Structural basis for the lipid asymmetry of the Gram-negative bacterial outer membrane

革兰氏阴性菌的特征是有一个由两层膜组成的细胞膜，即内(或细胞质)膜(IM)和外膜(OM)。这两个膜非常不同：内膜的两个小叶都含有磷脂(PL)，因此可以认为是对称的。相比之下，OM是一个不对称的双层结构，内层是PL，外层暴露在表面，几乎完全由脂多糖(LPS)组成。内毒素的性质和结构与磷脂酶有很大的不同，内毒素存在的主要后果是革兰氏阴性菌被一层极性层包围，形成了一层非常有效的中性和疏水分子的屏障。鉴于大多数药物具有中等疏水性，OM独特的不对称结构是革兰氏阴性菌对抗生素和其他有害化合物具有高度内在耐药性的一个主要因素。因此，从细菌的角度来看，OM的不对称性是非常重要的。然而，由于目前尚不清楚的原因，但可能是正常细胞生长的结果，PL可以在OM的外叶中积累。这些磷脂在脂多糖的海洋中形成了“岛屿”，增加了有毒化合物的渗透性。因此，需要从外叶中去除PL以恢复OM通透性屏障。MLA(维持脂质不对称)系统广泛存在于革兰氏阴性菌中，可能是维持OM脂质不对称的最重要系统。它由六种蛋白质组成，它们被认为构成了PL从OM到IM的“反向”运输系统。MlaA蛋白是MLA系统的OM成分，被认为是从外部OM小叶中选择性地提取PL。这是如何发生的还不清楚，主要是因为缺乏关于MLAA的结构性信息。我们已经(通过X射线结晶学)确定了MlaA蛋白的第一个结构，以了解MlaA是如何发挥作用的。这些初步数据非常有趣，因为它们表明MlaA具有独特的结构，但更重要的是，它们提供了关于功能的明确和可测试的线索。在本提案中，我们将使用这些结构作为起点来确定MlaA如何从OM外叶提取PL，并随后将它们转移到周质空间中的结合蛋白(MlaC)。我们将使用功能分析、生物物理方法、计算方法和X射线结晶学的跨学科方法。我们的项目将阐明革兰氏阴性菌中一个基本而重要的过程，并将揭示以革兰氏阴性病原体的MLA系统为靶点作为降低毒力和增强各种抗生素的前景。

Gram-negative bacteria are characterised by having a cell envelope consisting of two membranes, an inner (or cytoplasmic) membrane (IM) and an outer membrane (OM). The two membranes are very different: the two leaflets of the inner membrane both contain phospholipids (PL) and can therefore be considered symmetric. By contrast, the OM is an asymmetric bilayer with an inner leaflet of PL and an outer, surface-exposed leaflet composed almost exclusively of lipopolysaccharide (LPS). The properties and structure of LPS are very different from those of PL, and the main consequence of the presence of LPS is that Gram-negative bacteria are surrounded by a polar layer that forms a very effective barrier for neutral and hydrophobic molecules. Given that the majority of drugs are moderately hydrophobic, the unique, asymmetric structure of the OM is a major factor why Gram-negative bacteria have a high intrinsic resistance towards antibiotics and other harmful compounds. Thus, from the bacterium's point of view, the asymmetry of the OM is very important. However, due to reasons that are not yet clear but likely are a consequence of normal cell growth, PL can accumulate in the outer leaflet of the OM. Those PL form "islands" amid a sea of LPS that increase the permeability of noxious compounds. Thus, PL need to be removed from the outer leaflet to restore the OM permeability barrier.The Mla (maintenance of lipid asymmetry) system is widespread in Gram-negative bacteria and is likely the most important system for maintaining OM lipid asymmetry. It consists of six proteins that are thought to form a "reverse" transport system for PL from the OM to the IM. The protein MlaA is the OM component of the Mla system and is thought to selectively extract PL from the outer OM leaflet. How this happens is unclear, mainly due to a lack of structural information on MlaA. We have determined the first structures of MlaA proteins (by X-ray crystallography) to understand how MlaA functions. These preliminary data are very interesting in that they show that MlaA has a unique structure, but more importantly they provide clear and testable clues regarding function. In this proposal we will use the structures as starting points to determine how MlaA extracts PL from the OM outer leaflet and subsequently transfers them to a binding protein (MlaC) in the periplasmic space. We will use an interdisciplinary approach with functional assays, biophysical methods, computational approaches and X-ray crystallography. Our project will elucidate a fundamental and important process in Gram-negative bacteria and will inform on the prospect of targeting the Mla system of Gram-negative pathogens as a means to decrease virulence and to potentiate various antibiotics.