Identification of Antifungal Targets Using Proteomics

描述(由申请人提供)： 新生隐球菌是艾滋病患者的一种机会性真菌病原体。新生隐孢子菌是一种担子菌，与大多数其他真菌病原菌存在广泛的差异。最常见的临床表现是脑膜脑炎和肺隐球菌病。现有的针对麦角甾醇途径的抗真菌药物，由于固有的毒性、新出现的真菌耐药性以及终身治疗的要求，不足以继续安全有效的治疗。抗真菌治疗需要新的靶点。在感染过程中差异调节的蛋白质可能在发病机制中是必不可少的，并成为新的抗真菌疗法的良好靶点。在过去的十年里，人们利用分子生物学、遗传学、免疫学和生物化学的方法对新生葡萄球菌的发病机制进行了深入的研究。然而，使用传统方法只发现了少数新的抗真菌靶点。一项资助的新型隐球虫基因组计划使新方法成为可能。我们开发了一个利用二维凝胶电泳和MALDI-TOF分析的新生隐孢子虫蛋白质组系统。我们将重复分离和定量蛋白质，识别受特定条件影响的蛋白质，并确定这些蛋白质的特性。我们建议使用蛋白质组学来识别受特定体外条件影响的新生葡萄球菌蛋白质，这些条件模仿感染过程的一个方面。这些蛋白质将成为进一步研究动物模型中受影响蛋白质的比较的基础。编码这些蛋白质的基因将通过有针对性的基因破坏而突变，并使用小鼠模型测试它们在生存和发病中的作用。

DESCRIPTION (provided by applicant): Cryptococcus neoformans is an opportunistic fungal pathogen in patients with AIDS. C. neoformans is a basidiomycetes widely diverged from most other fungal pathogens. The most common clinical presentations are meningoencephalitis and pulmonary cryptococcosis. Available antifungal agents, directed against the ergosterol pathway, are inadequate for continued safe and effective therapy due to inherent toxicity, emerging fungal resistance, and the requirement for lifelong treatment. New targets for antifungal therapies are needed. Proteins that are differentially regulated during infection may be essential for pathogenesis and be good targets for novel antifungal therapies. Over the past decade, the pathogenesis of C. neoformans has been intensively studied using molecular biology, genetic, immunological and biochemical approaches. However, only a handful of new antifungal targets have been identified using traditional methods. A funded genome project for C. neoformans has made new approaches possible. We have developed a proteomic system for C. neoformans that utilizes 2-dimensional gel electrophoresis and MALDI-TOF analysis. We will reproducibly separate and quantitate proteins, identify proteins that are affected by specific conditions, and determine the identity of those proteins. We propose to use proteomics to identify C. neoformans proteins that are affected by specific in vitro conditions that mimic an aspect of the infection process. These proteins will form the basis for comparison for additional studies examining proteins that are affected in an animal model. The genes encoding these proteins will be mutated by targeted gene disruption and tested for their role in viability and pathogenesis using a mouse model.