Identification of Antifungal Targets Using Proteomics

描述（由申请人提供）： 新型隐球菌是艾滋病患者的一种机会致病真菌。C. neoformans是一种广泛不同于大多数其他真菌病原体的担子菌。最常见的临床表现是脑膜脑炎和肺隐球菌病。由于固有毒性、新出现的真菌耐药性和终身治疗的要求，针对麦角固醇途径的现有抗真菌剂不足以持续安全有效的治疗。需要抗真菌治疗的新靶点。在感染过程中差异调节的蛋白质可能是发病机制所必需的，并且是新型抗真菌治疗的良好靶点。近十年来，对C.利用分子生物学、遗传学、免疫学和生物化学方法对新生儿进行了深入研究。然而，只有少数新的抗真菌目标已被确定使用传统的方法。一个资助的C. neoformans使新的方法成为可能。我们建立了一个C.利用二维凝胶电泳和MALDI-TOF分析的新形式。我们将可重复地分离和定量蛋白质，鉴定受特定条件影响的蛋白质，并确定这些蛋白质的身份。我们建议使用蛋白质组学来鉴定C。这些蛋白质受到模拟感染过程的一个方面的特定体外条件的影响。这些蛋白质将构成在动物模型中检查受影响蛋白质的其他研究的比较基础。编码这些蛋白质的基因将通过靶向基因破坏进行突变，并使用小鼠模型测试其在生存力和发病机制中的作用。

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.