RUI: Mechanisms and physiological functions of bacterial sphingolipids

RUI：细菌鞘脂的机制和生理功能

• 批准号: 2224195
• 负责人: Eric Klein
• 金额: $ 75.66万
• 依托单位: Rutgers University Camden
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2224195&HistoricalAwards=false
• 关键词: RUI; Mechanisms; physiological; functions; bacterial

All cells are surrounded by a membrane which serves as a barrier between the contents of the cell and the outside world. In addition to their barrier function, membranes contribute to the mechanical stability of the cell. A major component of these membranes are lipids; these lipids have water-soluble heads that are oriented towards the outside of the membrane and oil-soluble tails facing the inside. There are numerous types of lipids that vary in their headgroups and tails, and the focus of this research is on sphingolipids. Sphingolipids are found throughout plants, fungi, and animals and were recently shown to be widespread in bacteria as well. Since the pathway for producing sphingolipids in bacteria was only recently discovered, there are many questions remaining about their synthesis and function. The goals of this project are to 1) determine the localization of the sphingolipid synthetic enzymes within bacteria, 2) identify the genes required for transporting the sphingolipids within the cell, and 3) use computational and genetic tools to understand how sphingolipids impact the structure and function of the bacterial outer membrane. The Broader Impacts of this project are to provide students with computational and quantitative skills necessary for modern biological research as well as stimulate excitement in pursuing STEM careers. Students at the LEAP Academy High School in Camden, NJ will have summer research internships in which they will learn bacterial genetics. Undergraduates from underrepresented minority groups will be recruited to the biology honor’s thesis program and graduate students in Computational and Integrative Biology will receive cross-disciplinary training in experimental and computational approaches for studying bacterial membranes.Sphingolipid are found ubiquitously in eukaryotes from fungi, to plants, to animals. By contrast, these lipids have been reported in only a small number of bacterial taxa. Despite the limited number of sphingolipid-producing bacteria, there is a wide range of physiological roles for sphingolipids including modulation of host-microbe interactions, protection from bacteriophage, bacterial life cycle and sporulation, and microbial predation. The recent identification of the complete pathway required for bacterial sphingolipid biosynthesis showed that sphingolipid synthesis is, in fact, far more widespread among bacteria than previously thought. The objectives of this project are to characterize the spatial organization of sphingolipid production and the mechanism of their transport to the outer membrane and computationally investigate how ceramides affect outer membrane functionality and biophysical properties. Specifically, a series of biochemical and genetic analyses will determine whether these enzymes are soluble or membrane-anchored, as well as whether their activity is in the cytoplasm versus the periplasm. The role of LptF/G and LptC homologues will be investigated for their role in sphingolipid transport. Lastly, Molecular Dynamics simulations (MD) will be used to assess lipid orientation, lipid clustering, and interactions between sphingolipids and membrane proteins and antibiotics. The predicted interactions identified by MD will be experimentally validated in C. crescentus. The expected findings will provide a vertical advance in the field of bacterial cell biology by contributing to critical basic research questions related to bacterial membrane synthesis and function.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.

所有细胞都被膜包围，膜作为细胞内容物与外界之间的屏障。除了其屏障功能外，膜还有助于细胞的机械稳定性。这些膜的主要成分是脂质。这些脂质具有面向膜外部的水溶性头部和面向内部的油溶性尾部。脂质有多种类型，其头部和尾部各不相同，本研究的重点是鞘脂。鞘脂存在于植物、真菌和动物中，最近显示在细菌中也广泛存在。由于细菌中产生鞘脂的途径最近才被发现，因此它们的合成和功能仍然存在许多问题。该项目的目标是 1) 确定鞘脂合成酶在细菌内的定位，2) 识别细胞内运输鞘脂所需的基因，3) 使用计算和遗传工具来了解鞘脂如何影响细菌外膜的结构和功能。该项目的更广泛影响是为学生提供现代生物学研究所需的计算和定量技能，并激发他们追求 STEM 职业的热情。新泽西州卡姆登 LEAP 学院高中的学生将进行暑期研究实习，学习细菌遗传学。来自代表性不足的少数群体的本科生将被招募参加生物学荣誉论文项目，计算和综合生物学的研究生将接受研究细菌膜的实验和计算方法的跨学科培训。鞘脂普遍存在于从真菌到植物到动物的真核生物中。相比之下，仅在少数细菌类群中报道了这些脂质。尽管产生鞘脂的细菌数量有限，但鞘脂具有广泛的生理作用，包括调节宿主-微生物相互作用、防止噬菌体、细菌生命周期和孢子形成以及微生物捕食。最近对细菌鞘脂生物合成所需的完整途径的鉴定表明，鞘脂合成实际上在细菌中比以前想象的要广泛得多。该项目的目标是表征鞘脂产生的空间组织及其运输到外膜的机制，并通过计算研究神经酰胺如何影响外膜功能和生物物理特性。具体来说，一系列生化和遗传分析将确定这些酶是可溶的还是膜锚定的，以及它们的活性是否在细胞质中而不是在周质中。将研究 LptF/G 和 LptC 同系物在鞘脂转运中的作用。最后，分子动力学模拟（MD）将用于评估脂质取向、脂质聚类以及鞘脂与膜蛋白和抗生素之间的相互作用。 MD 确定的预测相互作用将在 C. crecentus 中进行实验验证。预期的发现将通过为与细菌膜合成和功能相关的关键基础研究问题做出贡献，为细菌细胞生物学领域带来纵向进步。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。