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 nonmelanizing conditions; triglycerides (TGs), sterol esters (SEs), and polyisoprenoids (PPs) were the major constituents. Although no quantitative differences were found between melanized and nonmelanized 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 nonmelanized 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.
Highlights
Cryptococcus neoformans is a globally distributed opportunistic fungal pathogen and the primary causative agent of cryptococcosis, a frequently lethal infectious disease that results in ;180,000 deaths each year worldwide [1]
HRMAS NMR experiments conducted on melanin ghosts swelled with DMSO revealed 1H-13C through-bond connectivities that were surprisingly consistent with triglycerides (TGs)
It first allowed us to implement spectral editing techniques that discriminate between constituents based on variations in molecular motion: by preferentially detecting the signals arising from the mobile lipids and filtering out resonances from relatively rigid moieties such as polysaccharides, we were able to alleviate much of the spectral overlap that can compromise structural analyses in macromolecular biological systems
Summary
Cryptococcus neoformans is a globally distributed opportunistic fungal pathogen and the primary causative agent of cryptococcosis, a frequently lethal infectious disease that results in ;180,000 deaths each year worldwide [1]. C. neoformans cells isolated from individuals with active cryptococcal infections display robust pigment production [7,8,9], an unsurprising outcome considering that melanization is correlated with dissemination of infections and resistance to antifungal therapeutics [10, 11]. For these reasons, the study of C. neoformans melanogenesis can contribute directly to our understanding of the pathogenicity of this organism. Chitin and chitosan are not the only cellular constituents that associate tightly with melanin pigments—a substantial proportion of the nonpigment moieties found in melanin ghosts have been shown to be lipids, with largely unknown identities, origins, and physiological roles [13]
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