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From the Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, Maryland 21205
From the Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, Maryland 21205
the Department of Chemistry and Biochemistry, City College of New York and CUNY Institute for Macromolecular Assemblies, New York, New York 10031, the City University of New YorkPh.D. Programs in Biochemistry and
the Department of Microbiology and Immunology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461the CIC bioGUNE, 48160 Derio, Vizcaya, Spainthe Department of Preventive Medicine and Public Health and Microbiology, Autonoma University of Madrid, 28049 Madrid, Spain
From the Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, Maryland 21205
the Integrated Imaging Center, Department of Biology, Engineering in Oncology Center, and Institute for NanoBioTechnology, The Johns Hopkins University, Baltimore, Maryland 21218
the Department of Chemistry and Biochemistry, City College of New York and CUNY Institute for Macromolecular Assemblies, New York, New York 10031, the City University of New YorkPh.D. Programs in Biochemistry andChemistry, New York, New York 10016
To whom correspondence should be addressed: Dept. of Microbiology and Molecular Immunology, Bloomberg School of Public Health, The Johns Hopkins University, 615 N. Wolfe St., Rm. E5132, Baltimore, MD 21205. Tel.:410-955-3457; Fax:410-955-0105
From the Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, Maryland 21205
1 Both authors contributed equally to this work. 2 Recipient of a Johns Hopkins Malaria Research Institute fellowship. 3 Recipient of a fellowship award from the United States Department of Education Graduate Assistance in Areas of National Need (GAANN) Program in Biochemistry, Biophysics, and Biodesign at the City College of New York. 4 Supported in part by National Institutes of Health Grant AI115091 from NIAID, Spanish Ministry of Economy and Competitiveness Grant SAF2016-77433-R, CICbioGUNE through the Severo Ochoa Excellence Accreditation SEV-2016-0644, and is a “Ramon y Cajal” fellow from the Spanish Ministry of Economy and Competitiveness. 5 Supported by Hopkins Integrated Imaging Center and National Institutes of Health Grant 1S10OD012342 from NCRR for the acquisition of the Talos TEM.
Melanins are synthesized macromolecules that are found in all biological kingdoms. These pigments have a myriad of roles that range from microbial virulence to key components of the innate immune response in invertebrates. Melanins also exhibit unique properties with potential applications in physics and material sciences, ranging from electrical batteries to novel therapeutics. In the fungi, melanins, such as eumelanins, are components of the cell wall that provide protection against biotic and abiotic elements. Elucidation of the smallest fungal cell wall-associated melanin unit that serves as a building block is critical to understand the architecture of these polymers, its interaction with surrounding components, and their functional versatility. In this study, we used isopycnic gradient sedimentation, NMR, EPR, high-resolution microscopy, and proteomics to analyze the melanin in the cell wall of the human pathogenic fungus Cryptococcus neoformans. We observed that melanin is assembled into the cryptococcal cell wall in spherical structures ∼200 nm in diameter, termed melanin granules, which are in turn composed of nanospheres ∼30 nm in diameter, termed fungal melanosomes. We noted that melanin granules are closely associated with proteins that may play critical roles in the fungal melanogenesis and the supramolecular structure of this polymer. Using this structural information, we propose a model for C. neoformans’ melanization that is similar to the process used in animal melanization and is consistent with the phylogenetic relatedness of the fungal and animal kingdoms.
). These pigments carry out a vast array of functions in all biological kingdoms, including thermoregulation, radical scavenging, energy transduction, camouflage, invertebrate immunity, and sexual display (
). In the microbial world, melanins contribute to the virulence of many pathogenic microbes. Melanins are produced through the oxidation and polymerization of phenolic/indolic precursors; notably, these polymers are characterized by a strong negative charge, high molecular weight, and hydrophobic nature (
). In the fungal field, black-to-brown insoluble pigments designated as eumelanins confer greater fitness to melanotic species in vivo, thereby contributing to their virulence (
). In Cryptococcus neoformans, a facultative intracellular pathogen, melanized cells are able to modulate the immune response of the host through multiple mechanisms, e.g. altering cytokine profiles (
Down-regulation of the afferent phase of T cell-mediated pulmonary inflammation and immunity by a high melanin-producing strain of Cryptococcus neoformans.
). Recent synthesis of a soluble melanin (homogentesic acid and l-DOPA at a 1:1 ratio) demonstrated that it suppresses the production of cytokines and ROS under stimulation of fungal components thus verifying melanin's ability to modulate the immune system (
C. neoformans is a unique system for the study of melanin biology because, unlike other melanotic fungi, only exogenously provided catecholamines or diphenolic substrates, such as dihydroxyphenylalanine (l-DOPA), are utilized for melanin synthesis and not as an energy source (
). Consequently, it is possible to selectively label the melanin polymer with NMR or radioactive isotopes, a property that has been exploited for melanin structural studies (
). Melanization of C. neoformans results in the deposition of the polymer at the innermost surface of the cell wall, which becomes filled throughout over time (
). The pigment is composed of closely packed spherical particles ranging from 40 to 130 nm in diameter, which are found arranged in concentric layers in the cell wall (
Studies using isotopically-labeled precursors in conjunction with high-resolution solid-state NMR (ssNMR) revealed that the melanin polymer is likely to be covalently bonded to cell-wall chitin and was also found strongly associated with other nonpigment cellular moieties, including polysaccharides such as chitosan and vesicle and/or plasma membrane-derived lipids (
). These components serve as the scaffold for melanin synthesis. Indeed, C. neoformans’ strains with aberrant chitin and/or chitosan biosynthesis are unable to retain the pigment within the cell wall and display a leaky-melanin phenotype (
WdChs4p, a homolog of chitin synthase 3 in Saccharomyces cerevisiae, alone cannot support growth of Wangiella (Exophiala) dermatitidis at the temperature of infection.
). The concept of a fibrillary matrix upon which melanin is deposited extends to other species, including human eumelanin where it is known to also play a critical role in absorbing toxic oxidative melanin intermediates and to provide the elliptical shape to melanosomes (
In addition to the relevance of melanin to all biota and its great impact on fungal pathogenesis, this polymer has numerous potential applications in biophysics, material sciences, and even in the cosmetics and healthcare industries (
). The remarkable potential of melanin derives both from its chemical composition on the molecular level and its versatility as a component of the supramolecular structure within a cellular milieu. Investigation of the melanin structure is a notoriously challenging task due to the insoluble, amorphous, and heterogeneous nature of this architecturally complex polymer (
). Despite these challenges, over the past 2 decades a wealth of molecular level structural information has been uncovered using multiple nondestructive methodologies, among them are the following: X-ray scattering, ssNMR, EM, and atomic force microscopy (
). Current views of eumelanin molecular architecture posit that these pigments are composed of a common chemical motif and arranged as graphite-like π-stacked planar sheets of locally ordered monomers, mainly derivatives of 5,6-dihydroxyindole (DHI) (
), suggesting that the mode of synthesis can affect the final structure. A graphical schematic highlighting the current views on eumelanin synthesis and the relationship of melanin to the fungal cell wall, capsule, and cell body shows that melanin resides in the cell wall (Fig. 1).
Figure 1Current views about the synthesis of fungal eumelanins.A, catecholamine precursors like l-DOPA are oxidized by the polyphenol oxidase laccase to form dopaquinone, which undergoes further oxidation to form DHI or DHICA. DHI and DHICA form locally-ordered oligomers that in turn form planar stacks, presumably with stacking distances of about 4.5 Å (
). The higher order structure of eumelanin is considered “disordered,” because different planar structures are oriented in diverse geometries to one another resulting from diversity of DHI/DHICA oligomers stabilized by hydrogen bonding, cation–π, and van der Waals interactions (
). The aggregation of these simpler units leads to formation of the granular structure that we refer to as melanin granules, which have functional groups that interact with cell-wall and cell-membrane components such as lipids ((CH2)n, depicted in green), proteins, and polysaccharides (C=C, depicted in pink) (
). B, model representing the architecture of the plasma membrane represented by a lipid bilayer, which interacts with melanin nanoparticles that aggregate to form larger melanin granules (
Previous studies from our group have shown that the budding of melanized C. neoformans cells involves a decrease in the melanin thickness of the pigmented cell wall near the bud-site (
). We have hypothesized that the pigmented nanoparticles released into the extracellular space as part of the normal cell-wall–remodeling process are the smallest structural units of melanin found anchored to the cell wall. Hence, we have now investigated the physical properties, supramolecular structure, and protein content of these secreted melanin nanoparticles in the context of current knowledge of the cell-wall–associated fungal melanin.
Results
Prolonged acid hydrolysis of melanin ghosts yields melanin nanoparticles
Prior studies have shown that the solid yeast-shaped particles lacking all internal structures, designated as “melanin ghosts,” are composed of smaller spherical structures ranging in diameter from 40 to 130 nm (
). We hypothesized that these particles were held together by nonpigment cellular components, a supposition supported by NMR studies showing that C. neoformans ghosts contain tightly associated lipid moieties and cell-wall polysaccharides such as chitin (
). Because polysaccharides are typically susceptible to acid hydrolysis, we reasoned that prolonging the hydrochloric acid (HCl) incubation conducted during the melanin ghost isolation protocol would free the smaller spherical melanin particles for further analysis. Consistent with these expectations, increasing the incubation time of the HCl treatment led to significant morphological changes in the melanin ghosts samples. Scanning electron micrographs showed that ghosts, subjected to the prolonged acid incubation, initially appeared to be deformed as the relatively larger melanin particles, located close to the external surface, are released; over time, they took on a “hollowed out” appearance as the smaller melanin nanoparticles were subsequently liberated (Fig. 2, A–C). In addition, the roughness of the ghost surface increased with the prolonged acid treatment, suggesting that the removal of acid-labile nonpigment material exposes more of the underlying melanin particles. SEM visualization of material released during 4 h of HCl boiling revealed that melanin aggregates between 60 and 160 nm in diameter size, with the majority measuring around 60–80 nm, were surrounded by smaller granule-like melanin particles with diameters less than 60 nm (Fig. 2, D and E). Cross-sectional imaging of this suspended material showed a mix of granular nanoparticles ∼30 nm in diameter with variable electron density and degrees of consolidation (Fig. 2F). Further TEM analyses of these particles using negative staining, which involves coating the sample with a heavy metal stain that protects its structure from collapsing during drying and thus provides contrast for the detection of structural features, revealed images consistent with the notion that these nanospheres form tightly packed melanin aggregates (Fig. 2, G–I). Altogether, these results established that melanin ghosts of C. neoformans are composed of aggregated melanin nanoparticles arranged as small to large sized spherical particles, which are found embedded within an acid-labile matrix that produces a smooth appearance for these cell-sized structures.
Figure 2Fungal melanin nanoparticles are released by acid hydrolysis of C. neoformans’ melanin ghosts. SEM images of C. neoformans’ melanized cells subjected to standard melanin isolation but variable incubation time in 6 n HCl for 1 h (normal) (A), 24 h (B), or 4 days (C). Particles in solution released during 4 h of acid treatment were analyzed by multiple techniques. D, scanning electron micrographs showing nanoparticles (arrows) surrounding them. E, size distribution of released particle population determined by measuring diameters in scanning electron micrographs. Two hundred particles were counted. F, cross-sectional images of suspended acid-released nanoparticles showing variable electron density and degree of aggregation. Scale bars, 100 nm. G–I, acid-released particles recovered from C. neoformans strain 24067 analyzed by TEM using negative staining.
). However, in the various cellular systems where melanogenesis has been studied, melanin synthesis occurs within specialized vesicles known as melanosomes (
). During the budding process of melanized fungal yeasts, melanosomes in the cell wall are either degraded and/or laterally displaced so that the daughter cell can emerge (
). However, in synthetic systems, melanin formation is due to the auto-polymerization of precursor molecules, such as l-DOPA. Consequently, we hypothesized that the released melanosomes would be found in the supernatants of C. neoformans’ melanized cultures, possibly in association with extracellular vesicles. The culture supernatants from a C. neoformans’ WT strain (grown with or without l-DOPA) and a strain defective for melanin synthesis, ΔLAC1,2 mutant (Fig. 3, A–D, and G), were centrifuged at 100,000 × g, and the resulting pellets were analyzed. The solid material from each of the cell cultures, herein referred as crude material, was found to contain particles mostly around 100 nm in diameter as measured by dynamic light scattering (DLS) (Fig. 3, B, E, and H). As a control, we also analyzed the pelleted material from a culture containing l-DOPA and no fungal inoculum, observing a population of larger particles from 200 to 300 nm in diameter (Fig. 3K) that we attributed to melanin aggregates from l-DOPA auto-polymerization. Characterization of these materials by TEM using negative staining was also performed. The sample from C. neoformans grown with l-DOPA revealed the presence of electron-dense aggregated granular structures, henceforth called melanin granules (Fig. 3C). Similar aggregated structures were seen in the ΔLAC1,2 strain sample (Fig. 3I), whereas they were absent in the WT without l-DOPA (Fig. 3F). In the l-DOPA sample without C. neoformans cells, we observed irregular and nonspherical aggregates that did not resemble the melanin granules observed for the C. neoformans cultures (Fig. 3L) and that we attribute to l-DOPA auto-polymerization.
Figure 3Secreted vesicles and melanin granules from C. neoformans are found in culture supernatants.Top panel, cross-sectional view of representative cells by TEM. Scale bars, 500 nm. Middle panel, hydrodynamic diameter of vesicles and melanin granules measured by DLS. Bottom panel, representative micrographs by TEM of vesicles and melanin granules (often present as aggregates) using negative staining. A–C, C. neoformans grown in MM with l-DOPA. D–F, C. neoformans grown in MM without l-DOPA. G–I, C. neoformans’ ΔLAC1,2 strain grown in MM with l-DOPA. J, C. neoformans heat-killed grown MM with l-DOPA. K and L, MM with l-DOPA. Data represent three independent experiments.
Melanin has the unique ability to absorb almost every wavelength of light and, in contrast to most other natural chromophores, has a broadband monotonic absorption spectrum (
). Thus, we used UV-visible absorbance spectroscopy to verify whether the aggregated granular particles visualized by TEM are indeed melanin pigments. A monotonic curve was observed for the pelleted cellular material collected from C. neoformans cells grown in MM with l-DOPA (melanin granules and vesicles) but not for ΔLAC1,2 strain grown in MM with l-DOPA (l-DOPA aggregates and vesicles) nor for the fungal cells grown in MM lacking l-DOPA (vesicles) (Fig. 4A). DLS also yields a dispersity index, which is a measure of the dispersion (or spread) in the estimated hydrodynamic sizes of colloidal particles in a solution (
). We observed that melanin granules had lower dispersity than auto-polymerized l-DOPA (Fig. 4B). Furthermore, negative staining TEM allowed us to visualize spherical nanoparticles ∼200 nm in diameter, which were isolated from the supernatant of C. neoformans’ melanized cultures (Fig. 5, A and B), whereas melanin resulting from auto-polymerized l-DOPA particles showed minuscule structures with no specific pattern for their association (Fig. 5, C and D). These results demonstrated that C. neoformans’ extracellular vesicles along with melanin granules are found in the culture supernatant, whereas aggregated material seen in the ΔLAC1,2 strain has similar aggregation properties to auto-polymerized l-DOPA. Thus, melanin granules have unique colloidal characteristics, and these particles are distinct structures in size and morphology distinguishable from melanin resulting from auto-polymerized l-DOPA
Figure 4Secreted vesicles and melanin granules from C. neoformans are biologically synthesized.A, absorbance spectra of vesicles and melanin granules. Melanin granules and vesicles collected from C. neoformans grown in MM with l-DOPA (orange) have a broadband optical absorption curve characteristic of melanin. A curve for auto-polymerized l-DOPA is apparent in MM (purple), and no such curve is visible for vesicles collected from C. neoformans in MM lacking l-DOPA or for material collected from C. neoformans’ ΔLAC1,2 in MM with l-DOPA. B, dispersity index values of vesicles and melanin granules. Melanin granules in C. neoformans + l-DOPA showed low dispersity, suggesting that the population of melanin granules is highly monodisperse. l-DOPA aggregates had a broad polydisperse distribution. Representative data are from two independent experiments.
Figure 5C. neoformans' melanin granules are shown as highly monodisperse spherical nanoparticles. High-resolution TEM micrographs using a 1% phosphotungstic acid are shown. A and B, melanin granules found along with extracellular vesicles display a spherical shape. C and D, broadly polydispersed minute spherical l-DOPA particles resulted from auto-polymerization. D represents a close-up of rectangular area indicated in C. Representative images are from three experiments.
Divalent cations promote aggregation of C. neoformans' melanin granules
We studied the hydrodynamic size of melanin granule suspensions in the presence of monovalent (Na+) and divalent (Ca2+) salt concentrations to gain insight into their intramolecular interactions. The crude material recovered from the culture supernatants of C. neoformans cells grown with l-DOPA containing both melanin granules and vesicles (hereinafter referred to as crude melanin granules) aggregated at >0.01 m CaCl2, >0.1 m NaCl, and >10× PBS (Fig. 6, A–C). These results indicate that divalent cations promote melanin aggregation or fusion events at lower concentrations than monovalent cations. Alternatively, divalent cations likely promote particle aggregation by neutralizing negative charges on the surface of melanin (
Figure 6Divalent cations caused the aggregation of crude melanin granules at lower concentrations than monovalent cations. Size distribution of melanin granules and vesicles collected from C. neoformans grown in MM with l-DOPA were resuspended in the following: 1, 0.1, 0.01, and 0.001 m CaCl2 (A); 1, 0.1, 0.01, and 0.001 m NaCl (B); and 10, 1, and 0.1× PBS (C). Representative data are from two independent experiments.
). We hypothesized that in addition to these constituents, the cellular material recovered from melanized cell cultures would contain tightly-packed melanin granules of high molecular weight and low volume that could be separated from the vesicles and protein aggregates by exploiting their buoyancy. Thus, we analyzed the pelleted crude material from the supernatants of C. neoformans’ nonmelanized and melanized cultures using density gradient ultracentrifugation. A distinct dark band visible by examination with the naked eye was apparent toward the bottom of the tube for the melanized sample, corresponding to fraction 5 (Fig. 7A). TEM imaging of the collected fractions revealed vesicles primarily in fractions 1 and 2, for both samples (Fig. 7B); aggregated electron dense nanoparticles ∼30 nm in diameter, corresponding to the dimensions and appearance of melanin granules, were also apparent in fraction 5 of the melanized sample (Fig. 7B).
Figure 7Density gradient separation of melanin granules from extracellular vesicles.A, image of OptiPrepTM density gradient of ultrapelleted culture supernatants (crude material) from C. neoformans’ nonmelanized and melanized cultures grown in MM without or with l-DOPA, respectively. B, TEM images depicting fractions 1–5 collected from density gradient centrifugation. Melanin granules are visible in fraction 5 of the C. neoformans melanized sample. Representative data are from three independent experiments. Scale bars, 100 nm.
Melanin particles from ghosts and culture supernatants are similar
We entertained the hypothesis that melanin granules in C. neoformans’ culture supernatants were similar to those recovered from prolonged acid hydrolysis of melanin ghosts. Consequently, we sought to mechanically break down the melanin ghosts isolated from C. neoformans by extended ultrasonication, avoiding the chemical exposure. We found that ultrasonication of melanin ghosts for up to 12 min resulted in the release of particles in the range 0–200 nm measured by DLS (Fig. 8A). SEM visualization revealed that an ultrasonic pulse as short as 30 s led to fracture of the melanin ghosts characterized by jagged edges, which then turned into smaller round structures exhibiting a coarse and rough appearance as the process continued (Fig. 8B). The spherical melanin particles that resulted from extensive physical disruption of C. neoformans’ melanin ghosts showed a similar size distribution to the crude melanin granules in the culture supernatant. These data support the proposal that C. neoformans’ cell-wall melanin is composed of tightly packed melanin granules with a common substructure of melanin nanoparticles, which are also released to the extracellular environment.
Figure 8Sonic cavitation of melanin ghosts from C. neoformans reduces melanin to particles of ∼200 nm.A, size distribution of melanin particles by dynamic light scattering as sonication time increases. B, scanning electron micrographs showing melanin break down. Scale bar, 200 nm. Representative micrographs of three independent experiments.
To gain further insight into the structure of C. neoformans’ melanin granules, we used cryo-EM. Micrographs of fungal melanin granules isolated by buoyancy revealed a pattern of similarly sized round beads on a string (Fig. 9A). Each spherical nanoparticle, ∼200 nm in diameter, resembles a multilayered structure with well-defined edges but not a distinguishable lipid bilayer, suggesting that these structures may result from stepwise aggregation of smaller particles (Fig. 9B). We considered whether the structures visualized by cryo-EM images were water ice particles. We cannot unequivocally distinguish between these possibilities by this form of microscopy. Nevertheless, we noted that our putative melanin structures are relatively uniform, have dimensions comparable with those obtained melanin by other EM methods (Fig. S1), are translucent, less electron dense than ice, and do not express the geometric structures associated with water ice. Hence, we are confident that these are melanin granules.
Figure 9Ultrastructure of C. neoformans’ melanin granule suggests a multilayered superstructure.A, cryo-EMs exhibit melanin granules held together as beads on a string. B, spherical structure representative of the fundamental unit of C. neoformans’ cell-wall melanin ∼200 nm in diameter.
). Eumelanins can be identified based on the modification of their EPR signals by a number of agents, including light, pH, redox agents, and diamagnetic multivalent metal ions (
). The crude melanin granules recovered from the C. neoformans culture supernatant yielded a single slightly asymmetric EPR trace similar to the spectrum previously described for melanin ghosts (Fig. 10A) (
). Irradiation of fungal crude melanin granules with a 250-watt LED white light enhanced the EPR signal intensity 1.2 times, whereas doping with zinc ions increased it 5.3-fold. Alkaline pH had little or no effect on the EPR signal of crude melanin granules, but low pH was found to attenuate their EPR signal intensity, possibly due to changes in protonation of accessible ionizable groups. The melanin ghost EPR signal was insensitive to pH (Fig. 10B). Thus, these results demonstrated that melanin granules found in the culture media of C. neoformans, in the presence of l-DOPA, retain the paramagnetic properties characteristic of eumelanins.
Figure 10EPR spectroscopy analysis of C. neoformans' crude melanin granules.A, EPR of C. neoformans' crude melanin granules suspended in distilled water at pH 7 (blue); after irradiation for 20 min with white light from a 250-watt LED white lamp; and suspended in a solution of 0.1 m ZnCl2. B, EPR of C. neoformans' crude melanin granules suspended in distilled water at acidic pH adjusted with 1 n HCl (red); suspended in distilled water at pH 7 (blue); and suspended in distilled water at basic pH adjusted with 1 n NaOH (green).
C. neoformans' melanin is molecularly associated with non-polysaccharide cellular constituents
Previous ssNMR studies by our group have revealed that melanin ghosts isolated from C. neoformans cells are an assembly of the l-DOPA–derived melanin pigment and the cellular “scaffolding” moieties on which the pigment is deposited (
). Here, we again used ssNMR to determine whether the melanin granules that are present in the pelleted crude material from C. neoformans’ culture supernatants are also associated with nonpigment cellular constituents. To obtain a sample mass sufficient for ssNMR analysis, we took advantage of the previously reported C. neoformans’ mutant strain ST211A (GenBankTM accession number MK609896), which displays a “leaky melanin” phenotype; leaky melanin strain cells are able to synthesize melanin, but the cell wall is unable to the retain the pigments, and thus they leak into the extracellular space (
). The 13C cross-polarization magic-angle spinning (CPMAS) spectrum of the crude melanin granules obtained from the supernatant of a C. neoformans’ ST211A cell culture containing uniformly 13C-enriched glucose as the sole carbon source and l-DOPA (at natural abundance) is compared with that of isolated melanin ghosts in Fig. 11, A and B); this isotopic labeling scheme allows us to selectively observe melanin-associated cellular components without interfering signals from the melanin pigment. Whereas the most prominent peaks in the melanin ghosts’ CPMAS spectrum are centered at ∼30 ppm, likely arising from the aliphatic “tails” of vesicle-derived lipids functionalized during melanization, the crude melanin granules spectrum is dominated by signals in the region between ∼55 and 105 ppm that correspond to various types of polysaccharide ring carbons. The strong presence of polysaccharides in this crude preparation is unsurprising because our research group has previously demonstrated that capsular polysaccharides are synthesized intracellularly and transported to the extracellular space within vesicles (
). To test whether these polysaccharides are indeed capsular components that are not associated with the melanin granules, the crude sample was reexamined after 30 min of incubation in hot HCl (Fig. 11C). This relatively short treatment was sufficient to drastically diminish the polysaccharide content so that the signals in the ∼55–105-ppm region were of less relative intensity than observed for melanin ghosts, which had been subjected to an hour of HCl boiling in addition to other enzymatic and chemical treatments during sample preparation.
Figure 11ssNMR analyses of C. neoformans’ crude melanin granules demonstrated a weak association to polysaccharides and suggested the presence of peptides or proteins strongly “functionalized” during melanization.A and B,13C CPMAS spectra of crude melanin granules and melanin ghosts isolated from the leaky-melanin strain C. neoformans ST211A, showing dominant signals for polysaccharides (∼55–105 ppm) or lipids (∼30 ppm), respectively. C and D,13C CPMAS spectra of crude melanin granules and melanin ghosts post-acid treatment revealed that lipid signal (∼30 ppm) corresponding to fatty acids is intimately associated with melanin particles. E and F, 2D 13C–13C DARR spectra of crude melanin granules confirmed augmented polysaccharide content (region ∼55–105 ppm), although in melanin ghosts these constituents are less prominent. G and H, 2D 13C–13C DARR spectra of crude melanin granules and melanin ghosts post-acid treatment revealed that both samples are composed of the same cellular constituents and suggest the presence of lipids and possibly protein remnants (∼53 × 175 ppm) that might be strongly associated with the melanin polymer.
Further insight into the molecular identity of the cellular constituents that associate with melanin granules was obtained from 2D 13C–13C DARR spectra that display correlations between proximal carbon pairs equivalent to ∼1–2 bond lengths in distance. In analogy to our 13C CPMAS data, the DARR plot of the crude melanin granules displays a multitude of prominent cross-peaks corresponding to correlations between spatially-close polysaccharide ring carbons (∼55–105 ppm), whereas this region in the melanin ghosts 2D plot only contains a handful of correlations that we have previously attributed to the cell-wall scaffolding moieties (e.g. chitin) on which the pigment is deposited (Fig. 11, E and F) (
). No cross-peaks attributable to polysaccharides were observed for the crude melanin granules after the 30-min incubation in hot HCl, supporting our hypothesis that the polysaccharides were indeed superficially bound capsular components and that melanin granules released into the extracellular space are not tightly associated with cell-wall constituents as for melanin ghosts (Fig. 11G). Moreover, all of the spectral features displayed in the 2D DARR plot of the HCl-treated melanin granules were also observed in the melanin ghost spectrum. We interpreted this spectroscopic similarity to indicate that the 30-min HCl incubation was adequate to hydrolyze all cellular moieties that were not intimately associated with the melanin granules. The major difference between the two datasets is the presence of cross-peaks attributable to the polysaccharide scaffolding constituents in the melanin ghost DARR spectrum. To test the robustness of the association between these residual polysaccharides and the melanin pigment, we subjected the melanin ghosts to a series of treatments in boiling HCl using incrementally longer incubation times, and we monitored the peak intensities of the nonpigment constituents using 13C CPMAS experiments (Fig. S2). Increasing the incubation time from 2 to 4 h resulted in only minimal spectral differences, and thus we chose to further examine the 4-h HCl-treated melanin ghosts by acquiring a 2D DARR spectrum (Fig. 11H). Although the additional 4-h HCl treatment was effective in diminishing the signals corresponding to polysaccharides to undetectable levels (Fig. 11, F and H), the majority of other cross-peaks was preserved; furthermore, these cross-peaks were also retained in the DARR spectrum of the crude melanin granules after 30 min of HCl treatment (Fig. 11G). This comparison indicates that the two samples contain many of the same cellular constituents, which are likely strongly associated with the pigment and could play a role in melanin ultrastructure. Interestingly, the correlations common to both sample spectra suggest the presence of lipids and likely also protein remnants; the cluster of signals centered around ∼53 × 175 ppm is consistent with correlations between the α- and carbonyl-carbons of amino acids within a peptide bond. However, this later assignment is provisional and requires further testing.
Melanin-associated proteins are identified from C. neoformans' melanin granules
To assess the possibility that C. neoformans’ melanin nanoparticles are associated with, or possibly covalently bonded to, melanization-specific proteins, we performed a proteomic analysis on the isolated melanin granules comprising the visibly dark band (fraction 5) after density gradient centrifugation. To distinguish between proteins that are vesicle-associated and/or loosely bound from those chemically functionalized by the melanin nanoparticles, we included as controls fraction 5 samples collected from a C. neoformans culture grown in MM without l-DOPA as well as auto-polymerized l-DOPA. To ensure that sufficient material was available for LC-MS/MS analysis, 2–3 density gradient procedures were performed for each sample, and their respective fractions 5 were pooled together. Melanin granule-associated proteins were solubilized, reduced, carboxymethylated, and digested before LC-MS/MS measurements. Data analysis yielded the consistent identification of four proteins exclusively associated with C. neoformans’ melanin granules (Table 1). A bioinformatics analysis to examine the presence of predicted signal peptides and GPI anchors showed that three proteins had an N-terminal signal peptide, two of which (Cig1 and Blp1) were also found in extracellular vesicles (
). Only one protein, CNAG_05312, had both an N-terminal signal peptide and a GPI anchor indicative of plasma membrane association. These secreted peptides were previously reported to play critical roles in C. neoformans’ virulence (
Secretome profiling of Cryptococcus neoformans reveals regulation of a subset of virulence-associated proteins and potential biomarkers by protein kinase A.
Titan cells formation in Cryptococcus neoformans is finely tuned by environmental conditions and modulated by positive and negative genetic regulators.
). Taken together, these results revealed that C. neoformans’ melanin granules are indeed intimately associated with proteins that employ both a conventional and nonconventional secretory pathway for regulation and that may be involved with structural and/or functional properties of the cryptococcal eumelanin.
Table 1Identified proteins associated with C. neoformans melanin granules
Accession no.
Protein name
Molecular mass
Signal peptide
GPI-anchor
EV
Functional description
Refs.
kDa
CNAG_01653
Cytokine- inducing glycoprotein (Cig1)
30
Yes
No
Yes
Iron uptake via binding and transport of heme. Contributes to virulence
Secretome profiling of Cryptococcus neoformans reveals regulation of a subset of virulence-associated proteins and potential biomarkers by protein kinase A.
Titan cells formation in Cryptococcus neoformans is finely tuned by environmental conditions and modulated by positive and negative genetic regulators.
Secretome profiling of Cryptococcus neoformans reveals regulation of a subset of virulence-associated proteins and potential biomarkers by protein kinase A.
Based on the results obtained in this study, we propose a model of C. neoformans’ cell wall melanization that begins intracellularly and requires melanosome transport to the cell wall for their consolidation into melanin granules (Fig. 12). Melanin synthesis occurs within melanosomes (nanospheres 10–20 nm in diameter) enclosed in multivesicular bodies (MVBs) (∼450 nm in diameter) (Fig. 12A) and laccase-containing vesicles (∼80 nm in diameter) (Fig. 12B, top inset). These organelles may be signaled by the Qsp1 mature peptide to activate melanogenesis (
). Ongoing melanization is evidenced by the presence of melanosomes with variable degrees of aggregation and electron density within these double-layered compartments. The melanosomes aggregate into ∼60-nm particles (Fig. 12B, bottom inset), both within MVBs and in the cell cytoplasm, and are eventually transferred into the cell wall by events that are facilitated by the presence of invaginations in the plasma membrane (Fig. 12, C and D). These structures are characterized by finger-like protrusions that “scoop” melanosomes from the cytosol to transfer them to the cell wall. Further aggregation of the melanosomes into melanin granules (up to 200 nm in diameter) and anchoring within the cell wall could be augmented by vesicle materials, such as laccase and Blp1, respectively. Some of these vesicles are delivered to the cell wall within MVBs that fuse with the plasmatic membrane (Fig. 12E). Melanin granules’ concentric arrangement within the cell wall uses as a scaffold membranous sheets shown as multilaminated structures of cryptococcal cell walls (Fig. 12F). Finally, fungal melanosomes are held together by and anchored to cell-wall constituents (i.e. chitin, chitosan, and glucans) due to the following diverse interactions: (i) associative or covalent interactions between melanin-associated proteins and cell-wall components, and (ii) electrostatic interactions of oppositely charged melanin–chitosan pairs. As the fungal cell replicates, cell wall remodeling is required to allow budding; thus, the melanized scaffold is locally modified by secreted enzymes and allows the release of melanin granules (Fig. 12G) into the surrounding environment. A summary of C. neoformans’ melanin particles analyzed with multiple microscopic techniques is shown in Fig. 13.
Figure 12Illustrative model of melanization in C. neoformans. Melanogenesis in C. neoformans is characterized by multiple events that can be grouped into four phases: synthesis, transport, aggregation-deposition, and remodeling. Intracellular biosynthesis of the polymer occurs within melanosomes (nanospheres 10–20 nm in diameter) enclosed in MVBs (
). Active melanization is evidenced by the presence of melanosomes with variable degrees of pigmentation within these lipid bilayer compartments. Melanosomes forming aggregates ∼60 nm in diameter are also noticeable in the cell cytoplasm (B, bottom inset). Melanosomes are transported to the cell wall using an unconventional mechanism that involves invaginations of the plasma membrane (
). Finger-like protrusions of the plasma membrane scoop up melanosomes to transport them from the cytosol to the cell wall (C and D, white arrowheads). Once in the cell wall, aggregation of melanosomes up to 200-nm melanin granules might be promoted by laccase enzyme delivered to the cell wall via MVB fusion with the plasma membrane (
) (F, white arrows). Melanosomes anchor to the cell wall via multiple interactions: (i) associative and covalent interactions between them and cell-wall constituents (chitin, chitosan, glucans, and lipids), possibly mediated by Blp1-chitin and CNAG_05312-glucans/chitin/chitosan (
). Regular cell growth requires the cell-wall remodeling to allow budding inducing the melanin scaffold degradation/alteration via secretion of peptidases (
Integrated activity and genetic profiling of secreted peptidases in Cryptococcus neoformans reveals an aspartyl peptidase required for low pH survival and virulence.
Secretome profiling of Cryptococcus neoformans reveals regulation of a subset of virulence-associated proteins and potential biomarkers by protein kinase A.
), thus releasing melanin granules and string-like granules (G) into the extracellular environment. Removal of nonpigmented acid-labile cell-wall components exposes the concentric layered arrangement of the melanin granules within the cryptococcal cell wall from melanin ghosts (
Figure 13Summary of C. neoformans' melanin particles visualized with different microscopic approaches. These images are also found in Figure 2, Figure 9.
Elucidation of the smallest fungal cell-wall melanin particle that retains its characteristics from C. neoformans is essential for understanding how the pigment is assembled and interacts with other cellular components. Similarly, an appreciation of melanin supramolecular structure (
) is essential to understand how this material confers the fungal cell with unique biological properties beyond microbial virulence, such as photoprotection, energy harvesting, heat gain, thermoregulation, and metal scavenging, among others (
Previous structural studies on the molecular architecture of fungal eumelanins in the cell wall have focused on the so-called melanin “ghosts,” which are macromolecular structures that retain cellular morphology after hot acid digestion of melanized cells (
). Although such studies have been extremely informative in revealing chemical aspects of melanin composition, they focus on structures where the pigment remains organized into a cellular carcass of what was once a melanized cell. As such, this melanin polymer includes nonmelanin components that serve to hold the pigment together into structures with cell-like dimensions. In this study, we sought to go deeper in searching for the building block unit of the cell-wall melanin through complementary approaches that included prolonged acid digestion of melanin ghosts as well as to investigate the presence of such structures in culture supernatants. Both approaches produced much smaller melanin particles that were comparable in size and form. We then applied multiple techniques to study these particles; SEM, TEM, and cryo-EM were used to examine the surface and cross-sectional images of the melanin nanoparticles. DLS was used to analyze the size distribution of melanin particles in suspension and to probe their colloidal properties. Isopycnic centrifugation was used to isolate melanin granules based on their buoyancy. Spectroscopic methodologies (optical absorbance, EPR, and ssNMR) were used to probe the molecular properties of fungal eumelanins and to investigate their interaction with cell-wall components. Finally, proteomic analysis was conducted to identify proteins that maintained an intimate association with melanin granules. The results demonstrate the existence of relatively monodisperse melanin granules ranging from 40 to 200 nm in diameter, depending on the approach, that manifested a monotonic broadband absorption curve and stable EPR signal consistent with melanin. These particles are in turn composed of melanosomes, substructural nanospheres serving as the building unit of the fungal cell wall, functionally comparable with those described in mammalian melanization systems (
The small melanin particles derived from ghosts or culture supernatants are different from those obtained by auto-polymerization of l-DOPA into melanin, implying differences between the products of the biological catalytic and chemical reactions. Melanin ghosts can be broken down to smaller particles by either acid digestion or sonic cavitation, generating particles that are comparable in size to those released into the culture supernatant during C. neoformans' growth under melanizing conditions. The differences between C. neoformans-derived particles and those from auto-polymerized l-DOPA imply different synthesis, as fungal particles showed a unique electron microscopic appearance and size. We have shown previously that C. neoformans and synthetic melanins shared similar chemical groups in the aromatic core (
); however, differences between these materials could stem from the fact that the production of fungal particles is vesicle-associated and catalyzed by a laccase, whereas auto-polymerization is a spontaneous oxidative process that occurs in solution. Moreover, C. neoformans' melanin granules isolated from the extracellular media are spherically shaped and biologically synthesized particles of up to 200 nm in diameter, similar to those isolated from natural eumelanins such as Sepia officinales (
The fungal melanin granules demonstrated a close association with four proteins relevant to C. neoformans’ virulence: Cig1, Blp1, Qsp1, and CNI3590 macrophage-activating glycoprotein (Table 1). The mannoprotein Cig1 (CNAG_01653) plays a role in iron acquisition, and it is essential for virulence in a mutant lacking the ferroxidase for high-affinity uptake (
). The hypothetical protein containing a Barwin-like domain (Blp1) (CNAG_06346) is involved in capsule-independent anti-phagocytosis activity and is critical for successful colonization of mammalian lungs by C. neoformans. Blp1 is not essential for capsule formation and melanin production (
), suggesting a possible association of this protein with the cell-wall chitin. The quorum-sensing peptide (Qsp1) (CNAG_03012) is a secreted central signaling molecule that acts intracellularly to control virulence in C. neoformans and promote cell-wall function at high cell densities (
), which could explain the finding of this unique sequence in our proteomic analyses of melanin granules. CNI3590 (CNAG_05312) is a GPI-anchored protein and also a secreted factor associated with virulence that contains a carbohydrate-binding domain (
Secretome profiling of Cryptococcus neoformans reveals regulation of a subset of virulence-associated proteins and potential biomarkers by protein kinase A.
), suggesting its interaction with the plasma membrane and cell-wall components such as glucans, chitin, and chitosan. Furthermore, Cig1 and CNAG_05312 are positively regulated by the pH-responsive transcription factor Rim101, which in turns is induced by Pka1, a catalytic subunit of the cAMP/protein kinase A pathway (
Secretome profiling of Cryptococcus neoformans reveals regulation of a subset of virulence-associated proteins and potential biomarkers by protein kinase A.
). Pka1 is known to modulate the secretion of proteins associated with virulence and survival of C. neoformans in the host, as well as the expression of cell-wall–related genes (
Secretome profiling of Cryptococcus neoformans reveals regulation of a subset of virulence-associated proteins and potential biomarkers by protein kinase A.
). Similarly, Blp1, Qsp1, and CNAG_05312 are direct targets of three transcription factors required for C. neoformans’ virulence: Gat201, Gat204, and Liv3 (
), whereas deletion of GAT201 reduces the capsule size and severely attenuates virulence in a murine model but shows a hypermelanized colony phenotype (
) mutant strains exhibit defects in melanin or capsule production. Altogether, these data strongly support the notion that C. neoformans’ synthesis of melanin granules is a highly regulated process that leads to particles with unique morphological and compositional features, whose biogenesis might be subject to highly redundant gene networks. Although we cannot rule out that these proteins become associated with the melanin particles in culture supernatants, covalent association of melanin to proteins has been extensively demonstrated in mammalian melanogenesis (
). The association between C. neoformans’ melanin granules and these proteins may serve complex regulatory roles during the biopolymer synthesis and structural functions within the granular particle or the fungal cell-wall milieu.
C. neoformans’ melanin granules visualized by negative staining TEM and cryo-EM most likely are formed from the aggregation of melanosomes (∼30 nm in diameter), resembling the supramolecular structure described in Sepia melanin (
). Analysis of particles resulting from eumelanin synthesis via enzyme immobilization prompted the proposal of a mechanism of hierarchical buildup of melanin by the gradual aggregation of building block particles (type-A particles), which led to the formation of particles 200 nm in diameter (type-B particles) (
). Prior studies from our group noted that melanin ghosts are composed of smaller melanin granules arranged in concentric layers embedded within the fungal cell wall (
). Ghost structures revealed that the smallest melanin nanospheres with smooth appearance are seen on the surface of early buds. We propose that as the fungal cell ages, aggregated melanosomes giving rise to melanin granules (up to 200 nm in diameter) are embedded into the cell wall. These melanin particles display a layered concentric arrangement guided by membranous sheets within the cryptococcal cell wall, which becomes evident by the removal of nonpigment acid-labile cell-wall constituents. This supramolecular structure is also consistent with the findings of previous studies using NMR cryoporometry, which indicated that C. neoformans’ melanin layers have pores that become smaller with increased cell age and progressive melanization (
Moreover, our micrographs showed a close association between “finger-like” plasma membrane invaginations and melanin granules, suggesting that these membranes structures could be involved in transport from the cytosol to the fungal cell wall. In Saccharomyces cerevisiae, finger-like invaginations are sphingolipid-rich lateral domains termed MCP (membrane compartment of Pma1) (
Synthesis of sphingolipids with very long chain fatty acids but not ergosterol is required for routing of newly synthesized plasma membrane ATPase to the cell surface of yeast.
), most likely associated with an “inverted macropinocytosis” mechanism required for extracellular vesicle transport across the plasma membrane and into the cell wall (
). The exact nature of lateral membrane microdomains involved in the vesicular transport of C. neoformans and the role of lipids in this context remain to be elucidated.
Mammalian melanin synthesis is enzymatically driven and occurs in membrane-enclosed compartments called melanosomes, which prevents the exposure of most cellular components to toxic or deleterious reactants produced during the process (
). Structurally, eumelanosomes (most pigment content corresponds to eumelanin) are aggregated structures (type-B particles) of 95–307 nm in diameter, depending on the source, formed due to the previous assembly of 10–60-nm subunits of small nanoparticles (type-A particles) (
). Analogous to this system, fungal melanogenesis is also compartmentalized within a sphingolipid-enriched vesicle, depends on the laccase enzyme, and leads to the supramolecular buildup of cryptococcal eumelanin particles that are retained within the cell wall, where they provide protection against environmental stressors (
Our proposed model for melanization in C. neoformans is in consonance with previous observations by our group and others. We have identified organelles showing that pigment synthesis starts intracellularly within melanosomes, enclosed in double-layered compartments, which are then transferred to the cell wall to consolidate as melanin granules anchored within the cell wall. Several pieces of evidence are presented, which can be organized into a testable model. First, laccase is expressed within MVBs as well as in the cell wall (
). Next, aggregated melanosomes ∼60 nm in diameter (Fig. 12B, bottom inset) may rely on MCP-like regions of the plasma membrane that generate finger-like protrusions that mediate melanosomes’ traffic to the cell wall (Fig. 12, C and D). A previous study revealed that the major capsular component (GXM, glucoronoxylomannan) was found in association with endosome-like membranous structures possibly involved in GXM synthesis/transport (
). One of these highly conserved domains in S. cerevisiae is closely related to an unconventional mechanism for vesicle secretion termed inverted macropinocytosis (
). Finally, ongoing aggregation of melanin granules within the cell wall is supported by eumelanin biomimetic studies demonstrating that consolidation of type-B particles is due to high amounts of the laccase enzyme (
). Melanin-layered deposition on the cell wall follows the existence of membranous sheets described in early ultrastructural studies of the cryptococcal cell wall (
) (Fig. 12F); however, as we have demonstrated in this study, melanin particles are loosely associated with an acid-labile polysaccharide matrix that makes this layout within the melanized cell wall difficult to demonstrate. Nonetheless, the concentric layered arrangement of these granular particles ∼75 nm in diameter was exposed when analyzing melanin ghosts (
), possibly favored via electrostatic interactions between chitin/chitosan and melanin. Indeed, alterations in the cell-wall chitin/chitosan induced a leaky melanin phenotype (
). The recovery of melanin granules in the supernatant of the culture media is explained by normal cell-wall–remodeling required for cellular budding (Figs. 5, A and B, and 9, A and B), which involves local modifications of the melanin scaffold by secreted peptidases (
Integrated activity and genetic profiling of secreted peptidases in Cryptococcus neoformans reveals an aspartyl peptidase required for low pH survival and virulence.
Secretome profiling of Cryptococcus neoformans reveals regulation of a subset of virulence-associated proteins and potential biomarkers by protein kinase A.
) that lead to melanin granule detachment from the cell wall. The model does not address the source of strongly-associated lipids with melanin granules, but rather it provides new pieces of data that should encourage experiments to continue dissecting the phenomenon of fungal cell-wall melanization.
In summary, we note remarkable parallels between cryptococcal and mammalian melanization. We have assembled the available facts into a model for the cryptococcal melanogenesis process that can be tested in future studies. The resemblances in these two systems are perhaps not surprising given that animals and fungi are close relatives in the tree of life and that the chemistry of melanin synthesis is similar. In both systems, the melanization reaction is contained within a vesicular structure, which protects the host cell from the cytotoxicity of reaction intermediates. The isolation of melanin particles from culture supernatants promises to be a new source of melanin material for studies that further unlock the details of pigment formation and for raw materials in industrial applications that include radioprotection, light harvesting, and drug delivery, among various uses.
Experimental procedures
Strains
In this study, the following strains were used: C. neoformans strain H99 (serotype A), C. neoformans’ ΔLAC1,2 (H99 background), and C. neoformans’ ST211A (H99 background) kindly provided by Alexander Idnurm. Each strain was kept in 20% glycerol stocks at −80 °C and thawed on ice when needed for replication.
Cell growth and culture conditions
For fungal culture growth, a loop of the glycerol stock was inoculated into Sabouraud broth and incubated at 30 °C for 48 h. Experiments were performed using cultures grown for 10–14 days at 30 °C with continuous shaking at 120 rpm in defined chemical media (minimal media (MM): 15 mm dextrose, 10 mm MgSO4, 29.4 mm KH2PO4, 13 mm glycine, 3 μm thiamine-HCl, pH 5.5) with and without 1 mm l-DOPA.
Extended acid-hydrolysis of melanin ghosts
Melanin ghosts were isolated following the protocol previously described by Chatterjee et al. (
). Briefly, cells grown in MM with l-DOPA were collected by centrifugation and washed twice with phosphate buffer (pH 7.4). Melanized cells were then subjected to a multiple-step enzymatic and chemical treatment to remove the cell wall, proteins, and lipid components associated with the melanin polymer. Consequently, the end product is a hollowed particle coated by the acid-resistant pigment called a “melanin ghost” (
). The acid treatment step consists of incubation of the melanized particles in 6 n HCl at 80 °C for 1 h. For the extended acid hydrolysis, melanin ghosts were incubated for up to 4 days, including periodic additions of fresh acid solution. Aliquots at 1 and 24 h and 4 days were analyzed by SEM. The supernatant from the acid hydrolysis was also collected, visualized by transmission EM (TEM), and SEM. Its particle population size was estimated by measuring the diameter using scanning electron micrographs.
TEM
C. neoformans yeast cells or melanin particles were fixed in 2.5% (v/v) glutaraldehyde in 0.1 m sodium phosphate buffer (pH 7.3) for 24 h at 4 °C. Samples were encapsulated in 3% (w/v) low-melting-point–agarose prior to being processed in Spurr resin following a 24-h schedule on a Lynx tissue processor (secondary 1% osmium tetroxide fixation; 1% uranyl acetate contrasting; ethanol dehydration and infiltration with acetone/Spurr resin). Additional infiltration was conducted under vacuum at 60 °C before the samples were embedded in TAAB capsules (TAAB Laboratories, Amersham Biosciences, UK) and polymerized at 60 °C for 48 h. Semithin survey sections 0.5 μm thick were stained with 1% toluidine blue to identify the areas with the best cell density. Ultrathin sections (60–90 nm) were prepared with a Diatome diamond knife on a Leica UC6 ultramicrotome and stained with 2% uranyl acetate in 50% methanol, followed by lead citrate for examination with a JEOL 1200EX (Analytical Imaging Facility at Albert Einstein College of Medicine) or a Philips CM120 at 80 kV (Microscopy Facility at the Johns Hopkins School of Medicine). Negative staining of the fractions from OptiPrep density gradients was performed by adsorbing 10 μl of each fraction to glow-discharged 400 mesh ultra-thin carbon-coated grids (EMS CF400-CU-UL) for 2 min, followed by three quick rinses of TBS and staining with 1% uranyl acetate with 0.05% Tylose. Images were captured with an Gatan Orlus Digital 2K × 2K CCD camera or an AMT XR80 high-resolution (16-bit) 8-megapixel camera.
SEM
Melanin particles were suspended in PBS and incubated on coverslips coated with 0.01% polylysine and fixed with 2.5% glutaraldehyde in 0.1 m sodium phosphate buffer (pH 7.3) for 24 h at 4 °C. Cells and melanin particles were postfixed with 1% OsO4; dehydrated in 70, 90, 95, and 100% ethanol; and critical-point dried in CO2. The cells were sputter-coated with gold using an EMitech K550 Sputter Coater and viewed in a LEO/Zeiss Field-emission scanning electron microscope at a voltage of 10 kV.
DLS
The DLS techniques gives an estimate of the size and heterogeneity of a sample by measuring the intensity fluctuations of scattered light by particles in solution. Measurement of melanin particles by DLS was performed with a 90Plus instrument (Brookhaven Instruments). To assess the size of melanin ghosts after different sonication times, 500 μl of a melanin ghost suspension in distilled water (1 mg/ml) was sonicated with a horned sonicator at amplitude 5 for up to 12 min. Particle size of the suspension was measured at different time points. Data are expressed as the average of 10 runs of 1-min data collection each. To examine the colloidal properties as a function of salt concentration, 1, 0.1, 0.01, and 0.001 m solutions of NaCl and CaCl2 and 10, 1, 0.1, and 0.01× phosphate-buffered saline solutions were used to suspend 1 μl of vesicles/granules in 100 μl of the respective salt solution. Average hydrodynamic diameter was obtained as described above.
Isolation of melanin granules
The isolation of melanin granules from C. neoformans was performed in two phases: 1) isolation of a mixture of extracellular vesicles and melanin granules from the culture supernatant herein called crude melanin granules, and 2) isolation of melanin granules by density gradient. For the first phase, yeast cells were pelleted at 5,000 rpm (SLA rotor, Sorvall) for 30 min at 4 °C. The supernatant was filtered through a 0.22-μm pore filter (Millipore Sigma) and pelleted at 100,000 × g for 1 h at 4 °C, with a slow break (SW28 rotor, Beckman Coulter). The pellet was suspended in 1× Dulbecco’s phosphate-buffered saline (DPBS) without calcium and magnesium (Corning) and washed twice with the same buffer. The pellet (crude melanin granules) was suspended in ∼500 μl of 0.1× DPBS and stored at −80 °C until further analysis. For the second phase, OptiPrepTM solution (60%) dilutions were made in 10 mm HEPES, 0.85% NaCl, pH 7.4. The crude melanin granules were mixed with undiluted OptiPrepTM to make a 45% solution that was layered at the bottom of the gradient. The gradient was composed of 45, 35, 30, 25, 20, and 15% layers in the ratio 0.4:3:3:2:2:1 for a total volume of 3 ml. The gradient was spun at 50,000 rpm for 10 h (SW Ti55 rotor, Beckman Coulter) to allow the vesicles and granules to equilibrate at their respective densities. Five equal-volume fractions were collected from each tube.
Spectroscopic studies
UV-visible analysis
The absorption properties of crude vesicle samples were analyzed with UV-visible spectroscopy using a SpectraMax M2 microplate reader (Molecular Devices). The spectra were recorded in the wavelength range of 300–800 nm.
EPR
Suspensions of melanin samples (∼2 mg) in 150 μl of distilled water were sonicated for 1 min using a horned sonicator. Equal volumes of appropriate buffer (distilled water at pH 2.0, 7.0, 12.0, or 0.1 m ZnCl2) were added. Using a glass long-tip transfer pipette, each sample was placed in a 4-mm precision-bore quartz EPR tube and frozen by immersion in liquid nitrogen. The EPR spectra were recorded at 77 K using a Bruker EMX EPR spectrometer operating in X-band mode. Experimental parameters were as follows: modulation amplitude, 10 G; microwave power, 6.362 milliwatts; modulation frequency, 100 kHz; microwave frequency, 9.43 GHz; attenuation, 15 dB; number of scans averaged, 20. Data acquisition was performed using WinEPR software (Bruker). All spectra were obtained under identical instrumental conditions. Light irradiation of samples was carried out at room temperature for 20 min using a 250-watt cool white light operating at 4000 K.
ssNMR
Solid-state NMR experiments were carried out on a 600-MHz Varian (Agilent) DirectDrive2 (DD2) spectrometer using a 1.6-mm FastMAS probe (Agilent Technologies, Santa Clara, CA) with a magic-angle spinning (MAS) rate of 15.00 ± 0.02 kHz and a nominal spectrometer-set temperature of 25 °C. All spectra were acquired on 13C-enriched samples obtained from a C. neoformans’ ST211A cell culture containing [U-13C6]glucose and natural abundance l-DOPA as the sole carbon source and obligatory melanization precursor, respectively. Average post-lyophilization sample masses were ∼1–3 mg for the pelleted cellular material containing melanin granules and ∼5–7 mg for the melanin ghosts. The typical 90° pulse_lengths for the 13C CPMAS experiments were 1.65 μs for 1H and 2.25 μs for 13C, respectively. As described previously (
), 1H decoupling using the small-phase incremental alternation pulse sequence with a field strength of ∼180 kHz was applied during signal acquisition. The 2D 13C–13C through-space DARR correlation experiments were collected using a 50-ms mixing time with similar 1H and 13C 90° pulse-lengths and decoupling field strength as described for the 13C CPMAS experiments.
Cryo-EM
Five microliters of purified C. neoformans’ melanin granules (∼3 mg) was applied to a 300-mesh copper grid coated with a holey carbon film and vitreously frozen in liquid nitrogen-cooled ethane using an FEI Vitrobot Mark IV device. The sample was transferred to a Gatan 626/70° cryo-transfer holder and observed in an FEI Talos S200C transmission electron microscope operating at 200 kV. Images were collected with a Falcon 2 direct-electron detector camera and processed in Adobe Photoshop CS6 with only linear adjustments in brightness and contrast.
Proteomic analysis using MS
C. neoformans’ isolated melanin granules and the equivalent fraction from nonmelanized material (∼3 mg of each sample) were treated following a protocol described previously (
). Briefly, samples were subjected to in-solution digestion by resuspending in a solubilization buffer (8 m urea, 10% acetonitrile in 100 mm ammonium bicarbonate (pH 8.5)), flash-frozen, and thawed in a water bath at 30 °C for three rounds. Each sample was sonicated briefly on ice (10 s, horned sonicator at maximum amplitude) and mixed using a vortexer. Next, samples were reduced with 10 mm dithiothreitol (DTT) (Sigma) at 51 °C for 1 h and carboxyamido-methylated with 20 mm iodoacetamide (Sigma) for 45 min in the dark at room temperature. Subsequent digestions with endoprotease Lys-C (Roche Diagnostics) and trypsin (1:20 enzyme/protein ratio) for 6 and 10 h, respectively, were performed at room temperature. The reaction was stopped via addition of 10% trifluoroacetic acid (TFA) and incubation for 30 min at 37 °C. Finally, samples were centrifuged for 15 min at 20,800 × g; supernatants were individually transferred to low-protein–binding 1.5-ml tubes (Eppendorf) and completely dried in an acid-resistant CentriVap vacuum concentrator (Labconco). Peptides were resuspended in 40 μl of 0.1% TFA and stored at −80 °C until the mass spectrometric analysis could be performed. Digested samples were cleaned up on an Oasis HLB micro-elution plate and evaporated to dryness. The samples were taken up in 2% acetonitrile, 0.1% formic acid and then vortexed and centrifuged before placing into a 96-well plate. The samples were injected using an EasyLC into a QE-Plus (Thermo Fisher Scientific) mass spectrometer and eluted over a 90-min gradient from 100% buffer A (2% acetonitrile, 0.1% formic acid) to 100% buffer B (90% acetonitrile, 0.1% formic acid) with an MS resolution of 70,000 and an MS2 resolution of 35,000 running a top 15 DDA method. Target values for MS and MS2 were set at 3e6 and 1e5 at 100 and 150 ms, respectively, and a normalized collision energy of 28. Data were searched against the C. neoformans database (Broad Institute, November 2016) using PEAKS version 7 (Bioinformatics Solutions Inc., Waterloo, Ontario, Canada) using a peptide tolerance of 5 ppm and a fragment ion tolerance of 0.02 Da. Enzyme specificity was set to semi-specific trypsin with two missed cleavages allowed and variable carbamidomethyl-C, oxidation M, and deamidation N,Q. Search results were reported at a 1% FDR for all peptides.
In silico predictions
Bioinformatic analyses to identify the presence of signal peptide and a glycosylphosphatidylinositol (GPI) anchor were performed using SignalP version 5.0 (
E. C., R. V., R. J. C., R. E. S., and A. C. conceptualization; E. C., R. V., C. C., R. P.-R., and R. J. C. data curation; E. C., R. V., C. C., R. P.-R., R. J. C., R. N. C., R. E. S., and A. C. formal analysis; E. C., C. C., R. P.-R., R. E. S., and A. C. funding acquisition; E. C., R. V., C. C., R. P.-R., D. G., R. N. O., R. J. C., R. N. C., J. M. M., R. E. S., and A. C. validation; E. C., R. V., C. C., R. P.-R., D. G., R. N. O., R. J. C., R. N. C., and J. M. M. investigation; E. C., R. V., C. C., R. J. C., R. E. S., and A. C. visualization; E. C., R. V., C. C., R. P.-R., D. G., R. N. O., R. J. C., R. N. C., and J. M. M. methodology; E. C. and A. C. writing-original draft; E. C. and A. C. project administration; E. C., R. V., C. C., R. P.-R., R. N. O., R. J. C., R. N. C., J. M. M., R. E. S., and A. C. writing-review and editing; R. J. C., R. N. C., R. E. S., and A. C. supervision.
Acknowledgments
We thank Alexander Idnurm for kindly providing us with C. neoformans strain ST211A, Subhasish Chatterjee for useful discussions during the preliminary stages of this work, Carolina Coelho for critical reading of the manuscript, and Andre Nicola and Alexander Alanio for valuable discussion of experimental designs. We also gratefully acknowledge expertise and technical support from Barbara Smith (Microscopy Facility, School of Medicine, Johns Hopkins University) with transmission EM, SEM, and negative staining. We thank Joel Tang for technical support for data acquisition in connection with EPR analyses. City College of New York is supported by National Institutes of Health Grants PA200A120211 and PA200A150068. The NMR facilities used in this work are operated by City College and the CUNY Institute for Macromolecular Assemblies, with additional infrastructural support provided by Grant 8G12 MD007603 from the National Institute on Minority Health and Health Disparities of the National Institutes of Health.
Down-regulation of the afferent phase of T cell-mediated pulmonary inflammation and immunity by a high melanin-producing strain of Cryptococcus neoformans.
WdChs4p, a homolog of chitin synthase 3 in Saccharomyces cerevisiae, alone cannot support growth of Wangiella (Exophiala) dermatitidis at the temperature of infection.
Secretome profiling of Cryptococcus neoformans reveals regulation of a subset of virulence-associated proteins and potential biomarkers by protein kinase A.
Titan cells formation in Cryptococcus neoformans is finely tuned by environmental conditions and modulated by positive and negative genetic regulators.
Synthesis of sphingolipids with very long chain fatty acids but not ergosterol is required for routing of newly synthesized plasma membrane ATPase to the cell surface of yeast.
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