Melanin, melanin "ghosts," and melanin composition in Cryptococcus neoformans

Abstract

Melanin synthesis is associated with virulence for the pathogenic fungus Cryptococcus neoformans. Exposure of nonmelanized C. neoformans 24067 cells to 4 M guanidinium isothiocyanate followed by 6 M HCl at 100؇C resulted in complete solubilization of cellular structures. However, exposure of melanized C. neoformans 24067 to the same conditions produced a suspension of black particles. Analysis of black particles with transmission and scanning electron microscopy revealed cell "ghosts" with electron-dense walls. Electron spin resonance (ESR) spectroscopy of suspensions of black particles revealed a signal indicative of a stable free radical population. The magnitude of the ESR signal was increased by illumination, Zn 2؉ ions, and basic pH. Boiling the black particles produced only a small reduction in the magnitude of the ESR signal. These ESR changes are characteristic of melanin. Exposure of melanized black cells to 0.1 or 0.5% NaOCl bleached the cells white and abolished the ESR signal, consistent with melanin disruption. ESR spectroscopy was used to study the rate of stable free radical formation, taken as an index of melanization, of C. neoformans 24067 cells growing in medium with 1.0 mM L-dopa at 30 and 37؇C over the course of 14 days. Melanization was slower at 37؇C, but by day 14, cells grown at 30 and 37؇C had comparable levels of melanin. ESR spectroscopy of seven melanized C. neoformans strains revealed that some strains differed by up to eightfold in melanin content. The ESR spectra of C. neoformans 24067 grown in minimal medium containing either 1.0 mM catechol, dopamine, or L-dopa revealed large differences in signal amplitude. The relative melanin contents were 0.05, 0.45, and 1.0 for C. neoformans grown in media with catechol, dopamine, and L-dopa, respectively. Quantitative analysis revealed that melanin comprised 15.4% of the dry mass of the cell after 10 days of growth in medium containing 1.0 mM L-dopa. The results suggest (i) a useful protocol for isolating C. neoformans melanin, (ii) a role for melanin in maintaining cell wall integrity, (iii) disruption of melanin by NaOCl, (iv) strain differences in melanin content after growth in L-dopa, and (v) quantitative and/or qualitative differences in the melanin produced after growth in various substrates. MATERIALS AND METHODS Chemicals. Glucose, MgSO 4 , KH 2 PO 4 , HCl, guanidinium isothiocyanate, and NaOCl were purchased from Fisher Chemical Inc. (Fair Lawn, N.J.). Vitamin B 1 , L-dopa, catechol (1,2-benzenediol), dopamine (3-hydroxytyramine), sorbitol, and sodium citrate were purchased from Sigma Chemical Co. (Cleveland, Ohio). Novo-zyme 234 was purchased from BioPacific Inc. (Emoryville, Calif.). C. neoformans. Strains were maintained in Sabouraud dextrose agar (Difco Laboratories, Detroit, Mich.). Strain 24067 was obtained from the American Type Culture Collection (Rockville, Md.). Strains SB4A and SB6A were obtained from Eric Spitzer (Stony Brook, N.Y.). Strain NIH371 was obtained from J. E. Bennett (Bethesda, Md.). Strains J9A, J11A, and ...

Candida glabrata tryptophan‐based pigment production via the Ehrlich pathway

“…1A and data not shown). Furthermore, unlike in melanized fungi (Wang et al, 1996), treatment with guanidinium isothiocyanate and hydrochloric acid did not result in any residual particles or pigment 'ghosts' (not shown) suggesting that the tryptophan-induced pigment of C. glabrata has different characteristics compared with melanin. Similar to a recently described tryptophaninduced pigment in Cryptococcus gattii, this pigment therefore seems unrelated to melanins (Chaskes et al, 2008).…”

“…Furthermore, in M. grisea, C. neoformans and A. fumigatus, melanin has an important structural rolefor example during the production of appressoria, or by stabilizing the cell wall against mechanical and chemical stresses. This was most strikingly demonstrated by the presence of 'melanin ghosts' of C. neoformans after the destruction of the cell wall by very harsh conditions, including treatment with concentrated acids (Wang et al, 1996). In addition, melanin plays a role in the protection against antimycotic drugs (Wang and Casadevall, 1994b).…”

Supplement comprising of laccase and citric acid as an alternative for antibiotics: In vitro triggers of melanin production

“…The solution was then filtered through Whatman paper (45 μm) using a vacuum pump. The filter was dried completely in a desiccator before weighing to quantify melanin produced by the cells by adapting the method used by Wang et al and Sabiiti et al [26,27].…”

The contribution of melanin to microbial pathogenesis

“…The ability of C. neoformans to make pigments was discovered by F. Staib in the early 1960s when he noted that cryptococcal colonies in bird seed ( Guizotia abyssinica ) agar turned brown (Staib, 1962;1963). When grown in liquid culture with the addition of phenolic compounds, melanin comprises approximately 15% of the dry weight of C. neoformans (Wang and Casadevall, 1996a).…”