this study, we examined the expression of eight candidate ferric reductase genes and their contributions to iron acquisition as well as to ferric and cupric reductase activities. We found that loss of the FRE4 gene resulted in a defect in production of the virulence factor melanin and increased susceptibility to azole antifungal drugs. In addition, the FRE2 gene was important for growth on the iron sources heme and transferrin, which are relevant for proliferation in the host. Fre2 may participate with the ferroxidase Cfo1 of the high-affinity uptake system for growth on heme, because a mutant lacking both genes showed a more pronounced growth defect than the fre2 single mutant. A role for Fre2 in iron acquisition is consistent with the attenuation of virulence observed for the fre2 mutant. This mutant also was defective in accumulation in the brains of infected mice, a phenotype previously observed for mutants with defects in high-affinity iron uptake (e.g., the cfo1 mutant). Overall, this study provides a more detailed view of the iron acquisition components required for C. neoformans to cause cryptococcosis.

FIG 1

Domain structure of C. neoformans ferric reductases. Colored boxes indicate the ferric reductase transmembrane component-like domain (red box), the flavin adenine dinucleotide (FAD)-binding domain (blue box), and the ferric reductase NAD-binding domain (green box). An asterisk indicates the bis-heme motif comprising four conserved histidine residues. The length in number of amino acids is indicated adjacent to the sequences. Numbers in parentheses indicate the number of transmembrane regions.

FIG 2

Expression analysis of C. neoformans FRE transcript levels. (A) Relative expression of FRE genes in the WT strain in the presence of iron, as examined by qRT-PCR. RNA was extracted from cells incubated in YNB-LIM with no iron or with either 100 μM FeCl₃ or 10 μM hemin for 6 h. (B) Relative expression of FRE genes in the WT strain in the presence of copper, as examined by qRT-PCR. RNA was extracted from cells incubated in YNB-LCM with no copper or with 100 μM CuSO₄ for 6 h. The data were normalized using 18S rRNA as an internal control. The data are from three biological replicates, and the bars represent standard errors.

FIG 3

Reductase activities of freΔ mutants. (A) Ferric reductase activity of WT and freΔ mutant strains grown in defined LIM with no iron (−Fe) or with 100 μM FeCl₃ (+Fe). (B) Cupric reductase activity of WT and freΔ mutant strains grown in defined LCM with no copper (−Cu) or with 100 μM CuSO₄ (+Cu). The data are from three replicates, and the bars represent standard errors.

FIG 4

The fre4Δ mutant is defective in melanin production. (A) Melanin production in WT and freΔ mutant strains was compared by growing cells on l-DOPA plates for 2 days. (B) Melanin production in the fre4Δ mutant was restored to the WT level by addition of exogenous copper. Cells were grown on l-DOPA plates with no CuSO₄ (−Cu) or with 50 μM CuSO₄ (+Cu) for 2 days.

FIG 5

Fre2 is required for robust growth on hemin. Strains were first iron starved in YNB-LIM for 2 days, washed, and then transferred to either solid agar plates or liquid medium. (A) Tenfold serial dilutions of iron-starved strains were spotted on YPD, YNB-LIM, and YNB-LIM with different concentrations of FeCl₃ or hemin as indicated. Plates were incubated at 30°C for 2 days. (B) Iron-starved strains were inoculated into YNB-LIM with no iron or with different concentrations of FeCl₃ or hemin as indicated. Cultures were incubated at 30°C with shaking, and turbidity was measured every 12 h. Reduced growth of fre2Δ and fre201Δ mutants was consistently observed in several repeats of the experiments. (C) Tenfold serial dilutions of iron-starved strains were spotted on YNB-LIM and YNB-LIM with different concentrations of hemin as indicated. Plates were incubated at 30°C for 2 days.

FIG 6

Fre2 and Fre201 are required for robust growth on transferrin. Strains were first iron starved in YNB-LIM for 2 days, washed, and then inoculated into YNB-LIM with no transferrin or with 10 μM transferrin. Cultures were incubated at 30°C with shaking, and turbidity was measured every 12 h. Reduced growth of fre2Δ and fre201Δ mutants was consistently observed in several repeats of the experiments.

FIG 7

Fre2 is not required for hemin uptake. Strains were first iron starved in YNB-LIM for 2 days, washed, and then transferred to solid agar plates. Tenfold serial dilutions of iron-starved strains were spotted on YPD without or with 10 μM Ga-PPIX, YNB-LIM plus 100 μM FeCl₃, or YNB-LIM plus 10 μM hemin without or with 10 μM Ga-PPIX or 10 μM GaCl₃ as indicated. Plates were incubated at 30°C for 2 days.

FIG 8

Deletion of FRE4 increases susceptibility to antifungal drugs. (A) Tenfold serial dilutions of strains were spotted on YPD without or with the antifungal drugs as indicated. Plates were incubated at 30°C for 2 days. (B) Addition of exogenous hemin, FeCl₃, or CuSO₄ suppressed the antifungal drug susceptibility of the fre4Δ mutant. Tenfold serial dilutions of strains were spotted on YPD with fluconazole without or with an iron or copper source as indicated. Plates were incubated at 30°C for 2 days.

FIG 9

The fre2Δ mutant and the fre2Δ fre4Δ double mutants are attenuated in virulence. Thirteen BALB/c mice were infected intranasally with each of the strains indicated, and the survival of the mice was monitored over the time course indicated on the x axis. The difference in survival rates between the fre2Δ mutant and the WT strain was significant (P < 0.0001), as was the difference between the survival rates of the fre2Δ fre4Δ double mutants and the WT strain (P < 0.0001).

FIG 10

The fre2Δ mutant and the fre2Δ fre4Δ double mutants showed reduced fungal load in the brain in a mouse model of cryptococcosis. The distribution of fungal cells in the lungs and brains of mice was analyzed with three mice for each strain sacrificed at day 20 when WT-infected mice reached the endpoint. The dashed horizontal line indicates the detection limit of the assay, and the error bars indicate standard errors.