Glutathione metabolism in Candida albicans resistant strains to fluconazole and micafungin

Abstract

Currently available therapies for candidiasis are based on antifungal drugs belonging to azole and echinocandin families that interfere with different aspects of fungal metabolism. These drugs, beyond their specific effects, elicit also a cellular stress including an unbalance of redox state that is counteracted not only utilizing antioxidant species but also increasing the outcome export by transporters to detoxify the internal environment. These cellular actions are both based on the cytosolic concentration of reduced glutathione (GSH). In this paper we investigated the effects of two antifungal drugs fluconazole and micafungin on the redox state of the cell in C. albicans to understand if the resistance to these drugs is accompanied by variation of glutathione metabolism. Analyses of resistant strains showed a marked difference in glutathione contents in strains resistant to fluconazole (CO23RFLC) or micafungin (CO23RFK). In CO23RFLC, the total amount of glutathione was more than doubled with respect to CO23RFK thanks to the increased activity of γ-glutamilcysteine synthetase, the key enzyme involved in GSH synthesis. We demonstrated that the GSH increase in CO23RFLC conferred to this strain a clear advantage in counteracting oxidative toxic agents while assignment of other roles, such as a more efficient elimination of the drug from the cell, should be considered more speculative. As far as MCFG resistance is concerned, from our data a role of glutathione metabolism in supporting this condition is not evident. Overall our data indicate that glutathione metabolism is differently affected in the two resistant strains and that glutathione system may play an important role in the global organization of C.albicans cells for resistance to fluconazole. Such scenario may pave the way to hypothesize the use of oxidant drugs or inhibitors able to deplete reduced glutathione level as a novel approach, for counteracting the resistance to this specific antifungal drug.

Figure 1. Total glutathione in CO23, CO23RFK, CO23FLC, treated and untreated with FLC or MCFG.

The reported quantities were calculated using the average values for GSH and GSSG reported in Tab. 2.

Figure 2. Expression levels of C. albicans GLR1 and GSH1 genes determined by real-time PCR.

The expression level of the gene in the wild-type (CO23) or resistant (CO23RFK and CO23RFLC) strains, treated or untreated with FLC or MCFG, are represented as n-fold increase or decrease relative to the level of the untreated control strain (CO23). All bars represent mean±SD.

Figure 3. ROS production in sensitive and resistant strains.

The intracellular ROS contents is evaluated by flow cytometry using DHR123 staining. Fluorescence intensity is calculated as mean fluorescence channel. All bars represent mean±SD.

Figure 4. Analysis of oxidative effects in sensitive, CO23MCFG and CO23RFLC strains of C. albicans.

Approximately 10⁵ cells of CO23, CO23MCFG and CO23RFLC strains were serially diluted and spotted onto YPD plates treated or not with 4 mM H₂O₂ in absence (1) or in presence of GSH precursors 0.02 M glutamate, 0.02 M cysteine, 0.02 M, glycine (2) or with 5 mM BSO (L-buthionine-sulfoximine) (3).