Miltefosine Has a Postantifungal Effect and Induces Apoptosis inCryptococcusYeasts

ABSTRACTCryptococcusspp. 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 inCryptococcusis poorly understood. Here, we examined the effects of MFS onC. neoformansandC. gattiiyeasts (planktonic and biofilm lifestyles) to clarify its mechanism of action. MFS presented inhibitory and fungicidal effects against planktonicCryptococcuscells, with similar activities against dispersion biofilm cells, while sessile biofilm cells were less sensitive to MFS. Interestingly, MFS had postantifungal effect onCryptococcus, 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 inCryptococcusvia 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 againstC. neoformansandC. gattiiand warrant further studies to establish clinical protocols for MFS use against cryptococcosis.

N-Chlorotaurine Exhibits Fungicidal Activity against Therapy-Refractory Scedosporium Species and Lomentospora prolificans

ABSTRACTN-Chlorotaurine (NCT), a well-tolerated endogenous long-lived oxidant that can be applied topically as an antiseptic, was tested on its fungicidal activity againstScedosporiumandLomentospora, opportunistic fungi that cause severe infections with limited treatment options, mainly in immunocompromised patients. In quantitative killing assays, both hyphae and conidia ofScedosporium apiospermum,Scedosporium boydii, andLomentospora prolificans(formerlyScedosporium prolificans) were killed by 55 mM (1.0%) NCT at pH 7.1 and 37°C, with a 1- to 4-log10reduction in CFU after 4 h and a 4- to >6-log10reduction after 24 h. The addition of ammonium chloride to NCT markedly increased this activity. LIVE/DEAD staining of conidia treated with 1.0% NCT for 0.5 to 3 h increased the permeability of the cell wall and membrane. Preincubation of the test fungi in 1.0% NCT for 10 to 60 min delayed the time to germination of conidia by 2 h to >12 h and reduced their germination rate by 10.0 to 100.0%. Larvae ofGalleria mellonellainfected with 1.0 × 107conidia ofS. apiospermumandS. boydiidied at a rate of 90.0 to 100% after 8 to 12 days. The mortality rate was reduced to 20 to 50.0% if conidia were preincubated in 1.0% NCT for 0.5 h or if heat-inactivated conidia were used. Our study demonstrates the fungicidal activity of NCT against differentScedosporiumandLomentosporaspecies. A postantifungal effect connected with a loss of virulence occurs after sublethal incubation times. The augmenting effect of ammonium chloride can be explained by the formation of monochloramine.

Dynamic Interaction between Fluconazole and Amphotericin B against Cryptococcus gattii

ABSTRACTCryptococcus gattiiis the main pathogen of cryptococcosis in healthy patients and is treated mainly with fluconazole and amphotericin B. The combination of these drugs has been questioned because the mechanisms of action could lead to a theoretical antagonistic interaction. We evaluated distinct parameters involved in thein vitrocombination of fluconazole and amphotericin B againstCryptococcus gattii. Fourteen strains ofC. gattiiwere used for the determination of MIC, fractional inhibitory concentration, time-kill curve, and postantifungal effect (PAFE). Ergosterol quantification was performed to evaluate the influence of ergosterol content on the interaction between these antifungals. Interaction between the drugs varied from synergistic to antagonistic depending on the strain and concentration tested. Increasing fluconazole levels were correlated with an antagonistic interaction. A total of 48 h was necessary for reducing the fungal viability in the presence of fluconazole, while 12 h were required for amphotericin B. When these antifungals were tested in combination, fluconazole impaired the amphotericin B activity. The ergosterol content decreased with the increase of fluconazole levels and it was correlated with the lower activity of amphotericin B. The PAFE found varied from 1 to 4 h for fluconazole and from 1 to 3 h for amphotericin B. The interaction of fluconazole and amphotericin B was concentration-dependent and special attention should be directed when these drugs are used in combination againstC. gattii.