Antibiotic resistance in Gram-negative bacteria: structure and function of TolC-dependent multidrug efflux pumps

Bacterial diseases present an ominous threat throughout the world, causing great suffering, death, and economic cost, but for our lifetime antibiotics have protected us from the worst. Now however, the increasing prevalence of multidrug resistant bacteria threatens an apocalyptic scenario in which even trivial infections could become serious, often life threatening, and surgery and chemotherapy become nigh impossible. The severity of the problem is recognised by Chief Medical Officer Sally Davies who in the last year stated the problem is on a par with terrorism and global warming, and the World Health Organisation described the problem as "one of the three greatest threats to human health". A review commissioned by Prime Minister David Cameron suggested that without action now, antibiotic resistance could be responsible for 300 million premature deaths by 2050. It is therefore more important than ever to perform sophisticated studies on antibiotic (drug) resistance to learn how bacteria survive antibiotic treatment, and show us new ways to combat this threat.

In our laboratory we have over decades built up a step-wise programme of research in which we combine a range of powerful experimental approaches to study the biology of pathogenic (disease-causing) bacteria. Here we seek a continuation in funding to further our analyses of the machineries that bacteria build in their cell envelope to expel antibiotics underpinning the ability of bacteria like Salmonella, E. coli and Pseudomonas to become multidrug resistant. These drug efflux machineries are all made up of three components and span across the inner and outer lipid membranes and intervening space, the periplasm, and use energized transporters to bind and deliver or 'pump' their cargo of antibiotics to a cell exit duct called TolC which we have shown acts as a universal cell 'trash chute' opened to the outside of the cell. The third component, the periplasic adaptor, helps the transporter assemble and work with the TolC exit duct.

These tripartite multidrug efflux pumps differ in the detail of their specific components and inter-component interactions, and the range of antibiotics they bind and eject from the bacterial cell. We have succeeded in describing the structure and action of the TolC exit duct and adaptor components common to all the pumps, and defined their interactions with each other and the transporter in the most widespread class of drug efflux pump. This culminated in the first precise atomic view of a complete assembled multidrug efflux pump, and gave an insight into how it opens to allow efflux, and how this might be inhibited. We now aim to further refine our existing pump model, and exploit our hard-won expertise to shift focus to define the component structures and interactions in the two other classes of antibiotic pump that use different transporters and therefore assemble differently. We also aim to instigate new approaches to establish in detail how the pumps bind and translocate the drugs from the living bacterial cell. In these ways we hope to visualize how all the different drug efflux pumps assemble and operate within the bacterial cell envelope. In so doing we will offer a better understanding of the strategies bacteria employ to infect us and, resist chemotherapy, and by identifying weak points in the pumps open up new ways for us to inhibit and counteract them.

The increasing prevalence of bacterial multidrug resistance (MDR) presents a growing threat, and it is important to understand antibiotic resistance mechanisms and elicit ways to combat them.

We seek a continuation in funding from mid 2015 to extend our analyses of the tripartite efflux pumps that underpin MDR throughout Gram-negative bacteria like E.coli, Salmonella and Pseudomonas. These pumps comprise inner membrane transporters that bind and deliver antibiotics to the cell exit duct TolC that projects across the periplasm and outer membrane, assembly and function aided by the third component, the periplasic adaptor. There are three classes of tripartite MDR efflux pumps, differing in their transporters and the antibiotics they eject. By combining approaches, especially the crystallography and in vivo cross-linking of membrane proteins and complexes, we have described the structure and action of the exit duct and adaptor components common throughout the pumps, and defined their interactions with each other and the transporter in the widespread RND class of drug efflux pumps exemplified by E.coli AcrA-AcrB-TolC. This allowed us to model the first data-based atomic view of a complete assembled multidrug efflux pump, and provide insight into how the TolC periplasmic entrance opens to allow efflux, and how it might be inhibited. We aim to further refine our RND pump model, and shift focus to the component atomic structures and assembly of the two other distinct classes of antibiotic efflux pump, ABC and MFS, exemplified by E.coli MacA-MacB-TolC and EmrA-EmrB-TolC. We will also instigate approaches to establish how pumps bind and translocate antibiotics from live bacteria. In these ways we aim to visualize how all the drug efflux pumps assemble and operate within the cell envelope to offer a better understanding of the resistance mechanisms bacteria use to evade antibiotics, and also identify weak points in the pumps that may open up new ways for us to inhibit them

The greatest immediate beneficiaries for our work will be within the academic community. Our work provides fundamental understanding of efflux pump structure and function that is essential for understanding bacterial multidrug resistance (and other efflux processes) at the molecular level.

There is also potential for future impact on the private sector as the structures we intend to determine are anticipated to be vital to those seeking to develop inhibitors of these pumps that could later be developed for clinical use.

The work also benefits those seeking to use TolC-dependent pumps for biotechnological applications, providing fundamental knowledge on pumps that could be exploited by researchers currently involved, for example, in developing the protein-exporting TolC dependent tripartite pumps for use in large scale protein production systems, and/or seeking to co-opt these pumps for use in secreting biofuels from engineered E. coli.

We will also make significant societal impacts through training of researchers, developing new cohorts skilled in current molecular microbiology, structural biology and biochemical techniques. Researchers such as these are vital to the continued success of the UK's increasingly knowledge-led economy and to maintain the UK's status as a leading scientific nation. Such researchers are in high demand in the industrial sector, and previous members of our labs have taken up important positions in academia.

In addition to acquiring valuable technical expertise, the RAs employed on this project will work with the PI, Co-I, Cambridge University Personal and Professional Development Services and the University Careers Service to develop transferable professional skills (e.g. Leadership and Management Development, Presentation and Communication) that will equip them for transition to many employment sectors.

We will also be involved in raising public awareness of the problem of growing antibiotic resistance through dissemination of of our work, and engagement with local schools and University-led outreach events.

Finally, in the longer term, the fundamental framework of understanding reached on the structure and mechanism of bacterial efflux pumps will be essential in formulating the means to address the spiralling problem of growing antibiotic resistance among Gram negative bacteria.