Sharma Pradyumn, Parthasarathi Srividhya, Patil Nivedita, Waskar Morris, Raut Janhavi S, Puranik Mrinalini, Ayappa K Ganapathy, Basu Jaydeep Kumar
Unilever RD Bangalore, 64 Main Road, Whitefield, Bangalore 560066, India.
Langmuir. 2020 Aug 4;36(30):8800-8814. doi: 10.1021/acs.langmuir.0c01124. Epub 2020 Jul 20.
The bacterial cell envelope is a complex multilayered structure evolved to protect bacteria in hostile environments. An understanding of the molecular basis for the interaction and transport of antibacterial therapeutics with the bacterial cell envelope will enable the development of drug molecules to combat bacterial infections and suppress the emergence of drug-resistant strains. Here we report the successful creation of an in vitro supported lipid bilayer (SLB) platform of the outer membrane (OM) of , an archetypical Gram-negative bacterium, containing the full smooth lipopolysaccharide (S-LPS) architecture of the membrane. Using this platform, we performed fluorescence correlation spectroscopy (FCS) in combination with molecular dynamics (MD) simulations to measure lipid diffusivities and provide molecular insights into the transport of natural antimicrobial agent thymol. Lipid diffusivities measured on symmetric supported lipid bilayers made up of inner membrane lipids show a distinct increase in the presence of thymol as also corroborated by MD simulations. However, lipid diffusivities in the asymmetric OM consisting of only S-LPS are invariant upon exposure to thymol. Increasing the phospholipid content in the LPS-containing outer leaflet improved the penetration toward thymol as reflected in slightly higher relative diffusivity changes in the inner leaflet when compared with the outer leaflet. Free-energy computations reveal the presence of a barrier (∼6 ) only in the core-saccharide region of the OM for the translocation of thymol while the external O-antigen part is easily traversed. In contrast, thymol spontaneously inserts into the inner membrane. In addition to providing leaflet-resolved penetration barriers in bacterial membranes, we also assess the ability of small molecules to penetrate various membrane components. With rising bacterial resistance, our study opens up the possibility of screening potential antimicrobial drug candidates using these realistic model platforms for Gram-negative bacteria.
细菌细胞包膜是一种复杂的多层结构，进化而来是为了在恶劣环境中保护细菌。了解抗菌治疗药物与细菌细胞包膜相互作用和运输的分子基础，将有助于开发对抗细菌感染和抑制耐药菌株出现的药物分子。在此，我们报告成功创建了一种体外支持脂质双层（SLB）平台，该平台模拟了典型革兰氏阴性菌的外膜（OM），包含该膜完整的光滑脂多糖（S-LPS）结构。利用这个平台，我们结合荧光相关光谱（FCS）和分子动力学（MD）模拟来测量脂质扩散系数，并深入了解天然抗菌剂百里酚的运输情况。在内膜脂质构成的对称支持脂质双层上测得的脂质扩散系数显示，在百里酚存在的情况下有明显增加，MD模拟也证实了这一点。然而，仅由S-LPS组成的不对称OM中的脂质扩散系数在暴露于百里酚时不变。增加含LPS的外层小叶中的磷脂含量可改善对百里酚的渗透，这反映在内层小叶与外层小叶相比相对扩散系数变化略高。自由能计算表明，百里酚转运时仅在OM的核心糖区域存在一个屏障（约6），而外部O抗原部分很容易穿过。相比之下，百里酚能自发插入内膜。除了在细菌膜中提供小叶分辨的渗透屏障外，我们还评估了小分子穿透各种膜成分的能力。随着细菌耐药性的增加，我们的研究为使用这些针对革兰氏阴性菌的逼真模型平台筛选潜在抗菌药物候选物开辟了可能性。
