Using chemical glycobiology to further our understanding of the enzymatic processing of poly-N-acetylglucosamine (PNAG) in bacteria

Poly-N-acetylglucosamine (PNAG) is a polysaccharide that is found in the biofilms of several pathogenic species. As such it has been the subject of several studies since its discovery. However, synthesising long polysaccharides in vitro has proven difficult due to the challenges of carbohydrate chemistry, and as such access to pure PNAG of defined length and modification has not yet been achieved. This has led to there still being some gaps in the literature surrounding PNAG. The glycosyltransferase enzymes responsible for PNAG polymerisation in vivo are complex membrane proteins, and therefore unsuitable for in vitro synthesis. It has been shown that glycoside hydrolases can be mutated to perform the opposite reaction, creating what is known as a glycosynthase. Therefore, we proposed to screen mutants of a suitable PNAGase, such as DspB or a member of new class of potential staphylococciPNAGases, for glycosynthase activity using a set of successfully synthesised carbohydrate substrates alongside commercially available substrate. This strategy would have allowed us to use synthetic monosaccharide donors for enzymatic polymerisation of PNAG oligosaccharides which could be used for further study. However, these efforts failed due to the selected mutants of DspB not acting as glycosynthases, and the putative staphylococci PNAGases proving difficult to express without extensive method development that was outside the scope of this project.

We also aimed to develop a method to chemically modify PNAG in vitro. PNAG can be isolated in vivo, and can by modified by the selective incorporation of an azide at the reducing terminus of the PNAG polysaccharide, deacetylation, or desuccinylation. This modifiation of PNAG opens the door for many avenues of further research, such as bioconjugation to something antigenic potentially creating a vaccine candidate. Some initial evidence shows this modification might have been successful but further experiments are required to conclusively prove this