Examination of Acetylated Monosaccharides as Metabolic Probes in Bacteria

Bacterial glycans are validated antibiotic targets due to their crucial roles in supporting bacterial fitness and survival. The array of exclusively bacterial monosaccharides and their variable expression across bacterial species and serotypes present challenges in studying these structurally diverse molecules. Probes based on bacterial sugars have emerged as useful tools in metabolic labeling studies. Prior to the metabolic processing of probes by bacteria, most metabolic probes must be transported across the bacterial cell envelope. Probe acetylation has been used as one strategy to ease passive diffusion across the lipophilic cell membrane and relies on deacetylation by esterases within cells before subsequent metabolic processing into glycans is possible. However, inefficient probe deacetylation has the potential to yield artifactual labeling rather than physiological glycan labeling. Here, we systematically explored probe acetylation as a design criterion for metabolic labeling experiments in four bacterial species. Plesiomonas shigelloides, Vibrio vulnificus, and Helicobacter pylori exhibited a strong preference for metabolic incorporation of acetylated probes relative to unprotected probes, whereas Bacteroides fragilis incorporated both unprotected and acetylated probes at comparable levels. Curiously, only B. fragilis had sufficient esterase activity to quantitatively deacetylate a peracetylated monosaccharide probe in situ. These findings suggest the importance of validating acetylated probes on a case-by-case basis to ensure physiologically relevant bacterial glycan labeling.