Snyder D S, McIntosh T J
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Biochemistry. 2000 Sep 26;39(38):11777-87. doi: 10.1021/bi000810n.
Lipopolysaccharide (LPS), the primary lipid on the surface of Gram-negative bacteria, is thought to act as a permeability barrier, making the outer membrane relatively impermeable to hydrophobic antibiotics, detergents, and host proteins. Mutations in the LPS biosynthetic apparatus increase bacterial susceptibility to such agents. To determine how this increased susceptibility is mediated, we have correlated antibiotic susceptibilities of rough (antibiotic resistant) and deep rough (antibiotic susceptible) bacterial strains with antibiotic permeabilities and fluorescent probe binding kinetics for bilayers composed of LPS purified from the same strains. Bilayer permeabilities of two hydrophobic beta-lactam antibiotics were measured by encapsulating the appropriate beta-lactamases in large unilamellar vesicles. In the presence of MgCl(2), permeabilities of LPS bilayers from rough and deep rough bacteria were similar and significantly lower than those of bacterial phospholipids (BPL). Addition of BPL to the LPS bilayers increased their antibiotic permeability to approximately the level of the BPL bilayers. Binding rates of the fluorescent probe bis-aminonaphthylsulfonic acid (BANS) were 2 orders of magnitude slower for both rough and deep rough LPS bilayers compared to that of bilayers composed of BPL or mixtures of LPS and BPL. On the basis of these results and the observation that deep rough bacteria have higher levels of phospholipid on their surface than do rough bacteria (Kamio, Y., and Nikaido, H. (1976) Biochemistry 15, 2561-2569), we argue that the high susceptibility of deep rough bacteria is due to the presence of phospholipids on their surface. Experiments with phospholipid bilayers showed that the addition of PEG-lipids (containing covalently attached hydrophilic polymers) had little effect on permeability and binding rates, whereas the addition of cholesterol reduced permeability and slowed binding to levels approaching those of LPS. Therefore, we argue that the barrier provided by LPS is primarily due to its tight hydrocarbon chain packing (Snyder et al., (1999) Biochemistry 38, 10758-10767) rather than to its polysaccharide headgroup.
脂多糖（LPS）是革兰氏阴性菌表面的主要脂质，被认为起着渗透屏障的作用，使外膜对疏水性抗生素、去污剂和宿主蛋白相对不透。LPS生物合成装置中的突变会增加细菌对这些物质的敏感性。为了确定这种增加的敏感性是如何介导的，我们将粗糙型（抗生素抗性）和深粗糙型（抗生素敏感）细菌菌株的抗生素敏感性与由从相同菌株纯化的LPS组成的双层膜的抗生素渗透性和荧光探针结合动力学进行了关联。通过将适当的β-内酰胺酶包封在大单层囊泡中来测量两种疏水性β-内酰胺抗生素的双层膜渗透性。在MgCl₂存在下，粗糙型和深粗糙型细菌的LPS双层膜的渗透性相似，且显著低于细菌磷脂（BPL）的渗透性。向LPS双层膜中添加BPL会使其抗生素渗透性增加到大致与BPL双层膜相同的水平。与由BPL或LPS与BPL的混合物组成的双层膜相比，粗糙型和深粗糙型LPS双层膜的荧光探针双氨基萘磺酸（BANS）的结合速率均慢2个数量级。基于这些结果以及深粗糙型细菌表面的磷脂水平高于粗糙型细菌的观察结果（神尾洋、西户泰夫（1976年）《生物化学》15卷，2561 - 2569页），我们认为深粗糙型细菌的高敏感性是由于其表面存在磷脂。磷脂双层膜实验表明，添加聚乙二醇脂质（含有共价连接的亲水性聚合物）对渗透性和结合速率影响很小，而添加胆固醇会降低渗透性并减缓结合速率，使其接近LPS的水平。因此，我们认为LPS提供的屏障主要是由于其紧密的烃链堆积（斯奈德等人，（1999年）《生物化学》38卷，10758 - 10767页），而不是由于其多糖头部基团。
