Mapping the interactions within multidrug efflux pump assemblies.

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.

In Gram-negative bacteria tripartite efflux-pumps expel wide range of noxious compounds from the cell including antibiotics and thus are central contributors to multidrug resistance. Spanning both membranes these systems are composed of 3 components: the Inner Membrane (IMPs); the Outer Membrane Proteins (OMPs) and Membrane-Fusion Proteins (MFPs). Although high-resolution structural data is available for each protein in isolation, very little is known how the pumps actually assemble into their functional state. Despite their key role in drug resistance, no currently approved drugs target the pumps and the potential of interference with the OMP-MFP assembly has not been explored.

This project will provide detailed mapping of the OMP-MFP interfaces underlining pump assembly and test the potential of competitive inhibition of OMP-MFP interaction as a novel antimicrobial strategy. To this end we will map protein-protein interfaces using a unique approach, X-ray radiolytic footprinting (XRF) applicability of which to membrane proteins we recently demonstrated. Once identified, we will use a combination of site-directed mutagenesis of both OMP and MFP interfaces with functional and biophysical analyses to identify residues critical for the pump assembly. Recently we showed that the MFP binds the OMP in an energy-independent fashion driving its opening, while isolated MFP hairpin-domains bind the OMP with up-to 100x higher affinity than the full-length MFP, but are unable to complement pump function. Taking advantage of these findings and the residue-specific data derived from the XRF, we will design full-length and truncated MFP-derivatives and test their ability to outcompete native MFPs in vivo to effectively decouple the pumps. This integrative approach will clarify the role of the MFPs in pump assembly allowing to validate the MFP-OMP interaction as a potential drug target providing high-impact results and new tools in the in the fight against multi drug resistance

This study addresses directly the rising bacterial multidrug resistance (MDR), one of principal threats facing humanity and will benefit both academic and non-academic groups. It fits squarely into the BBSRC strategic priority area of "Antimicrobial resistance" and specifically the BBSRC call for need in development of strategies to mitigate its effects e.g. through novel antimicrobials. By studying a fundamental area of microbiology - efflux via tripartite efflux pumps - it will provide deeper understanding of the mechanisms underlying resistance, allowing development of a novel mitigation strategy for non-specific drug resistance based on the inhibition of their action. The basic science developed will be applicable to all Gram-negative organisms, underpinning the creation of genuinely novel class of antimicrobials for animal, human and plant use. Validating OMP-MFP interaction as a potential drug target provides completely novel approach to design of pump inhibitors, relevant to all species, providing wide translational potential for future research and great economic value.
Impact is expected to come from several main streams:
1. Increase of fundamental knowledge of pump function, benefitting wider academic community.
2. Translational potential from the conceptual validation of a novel strategy for inhibition of pump function at OMP-MFP level, influencing future drug design in both human and veterinary medicine.
3. Methodological development in radiolytic footprinting (XRF) advancing our capabilities for studying membrane proteins.
4. Forging strategic international collaborations and bringing capability not currently available in the UK to increase competitiveness and provide high-tech training for the participants.
Who will benefit from this research and how?
1. Academics on national and international levels working on MDR and structural biology. Anticipated benefits include increased basic knowledge and sharing of methodologies.
2. Students/early career researchers: My teaching involvement at UoB provides a key opportunity to engage students, allowing their exposure to cutting-edge research and experimental techniques.
3. Research Staff: These users will benefit in terms of training in specific research techniques and also general professional and transferrable skills.
4. Translational potential: increased knowledge of central drug tolerance mechanisms will ultimately inform strategies intervening in the treatment of MDR pathogens. By specifically testing the feasibility of the interfering with pump assembly as a viable antimicrobial strategy for the first time we provide a major step into this direction. Detailed interaction maps generated by this research will be an important departure point for rational drug-design.
5. Wider socio-economic impacts: The potential impact of understanding and ultimately manipulating MDR mechanisms is difficult to underestimate e.g. according to the authoritative Government review http://amr-review.org, MDR claimed over 700k lives globally as of 2013, a number projected to spiral out to over 10M by 2050, with major societal and economic costs resulting in a reduction of up to 3.5% in the Gross Domestic Product (GDP) per country, and estimates totaling a staggering $100 trillion for the global economy over the next 3 decades.
6. Methodology development: We will provide novel tools to study membrane proteins representing over 60% of all drug targets. Economy of scale in required sample size will have an important impact enabling to address difficult to produce targets.
7. Increasing the competitiveness of UK science by allowing access to know-how and unique state-of-the-art experimental set-ups without analogues in Europe and forging new International collaborations.
8. Public. We will engage wider non-academic users with research directions and outputs from the study, increasing basic knowledge and understanding of research undertaken through the support of BBSRC.