Nothing wasted: Peptidoglycan recycling in mycobacteria

Mycobacterium tuberculosis is a living paradox. Amongst bacteria, it possesses one of the single most complex and carbon-rich cell walls and yet it thrives in the decidedly nutrient poor environment of the human lung. This is made even more impressive considering the fact that components of the mycobacterial cell wall are potent triggers of the innate immune system. Therefore, to support stable growth in their host, these bacteria must be exquisitely efficient with their available nutrients and be able to hide themselves from the immune system.

The innermost component of the mycobacterial cell wall is a complex macromolecule called peptidoglycan (PG). While textbook illustrations of PG suggest that it is a static bag-like structure, in reality it is a heavily modified and dynamic molecule that prevents the cell from exploding while simultaneously giving it shape. Throughout the life cycle of the bacterium, PG must constantly and carefully be cut and remodeled to support growth and division. Researchers have long studied PG, chiefly because of its essential nature. Indeed, the first clinical antibiotic, penicillin, specifically targets the biosynthesis of PG. While much of this biosynthesis has been understood for many years, relatively little is known about what happens to PG after it is built. This is even truer for the PG of M. tuberculosis, which is a global pathogen and the second leading killer of people amongst infectious diseases. For example, nothing is known about the fate of mycobacterial PG fragments once lytic enzymes have cut them from the cell wall. This is an important question because as mentioned above, these PG fragments are potent stimulators of the immune system and command a great deal of scarce resources to make. Intriguingly, these same PG fragments are capable of triggering resuscitation of the bacterium from the dormant phase of its lifestyle. Dormant M. tuberculosis is capable of persisting undetected for years inside of its host before producing an active infection. This severely complicates control of the disease and associated outbreaks, because even if they are detected, dormant M. tuberculosis are exceptionally antibiotic resistant. These points make it clear that an understanding of the fate of M. tuberculosis cell wall fragments is needed. In some bacteria complex PG recycling systems have been described which work by bringing PG fragments back into the cell to be reused. In all cases observed to-date this recycling system is important to the ability of the bacterium to infect its host. Despite this, PG recycling has never been investigated in M. tuberculosis. It is important that this pathway be investigated because it is very likely that the elements of a mycobacterial PG recycling system are novel antibiotic targets.

During this Fellowship I will use new and exciting techniques to demonstrate that mycobacteria recycle their PG. I will take a high-resolution approach by exploring the precise structure of the PG fragments that are being recycled. I will then determine the cellular machinery that is required for PG recycling and characterise the key proteins involved in the pathway. Finally, I will examine the relationship between tuberculosis resuscitation and PG recycling. This work will address the M. tuberculosis paradox and show how it is able to balance its nutritional and growth requirements with its nutrient poor environment and the need to hide itself from our immune system.

Almost nothing is known about the fate of peptidoglycan (PG) metabolites generated during the maturation and break down of the mycobacterial cell wall. This lack of understanding is partially due to the complexity of the mycobacterial cell wall and the technical limitations associated with studying a metabolite that cannot be purchased commercially in significant quantities. It is my hypothesis that at least some of these muropeptides are recycled by the bacterium despite the fact that the genome of Mycobacterium tuberculosis does not encode homologs of known PG-recycling genes. I will demonstrate and investigate this important pathway using a combination of novel methods. First, I will use metabolic labelling of PG with 14[C] GlcNAc to generate radiolabelled PG. Following this I will combine graphitized carbon solid-phase extraction and a new rapid soluble-PG extraction method to isolate structurally defined, radio-labelled muropeptides. These will be used in uptake assays with Mycobacterium bovis BCG and M. tuberculosis to demonstrate that these organisms are capable of re-using soluble muropeptides. My preliminary data shows that this is the case. Next I will use specific PG cleaving enzymes to probe the substrate specificity for this pathway by generating a library of structurally defined muropeptides. With PG-recycling firmly established I will probe the genetic and proteomic requirements for this process by screening a transposon mutant library for its ability to grow on N-acetylmuramic acid, a sugar that is unique to PG. This will be complemented using targeted knock-outs and phenotypic analysis using fluorescent PG labels. Biochemical and structural analysis of the proteins involved in this pathway will yield important insights into a completely unknown pathway. Finally, the relationship between PG recycling and dormancy will be explored. This novel work will uncover a completely novel biochemical pathway in this important human pathogen.

Impact Summary

This Fellowship will increase our knowledge of basic TB biology and Mycobacterium tuberculosis physiology and generate data that will lead to new TB drug targets and new compounds for hit-to-lead optimisation. The most far-reaching impact of the work would therefore be to translate this basic research into new drugs for treating TB. In 2013, 9 million new cases of TB were reported and 1.5 million people died from the disease. Reducing these high morbidity and mortality rates by developing new, more efficacious drugs, which are also able to target increasingly drug-resistant strains of M. tuberculosis, would not only have a major and far-reaching global impact upon the health and well-being of society but since the disease mostly affects young adults in their productive years, would also have significant economic impact, particularly in those developing countries where the disease is most prevalent.

The introduction of a new TB drug on to the market and realising its associated potential for impacting upon the health of society and the global economy needs to be viewed on at least a 10-20-year timescale, which is therefore beyond the duration of this 3-year Fellowship; however, by the end of this project, I aim to have identified novel targets and will be looking to develop these by exploring new funding opportunities in succession planning as I transition to an independent academic research position. The most realistic avenue for this to be achieved will be through the identification of an industry partner. The host-group supervisor has an ongoing collaboration with GlaxoSmithKline (GSK) and so with Professor Besra as a link I will have an excellent opportunity to engage with GSK to explore promising targets at the conclusion of this Fellowship.

In the short to medium term, the proposed research will impact most on the international scientific community, who will benefit from our improved understanding of TB biology and Mycobacterium tuberculosis physiology and the disclosure of a completely novel biochemical pathway, which will open up new directions and avenues for study. Successful completion of this project will put me at the forefront of a new area of TB research, which will position me well for future funding and collaboration.

The University of Birmingham has made a significant investment to support research in the life sciences; thus a successful Fellowship would provide a valuable opportunity to build on this strong platform in garnering additional funding to establish itself as an international research centre of excellence for biomedical research that is able to attract the best researchers from across the globe. This in turn will feed into the University's undergraduate teaching programmes, be it in offering final-year research projects in exciting interdisciplinary science in internationally leading groups, to providing a catalyst for developing new degree programmes that are attractive to high-quality students from both home and overseas.

Whilst the public will benefit in the long term from the development of new TB treatments, over the duration of this Fellowship, I will engage the public in various ways (outreach activities, social media, internet) to disseminate its findings and highlight the importance of scientific research and in particular the growing problem of TB.