Molecular Basis for Substrate Recognition of Outer Membrane Proteins of the Human Pathogen Pseudomonas Aeruginosa

Bacterial membranes are notoriously impermeable - this is one of the reasons they are so difficult to beat with currently available antibiotics. This is leading to the widespread development of so-called 'superbugs'; bacteria that are resistant to many different antibiotics. This phenomenon provides modern healthcare with a serious problem. Pseudomonas aeruginosa is a human, animal and plant pathogen that is resistant to many different antibiotics. In humans, it tends to target those with already compromised immune systems, and is responsible for a large proportion of hospital acquired infections.

Before we can embark on rational drug design to combat Pseudomonas aeruginosa, we must understand the routes via which the bacterial cell can be penetrated. Given that nature has had the benefit of years of evolution, it makes sense to exploit this by first trying to understand how molecules necessary for the survival of the bacterium, enter it. In this project we will use a range of computational methods supported by electrophysiology experiments by our collaborators, to elucidate the molecular pathways taken by dipeptides to enter the cell through the OprD protein. This protein is one member of the largest family of substrate-specific channel proteins located within the outer membrane of Pseudomonas aeruginosa.

Recently reported studies reveal that using a combination of antibiotics (combination therapy) can be more effective than administering just one type. However the molecular-level consequences of combination therapy are currently completely unknown. Thus, in addition to studying OprD under 'normal' conditions, we will also explore how the protein behaves when its local environment is altered by polymyxin B1- an antibiotic.

The development of bacterial resistance to antibiotics for humans, animals and plants is a major problem for modern healthcare. This has recently been highlighted by a World Health Organization report, 2014 (ISBN: 978 92 4 156474 8) which states: "resistance to common bacteria has reached alarming levels in many parts of the world and that in some settings, few, if any, of the available treatment options remain effective for common infections"

The overall aim is to facilitate the rational design of novel antibiotics. To do this, we must lay the molecular-level groundwork. This project aims to do just that by focussing on the Gram-negative bacterium, Pseudomonas aeruginosa. We will employ a combination of molecular modelling, docking, steered molecular dynamics, umbrella sampling and straightforward equilibrium molecular dynamics to elucidate the molecular recognition pathways utilised by dipeptides as they permeate through the protein, OprD to get across the outer membrane of Pseudomonas aeruginosa. The combination of methods we plan to use will provide us with information about individual molecular interactions that mediate the protein-substrate recognition and subsequent permeation processes including quantitative information regarding the relative energetics involved. Thus this project will constitute one of the most detailed computational studies of a bacterial outer membrane protein.

Our calculations will be backed up by experimental data from electrophysiology studies by our colleagues in Bremen. The bi-directional flow of information between theory and experiment will serve to improve the theoretical models and also guide future experimental studies.

The science represented by this proposal will have an impact on the academic and private and public sectors, facilitated by our plans for dissemination, outreach and public engagement.


Public sector:
The impact on the public sector will largely be in the context of education. We have recently started a new initiative to improve visualisation of academic posters to render them more interactive and hence ideal for educational purposes. This is initiative is supported by RIM, who are providing Blackberry hardware for SK to produce prototype posters than incorporate touch screen technology for interactive visualisation of simulation data. This project will provide data for such educational posters. Furthermore we propose to fully develop our prototype 'arcade machine' for running interactive molecular dynamics simulations for 'hands-on' demonstrations. We will visit schools to give talks and demonstrations throughout the duration of the project.


Commercial sector:
There is scope for commercialisation of the interactive posters as well as the software for searching the database of simulation movies. Novel antibiotics will of course have an impact for the commercial sector, and we plan to work closely with pharmaceutical companies to realise this impact as efficiently as possible. Realistically, the development of new drugs will require computational and experimental work beyond the current project.


Staff working on this project will develop skills in: a range of cutting-edge molecular simulation methodologies, including docking, free-energy calculations, steered molecular dynamics simulations as well as experience of working with complex bacterial membrane models. We are one of only 2 or 3 laboratories in the world with this expertise, thus this represents an excellent training opportunity for the postdoc. The postdoc will be required to interact with experimental colleagues and therefore will learn valuable communication, collaboration and interpretation skills.

In summary, the results of our proposed project will underpin the development of novel antibiotics, in particular those that are potent against Pseudomonas aeruginosa, thus the project will contribute to enhancing the quality of life and health. Commercialisation of the antibiotics will improve the economic competitiveness of the UK. The realistic timescales to achieving the full impact of the project will be a few years after its completion given we propose to first establish the fundamental principles of protein-substrate recognition, it will take time for these to be translated into viable therapeutics. Thus this project represents impact that will be realised in the long-term and will be long-lasting.