A defect in iron uptake enhances the susceptibility of Cryptococcus neoformans to azole antifungal drugs
Highlights
► Altered antifungal susceptibility of a mutant lacking CFO1 was investigated. ► Iron uptake plays a key role in antifungal susceptibility of Cryptococcus neoformans. ► Iron uptake pathway could be used as a novel target for combination therapy.
Introduction
Cryptococcus neoformans is a basidiomycete fungal pathogen of humans that frequently causes life-threatening fungal meningitis in immunocompromised individuals. Cryptococcal meningitis associated with AIDS is fatal in the absence of antifungal therapy and the mortality rate can be as high as 40% in sub-Saharan Africa (Hakim et al., 2000). Non-HIV-associated cryptococcosis has also become an important issue, especially among patients with organ transplantation or long-term anticancer therapy (Bicanic and Harrison, 2004). A limited number of antifungal drugs are available to treat cryptococcosis with the polyene drug amphotericin B and azole drugs such as fluconazole being the most widely used. Amphotericin B interacts directly with sterols in the fungal membrane and this results in the production of pores that alter permeability and cause cytoplasmic leakage leading to cell death (Ghannoum and Rice, 1999, Lupetti et al., 2002). Although amphotericin B is considered the most effective antifungal drug, severe side effects such as nephrotoxicity limit its clinical use. To reduce side effects, liposomal forms of amphotericin B have been developed. Moreover, combination therapy with amphotericin B and 5-flucytosine has been established to treat cryptococcal meningitis (Bennett et al., 1979).
Besides amphotericin B, azole antifungals are also widely used in the treatment of cryptococcal infections. Fluconazole, in particular, has been the agent of choice for the long term management of cryptococcosis because of its clinical efficacy and safety (Zonios and Bennett, 2008). Azole antifungal drugs such as fluconazole are generally considered fungistatic and act by inhibiting the ergosterol biosynthetic enzyme lanosterol 14α-demethylase (Erg11), which belongs to the hemoprotein cytochrome P450 protein family. Inhibition of Erg11 interferes with the conversion of lanosterol to ergosterol, a membrane component, and this leads to accumulation of 14-α-methylsterols that cause membrane disorganization, cytoplasmic leakage and growth arrest (Ghannoum and Rice, 1999). Although they are effective, long-term use of azole antifungal drugs often leads to the emergence of resistance that causes treatment failure and recurrence of disease. The mechanisms of resistance to azole antifungal drugs have been extensively studied in another well-known pathogenic yeast, Candida albicans. In C. albicans, several mechanisms result in the emergence of resistance: (1) reduced intracellular accumulation of azoles, which is often associated with the up-regulation of genes encoding efflux pumps; (2) increased levels of the azole cellular target by altered regulation; (3) alteration in sterol synthesis, which is usually achieved by a deficiency in the sterol Δ5,6-desaturase encoded by ERG3 and; (4) decreased affinity of azoles to their target due to mutations in ERG11 (Lupetti et al., 2002). Moreover, a role of Hsp90 in evolution of azole resistance has been demonstrated (Cowen et al., 2006, Cowen and Lindquist, 2005).
Azole resistant strains have been reported for C. neoformans, although few studies have examined the mechanisms of azole resistance. One example is a recent study that employed comparative genome hybridization to reveal that the formation of disomic chromosomes carrying ERG11 or a gene for an efflux pump caused increased resistance (Sionov et al., 2010). Furthermore, another recent study reported that mutations in the ERG11 gene were associated with azole resistance (Sionov et al., 2011). The emergence of resistance can be countered by combination antifungal therapy and this approach has been successfully applied to cryptococcosis through the use of amphotericin B in combination with 5-flucytocine (Wirk and Wingard, 2008). Combinations of drugs that target new functions also hold promise for expanding antifungal therapeutic options. For example, the combination of Hsp90 inhibition and micafungin, a member of the echinocandin class of inhibitors of fungal 1,3-β-D-glucan synthase, has shown enhanced fungicidal effect (Singh et al., 2009).
Interference with iron acquisition by pathogens may yield new opportunities for combination therapy. For C. neoformans, the high-affinity reductive iron uptake pathway comprised of the iron permease Cft1 and the ferroxidase Cfo1 plays an important role in virulence. Mutants deficient in the pathway are not able to utilize iron from transferrin and display significant attenuation of virulence in a mouse model of cryptococcosis (Jung et al., 2009, Jung et al., 2008). Moreover, our previous data suggested that the high-affinity reductive iron uptake pathway influences susceptibility to azole antifungal drugs in C. neoformans (Jung et al., 2009). For example, a mutant lacking CFT1 shows increased susceptibility to miconazole, and a mutant lacking CFO1 displays increased susceptibility to fluconazole (Jung et al., 2009, Jung et al., 2008). These results support the possibility of a novel combination therapy that uses currently available azole drugs and inhibition of the high-affinity reductive iron uptake pathway. This idea prompted our current study of the mechanism of altered azole susceptibility for the cfo1 mutant. Our approach was to examine the transcriptome of the cfo1 mutant grown in the presence or absence of fluconazole using deep RNA sequencing (RNA-Seq) and to compare it with the transcriptome of the wild-type strain. We found that the expression of genes encoding iron-dependent enzymes including those required for ergosterol biosynthesis and respiration were significantly changed by deletion of CFO1. Moreover, our data suggested that reduced expression of genes required for mitochondrial functions such as respiration and Fe–S cluster synthesis made important contributions to the increased fluconazole susceptibility of the cfo1 mutant. We also found a consistent pattern of regulation in which these genes were transcriptionally regulated by the iron-regulatory proteins Hap3 and HapX; mutations in these regulators also influenced azole susceptibility in C. neoformans.
Section snippets
Strains and growth conditions
Strains used in this study were C. neoformans var. grubii H99 and a cfo1 mutant of the same strain. The mutant was constructed previously and complementation demonstrated that the phenotypes of the strain were due to the deletion of CFO1 (Jung et al., 2009). Cells were routinely grown in YPD medium (1% yeast extract, 2% bacto-peptone and 2% glucose) or YNB medium (yeast nitrogen base, Difco) with 2% glucose. YPG medium (1% yeast extract, 2% bacto-peptone and 2% galactose) was used to induce
Deletion of CFO1 increases susceptibility and reduces tolerance to azole antifungal drugs
We previously reported that deletion of the CFO1 gene encoding the ferroxidase component of the high-affinity reductive iron uptake system increased the susceptibility of C. neoformans to the antifungal drug fluconazole. We hypothesized that a possible underlying mechanism could be low intracellular iron and a concomitant deficiency in heme synthesis resulting in reduced activity of heme-containing enzymes for ergosterol biosynthesis (e.g., Erg11) (Jung et al., 2009). To begin to test this
Discussion
In this study, we examined phenotypic and gene expression changes in the cfo1 mutant lacking the ferroxidase for the high-affinity iron uptake system in C. neoformans to obtain a more comprehensive view of the reasons underlying increased fluconazole susceptibility in the mutant. Initially, we found that fluconazole tolerance was significantly reduced by deletion of CFO1 and that the normally fungistatic activity of fluconazole became fungicidal upon loss of Cfo1. Similar results have been
Conclusions
A C. neoformans mutant lacking the ferroxidase Cfo1 displayed decreased intracellular iron levels and increased susceptibility to several azole drugs. A combination of transcriptional profiling, mutant analysis, overexpression studies and the use of inhibitors demonstrated that the altered fluconazole susceptibility of the cfo1 mutant was caused by decreased respiration in combination with other mitochondrial functions such as redox homeostasis and Fe–S cluster synthesis. Furthermore, we
Acknowledgments
We thank Kyunghwan Han for help with CFU analysis, and Byung Hee Kim and Kyoung Kyu Kang for help with ICP analysis. This work was supported by awards from the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology 2012-0004062 (W.H.J.) and 2011-0029843 (W.H.J., Y.L), and the National Institutes of Health R01 AI053721 (J.W.K.).
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