Solid-state NMR Reveals the Carbon-based Molecular Architecture of Cryptococcus neoformans Fungal Eumelanins in the Cell Wall

Abstract

Background: Melanin is a poorly understood fungal virulence factor. Results: 2D 13 C-13 C correlation solid-state nuclear magnetic resonance reveals the carbon-based molecular architecture of intact melanin pigment assemblies in Cryptococcus neoformans. Conclusion: Polysaccharide cell-wall components form a scaffold for layered deposition of aromatic-based pigment assemblies. Significance: Deciphering macromolecular interactions that drive melanin pigment assembly in fungal cell walls facilitates the development of drug delivery materials.

The structural unit of melanin in the cell wall of the fungal pathogenCryptococcus neoformans

“…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 ∆LAC1,2 strain grown in MM with L-DOPA (L-DOPA aggregates and vesicles) nor for the fungal cells grown in MM lacking L-DOPA (vesicles) (Figure 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 (15,16). We observed that melanin granules had lower dispersity than autopolymerized L-DOPA ( Figure 4B).…”

“…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 (21,49). We hypothesized that these particles were held together by non-pigment cellular components, a supposition supported by NMR studies showing that C. neoformans ghosts contain tightly associated lipid moieties and cellwall polysaccharides such as chitin (16,19). Since 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.…”

“…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 (11)(12)(13). Consequently, it is possible to selectively label the melanin polymer with NMRor radioactive isotopes, a property that has been exploited for melanin structural studies (14)(15)(16)(17)(18)(19). 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 (20).…”

“…Studies using isotopically-labeled precursors in conjunction with high resolution solid-state nuclear magnetic resonance (ssNMR), revealed that the melanin polymer is likely to be covalently bonded to cell-wall chitin and also found strongly associated with other non-pigment 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.…”

“…Investigation of the melanin structure is a notoriously challenging task due to the insoluble, amorphous, and heterogeneous nature of this architecturally complex polymer (41). Despite these challenges, over the past two decades a wealth of molecular level structural information has been uncovered using multiple nondestructive methodologies among them: X-ray scattering, ssNMR, electron microscopy, and atomic force microscopy (16,(42)(43)(44)(45)(46). Current views of eumelanin molecular architecture posit that these pigments are composed of a common chemical motif and arranged as graphite-like πstacked planar sheets of locally ordered monomers, mainly derivatives of 5,6dihydroxyindole (DHI) (47,48), which give rise to subunit nanoparticles that then undergo spontaneous aggregation until submicron particles are formed (45,46).…”