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

该项目的目标是了解密切相关的真菌物种在营养饥饿时基因组的变化，以及这些微妙的基因组差异如何改变每个物种的行为。这项工作研究了硫胺素信号转导（THI）途径在酵母菌种光滑念珠菌，酿酒酵母，和其他相关物种，以确定是否途径功能是可预测的基因组序列。硫胺素（维生素B1）是所有生物体代谢糖所必需的，在大多数微生物中从头合成，并且它是所有动物的饮食需求。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，重点是两个物种-酿酒酵母（或芽殖酵母）和光滑念珠菌。使用这种详细的表征，研究人员将使用这些信息来预测未表征的酵母物种将如何应对硫胺素饥饿。该项目将通过培训现代分子生物学技术来教育和激励下一代科学家。从事该项目的本科生平均将花费两年半的时间进行研究，将对他们的项目负责，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需要硫胺素来生长，不像许多酵母物种可以从头合成它。在硫胺素饥饿期间，S.酿酒酵母使用转录调节因子Thi 2、Thi 3和Pdc 2上调~10个基因（称为THI基因）。C. glabrata缺乏1）硫胺素生物合成途径的一半，2）硫胺素再循环所需的祖先磷酸酶，和3）保守的转录因子Thi 2。由于这些物种在如何获得和合成硫胺素方面存在差异，因此需要详细了解两种物种中的THI途径，以使用基因组序列来预测未表征的测序酵母物种的行为，并更普遍地了解THI途径的进化。本计画将利用启动子截短与融合实验来鉴定硫胺素饥饿反应所必需的DNA元件。在识别出TREs（或硫胺素响应元件）后，研究人员将确定这些TREs是否足以进行硫胺素饥饿调节;将使用SEL-seq方法来识别TREs的序列决定簇;使用ChIP-seq来确认Pdc 2与TREs的结合;并确定两种物种S的调节共性。cerevisiae和C.光滑的在对这两个物种进行详细描述后，研究人员将重新构建C。glabrata途径进入S.酿酒酵母（和相反-S.酿酒酵母THI途径进入C. glabrata）。这种重建将使研究人员能够验证THI基因调控所需的所有成分都已鉴定，并测试其他进化相关物种（专注于Nakaseomyces进化枝）结果的预测能力，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物种1）可以合成硫胺素，2）改变其回收硫胺素的能力，和3）在硫胺素不存在的情况下，可能已经找到了其他进化的生存解决方案。该奖项反映了NSF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进行评估，被认为值得支持的搜索.

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.