Abstract
Cryptococcus species are major fungal pathogens responsible for life-threatening infections in approximately a million individuals globally each year, with alarmingly high mortality rates. These fungi are distinguished by a distinctive cell wall architecture further reinforced by two virulence-associated layers, melanin and capsule, rendering them insensitive to antifungal agents targeting the cell wall, such as echinocandins. The molecular interplay between these three biomolecular layers remains poorly understood. Here, we employ solid-state NMR spectroscopy to examine intact cells of both wild-type and capsule-deficient strains of C. neoformans, along with melanized cells. High-resolution (13)C and (1)H data revealed five distinct structural forms of α-1,3-glucans that play versatile roles in forming the rigid cell wall scaffold by interacting with chitin microfibrils and chitosan, and in stabilizing the mobile matrix by associating with β-1,6-glucan and a small fraction of β-1,3-glucan. Two primary forms of α-1,3-glucans were distributed throughout the wall, capable of hosting melanin deposition in the inner domain and capsule attachment on the cell surface. These findings offer a paradigm shift in understanding the cryptococcal cell wall and its interaction with two key virulence factors on opposite sides, raising critical biochemical questions that could inform the development of more effective antifungal treatments for cryptococcosis.