title: Effects of melanin upon susceptibility of Cryptococcus to antifungals.
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.
authors.affiliation: Department of Microbiology, Meiji Pharmaceutical University, Kiyose, Tokyo 204-8588, Japan. ikeda@my-pharm.ac.jp
authors.forename: Reiko
authors.initials: R
authors.lastname: Ikeda
authors.forename: Takashi
authors.initials: T
authors.lastname: Sugita
authors.forename: Eric S
authors.initials: ES
authors.lastname: Jacobson
authors.forename: Takako
authors.initials: T
authors.lastname: Shinoda
mesh_terms: D000666:Amphotericin B / Q000378:metabolism / Q000494:pharmacology / Q000633:toxicity; D000935:Antifungal Agents / Q000494:pharmacology*; D003454:Cryptococcus / Q000145:classification / Q000187:drug effects* / Q000472:pathogenicity*; D003455:Cryptococcus neoformans / Q000187:drug effects / Q000472:pathogenicity; D025141:Drug Resistance, Fungal; D015725:Fluconazole / Q000378:metabolism / Q000494:pharmacology / Q000633:toxicity; D005456:Fluorescent Dyes / Q000378:metabolism; D008543:Melanins / Q000502:physiology*; D008826:Microbial Sensitivity Tests
chemical_list: D000935:Antifungal Agents; D005456:Fluorescent Dyes; D008543:Melanins; D000666:Amphotericin B; D015725:Fluconazole