Fam, K. T.; Chodisetti, P. K.; Wang, Z.; Homer, J. A.; Smedley, C. J.; Kitamura, S.; Silva, B.; Xiong, Y.; Hansel-Harris, A.; Holcomb, M.; Babarinde, S.; Van Tyne, D.; Wilson, I. A.; Forli, S.; Cravatt, B.; Park, D.; Wolan, D. W.; Moses, J. E.; Hang, H. C.

Vancomycin-resistant Enterococcus faecium (VREfm) is a leading cause of healthcare-associated infections globally and demands new approaches for treatment. Here we show that genetic and pharmacological inactivation of a highly conserved NlpC/P60 peptidoglycan hydrolase, secreted antigen A (SagA), enhanced antibiotic susceptibility in VREfm ex vivo and in vivo. Genetic deletion of sagA impaired VREfm peptidoglycan remodeling, growth and increased antibiotic susceptibility, which notably also restored vancomycin sensitivity in VREfm. We then identified first-in-class covalent NlpC/P60 peptidoglycan hydrolase inhibitors and demonstrated that pharmacological inactivation of SagA activity also impaired peptidoglycan remodeling and promoted vancomycin susceptibility across distinct VREfm clinical isolates. Our study reveals peptidoglycan hydrolases are druggable targets whose inhibition boosts the efficacy of antibiotics against VREfm.