Bacterial antibiotic resistance: structure, mechanism and inhibition of ABC transporters responsible for drug efflux and cell wall biogenesis.

Bacterial diseases impose a terrible burden of suffering and death throughout the world but for the past 70 years antibiotics have shielded us from the worst. However, we now face serious peril as the development of antibiotic resistance may, in the words of recent Chief Medical Officer Professor Dame Sally Davies, "kill us before climate change." Increased resistance threatens to revert modern healthcare to a pre-antibiotic era where minor infections become life threatening and even routine surgical procedures carry significant risk. The severity of the problem is acknowledged globally by the World Health Organization, which has initiated coordinated efforts to address this global emergency, which unchecked will lead to millions of premature deaths.

Bacteria like E. coli and Salmonella are surrounded by a complex cell envelope consisting of two lipid membranes, which provides a formidable barrier to the entry of many antibiotics. Resistance to antibiotics is further established by dedicated membrane machineries that recognise antibiotics and eject them from the cell before they have an opportunity to act. These drug efflux pumps are composed of three separate components: an inner transporter protein (of which three different classes exist) which recognizes and passes drugs to a TolC "trash chute" open to the cell exterior, and a critical adaptor protein which controls the assembly of this molecular machine.

Our laboratory has over 30 years built a research program tackling the molecular mechanisms by which bacteria exhibit antibiotic resistance. We have succeeded in describing the atomic structure and action of the TolC exit duct, and the adaptor proteins. We have also atomically defined their interactions with each other, and the transporter component in the most widespread class of these efflux pumps. This culminated in the first precise atomic view of a complete assembled multidrug efflux pump, provided insight into how it opens to allow expulsion, and how this might be inhibited.
More recently, we described the structure of a previously uncharacterised transporter component of a pump revealing a novel structure and atypical mechanism of drug efflux. A related, but distinct, transporter acting in concert with chaperone proteins uses the same architecture to transport lipoproteins, vital components of the protective cell envelope, to the outer membrane. Without this system functioning properly, bacteria cannot survive, making it an attractive target for therapeutic intervention. We seek a continuation of funding for our work, to study these related transporters in drug efflux and lipoprotein transport. By understanding how they recognise the molecules they transport and interact with their partner proteins we will develop a better understanding of the molecular mechanisms by which bacteria resist antibiotics and raise the possibility of counteracting them.

The emergence of antimicrobial resistant (AMR) bacteria is a well-recognized threat to human health through increased incidence, severity, and persistence of infections. In Gram-negative pathogens like E. coli and P. aeruoginosa, AMR is underpinned in two major ways: the robust outer membrane barrier which prevents many antibiotics reaching their intracellular targets and tripartite efflux pumps which eject drugs from the cell. These pumps consist of an inner membrane (IM) transporter, a periplasmic adaptor protein and the TolC exit duct. Previously, we have determined the structures and interactions of several pump components. More recently, we have revealed the structure of the IM transporter MacB bound to ATP, revealing a novel architecture and mechanism of action where cytoplasmic ATP hydrolysis is coupled to conformational changes in the periplasm, a process we have termed mechanotransmission. Topologically-related to MacB, the LolCDE transporter mediates the extraction of lipoproteins from the inner membrane and their subsequent transfer to the periplasmic chaperone, LolA. Lipoproteins are finally inserted into the outer membrane by LolB. Disruption of the Lol pathway at any stage has been shown to lead to cell death. As such, and with no human ortholog in existence, this system presents an attractive target for the identification, and synthesis of novel antimicrobials.

We aim, through our breadth of experience in microbiology, biochemistry, biophysical and structural methods to understand the molecular mechanisms underpinning both antibiotic efflux by MacB tripartite pumps, and lipoprotein trafficking by the Lol system. While the two core transporters bear an overall similarity in fold and likely share the same mechanotransmissive mechanism, they play divergent roles, mediating drug efflux and outer membrane biogenesis. Our studies will further the development of therapeutics capable of interfering with both.

Academic researchers will be the primary beneficiaries of this research proposal. Our work will positively impact on the academic community, providing a fundamental understanding on the structure and mechanism of bacterial translocation of small molecules and proteins across membranes. Our proposed areas of study play a vital role in multidrug resistance, and pathogen viability, both of which are essential areas of scientific focus for multiple academic groups worldwide.

Another noteworthy beneficiary of our publicly deposited structures will be private biotech companies seeking to develop inhibitors of efflux pumps and the lipoprotein trafficking pathway. Access to our existing, and forthcoming structural data will be of significant benefit in these pursuits. Insights into the operation of the Lol trafficking system will also benefit those seeking to optimise the production of recombinant lipoprotein based vaccines.

We will make significant societal impacts through the training of scientists at early stages of their career, preparing skilled people for academic and/or industrial professions. Our laboratory possesses a long-term expertise in studying protein function using molecular microbiology, biochemical and structural techniques which renders it ideally positioned to prepare researchers for professional positions in both academia and industry.

Finally, we will continue to promote our work to non-specialised audiences at outreach events and local schools. In so doing, we will raise public awareness of the problem of bacterial multidrug resistance and emphasise the importance of responsible antibiotic use and the vital need for innovative therapeutic solutions.