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

革兰氏阴性菌的外膜为这些细菌的渗透提供了物理屏障。离子、营养物质、代谢物以及抗生素等有毒物质进入细胞的基本途径是由外膜上的毛孔提供的。铜绿假单胞菌是一种不同寻常的多用途生物，已成为我们社会中主要的机会致病菌。异常低透性的外膜可能导致其固有阻力。与许多其他革兰氏阴性菌不同，铜绿假单胞菌缺乏大量的一般扩散孔，依赖于狭窄的底物特异性通道来获取其生长和代谢所需的营养物质或代谢物。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家族的一个孔是磷酸饥饿诱导的OprP孔，它含有磷酸的结合位点。在项目的第一阶段，使用电生理学和分子动力学模拟分析了OprP野生型和精氨酸阶梯的几个突变体。此外，在项目第一阶段的初步研究中，对二磷酸特异性且与OprP高度相似的OprO孔进行了研究，包括其晶体结构。OprP和OprO的结构分析揭示了两个关键的差异在中央收缩区。构建了OprP的双突变体，其选择性与OprO相近。反向双突变体仍然缺失，需要解决。此外，该项目的第一阶段侧重于OprP中精氨酸阶梯的突变体，第二阶段将重点关注OprP和OprO中的赖氨酸簇，这对各自的选择性都很重要。下一步，我们将通过脂质双分子层实验和分子动力学模拟来研究含磷抗生素磷霉素和磷米霉素通过磷酸特异性孔OprP和OprO的易位。OprP和OprO孔是这些药物分子运输的明显候选者。该研究还将扩展到不对称膜与铜绿假单胞菌的LPS在一个小叶。此外，计划开发用于通道理论分析的工具，并测试最近开发的混合方法的适用性，其中仅在全原子水平上对系统的重要部分进行建模。

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.