Cryptococcus neoformans and Cryptococcus gattii are the etiological agents of cryptococcosis, a disease with a high incidence among severely immunocompromised patients. Amphotericin B is the standard antifungal to treat the neuronal form of cryptococcosis, but its use has been limited. Thus, the search for new therapeutic options is necessary for the treatment of this mycosis. Previous studies had shown miltefosine (MFS) as an alternative antifungal drug for several pathogenic fungi including Cryptococcus spp., however, its mechanism of action on this fungus is poorly understood. The aim of this study was to characterize the fungicidal effects of MFS on Cryptococcus.
The broth microdilution assay was performed to determine the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of MFS on C. neoformans (H99 and CAP59) and C. gattii (ATCC 56990). Subsequent assays were performed to evaluate the post-antifungal effect (PAFE) of MFS, yeast viability, plasma membrane permeability, drug interaction with exogenous ergosterol and yeast morphological alterations. Yeasts of C. neoformans H99 untreated or treated with fungicidal concentrations of MFS (4x and 8x MIC) were analyzed by flow cytometry or fluorescence microscopy using propidium iodide, Rhodamine123, DCFH-DA, TUNEL, and Hoechst 33342 as fluorescent markers. 
We confirmed the inhibitory and fungicidal activity of MFS on planktonic cells (0.5 - 2.0 µg/ml) and also observed that the dispersion cells from Cryptococcus biofilms exhibited similar MFS susceptibility to their planktonic counterpart. In contrast, MFS did not present inhibitory effect on sessile cells from biofilms at concentrations up to 16 µg/ml. Interestingly, MFS showed post-antifungal effect on Cryptococcus spp. that lasted for up to 8.15 h suggesting prolonged MFS inhibitory activity even when the drug pressure is no longer present, and this effect is associated with the fungicidal effect. Morphological changes were observed by light and transmission electron microscopies after exposure of the yeasts to MFS, such as a significant reduction of the mucopolysaccharide capsule thickness , plasma membrane irregularity, and presence of swollen mitochondria. In addition, treatment with fungicidal concentrations led to significant extravasation of DNA and proteins through the membrane. Conversely, in the competition assay with ergosterol we observed a reduction in the antifungal activity of MFS. Thus, the increased permeability in the plasma membrane (Figure 1A) may be related with a direct interaction of MFS with the ergosterol in the plasma membrane. Also, our flow cytometry results showed that MFS interferes with the mitochondrial potential (Figure 1B) leading to increased ROS production (Figure 1C), and DNA fragmentation (Figure 1D) and DNA condensation (Figure 1E) as final consequence. Amphotericin B, an antifungal gold standard, was used to compare with MFS. 
Together, our results suggest that MFS acts on the membranes of Cryptococcus spp. yeasts leading to two important events: increased plasma membrane permeability and a deleterious effect on the mitochondrial activity inducing cell death by apoptosis; both effects corroborating to the MFS fungicidal activity. These findings reinforce the potential use of MFS as an alternative treatment for cryptococcosis.

Miltefosine shows fungicidal effect on Cryptococcus spp. by apoptosis mechanism.