Cryptococcus neoformans is a leading cause of central nervous system infections in AIDS patients, and fully satisfactory methods for treating and preventing these infections are not yet available. We have shown that C. neoformans produces large amounts of the hexitol D-mannitol in culture and in infected animals. Although the functions of D-mannitol in C. neoformans are not yet known, the substantial amounts of energy and nutritional resources expended on D-mannitol biosynthesis suggest that D- mannitol if functionally important. As well, other fungi utilize D- mannitol and other polyols for storing energy and reducing equivalents and for regulating intracellular tonicity, pH and NADPH levels. The D-mannitol produced within infected tissues by C. neoformans may also contribute more directly to pathogenesis by causing brain edema (a characteristic of cryptococcal meningitis) or by scavenging free hydroxyl radicals (thus interfering with oxidative killing by host phagocytes). Since D-mannitol is not a produce of mammalian metabolism, the enzymes that catalyze D- mannitol metabolism in C. neoformans are potential therapeutic targets. The overall goal of this project is to conduct basic studies of D-mannitol metabolism in C. neoformans, especially as it relates to pathogenesis and to possible future therapies.
Specific Aim 1 is to identify, purify and characterize the enzymes that catalyze D-mannitol biosynthesis and catabolism in C. neoformans.
Specific Aim 2 is to sequence and analyze the gene that encodes the NAD-dependent D-mannitol dehydrogenase (NAD-MDH or MPD will be constructed by gene disruption and replacement, and mutants unable to utilize and/or produce D-mannitol will be generated by classical mutagenesis and selection.
In Aim 5, mutants obtained in Aim 4 will be characterized phenotypically and enzymatically. In addition, we will study the abilities of these mutants i) to resist killing by host phagocytes and their oxidative products and ii) to cause brain edema and/or death in experimentally-infected animals.
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