An in vitro gram-negative double bilayer envelope mimetic

The gram-negative bacterial envelope is a complex structure composed of two membranes and a peptidoglycan layer that together maintain the cell's integrity and shape. Despite extensive knowledge of its composition, replicating this system in vitro remains a challenge, particularly for trans-envelope studies, as existing models focus on either the outer or inner membrane alone. The study of transport processes between these membranes, especially biogenic transport responsible for outer membrane (OM) formation, is therefore limited. One key system, the PqiABC complex, is believed to facilitate glycerophospholipid (GPL) transport by forming a trans-envelope tunnel, tethering the two membranes. Previous work by us has shown that PqiABC can be reconstituted and used to construct a double bilayer architecture on a planar surface. In this proposal, we aim to utilise a non-functional variant of PqiABC as a scaffold to study other trans-envelope biogenic processes. As a proof of concept, by integrating the Maintenance of Outer Membrane Lipid Asymmetry (Mla) pathway, which regulates GPL distribution between the membranes, we will investigate its activity in this controlled mimetic system. This approach has the potential to significantly advance the accuracy of in vitro studies of gram-negative envelope biology, enabling more precise investigations into membrane dynamics and transport processes.

革兰氏阴性菌包膜是一种复杂结构，由两层膜与一层肽聚糖层共同构成，维持细胞的完整性与形态。尽管人们对其组成已有深入了解，但在体外复制该系统仍是一项挑战——尤其是对于跨包膜研究而言，现有模型仅聚焦于外膜或内膜中的单一膜层。因此，关于这些膜之间转运过程（尤其是负责外膜（OM）形成的生物源转运）的研究受到限制。PqiABC复合物作为关键系统，被认为可通过形成跨包膜通道并连接两层膜来促进甘油磷脂（GPL）的转运。我们先前的研究表明，PqiABC可被重组并用于在平面上构建双双层膜结构。在本研究方案中，我们旨在利用PqiABC的非功能变体作为支架，研究其他跨包膜生物发生过程。作为概念验证，我们将整合外膜脂质不对称维持（Mla）通路（该通路调控膜间GPL分布），并在这一受控模拟系统中研究其活性。该方法有望显著提升革兰氏阴性菌包膜生物学体外研究的准确性，从而为膜动力学及转运过程的更精准探究提供可能。