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

项目总结/摘要 新型隐球菌是一种机会致病真菌，具有很高的致死率。其主要 毒力因子主要由充分表征的聚糖组成;然而，涉及的酶促步骤 在合成这些聚糖中的作用在很大程度上未知。拟议研究的长期目标是确定 该微生物聚糖合成途径中的未知步骤，由糖基转移酶（GT）介导， 对致病力很重要的基因我建议采取两种相辅相成的办法来实现这一目标。一是 GT基因缺失突变体的毒力将与野生型C进行比较。新形式主义者优先考虑候选人 为了学习缺乏最感兴趣的GT的突变体将进行表型评估，以提示生物学作用 的GT，并分析其聚糖的缺陷，以表明特定的生化作用的GT。同时， 将重组表达感兴趣的GT，并使用新的生物化学方法测定其酶活性。 测定。总之，这些方法将确定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理解，并可能揭示新的药物靶点。通过 成功完成拟议的研究，以及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，培养适合未来学术生涯的专业技能， 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，露丝L. Kirschstein博士前个人国家研究服务奖将使PI 发展成为一个独立的科学家，以符合其使命。

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.