D-MANNITOL METABOLISM IN CRYPTOCOCCUS NEOFORMANS

新生隐球菌是引起中枢神经系统 艾滋病患者的感染，以及完全令人满意的治疗方法， 和预防这些感染的方法还不存在。 我们已经证明 C.在培养物中，新变型产生大量己糖醇D-甘露醇 在受感染的动物中。 虽然D-甘露醇在C. 目前尚不清楚，大量的能量和 D-甘露醇生物合成所消耗的营养资源表明，D-甘露醇生物合成所消耗的营养资源， 甘露醇（如果功能重要）。 同样，其他真菌也利用D- 甘露醇和其他多元醇，用于储存能量和减少当量， 用于调节细胞内张力、pH和NADPH水平。 D-甘露醇 由C.新生儿也可能贡献更多 通过引起脑水肿（脑水肿的特征）直接导致发病机制。 隐球菌性脑膜炎）或通过清除自由羟基（因此 干扰宿主吞噬细胞的氧化杀伤）。 自从D-甘露醇以来 不是哺乳动物新陈代谢的产物，催化D- 甘露醇代谢新生儿是潜在的治疗靶点。 本项目的总体目标是进行D-甘露醇的基础研究 C.新生儿，特别是因为它涉及到发病机制， 未来可能的治疗方法。 具体目标1是鉴定、纯化和 表征催化D-甘露醇生物合成的酶， 卡地拉C.新人类 具体目标2是测序和分析 编码NAD依赖性D-甘露醇脱氢酶（NAD-MDH或 MPD将通过基因破坏和替换以及突变体来构建 不能利用和/或生产D-甘露醇的药物将由经典的 诱变和选择。 在目标5中，将在目标4中获得的突变体 在表型和酶方面进行表征。 此外，我们将研究 这些突变体i）抵抗宿主吞噬细胞杀伤的能力， 它们的氧化产物和ii）引起脑水肿和/或死亡， 实验感染的动物。

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.