Dissecting the structure, dynamics and mechanism of drug efflux pumps associated with antimicrobial resistance

Antimicrobial resistance is a major global health concern. In the EU alone there are ~25,000 deaths per year directly associated with drug resistant bacteria, costing an estimated €1.5 billion in extra healthcare costs and loss of productivity. Unchecked, the global death toll is predicted to exceed 10 million per year, costing over 100 trillion dollars in loss of productivity by 2050. As well as the need for new drug discovery, developing the means to combat resistance itself, by inhibiting known resistance mechanisms, is a promising approach, as it could breathe new life into drugs currently rendered ineffective and prolong the effectiveness of newly developed drugs.

One of the most effective mechanisms bacteria have developed to resist the effects of antimicrobial agents is to pump them out of the cell before they can do any damage. Multidrug resistance transporters are transmembrane molecular machines responsible for the majority of drug efflux in bacteria. Single multidrug resistance transporters can expel a huge range of structurally diverse substrates, thus undermining the efficacy of a wide variety of drugs in one fell swoop. Understanding the mechanisms of drug efflux transporters, and ways of inhibiting them, will increase both the efficacy and longevity of current and future antimicrobial agents.

This project will focus on understanding the molecular mechanisms of secondary active drug efflux pumps; in particular those from the multidrug and toxic compound extrusion (MATE) family of transporters. MATEs confer resistance to several pathogenic bacteria, including Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA) and Clostridium difficile. Much is known about the structure of the MATE transporters from crystallographic studies; yet the mechanism by which these transporters pump drugs across the membrane remains elusive. The goal of this project is to probe the structure, function and dynamics of several members of the MATE family to understand how they work. The project will focus on three major questions; how do these proteins recognise such a diverse range of drugs; how do they harness an energy source and couple it to drug pumping, and what conformational changes are required for this process? To do this, the structure and activity of the MATE transporters will be probed using assays in bacterial cells as well as with purified protein in detergent solution and reconstituted into proteoliposomes (purified protein inserted into artificial membranes). A suite of molecular biology methods, as well as biochemical and biophysical tools, including spectrofluorometry, chemical crosslinking, and advanced in vitro transport assays, will be used to elucidate the mechanism of drug efflux.

A thorough mechanistic understanding how these transporters work will give us a better understanding of this important antimicrobial resistance mechanism and potentially provide a basis for future drug design.