Overcoming antibiotic resistance by studying antibiotic hypersensitivity

Lead Research Organisation: University of York
Department Name: Biology


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.

Technical Summary

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.

Planned Impact

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.
Description We now understand the components of the complete pathway leading to sugar attachment to proteins (protein O-glycosylation) in the innately antibiotic resistant bacterium, Streptomyces coelicolor.
We have identified 24 proteins located on the surface of the bacterium that are modified by sugars.
Disruption at any stage of the protein modification pathway leads to hypersensitivity of the bacterium to multiple antibiotics and retarded growth.
Multiple lines of evidence indicate that the protein modification by sugars is required for normal protein function.
Mitigation of the growth defects sustained through disruption of the protein modification pathway occurs through remodeling bacterial metabolism.
Work by others showed that disrupting the equivalent protein modification pathway in Mycobacterium tuberculosis leads to a non-virulent phenotype and/or lethality; as Streptomyces strains lacking protein glycosylation are viable, they are a good model to understand protein O-glycosylation in bacteria.
Exploitation Route Protein modification by sugars in bacteria is poorly understood and we do not consider its implications when we study the function of proteins. Our research highlights the role of protein modification.
Our research could be taken forward to understand in detail the role of modified proteins in the construction of the peptidoglycan cell wall - the target of multiple antibiotics.
We envisage that the protein glycosylation pathway could be a viable target for the rational design of co drugs that could be used alongside existing antibiotics for use against Mycobacterium tuberculosis.
Sectors Healthcare

Description Our findings will be used to support a new bid (for EU funding) to continue research on developing strains to convert low value sterols to high value steroids.
First Year Of Impact 2017
Sector Manufacturing, including Industrial Biotechology
Impact Types Economic

Description James Burgess scholarship
Amount £90,000 (GBP)
Organisation Burgess Road Surgery 
Sector Public
Country United Kingdom
Start 10/2016 
End 09/2020
Description NPRONET-NIBB 
Organisation University of Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution Management board member, scoring pump priming applications.
Collaborator Contribution NPRONET provide a network of contacts from academia and industry for our research
Impact NPRONET workshops and pump priming have stimulated networking and research on natural products.
Start Year 2013
Description Delivery the Leeds Darwin Day lecture to the British Humanist Association 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact British Humanist society invited me to deliver a lecture entitled 'Back to the Dark Ages; Evolution and antibiotic resistance'. This was a 1 hour lecture followed by 30 minutes of Q&A.
Year(s) Of Engagement Activity 2017
URL https://humanism.org.uk/2017/02/09/is-humanity-heading-back-to-the-dark-ages-professor-maggie-smith-...
Description Opera North/University of Leeds sandpit 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Media (as a channel to the public)
Results and Impact Opera North/University of Leeds 'Sandpit' event. This activity was to stimulate the idea of dramatising the notion of the 'end of the antibiotics era'. My role in the workshop was just to answer questions about the science of antibiotics.

The outcome of the sandpit was a show entitled 'Transmission' that has been shown at at least 2 venues; one of these was within the University of York during the 'Festival of ideas'. This show itself - a dance - was entirely the work of Becs Andrews and Mike Brockhurst (University of York).
Year(s) Of Engagement Activity 2013
Description Pint of Science 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact an evening talking about the micro biome and antibiotics. The evening increased the debate on antibiotic resistance
Year(s) Of Engagement Activity 2015
URL https://pintofscience.co.uk/event/resistance-and-virulence/
Description Yornight 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact And evening of talks on the value of antibiotics, the micro biome and antibiotic resistance. Sparked discussion
Year(s) Of Engagement Activity 2015
URL http://yornight.com/2015/activities/medical-society/
Description sky news 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Sky news filmed the research lab and interviews on the subject of AMR
Year(s) Of Engagement Activity 2015
URL http://www.gettyimages.co.uk/detail/video/shows-interior-shots-research-scientists-working-in-a-lab-...