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.

Reactive oxidant species induced by antifungal drugs: identity, origins, functions, and connection to stress-induced cell death

“…Evidence of ROS formation in response to azole exposure has been reported in both yeasts and molds ( Table 1 ). Interestingly, in Candida , the most robust and reproducible evidence for ROS formation has been obtained in response to fungicidal azoles: miconazole and itraconazole ( Kobayashi et al., 2002 ; Francois et al., 2006 ; Belenky et al., 2013 ; Lee and Lee, 2018 ; Muñoz-Megías et al., 2023 ). In contrast, investigating whether C. albicans induces ROS in response to fluconazole, which is fungistatic in Candida , has produced conflicting results ( Kobayashi et al., 2002 ; Francois et al., 2006 ; Belenky et al., 2013 ) ( Table 1 ).…”

“…Additionally, the same study reported that although the echinocandin caspofungin also elicited robust ROS induction (at least as high as amphotericin B), no mitochondrial membrane peroxidation was observed during caspofungin treatment (lipid peroxidation and Yap1 localization were not reported for caspofungin) ( Shekhova et al., 2017 ). Furthermore, although in C. parapsilosis itraconazole induced ROS production, there was no concomitant increase in the activities of either catalase or superoxide dismutase, two of the primary enzymes responsible for detoxifying cellular ROS ( Muñoz-Megías et al., 2023 ). Finally, we have reported that although the echinocandins caspofungin and micafungin elicit robust ROS induction in N. glabratus , we detected no concomitant increases in lipid peroxidation or induction of oxidative genes ( Garcia-Rubio et al., 2021 ).…”

“…Several studies have explicitly posited that the ROS induced by antifungal drugs are of mitochondrial origin ( Mesa-Arango et al., 2014 ; Shekhova et al., 2017 ; Muñoz-Megías et al., 2023 ). A key piece of evidence supporting that conclusion was that the addition of rotenone, an inhibitor of mitochondrial complex I, significantly reduced the levels of ROS induced by amphotericin B and itraconazole in A. fumigatus ( Shekhova et al., 2017 ), itraconazole in C. parapsilosis ( Muñoz-Megías et al., 2023 ), and amphotericin B in C. tropicalis ( Mesa-Arango et al., 2014 ). All of these fungi contain the canonical mitochondrial complex I, so the effect of rotenone could be explained due to its known inhibition of that complex, resulting in reduced electron leakage and ROS formation.…”