Pigment production on L-tryptophan medium by Cryptococcus gattii and Cryptococcus neoformans.

In recent years strains previously grouped within Cryptococcus neoformans have been divided into two species C. neoformans and C. gattii, with Cryptococcus neoformans comprising serotypes A, D, and AD and C. gattii comprising serotypes B and C. Cryptococcus neoformans have also been subdivided into two varieties C. neoformans var. grubii, serotype A, and C. neoformans var. neoformans, serotype D. We analyzed the growth and pigment production characteristics of 139 strains of Cryptococcus spp. in L-tryptophan containing media. Nearly all strains of Cryptococcus, including each variety and serotype tested produced a pink water-soluble pigment (molecular weight of 535.2 Da) from L-tryptophan. Consequently, the partial separation of the species was based on whether the pink pigment was secreted into the medium (extracellular) or retained as an intracellular pigment. On L-tryptophan medium C. neoformans var. grubii and serotype AD produced a pink extracellular pigment. In contrast, for C. gattii, the pink pigment was localized intracellularly and masked by heavy production of brown pigments. Pigment production by C. neoformans var. neoformans was variable with some strains producing the pink extracellular pigment and others retained the pink pigment intracellularly. The pink intracellular pigment produced by strains of C. neoformans var. neoformans was masked by production of brown pigments. Cryptococcus laccase mutants failed to produce pigments from L-tryptophan. This is the first report that the enzyme laccase is involved in tryptophan metabolism. Prior to this report Cryptococcus laccase produced melanin or melanin like-pigments from heterocyclic compounds that contained ortho or para diphenols, diaminobenzenes and aminophenol compounds. The pigments produced from L-tryptophan were not melanin.

Growth and pigment production on D-tryptophan medium by Cryptococcus gattii, Cryptococcus neoformans, and Candida albicans.

Given the increasing prevalence of cryptococcosis caused by Cryptococcus gattii (serotypes B and C) strains, there is a need for rapid and reliable tests that discriminate C. gattii from Cryptococcus neoformans (serotypes A, D, and AD). Seventy-two C. neoformans strains, sixty-seven C. gattii strains, and five Candida albicans strains were analyzed for their ability to grow and produce pigment on minimal D-tryptophan D-proline (m-DTDP) medium, on yeast carbon base D-tryptophan D-proline (YCB-DTDP) medium, and on fructose D-tryptophan glycine (m-FDTG) medium. Of the C. gattii and C. neoformans isolates, 94% and 0% grew on m-DTDP agar, respectively, and 98% and 0% grew in YCB-DTDP medium, respectively. C. gattii produced large amounts of brown intracellular pigment(s) on m-DTDP agar and smaller amounts of yellow-brown (amber) extracellular pigment(s). C. albicans grew on both media and produced a pink photoactivated pigment on m-DTDP agar. C. gattii produced large amounts of brown intracellular pigments on the differential medium m-FDTG, whereas C. neoformans produced smaller amounts of the brown pigments and C. albicans produced a pink pigment. The pigments produced by C. gattii from D-tryptophan were distinct and were not related to melanin formation from 3,4-dihydroxyphenylalanine. Thin-layer chromatography of the methanol-extracted C. gattii cells detected four different pigments, including brown (two types), yellow, and pink-purple compounds. We conclude that tryptophan-derived pigments are not melanins and that growth on m-DTDP or YCB-DTDP agar can be used to rapidly differentiate C. gattii from C. neoformans.

Cryptococcus neoformans laccase catalyses melanin synthesis from both D- and L-DOPA.

The human fungal pathogen Cryptococcus neoformans produces melanin in the presence of various substrates, including the L enantiomer of 3,4-dihydroxyphenylalanine (DOPA). The enzyme laccase catalyses the formation of melanin by oxidizing L-DOPA, initiating a series of presumably spontaneous reactions that ultimately leads to the polymerization of the pigment in the yeast cell wall. There, melanin protects the cell from a multitude of environmental and host assaults. Thus, the ability of C. neoformans to produce pigments from a variety of available substrates is likely to confer a survival advantage. A number of C. neoformans isolates of different serotypes produced pigments from D-DOPA, the stereoisomer of L-DOPA. Acid-resistant particles were isolated from pigmented C. neoformans cells grown in the presence of D-DOPA. Biophysical characterization showed the particles had a stably detectable free-radical signal by EPR, and negative zeta potential, similar to L-DOPA-derived particles. No major differences were found between L- and D-DOPA ghosts in terms of binding to anti-melanin antibodies, or in overall architecture when imaged by electron microscopy. C. neoformans cells utilized L- and D-DOPA at a similar rate. Overall, our results indicate that C. neoformans shows little stereoselectivity for utilizing DOPA in melanin synthesis. The ability of C. neoformans to use both L and D enantiomers for melanization implies that this organism has access to a greater potential pool of substrates for melanin synthesis, and this could potentially be exploited in the design of therapeutic inhibitors of laccase.

Induction by Klebsiella aerogenes of a melanin-like pigment in Cryptococcus neoformans.

While studying the interaction of Cryptococcus neoformans with Dictyostelium discoideum, we noticed that yeast colonies in agar with a feeder lawn of Klebsiella aerogenes were brown. This finding was intriguing because C. neoformans colonies are not pigmented unless they are provided with precursors for melanization. Strains of all C. neoformans serotypes produced brown pigment in response to K. aerogenes at 22, 30, and 37 degrees C. Pigment production required fungal laccase and was suppressed by high concentrations of glucose. Treatment of brown cells with guanidinium isothiocyanate and hot concentrated HCl yielded particulate material that had the physical and chemical characteristics of melanins. No pigment formation was observed when C. neoformans was exposed to live Escherichia coli or heat-killed K. aerogenes. Analysis of K. aerogenes supernatants revealed the presence of dopamine, which can be a substrate for melanin synthesis by C. neoformans. Our findings illustrate a remarkable interaction between a pathogenic fungus and a gram-negative bacterium, in which the bacterium produces a substrate that promotes fungal melanization. This observation provides a precedent that could explain the source of a substrate for C. neoformans melanization in the environment.