Melanin and virulence in Cryptococcus neoformans
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Cryptococcus neoformans is an opportunistic fungal pathogen. C. neoformans has a phenoloxidase enzyme system which catalyzes black pigment formation from catechol substrates such as L-dopa. Black pigment production is associated with virulence. We hypothesized that: (I) the black pigment is a type of melanin; (II) the black pigment contributes to virulence by scavenging free radicals generated by immune effector cells; (III) pigmentation protects C. neoformans against the antifungal effects of macrophages; (IV) pigmentation of C. neoformans increases the resistance of C. neoformans to fungicidal effect of amphotericin B and ultraviolet (UV) radiation; (V) pigmentation of C. neoformans affects susceptibility to the melanin-binding drugs such as chloroquine, paraquat and trifluoperazine.;These hypotheses were tested during my Ph.D. training and data were obtained to support each of them. Transmission electron microscopy showed electron-dense cell walls, and electron spin resonance (ESR) spectroscopy revealed a stable free-radical population only in pigmented cells. The black pigment was isolated from pigmented cells by combined treatment with 4 M guanidinium isothiocyanate and boiling 6 M HCl. ESR spectroscopy of the black pigment particles revealed that black particles contained a stable free radical population. Characterization of the ESR signals indicated that the black pigment was a type of melanin. Melanized C. neoformans cells were less susceptible to chemically-generated nitrogen- and oxygen-derived radicals than non-melanized C. neoformans cells. Increases in the intensity of the ESR signals of melanized cells in solutions containing chemically generated oxygen- and nitrogen-derived radicals indicated electron transfer to or from melanin. Melanized cells were more resistant to antibody-mediated phagocytosis and the antifungal effects of murine macrophages than non-melanized cells. Furthermore, C. neoformans strains differed in melanin content after growth in media with L-dopa, and there was an inverse relation between melanin content and strain susceptibility to macrophages. Melanized C. neoformans cells were less susceptible to fungicidal effect of amphotericin B and UV radiation than non-melanized C. neoformans. Melanized C. neoformans cells were more susceptible to fungicidal effect of trifluoperazine and paraquat than non-melanized cells. Chloroquine had no antifungal activity against either melanized or non-melanized C. neoformans cells. Our data provide strong evidence that melanin is a virulence factor in C. neoformans and indicate that melanin contributes to virulence by protecting fungal cells against attack by immune effector cells. Melanization of C. neoformans might contribute to the difficulty in eradicating C. neoformans infection with amphotericin B and protect the fungus in the environment against solar radiation. Furthermore, our data suggest that strategies which target melanin or the melanin biosynthetic pathway may be productive for antifungal-drug discovery.