Chitosan biosynthesis and virulence in the human fungal pathogenCryptococcus gattii

Abstract

AbstractCryptococcus gattiiR265 is a hyper-virulent fungal strain responsible for the major outbreak of cryptococcosis in Vancouver Island of British Columbia in 1999. It differs significantly fromC. neoformansin its natural environment, its preferred site in the mammalian host, and in the nature and mode of pathogenesis. Our previous studies inC. neoformanshave shown that the presence of chitosan, the deacetylated form of chitin, in the cell wall attenuates inflammatory responses in the host, while its absence induces robust immune responses, which in turn facilitate clearance of the fungus and induces a protective response. The results of the present investigation reveal that the cell wall ofC. gattiiR265 contains 2-3-fold higher amount of chitosan compared to that ofC. neoformans. The genes responsible for the biosynthesis of chitosan are highly conserved in the R265 genome; the roles of the three chitin deacetylases (CDA) have however, been modified. To deduce their roles, single, double and a tripleCDAdeletion strains were constructed in a R265 background and were subjected to mammalian infection studies. UnlikeC. neoformanswhere Cda1 has a discernible role in fungal pathogenesis, in R265 Cda3 is critical for virulence. Deletion of eitherCDA3alone (cda3Δ) or in combination with eitherCDA1(cda1Δ3Δ) orCDA2(cda2Δ3Δ) or both (cda1Δ2Δ3Δ) rendered the yeast cells avirulent and were cleared from the infected host. Moreover, thecda1Δ2Δ3Δstrain of R265 induced a protective response to a subsequent infection with R265. These studies shed more light into the regulation of chitosan biosynthesis ofC. gattiiand its subsequent effect on fungal virulence.ImportanceThe fungal cell wall is an essential organelle whose components provide the first line of defense against host-induced antifungal activity. Chitosan is one of the carbohydrate polymers in the cell wall that significantly affects the outcome of host-pathogen interaction. Chitosan-deficient strains are avirulent, implicating chitosan as a critical virulence factor.C. gattiiR265 is an important fungal pathogen of concern due to its ability to cause infections in individuals with no apparent immune dysfunction and an increasing geographical distribution. Characterization of the fungal cell wall and understanding the contribution of individual molecules of the cell wall matrix to fungal pathogenesis offers new therapeutic avenues for intervention. In this report, we show that theC. gattiiR265 strain has evolved alternate regulation of chitosan biosynthesis under both laboratory growth conditions and during mammalian infection compared to that ofC. neoformans.