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

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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.