Polysaccharide capsule export in the fungal pathogen Cryptococcus neoformans

描述（由申请人提供）：多糖胶囊出口的真菌病原体新型隐球菌摘要新型隐球菌是一种机会致病菌，负责危及生命的疾病，在个人谁是免疫功能低下，由于艾滋病或其他条件。隐球菌的主要毒力因子是围绕其细胞壁的广泛多糖荚膜。这种胶囊是真菌引起疾病所必需的。关于胶囊成分的细胞内形成的信息很少，也没有关于它们如何离开细胞并与其表面结合的信息。在这个R21应用程序中，我们建议开发新的方法来探索这些基本问题，使用尚未应用于C的细胞生物学方法。新人类具体地说，我们建议直接研究细胞内囊物质的性质，利用我们已经工程化的突变株来积累含有囊相关多糖的分泌囊泡。目的研究含有囊泡前体的分泌囊泡的纯化方法。目的二提出从这些囊泡中分离出与囊泡相关的多糖，纯化它们，并应用分析方法来确定它们的聚糖结构。根据胶囊前体的具体性质，实验可能开始以解决与相关聚糖相关的任何非碳水化合物组分的存在。将用于实现这些目标的方法包括亚细胞分级分离、电子显微镜、生化酶测定、免疫检测、差异提取和沉淀、各种色谱法和聚糖分析。这些探索性研究将对该领域产生重大影响，因为它们将解决我们对该生物体主要毒力因子的理解中的关键空白，为研究C.新形式，并打开令人兴奋的领域，为调查这一重要的病原体。/相关性这项研究与公共卫生高度相关，因为所研究的微生物会导致严重的人类疾病，而目前的治疗方法不足以治疗这些疾病。待研究的过程是新型隐球菌致病能力的基础，因此了解和抑制它可能会促进隐球菌感染的治疗。此外，这项工作将有助于基础科学知识，这将影响病原微生物领域以及生物学的其他领域。

DESCRIPTION (provided by applicant): Polysaccharide Capsule Export in the Fungal Pathogen Cryptococcus neoformans ABSTRACT Cryptococcus neoformans is an opportunistic pathogen, responsible for life-threatening disease in individuals who are immunocompromised due to AIDS or other conditions. The main virulence factor of Cryptococcus is an extensive polysaccharide capsule that surrounds its cell wall. This capsule is required for the fungus to cause disease. Little information is available about the intracellular formation of capsule components, and none about how they exit the cell and become associated with its surface. In this R21 application we propose to develop new approaches to exploration of these fundamental questions, using cell biological methods that have not yet been applied to C. neoformans. Specifically, we propose to directly investigate the nature of intracellular capsular material, taking advantage of a mutant strain we have engineered to accumulate secretory vesicles containing capsule- related polysaccharides. Aim I proposes to develop methods to purify and characterize the secretory vesicles that contain capsule precursors. Aim II proposes to isolate capsule-related polysaccharides from these vesicles, purify them, and apply analytical methods to define their glycan structures. Depending on the specific nature of the capsule precursors, the experiments may begin to address the presence of any non-carbohydrate components associated with the relevant glycans. The approaches that will be used to achieve these goals include subcellular fractionation, electron micros- copy, biochemical enzyme assays, immuno-detection, differential extraction and precipitation, a variety of chromatography methods, and glycan analysis. These exploratory studies will have a major impact on the field, as they will address a critical gap in our understanding of this organism's major virulence factor, break new ground in the methods available for study of C. neoformans, and open exciting areas for investigation of this important pathogen. / RELEVANCE This research is highly relevant to public health because the organism being investigated causes serious human illness, for which current therapies are not adequate. The process to be studied is fundamental to the ability of Cryptococcus neoformans to cause disease, so understanding and inhibiting it may advance treatment of cryptococcal infection. Further, this work will contribute to basic science knowledge, which will impact the area of pathogenic microbes as well as other areas of biology.