Elucidating the membrane properties regulating antimicrobial peptidebinding to bacterial vesicles

项目摘要 抗菌肽(AMPs)，如LL-37，已被认为是潜在的下一代 抗生素由于其结合和破坏细菌细胞膜的能力。然而，它们的使用有 受到几个因素的限制，包括与细菌外膜囊泡结合而使其失活 (OMVS)。OMVS来源于革兰氏阴性细菌的外膜，因此 具有与OM相似的膜成分。OMV在物理上是异类的，并且 化学性质，包括大小、电荷、脂肪和蛋白质组成，即使在OMV中也是如此 由同一亲本细菌产生的。研究蛋白质/肽相互作用的传统方法 OMV使用批量测量，其结果是可能掩盖OMV影响的集合平均 异质性对这些互动产生了影响。我们已经开发了几种方法，使我们能够研究 在单囊泡和亚群水平上的OMV中的蛋白质-脂质相互作用，我们将在这里使用 该项目旨在揭示调节LL-37与OMV结合的特定属性。我们将首先使用脂质体 用受控成分确定囊泡大小、电荷、脂饱和度和脂多糖的作用 在LL-37绑定中的组成。接下来，我们将使用不同大小和费用的OMV来关联LL-37 亲和力与OMVS保护细菌细胞免受LL-37活性的能力有关。最后，我们将调查 当细菌暴露于LL-37时，OMV的特性如何变化以及这些变化是如何变化的 影响IL-37与OMV的结合。本科生和研究生将高度参与所有 建议的研究，调查人员致力于指导这些学生最大限度地提高他们的 培养技术、批判性思维和沟通能力。我们预计结果会是 从这个项目产生的将为未来的设计提供更有效的AMP。

Project Summary Antimicrobial peptides (AMPs), such as LL-37, have been proposed as potential next-generation antibiotics due to their ability to bind to and disrupt the bacterial cell membrane. However, their use has been limited by several factors, including inactivation by binding to bacterial outer membrane vesicles (OMVs). OMVs are derived from the outer membrane (OM) of Gram-negative bacteria and therefore have a similar membrane composition as the OM. OMVs are heterogeneous in their physical and chemical properties, including size, charge, and lipid and protein composition, even among OMVs produced by the same parent bacterium. Traditional methods to study protein/peptide interactions with OMVs use bulk measurements, which result in ensemble averages that can obscure the influence OMV heterogeneity has on these interactions. We have developed several approaches that enable us to study protein-lipid interactions in OMVs on single-vesicle and subpopulation levels, which we will use in this project to uncover the specific properties that regulate LL-37 binding to OMVs. We will first use liposomes with controlled compositions to identify the roles of vesicle size, charge, lipid saturation, and LPS composition in LL-37 binding. Next, we will use OMVs with varying sizes and charges to relate LL-37 affinity to the OMVs’ ability to protect bacterial cells from the activity of LL-37. Finally, we will investigate how the properties of OMVs change upon exposure of the bacteria to LL-37 and how these changes affect LL-37 binding to OMVs. Undergraduate and graduate students will be highly involved in all of the proposed studies, and the Investigators are committed to mentoring these students to maximize their development of technical, critical thinking, and communication skills. We anticipate that the results generated from this project will inform the future design of more effective AMPs.