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

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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.