Abstract
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.
Highlights
Melanins are synthesized macromolecules that are found in all biological kingdoms
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 [16, 19]
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
Summary
The structural unit of melanin in the cell wall of the fungal pathogen Cryptococcus neoformans. 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. 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 cellwall chitin and was found strongly associated with other nonpigment cellular moieties, including polysaccharides such as chitosan and vesicle and/or plasma membrane-derived lipids [16, 19]. These components serve as the scaffold for melanin synthesis. We have 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
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