The antifungal effect induced by itraconazole in Candida parapsilosis largely depends on the oxidative stress generated at the mitochondria

Abstract

In Candida parapsilosis, homozygous disruption of the two genes encoding trehalase activity increased the susceptibility to Itraconazole compared with the isogenic parental strain. The fungicidal effect of this azole can largely be counteracted by preincubating growing cells with rotenone and the protonophore 2,4-Dinitrophenol. In turn, measurement of endogenous reactive oxygen species formation by flow cytometry confirmed that Itraconazole clearly induced an internal oxidative stress, which can be significantly abolished in rotenone-exposed cells. Analysis of the antioxidant enzymatic activities of catalase and superoxide dismutase pointed to a moderate decrease of catalase in trehalase-deficient mutant cells compared to the wild type, with an additional increase upon addition of rotenone. These enzymatic changes were imperceptible in the case of superoxide dismutase. Alternative assays with Voriconazole led to a similar profile in the results regarding cell growth and antioxidant activities. Collectively, our data suggest that the antifungal action of Itraconazole on C. parapsilosis is dependent on a functional mitochondrial activity. They also suggest that the central metabolic pathways in pathogenic fungi should be considered as preferential antifungal targets in new research.

Keywords: Candida parapsilosis; Dinitrophenol; Mitochondria; ROS; Rotenone; Trehalase.

Fig. 1

Effect of homozygous disruption of the two genes encoding trehalase activity in C. parapsilosis on cell susceptibility to Itraconazole (ITC) and Voriconazole (VRC). A pre-inoculum of both the parental strain of C. parapsilosis (Cp) and the trehalase-deficient null mutant atc1Δ/ntc1Δ were cultured overnight in YPD medium, diluted to an OD_{600 nm} of 0.3 in the same medium and further incubated at 37°C. Cellular growth was measured turbidimetrically by recording the changes in OD_{600 nm} during 24 h. Symbols: (■) Control, (▲) ITC (0.3 µg/ml), (○) VRC (0.3 µg/ml), (▼) (ITC + rotenone)

Fig. 2

Protective action of rotenone and DNP on C. parapsilosis cell viabilitiy against the antifungal toxicity induced by addition of Itraconazole (ITC). YPD-grown exponential cells of the two cell types under study, were preincubated with either rotenone (0.156 mM) or DNP (0.5 mM) for 1 h at 37°C, and immediately treated with ITC (0.3 µg/ml) for 1 h or 10 h A and B or only 10 h C. Identical samples were then harvested, diluted appropriately, and spread on YPD plates. The percentage of cell survival was determined by CFU counting. The data shown are the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.01 represent statistically significant differences with respect to an untreated control according to Mann–Whitney U test. B Tenfold cell suspensions were spotted on YPD plates (5 µl), which were incubated at 37°C and scored after 48 h

Fig. 3

Level of intracellular ROS production after treatment with Itraconazol and protective role of rotenone in C. parapsilosis. Equivalent aliquots of YPD-grown exponential cultures of the two strains were incubated for 1 h in the absence or presence of rotenone (0.156 mM) at 37°C, followed by 1 h of treatment with ITC (0.3 µg/ml). ROS were quantified by flow cytometry using DHF in Cp (upper row histograms) and atc1Δ/ntc1Δ (lower row histograms) strains. The histograms display the cell number with respect to the green fluorescence intensity (FL1). A positive marker for acute oxidative stress (50 mM H₂O₂) was also introduced

Fig. 4

Effect of rotenone on the ITC-induced changes in enzymatic activity shown by the antioxidant enzymes catalase (A) and superoxide dismutase (SOD) (B) in C. parapsilosis Cp and atc1Δ/ntc1Δ strains. Exponential cultures grown in YPD were subjected to the indicated ITC treatment for 1 h with or without preincubation with rotenone. A sample treated with Voriconazole (VRC) was also included. The data shown are the mean ± SD of three independent experiments. *P < 0.05, represents statistically significant differences with respect to an untreated control according to Mann–Whitney U test or between the samples indicated with the brackets