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其基因组中有多大程度上因营养饥饿而发生变化，以及这些细微的基因组差异如何导致每个物种的行为发生变化。这项工作研究了硫胺素信号转导途径(THI)在酵母物种光滑假丝酵母、酿酒酵母和其他相关物种中的作用，以确定该途径的功能是否可以从基因组序列预测。硫胺素(维生素B1)是所有生物代谢糖所必需的，在大多数微生物中是从头合成的，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，包括本科生和硕士研究生，将详细描述维生素B1是如何在不同的酵母物种中合成的，重点是两个物种--酿酒酵母(或萌芽酵母)和光滑假丝酵母。利用这种详细的特性，研究人员将利用这些信息来预测未确定特性的酵母物种将如何应对硫胺素饥饿。该项目将通过对下一代科学家进行现代分子生物学技术培训来教育和激励他们。从事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，因为硫胺素对生长是必不可少的，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。光滑假单胞菌需要为生长提供硫胺素，不像许多酵母物种可以从头合成硫胺素。在硫胺饥饿过程中，酿酒酵母利用转录调控因子Thi2、Thi3和Pdc2上调~10个基因(称为Thi基因)。光滑毛藻缺少1)硫胺素生物合成途径的一半，2)硫胺素循环所需的祖先磷酸酶，以及3)保守的转录因子Thi2。由于这两个物种在获取和合成硫胺素的方式上存在差异，因此需要详细了解这两个物种中的Thi途径，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，并更全面地了解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来鉴定硫胺素饥饿反应所必需的DNA元件。在确定了TRES(或硫胺素反应元件)后，研究人员将确定这些TRE是否足以调节硫胺饥饿；将使用SEL-SEQ方法来鉴定TRES的序列决定因素；使用CHIP-SEQ来确认Pdc2与TRES的结合；并确定两个物种酿酒酵母和光滑链球菌调控的共同点。在对这两个物种进行了详细的鉴定后，研究人员将重建光面葡萄球菌进入酿酒酵母的途径(以及反向-酿酒酵母进入光面葡萄球菌的途径)。这种重组将允许研究人员验证调控Thi基因所需的所有成分已经被识别，并在其他进化相关物种(重点是Nakasemycesclade)中测试结果的预测能力，这些物种只知道DNA序列。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，2)它们回收硫胺的能力发生了变化，3)可能已经找到了在没有硫胺的情况下生存的其他进化解决方案。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.