The Mycobacterium tuberculosis Cell Envelope: unravelling complex cell wall assembly, degradation and re-cycling pathways

Tuberculosis (TB) is a bacterial infection caused by the tubercle bacillus. Worldwide TB remains the leading bacterial cause of mortality and morbidity. In 2016, the WHO reported 10.4 million new cases of TB, with 1.7 million people dying from TB that year. Predominantly a disease of poverty, TB affects young adults in their productive years and hence also carries a large economic burden. This picture is highlighted by populations living in low and middle income countries (LMICs), where the burden of TB is most prevalent, with high morbidity and mortality rates, such as in India (510,000 p.a.), Indonesia (130,000 p.a.), China (38,000 p.a.), Nigeria (240,000 p.a.) and South Africa (97,000 p.a.). Therefore, introducing a new TB drug onto the market and its impact upon the health and global economy for these LMICs represents an urgent healthcare challenge, which needs to be viewed on at least a 10-15-year timescale.

Our application seeks support for research into the cell envelope of Mycobacterium tuberculosis. All bacterial cells are enclosed in a cell wall or cell envelope. This is a dense layer of covalently-linked molecules around the cellular membrane, protecting the organism from its immediate environment. The cell wall of M. tuberculosis is very distinctive, differing from other bacteria in containing an exceptional amount of unique lipids (fats) and sugars. This high content of lipids and sugars makes the M. tuberculosis cell wall impermeable to most antibiotics. At the same time, the organism is very vulnerable if the cell wall has defects. Therefore, some existing TB antibiotics interfere with the synthesis of cell wall components, and for the same reason many TB drug development efforts focus on the cell wall.

Our research will concentrate on learning more about the biology surrounding the M. tuberculosis cell wall. Firstly, building on our previous studies on lipid synthesis, we will examine enzyme systems that degrade lipids. This is important because M. tuberculosis has limited access to nutrients and has developed strategies to recycle molecular components for new uses. Secondly, we will study how M. tuberculosis assembles sugar-like polymers, such as arabinogalactan and the key virulence factor, lipoarabinomannan, outside of its cell membrane. Our third aim is to understand a range of enzymes that appear to play a role in remodelling peptidoglycan, a molecular 'mesh' that is commonly found in bacteria, but has distinct features in M. tuberculosis. Fourthly, we will use small-scale mechanical probes to clarify how the bacteria are constructed and help explain how interactions of the key building blocks influence virulence.

In characterising the enzymes and proteins involved in these biological processes, we aim to find proteins that are 'good' drug targets, for instance because they are unique to this organism or because the cell cannot compensate when they are inactivated by a drug. To achieve this goal, our research will rest on three fundamental research pillars: 1) the identification of essential mycobacterial proteins and how the interaction between the protein target and an inhibitor can be improved; 2) determine how and why these compounds kill bacilli, their mode of action; and 3) develop our partnerships with laboratories in LMICs, such as the Institute of Materia Medica (IMM), Beijing, China and the Indian Institute of Science (IISc), Bangalore, India, and through our industrial links with GlaxoSmithKline Diseases of the Developing World (GSK DDW), Madrid, Spain, develop drug discovery projects to turns 'hits' into 'leads' and ultimately into new TB-drugs. Thus, this proposal offers the opportunity to tackle a range of fundamental questions about an organism that has puzzled microbiologists ever since its discovery by Robert Koch in 1882, that can be used to treat a debilitating disease which represents one of the most pressing healthcare challenges for Society in the 21st Century.

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 studies are aimed at coordinating a continued investigation into the basic biochemistry and physiology, and drug targeting potential of the M. tuberculosis cell envelope. The application will focus on the following key components of this complex; arabinogalactan (AG), lipoarabinomannan (LAM) and peptidoglycan (PG), and in addition we will continue to explore key physical properties of the mycobacterial cell envelope. Firstly, we will build on our previous studies on lipid biosynthesis and investigate the role of echA proteins in lipid degradation and re-cycling pathways. Secondly, we will extend our characterisation of the assembly of the complex AG matrix, with an emphasis on the role of glycosyltransferases and transporters, as well as the related enzymes leading to the key immunogenic cell envelope component, LAM. Thirdly, 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. Fourthly, we investigate the physicochemical properties of the mycobacterial cell envelope. Addressing these topics will provide fundamental knowledge on M. tuberculosis cell envelope assembly, degradation and recycling pathways. In addition, it is anticipated that the above functional and structural studies, will ultimately prove useful for screening for inhibitors, potentially generating valuable hits and leads, ultimately leading towards new anti-TB drugs. In the longer-term, these studies will offer key translational opportunities for improved therapies and prevention strategies, particularly for populations living in low and middle income countries (LMIC) where the burden of TB is greatest.

Impact on the Treatment of Disease:This Programme Grant will increase our knowledge of tuberculosis (TB) biology,whilst delivering new TB drug targets for future drug-discovery studies.The most far-reaching impact of the work would therefore be the introduction of new drugs for treating drug-resistant TB strains.Since,TB mostly affects young adults in their productive years,new TB therapies can be expected to have a significant economic impact,particularly for populations living in low and middle income countries (LMICs), where the burden of TB is most prevalent,such as in India (2.8 million p.a.), Indonesia (1 million p.a.),China (0.9 million p.a.),Nigeria (0.4 million p.a.),Pakistan (0.5 million p.a.) and South Africa (0.4 million p.a.).Introducing a new TB drug onto the market for these LMICs, and its impact upon their health and global economy,needs to be viewed on at least a 10-15-year timescale.
Whilst, the introduction of a new TB drug extends beyond the duration of this application,by the end of the project,we aim to have addressed key topics that will provide fundamental knowledge on M. tuberculosis cell envelope assembly and the identification of new TB drug targets,which will ultimately prove useful for screening for new inhibitor-target pairs,potentially generating valuable hits and leads,leading towards the identification of new anti-TB drugs.
We will look to develop our new TB targets by securing translational funding through succession planning activities with our key partners in LMICs and industry, such as the Institute of Materia Medica (IMM),Beijing,China,the Indian Institute of Science (IISc),Bangalore,India,and with GlaxoSmithKline Diseases of the Developing World (GSK DDW),Madrid,Spain.In this context,our established strategic partnerships with IMM and GSK DDW,has provided proof-of-concept research models for taking natural products and hits identified from screening activities in China and Spain, and exploiting complementary expertise from the University of Birmingham (UoB),to develop lead-like molecules targeting cell wall biosynthetic enzymes.
Who will benefit from this research: In the shorter term, the international scientific community and potential future beneficiaries, such as industry will benefit from our improved understanding of TB biology and the disclosure of new TB drug targets. In developing high-impact science, the investigators will ensure they maintain their internationally-leading research profiles, whilst those researchers working at the bench will benefit from supervision from leading scientists and academic training visits from our collaborators, such as those from IMM, IISc and GSK DDW, which will foster inter-disciplinary research and open up career opportunities for them in academia and industry.
How the researchers will benefit from this project: In addition to group meetings, regular meetings with the researchers will be used to set goals, trouble-shoot and review progress.The UoB will offer mentoring schemes, which will ensure that the researchers continuing professional development training needs are identified efficiently and developed effectively.
Other academic beneficiaries and commercial exploitation.The research outputs from this project will impact the international scientific community,both from academia and industry,who will benefit from our improved understanding of mycobacterial biology and the disclosure of new TB drug targets.Commercial exploitation will be pursued through the Research and Technology Transfer Unit based at the UoB.
The wider public. Whilst, LMICs with high prevalence rates will be the benefit in the longer-term from the development of new TB drugs,over the duration of this project,the research team will engage the general public through outreach activities and social media to disseminate its findings and highlight the urgent need to develop new TB treatments and address the growing issue of antibiotic resistance.