Evaluating the Bilayer-Couple Model of Outer Membrane Vesicle Biogenesis Using Novel Asymmetric Membrane Templates

使用新型不对称膜模板评估外膜囊泡生物发生的双层耦合模型

基本信息

批准号：
9199067
负责人：
Jeffrey Schertzer
金额：
$ 18.06万
依托单位：
STATE UNIVERSITY OF NY,BINGHAMTON
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-01-01 至 2018-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9199067
关键词：
Address Antibiotic Resistance Architecture Area Bacteria Biochemical Biogenesis Biological Biophysical Process Caliber Cell Communication Cells Characteristics Communication Complement Computer Simulation Development Disease Drug Delivery Systems Equilibrium Fluorescence Microscopy Foundations Goals Gram-Negative Bacteria Health Horizontal Gene Transfer Immune Evasion Immune system In Vitro Lipids Liposomes Mediating Membrane Membrane Biology Membrane Lipids Microbial Biofilms Microfluidics Modeling Molecular Monitor Mono-S Organism Pathogenesis Phospholipids Physiological Play Population Process Pseudomonas Pseudomonas aeruginosa Quinolones Research Role Signal Transduction Sorting - Cell Movement Structure System Techniques Technology Testing Time Toxin Vesicle Virulence Factors antimicrobial biophysical model computerized tools experimental study flexibility human disease insight intercalation killings microbial molecular dynamics novel novel strategies particle physical property preference public health relevance response simulation small molecule synthetic construct trafficking vaccine development

项目摘要

DESCRIPTION (provided by applicant) Bacterial secretion has long been recognized as an essential facet of microbial pathogenesis and human disease. One important but poorly understood system, which is ubiquitous among Gram-negative organisms, involves packaging cargo into small outer membrane derived vesicles (OMVs). Numerous virulence factors have been found to be transported in this way, and delivery by OMVs often results in increased potency. OMVs have also been implicated in the killing of host cells and competing bacteria, avoidance of and interference with the immune system, horizontal gene transfer mediating antibiotic resistance, biofilm formation, and trafficking small molecule communication signals. Remarkably, little is known about how these versatile structures are formed or how their cargo is selected and packaged. To address this, our team proposed the Bilayer-Couple Model where intercalation of self-produced small molecules into the outer membrane drives the induction of membrane curvature to initiate OMV formation. This is a biochemical/biophysical model that followed the discovery by our team and colleagues that the Pseudomonas Quinolone Signal (PQS) is packaged within and drives biogenesis of OMVs in Pseudomonas aeruginosa. In developing this model, we encountered a problem that is common in membrane biology: while all biological membranes contain asymmetric lipid distributions (leaflet vs. leaflet), it was impossible to generate a useful quantiy of in vitro liposomes matching these characteristics. Thus, weakly-relevant surrogates had to be used. Recently, our team has developed a novel approach for constructing synthetic asymmetric vesicles possessing a bilayer architecture that is more physiologically accurate than any other available system. Our approach utilizes microfluidic technology to build vesicles with controlled size, membrane asymmetry, uniformity, and luminal content. These vesicles are the ideal system to experimentally test the predictions of the Bilayer-Couple Model. To gain a greater physical insight in complement to experiments, we also propose to create the first-ever atomistic molecular dynamics and mesoscopic dissipative particle dynamics simulation of the bacterial outer membrane to discover the specific interactions between PQS and physiological-relevant asymmetric membranes. In particular, this model will help elucidate the detailed dynamics of PQS insertion into the outer membrane, its orientation PQS vs. surrounding lipids in the leaflet and whether its own physical properties direct its observed packaging into OMVs. Using leading edge experimental and computational tools, this proposal will address fundamental aspects of OMV formation, including (1) how PQS interacts with and alters the structure of the outer membrane, (2) whether these interactions are sufficient to initiate OMV formation, and (3) whether PQS itself may contribute to its accumulation in OMVs as cargo. The fundamental mechanistic foundations established through this study will have implications in many aspects of health research, potentially enabling applied topics such as vaccine development and drug delivery, for which OMVs are rapidly becoming exciting candidates.

描述（由申请人提供）长期以来，细菌分泌一直被认为是微生物发病机理和人类疾病的重要方面。一个重要但知之甚少的系统，它在革兰氏阴性生物中无处不在，涉及将货物包装到小的外膜衍生蔬菜中（OMV）。已经发现许多病毒因素是通过这种方式运输的，OMV的传递通常会增加效力。 OMV也与宿主细胞和竞争细菌的杀死，避免和干扰免疫系统，水平基因转移介导抗生素耐药性，生物膜形成以及运输小分子通信信号。值得注意的是，这些多功能结构是如何形成或如何选择和包装的，知之甚少。为了解决这个问题，我们的团队提出了双层组模型，其中自生产的小分子在外膜中的插入驱动膜曲率诱导以启动OMV形成。这是一个生化/生物物理模型，遵循我们的团队和同事发现假单胞菌信号（PQS）的发现，并驱动铜绿假单胞菌中OMV的生物发生。在开发该模型时，我们遇到了一个在膜生物学中常见的问题：虽然所有生物膜都包含不对称的脂质分布（小叶与小叶），但不可能产生与这些特征相匹配的体外脂质体的有用量化。这是必须使用弱相关的替代物。最近，我们的团队开发了一种新颖的方法，用于构建具有双层体系结构的合成不对称蔬菜，该蔬菜比任何其他可用系统都更准确。我们的方法利用微流体技术来建造具有控制尺寸，膜不对称，均匀性和腔内含量的蔬菜。这些蔬菜是实验测试双层组模型预测的理想系统。为了在实验完成时获得更大的物理见解，我们还建议对细菌外膜的第一个原子分子动力学和介质耗散粒子动力学模拟进行介绍，以发现PQS与物理相关的非对称不对称膜之间的特定相互作用。特别是，该模型将有助于阐明PQS插入外膜的详细动力学，其方向PQS与传单中的脂质周围的脂质以及其自身的物理特性是否将其观察到的包装引导到OMV中。使用前沿实验和计算工具，该提案将解决OMV形成的基本方面，包括（1）PQS如何与外膜相互作用并改变外膜的结构，（2）这些相互作用是否足以启动OMV形成，以及（3）PQ本身是否可以在OMV中积累OMV。通过这项研究建立的基本机械基础将对健康研究的许多方面产生影响，这可能会启用应用主题，例如疫苗开发和药物输送，而OMV迅速成为令人兴奋的候选人。