Activity-Based Protein Profiling Identifies an α‑Amylase Family Protein Contributing to the Virulence of Methicillin-Resistant Staphylococcus aureus

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.