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

革兰氏阴性细菌外膜脂质不对称的结构基础

• 批准号: BB/R004366/1
• 负责人: Bert Van Den Berg
• 金额: $ 50.06万
• 依托单位: Newcastle University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FR004366%2F1
• 关键词: Structural; basis; lipid; asymmetry; Gram

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.

革兰氏阴性菌的特征在于具有由两个膜组成的细胞被膜，即内膜（或细胞质）（IM）和外膜（OM）。这两种膜非常不同：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，具有PL的内小叶和几乎完全由脂多糖（LPS）组成的外表面暴露小叶。LPS的性质和结构与PL的性质和结构非常不同，LPS存在的主要后果是革兰氏阴性菌被极性层包围，该极性层对中性和疏水分子形成非常有效的屏障。鉴于大多数药物是适度疏水的，OM的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内在抗性的主要因素。因此，从细菌的角度来看，OM的不对称性非常重要。然而，由于尚不清楚的原因，但可能是正常细胞生长的结果，PL可积聚在OM的外瓣叶中。这些PL在LPS的海洋中形成“岛屿”，增加了有毒化合物的渗透性。因此，PL需要从外小叶中移除以恢复OM渗透屏障Jla（脂质不对称性维持）系统广泛存在于革兰氏阴性菌中，并且可能是维持OM脂质不对称性的最重要系统。它由六种蛋白质组成，这些蛋白质被认为形成PL从OM到IM的“反向”运输系统。蛋白质MlaA是Mla系统的OM组分，并且被认为选择性地从外部OM小叶提取PL。这是如何发生的尚不清楚，主要是由于缺乏MlaA的结构信息。我们已经确定了MlaA蛋白的第一个结构（通过X射线晶体学），以了解MlaA如何发挥作用。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。在这个建议中，我们将使用的结构作为出发点，以确定如何MlaA提取PL从OM外小叶，随后将其转移到周质空间中的结合蛋白（MlaC）。我们将使用功能测定，生物物理方法，计算方法和X射线晶体学的跨学科方法。我们的项目将阐明革兰氏阴性菌的一个基本和重要的过程，并将告知革兰氏阴性病原体的Mla系统作为降低毒力和增强各种抗生素的手段的前景。

项目成果

Acquisition of ionic copper by the bacterial outer membrane protein OprC through a novel binding site.

• DOI: 10.1371/journal.pbio.3001446
• 发表时间: 2021-11
• 期刊: PLoS biology
• 影响因子: 9.8
• 作者: Bhamidimarri SP;Young TR;Shanmugam M;Soderholm S;Baslé A;Bumann D;van den Berg B
• 通讯作者: van den Berg B