Mapping the interactions within multidrug efflux pump assemblies.

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Multidrug-resistant bacterial infections are one of principal challenges facing medicine today. A group of bacteria known as the Gram-negative are particularly resistant to the action of antibiotics, as they have evolved a secondary membrane around their cells, preventing easy entry of the antibiotics. No new antibiotics targeting this group have been developed for over 40 years and the need to find and exploit novel bacterial weakness points is of great importance for both human and veterinary medicine. One of the central mechanisms underlying their multidrug resistance, is the action of the of the so-called multidrug-efflux pumps. These assemble from 3 components, spanning the double membrane and pumping out antibiotics, lowering their effective concentration in the cell thus rendering them ineffective. While pumps are very diverse, they share a common outer membrane protein (OMP). Deactivation or removal of OMPs dramatically increases bacterial sensitivity to antibiotics, suggesting that targeting the OMP may be an effective therapeutic approach. Yet none of the currently available drugs target the pump assembly process. Partially this is due to lack of information on the interactions between pump components to design targeted inhibitors. This project specifically aims to close the gap in our understanding of pump intercomponent interactions with the view of disrupting their assembly.The study of full pump assemblies has been hindered by their complexity and transient association of their elements, preventing effective usage of standard structural approaches such as X-ray crystallography.Here, we will overcome these bottlenecks by innovative usage of multiple approaches. The first is the application of the novel technique of X-ray Radiolytic Footprinting (XRF), allowing to study transient and heterogeneous proteins in solution. XRF is a mapping technique, which is based on oxidation of the surface-exposed parts of the protein by usage of highly reactive hydroxyl radicals. These radicals are created by splitting water molecules in solution by usage of high-energy X-rays. The pattern of oxidation is detected by the molecular weight differences of fragmented proteins using a technique called mass-spectrometry. Comparison of the modification of a given protein on its own with the modification obtained in the presence of its binding partner reveals zones of protection, or footprints, corresponding to the interaction surfaces between the proteins. By using this approach we will map the tripartite pump complex and will use the information to specifically target the binding interfaces by mutagenesis to disrupt the association of the OMP with the rest of the pump. We will characterise the effect of these mutations on antibiotic resistance of the cells to identify crucial residues.Furthermore, we will use modified MFP proteins with truncated and scrambled hairpins to dissect their role in the assembly of the pump, allowing to distinguish between the two currently contradictory models of assembly. Recently, we have shown that the hairpin-domain of the MFP binds the OMP with higher affinity than the corresponding full-length protein. Furthermore it binds in an energy-independent fashion. However, unlike the full-length protein the binding of the hairpin does not produce a functional pump. Here, we will exploit these findings by further engineering stabilising interactions of the hairpin with its target OMP, and will test its capability to outcompete native MFPs and inhibit the function of the pump in vivo. This project will further our fundamental understanding of the pump assembly and settle the long-standing debate on the mode of MFP-OMP interaction. Demonstrating competitive inhibition of the MFP-OMP binding interface as viable strategy will pave the way to future design of a completely novel class of drugs targeting the assembly process providing a powerful tool in the fight against drug-resistance.