Peptidoglycan release and recycling in pathogenic mycobacteria.

The UN and WHO have both recognised tuberculosis (TB) as a major driver of poverty with drug-resistant forms of the disease causing high mortality rates. Drug-resistant strains have poor treatment options, and TB infection is compounded by frequent co-infection with HIV. These factors contributed to TB killing approximately 1.8 million people last year. Despite global efforts, this problem is only getting worse. The percentage of TB cases reported to be resistant to rifampicin, a front-line antibiotic rose from 31% in 2015 to 41% in 2016. This is an antibiotic resistance crisis and new therapeutic options are urgently needed. One of the most efficient ways to address this crisis is to rehabilitate existing medicines. For years beta-lactam antibiotics have not been used in TB treatment due to its endogenous broad-spectrum beta-lactamase. Recent studies, including a clinical trial have changed this view. When combined with a beta-lactamase inhibitor, beta-lactam antibiotics perform as well as existing therapeutics. Despite this, we know relatively little about the metabolism of their either eventual target, peptidoglycan in Mycobacterium tuberculosis, or how M. tuberculosis uses PG to modulate the host immune system.

M. tuberculosis is the causative agent of the TB and generates one of the most complex cell walls amongst bacteria. The mycobacterial cell wall is comprised of three main layers including peptidoglycan, arabinogalactan and mycolic acids. A complex cell wall creates constraints on growth and division, which must be overcome through careful remodelling of the cell wall during division or the insertion of envelope spanning structures. This process necessarily leads to the production of small molecule metabolites, which are known to be recognised by the immune system.

Peptidoglycan is a combination of long polysaccharides with a repeating disaccharide unit cross-linked through short peptides. This mesh-like structure gives the organism shape and protects it from numerous stresses. During my BBSRC Future Leader Fellowship, I have shown that mycobacteria generate two distinct forms of peptidoglycan fragments during growth as a consequence of a novel peptidoglycan recycling system. Immunostimulatory disaccharide-peptide conjugates activate host NOD receptors, while disaccharides stripped of their stem peptide serve as signals to the bacterium itself driving growth and antibiotic resistance.

Peptidoglycan metabolism can be thought of as a cycle, with synthesis, remodelling, degradation and recycling intimately associated. In this BBSRC David Phillips Fellowship I will genetically and biochemically dissect these pathways using a multi-disciplinary approach. By employing unbiased genetic screens and our body's ability to sense peptidoglycan fragments I will uncover for the first time the genetic and mechanistic basis of peptidoglycan stem-peptide recycling in pathogenic mycobacteria. I will go on to dissect the molecular basis of peptidoglycan biosynthesis, remodelling and degradation so that we might better understand this important and druggable pathway in mycobacteria, with the long-term aim of reducing the burden of antimicrobial resistance.

Most bacteria wrap themselves in a cell wall which protects them from a wide range of stresses. Textbooks present this wall as a static, un-changing structure. This is completely false. To grow, divide and carry out metabolic operations the cell wall must be a dynamic and plastic structure. This is true of all walled bacteria but represents a unique challenge to Mycobacterium tuberculosis (Mtb) which has a complex, tripartite cell wall. The peptidoglycan (PG) layer of mycobacteria is covalently bound to a large polysaccharide called arabinogalactan. This layer is then esterified by mycolic acids which along with free glycolipids form the outer membrane of Mtb. This means that at each stage of growth and division the bacteria need to manipulate all of these layers by cutting and remodelling. This process generates a diverse array of small molecules including fragments of PG.

I have previously shown that pathogenic mycobacteria, including Mtb recycle these PG fragments and that this process is important for antimicrobial resistance and growth in macrophages. During this David Phillips Fellowship I will elucidate the mechanism by which mycobacteria recycle their stem peptides by harnessing our bodies' PG sentinels, the NOD-receptors. To complete this pathway, I will uncover the molecular basis of the cleavage of a key bond in PG, the D-lactyl-ether of MurNAc. Next, I will determine the molecular basis of PG degradation in Mtb, which will allow us to identify the fragments produced by the bacterium that feed PG-fragment recycling. Finally, to complete the cycle I will establish the biosynthetic and inhibitory potential of the penicillin binding protein complement from Mtb. Together, these data will provide an unprecedented window into the details of PG biosynthesis, remodelling, degradation and recycling in this global pathogen. This will support future drug discovery efforts and help us understand how these bacteria modulate or evade sensing by the immune system.

Tuberculosis (TB) is a major driver of poverty, afflicting young people disproportionately in their most productive years. This burden is increasing with the rise of antimicrobial resistance, including multi- and extensively-drug resistant strains of Mycobacterium tuberculosis. Any efforts to alleviate this disease could have long term health and economic impacts, especially in developing nations. Peptidoglycan (PG) metabolism in mycobacteria is an important field of research that is gaining in profile. By developing a molecular understanding of this crucial pathway, we will be able to inform future drug discovery efforts. As such, the single greatest impact of this work would be felt with the development of a novel antimycobacterial drug. A key consideration here is that the timeline of bringing a drug to market from an initial hit is on the scale of 10-20 years. Despite this lag, one of the substantial benefits of this Fellowship is that I propose to characterise a system for which drug scaffolds already exist. This can accelerate the use of beta-lactam antibiotics in mycobacterial disease treatment which could have a much more immediate impact.

Another aspect of this project is in non-tuberculosis mycobacterial diseases. For example, Mycobacterium avium subspecies paratuberculosis (MAP) is a major health threat in the farming industry causing an estimated £13 million in losses each year. It is possible that treatments that are less effective at treating TB, or have poor side-effects in humans will be useful in farming. Intriguingly, MAP is also associated with human Crohn's disease in a manner that is thought to be NOD-dependent. Insights into PG fragment recycling in TB may be applicable in this disease as well, which could have substantial impacts in human healthcare.

The second group for which a significant impact would be felt is the UK research community. This will be felt in two ways aside from those listed in the academic beneficiaries statement. First, by awarding me this David Phillips Fellowship the BBSRC will be further investing in a centre of research excellence at the University of Birmingham. This investment will help to enhance Birmingham's research-driven teaching programmes which will develop the next generation of UK bioscience innovators. Secondly, within the immediate timescale of this Fellowship I will be involved in the training of a research technician and a PhD student (provided by the School of Biosciences) who will gain or enhance their expertise in a variety of techniques which will position them to contribute in meaningful ways to the growing bioeconomy regardless of their career aspirations.

The final group of beneficiaries is the public. I am currently engaged with several outreach activities including the Access 2 Birmingham program, the IMI Summer School program, and University Open Days. In these capacities I have sought to share my research in an accessible way so that people have an understanding of how we are using their money to tackle complex and important problems. As another means of public engagement, I am active on social media in promoting science and good science practises as well as my own research outputs.

This David Phillips Fellowship will deliver excellent short-term impacts and has the potential to make exceptional long-term impacts on human health by addressing the BBSRC priority area of antimicrobial resistance. Through this David Phillips Fellowship the BBSRC will be able to improve our fundamental understanding of an important human pathogen, combat antimicrobial resistance and contribute to the bioeconomy.