Several enzymes involved in the synthesis of complex sphingolipids have been shown to affect the virulence of Cryptococcus neoformans; however, at this time the enzymes which break down these complex sphingolipids have not been characterized in pathogenic fungi. In this study, we investigated the role of the enzyme inositol phospho sphingolipid-phospholipase C (Isc1), which generates phytoceramide from the hydrolysis of fungal sphingolipids, in the pathophysiology of C. neoformans. We demonstrated that genetic deletion of this enzyme decreased fungal dissemination to the brain in an immunocompetent murine model of cryptococcal infection. Histological observations of pulmonary tissues from infected mice revealed that the Delta isc1 mutant appeared exclusively extracellularly and was more heavily encapsulated compared to a wild type strain. In vitro co-culture studies confirmed the Deltaisc1 mutant had poorer intracellular survival within macrophages compared to control strains with a functional ISC1 gene. The Deltaisc1 mutant was also found to be more sensitive to acidic, oxidative, and nitrosative stresses. We hypothesize that the Deltaisc1 strain is more sensitive to the antifungal effects of macrophages, and selective pressure by the host induces the Delta isc1 mutant to mount stronger antiphagocytic defenses, such as the production of a larger polysaccharide capsule. The larger capsule, while protecting the pathogen from phagocytes, also limits the ability of the Delta isc1 to establish infection in the brain. This is the first study to show that Isc1 plays an important role in pathogenesis by enhancing fungal survival within phagocytes and the first to demonstrate a correlation between alveolar macrophage fungicidal capacity and protection against cryptococcal dissemination to the central nervous system.
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