Mapping the interactions within multidrug efflux pump assemblies.

Lead Research Organisation: University of Essex
Department Name: Life Sciences

Abstract

Multidrug-resistant bacterial infections are one of principal challenges facing medicine today. A group of bacteria known as the Gram-negative are particularly resistant to the action of antibiotics, as they have evolved a secondary membrane around their cells, preventing easy entry of the antibiotics. No new antibiotics targeting this group have been developed for over 40 years and the need to find and exploit novel bacterial weakness points is of great importance for both human and veterinary medicine. One of the central mechanisms underlying their multidrug resistance, is the action of the of the so-called multidrug-efflux pumps. These assemble from 3 components, spanning the double membrane and pumping out antibiotics, lowering their effective concentration in the cell thus rendering them ineffective. While pumps are very diverse, they share a common outer membrane protein (OMP). Deactivation or removal of OMPs dramatically increases bacterial sensitivity to antibiotics, suggesting that targeting the OMP may be an effective therapeutic approach. Yet none of the currently available drugs target the pump assembly process. Partially this is due to lack of information on the interactions between pump components to design targeted inhibitors.

This project specifically aims to close the gap in our understanding of pump intercomponent interactions with the view of disrupting their assembly.
The study of full pump assemblies has been hindered by their complexity and transient association of their elements, preventing effective usage of standard structural approaches such as X-ray crystallography.
Here, we will overcome these bottlenecks by innovative usage of multiple approaches. The first is the application of the novel technique of X-ray Radiolytic Footprinting (XRF), allowing to study transient and heterogeneous proteins in solution. XRF is a mapping technique, which is based on oxidation of the surface-exposed parts of the protein by usage of highly reactive hydroxyl radicals. These radicals are created by splitting water molecules in solution by usage of high-energy X-rays. The pattern of oxidation is detected by the molecular weight differences of fragmented proteins using a technique called mass-spectrometry. Comparison of the modification of a given protein on its own with the modification obtained in the presence of its binding partner reveals zones of protection, or footprints, corresponding to the interaction surfaces between the proteins.
By using this approach we will map the tripartite pump complex and will use the information to specifically target the binding interfaces by mutagenesis to disrupt the association of the OMP with the rest of the pump. We will characterise the effect of these mutations on antibiotic resistance of the cells to identify crucial residues.

Furthermore, we will use modified MFP proteins with truncated and scrambled hairpins to dissect their role in the assembly of the pump, allowing to distinguish between the two currently contradictory models of assembly.
Recently, we have shown that the hairpin-domain of the MFP binds the OMP with higher affinity than the corresponding full-length protein. Furthermore it binds in an energy-independent fashion. However, unlike the full-length protein the binding of the hairpin does not produce a functional pump. Here, we will exploit these findings by further engineering stabilising interactions of the hairpin with its target OMP, and will test its capability to outcompete native MFPs and inhibit the function of the pump in vivo.

This project will further our fundamental understanding of the pump assembly and settle the long-standing debate on the mode of MFP-OMP interaction. Demonstrating competitive inhibition of the MFP-OMP binding interface as viable strategy will pave the way to future design of a completely novel class of drugs targeting the assembly process providing a powerful tool in the fight against drug-resistance.

Technical Summary

In Gram-negative bacteria tripartite efflux-pumps expel wide range of noxious compounds from the cell including antibiotics and thus are central contributors to multidrug resistance. Spanning both membranes these systems are composed of 3 components: the Inner Membrane (IMPs); the Outer Membrane Proteins (OMPs) and Membrane-Fusion Proteins (MFPs). Although high-resolution structural data is available for each protein in isolation, very little is known how the pumps actually assemble into their functional state. Despite their key role in drug resistance, no currently approved drugs target the pumps and the potential of interference with the OMP-MFP assembly has not been explored.

This project will provide detailed mapping of the OMP-MFP interfaces underlining pump assembly and test the potential of competitive inhibition of OMP-MFP interaction as a novel antimicrobial strategy. To this end we will map protein-protein interfaces using a unique approach, X-ray radiolytic footprinting (XRF) applicability of which to membrane proteins we recently demonstrated. Once identified, we will use a combination of site-directed mutagenesis of both OMP and MFP interfaces with functional and biophysical analyses to identify residues critical for the pump assembly. Recently we showed that the MFP binds the OMP in an energy-independent fashion driving its opening, while isolated MFP hairpin-domains bind the OMP with up-to 100x higher affinity than the full-length MFP, but are unable to complement pump function. Taking advantage of these findings and the residue-specific data derived from the XRF, we will design full-length and truncated MFP-derivatives and test their ability to outcompete native MFPs in vivo to effectively decouple the pumps. This integrative approach will clarify the role of the MFPs in pump assembly allowing to validate the MFP-OMP interaction as a potential drug target providing high-impact results and new tools in the in the fight against multi drug resistance

Planned Impact

This study addresses directly the rising bacterial multidrug resistance (MDR), one of principal threats facing humanity and will benefit both academic and non-academic groups. It fits squarely into the BBSRC strategic priority area of "Antimicrobial resistance" and specifically the BBSRC call for need in development of strategies to mitigate its effects e.g. through novel antimicrobials. By studying a fundamental area of microbiology - efflux via tripartite efflux pumps - it will provide deeper understanding of the mechanisms underlying resistance, allowing development of a novel mitigation strategy for non-specific drug resistance based on the inhibition of their action. The basic science developed will be applicable to all Gram-negative organisms, underpinning the creation of genuinely novel class of antimicrobials for animal, human and plant use. Validating OMP-MFP interaction as a potential drug target provides completely novel approach to design of pump inhibitors, relevant to all species, providing wide translational potential for future research and great economic value.
Impact is expected to come from several main streams:
1. Increase of fundamental knowledge of pump function, benefitting wider academic community.
2. Translational potential from the conceptual validation of a novel strategy for inhibition of pump function at OMP-MFP level, influencing future drug design in both human and veterinary medicine.
3. Methodological development in radiolytic footprinting (XRF) advancing our capabilities for studying membrane proteins.
4. Forging strategic international collaborations and bringing capability not currently available in the UK to increase competitiveness and provide high-tech training for the participants.
Who will benefit from this research and how?
1. Academics on national and international levels working on MDR and structural biology. Anticipated benefits include increased basic knowledge and sharing of methodologies.
2. Students/early career researchers: My teaching involvement at UoB provides a key opportunity to engage students, allowing their exposure to cutting-edge research and experimental techniques.
3. Research Staff: These users will benefit in terms of training in specific research techniques and also general professional and transferrable skills.
4. Translational potential: increased knowledge of central drug tolerance mechanisms will ultimately inform strategies intervening in the treatment of MDR pathogens. By specifically testing the feasibility of the interfering with pump assembly as a viable antimicrobial strategy for the first time we provide a major step into this direction. Detailed interaction maps generated by this research will be an important departure point for rational drug-design.
5. Wider socio-economic impacts: The potential impact of understanding and ultimately manipulating MDR mechanisms is difficult to underestimate e.g. according to the authoritative Government review http://amr-review.org, MDR claimed over 700k lives globally as of 2013, a number projected to spiral out to over 10M by 2050, with major societal and economic costs resulting in a reduction of up to 3.5% in the Gross Domestic Product (GDP) per country, and estimates totaling a staggering $100 trillion for the global economy over the next 3 decades.
6. Methodology development: We will provide novel tools to study membrane proteins representing over 60% of all drug targets. Economy of scale in required sample size will have an important impact enabling to address difficult to produce targets.
7. Increasing the competitiveness of UK science by allowing access to know-how and unique state-of-the-art experimental set-ups without analogues in Europe and forging new International collaborations.
8. Public. We will engage wider non-academic users with research directions and outputs from the study, increasing basic knowledge and understanding of research undertaken through the support of BBSRC.

Publications

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Description A note: The project is currently active with over 8 months still remaining (till early December 2019). The project was suspended for a while associated with the untimely departure of the post-doctoral research associate (PDRA) associated with the grant, from November 2017 to mid-April 2018. The work on the grant has been resumed with the recruitment of a new PDRA. While we have encountered some issues with reproducibility of the data generated during the first half of the project, it is now fully on track.

Prior to the departure of the first research associate we have made notable progress - namely, we have established a pattern of recognition between the proteins within the multidrug-efflux pump assemblies. These pumps are responsible for active pumping of antibiotic substances out of the bacterial cell and we are hopeful that this pattern of recognition between main participating proteins can be exploited for future therapeutic use - namely the outer membrane proteins (OMPs) of TolC family and periplasmic adaptor proteins (PAPs).
For that purpose we have created a number of mutants of the two main partner groups of proteins and following the characterization of their impact on the functional properties of the pumps we have developed a novel model of outer membrane channel gating in Neisseria that is being further corroborated and validated with the help of the new structure (see below).
One of the key outputs of this grant is a result of a productive collaboration with the group of Prof. Ed Yu (Case Western Reserve University, USA) with whom we have obtained crystal diffraction data and have refined a new crystal structure of an outer membrane protein from Neisseria. We are currently finalizing the results for publication pending for some additional control experiments.

Another major outcome has been a result of a newly-initiated collaboration with the group of Dr Jessica Blair (U. of Birmingham, UK) which capitalized both on the structural modelling which we have advanced and our mutagenesis work developed within the framework of this project. The work has resulted in first time identification of the binding-residues involved in transporter - PAP interaction in Salmonella and hinted at novel mechanisms of recognition between these proteins, providing a framework for further targeting of their interaction. This work is currently submitted for publication in a leading journal with Dr Bavro as a joint corresponding author.

The third output has come from a further investigation into the Salmonella outer membrane proteins (OMPs) discussed above and LPS involvement in modulating and restricting the immunological protection afforded by antibodies that target these surface structures. In collaboration with the groups of Prof. Adam Cunnigham (U.of Birmingham), Prof. Jeremy Lakey (U. of Newcastle) and Dr. J.C. Gumbart (Georgia Institute of Technology, USA) we have developed a novel predictive model of antibody binding to the surface antigens that may inform future vaccine design. This highly impactful research is currently under consideration in a leading journal with Dr. Bavro as a joint corresponding author.
Exploitation Route At this stage we still do not have any published results that can be directly taken forward by others. We anticipate to be able to deliver new structural (and by proxy) functional understanding of the process of assembly of the multidrug efflux pumps, allowing specific targeting of the protein-protein interfaces involved. A central part of our proposal is the demonstration of the proof of principle of exploiting interference with pump assembly in vivo to impact antibiotic sensitivity in Gram-negative bacteria. If successful this will open the possibility of design of targeted inhibitors of assembly, which would be useful both as research tools and will potentially have much wider therapeutic application. Control of pump function is of interest not only for the field of antibiotic resistance, but also to biotechnology and pharmaceutical industry e.g. for bioethanol production and protein secretion modulation.
Importantly, our work with Prof. Cunningham on the antibody recognition of the cell-surface antigens in Gram-negative bacteria will have a clear impact on the development of future vaccine candidates.
Sectors Agriculture, Food and Drink,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology,Other

 
Description PVC (Research) Infrastructure Fund 2017/18
Amount £100,000 (GBP)
Organisation University of Essex 
Sector Academic/University
Country United Kingdom
Start 07/2018 
 
Description Mapping of PAP-RND pump interfaces - Dr Jessica Blair 
Organisation University of Birmingham
Country United Kingdom 
Sector Academic/University 
PI Contribution As a part of this collaboration my group has provided access to prior data; equipment and facilities as well as materials - including our mutagenesis libraries. We have provided bioinformatic and structural biology expertise, along with molecular docking approaches to guide and rationalise mutagenesis work. We have generated and tested a number of novel mutants in vivo and in vitro. We have provided intellectual input throughout guiding the direction of the joint collaborative effort. We are involved in joint delivery of the research outputs and manuscript preparation.
Collaborator Contribution As a part of this collaboration Dr Blair's group has provided access to prior data; equipment and facilities as well as materials. Dr Blair's group has provided training for the PDRA involved in the project. They have contributed microbiological and in vivo characterisation of the strains we have designed and produced and contributed to optimisation of protocols involved. They have provided intellectual input throughout guiding the direction of the joint collaborative effort. Dr Blair's group are involved in joint delivery of the research outputs and manuscript preparation.
Impact The work has resulted in first time identification of the binding-residues involved in transporter - PAP interaction in Salmonella and hinted at novel mechanisms of recognition between these proteins, providing a framework for further targeting of their interaction. This work is currently submitted for publication in a leading journal with Dr Bavro as a joint correspondent author.
Start Year 2018
 
Description Matthias Winterhalter 
Organisation Jacobs University Bremen
Country Germany 
Sector Academic/University 
PI Contribution With the help of the team of Prof. Winterhalter are characterising a number of mutations introduced into the outer membrane proteins and periplasmic adapter proteins (PAPs) using elecrophysiology approaches.
Collaborator Contribution Our team is providing the protein constructs and mutagenesis libraries of outer membrane proteins and periplasmic adapter proteins (PAPs) which form part of the tripartite efflux pumps, while the group of Prof. Winterhalter provides incorporation into lipid bilayers and in vitro characterisation of these proteins using electrophysiology.
Impact This collaboration is in the early stages of development and we are still awaiting the results of the pilot experiments.
Start Year 2017
 
Description Prof. Adam Cunningham - University of Birmingham 
Organisation University of Birmingham
Department Institute of Immunology and Immunotherapy
Country United Kingdom 
Sector Academic/University 
PI Contribution Our group is providing our expertise in structural biology and modelling. We are providing access to data and computing facilities, as well as protein purification and crystallisation facilities. We provide intellectual input throughout in guiding collaborative research as well as reporting the outputs.
Collaborator Contribution Prof. Cunningham's group is providing expertise in immunology, including access to in vivo mouse models of infection, antibody libraries and in vitro assays. They are providing access to data and facilities including access to in vivo mouse models of infection, antibody libraries and in vitro assays. They provide intellectual input throughout in guiding collaborative research as well as reporting the outputs.
Impact In collaboration with the groups of Prof. Adam Cunnigham (U.of Birmingham), as well as Prof. Jeremy Lakey (U. of Newcastle) and Dr. J.C. Gumbart (Georgia Institute of Technology, USA) we have developed a novel predictive model of antibody binding to the surface antigens that may inform future vaccine design. This highly impactful research is currently under consideration in a leading journal with Dr. Bavro as a joint correspondent author. This is a multidisciplinary collaboration with each partner providing expertise in different disciplines: Dr Bavro - Structural biology of membrane proteins; Molecular modelling; Prof. Cunningham - Immunology and in vivo models of Samonella infection; Prof. Lakey - LPS chemistry and Gram-negative membrane biology; Dr. J.C.Gumbart - Atomistic molecular dynamics simulations;
Start Year 2017
 
Description Prof. Ed Yu 
Organisation Case Western Reserve University
Country United States 
Sector Academic/University 
PI Contribution We have collaborated with the team of Prof. Edward Yu (Case Western Reserve University) on solving of the 3D structure of a novel membrane protein, which is part of the TolC family. The structure provides new insight into the functioning of the family and will form a centre piece of a manuscript, currently in preparation.
Collaborator Contribution Our team have performed essential functional data, in vivo and in vitro characterisation of the protein, structural validation and data analysis work, while the partners from Case Western have provided the data collection and crystallisation facilities. This collaboration is currently ongoing.
Impact The collaboration is currently ongoing. We have recently solved a crystal structure of a membrane protein, which will be deposited into the PDB databank (www.rcsb.org/) and we are preparing a manuscript describing it.
Start Year 2016
 
Description Victor Lin 
Organisation National Taiwan Ocean University
PI Contribution We have contributed molecular modelling and data analysis for predicting the structure of a novel inner-membrane transporter associated with the drug efflux of macrolide antibiotics. Our team also performed a number of antibiotic sensitivity and efflux assays, which has resulted in the first characterisation of a novel function of the transporter protein. We have jointly contributed to writing a manuscript describing the findings, which is currently in submission.
Collaborator Contribution The team of Assoc. Prof. Hong-Ting Lin, PhD from the National Taiwan Ocean University has been instrumental in identifying the activity of the transporter protein described, creating a number of expression constructs and initial characterisation of the efflux function of the protein. The team of Prof. Lin has jointly contributed to the writing of the manuscript describing the findings.
Impact The collaborative work with Dr Lin's lab has so far resulted in a publication of the following manuscript: https://www.ncbi.nlm.nih.gov/pubmed/29584668
Start Year 2017
 
Description Cafe Scientifique - Public lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Over 60 people attended for a Cafe Scientifique public-engagement in science session in Colchester titled "How do bacteria evade antibiotics?"
Year(s) Of Engagement Activity 2019
URL http://www.cafescientifique.org