D-MANNITOL METABOLISM IN CRYPTOCOCCUS NEOFORMANS

新生隐球菌是中枢神经系统的主要致病因素 艾滋病患者的感染，以及完全令人满意的治疗方法 目前还没有预防这些感染的方法。我们已经证明了 新生葡萄球菌在培养中产生大量己糖醇D-甘露醇 在感染的动物身上也是如此。虽然D-甘露醇在C. 新生物尚不为人所知，大量的能量和 花费在D-甘露醇生物合成上的营养资源表明，D-甘露醇 甘露醇如果功能重要的话。同样，其他真菌也利用D- 甘露醇和其他用于储存能量和减少当量的多元醇和 用于调节细胞内张力、pH和NADPH水平。D-甘露醇 新生假单胞菌在感染组织中产生的作用也可能更大。 通过引起脑水肿直接致病(脑水肿的一个特征 隐球菌性脑膜炎)或通过清除自由基(因此 干扰宿主吞噬细胞的氧化杀死)。由于D-甘露醇 不是哺乳动物新陈代谢的产物，这种酶催化D- 新生假单胞菌的甘露醇代谢是潜在的治疗靶点。 该项目的总体目标是进行D-甘露醇的基础研究 新生葡萄球菌的代谢，特别是与发病机制和 未来可能的治疗方法。具体目标1是识别、提纯和 表征催化D-甘露醇生物合成的酶和 新生葡萄球菌的分解代谢。具体目标2是测序和分析 编码依赖NAD的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-甘露醇将由经典的 诱变和选择。在目标5中，在目标4中获得的突变体将是 表型和酶学特征。此外，我们还将研究 这些突变体的能力I)抵抗宿主吞噬细胞的杀伤和 他们的氧化产物和ii)导致脑水肿和/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.