Activity-based protein profiling of serine hydrolases and penicillin-binding proteins in Enterococcus faecium

Area of Science:

• Microbiology
• Chemoproteomics
• Drug Discovery

Background:

• Enterococcus faecium is an opportunistic pathogen with increasing antimicrobial resistance, necessitating novel therapeutic targets.
• Vancomycin-resistant strains pose a significant treatment challenge.
• Serine hydrolases are a diverse enzyme family with underexplored potential as drug targets.

Purpose of the Study:

• To identify novel druggable targets in Enterococcus faecium using activity-based protein profiling (ABPP).
• To investigate serine hydrolase activity across different Enterococcus strains and conditions.
• To characterize previously unannotated serine hydrolases in vancomycin-resistant E. faecium.

Main Methods:

• Utilized activity-based protein profiling (ABPP) with functionalized covalent inhibitors.
• Employed gel-based ABPP with Bocillin-FL to assess penicillin-binding protein (PBP) activity.
• Used a broadly reactive fluorescent probe (fluorophosphonate-TMR) for serine hydrolase profiling.
• Identified targets via enrichment with a biotinylated probe and liquid chromatography-mass spectrometry.

Main Results:

• Detected strain- and condition-dependent variations in PBP activities.
• Observed high serine hydrolase activity similarity in E. faecium clade A1 strains, with greater variation in A2 and E. lactis.
• Identified 11 novel, largely uncharacterized serine hydrolases (e.g., α,β-hydrolases, SGNH-hydrolases, phospholipases, amidases, peptidases).
• These targets are druggable and accessible in live vancomycin-resistant E. faecium E745.

Conclusions:

• ABPP is effective for identifying active enzymes in E. faecium.
• Discovered novel serine hydrolase targets with potential roles in E. faecium viability.
• These targets represent promising candidates for developing new antimicrobial therapies against resistant strains.