Development of Fluorogenic Probes to Detect Glycan-Degrading Enzymes in Bacteria

Bacterial fitness and survival depend on the biosynthesis and maintenance of cell-envelope glycans. Bacterial glycans are composed of over 700 monosaccharides, and their construction, tailoring, recycling, and degradation require the choreographed action of myriad glycosyltransferases and glycosidases. Because of the structural diversity and complexity of bacterial glycans, coupled to limited methods to profile bacterial glycosidase activity, the function and existence of glycosidases that degrade most bacterial glycans are unknown. Taking a cue from the successful use of fluorogenic glycosides based on ubiquitous monosaccharides to detect glycosidase activity in cells and tissues, we sought to expand this approach to detect glycosidase activity in bacteria. Fluorogenic probes based exclusively on bacterial amino and deoxy sugars N-acetyl-d-fucosamine and 2,4-diacetamido-2,4,6-trideoxy-d-galactose were developed, and their activation was directly compared to a d-glucose-based fluorogenic probe in bacterial and mammalian cells. The species-selective utilization of these glycosidase probes, coupled to the difference in relative magnitude of glycosidase activity observed, provides important insights into these relatively unexplored glycan-processing enzymes. The use of these fluorogenic probes expands the toolkit for detecting glycan-degrading enzymes and offers an approach to enhancing a broader understanding of bacterial glycan metabolism and its diversity across different species.