| The family of carbohydrates called sialic acids was long thought to exclusively occur in glycoproteins and glycolipids of vertebrates, but has since also been found in many microbes. Especially symbiotic and pathogenic microbes associated with the human gut express a wide array of unique sialic acids. The roles microbial sialic acids (MSAs) play in the functioning of gut-associated microbes are currently not well understood. With this proposal I aim to develop molecular tools and use them to investigate the glycobiology of MSAs in two relevant gut-associated microbes. The proposed molecular tools consist of defined MSA glycosides and functional derivatives thereof, which will be used to probe the proteins that interact with MSAs during their life-cycle: glycosyltransferases, lectins and glycosidases. Therefore the initial challenge in this project will be to develop a robust and flexible synthetic route towards the MSA monosaccharides. Next, I will explore their virtually uncharted glycosylation chemistry. The hereby produced glycosides will be incorporated in a microarray and used to discover the microbial lectins that bind MSAs. Another key part of the proposal will be to extend the developed chemistry to produce a novel panel of labeled enzyme substrates, inhibitors and activity-based probes. This panel of functional MSA derivatives will be evaluated, optimized if needed and then used to probe the unexplored glycosidases and glycosyltransferases. Using labeled sialic acids as substrates I will be able to detect and study their enzyme activity in microbial cultures and lysates. The inhibitors will be evaluated on the detected glycosidase activities and with activity-based probes I will localize and identify these activities. The results from the glycobiology study and the developed chemistry and panel of molecular tools will prove highly useful in starting to reveal the roles of MSAs in both pathogenic and symbiotic gut-associated microbes. |
