Molecular architecture of Cryptococcus cell walls reveals species-specific chitosan-dependent remodeling

Abstract Cryptococcus neoformans and Cryptococcus gattii are fungal pathogens that cause life-threatening infections, including cryptococcal meningitis. A distinctive feature of the cryptococcal cell wall is the extensive deacetylation of chitin to chitosan, a modification that is essential for virulence but whose structural role in cell-wall organization remains poorly understood. Here, we analyze the cell walls of wild-type strains of both species and their avirulent chitosan-deficient mutants, which serve as vaccine candidates. Loss of chitosan disrupted cell morphology and altered cell-wall ultrastructure, with more pronounced defects in C. neoformans . Solid-state NMR reveals that aggregated α-(1→3)-glucans form the principal rigid domain of the cell wall in both species and are closely associated with chitin microfibrils, whereas surrounding β-glucans and mannoproteins constitute a more dynamic matrix. Chitosan modulates hydration and flexibility, and its loss increases chitin exposure and triggers species-specific remodeling of the polysaccharide network. In C. neoformans , chitosan depletion increased α-(1→3)-glucan content and reduced β-glucan levels, whereas C. gattii selectively lost one α-(1→3)-glucan subtype while maintaining β-glucan levels. Although capsule production remained intact, chitosan deficiency altered glucuronoxylomannan linkage patterns and mannoprotein composition. These findings reveal how chitosan organizes cryptococcal cell-wall architecture and highlight distinct structural adaptation strategies among pathogenic Cryptococcus species.