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RUI: Characterization of thiamine signal transduction pathway in Candida glabrata and other Ascomycetes

RUI：光滑念珠菌和其他子囊菌中硫胺素信号转导途径的表征

基本信息

批准号：
1921632
负责人：
Dennis Wykoff
金额：
$ 56.1万
依托单位：
Villanova University
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-01 至 2023-07-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1921632&HistoricalAwards=false
关键词：
RUI Characterization thiamine signal transduction

项目摘要

The goal of this project is to understand how closely related fungal species have changes in their genome specific to nutrient starvation, and how those subtle genomic differences allow for changes in each species' behavior. This work studies the thiamine signal transduction (THI) pathway in the yeast species Candida glabrata, Saccharomyces cerevisiae, and other related species, to determine if pathway function is predictable from genomic sequence. Thiamine (vitamin B1) is required by all living organisms to metabolize sugars, is synthesized de novo in most microorganisms, and it is a dietary requirement in all animals. In this project, researchers at Villanova University, including undergraduate and Master's students, will characterize in detail how vitamin B1 is synthesized in various yeast species, with a focus on two species - Saccharomyces cerevisiae (or budding yeast) and Candida glabrata. Using this detailed characterization, the investigators will then use this information to develop predictions as to how uncharacterized yeast species will behave in response to thiamine starvation. This project will educate and motivate the next generation of scientists by training them in modern molecular biological techniques. Undergraduates working on this project will, on average, spend over two and a half years working on their research, will take responsibility for their projects and present their data at professional meetings and in scientific journals, giving them a rigorous foundation to continue STEM studies after graduation. In the long-term, because thiamine is essential for growth, gaining a deep understanding of how thiamine is made and acquired will be critical for the development of interventions to prevent fungal growth.C. glabrata requires thiamine to be supplied for growth, unlike many yeast species that can synthesize it de novo. During thiamine starvation, S. cerevisiae upregulates ~10 genes (called THI genes) using the transcriptional regulators Thi2, Thi3, and Pdc2. C. glabrata lacks 1) one half of the thiamine biosynthetic pathway, 2) the ancestral phosphatase required for thiamine recycling, and 3) the conserved transcription factor Thi2. Because these species have differences in how they acquire and synthesize thiamine, a detailed understanding of the THI pathway in both species is required to use genomic sequence to predict behaviors of uncharacterized, sequenced yeast species, and to understand the evolution of the THI pathway more generally. This project will identify DNA elements essential for the thiamine starvation response using promoter truncation and promoter fusion experiments. After identification of TREs (or thiamine responsive elements), the researchers will determine whether these TREs are sufficient for thiamine starvation regulation; will use a SEL-seq approach to identify the sequence determinants of the TREs; use ChIP-seq to confirm Pdc2 binding to the TREs; and determine the commonalities of regulation with the two species S. cerevisiae and C. glabrata. After a detailed characterization of these two species, the researchers will reconstitute the C. glabrata pathway into S. cerevisiae (and the reverse - the S. cerevisiae THI pathway into C. glabrata). This reconstitution will allow investigators to validate that all components necessary for regulation of THI genes have been identified, and to test the predictive power of the results in other evolutionarily related species (focusing on the Nakaseomyces clade) where only DNA sequence is known. When this work is completed, researchers should be able to predict which yeast species 1) can synthesize thiamine, 2) are altered in their ability to recycle thiamine, and 3) may have come to other evolutionary solutions to surviving when thiamine is not present.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

这个项目的目标是了解密切相关的真菌物种在它们的基因组中对营养饥饿的特异性变化，以及这些微妙的基因组差异如何允许每个物种的行为发生变化。本研究研究了念珠菌（Candida glabrata）、酿酒酵母（Saccharomyces cerevisiae）和其他相关物种的硫胺素信号转导（THI）途径，以确定该途径的功能是否可以从基因组序列中预测。硫胺素（维生素B1）是所有生物代谢糖所必需的，在大多数微生物中都是从头合成的，也是所有动物的膳食需求。在这个项目中，维拉诺瓦大学的研究人员，包括本科生和硕士生，将详细描述维生素B1是如何在各种酵母物种中合成的，重点是两种物种-酿酒酵母（或出芽酵母）和面露假丝酵母。利用这些详细的特征，研究人员将利用这些信息来预测未被描述的酵母物种对硫胺素饥饿的反应。这个项目将通过训练下一代科学家掌握现代分子生物学技术来教育和激励他们。参与该项目的本科生平均将花费超过两年半的时间进行研究，将负责他们的项目，并在专业会议和科学期刊上展示他们的数据，为他们毕业后继续STEM研究奠定坚实的基础。从长远来看，由于硫胺素对生长至关重要，因此深入了解硫胺素是如何产生和获得的，对于开发预防真菌生长的干预措施至关重要。Glabrata需要提供硫胺素来生长，不像许多酵母菌可以从头合成硫胺素。在硫胺素饥饿期间，酿酒酵母通过转录调控因子Thi2、Thi3和Pdc2上调约10个基因（称为THI基因）。C. glabrata缺乏1)一半的硫胺素生物合成途径，2)硫胺素循环所需的祖传磷酸酶，3)保守的转录因子Thi2。由于这些物种在获取和合成硫胺素的方式上存在差异，因此需要对这两个物种的THI途径进行详细的了解，以便使用基因组序列来预测未表征的、已测序的酵母物种的行为，并更广泛地了解THI途径的进化。该项目将通过启动子截断和启动子融合实验确定硫胺素饥饿反应所需的DNA元素。在鉴定出TREs（或硫胺素响应元件）后，研究人员将确定这些TREs是否足以调节硫胺素饥饿；将使用SEL-seq方法来确定TREs的序列决定因素；使用ChIP-seq确认Pdc2与TREs的结合；并确定了酿酒葡萄和光叶葡萄两种间的调节共性。在对这两个物种进行详细表征后，研究人员将重建C. glabrata途径到S. cerevisiae（反过来- S. cerevisiae THI途径到C. glabrata）。这种重组将使研究人员能够验证THI基因调控所需的所有成分已经被确定，并测试结果在其他进化相关物种（重点是Nakaseomyces分支）中的预测能力，其中只有DNA序列是已知的。当这项工作完成后，研究人员应该能够预测哪些酵母菌种类1)可以合成硫胺素，2)改变了它们循环利用硫胺素的能力，以及3)当硫胺素不存在时，它们可能会找到其他的进化解决方案来生存。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。