Award details

Bacterial oligosaccharyltransferase for glycoengineering and vaccine development

Reference	BB/F009321/1
Principal Investigator / Supervisor	Professor Brendan Wren
Co-Investigators / Co-Supervisors
Institution	London Sch of Hygiene and Trop Medicine
Department	Infectious and Tropical Diseases
Funding type	Research
Value (£)	205,586
Status	Completed
Type	Research Grant
Start date	01/04/2008
End date	31/03/2011
Duration	36 months

Abstract

Glycan-containing biomolecules are ubiquitous and although involved in diverse processes ranging from immune recognition to cancer development their significance remains underestimated. Furthermore, and in contrast to polypeptides and nucleic acids, glycoproteins have escaped biotechnological applications. However our demonstration of glycoprotein biosynthesis in the model bacterium Escherichia coli, through the activity of a novel C. jejuni oligosaccharyltransferase (CjPglB), is a significant advance (Wacker et al, Science 2002). Recently, along with our collaborators we have demonstrated that CjPglB has a relaxed specificity enabling transfer of diverse polysaccharides, including lipopolysaccharide and capsules, to proteins with appropriate D/E-X-N-Z-S/T consensus sequons. However, CjPglB is unable to transfer all glycans; one requirement is for an acetamido group at the C-2 position of the reducing end sugar of the glycan. Thus before the production of diverse protein-polysaccharide conjugates in E. coli can be fully realised, a wider specificity of the PglB enzyme is required. To this end, we propose a dual strategy of mining further bacterial pglB orthologues combined with directed evolution approaches to explore the specificity range of characterised oligosaccharyl transferases. Additionally, we will attempt structural characterisation of CjPglB to provide mechanistic information on oligosaccharide transferase activity. As test cases we will determine if capsules from bacterial pathogens Streptococcus pneumoniae and Burkholderia pseudomallei, that lack an acetamido group at the C-2 position, can be transferred to an appropriate acceptor protein by the novel or modified PglBs. If successful these studies could radically improve the production and range of novel protein glycoconjugates for vaccines. Irrespective of vaccine development, we are convinced that the studies will be important for the development of glycoengineering in both basic and applied research.

Summary

Vaccination has been incredibly successful in reducing the burden of infectious diseases. Examples of successful vaccines include those against the deadly bacteria Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae. The basis for these vaccines is a complex sugar structure, known as the capsule, which covers bacterial cells protecting them from immune attack. In order to evoke an appropriate immune response, vaccines against these bacteria consist of capsule linked to a protein carrier forming a glycoprotein or glycoconjugate. However, despite the success of these glycoconjugate vaccines they have major drawbacks in terms of the technical difficulties in purifying the capsule material from bacterial cells and then conjugating the capsule to carrier proteins. Additionally, capsules are often highly variable, and the specific immunity elicited by immunisation with one type of capsule will not protect against bacteria with different capsule structures. Thus as new disease strains emerge (e.g. from selective pressure by large scale vaccination regimes) the existing vaccines become ineffective. An inexpensive, rapid and flexible method for glycoconjugate vaccine production would enable a more effective response to the emergence of new pathogenic bacterial strains with different capsule structures. One such approach is to produce glycoconjugate vaccines in the genetically tractable bacterium Escherichia coli. E. coli is already used as a 'cellular factory' to produce large amounts of proteins; however, until recently it has not been possible to generate glycoproteins in this bacterium. That could now change. We have recently identified and characterised a gene cluster (pgl) which is responsible for the synthesis of glycoproteins in the bacterial food-borne pathogen, Campylobacter jejuni. We have been able to transfer the segment of DNA containing the pgl genes into E. coli to produce recombinant glycoproteins, thus opening up the field of glycoengineering. The key enzyme in the C. jejuni pathway that couples proteins to sugars is the oligosaccharyltransferase protein termed CjPglB. Although CjPglB can transfer many sugar structures unfortunately there are many structures from various pathogens that it cannot. Essentially, the end of the glycostructure that is attached to the protein by CjPglB, must have a sugar unit with a specific configuration - an acetamido group at the C-2 position of the sugar at the reducing end of the glycan. This severely limits the potential applications of this technology. Indeed many capsules of pathogenic bacteria do not have this configuration and therefore CjPglB could not be used to produce glycoconjugate vaccines for protection against these bacteria. In this study we propose a number of strategies to overcome this problem. We will seek to identify or engineer alternative PglB proteins that will have a modified specificity for different glycostructures. We will use a dual approach of seeking alternative PglBs from other bacteria that may naturally have a different specificity to the original CjPglB, and also a mutagenesis approach to alter the enzymatic specificity of CjPglB. To ascertain if the specificity of the natural and mutated PglBs have been altered we will test separate capsular polysaccharides from the important pathogens Streptococcus pneumoniae and Burkholderia pseudomallei to determine if the respective capsules can now be coupled/conjugated to an appropriate carrier protein. The new recombinant glycoconjugates in E. coli will be ideal vaccine candidates that can be readily purified and tested. The glycoengineering principles to be pioneered in this study could be applied generically to the design of other glycoconjugate and combination vaccines. Irrespective of vaccine development, this new and emerging technology will be of direct importance to scientists interested in basic research and in applied research in glyco-biotechnology.

Committee	Closed Committee - Engineering & Biological Systems (EBS)
Research Topics	Animal Health, Immunology, Microbiology, Structural Biology, Synthetic Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

Associated awards:

BB/F009496/1 Bacterial oligosaccharyltransferase for glycoengineering and vaccine development

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