Cryptococcus neoformans melanization incorporates multiple catecholamines to produce polytypic melanin

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. 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. Cryptococcus neoformans is an encapsulated, free-living fungal organism that can establish opportunistic infections in both plant and animal hosts (1Springer D.J. Mohan R. Heitman J. Plants promote mating and dispersal of the human pathogenic fungus Cryptococcus.PLoS One. 2017; 12e0171695Crossref PubMed Scopus (20) Google Scholar). C. neoformans is broadly prevalent in the environment and colonizes a variety of ecological niches, which notably include soil and bird guano (2Lin X. Heitman J. The biology of the Cryptococcus neoformans species complex.Annu. Rev. Microbiol. 2006; 60: 69-105Crossref PubMed Scopus (280) Google Scholar). Despite ubiquitous exposure of the human population, C. neoformans rarely causes disease in individuals with intact immunity as the infection is either cleared or becomes asymptomatically latent (3Coelho C. Bocca A.L. Casadevall A. The intracellular life of Cryptococcus neoformans.Annu. Rev. Pathol. 2014; 9: 219-238Crossref PubMed Scopus (77) Google Scholar). However, C. neoformans poses a serious threat to those with compromised immunity resulting from disease or medical treatment, especially AIDS patients, and is estimated to cause 180,000 deaths annually (4Rajasingham R. Smith R.M. Park B.J. Jarvis J.N. Govender N.P. Chiller T.M. Denning D.W. Loyse A. Boulware D.R. Global burden of disease of HIV-associated cryptococcal meningitis: An updated analysis.Lancet Infect. Dis. 2017; 17: 873-881Abstract Full Text Full Text PDF PubMed Scopus (887) Google Scholar). Cryptococcal infection commences with the inhalation of desiccated fungal cells or spores that become deposited in the lungs, which can result in cryptococcal pneumonia for immunocompromised individuals (5May R.C. Stone N.R.H. Wiesner D.L. Bicanic T. Nielsen K. Cryptococcus: From environmental saprophyte to global pathogen.Nat. Rev. Microbiol. 2016; 14: 106-117Crossref PubMed Scopus (212) Google Scholar). Progression of the primary infection can occur through extrapulmonary dissemination as yeast cells dispersed through the bloodstream are able to cross the blood–brain barrier and take up residence in brain tissue (6Chrétien F. Lortholary O. Kansau I. Neuville S. Gray F. Dromer F. Pathogenesis of cerebral Cryptococcus neoformans infection after fungemia.J. Infect. Dis. 2002; 186: 522-530Crossref PubMed Scopus (159) Google Scholar, 7Chang Y.C. Stins M.F. McCaffery M.J. Miller G.F. Pare D.R. Dam T. Paul-Satyaseela M. Kim K.S. Kwon-Chung K.J. Cryptococcal yeast cells invade the central nervous system via transcellular penetration of the blood-brain barrier.Infect. Immun. 2004; 72: 4985-4995Crossref PubMed Scopus (175) Google Scholar). The ensuing meningoencephalitis that is typical of this aggressive stage of cryptococcosis, if left untreated, is almost always fatal (8Perfect J.R. Dismukes W.E. Dromer F. Goldman D.L. Graybill J.R. Hamill R.J. Harrison T.S. Larsen R.A. Lortholary O. Nguyen M.-H. Pappas P.G. Powderly W.G. Singh N. Sobel J.D. Sorrell T.C. Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the Infectious Diseases Society of America.Clin. Infect. Dis. 2010; 50: 291-322Crossref PubMed Scopus (1598) Google Scholar). Although concerted efforts are underway to develop an effective vaccine (9Datta K. Pirofski L. Towards a vaccine for Cryptococcus neoformans: Principles and caveats.FEMS Yeast Res. 2006; 6: 525-536Crossref PubMed Scopus (41) Google Scholar), there is currently no reliable means of preventing infection and even the most current treatment strategies that employ a combined antifungal regimen only decrease 10-week mortality rates to 24% (10Iyer K.R. Revie N.M. Fu C. Robbins N. Cowen L.E. Treatment strategies for cryptococcal infection: Challenges, advances and future outlook.Nat. Rev. Microbiol. 2021; 19: 454-466Crossref PubMed Scopus (18) Google Scholar). Thus, an improved understanding of factors that contribute to fungal virulence is vital to our efforts in combating this harmful pathogen. Among the adaptations that safeguard C. neoformans from harsh conditions both in the environment and during host infection is the deposition of a layer of melanin in the fungal cell wall. Melanins are a diverse group of pigments, typically black or dark brown in color, which are formed by the oxidative polymerization of phenolic or indolic precursors (11Eisenman H.C. Casadevall A. Synthesis and assembly of fungal melanin.Appl. Microbiol. Biotechnol. 2012; 93: 931-940Crossref PubMed Scopus (373) Google Scholar). They are notorious for their extreme recalcitrance, structural complexity, and unique biophysical properties, such as the ability to absorb a wide spectrum of UV light. Melanin pigments are widely employed throughout the biosphere, serving such diverse functions as camouflage, thermal modulation, and protection from radiation (12Hill H.Z. The function of melanin or six blind people examine an elephant.Bioessays. 1992; 14: 49-56Crossref PubMed Scopus (259) Google Scholar); in pathogenic fungi, they are linked to increased virulence (13Casadevall A. Rosas A.L. Nosanchuk J.D. Melanin and virulence in Cryptococcus neoformans.Curr. Opin. Microbiol. 2000; 3: 354-358Crossref PubMed Scopus (213) Google Scholar) and drug resistance (14Nosanchuk J.D. Casadevall A. Impact of melanin on microbial virulence and clinical resistance to antimicrobial compounds.Antimicrob. Agents Chemother. 2006; 50: 3519-3528Crossref PubMed Scopus (272) Google Scholar). In C. neoformans cells, melanin is synthesized from phenolic compounds in small vesicles called melanosomes that are transported and subsequently deposited within the cell wall (15Eisenman H.C. Frases S. Nicola A.M. Rodrigues M.L. Casadevall A. Vesicle-associated melanization in Cryptococcus neoformans.Microbiology. 2009; 155: 3860-3867Crossref PubMed Scopus (96) Google Scholar). There, the melanin particles assemble into larger granules that become tightly interwoven with the constituents comprising the cell wall, which consequently thickens over time (16Eisenman H.C. Nosanchuk J.D. Webber J.B.W. Emerson R.J. Camesano T.A. Casadevall A. Microstructure of cell wall-associated melanin in the human pathogenic fungus Cryptococcus neoformans.Biochemistry. 2005; 44: 3683-3693Crossref PubMed Scopus (103) Google Scholar). In the environment, melanization is credited with protecting C. neoformans from electromagnetic radiation, temperature stresses, and amoeba (17Cordero R.J.B. Casadevall A. Functions of fungal melanin beyond virulence.Fungal Biol. Rev. 2017; 31: 99-112Crossref PubMed Scopus (135) Google Scholar, 18Fu M.S. Liporagi-Lopes L.C. dos Santos Júnior S.R. Tenor J.L. Perfect J.R. Cuomo C.A. Casadevall A. Amoeba predation of Cryptococcus neoformans results in pleiotropic changes to traits associated with virulence.bioRxiv. 2020; ([preprint])https://doi.org/10.1101/2020.08.07.241190Crossref Scopus (0) Google Scholar). During infection, melanization helps shield C. neoformans cells from engulfment and killing by host macrophages (19Wang Y. Aisen P. Casadevall A. Cryptococcus neoformans melanin and virulence: Mechanism of action.Infect. Immun. 1995; 63: 3131-3136Crossref PubMed Google Scholar, 20Blasi E. Barluzzi R. Mazzolla R. Tancini B. Saleppico S. Puliti M. Pitzurra L. Bistoni F. Role of nitric oxide and melanogenesis in the accomplishment of anticryptococcal activity by the BV-2 microglial cell line.J. Neuroimmunol. 1995; 58: 111-116Abstract Full Text PDF PubMed Scopus (55) Google Scholar, 21Liu L. Tewari R.P. Williamson P.R. Laccase protects Cryptococcus neoformans from antifungal activity of alveolar macrophages.Infect. Immun. 1999; 67: 6034-6039Crossref PubMed Google Scholar) and contributes to the severity of infection by promoting neurotropism (22Polacheck I. Hearing V.J. Kwon-Chung K.J. Biochemical studies of phenoloxidase and utilization of catecholamines in Cryptococcus neoformans.J. Bacteriol. 1982; 150: 1212-1220Crossref PubMed Scopus (109) Google Scholar, 23Lee S.C. Dickson D.W. Casadevall A. Pathology of cryptococcal meningoencephalitis: Analysis of 27 patients with pathogenetic implications.Hum. Pathol. 1996; 27: 839-847Crossref PubMed Scopus (146) Google Scholar). As melanization is one of two primary virulence factors for C. neoformans, the disruption of this process, perhaps by inhibiting the laccase enzyme responsible for catalyzing melanin synthesis, has been widely recognized as a potential therapeutic target (24Coelho C. Casadevall A. Cryptococcal therapies and drug targets: The old, the new and the promising.Cell. Microbiol. 2016; 18: 792-799Crossref PubMed Scopus (57) Google Scholar). A defining feature of the C. neoformans laccase enzyme is its inability to produce melanin pigments from endogenously synthesized compounds such as tyrosine, the precursor most commonly used by other melanotic organisms (25Williamson P.R. Biochemical and molecular characterization of the diphenol oxidase of Cryptococcus neoformans: Identification as a laccase.J. Bacteriol. 1994; 176: 656-664Crossref PubMed Scopus (289) Google Scholar). Instead, C. neoformans draws on exogenous sources of substrates, producing melanin pigments from a wide variety of phenolic compounds found in natural habitats and in animal hosts, as evidenced by the isolation of melanized cells from both natural isolates (26Nosanchuk J.D. Rudolph J. Rosas A.L. Casadevall A. Evidence that Cryptococcus neoformans is melanized in pigeon excreta: Implications for pathogenesis.Infect. Immun. 1999; 67: 5477-5479Crossref PubMed Google Scholar) and infected host tissues (27Nosanchuk J.D. Valadon P. Feldmesser M. Casadevall A. Melanization of Cryptococcus neoformans in murine infection.Mol. Cell. Biol. 1999; 19: 745-750Crossref PubMed Scopus (102) Google Scholar, 28Nosanchuk J.D. Rosas A.L. Lee S.C. Casadevall A. Melanisation of Cryptococcus neoformans in human brain tissue.Lancet. 2000; 355: 2049-2050Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Indeed, the animal nervous system serves as a rich source of precursors in the form of catecholamines, nitrogen-containing diphenolic compounds, which include the neurotransmitters dopamine, epinephrine, and norepinephrine. C. neoformans melanization has been studied in vitro by culturing cells in media supplemented with each of these compounds and more commonly, with 3,4-Dihydroxy-L-phenylalanine (L-DOPA), the immediate biosynthetic precursor of dopamine. Considerable insight into the properties of C. neoformans melanin has been gained by applying biophysical techniques such as solid-state nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) to analyze melanin containing particles derived from such cultures (29Chatterjee S. Prados-Rosales R. Frases S. Itin B. Casadevall A. Stark R.E. Using solid-state NMR to monitor the molecular consequences of Cryptococcus neoformans melanization with different catecholamine precursors.Biochemistry. 2012; 51: 6080-6088Crossref PubMed Scopus (30) Google Scholar, 30Chatterjee S. Prados-Rosales R. Itin B. Casadevall A. Stark R.E. Solid-state NMR reveals the carbon-based molecular architecture of Cryptococcus neoformans fungal eumelanins in the cell wall.J. Biol. Chem. 2015; 290: 13779-13790Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 31Chatterjee S. Prados-Rosales R. Tan S. Phan V.C. Chrissian C. Itin B. Wang H. Khajo A. Magliozzo R.S. Casadevall A. Stark R.E. The melanization road more traveled by: Precursor substrate effects on melanin synthesis in cell-free and fungal cell systems.J. Biol. Chem. 2018; 293: 20157-20168Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). These analyses have indicated that the pigments produced by C. neoformans from each of these precursors are indeed melanins. They have also revealed distinct differences in molecular structure and EPR signal intensity, but the implications of these differences with respect to virulence remain unclear. Whereas previous studies of C. neoformans melanin have largely employed L-DOPA as the precursor (reviewed in (32Nosanchuk J.D. Stark R.E. Casadevall A. Fungal melanin: What do we know about structure?.Front. Microbiol. 2015; 6: 1463Crossref PubMed Scopus (138) Google Scholar)), the low abundance of this compound in host tissues prompted us to question how faithfully L-DOPA melanin produced in culture reflects the structure and function of “natural” melanin produced during the course of C. neoformans infection. The progression of cryptococcal disease ultimately results in brain dissemination, which provides a rich and varied source of catecholamines, thereby presenting the potential for simultaneous incorporation of multiple precursors. Although acid-resistant melanin particles can be isolated from infected human and animal tissue (28Nosanchuk J.D. Rosas A.L. Lee S.C. Casadevall A. Melanisation of Cryptococcus neoformans in human brain tissue.Lancet. 2000; 355: 2049-2050Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 33Rosas A.L. Nosanchuk J.D. Feldmesser M. Cox G.M. McDade H.C. Casadevall A. Synthesis of polymerized melanin by Cryptococcus neoformans in infected rodents.Infect. Immun. 2000; 68: 2845-2853Crossref PubMed Scopus (116) Google Scholar), the limited quantity of melanized material that can be recovered from animal models of infection has precluded extensive biophysical analyses. To circumvent this obstacle, we sought to develop a cell culture system that would approximate the repertoire of catecholamine precursors present in the human brain. Drawing on previous neurochemical studies that quantified catecholamines isolated from postmortem human brains (34Herregodts P. Michotte Y. Ebinger G. Regional differences in the distribution of norepinephrine and epinephrine in human cerebral cortex: A neurochemical study using HPLC and electrochemical detection.Neurosci. Lett. 1989; 98: 321-326Crossref PubMed Scopus (13) Google Scholar, 35Herregodts P. Ebinger G. Michotte Y. Distribution of monoamines in human brain: Evidence for neurochemical heterogeneity in subcortical as well as in cortical areas.Brain Res. 1991; 542: 300-306Crossref PubMed Scopus (28) Google Scholar), we melanized C. neoformans in a mixture of three catecholamines according to their relative brain proportions of 60% dopamine, 33% norepinephrine, and 7% epinephrine. Here we describe the isolation and analysis of melanin produced from this brain catecholamine mixture (BCM) and provide evidence that C. neoformans can incorporate multiple precursors simultaneously during melanin synthesis. BCM is found to be comparable to L-DOPA melanin in its ability to protect C. neoformans from sources of cellular damage such as UV radiation and free oxygen radicals. Despite these functional similarities, the ability of C. neoformans to utilize a mixture of chemically distinct precursors during melanin synthesis implies architectural heterogeneity for this polymer depending on the local abundance of precursors and significantly complicates its structural characterization. We sought to approximate the melanization process during infection by growing C. neoformans in a mixture of catecholamines representative of human brain proportions (BCM). A key consideration in this effort was the selection of a laboratory strain variant that would be capable of robust melanin production from BCM at the human body temperature of 37 °C. Several variants derived from the widely used H99 strain of C. neoformans var. grubii (H99O), including H99E, H99C, and H99W, display reduced virulence in mice and decreased melanization on L-DOPA agar as a result of laboratory passage (36Janbon G. Ormerod K.L. Paulet D. Byrnes 3rd, E.J. Yadav V. Chatterjee G. Mullapudi N. Hon C.-C. Billmyre R.B. Brunel F. Bahn Y.-S. Chen W. Chen Y. Chow E.W.L. Coppée J.-Y. et al.Analysis of the genome and transcriptome of Cryptococcus neoformans var. grubii reveals complex RNA expression and microevolution leading to virulence attenuation.PLoS Genet. 2014; 10e1004261Crossref PubMed Scopus (244) Google Scholar). Conversely, variants H99S, H99F, and KN99α/a that were derived from passage of H99 through the rabbit central nervous system model are more virulent and produce more melanin when grown on L-DOPA (36Janbon G. Ormerod K.L. Paulet D. Byrnes 3rd, E.J. Yadav V. Chatterjee G. Mullapudi N. Hon C.-C. Billmyre R.B. Brunel F. Bahn Y.-S. Chen W. Chen Y. Chow E.W.L. Coppée J.-Y. et al.Analysis of the genome and transcriptome of Cryptococcus neoformans var. grubii reveals complex RNA expression and microevolution leading to virulence attenuation.PLoS Genet. 2014; 10e1004261Crossref PubMed Scopus (244) Google Scholar). We compared melanization by H99O and KN99α on agar supplemented with BCM as well as the individual precursors DA, NE, and L-DOPA at 30 °C and 37 °C (Fig. 1A). The well-characterized H99-derived ΔLAC1 strain deficient in Laccase-1, the primary enzyme required for melanization, was included as a negative control. C. neoformans laccase production and activity has been shown previously to decrease with increasing temperature (37Jacobson E.S. Emery H.S. Temperature regulation of the cryptococcal phenoloxidase.J. Med. Vet. Mycol. 1991; 29: 121-124Crossref PubMed Scopus (40) Google Scholar, 38Jacobson E.S. Compton G.M. Discordant regulation of phenoloxidase and capsular polysaccharide in Cryptococcus neoformans.J. Med. Vet. Mycol. 1996; 34: 289-291Crossref PubMed Scopus (26) Google Scholar). Accordingly, we observed a slower rate of melanin production and a diminished amount of overall pigmentation for both H99O and KN99α grown at 37 °C compared with 30 °C, regardless of which precursors were provided. Whereas H99O melanization was delayed compared with that of KN99α at 30 °C, similar degrees of pigmentation were observed for the two strains after incubation for 48 h (Fig. 1A, left panel). However, H99O cells showed significantly less pigmentation than KN99α after 48 h at 37 °C (Fig. 1A, right panel; Fig. 1B) and only achieved equivalent melanization levels after an additional 2 days of growth (data not shown). Melanization by KN99α on BCM-supplemented agar was comparable to the pigmentation observed with each of the individual catecholamines at 30 °C (Fig. 1C, left graph) and was more efficient than NE alone at 37 °C (Fig. 1C, right graph). Given the increased melanin production of KN99α compared to H99O when grown in the presence of BCM at the human body temperature of 37 °C, the KN99α variant of C. neoformans was chosen for all subsequent analyses. To further characterize the melanin produced by C. neoformans when provided with a mixture of catecholamine precursors, KN99α was melanized in liquid culture for 10 days at 30 °C, which is the temperature of optimal growth, in MM supplemented with BCM (0.6 mM DA/0.33 mM NE/0.07 mM E) or either 1 mM DA or 1 mM NE. Pigmentation intensity increased more rapidly for cells grown in BCM compared with DA initially but both cultures reached maximal melanization levels after 5 days (Fig. 2A). The culture grown in NE melanized more slowly but eventually reached an intensity comparable to the other two cultures after 10 days (Fig. 2A). Melanization was also achieved for cultures grown in MM supplemented with BCM, DA, or NE at the human body temperature of 37 °C, although pigment production occurred more slowly and at a comparable rate for all three cultures (Fig. 2B). To obtain melanin for biophysical analysis, cells from each culture were harvested and subjected to enzymatic cell wall digestion, protein denaturation and hydrolysis, lipid extraction, and finally boiling in concentrated HCl. In each case, acid-resistant material was recovered that, upon examination by light microscopy, was verified to be dark, yeast-shaped and yeast-sized silhouettes referred to as melanin “ghosts” (Fig. 2C). These particles were also confirmed to be nonviable since plating >1 × 107 particles on SAB agar yielded no colonies. Microscopic examination revealed differences in the physical appearance of particles derived from each culture. The melanin ghost particles isolated from the DA control culture grown at 30 °C were darkly pigmented (Fig. 2C) and similar in appearance to those isolated previously from cultures melanized in L-DOPA at 30 °C (39Garcia-Rivera J. Eisenman H.C. Nosanchuk J.D. Aisen P. Zaragoza O. Moadel T. Dadachova E. Casadevall A. Comparative analysis of Cryptococcus neoformans acid-resistant particles generated from pigmented cells grown in different laccase substrates.Fungal Genet. Biol. 2005; 42: 989-998Crossref PubMed Scopus (35) Google Scholar). Fewer intact particles were recovered from the corresponding NE control culture, and they tended to be small and pale (Fig. 2C); there was an overall lower yield of acid-resistant material (70 mg/l) compared with that of DA (130 mg/l). The BCM melanin ghosts from the culture grown at 30 °C were intermediate between the two controls both in appearance, with a mixture of intact dark particles and more fragile, pale particles (Fig. 2C), and in terms of the yield of material (100 mg/l). Ultrastructural examination of melanin ghost particles using transmission electron microscopy revealed “empty shells” with no evidence of membrane-bound organelles or polysaccharide capsule that were visible in the corresponding pretreatment samples (Fig. 2D). Melanin ghost particles isolated from cultures melanized at 37 °C were smaller and reduced in yield by several-fold (37 mg/l for DA and 14 mg/l for BCM and NE) compared with those isolated at 30 °C. The reduced yield is consistent with lower expression of laccase enzyme at 37 °C (38Jacobson E.S. Compton G.M. Discordant regulation of phenoloxidase and capsular polysaccharide in Cryptococcus neoformans.J. Med. Vet. Mycol. 1996; 34: 289-291Crossref PubMed Scopus (26) Google Scholar). An even smaller yield of intact acid-resistant particles (<1 in 106 of the total number of cells) was recovered from KN99α cells grown at 30 °C on agar plates containing brain tissue extract (Fig. 2E). In light of this limitation, the robust pigment production achieved in our large-scale BCM-supplemented culture system, especially at 30 °C, supports its utility for acquiring melanin that approximates that produced in human brain tissue but in sufficient quantities to permit structural characterization. Given the strong resemblance of the particles derived from C. neoformans grown in BCM to melanin ghosts, we sought to characterize the molecular profile of this pigmented material. Solid-state NMR (ssNMR) analysis was implemented to examine its molecular architecture and compare it with that of melanin ghost material isolated from cells grown with either of the two major constituent precursors, DA or NE. The 1D 13C cross-polarization magic-angle spinning (CPMAS) spectra of each of these three samples clearly displayed all of the spectral features that are characteristic of melanin ghosts (Fig. 3) (29Chatterjee S. Prados-Rosales R. Frases S. Itin B. Casadevall A. Stark R.E. Using solid-state NMR to monitor the molecular consequences of Cryptococcus neoformans melanization with different catecholamine precursors.Biochemistry. 2012; 51: 6080-6088Crossref PubMed Scopus (30) Google Scholar, 30Chatterjee S. Prados-Rosales R. Itin B. Casadevall A. Stark R.E. Solid-state NMR reveals the carbon-based molecular architecture of Cryptococcus neoformans fungal eumelanins in the cell wall.J. Biol. Chem. 2015; 290: 13779-13790Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 31Chatterjee S. Prados-Rosales R. Tan S. Phan V.C. Chrissian C. Itin B. Wang H. Khajo A. Magliozzo R.S. Casadevall A. Stark R.E. The melanization road more traveled by: Precursor substrate effects on melanin synthesis in cell-free and fungal cell systems.J. Biol. Chem. 2018; 293: 20157-20168Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar), notably the group of broad, overlapping peaks located between ∼110 and 160 ppm. These peaks correspond to the various aromatic carbons that comprise the inherently heterogeneous melanin polymer: although many of the carbons are chemically similar, they differ in both their bonding patterns and local chemical environments and consequently resonate at slightly different frequencies. This results in a multitude of signals that overlap in the aromatic-carbon spectral region, forming what visually appears as a single, broad peak and serves as a telltale indicator of the presence of melanin pigments. Other characteristic features of the cellular components of melanin ghosts were also observed in all three sample spectra: each of the spectral regions between each ∼10 to 40 ppm and ∼50 to 110 ppm displayed groups of peaks, which are attributable to long-chain fatty acids within triacylglycerols and polysaccharide-ring carbons, respectively (40Chrissian C. Camacho E. Fu M.S. Prados-Rosales R. Chatterjee S. Cordero R.J.B. Lodge J.K. Casadevall A. Stark R.E. Melanin deposition in two Cryptococcus species depends on cell-wall composition and flexibility.J. Biol. Chem. 2020; 295: 1815-1828Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). The exceptional recalcitrance of melanin pigments, which in C. neoformans are deposited and intimately interwoven throughout the cell wall, protects this structure from the harsh treatments performed during melanin isolation. Hence, this process yields melanized cell walls (i.e., melanin “ghosts”), which contain remnants of the polysaccharide scaffold in addition to trapped neutral lipids, rather than only the purified fungal pigments. Melanin ghost spectra therefore characteristically display ssNMR signals that correspond to nonpigment cellular remnants in addition to those that correspond to the melanin pigment itself. The fact that these signals were observed in all three sample spectra confirmed that the black particles recovered from C. neoformans cell cultures grown with BCM are indeed melanin ghosts. Our ssNMR analysis also provided insight into whether C. neoformans is able to incorporate more than one melanization precursor into the pigment architecture when provided with multiple substrates. The heterogenous nature of the melanin