Insight into the Role of Metal Homeostasis in Fungal Adaptation to Drug Stress

Abstract

Maintenance of metal homeostasis is critical to cell survival due to the multitude of cellular processes that depend on one or more metal cofactors. We show that the opportunistic fungal pathogen Candida albicans becomes sensitized to both Cu limitation and Cu elevation during exposure in liquid culture to the antifungal drug fluconazole, a widely prescribed antifungal agent. Cu supplementation reduces tolerance of C. albicans to fluconazole in a way that does not require formation of a Cu–fluconazole complex. Rather, our data point to a less obvious relationship between drug stress and Cu availability that gives rise to metal-mediated outcomes of drug treatment. qRT-PCR, EPR, fluorescence, and ICP-MS studies demonstrate that C. albicans extensively remodels its metal homeostasis networks to respond to treatment with fluconazole. These adaptation strategies include increased Cu import and storage, increased retention of Fe, Mn, and Zn, altered utilization of Cu- and Mn-dependent enzymes, mobilization of intracellular Fe stores, and increased production of the heme prosthetic group utilized by the enzyme target of fluconazole. Furthermore, RNA-seq analysis reveals that co-treatment with fluconazole and Cu gives rise to unique patterns of gene expression that illustrate the profound impacts of small fluctuations in Cu availability on the transcriptomic response to fluconazole stress. Finally, we show that fluconazole causes substantial changes to the metalloproteome of C. albicans, most notably to the levels of Cu proteins. The findings offer a new perspective for thinking about fungal response to drug stress that pushes cells out of their metal homeostatic zones, leading them to enact metal-associated adaptation mechanisms to restore homeostasis to survive.