Investigation of stochastic variations in growth rate as the mechanism of drug tolerance in Mycobacterium tuberculosis

Mycobacterium tuberculosis is a major pathogen of man and animals. Drug treatment is available for human disease but it takes six months, which is impractical in developing world settings where TB is most common. Consequent non-compliance with treatment regimes leads to the emergence of drug-resistance. This is now a major world-wide problem with practically incurable 'extreme drug-resistant' strains appearing in many countries, including the UK. Treatment would be much more effective if treatment regimes could be shortened. The principle reason why treatment regimes have to be so prolonged is that that a sub-population of cells in lesions are relatively tolerant to the antibiotic, a phenomenon termed phenotypic tolerance or non-inherited antibiotic resistance. The mechanism of phenotypic tolerance is currently unknown but we have recently discovered that it is associated with slow growth in M. tuberculosis. We have recently shown, through theoretical studies, that random variations of growth rate in individual bacteria cells may the mechanisms for generating persisters. This project is to investigate this hypothesis in an experimental system. The results will provide a new understanding of the mechanistic basis of phenotypic tolerance that is likely to provide new targets and opportunities to interfere with the resistance mechanisms and thereby increase efficacy of TB treatment.

Mycobacterium tuberculosis (Mtb) is a major pathogen of man and animals. Drug treatment takes six months or more because the pathogen exhibits phenotypic tolerance to the antibiotic regime in vivo. The key signature of antibiotic tolerance is the biphasic kill curve which is obtained when growing bacteria are exposed to antibiotic. The killing rate is initially rapid but then slows down. This tail of slowly killed bacteria is responsible for the phenomenon of drug tolerance. However, the mechanism by which bacteria become tolerant is completely unknown. The most popular theory involves phenotypic switching of cells between two or more states (bistability) corresponding to normal and antibiotic-tolerant cells. However, there are many problems with this theory, for instance, that it has not been possible to engineer cells in pure normal or persistent states. We have developed an alternative persistence hypothesis whose starting point is the well-established fact that antibiotic killing of bacteria is growth rate-dependent. We then consider cells in which growth rate is controlled by a single gene and demonstrate that such a system would generate biphasic kill curves if the gene is expressed at low level and thereby subject to stochastic noise. The aim of this project is to investigate this hypothesis for the TB bacillus. We will engineer cells whose growth rate is controlled by a gene with a range of promoter strengths. We will investigate population variation in gene expression, growth rate and antibiotic killing by single cell studies and differences in metabolism by 13C-metabolic flux analysis. The studies may lead to develop of new therapeutic strategies that target the ability of the pathogen to enter or maintain itself in the persistent (drug-tolerant) state.

This research will investigate a problem of huge importance for tuberculosis control, drug tolerance. TB affects about 15 million people in the world today causing nearly 3 million deaths. The research will therefore benefit our understanding of this devastating disease and lead to new therapeutics. The market for antituberculous drugs is estimated to be USD 612-670 million annually. In the UK there are several pharmaceutical companies that that have an interest in development of novel antuberculous drugs, such GSK and AstraZeneka. Many biotech companies are involved in the development of new antibiotics. We plan to engage with potential industrial partners in a number of ways. Firstly, we will use our existing contacts. Professor McFadden and co-I's have considerable experience interacting with industry. For instance, Professor McFadden is currently PI of a collaborative research project with Sanofi Pasteur to develop new meningococcal vaccines. The University of Surrey degree structure also provides additional routes to engagement with potential partners as most of our students spend one year in a professional training placements, often in an industrial research laboratory. For instance, bioscience students are currently placed in GSK and the company Biocompatibles (Farnham, Surrey) involved in the development of drug delivery vehicles. The applicants make regular visits of students at these placements, providing opportunities for discussing this project with industrial scientists who may be interested in partnering the work. The research will also benefit the pharmaceutical more generally as drug-tolerance is a major problem in many other diseases such as those caused by staphylococci and streptococci. By leading to new drugs capable of controlling infectious diseases more effectively, the research will therefore benefit the UK and European public and worldwide. The project will also contribute to the training of interdisciplinary scientists capable of pursuing a career in the fast-moving field of systems biology.