Overcoming antibiotic resistance by studying antibiotic hypersensitivity

Antibiotics have saved millions of lives since their discovery. Antibiotics kill pathogenic bacteria by targeting an essential metabolic process. Pathogenic bacteria can protect themselves from antibiotics by altering or providing a new target that no longer binds the antibiotic, destroying the antibiotic or pumping the antibiotic away from the target. Antibiotic resistance is now a serious problem in treating diseases caused by pathogenic bacteria such that in the EU 25,000 people die annually from untreatable infections. Globally there are 440,000 new cases annually of multidrug resistant tuberculosis resulting in 150,000 deaths. Solutions to the problem of antibiotic resistant bacteria are being sought on several fronts including better control over the use of existing antibiotics, the discovery and development of new antibiotics and antibacterial strategies that do not rely on antibiotics such as phage therapy, bacteriocins, antibacterial peptides and vaccines.
A hitherto underexplored but potentially exciting approach is to use combination therapy in which two or more drugs are used simultaneously and act synergistically to kill antibiotic resistant bacteria. This approach has been used for combating HIV and tuberculosis for some time. However combination therapy need not always involve two antibiotics; one of the drugs used may not itself have anti-microbial activity but potentiates the activity of the antibiotic. A well-known potentiator that has been taken by most people is clavulanic acid, an inhibitor of the enzyme beta-lactamase that destroys beta-lactam antibiotics such as penicillin.
How many potentiator targets are there and how do we find them? There is evidence that there are hundreds of potentiator targets in bacteria of varying efficacy and that might act against different types of antibiotics. This evidence comes from measuring the antibiotic sensitivity in bacteria that have single gene mutations; those mutants with greater sensitivity to an antibiotic compared to a strain with an intact gene (the parent) indicate that the mutated gene or its consequences on metabolic processes is a potentiator target.
We have isolated mutants in a bacterium, Streptomyces coelicolor (a relative of Mycobacterium tuberculosis) that are hypersensitive to a subset of antibiotics including two antibiotics that are so-called 'last resort' antibiotics for some pathogenic bacteria. The mutations lie in enzymes required to modify proteins being localised to the outside of the cell with sugars. Knocking out this modification system may have a variety of consequences on metabolic processes, all unknown at present. We hypothesise that if we understand what these consequences are at the metabolic level, we can identify rational targets for potentiators and, in some cases, undermine their resistance mechanisms. Our first objective is to ask whether mutations in the protein modification system in related bacteria are also hypersensitive to establish whether our observations are general, and to initiate screens for potentiator chemicals in collaboration with NovaBiotics Ltd and the Marine Biodiscovery Centre in Aberdeen. Second we plan to determine what major metabolic changes have occurred in the mutants compared to the parent strain by studying the proteins that might be affected by modification in the cell surface and by measuring changes in gene expression. Third we plan to identify what genetic changes need to happen to the hypersensitive strains to make them resistant again and this will point to both an explanation of the hypersensitivity and how resistance to potentiators might arise. At the end of this project we hope to be in a position where we can start screening for potentiators for use with antibiotics that act against Mycobacterium tuberculosis and some vancomycin resistant pathogens.

One approach to extending the use of antibiotics is to use combination therapy in which two or more drugs are used simultaneously and act synergistically to kill pathogens containing either innate or acquired resistance mechanisms. In combination therapy it is not always necessary that both drugs have anti-microbial activity; one of the drugs may simply potentiate the activity of the antibiotic. Recently large scale phenotyping of mutant libraries have revealed many possible targets for potentiators. Mutations in genes, sometimes housekeeping genes, can lead to enhanced sensitivity to antibiotics. Thus antibiotic plus potentiator used in combination should have the same effect on antibiotic sensitivity as antibiotic plus mutation.
We have isolated mutants in S. coelicolor that are hypersensitive to a subset of antibiotics. The mutants have defects in one of two genes that encode enzymes for a protein O-glycosylation pathway. Our hypothesis is that bacteria that lack the protein glycosylation pathway have suffered an alteration in one or more cellular processes that leads to increased susceptibility to certain antibiotics. The aim of this project is to identify the physiological changes in the glycosylation mutants so that we can apply this knowledge to potentiator discovery for combination therapy. We will develop robust screens for potentiators in a variety of organisms that could be used in high throughput screening. The physiological differences between the parent and mutant strains will be examined by global gene expression and analysis of the cell membrane and cell wall proteomes. Processes that are defective in glycosylation mutants may be mediated by glycoproteins and we will identify which proteins are glycosylated. Second site suppressors of antibiotic hypersensitivity will be characterised to identify genetic changes that compensate for hypersensitivity and to provide insight into how resistance to potentiators might arise.

This project addresses the search for novel drugs that can be used against antibiotic resistant pathogens. There are therefore opportunities for scientists, clinicians, industry, and the general public to benefit from his research.
1. Communication to beneficiaries
As for academic beneficiaries, scientists from the pharmaceutical and the biotechnology industries and clinicians will read about our work from our peer reviewed publications and from conference presentations. This application has already attracted interest (see letter of support) from Novobiotics Ltd, a University of Aberdeen spin-out company interested in novel anti-microbials. In association with the Kosterlitz Centre for Therapeutics at the University of Aberdeen we will seek out more industrial partners through direct communications. The University of Aberdeen Research and Innovation Unit will work with us and any collaborators to negotiate fair intellectual property arrangements should any arise from this project.
Communication of the work to the public will be through the Communications Team at the University of Aberdeen whose press releases are frequently then taken up in local and national newpapers, TV channels and web sites. I aim to publicise my research at the Café Scientifique in Aberdeen and through my web site. More broadly I organise an annual Schools microbiology lecture (running now for 6 years) so that up to 300, 15-17 year olds have the opportunity of hearing exciting developments in microbiology from very eminent scientists.
2. What are the benefits?
To industry, medicine and in the veterinary sciences we will provide knowledge and proof of new approaches for combating antibiotic resistant infection. Taking the research to the next step i.e. high through put screening, validation, animal models and clinical trials will clearly required involvement from these groups of people and hence the importance of communication. The benefits of taking the research on to this level will be economic as well as quality of life and wellbeing, which will affect industry and the general public. Communication of the work to the public also imparts benefits in terms of education, understanding and accountability.