Novel cell wall-associated genes that enable Cryptococcus neoformans to evade dectin-1-mediated innate immune recognition

The fungal pathogen Cryptococcus neoformans contains about 200 µg of β-1,3-glucan (1,3BG) per 1 mg of dry cell weight when grown under standard culture conditions (YPD medium at 30 °C under aerobic conditions). However, 1,3BG exposure is tightly suppressed, even in capsule-deficient strains, allowing the fungus to evade recognition by the immune receptor dectin-1 and anti-1,3BG antibodies. Although other pathogenic fungi mask 1,3BG with α-1,3-glucan (1,3AG) to evade dectin-1 recognition, the factors responsible for 1,3BG masking and dectin-1 evasion in C. neoformans remain incompletely understood. To identify capsule-independent 1,3BG masking and dectin-1-evasion factors, we generated a series of cell wall-related gene deletion strains in the capsule-deficient strain cap59 Δ by using CRISPR-Cas9 and screened for mutants that failed to evade dectin-1 binding. We found eight deletants ( cap59 Δ/ mpk1 Δ, cap59 Δ/ chs3 Δ, cap59 Δ/ kre5 Δ, cap59 Δ/ crz1 Δ, cap59 Δ/ kre6 Δ/ skn1 Δ, cap59 Δ/ hxl1 Δ, cap59 Δ/ uge1 Δ, and cap59 Δ/ ugt1 Δ) that exhibited increased binding to dectin-1 or anti-1,3BG antibody, or both. As a similar phenotype was not observed in cap59 Δ/ ags1 Δ, 1,3AG-mediated masking of 1,3BG appears to play a limited role in C. neoformans . These eight deletants induced significantly greater secretion of IL-6 and IL-1β from dendritic cells (DCs) than did cap59 Δ. This enhanced inflammatory response was markedly attenuated in dectin-1-deficient DCs, indicating that the increased immunogenicity was driven by 1,3BG exposure and subsequent dectin-1 recognition. Collectively, these findings demonstrate that multiple genes involved in maintaining cell wall integrity—including those involved in β-1,6-glucan and chitosan biosynthesis—are essential for regulating 1,3BG exposure and enabling C. neoformans to evade dectin-1-mediated immune recognition.