Polysaccharide capsule export in the fungal pathogen Cryptococcus neoformans

描述(申请人提供)：多糖胶囊出口真菌病原体新生隐球菌摘要新生隐球菌是一种机会性病原体，对因艾滋病或其他疾病而免疫受损的人造成危及生命的疾病。隐球菌的主要毒力因素是围绕其细胞壁的广泛的多糖膜。这种胶囊是真菌致病所必需的。几乎没有关于胶囊成分在细胞内形成的信息，也没有关于它们如何离开细胞并与其表面相关联的信息。在R21的应用中，我们建议开发新的方法来探索这些基本问题，使用尚未应用于新生葡萄球菌的细胞生物学方法。具体地说，我们建议利用我们设计的一种突变菌株来积累含有胶囊相关多糖的分泌囊泡，从而直接调查细胞内胶囊材料的性质。目的建立一种纯化和鉴定含有胶囊前体的分泌囊泡的方法。目的II建议从这些囊泡中分离、纯化与胶囊相关的多糖，并应用分析方法确定其糖链结构。根据胶囊前体的特定性质，实验可能会开始处理与相关多糖相关的任何非碳水化合物成分的存在。实现这些目标的方法包括亚细胞分离、电子显微镜、生化酶分析、免疫检测、差示提取和沉淀、各种层析方法和多糖分析。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这种生物的主要毒力因素的理解中的一个关键差距，在可用于研究新生葡萄球菌的方法上开辟新的天地，并为研究这种重要的病原体开辟令人兴奋的领域。这项研究与公共卫生高度相关，因为被调查的生物体会引起严重的人类疾病，而目前的治疗方法并不充分。待研究的过程是新生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.