RUI: Mechanisms and physiological functions of bacterial sphingolipids

所有细胞都被一层膜所包围，该膜是细胞内容物与外部世界之间的屏障。除了屏障功能外，膜还有助于细胞的机械稳定性。这些膜的主要成分是脂类；这些脂类有面向膜外部的水溶性头部和面向内部的油溶尾部。有许多类型的脂在它们的头部和尾部有不同的变化，这项研究的重点是鞘磷脂。鞘脂在植物、真菌和动物中都有发现，最近被证明在细菌中也很普遍。由于在细菌中产生鞘脂的途径是最近才发现的，所以关于它们的合成和功能还存在许多问题。该项目的目标是1)确定鞘脂合成酶在细菌中的定位，2)确定在细胞内运输鞘脂所需的基因，以及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量化技能，并激发学生追求STEM职业的兴奋。新泽西州卡姆登的LEAP学院高中的学生将进行暑期研究实习，在实习中他们将学习细菌遗传学。来自少数族裔的本科生将被招募到生物学荣誉的论文项目中，计算和整合生物学的研究生将接受跨学科的培训，研究细菌膜的实验和计算方法。鞘磷脂广泛存在于真核生物中，从真菌到植物，再到动物。相比之下，这些脂类只在少数细菌类群中被报道过。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，包括调节宿主与微生物的相互作用、保护细菌免受噬菌体的侵袭、细菌生命周期和孢子形成以及微生物的捕食。最近对细菌鞘脂生物合成所需完整途径的鉴定表明，事实上，鞘脂合成在细菌中的分布比之前认为的要广泛得多。该项目的目标是描述神经鞘脂脂产生的空间组织及其向外膜的运输机制，并通过计算研究神经酰胺如何影响外膜功能和生物物理性质。具体地说，一系列的生化和遗传分析将确定这些酶是可溶性的还是膜锚定的，以及它们的活性是在细胞质中还是在周质中。LptF/G和LptC同系物在鞘磷脂转运中的作用将被研究。最后，分子动力学模拟(MD)将被用来评估脂类取向、脂簇以及鞘磷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新月形珊瑚中得到实验验证。这些预期的发现将通过对与细菌膜合成和功能相关的关键基础研究问题的贡献，在细菌细胞生物学领域取得垂直进展。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。

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.