A conserved protein O-glycosylation pathway in the Burkholderia genus essential for bacterial fitness and antigenicity in humans

The Burkholderia genus includes bacteria widely distributed around the planet, which can survive in diverse environments and in association with diverse hosts. Some of the Burkholderia species are particularly dangerous to humans, as they cause disseminated and often lethal infections such as melioidosis (B. pseudomallei) and glanders (B. mallei), and are also classified as category B organisms due their potential use as biological warfare agents. Other Burkholderia species (e.g. B. cenocepacia and B. multivorans), cause debilitating lung infections in cystic fibrosis patients. On the other hand, Burkholderia species are highly useful for bioremediation, plant growth promotion, and pest biocontrol. Preventing Burkholderia infections in susceptible people will eliminate the threat of Burkholderia for humans and make it possible to better exploit the multiple beneficial aspects of these bacteria. We have characterised a protein glycosylation pathway conserved in all Burkholderia that allows the possibility to develop a universal Burkholderia vaccine.

The Burkholderia protein glycosylation pathway is encoded by genes conserved in all Burkholderia species, and consists of proteins involved in stitching sugars together in a particular sequence to form an oligosaccharide molecule, which is then incorporated to several bacterial proteins that are located on the bacterial cell envelope. We are in the process to establishing the carbohydrate structure of the oligosaccharide attached to at least 23 Burkholderia proteins, and have elucidated the genes required for oligosaccharide assembly and export. Our research team also discovered that sera from Burkholderia-infected patients suffering from cystic fibrosis (B. cenocepacia and B. multivorans), melioidosis (B. pseudomallei) and glanders (B. mallei) have antibodies that specifically recognize a glycosylated protein purified from B. cenocepacia, indicating that glycosylated Burkholderia proteins are perceived by the human immune system. To test whether immunization with a Burkholderia glycosylated protein stimulates a protective immune response, groups of mice were immunized against a glycosylated protein purified from B. cenocepacia, which was also mixed with Alum, a clinically used vaccine antigen. Mice were protected from infection upon intraperitoneal challenge with B. multivorans, also indicating that the vaccine elicits cross-protection against different Burkholderia species.

This proposal underpins fundamental studies at the forefront of microbial glycobiology, molecular biology and glycochemistry research with the goals to: (i) Decode the functions of the enzymes involved in the Burkholderia protein glycosylation pathway and elucidate the molecular structure of the oligosaccharide glycan; (ii) Elucidate the mechanism behind the physiological alterations due to loss of protein glycosylation in bacteria; and (iii) Determine the structure function of the oligosaccharyltransferase PglL to enable biotechnological applications through glycoengineering approaches. Aligned to the BBSRC roadmap, this innovative project rises to the challenges of finding novel means to deal with dangerous opportunistic pathogens by advancing biotechnological research elucidating the protein glycosylation system in Burkholderia and exploiting this knowledge to develop wide-spectrum vaccine. It also fits well with the need to find alternatives to antibiotics for the control of multidrug resistant bacteria, such as the Burkholderia, by exposing new ways to prevent infection by these bacteria in susceptible patients.

This proposal underpins fundamental studies at the forefront of microbial glycobiology, molecular biology and glycochemistry research. We discovered a novel protein O-glycosylation (ogc) pathway conserved in all members of the Burkholderiaceae. Our objectives are to: (i) Decode the functions of the enzymes involved in the Burkholderia protein glycosylation pathway and elucidate the molecular structure of the oligosaccharide glycan, (ii) Elucidate the mechanism behind the physiological alterations due to loss of protein glycosylation in bacteria, and (iii) Determine the structure-function of the oligosaccharyltransferase PglL to enable biotechnological applications through glycoengineering approaches.

The following specific aims will be carried out:
1. To functionally characterise the ogc gene cluster and determine the structure of the oligosaccharide glycan: 1.1 functional assignments of each ogc gene and corresponding proteins in B. cenocepacia; and 1.2 structural characterisation of the oligosaccharide glycan in B. cenocepacia and other Burkholderia species.

2. To investigate the role of protein glycosylation in bacterial physiology and establish its molecular mechanism: 2.1. global proteomics and transcriptomics analyses; 2.2. investigation of the degradome in the O-glycosylation mutants.

3. Structure function of PglB and glycoengineering approaches based on the Burkholderia protein glycosylation system: 3.1 Construction of a topological model for PglL and identification of functional residues; 3.2. Expression of PglL and Ogc proteins in E. coli to reconstruct the glycosylation system and investigate PglL substrate requirements for possible glycoengineering approaches.

This combined fundamental research and applied proposal will result in novel and significant contributions to UK science and UK knowledge economy, which are outlined as follows:

1) Publication of the research in peer-reviewed, open access journals. Publications will be complemented by presentation of data at national and international scientific conferences, as well as other appropriate venues involving dissemination of vaccine strategies to curtail Burkholderia infections which gather multidisciplinary scientists, public health, government and business representatives.

2) Our proposal provides fundamental research that has potential relevance to the pharmaceutical industry (biopharmaceuticals, antibiotic resistance). The common theme concerning public benefit arising from this research is the potential to lead to life-enhancing applications when antibiotic resistance becomes more widespread. The problem of antibiotic resistance is even more critical in the case of infections by opportunistic pathogens.

3) Our proposal provides a foundation to exploit the protein O-glycosylation pathway in Burkholderia for glycoengineering preventative (e.g. vaccine) or diagnostic (e.g. complement fixatino test for glanders in horses) approaches.

4) During the course of our grant proposal we envisage a patent filed, which will protect the IP on the uniqueness of the glycan trisaccharide for vaccine development. We will engage potential companies to explore potential research partnerships that will enable additional funding (Innovation UK, Wellcome Trust, POC-NI). We will also establish collaborations with other QUB researchers (e.g. Prof Ryan Donnelly in the School of Pharmacy) to include novel delivery strategies via micro-needles or liposomal formulations, which will enrich the commercial value of our vaccine. Finally, we will also explore contract research grant agreements with the UK Department of Defense and the US DoD enabled by the need to safeguard the bio-warfare potential of some Burkholderia types.

5) This project will contribute to the career development of the PDRA and Research Technician appointed to the project by ensuring additional training and experience. Similar skills training will be made available to other participants in PhD, MSc and undergraduate positions.

6) The relevance of this research to the biopharmaceutical and biotechnology sectors will benefit public engagement to inform and educate the public about opportunities arising from a knowledge-based economy and to educate about science in general. We intend to take full advantage of available opportunities to do so through Queen's outreach activities and the media. Furthermore, we will use available opportunities to disseminate information about published research success to the general media through the Press and Publicity Units at Queen's.