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Effects of melanin upon susceptibility of Cryptococcus to antifungals
Reiko Ikeda1, Takashi Sugita, Eric S Jacobson
1Department of Microbiology, Meiji Pharmaceutical University, Kiyose, Tokyo 204-8588, Japan. ikeda@my-pharm.ac.jp
Abstract:
Melanin is a recognized virulence factor in Cryptococcus neoformans; several pathogenetic mechanisms have been suggested. We studied melanin as an antifungal resistance factor. The growth of laccase-active strains of C. neoformans and C. albidus in L-DOPA resulted in the production of black pigment. The formal minimal inhibitory concentrations (MICs) of amphotericin B and fluconazole were not changed by melanization. However, when we examined those wells which contained inhibited cells, we found live cells only in wells containing melanized C. neoformans. In contrast, melanization did not protect C. albidus from killing by amphotericin B. In an amphotericin B time-kill study of C. neoformans, significantly more melanized cells than non-melanized survived for the first few hours. Fluorescence microscopy and flow cytometry analyses showed that fewer melanized cells were stained with the fluorescent dye MitoRed. Incubation of MitoRed (the model) or amphotericin B with melanin extracted from C. neoformans decreased the free concentrations of these substances. Fluconazole, in contrast, was not removed from solution by melanin. This suggests that neoformans cryptococcal melanin deposited amphotericin B in the cell wall binds, reducing its effective concentrations.
Insights
Cryptococcus neoformans melanin enhances fungal survival against amphotericin B by binding the drug. Melanization protects yeast cells, suggesting melanin is an antifungal resistance factor.
Area of Science:
- Mycology
- Medical Mycology
- Antifungal Research
Background:
- Melanin is a known virulence factor in Cryptococcus neoformans.
- The role of melanin in antifungal resistance remains incompletely understood.
- Investigating melanin's impact on antifungal drug efficacy is crucial for understanding cryptococcal pathogenesis.
Purpose of the Study:
- To investigate the role of melanin as a factor in antifungal resistance in Cryptococcus neoformans.
- To determine if melanization affects the susceptibility of C. neoformans to amphotericin B and fluconazole.
- To elucidate the mechanism by which melanin might confer resistance.
Main Methods:
- Melanization of Cryptococcus neoformans and Cryptococcus albidus using L-DOPA.
- Determination of minimal inhibitory concentrations (MICs) for amphotericin B and fluconazole.
- Time-kill studies with amphotericin B.
- Fluorescence microscopy and flow cytometry using MitoRed dye.
- In vitro studies of drug-melanin interactions.
Main Results:
- Melanization did not alter the MICs of amphotericin B or fluconazole.
- Melanized C. neoformans showed increased survival against amphotericin B in time-kill studies.
- Melanin extracted from C. neoformans reduced the effective concentration of amphotericin B and MitoRed in solution.
- Melanin did not bind or reduce the concentration of fluconazole.
- Fewer melanized cells were stained with MitoRed, indicating reduced drug uptake.
Conclusions:
- Cryptococcal melanin acts as an antifungal resistance factor by binding amphotericin B.
- Melanin reduces the effective concentration of amphotericin B, leading to increased yeast survival.
- This mechanism highlights a novel pathway for antifungal drug inactivation by fungal melanin.