Collaborative Research: Chemical Tools to Elucidate Glycolipid Biosynthesis and Transport in the Corynebacterineae

在化学系生命过程化学(CLP)计划的支持下，中央密歇根大学的本杰明·斯沃茨教授和科罗拉多州立大学的玛丽·杰克逊教授正在开发化学工具来研究一类复杂的糖脂，或糖联脂分子，这些分子存在于棒状杆菌亚目数百种细菌的细胞膜中。这些糖脂被称为磷脂酰肌醇甘露糖苷(PIMs)，在细菌的细胞防御、完整性和免疫活性中发挥着重要作用。虽然PIM和相关分子是棒状杆菌所特有的，并对这些生物独特的生理和致病特性做出了贡献，但这些糖脂在细菌细胞中如何构造和组织的许多方面仍然未知。这样的知识差距仍然存在，因为使用传统技术研究PIM具有挑战性，而且缺乏分析它们的现代工具。该项目寻求开发新的化学生物学工具，以允许在细胞环境中对PIM和相关糖脂进行特定标记和跟踪。这个工具箱的应用可以更好地了解棒状杆菌是如何构建其细胞膜的。从事这项研究的本科生、研究生和博士后研究员将获得有机合成、化学生物学和微生物学的跨学科培训。为了促进跨学科培训和交流，两所大学的学生和博士后将定期举行虚拟会议，讨论该项目。该项目将被整合到一个推广计划中，向社区大学生介绍化学细菌学研究，并鼓励他们在STEM(科学、技术、工程和数学)方面继续教育。磷脂酰肌醇甘露糖苷(PIM)及其高度糖基化的衍生物，脂甘露聚糖(LM)和脂阿拉伯甘露聚糖(LAM)，很难用传统的生物化学和分子生物学技术进行研究，因为它们由高度复杂的非基因编码的脂和糖组成。为了解决这个问题，这个研究项目试图开发基于合成碳水化合物的探针，利用天然和基因工程的代谢途径将化学标签引入活菌中的PIM、LM和LAM。荧光标记的糖脂分子将在整个细胞中进行测试，这可能提供一种在细胞环境中专门跟踪糖脂的方法。该项目中开发的探针有望提供以下信息：(I)活细菌细胞中PIM、LM和LAM结构的时空动力学；以及(Ii)负责PIM跨细菌内膜运输的蛋白质(S)的身份。这个项目的发现有望提供对糖脂生物合成和动力学的更好的理解，这可以为棒状杆菌亚目中的细菌的生理学提供洞察力。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。

With the support of the Chemistry of Life Processes (CLP) program in the Division of Chemistry, Professors Benjamin Swarts of Central Michigan University and Mary Jackson of Colorado State University are developing chemical tools to investigate a class of complex glycolipids, or sugar-linked lipid molecules, that are present in the cell envelope of hundreds of bacterial species from the suborder Corynebacterineae. These glycolipids, which are referred to as phosphatidylinositol mannosides (PIMs), play essential roles in bacterial cellular defense, integrity, and immunological activity. While PIMs and related molecules are unique to the Corynebacterineae and contribute to these organisms’ distinctive physiological and pathogenic characteristics, many aspects of how these glycolipids are constructed and organized in bacterial cells remain unknown. Such knowledge gaps remain because PIMs are challenging to study using traditional techniques and there is a lack of modern tools for analyzing them. This project seeks to develop new chemical biology tools to allow specific tagging and tracking of PIMs and related glycolipids in cellular contexts. Applications of this toolbox could provide a better understanding of how the Corynebacterineae construct their cell envelope. Undergraduate and graduate students and a postdoctoral fellow working on this research will acquire interdisciplinary training in organic synthesis, chemical biology, and microbiology. To promote interdisciplinary training and communication, students and postdocs at both institutions will meet regularly virtually to discuss the project. This project will be integrated into an outreach program to introduce community college students to chemical bacteriology research and to encourage their continued education in STEM (science, technology, engineering and mathematics). The tools generated in this project are expected to be broadly applicable and support other scientists pursuing research on the Corynebacterineae.Phosphatidylinositol mannosides (PIMs) and their highly glycosylated derivatives, lipomannan (LM) and lipoarabinomannan (LAM), are difficult to investigate using traditional techniques of biochemistry and molecular biology because they consist of highly complex, non-genetically encoded lipids and sugars. To address this problem, this research project seeks to develop synthetic carbohydrate-based probes that exploit native and genetically engineered metabolic pathways to introduce chemical tags into PIMs, LM, and LAM in live bacteria. Fluorescently labeled glycolipid molecules will be tested in whole cells, potentially providing a means to track the glycolipids specifically in a cellular context. The probes developed in this project are expected to provide information on (i) the spatiotemporal dynamics of PIM, LM, and LAM construction in live bacterial cells; and (ii) the identity of the protein(s) that is/are responsible for transport of PIMs across the bacterial inner membrane. The findings from this project are expected to provide an improved understanding of glycolipid biosynthesis and dynamics, which could provide insight into the physiology of bacteria in the Corynebacterineae suborder.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.