A NOVEL GLYCAN SYNTHETIC ACTIVITY IN CRYPTOCOCCUS NEOFORMANS

描述(由申请人提供)：新生隐球菌是一种机会性的病原体，对因艾滋病或其他疾病而免疫功能受损的人造成危及生命的疾病。目前对隐球菌病的治疗是不够的。真菌多糖结构为特定的化疗提供了吸引人的潜在靶点，因为它们与宿主生物中的那些有很大的不同。新生隐孢子虫中的碳水化合物结构修饰蛋白质和脂肪，组成细胞壁的大部分，并形成包裹在细胞壁周围的精致多糖膜，这是毒力所必需的。值得注意的是，囊壁和细胞壁是哺乳动物细胞中完全不存在的关键结构，细胞壁已经被证实是药物靶点。我们在新生葡萄球菌中发现了一种新的糖基化酶，它将木糖-磷酸从UDP-木糖转移到甘露糖残基。这次活动在性质上是史无前例的。在R21的这一应用中，我们提出了该酶在隐球菌生物学中重要的假设，并着眼于新的功能和筛选方法的发展来探索其生化活性。正如目标一所建议的，蛋白质的生化研究将涉及观察到的活性的反应物和动力学，以及关键残留物和改进的分析方法。这种酶的细胞环境将通过对AIM II中的定位和潜在结合伙伴的研究来确定。最后，将使用缺乏这种活性的基因工程细胞来解决这种独特的蛋白质在生物学中的作用。这些方法将与亲本菌株进行比较，以评估AIM III中糖基化和相关表型的变化。方法将包括生化分析、糖链分析、表位标记、成像、代谢放射性标记和多肽分析。这一多方面的策略将阐明一种独特蛋白质的活性，并提供对真菌生物学和多糖合成的基本理解。 公共卫生相关性：这项研究与公共卫生高度相关，因为被调查的生物体会导致严重的人类疾病，而目前的治疗方法并不充分。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的研究和生物学的其他领域。

DESCRIPTION (provided by applicant): Cryptococcus neoformans is an opportunistic pathogen, responsible for life-threatening disease in individuals who are immunocompromised due to AIDS or other conditions. Current therapy for cryptococcosis is inadequate. Fungal glycan structures offer appealing potential targets for specific chemotherapy, because they differ substantially from those found in host organisms. Carbohydrate structures in C. neoformans modify proteins and lipids, compose most of the cell wall, and form an elaborate polysaccharide capsule that surrounds the cell wall and is required for virulence. Notably, the capsule and cell wall are critical structures that are completely absent from mammalian cells, and the cell wall has already been validated as a drug target. We have discovered a novel glycosylating enzyme in C. neoformans, which transfers xylose-phosphate from UDP-xylose to mannose residues. This activity is unprecedented in nature. In this R21 application, we propose to pursue the hypothesis that this enzyme is important in cryptococcal biology, and to explore its biochemical activity with an eye to novel function and the development of screening assays. As proposed in Aim I, biochemical studies of the protein will address the reactants and kinetics of the observed activity, as well as critical residues and improved assays. The cellular context of the enzyme will be determined by studies of localization and potential binding partners in Aim II. Finally, the role of this unique protein in biology will be addressed using cells genetically engineered to lack the activity. These will be compared to parental strains to assess alterations in glycosylation and related phenotypes in Aim III. Methods will include biochemical assays, glycan analysis, epitope-tagging, imaging, metabolic radiolabeling, and peptide analysis. This multi-faceted strategy will elucidate the activity of a unique protein and provide fundamental understanding of fungal biology and glycan synthesis. PUBLIC HEALTH 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. Further, this work will contribute to basic science knowledge, which will impact the study of pathogenic microbes and other areas of biology.