The Mycobacterium tuberculosis Cell Envelope: unravelling complex cell wall assembly and the identification of potential new drug targets

Mycobacterium tuberculosis is the cause of tuberculosis (TB) and is the leading bacterial burden of mortality and morbidity in the world. One third of the world's population is infected with TB, and in conjunction with HIV represents a serious problem that urgently needs addressing. TB is a disease of poverty and mostly affects young adults in their productive years, mostly in the developing world. The most recent report from the World Health Organisation states that in 2011, 8.8 million new cases of TB were reported and that 1.4 million people died from TB, corresponding to approximately 4000 deaths daily. In the UK, there were 7000 new cases of TB reported, mostly in large cities, and 75% of cases were in individuals not born in the UK. It can be argued that, globally, M. tuberculosis is the single most important infectious agent affecting mankind. Bacteria are enclosed in a cell wall which protects the organism from its immediate environment. Moreover, fortuitously, they also represent important targets for drugs like penicillin that can be used to treat bacterial infections. However, M. tuberculosis has a distinctive cell wall that differs in composition from that of other bacteria; in particular it contains an exceptional amount of unique lipids (fats), sugars, and a mesh-like structure. Although, there are drugs that specifically affect the M. tuberculosis cell wall, the current treatment for TB lasts 6 months and is potentially toxic to patients who often cease treatment early. Moreover, the efficacy of treatment is threatened by the emergence of multi-drug and extensively-drug resistant (MDR and XDR) strains of M. tuberculosis. The proposed research will increase our knowledge as to the basic biology surrounding the M. tuberculosis 'cell envelope' and has the potential to generate data that will lead to the discovery of novel drug targets and potentially the characterisation of new 'hit-to-lead' chemical entities with a defined mode of action. Firstly, we will build on our previous studies on the synthesis of the major 'fats' of the cell envelope, termed mycolic acids and the mode of action of specific inhibitors. Secondly, we will extend our characterisation of the enzymes involved in the assembly of the 'sugars' which make up the key polysaccharides found in M. tuberculosis, which includes the macromolecules arabinogalactan, lipoarabinomannan and alpha-glucan. Thirdly, we will integrate our studies on peptidoglycan synthesis, the underlying molecular 'mesh' that covers the cell, into our framework of studies centered on the M. tuberculosis 'cell envelope'. The research in this application will increase our knowledge into the basic biology of the tubercle bacillus and has the potential to impact upon human health, with wide-reaching benefits both nationally and internationally. In particular, our proposed studies will have the potential to identify novel drug targets and aid in the development of new anti-tubercular compounds.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), has a unique cell envelope, which accounts for its unusual low permeability and hence, contributes to resistance against common antibiotics. Our integrated programme of studies is aimed at coordinating a continued investigation into the basic biochemistry and physiology, and drug targeting potential of the mycolyl-arabinogalactan-peptidoglycan (mAGP) complex of M. tuberculosis, including key aspects related to the essential immunogens, lipoarabinomannan (LAM) and alpha-glucan. Firstly, we will build on our previous studies on mycolate biosynthesis and the mode of action of mycolate targeted inhibitors, such as isoxyl and SQ109, thus providing a focus on the biochemical and structural characterisation of key enzymatic components of the fatty acid/mycolic acid metabolism and catabolism pathways. Secondly, we will extend our characterisation of the assembly of the complex AG matrix, with an emphasis on the role of glycosyltransferases, as well as related enzymes leading to the synthesis of the key immunogenic cell envelope components, LAM and alpha-glucan. Finally, we will integrate our studies on the cross-linked network of PG, following a multi-pronged approach that will involve the detailed genetic, biochemical and structural characterisation of key M. tuberculosis PG biosynthetic enzymes. Addressing these topics will provide fundamental knowledge on M. tuberculosis cell envelope assembly. In addition, it is anticipated that the above basic functional and structural studies will ultimately prove useful in the identification and detailed mode of action studies of inhibitors, emerging from whole cell 'hit-to-lead' screening studies, such as benzothiazinones (BTZ). All of the above investigations will exploit the full power of a multi-disciplinary approach using biochemistry, chemistry, immunology and molecular biology.

Who will benefit from this research?

The research in this application will increase our knowledge in basic biomedical science and has the potential to impact upon human health, with wide-reaching benefits both nationally and internationally. The proposed research will increase our knowledge as to the basic biology surrounding M. tuberculosis 'cell envelope physiology' and has the potential to generate data that will lead to the discovery of new drug targets and the characterisation of the mode of action of new 'hit-to-lead' chemical entities. Effective communication between the PI and immediate beneficiaries (PDRAs, PIs research group, and the TB research community) is critical to maximising outputs from this research. It is also important in ensuring that potential future beneficiaries (industry, health services and the wider public) are able to access, engage with and potentially exploit our research findings. That fact that the proposed research involves work with the pathogen that causes TB, makes this particularly relevant in terms of impact.

How will they benefit from this research?

Career development of the PDRAs. Regular meetings, in addition to laboratory meetings, will be held with the PDRA to set goals and for trouble-shooting, and if necessary revising the experimental approach. This will ensure that outputs are maximised and the research is focussed. Additionally, for the personal development of the PDRAs, the School of Biosciences has a mentoring scheme termed 'Personal Best' that ensures that the PDRAs training needs and career opportunities are identified. Career Training presentations are also regularly held in the University to bolster the professional development of early career stage researchers.

Commercial exploitation of outputs. The University has support and training available to ensure that opportunities arising from the research with commercial opportunities are taken and potential investors identified and informed. For commercial exploitation of results, the PI will engage with University Research and Commercial Services and the University-based Alta Innovations Ltd for matters relating to intellectual assets and potential patent applications. In addition to this, advice will also be sought regarding exploitation of outputs through collaborations with Industry, including the possibility of CASE studentships. In addition, involvement in the Tuberculosis Drug Discovery Consortium (TBD-UK), an integrated UK TB drug discovery consortium that has a strong working relationship with the UK government and policy makers, and collaboration with the Health Protection Agency, will enable outcomes of the research to be disseminated to policy makers and non-profit organisations, such as the TB Alliance and the Stop-TB partnership, to eliminate TB as a public health concern.

Management and involvement in impact activities. The PI will also ensure that the PDRAs can attend all professional development opportunities offered within the University including career development and fellowship application courses. Impact activities that target collaborations, commercial exploitation of results and the wider public will be carried out by the PI.

Deliverables and milestones. Immediate impact activities will be measured in the number of publications, invited talks at conferences, new collaborations and the use of data generated for further applications and career progression of the PDRAs. These are expected to take place during the time of the proposed application. Long term impacts like collaborations with industry and commercial exploitation of findings are likely to occur beyond the timeline of the application. If we are successful in identifying new targets for anti-TB drug development from these studies, this will be a key starting point for a long process that could in the future lead to the development of drugs against these novel targets and eventually have an impact on national and global health.