Exploring the three novel enzymes involved in Burkholderia pseudomallei capsular polysaccharide biosynthesis

In many less developed parts of the world, bacteria that are not found in the Europe cause disease in large numbers of people. Because these diseases are uncommon in industrialised societies, little research has been carried out into finding appropriate cures for the diseases. Furthermore, some of these bacteria are not affected by many of the antibiotic medicines that have been developed for treating diseases common in the West. This leaves many people at risk from infections that are poorly understood, hard to treat, and uneconomic for pharmaceutical companies to research. One example of this is the bacterium Burkholderia pseudomallei, which causes the disease melioidosis. This is a common disease in many parts of South-East Asia and Northern Australia; in Thailand, it is a leading cause of community-acquired infection. The bacterium can stay dormant in a human for long periods of time (62 years has been known), and causes disease when the affected individual becomes weakened. The bacteria are resistant to the majority of antibiotics, and even the few antibiotics that are available require long term use to be effective. Between one-fifth and one half of all patients diagnosed with the most severe forms of melioidosis go on to die, depending on where they are treated. Because of this high mortality rate, melioidosis is seen as a potential bioterror threat. There is consequently a strong desire to generate a vaccine, both to protect populations at risk from natural exposure to the bacterium, and to mitigate the threat of bioterrorism. B. pseudomallei has a coating layer of sugars that serve to protect the bacterium from the human immune system. The presence of these sugars is required for the bacteria to be fully infectious, and so they make a good candidate for the development of vaccines: loss of the sugars would make the bacteria far less infectious, and it is unlikely that the bacteria will be able to radically alter their sugars, so resistance is unlikely. However, it has proved difficult to obtain a sufficiently pure source of the sugars from bacteria to prepare vaccines. The bacteria produce a mixture of two main multi-sugar products as part of this coat; a better understanding of how they achieve this would inform attempts to make better preparations of the coat, and might lead to new methods for industrial production of the sugar. This work will examine the mechanisms that the bacteria use to produce one of the multi-sugar coating molecules. Each of the chemical steps that the bacteria use is carried out by a different protein. Most of the proteins responsible have been clearly identified and characterised; however, three proteins are required to make the sugar coat whose role is unclear. No proteins like these have been studied in the past. We will prepare each of these in a safe bacterium, and assess how they alter the structure of parts of the sugar coat whose origins are known. By examining the products, we hope to understand why they are needed by the bacterium. This will allow us to produce a clear pathway for the chemical steps that produce the final sugars. Following this, we will determine the three dimensional atomic structure of each protein, both on its own and together with the chemicals that it acts on. By so doing, we hope to understand exactly how the chemical steps take place. We will confirm this by altering the proteins so that they are no longer able to carry out the chemical steps; and by showing that B. pseudomallei carrying these alterations are unable to make the sugar coat in its natural form. By carrying out these studies, we expect to generate precise information about the production of the B. pseudomallei coating sugar. This will be used to design improved methods for preparing sugars for vaccines, and may highlight potential drug targets. We will demonstrate how these three unusual proteins work, so that they can then be applied for use in other processes by industry or academics.

The development of vaccines against Burkholderia pseudomallei based on the capsular polysaccharide of this organism have been thwarted by the difficulties in preparing homogenous polysaccharide. The biosynthetic cluster for this capsule has been well established, and the functions of many of the genes included are clear. However, three of the genes do not show significant sequence identity to any other characterised gene, and the proteins produced have no unequivocal structural homologues. These genes (wcbF, wcbG, wcbI) are likely to encode enzymes that are required for missing functions in the biosynthesis of the capsule. Each gene will be expressed in E. coli, and the purified protein assayed for the capacity to bind a variety of potential substrates and cofactors. A key substrate will be the purified capsule intermediate sugar, GDP-6-deoxy-b-D-manno-heptopyranose (GDP-dDHep), which has been successfully isolated in my laboratory. Assays will be based on thermal denaturation and isothermal calorimetry. When potential substrates have been identified, activity on GDP-dDHep will be assessed (using HPLC and mass spectrometry of products). Further insight into the mechanism of activity will be provided by determining the structure of each enzyme by X-ray crystallography. Co-crystal or soaked structures will be also solved so that the structural basis for the activity of each enzyme can be identified. Finally, point mutants will be made in each protein to confirm that the mutation of putative catalytic residues reduces or abolishes activity. To illustrate that the in vitro data reflect the biological state, inactive mutant proteins will be added to deletion mutants of each enzyme, to demonstrate that these inactive mutants cannot rescue the capsule phenotype. These data will establish the function of these three novel proteins; and further complete the synthetic pathway of the B. pseudomallei capsule, providing a starting point for ex vivo capsule synthesis.

This study will benefit those outside the academic research community by contributing to the development of polysaccharides for vaccines against Burkholderia pseudomallei. This bacterium causes melioidosis - an infectious disease which is of public health importance in endemic areas (particularly Thailand and northern Australia) and is considered to be a potential bioterrorism threat (listed on Schedule B of the USA's Department of Health and Human Services Centers for Disease Control and Prevention list of bioterror threats). B. pseudomallei is resistant to many antibiotics and no vaccine currently exists. Consequently, there is an urgent public effort in both the UK and the USA to develop effective strategies for controlling this agent to mitigate against its use in bioterrorism. Hence, we have identified two key partners for impact activity: the UK's Defence Science and Technology Laboratory (Dstl) and the USA's Center for Disease Control and Prevention (CDC). A formal link has been established with Dstl. We also wish to engage with the USA's CDC in order to maximize the potential opportunities for commercialization of the work. We will engage with these key partners specifically by the following actions: - Establish an impact group comprising the PI, PDRA, staff from the University of Exeter's Research & Knowledge section and a representative from Dstl. - Provide two week-long secondments for the PI & PD to spend time in the Dstl labs. These secondments will provide opportunities to exchange skills, to disseminate preliminary results, and build research links. - Establish a network with CDC scientists by visiting laboratories that have an interest in this field of research. The objective of this engagement will be to support work leading to the future biosynthesis of an analogue of the B. pseudomallei capsular polysaccharide. This will allow this molecule to be used in vaccine trials as part of a conjugate vaccine. This will potentially expedite the development of an effective vaccine against B. pseudomallei. A realistic timescale for the full exploitation of this work will be 5-8 years from the end of the project, given the time taken to complete the development of a vaccine suitable for use by humans. Hence, the work will be of considerable potential benefit to promoting improved healthcare in endemic regions and acting proactively against potential security threats. The University of Exeter has well-established mechanisms for exploiting potential commercial opportunities from this work. The School of Biosciences works closely with the University's Research & Knowledge Transfer service, which has expertise in IP, patenting, non-disclosure agreements, and commercialisation. A strategy to protect the IP generated and develop potential commercialisation will be adopted. We also intend to use the study to promote bioscience as a career option for school students and develop a project website. The PI has worked in industry and is fully committed to developing the impact of this work. The PI will have responsibility for the impact activity within the project with support from the PDRA. This will provide an opportunity to build important networks for a new researcher. We will provide the following support to the PI: - Provide a mentor to support impact activities. - Provide career-development opportunities recognised in the commercial sector by means of secondments. The PI will be supported by the Research & Knowledge Transfer section in relation to IP and commercialisation activities. The web component of the project will be supported by the School of Biosciences Web Marketing Officer.