Molecular Mechanisms of Lipopolysaccharide Transport Driven by ABC Transporters

摘要 脂多糖（LPS）存在于大多数革兰氏阴性菌的外膜中， 在构建细菌在恶劣环境中生存的适当细胞包膜方面起着关键作用。的 LPS在外膜中的紧密包装产生电荷和糖的网络，其选择性地 允许营养分子进入，同时限制包括清洁剂在内的有毒化合物的渗透 和抗生素。由于其在细菌膜屏障的生物发生中的关键重要性， 生物合成和转运途径是开发新型抗生素的特别令人感兴趣的靶点。 LPS在宿主-病原体相互作用中也是至关重要的，并且作为天然免疫的有效激活剂起作用。 动物的反应。LPS是一种复杂且高度可变的糖脂，由脂质A部分组成， 核心寡糖和长链O-抗原多糖。脂质A和核心的结构 寡糖是相对保守的，大概是由于它们在维持蛋白质完整性方面的作用。 渗透屏障相反，LPS的O-抗原显示高变结构，这是一致的。 与外界环境相互作用和宿主防御的功能。 革兰氏阴性菌将大量的能量和复杂的蛋白质机制用于 LPS分子的有效和适当的生产、运输和组装。LPS的合成开始 在细胞质和内膜之间的界面处，导致脂质A-核心的产生 低聚糖，也称为粗糙LPS，其存在于内膜的内小叶中。粗糙 LPS通过ATP结合盒（ABC）转运蛋白MsbA翻转穿过内膜。的 周质小叶中的粗糙LPS进一步加入各种长度和形式的O-抗原， 成为一个“光滑”的LPS。对于LPS穿过周质并转运到外膜，7个 称为LptA-G的蛋白质参与其中。几条证据汇集在一起，提出了一个模型，其中 Lpt蛋白形成连接两个膜的连续桥。ABC运输机，形成 LptB 2FG，被认为是从内膜提取LPS分子，并将它们转运到 紧密相关的双位点LptC，以及周质LptA。多个LptA蛋白可以形成连续的 桥到达外膜中的LptDE复合物，其介导LPS插入到 外膜的外部小叶。在这里，我们提出了一系列的结构和功能的研究， 使用各种生化和冷冻-EM技术的LPS转运蛋白机器。分子 了解LPS转运途径的功能和调节将有助于 了解许多革兰氏阴性菌外膜的生物起源，也有助于 直接针对细菌膜屏障的新型抗生素的开发。

ABSTRACT Lipopolysaccharide (LPS) is present in the outer membrane of most Gram-negative bacteria, and plays a key role in constructing a proper cellular envelope for bacteria to survive in harsh environments. The tight packing of LPS in the outer membrane generates a network of charges and sugars, which selectively allow the entry of nutrient molecules, while limit the penetration of toxic compounds including detergents and antibiotics. Due to its critical importance in the biogenesis of bacterial membrane barrier, LPS biosynthesis and transport pathway is a particularly interesting target for developing novel antibiotics. LPS is also crucial in the host-pathogen interactions, and functions as a potent activator of innate immune response in the animals. LPS is a complex and highly variable glycolipid, composed of a lipid A moiety, a core oligosaccharide and a long-chain O-antigenic polysaccharide. The structure of lipid A and core oligosaccharide are relatively conserved, presumably due to their roles in maintaining the integrity of permeability barrier. In contrast, the O-antigen of LPS shows hypervariable structures, which is consistent with their functions in interacting with the outside environment and host defense. Gram-negative bacteria devote a large amount of energy and a sophisticated protein machinery to the efficient and proper production, transport and assembly of LPS molecules. The synthesis of LPS starts at the interface between the cytoplasm and the inner membrane, leading to the generation of lipid A-core oligosaccharide, also called rough LPS, which resides in the inner leaflet of the inner membrane. Rough LPS is flipped across the inner membrane by an ATP binding cassette (ABC) transporter, MsbA. The rough LPS in the periplasmic leaflet is further added with various lengths and forms of O-antigen, becoming a “smooth” LPS. For the LPS transport across the periplasm and to the outer membrane, seven proteins named as Lpt A-G are involved. Several lines of evidence converge to suggest a model, in which the Lpt proteins form a continuous bridge connecting the two membranes. The ABC transporter, formed as LptB2FG, is thought to extract the LPS molecules from the inner membrane, and transport them to the tightly associated bitopic LptC, and to the periplasmic LptA. Multiple LptA proteins may form a continuous bridge to reach the LptDE complex in the outer membrane, which mediates the LPS insertion into the outer leaflet of the outer membrane. Here we propose a series of structural and functional studies on the LPS transport protein machinery using a variety of biochemical and cryo-EM techniques. A molecular understanding on the function and regulation of the LPS transport pathway will contribute to the understanding of the biogenesis of the outer membrane of many Gram-negative bacteria, and also aid the development of novel antibiotics that directly target the bacterial membrane barrier.