Cryptococcus neoformans melanization incorporates multiple catecholamines to produce polytypic melanin

Abstract

Melanin is a major virulence factor in pathogenic fungi that enhances the ability of fungal cells to resist immune clearance. Cryptococcus neoformans is an important human pathogenic fungus that synthesizes melanin from exogenous tissue catecholamine precursors during infection, but the type of melanin made in cryptococcal meningoencephalitis is unknown. We analyzed the efficacy of various catecholamines found in brain tissue in supporting melanization using animal brain tissue and synthetic catecholamine mixtures reflecting brain tissue proportions. Solid-state NMR spectra of the melanin pigment produced from such mixtures yielded more melanin than expected if only the preferred constituent dopamine had been incorporated, suggesting uptake of additional catecholamines. Probing the biosynthesis of melanin using radiolabeled catecholamines revealed that C. neoformans melanization simultaneously incorporated more than one catecholamine, implying that the pigment was polytypic in nature. Nonetheless, melanin derived from individual or mixed catecholamines had comparable ability to protect C. neoformans against ultraviolet light and oxidants. Our results indicate that melanin produced during infection differs depending on the catecholamine composition of tissue and that melanin pigment synthesized in vivo is likely to accrue from the polymerization of a mixture of precursors. From a practical standpoint, our results strongly suggest that using dopamine as a polymerization precursor is capable of producing melanin pigment comparable to that produced during infection. On a more fundamental level, our findings uncover additional structural complexity for natural cryptococcal melanin by demonstrating that pigment produced during human infection is likely to be composed of polymerized moieties derived from chemically different precursors.

Keywords: Cryptococcus neoformans; catecholamine; cell wall; dopamine; fungi; melanin; reactive oxygen species; solid-state NMR; virulence factor.

Figure 1

Melanization of C. neoformans grown in the presence of different precursors.A, representative images of C. neoformans strains KN99α, H99O, and H99-ΔLAC1 that were spotted on agar supplemented with a mixture of 0.6 mM dopamine, 0.33 mM norepinephrine, and 0.07 mM epinephrine (BCM), 1 mM Dopamine (DA), 1 mM norepinephrine (NE), 1 mM L- DOPA (L-D), or minimal media only control (MM) and allowed to melanize at the indicated temperatures for 24 and 48 h. B, quantified H99O pigmentation expressed as a fraction of that measured for KN99α after 48 h at 37 °C showing significantly reduced melanization for H99O compared with KN99α under all conditions tested. C, quantification of KN99α pigmentation relative to the ΔLAC1 negative control after 48 h at 30 °C (left graph) and 37 °C (right graph). Statistical significance for this and all subsequent analyses was calculated using an ordinary one-way ANOVA (ns = not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). BCM, brain catecholamine mixture; MM, minimal media.

Figure 2

Melanin ghosts from KN99α melanized in pure or tissue-extracted catecholamines. Color photographs (left) and quantification (right) of relative pigment levels of 2 ml samples taken from KN99α cultures grown for the indicated number of days in minimal media supplemented with a mixture of 0.6 mM dopamine, 0.33 mM norepinephrine, and 0.07 mM epinephrine (BCM), 1 mM dopamine (DA), or 1 mM norepinephrine (NE) at either 30 °C (A) or 37 °C (B). C, bright field images of melanized cells prior to harvesting and acid-resistant melanin “ghost” particles recovered from each culture. D, transmission electron micrographs of melanized cells and melanin ghost particles from cultures supplemented with the indicated precursors at 30 °C. E, bright field images of cells melanized on agar plates supplemented with lamb brain catecholamine extract. Scale bars are 5 μm and 1 μm for light and electron microscopy, respectively. BCM, brain catecholamine mixture.

Figure 3

Solid-state NMR spectra of melanin “ghosts” from C. neoformans cells grown with DA, NE, or BCM. 1D ¹³C cross-polarization (CPMAS) ssNMR spectra of melanin ghosts isolated from C. neoformans KN99α cultures grown for 10 days at 30 °C in minimal media containing either 1 mM dopamine (DA, top), 1 mM norepinephrine (NE, bottom) or in a “brain catecholamine mixture” of 0.6 mM dopamine, 0.33 mM norepinephrine, and 0.07 mM epinephrine (BCM, middle). The spectral region shaded in red (10–40 ppm) displays peaks primarily corresponding to the aliphatic carbons of lipids, the region in blue (50–110 ppm) to polysaccharide-ring carbons, and the region in yellow (110–160 ppm) to the aromatic carbons of the melanin pigment. The unshaded region (165–190 ppm) displays several overlapping peaks corresponding to the carbonyl carbons in all three constituent types. BCM, brain catecholamine mixture; CPMAS, cross-polarization magic-angle spinning.

Figure 4

Incorporation of radiolabeled precursors into melanin.A, scatter plot graph (upper panel) of the amount of radioactivity retained in cell pellets of KN99α and ΔLAC1 cultures supplemented with 2 μCi ³H-NE (0.0001 mM) in the absence or presence of the indicated concentration of unlabeled catecholamines as expressed relative to cultures with ³H-NE only (n = 2). Panel below graph displays representative regions taken from color photographs of each culture. B, radioactivity measured in acid-resistant melanin ghost particles as a percentage of the radioactivity in the samples before acid treatment. Cultures were melanized in minimal media supplemented with 0.0001 mM ³H-NE -NE or ³H-DA and with 1 mM unlabeled catecholamines as indicated (n = 2).

Figure 5

Comparison of melanization conditions for resistance to cellular damage.A, percent survival relative to untreated controls for melanized and nonmelanized (MM) KN99α plated on SAB agar plates and then exposed to 15,000 μJ/cm² UV radiation (n = 5). B, survival of KN99α melanized with the indicated catecholamines or a nonmelanized control (MM) was determined as the number colony forming units (CFU) for cultures exposed to oxygen-derived radicals for 5 h at 37 °C as a percentage of the CFU for untreated controls (n = 3). Melanization conditions were 0.6 mM dopamine, 0.33 mM norepinephrine, and 0.07 mM epinephrine (BCM), 1 mM dopamine (DA), 1 mM norepinephrine (NE), and 1 mM 3,4-Dihydroxy-L-phenylalanine (L-D). BCM, brain catecholamine mixture; MM, minimal media.