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Title	Regulation of the virulance of Cryptococcus neoformans by the sphingolipid pathway : a novel signaling cascade mediated by diacylglycerol (DAG) and protein kinase C1 (Pkc1)
Author	Heung, Lena Jachuan
Date	2005
Affiliation/College	College of Graduate Studies
Department	Biochemistry and Molecular Biology
Extent	ix, 87 leaves ill.; 29 cm
Source	QU 141 H593r 2005
Subject.MeSH	Academic Dissertations
Collection	MUSC Theses and Dissertations
Contributing Institution	Waring Historical Library (MUSC)
Rights	Copyright is held by the author.
Language	English
Type	Text
Format	application/pdf
Media Type	Manuscripts
Resource Identifier	mtd2005_heung_lj
Abstract	The sphingolipid biosynthetic pathway generates bioactive molecules crucial to the regulation of mammalian and fungal physiological and pathobiological processes. In previous studies (Luberto, C., Toffaletti, D. L., Wills, E. A., Tucker, S. C., Casadevall, A., Perfect, J. R. Hannun, Y. A., and Del Poeta, M. (2001) Genes Dev. 15, 201-212), it was demonstrated that an enzyme of the fungal spQingolipid pathway, inositolphosphoryl ceramide synthase 1 (Ipcl), regulates activity of the cell wall-associated enzyme laccase which produces melanin, a black pigment required for the pathogenic fungus Cryptococcus neoformans to cause disease. The goal of the present study was to investigate the mechanism by which Ipc 1 regulates melanin production. Because Ipc 1 catalyzes the production of diacylglycerol (DAG), a physiological activator of the classical and novel isoforms of mammalian protein kinase C (PKC), and since it has been suggested that PKC is required for melanogenesis in mammalian cells, it was examined whether Ipcl regulates melanin in C. neoformans through the production ofDAG and the subsequent activation ofPkcl, the fungal homolog of mammalian PKC. The results show that modulation ofIpcl regulated the levels ofDAG in C. neoformans cells. Next, C. neoformans Pkc 1 was demonstrated to be a DAG-activated kinase, and inhibition of Pkc 1, by both pharmacological and genetic approaches, abolished melanin formation inc. neoformans. To explore the mechanism by which Pkcl is regulated by DAG, the putative DAG-binding Cl domain was deleted from C. neoformans Pkcl. The Cl deletion not only abolished the activation ofPkcl by DAG, but also repressed laccase activity and melanin production. Finally, the data indicate that the biological effects observed in the C 1 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 the regulation of Pkc 1 and laccase by the sphingolipid pathway in C. neoformans, with considerable implications for understanding and targeting the Ipcl-DAG-Pkcl-Iaccase cascade as a regulator of virulence of this important human pathogen.

Regulation of the virulance of Cryptococcus neoformans by the sphingolipid pathway : a novel signaling cascade mediated by diacylglycerol (DAG) and protein kinase C1 (Pkc1)

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