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Insight into antibiotic resistance of pathogenic bacteria via structural studies of a multidrug transporter

Reference	BB/K014226/1
Principal Investigator / Supervisor	Dr Christopher Law
Co-Investigators / Co-Supervisors
Institution	Queen's University of Belfast
Department	Sch of Biological Sciences
Funding type	Research
Value (£)	328,170
Status	Completed
Type	Research Grant
Start date	01/09/2013
End date	31/08/2016
Duration	36 months

Abstract

We aim to solve the high-resolution 3D structure, and to characterise in detail the molecular mechanism of transport and substrate polyspecificity of the novel E. coli MDR transporter MdtM. To understand the substrate polyspecificity and conformational changes upon substrate binding in the transport cycle of MdtM, we aim to solve the crystal structure of MdtM with bound substrate by X-ray crystallography. We already have grown crystals of MdtM that diffract to 8.5 Angstrom resolution and these will be optimized. Crystals of MdtM in complex with antimicrobial substrate will also be grown. In the structure of the complex, those residues in direct contact with substrate are probably involved in determining the transporter's specificity. Comparison of MdtM crystal structures, with and without substrate bound, will reveal-substrate induced conformational changes during the transport cycle. We will also determine the component of the proton electrochemical gradient that drives MdtM-mediated efflux, and the electrogenicity of the transport reaction. Preliminary data from whole cell EtBr efflux assays support the notion of MdtM as an antiporter that utilises components of the proton electrochemical gradient as the driving force. We will determine the component that energises efflux of differently charged substrates across the inner membrane. Acidic residues play crucial roles in multidrug and/or proton binding in a variety of proteins. Our homology model of MdtM in occluded conformation reveals a network of acidic residues, located in the N-terminal half of the protein, that spans the membrane and could be involved in forming a proton relay. We will test the role of these residues in transport by individually mutating them and performing substrate-binding and transport assays. Preliminary data suggest that MdtM may function in protecting E. coli from the effects of bile salts; this potential function will be investigated.

Summary

Antibiotic resistance is a major global health concern. One of the mechanisms that infectious bacteria have evolved to resist the effects of antibiotics is that of multidrug efflux. This process is mediated by molecular pumps, made of protein, that are embedded in the fatty membranes that surround the bacterial cell. An understanding of the structure and mechanism of these membrane proteins is vital if we are to understand how they recognise and transport their antibiotic substrates and, in turn, use that knowledge to our advantage in the fight against bacterial infections. Although membrane proteins are important biological molecules that represent about one third of all the proteins made by a typical cell, and nearly half of them are targets of drugs on the market today, the structures of relatively few membrane proteins have been solved. One of the reasons for this is the challenge of working with proteins that are embedded in the oil-like environment of biological membranes. This means they first need to be isolated from their membrane environment using detergents before they can be highly purified to free them of contaminants. The purified protein-detergent complex then needs to be crystallised and exposed to X-rays in order to obtain a detailed, three-dimensional atomic structure. We have isolated, purified and grown crystals of a membrane protein from E. coli that plays a role in the transport of a broad range of antibiotics out of the cell, thereby contributing to multidrug resistance. This is a vital first step towards solving the structure of this transporter protein using a technique called X-ray crystallography. The structural information gained will provide insight into how these proteins recognise and bind to a diverse range of antibiotic substrate molecules and how they use energy to drive those substrates out of the cell. This information, in combination with biochemical studies designed to test if the protein performs other physiologically relevant functions, could be of use in the fight against antibiotic resistance in harmful bacteria.

Impact Summary

Antimicrobial resistance is a global health concern that has emerged as a result of the use, and particularly the misuse, of antibiotics. Apart from hampering the control of infectious diseases it threatens a return to the pre-antibiotic era, increases the cost of health care, jeopardises societal health care gains and damages economies. The health burden related to antibiotic resistant microorganisms will inevitably increase in the coming decades. Antimicrobial resistance in bacteria is principally a consequence of the activity of multidrug transporters that can efflux a wide range of structurally dissimilar antimicrobial compounds from the cell. The elucidation of the structure and biochemical characterisation of the MdtM multidrug resistance transporter from E. coli in a substrate-bound conformation will primarily benefit the academic community. However, high-resolution 3-D structural information on MdtM would also be of value to the UK industrial sector since it will provide vital information on multidrug substrate recognition and efflux of antibiotics. Potential beneficiaries of this research include amongst others: 1. Public Sector: Government agencies in the UK such as the NHS. 2. Industry 3. Charities involved in antimicrobial research such as the British Society for Antimicrobial Chemotherapy. 4. Schools: local sixth-form students with an interest in the life sciences and medicine. 5. Academia: UK and overseas universities, including QUB's medical and life sciences students at the undergraduate, masters and doctoral levels. Although the research is of a basic nature and unlikely to have any direct clinical or industrial impact, because it is designed to elucidate the structure(s) and mechanism(s) of a representative of an important family of membrane transporters involved in multidrug efflux it does have the capacity to focus attention on the phenomenon of antimicrobial resistance and misuse of antimicrobials within the health, veterinary and agri-food sectors. Charitable groups with an interest in antimicrobial chemotherapy may benefit from the greater understanding of the drug efflux process. Additionally, this knowledge may help identify ways in which the mechanisms of multidrug efflux could be modified by drugs. Consequently, by feeding knowledge into the pharmaceutical industry, the proposed research has the potential to help ensure that the UK maintains its strong commercial, economic and industrial position. At a local level, students of Queen's University Belfast will benefit by improved, research-informed teaching excellence and in the resources available to them by the host institution's strengthened research position. For example, the programme of work will generate suitable projects for final year undergraduate laboratory dissertations and projects for postgraduate students taking the newly created MSc in Biotechnology. Local sixth form students will also benefit through increased access to work experience opportunities and the inspiration and motivation that research excellence fosters in the young mind; I am an active participant in the School of Biological Sciences work shadowing scheme aimed at enthusing school students to consider a career in the life sciences. Enhancing research capacity is an important aspect of the proposal and will be met by the provision of high quality training and career development of the appointed PDRA. Queen's University offers a number of training courses and workshops aimed specifically at contract research staff. Attendance at these courses would complement the extensive lab-based training in membrane protein structural biology that the PDRA will receive and will develop the transferable skills necessary to enable engagement with, and movement between, different sectors. Research capacity will be further enhanced by the collaboration in place with Dr. Newstead, an internationally recognised scientist in the field of membrane protein crystallography.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Microbiology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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