Solid-state NMR spectroscopy identifies three classes of lipids inC. neoformansmelanized cell walls and whole fungal cells

Abstract

A primary virulence-associated trait of the opportunistic fungal pathogen Cryptococcus neoformans is the production of melanin pigments that are deposited into the cell wall and interfere with the host immune response. Previously, our solid-state NMR studies of isolated melanized cell walls (melanin ‘ghosts’) revealed that the pigments are strongly associated with lipids, but their identities, origins, and potential roles were undetermined. Herein, we exploited spectral editing techniques to identify and quantify the lipid molecules associated with pigments in melanin ghosts. The lipid profiles were remarkably similar in whole C. neoformans cells, grown under either melanizing or non-melanizing conditions; triglycerides (TGs), sterol esters (SEs) and polyisoprenoids (PPs) were the major constituents. Although no quantitative differences were found between melanized and non-melanized cells, melanin ghosts were relatively enriched in SEs and PPs. In contrast to lipid structures reported during early stages of fungal growth in nutrient-rich media, variants found herein could be linked to nutrient stress, cell aging, and subsequent production of substances that promote chronic fungal infections. The fact that TGs and SEs are the typical cargo of lipid droplets suggests that these organelles could be connected to C. neoformans melanin synthesis. Moreover, the discovery of PPs is intriguing because dolichol is a well-established constituent of human neuromelanin. The presence of these lipid species even in non-melanized cells suggests that they could be produced constitutively under stress conditions in anticipation of melanin synthesis. These findings demonstrate that C. neoformans lipids are more varied compositionally and functionally than previously recognized.

Unconventional Constituents and Shared Molecular Architecture of the Melanized Cell Wall of C. neoformans and Spore Wall of S. cerevisiae

“…The absence of signals from the DOPA-derived pigment allows the unambiguous identification of the cellular constituents that make up this melanin scaffold. In particular, the signals shaded in pink have been shown to arise from chitin and chitosan polysaccharides [ 21 ], whereas those shaded in yellow have been attributed to the methyl and methylene carbons within the fatty acyl chains of triglycerides [ 27 ].…”

“…This strategy serves to spread the spectral features into a second spectroscopic dimension and moreover, to identify nearby pairs of carbons within the macromolecular assemblies. Whereas C. neoformans melanin ghosts have been studied extensively using a variety of homo- and heteronuclear multi-dimensional ssNMR techniques [ 24 , 27 , 42 , 44 , 45 ], only 1D 13 C ssNMR data have been reported for S. cerevisiae spore walls [ 34 ]. Thus, the comprehensive identifications made previously for melanin ghosts were used to guide spectroscopic analysis of the 2D DARR results for spore walls.…”

“…The final striking similarity witnessed between these 2D spectra pertains to a group of cross-peaks that, in C. neoformans melanin ghosts, have been determined to arise from proximal carbon correlations within triglycerides that have unsaturated fatty acid (FA) acyl chains [ 27 ]. Although the diagnostic triglyceride glycerol carbon peaks (~62 and 69 ppm) are obscured in this experiment by overlap with polysaccharide ring carbon signals, several key cross-peaks that are hallmarks of unsaturated FAs are clearly identifiable in both DARR spectra [ 51 , 52 ].…”

“…Loss of this enzyme, which synthesizes a lipid-droplet localized pool of polyisoprenoids, results in loss of the chitosan and dityrosine layers of the spore wall [ 70 ]. This property provides an additional link between the lipid droplets and spore wall assembly, and it might also offer an additional parallel between spore walls and melanin ghosts; although polyisoprenoids are not evident in our ssNMR spectra of the S. cerevisiae outer spore wall, they have recently been identified in C. neoformans melanin ghosts through the implementation of ssNMR experiments that select for mobile constituents such as lipids [ 27 , 71 ].…”

“…A variety of ssNMR techniques have been employed to analyze these samples, demonstrating that the melanin pigments produced by C. neoformans are indeed strongly associated with both chitin and chitosan [ 21 ]. However, the pigments are not exclusively attached to polysaccharides; several other cellular constituents have also been identified in this melanization scaffold, including substantial quantities of neutral lipids, which are primarily triglycerides [ 27 ].…”