A defect in iron uptake enhances the susceptibility of Cryptococcus neoformans to azole antifungal drugs

Abstract

The high-affinity reductive iron uptake system that includes a ferroxidase (Cfo1) and an iron permease (Cft1) is critical for the pathogenesis of Cryptococcus neoformans. In addition, a mutant lacking CFO1 or CFT1 not only has reduced iron uptake but also displays a markedly increased susceptibility to azole antifungal drugs. Altered antifungal susceptibility of the mutants was of particular interest because the iron uptake system has been proposed as an alternative target for antifungal treatment. In this study, we used transcriptome analysis to begin exploring the molecular mechanisms of altered antifungal susceptibility in a cfo1 mutant. The wild-type strain and the cfo1 mutant were cultured with or without the azole antifungal drug fluconazole and their transcriptomes were compared following sequencing with Illumina Genome Analyzer IIx (GAIIx) technology. As expected, treatment of both strains with fluconazole caused elevated expression of genes in the ergosterol biosynthetic pathway that includes the target enzyme Erg11. Additionally, genes differentially expressed in the cfo1 mutant were involved in iron uptake and homeostasis, mitochondrial functions and respiration. The cfo1 mutant also displayed phenotypes consistent with these changes including a reduced ratio of NAD+/NADH and down-regulation of Fe-S cluster synthesis. Moreover, combination treatment of the wild-type strain with fluconazole and the respiration inhibitor diphenyleneiodonium dramatically increased susceptibility to fluconazole. This result supports the hypothesis that down-regulation of genes required for respiration contributed to the altered fluconazole susceptibility of the cfo1 mutant. Overall, our data suggest that iron uptake and homeostasis play a key role in antifungal susceptibility and could be used as novel targets for combination treatment of cryptococcosis. Indeed, we found that iron chelation in combination with fluconazole treatment synergistically inhibited the growth of C. neoformans.

UPC2 Is Universally Essential for Azole Antifungal Resistance in Candida albicans

“…This phenotype reverted upon the addition of iron to the medium. A similar observation has been made for Cryptococcus neoformans, where disruption of the genes encoding the ferroxidase (CFO1) and an iron permease (CFT1) of the high-affinity reductive iron uptake system resulted in increased susceptibility to azole antifungals (52). In the present study, we found that when UPC2 is intact, fluconazole exposure results in the upregulation of four genes associated with high-affinity iron uptake (FTR1, FET3, FET34, and FRP1).…”

The lysine biosynthetic enzyme Lys4 influences iron metabolism, mitochondrial function and virulence in Cryptococcus neoformans

“…Our previous transcriptome data showed that the gene CNAG_02565 was significantly downregulated in the cfo1 mutant, which suggested a possible connection between the gene function and the iron homeostasis and metabolism in C. neoformans [12]. The protein sequence of CNAG_02565 is highly homologous to Lys4, an enzyme in the lysine biosynthesis pathway in S. cerevisiae , with 64% similarity and 52% identity [21].…”

“…The protein encoded by S. cerevisiae LYS4 contains amino acid residues associated with an iron-sulfur (Fe–S) cluster and shows evolutionary conservation with the aconitase family of proteins [3,11]. The S. cerevisiae ortholog of LYS4 in C. neoformans was initially found in our transcriptome analysis to identify differentially expressed genes in a mutant lacking CFO1 , the gene encoding a ferroxidase for high-affinity iron transport at the plasma membrane [12]. The expression of LYS4 was significantly down-regulated in the cfo1 mutant, suggesting that Lys4 function may be associated with iron metabolism in C. neoformans .…”

Defects in Phosphate Acquisition and Storage Influence Virulence of Cryptococcus neoformans

“…Cellular levels of P, Na, Fe, and Zn were determined by inductively coupled plasma-atomic emission spectroscopy (ICP-AES) as described elsewhere (41). Briefly, cells were grown in YPD at pH 7.0, washed twice with water, frozen, and lyophilized.…”