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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - PACEMech (The structure and molecular mechanism of transport proteins within the PACE family of multidrug efflux pumps)

Teaser

Summary

What was the problem/issue being addressed?
Bacterial pathogens that infect the most vulnerable patients in hospitals worldwide are becoming increasingly resistant to antibiotics. Drug efflux is a primary mechanism of resistance intrinsic to bacteria and is mediated by integral membrane transport proteins. These proteins act as â€˜pumpsâ€™ and lower the concentration of antimicrobial drugs within a cell by moving them across the cell membrane and out of the cell. Most drug efflux pumps recognise a wide range of structurally dissimilar antimicrobial compounds. Consequently, the increased expression of just one of these â€˜multidrugâ€™ efflux pumps in a bacterial strain in response to antimicrobial selective pressures can result in resistance to a swathe of antibiotics.
We recently identified a new family of bacterial multidrug efflux pumps, which we called the Proteobacterial Antimicrobial Compound Efflux (PACE) family of efflux pumps. This was the first new family of drug efflux pumps to be described in 15 years. Members of the PACE family confer efflux mediated resistance to structurally distinct biocides and are highly conserved in the genomes of several major human pathogens including Acinetobacter, Pseudomonas, Klebsiella, Salmonella and Burkholderia species. Due to their recent identification, we had little mechanistic or structural information for proteins classified in the PACE family. This proposal aimed to provide fundamental details of the transport reaction and structure of PACE family proteins, with the view that this information may be used in future drug development projects to interfere with their resistance functions.
Why was it important for society?
Drug resistance in bacterial pathogens is one of the major challenges to human health worldwide. The occurrence of multidrug and pan-drug resistant bacterial pathogens in hospitals continues to rise.In 2017, the World Health Organisation (WHO) published its first ever list of drug resistant bacterial pathogens for which new antibiotics are urgently needed to guide drug development efforts. All of the pathogens in the top category (Priority 1 â€œCRITICALâ€ targets) were Gram-negative bacteria, including strains of Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacteriaceae. PACE family proteins are encoded by all of the Priority 1 pathogens listed by the WHO and could be contributing to their high levels of antimicrobial resistance.
What were the overall objectives and what conclusions have been made?
This project applied a combination of sophisticated research technologies available in the Astbury Centre for Structural and Molecular Biology at the University of Leeds, and drew on the diverse collective expertise of the Fellow, Dr Karl Hassan, the Fellowship Supervisor, Prof Peter Henderson, and other Faculty members within the Astbury Centre. The broad research objectives were:
1) To investigate the transport mechanism of PACE family transport proteins. Many elements of PACE transport function needed to be clarified. For example, active transport proteins require energy to mediate the transport of substrates against a concentration gradient (usually chemical energy or the energy held in another concentration gradient). The energy source for PACE proteins was unknown. Additionally, the spectrum of compounds that can be transported by PACE family proteins had not been fully elucidated. It has been shown that several biocides are transported, but we suspected that PACE proteins also recognised other substrates. Finally, previous experiments of PACE family proteins had been conducted in whole bacterial cells. Therefore, it had been questioned whether they were able to function independently in the absence of all other cellular proteins.
2) to generate structural information for a PACE family pump. Structural information for a catalytic protein provides huge insights into how it operates and how we might be able to influence or perturb its function. Trans

Work performed

During the project we made significant advances in our understanding of PACE family proteins, particularly in their transport mechanism. As a result of this work we now have detailed knowledge of their physiological functions, we demonstrated for the first time that they are able to function independently, and we showed that they are energised by the proton-motive-force, using protons as counter ions for substrate efflux via an antiport mechanism.
Some of the functional data obtained during the project have been detailed in a recently accepted paper (Hassan et al., Research in Microbiology, Accepted; https://doi.org/10.1016/j.resmic.2018.01.001; online 2 February 2018). The remaining results, including the highly significant discoveries of the physiological function of PACE pumps, their transport self-sufficiency and their mode of energisation will be described in a manuscript that is currently in preparation for submission.
We initiated structural investigations of PACE proteins in collaboration with research groups at the University of Leeds, using membrane protein crystallography and single particle electron microscopy. The preliminary data obtained were encouraging with respect to the possibility of obtaining a good resolution structure for a PACE protein.

Final results

The results of the project may be translated into novel therapies for treating drug resistant infections, and subsequently have large impacts on health and economies. There is significant interest in developing inhibitor compounds for multidrug efflux proteins, such as those within the PACE family. An inhibitor compound could be co-administered to augment the activities of antimicrobials to treat or prevent drug resistant infections. Additionally, the inhibition of drug efflux pumps has been shown to stall the development of additional, clinically-relevant levels of drug resistance, via alternative resistance mechanisms synergistic to efflux. Consequently, the deployment of a PACE inhibitor could reduce rates of infection and slow the development of additional levels of drug resistance in Gram-negative bacterial pathogens. The results of this work lay a foundation for understanding the parts of PACE proteins that are likely to constitute the substrate and proton binding sites, and the types of substrates that can bind and interact at these sites. This information is significant in determining the types of compounds that should be screened for novel inhibitors.
During the Fellowship, Dr Hassan was appointed as a full time Senior Lecturer at the University of Newcastle, Australia. This was a key ambition of Dr Hassan and highlights the esteem of the Fellowship, as well as the outstanding quality of the research training that was obtained in the host laboratory.