Cryptococcus neoformans/Cryptococcus gattii species complex melanized by epinephrine: Increased yeast survival after amphotericin B exposure

Abstract

Cryptococcus neoformans/Cryptococcus gattii complex species are etiological agents of cryptococcosis, a systemic mycosis that cause respiratory infection and meningoencephalitis. To establish the infection, these yeasts produce virulence factors, such as melanin, which contribute to pathogenicity and antifungal tolerance. The aim of this study was to investigate melanin production by the C. neoformans/C. gattii complex in the presence of different precursors of melanogenesis and evaluate the effect of melanization on the antifungal susceptibility of these species to fluconazole, flucytosine and amphotericin B. Epinephrine, norepinephrine, dopamine and caffeic acid were used as substrates for melanin production, and l-dopa was used as positive control. The susceptibility of melanized strains (n = 6), after exposure to epinephrine or l-dopa, was evaluated by broth microdilution assay, and non-melanized strains were used as control. The antifungal activity of amphotericin B against melanized strains was also investigated by time kill assay. All Cryptococcus spp. strains produced melanin after exposure to the tested substrates. After exposure to epinephrine, minimum inhibitory concentration (MIC) ranges were 1–8 μg/mL for fluconazole, 2–8 μg/mL for flucytosine and 0.125–1 μg/mL for amphotericin B, while, after exposure to l-dopa, MIC ranges were 2–8 μg/mL for fluconazole, 4–8 μg/mL for flucytosine, and 0.125–0.5 μg/mL for amphotericin B. Similar results were observed for non–melanized strains. The production of melanin after exposure to epinephrine was higher than that induced by l-dopa. Melanized cells of both species were more tolerant to amphotericin B than the non-melanized control, emphasizing the importance of melanin production for fungal virulence.