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，细菌分泌一直被认为是微生物致病和人类疾病的一个重要方面。一个重要但知之甚少的系统，在革兰氏阴性生物中普遍存在，涉及将货物包装成小的外膜衍生囊泡(OMV)。已发现许多毒力因子是通过这种方式运输的，而OMV的输送往往会导致效力的增强。OMVS还与杀死宿主细胞和竞争细菌、避免和干扰免疫系统、水平基因转移介导抗生素耐药性、生物被膜形成和运输小分子通讯信号有关。值得注意的是，人们对这些多功能结构是如何形成的，或者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，在该模型中，自产生的小分子插入外膜驱动膜曲率的诱导，以启动OMV的形成。这是一个生化/生物物理模型，此前我们的团队和同事发现，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。在开发这个模型时，我们遇到了膜生物学中常见的一个问题：尽管所有生物膜都含有不对称的脂质分布(叶状物与叶状物)，但不可能产生与这些特性相匹配的大量体外脂质体。因此，必须使用相关性较弱的代理人。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，这种结构在生理上比任何其他可用的系统都更准确。我们的方法利用微流控技术来构建具有可控大小、膜不对称性、均匀性和管腔含量的囊泡。这些囊泡是对双层耦合模型的预测进行实验测试的理想系统。为了更好地补充实验的物理意义，我们还提出了首次建立细菌外膜的原子分子动力学和介观耗散粒子动力学模拟，以发现PQS与生理相关的不对称膜之间的特定相互作用。特别是，这个模型将有助于阐明PQS插入外膜的详细动力学，它在小叶中的取向与周围脂质的关系，以及它自身的物理性质是否指导其观察到的包装进入OMV。利用前沿的实验和计算工具，这一建议将讨论OMV形成的基本方面，包括(1)PQS如何与外膜相互作用并改变其结构，(2)这些相互作用是否足以启动OMV的形成，以及(3)PQS本身是否可能有助于其作为货物在OMV中积累。通过这项研究建立的基本机制基础将对卫生研究的许多方面产生影响，可能使疫苗开发和药物输送等应用主题成为可能，而OMV正迅速成为这些领域令人兴奋的候选者。

 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.