Discerning the roles of novel glycosyltransferases in the pathogenic yeast Cryptococcus neoformans

项目摘要/摘要 新生隐球菌是一种致死率较高的机会性真菌病原体。它的主要特点 毒力因子主要由具有良好特性的多聚糖组成；然而，涉及的酶步骤 这些多糖的合成在很大程度上是未知的。拟议研究的长期目标是确定 在糖基转移酶(GTS)的介导下，这种生物体的糖合成途径中的未知步骤， 对致死性很重要。我提出两种相辅相成的方法来实现这一目标。首先， GT基因缺失突变体的毒力将与野生型新生葡萄球菌的毒力进行比较，以确定候选菌株的优先顺序 为了学习。缺乏GT的突变体将被评估表型，以暗示生物学作用 对GT及其多糖的缺陷进行分析，以表明GT的特定生化作用。同时， 将重组表达感兴趣的GTS，并使用新的生化方法测定其酶活性 化验。这些方法将共同定义GT在毒力中的重要作用，有助于 我们对这种病原体的基本生物学的了解，并可能揭示新的药物靶点。通过 成功完成拟议的研究，加上额外的正式和非正式培训，PI将 熟练掌握分子微生物学、糖生物学和蛋白质生物化学。此外，PI 将通过在研讨会上展示原创作品来培养适应未来学术界职业生涯的专业技能， 会议，并以书面形式出版。通过帮助她参加这些博士前培训 机会，Ruth L.Kirschstein博士前个人国家研究服务奖将使PI 作为一名独立的科学家发展，与其使命保持一致。

PROJECT SUMMARY/ABSTRACT Cryptococcus neoformans is an opportunistic fungal pathogen with a high mortality rate. Its major virulence factors are primarily composed of well-characterized glycans; however, the enzymatic steps involved in synthesizing these glycans are largely unknown. The long-term objective of the proposed research is to define unknown steps in glycan synthesis pathways of this organism, mediated by glycosyltransferase enzymes (GTs), that are important for virulence. I propose two complementary approaches to achieve this objective. First, the virulence of GT gene deletion mutants will be compared to that of wild-type C. neoformans to prioritize candidates for study. Mutants lacking GTs of greatest interest will be assessed phenotypically, to suggest biological roles of the GTs, and their glycans analyzed for defects, to suggest specific biochemical roles of the GTs. In parallel, GTs of interest will be recombinantly expressed and their enzymatic activity determined using novel biochemical assays. Together, these approaches will define the roles of GTs that are important in virulence, contributing to our understanding of the basic biology of this pathogen and potentially revealing novel drug targets. Through the successful completion of the proposed research, together with additional formal and informal training, the PI will become technically adept in molecular microbiology, glycobiology, and protein biochemistry. In addition, the PI will cultivate professional skills tailored to a future career in academia by presenting original work at seminars, conferences, and in written form for publication. By facilitating her participation in these predoctoral training opportunities, the Ruth L. Kirschstein Predoctoral Individual National Research Service Award will enable the PI to develop as an independent scientist, in keeping with its mission.