Kyong Tkhe Fam, Pavan Kumar Chodisetti, Zifei Wang, Joshua A. Homer, Christopher J. Smedley, Seiya Kitamura, B.C. Silva, Yijun Xiong, Althea Hansel-Harris, Matthew Holcomb, Simeon Babarinde, Adrianna M. Turner, Daria Van Tyne, Ian A. Wilson, Stefano Forli, Benjamin F. Cravatt, Donghyun Park, Dennis W. Wolan, John E. Moses, Howard C. Hang
Abstract 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 vancomycin susceptibility of VREfm ex vivo and in vivo . Notably, genetic deletion of sagA impaired VREfm peptidoglycan remodeling, growth and increased the activity of vancomycin. 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 increased the efficacy of vancomycin across genetically distinct VREfm clinical isolates. Our study reveals peptidoglycan hydrolases are druggable targets whose inactivation improves the efficacy of vancomycin against VREfm.