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

Abstract

The fungal pathogen Cryptococcus neoformans contains about 200 {micro}g of {beta}-1,3-glucan (1,3BG) per 1 mg of dry cell weight when grown under standard culture conditions (YPD medium at 30 {degrees}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 -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{Delta} by using CRISPR-Cas9 and screened for mutants that failed to evade dectin-1 binding. We found eight deletants (cap59{Delta}/mpk1{Delta}, cap59{Delta}/chs3{Delta}, cap59{Delta}/kre5{Delta}, cap59{Delta}/crz1{Delta}, cap59{Delta}/kre6{Delta}/skn1{Delta}, cap59{Delta}/hxl1{Delta}, cap59{Delta}/uge1{Delta}, and cap59{Delta}/ugt1{Delta}) that exhibited increased binding to dectin-1 or anti-1,3BG antibody, or both. As a similar phenotype was not observed in cap59{Delta}/ags1{Delta}, 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-1{beta} from dendritic cells (DCs) than did cap59{Delta}. 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 {beta}-1,6-glucan and chitosan biosynthesis--are essential for regulating 1,3BG exposure and enabling C. neoformans to evade dectin-1-mediated immune recognition. HighlightsO_LIWe identified new capsule-independent {beta}-1,3-glucan-masking genes in C. neoformans C_LIO_LIGene deletants had higher dectin-1 deposition than parental acapsular cap59{Delta} C_LIO_LIDeletion of these genes enhanced IL-6 and IL-1{beta} secretion by dendritic cells C_LIO_LIThe enhanced cytokine response was suppressed in dendritic cells lacking dectin-1. C_LIO_LIDeletant strains may serve as new whole-cell antigens for cryptococcal vaccines C_LI