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

 描述（由申请人提供） 长期以来，细菌分泌物被认为是微生物致病和人类疾病的重要方面。一种重要但知之甚少的系统，其在革兰氏阴性生物中普遍存在，涉及将货物包装到小的外膜衍生囊泡（OMV）中。已经发现许多毒力因子以这种方式运输，并且通过OMV递送通常导致增加的效力。OMV还涉及杀死宿主细胞和竞争细菌、避免和干扰免疫系统、介导抗生素抗性的水平基因转移、生物膜形成和运输小分子通讯信号。值得注意的是，人们对这些多功能结构是如何形成的，以及它们的货物是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耦合模型，其中将自产的小分子插入外膜中，驱动膜弯曲的诱导，以启动OMV的形成。这是一个生物化学/生物物理模型，遵循我们的团队和同事的发现，即假单胞菌喹诺酮信号（PQS）被包装在铜绿假单胞菌中并驱动OMV的生物发生。在开发该模型时，我们遇到了膜生物学中常见的问题：虽然所有生物膜都含有不对称的脂质分布（小叶与小叶），但不可能产生与这些特征匹配的有用量的体外脂质体。因此，必须使用弱相关的替代品。最近，我们的团队开发了一种新的方法来构建具有双层结构的合成不对称囊泡，这种结构比任何其他可用的系统在生理上都更准确。我们的方法利用微流体技术来构建具有可控尺寸、膜不对称性、均匀性和腔内容物的囊泡。这些囊泡是实验测试双层耦合模型预测的理想系统。为了获得更大的物理洞察力，以补充实验，我们还建议创建有史以来第一个原子分子动力学和介观耗散粒子动力学模拟的细菌外膜，以发现PQS和生理相关的非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，其取向PQS与小叶中周围脂质的关系，以及其自身的物理性质是否将其观察到的包装引导到OMV中。利用先进的实验和计算工具，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）PQS本身是否有助于其作为货物在OMV中的积累。通过这项研究建立的基本机制基础将在健康研究的许多方面产生影响，可能使应用主题，如疫苗开发和药物输送，其中OMV正在迅速成为令人兴奋的候选人。

 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.