Molecular Mechanisms of Lipopolysaccharide Transport Driven by ABC Transporters

摘要 内毒素存在于大多数革兰氏阴性细菌的外膜中，并发挥作用 在为细菌在恶劣环境中生存构建适当的细胞膜方面起着关键作用。这个 脂多糖在外膜中紧密堆积，产生一个电荷和糖的网络，选择性地 允许营养分子进入，同时限制包括洗涤剂在内的有毒化合物的渗透 还有抗生素。由于其在细菌膜屏障生物发生中的关键作用，内毒素 生物合成和转运途径是开发新型抗生素的一个特别有趣的目标。 内毒素在宿主与病原体的相互作用中也是至关重要的，并作为天然免疫的有效激活剂发挥作用。 动物的反应。脂多糖是一种复杂的高度可变的糖脂，由脂A部分组成， 一种核寡糖和一种长链O-抗原性多糖。类脂A和核心的结构 寡糖相对保守，可能是因为它们在维持 渗透性屏障。相反，内毒素的O-抗原呈现出高度可变的结构，这是一致的 具有与外界环境相互作用和宿主防御的功能。 革兰氏阴性细菌将大量能量和复杂的蛋白质机制投入到 高效、正确地生产、运输和组装脂多糖分子。脂多糖的合成开始 在细胞质和内膜之间的交界处，导致类脂A-核心的产生 低聚糖，也称为粗脂多糖，存在于内膜的内叶中。粗略的 脂多糖通过三磷酸腺苷结合盒(ABC)转运体MSBA翻转穿过内膜。这个 在周质小叶中进一步加入不同长度和形式的O-抗原， 成为一个“顺畅”的内毒素。对于内毒素跨周质和外膜的运输，7 被命名为LPT A-G的蛋白质也参与其中。几条证据汇聚成一种模型，在这种模型中， LPT蛋白形成了连接两层膜的连续桥梁。ABC运输机，形成了 作为LptB2FG，被认为是从内膜中提取内毒素分子，并将它们输送到 紧密结合的位位LptC和周质LPTA。多个LPTA蛋白可能形成一个连续的 桥到达外膜上的LptDE复合体，它介导内毒素插入到 外膜的外小叶。在这里，我们提出了一系列的结构和功能的研究 内毒素利用各种生化和低温EM技术运输蛋白质机械。一种分子 了解内毒素转运途径的功能和调控将有助于 了解许多革兰氏阴性细菌外膜的生物发生，并帮助 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.