Miltefosine Has a Postantifungal Effect and Induces Apoptosis in Cryptococcus Yeasts

Abstract

<i>Cryptococcus</i> spp. are common opportunistic fungal pathogens, particularly in HIV patients. The approved drug miltefosine (MFS) has potential as an alternative antifungal against cryptococcosis; however, the mechanism of action of MFS in <i>Cryptococcus</i> is poorly understood. Here, we examined the effects of MFS on <i>C. neoformans</i> and <i>C. gattii</i> yeasts (planktonic and biofilm lifestyles) to clarify its mechanism of action. MFS presented inhibitory and fungicidal effects against planktonic <i>Cryptococcus</i> cells, with similar activities against dispersion biofilm cells, while sessile biofilm cells were less sensitive to MFS. Interestingly, MFS had postantifungal effect on <i>Cryptococcus</i>, with a proliferation delay of up to 8.15 h after a short exposure to fungicidal doses. MFS at fungicidal concentrations increased the plasma membrane permeability, likely due to a direct interaction with ergosterol, as suggested by competition assays with exogenous ergosterol. Moreover, MFS reduced the mitochondrial membrane potential, increased reactive oxygen species (ROS) production, and induced DNA fragmentation and condensation, all of which are hallmarks of apoptosis. Transmission electron microscopy analysis showed that MFS-treated yeasts had a reduced mucopolysaccharide capsule (confirmed by morphometry with light microscopy), plasma membrane irregularities, mitochondrial swelling, and a less conspicuous cell wall. Our results suggest that MFS increases the plasma membrane permeability in <i>Cryptococcus</i> via an interaction with ergosterol and also affects the mitochondrial membrane, eventually leading to apoptosis, in line with its fungicidal activity. These findings confirm the potential of MFS as an antifungal against <i>C. neoformans</i> and <i>C. gattii</i> and warrant further studies to establish clinical protocols for MFS use against cryptococcosis.