Miltefosine exhibits fungicidal activity through oxidative stress generation and Aif1 activation in Candida albicans.

Invasive candidiasis is the most common and serious fungal disease worldwide, and the development of antifungal drug resistance in Candida species has become an emerging problem. Miltefosine approved as an orphan drug for the therapy of invasive Candida infections by the United States Food and Drug Administration, has broad-spectrum antifungal activity, but its mechanism of action is unclear. Here, we evaluated the antifungal drug susceptibility of azole-resistant Candida species isolates and found that miltefosine showed good activity, with a geometric mean value of 2 μg/mL. We confirmed that miltefosine increases intracellular ROS production and induces apoptosis in C. albicans. We then performed RNA sequencing (RNA-Seq) analysis and iTRAQ-labeling-based quantitative proteomic mass spectrometry analysis. We identified Aif1 and the oxidative stress pathway involved in miltefosine-mediated apoptosis using global transcriptomic and proteomic combined screening. Miltefosine increased the mRNA and protein expressions of Aif1. We examined the localization of Aif1 using confocal microscopy and found that the GFP-Aif1 fusion protein was translocated from the mitochondria to the nucleus when sensing miltefosine. Then, we constructed the pex8 Δ/Δ strain and found that the minimum inhibitory concentration of miltefosine decreased four-fold (from 2 to 0.5 μg/mL) and the intracellular ROS significantly increased after knocking out the PEX8 gene. Moreover, we found that miltefosine triggers Hog1 phosphorylation. These findings indicate that Aif1 activation and the Pex8-mediated oxidative stress pathway are the mechanisms of action of miltefosine on C. albicans. Our results provide clues for us to understand the mechanisms by which miltefosine acts on fungi.