In search of new putative antimicrobial drug targets in methicillin-resistant Staphylococcus aureus, we aimed to identify and characterize retaining glycosidase activities in this bacterial pathogen. Using activity-based protein profiling (ABPP), a panel of 7 fluorescent probes was screened to detect activities of diverse retaining glycosidase families. Based on this, a cocktail of 3 biotinylated probes (targeting α-glucosidases, β-galactosidases and α-fucosidases) was used for target enrichment and three glycoside hydrolase family proteins were identified by mass-spectrometry: 6-phospho-β-glucosidase (BglA), α-amylase family protein trehalase C (TreC), and autolysin (Atl). The physiological relevance of previously uncharacterized BglA and TreC was addressed in CRISPRi and inhibitor studies with the putative TreC inhibitor α-cyclophellitol-aziridine. Silencing of treC did not affect bacterial growth in rich media, but reduced biofilm formation in vitro, and attenuated virulence during Galleria mellonella infection, warranting future investigations into the biochemical function of this enzyme.

In search of new putative antimicrobial drug targets in methicillin-resistant Staphylococcus aureus, we aimed to identify and characterize retaining glycosidase activities in this bacterial pathogen. ...