O2-3 Metabolic Glycoengineering: A strategy to label Brucella abortus cell envelope with synthetic analogues of D-mannose

Abstract

Metabolic Glycoengineering (MGE) is a powerful tool for the study of biomolecules in living systems. This technique consists in manipulating the metabolism of living cells, prokaryotic or eukaryotic, in order to modulate their glycosylation. MGE can be used to modify natural glycans, disrupt glycan biosynthesis and probe metabolic flux inside cells. An interesting approach is to install unnatural monosaccharides, with subtlemodifications, into cellular glycans. We aim to chemically modify the bacterial cell surfaces by targeting the cell wall with unnatural clickable sugars that are metabolically transformed and incorporated. The metabolic incorporation into glycans pathway can be visualized by chemical probes, for instance fluorescent reporters, that could react with the unnatural carbohydrate via biorthogonal click reactions. In this project, the strategy is to explore the metabolic route of D-mannose because, to the best of our knowledge, clickable D-mannose derivatives have never been described for such an approach in the literature. Moreover, D-mannose is present only in some bacterial cell surface, which could lead to a method with a high specificity. We performed a multi- step stereoselective synthesis of clickable D-mannose derivative. In this study, we showed that D-mannose derivatives can be used to fluorescently label Brucella abortus cell envelope in a specific manner. Furtherinvestigations showed that the unnatural monosaccharide is incorporated in the lateral branch of the core of the lipopolysaccharide, since a wadC mutant is unable to incorporate the modified mannose, while a gmd mutant is still incorporating it. In contrast to what has been previously described, we here labelled the entire cell envelope and not only the growing pole of the bacterium, which suggests a putative remodeling of the bacterial surface of Brucella abortus.