Abstract
The fungus Cryptococcus neoformans is an opportunistic human pathogen that causes a life-threatening meningoencephalitis by expression of virulence factors such as melanin, a black pigment produced by the cell wall-associated enzyme laccase. In previous studies (Heung, L. J., Luberto, C., Plowden, A., Hannun, Y. A., and Del Poeta, M. (2004) J. Biol. Chem. 279, 21144-21153) we proposed that the sphingolipid enzyme inositol-phosphoryl ceramide synthase 1 (Ipc1) regulates melanin production through the generation of diacylglycerol (DAG), which was found to activate in vitro protein kinase C1 (Pkc1). Here, we investigated the molecular mechanisms by which DAG regulates Pkc1 in vivo and the effect of this regulation on laccase activity and melanin synthesis. To this end we deleted the putative DAG binding C1 domain of C. neoformans Pkc1 and found that the C1 deletion abolished the activation of Pkc1 by DAG. Deletion of the C1 domain repressed laccase activity and, consequently, melanin production. Finally, we show that these biological effects observed in the C1 deletion mutant are mediated by alteration of cell wall integrity and displacement of laccase from the cell wall. These studies define novel molecular mechanisms addressing Pkc1-laccase regulation by the sphingolipid pathway of C. neoformans, with important implications for understanding and targeting the Ipc1-Pkc1-laccase cascade as a regulator of virulence of this important human pathogen.
Highlights
Cryptococcus neoformans is a fungal pathogen that infects humans via the respiratory tract
Lipids Modulated by inositolphosphoryl ceramide synthase 1 (Ipc1) Regulate Activation of C. neoformans protein kinase C1 (Pkc1)—Because our previous studies indicated that Ipc1 modulates DAG and phytoceramide levels in C. neoformans cells and that Pkc1 activity is regulated by DAG and phytoceramide in vitro (15), we investigated whether modulation of Ipc1 in C. neoformans cells regulates the activity of Pkc1
When Pkc1 was treated with lipids from cells in which Ipc1 was down-regulated (GAL7::IPC1 grown on glucose), kinase activity decreased by 30.7% relative to treatment with lipids from the wild type cells grown in glucose (p Ͻ 0.05)
Summary
Cryptococcus neoformans is a fungal pathogen that infects humans via the respiratory tract. A recent study by Noverr et al (8) has demonstrated that laccase is required for the dissemination of C. neoformans from the lung to the brain, suggesting a role for melanin in the establishment of infection and in the progression of disease. The putative Ipc1-DAG-Pkc pathway is the first evidence that the sphingolipid pathway is crucial to the regulation of fungal virulence. Whereas biochemical data suggests that C. neoformans Pkc is activated by DAG (15), fungal Pkc homologs from S. cerevisiae and Candida albicans are not regulated by DAG (20 –22). Mammalian PKC isoforms regulated by DAG have conserved residues in the C1 domain required for DAG activation (Pro, Gly, and Gln27) as well as five hydrophobic residues surrounding the DAG-binding site (positions 8, 13, 20, 22, and 24) (23). The three DAG-binding site residues and four of the five hydrophobic residues are
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