A novel glucan pathway for immune system evasion by pathogens and carbon management in differentiating bacteria

We have recently discovered a new metabolic pathway widespread in bacteria called the GlgE pathway. This pathway involves four enzymes that make a glucose polymer (glucan) that is utilised by different bacteria to suit their lifestyles. Mycobacterium tuberculosis is the causative agent of tuberculosis, which kills almost 2 million people each year. The GlgE pathway exists in this pathogenic bacterium and we have identified that blocking the defining enzyme of this pathway, GlgE, leads to death of the bacterial cells. There are links between the GlgE pathway and a glucan present in the capsule of this pathogen. The capsule coats the outer surface of the bacterium and helps it to evade the immune system. This contributes to persistence of the pathogen in patients leading to therapies that take many months to complete. Targeting the production of capsular glucan could therefore offer the potential to shorten the duration of treatments. Interestingly, the related bacterium Streptomyces coelicolor uses the GlgE pathway for a very different purpose. This is a soil-dwelling organism that has been extensively studied because it makes antibiotics and has a complex life cycle involving spore formation. The GlgE pathway appears to be involved in the management of the carbon it obtains from its environment. It seems to use the GlgE pathway to help mobilise and store carbon in the form of glucan at appropriate times throughout its lifecycle. We hypothesise that the GlgE glucan product has a specialised centipede-like structure in order that it can be exported more easily to the outside surface of the mycobacterial cell. Furthermore, GlgE glucan can be structurally differentiated from other glucans, such as glycogen that has a tree-like structure. The GlgE glucan will be defined by GlgE, which makes the linear polymer backbone, and GlgB, which introduces branches. We have determined the three-dimensional structure of GlgE. This will allow us to identify what is special about its structure such that it controls the properties of the glucan product in the context of the branching enzyme. This will involve mutating the GlgE enzyme and measuring the effect of these changes on enzyme activity. We will also determine the structures of these variant enzymes with various glucans bound to them. We will determine the branch lengths generated by GlgB and how GlgB works together with GlgE to make the overall glucan structure. We have evidence for how GlgE could be regulated by phosphorylation that is distinct from other similar metabolic pathways and we will test whether this is indeed the case. Knowledge of how the GlgE pathway operates and is regulated is important in the development of novel therapies against tuberculosis.

We have discovered a new enzyme (GlgE) and metabolic pathway for alpha-glucan biosynthesis. The GlgE pathway genes exist in 14% of all completed bacterial genomes. In Mycobacterium tuberculosis, this pathway is linked to human immune system evasion associated with a polysaccharide capsule and we have already genetically validated GlgE to be a drug target. Furthermore, it would appear that this pathway has been adapted for very different lifestyles in other bacteria. In the differentiating bacterium and antibiotic producer Streptomyces coelicolor, the pathway appears to be involved in developmentally regulated carbon management that is distinct from the classical glycogen pathway. Building on our recently determined structure of GlgE and evidence from our collaborator, V. Molle (France), for the specific phosphorylation of GlgE by the Ser/Thr kinase PknB, we will establish how the GlgE pathway generates a glucan distinct from glycogen that is suitable for cellular export and carbon management and how the GlgE pathway is regulated. We will test hypotheses at the protein structural level regarding how GlgE controls glucan structure as it is elongated by GlgE and branched by GlgB. We will also test the hypothesis that GlgE is regulated by specific phosphorylation. The methods employed to meet these objectives are well established. These include X-ray crystallography and site-specific enzyme mutagenesis coupled with glucan production in vitro and in vivo, enzyme assays and glucan analyses using mass spectrometry, capillary electrophoresis, dynamic light scattering and analytical ultracentrifugation. This fundamental biological knowledge will provide important new information of relevance to the search for novel therapies against tuberculosis and to an understanding of the management of carbon in differentiating bacteria that produce antibiotics.

This proposal maps onto the BBSRC Strategic Plan 2010-2015 strategic research priority 3, Basic Bioscience Underpinning Health. Specifically, the proposed research addresses 'fundamental ... biology leading to potential new antimicrobial drugs' such that 'chemical biology, biochemistry and biophysics drive the discovery and validation of new drug targets leading to more effective and/or selective pharmaceuticals'. Thus, the main direct beneficiaries of the proposed fundamental research will ultimately be patients suffering from tuberculosis and other diseases and perhaps farmers protecting their herds from bovine tuberculosis. This would be delivered through the pharmaceutical and veterinary medicine industries. The current therapies for tuberculosis are decades old. Human tuberculosis is a global problem with ~2 million deaths per annum, 13 million active cases of tuberculosis and a staggering one third of the population carrying the bacterium. This disease therefore also causes a significant drain on developing world economies. Although anti-tuberculosis drugs are unlikely to make lots of money for pharma, the global economy would benefit significantly trough such indirect routes. The incidence of tuberculosis within the UK is small (~8,000 cases per annum), but the spread of extensively drug resistant strains from Africa and the edge of Eastern Europe increases its threat in the UK in the medium term. Bovine tuberculosis is a significant problem within the UK and beyond. Being able to control this disease would benefit farmers and vets with implications for wildlife management associated with bovine tuberculosis in wild animals. Managing the development and use of drugs against tuberculosis would require significant input from policy makers. The proposed work is focussed on science excellence and fundamental discoveries. All tuberculosis-related aspects of the project will continue to be in collaboration. We will extend our engagement with organisations involved in tuberculosis therapeutics, such as TB Drug Discovery UK, the Global Tuberculosis Alliance and others, backed up by our existing patent application and Nature Chemical Biology publication. Getting a drug onto market is recognised to require ~10 years and billions of pounds, but tuberculosis is certainly a worthy target. The principle investigator has worked in industry in the past and has recently attracted industrial funding for another unrelated carbohydrate enzyme project, providing valuable experience in bridging the gap between fundamental science and the commercial sector. Our intellectual property is being managed by PBL Technologies together with the Albert Einstein College of Medicine and the Howard Hughes Medical Institute, with whom we made our initial discoveries. PBL Technologies has a strong track record in working with the John Innes Centre and our core intellectual property on GlgE is shared with our collaborating American institutions. In addition, uncovering the details of how bacteria handle carbon through glucans could have significant implications regarding the fermentation of a wide variety of industrially important organisms with a wide breadth of utility, including biofuel production, as well as more traditional uses (e.g. Corynebacterium glutamicum used for the production of amino acids, nucleotides and steroids as well as for food uses). The staff associated with this project will be carrying out fundamental research within the context of not only an important area of biology but also human and animal diseases. The knowledge and skills acquired by the staff will therefore be beneficial to their careers. We will reach beyond the above beneficiaries with press releases. We have already worked with our John Innes Centre communication colleagues to publicise our original discovery to the press through the Science Media Centre and will be able to use this route again to publicise future advances.