Ion and antibiotics transport through the phosphate specific outer membrane porins OprP and OprO

离子和抗生素通过磷酸盐特异性外膜孔蛋白 OprP 和 OprO 运输

• 批准号: 190764917
• 负责人: Professor Dr. Roland Benz
• 金额: --
• 依托单位: Department of Physics and Earth Sciences
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2011
• 资助国家: 德国
• 起止时间: 2010-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/190764917?language=en
• 关键词: Ion; antibiotics; transport; through; phosphate

The outer membrane of Gram-negative bacteria serves as a physical barrier for the penetration into these bacteria. Essential pathways for the flow of ions, nutrients and metabolites but also noxious substances such as antibiotics into the cell are provided by pores in the outer membrane. The bacterium Pseudomonas aeruginosa is an unusually versatile organism that has become a major opportunistic pathogen in our society. An unusually low permeable outer membrane presumably causes its intrinsic resistance. Unlike many other Gram-negative bacteria, P. aeruginosa lacks large numbers of general diffusion porins and relies on narrow and substrate-specific channels for the acquisition of nutrients or metabolites required for its growth and metabolism. Thus, also antibiotics molecules need to pass these narrow and substrate-specific channels. One of the porins of the OprD family is the phosphate-starvation inducible OprP pore that contains a binding site for phosphate. In the first phase of the project, the OprP wildtype and several mutants of the arginine ladder have been analyzed using electrophysiology and molecular dynamics simulations. Furthermore, the OprO pore which is specific for diphosphate and highly similar to OprP has been investigated in an initial study in the first phase of the project including its crystal structure. A structural analysis of OprP and OprO revealed two crucial differences in the central constriction region. A double mutant of OprP was created which has selectivity features close to those of OprO. The reverse double mutant is still missing and needs to be tackled. Moreover, while the first phase of the project concentrated on mutants in the arginine ladder in OprP, the second phase will focus on the lysine clusters in both OprP and OprO, which are important for the respective selectivity. In a next step, the translocation of the phosphate-containing antibiotics fosfomycin and fosmidomycin through the phosphate-specific pores OprP and OprO shall be investigated in lipid bilayer experiments and in molecular dynamics simulations. The OprP and OprO pores are obvious candidates for the transport of these drug molecules. The studies will also be extended to asymmetric membranes with LPS of P. aeruginosa in one leaflet. Moreover, it is planned to developed tools for the theoretical analysis of channels and to test the applicability of a recently developed hybrid approach in which only the important parts of the systems are modelled on an all-atom level.

革兰氏阴性菌的外膜作为渗透进入这些细菌的物理屏障。外膜上的孔提供了离子、营养物质和代谢物以及抗生素等有毒物质流入细胞的基本途径。铜绿假单胞菌是一种非常通用的生物体，已成为我们社会中的主要机会致病菌。一种异常低渗透性的外膜可能导致其内在的阻力。与许多其他革兰氏阴性细菌不同，铜绿假单胞菌缺乏大量的一般扩散孔蛋白，并依赖于狭窄的和底物特异性的通道来获取其生长和代谢所需的营养物质或代谢产物。因此，抗生素分子也需要通过这些狭窄的底物特异性通道。OprD家族的孔蛋白之一是磷酸盐饥饿诱导的OprP孔，其含有磷酸盐结合位点。在该项目的第一阶段，OprP野生型和精氨酸梯形的几个突变体进行了分析，使用电生理学和分子动力学模拟。此外，在项目第一阶段的初步研究中，研究了对二磷酸盐具有特异性且与OprP高度相似的OprO孔，包括其晶体结构。OprP和OprO的结构分析揭示了中央收缩区域的两个关键差异。产生OprP的双突变体，其具有接近OprO的选择性特征。反向双突变体仍然失踪，需要解决。此外，虽然该项目的第一阶段集中在OprP中精氨酸梯的突变体上，但第二阶段将集中在OprP和OprO中的赖氨酸簇上，这对各自的选择性很重要。在下一步中，将在脂质双层实验和分子动力学模拟中研究含磷酸盐抗生素磷霉素和磷霉素通过磷酸盐特异性孔OprP和OprO的易位。OprP和OprO孔是用于运输这些药物分子的明显候选物。研究还将扩展至单瓣叶中含有铜绿假单胞菌LPS的不对称膜。此外，它计划开发工具的理论分析渠道和测试的适用性，最近开发的混合方法，其中只有系统的重要部分是在全原子水平上建模。