Antibiotic-adjuvants abolish resistance conferred by the Staphylococcus aureus erythromycin resistance methyltransferase in an Escherichia coli model

Abstract

Enzyme-mediated resistance is among the main strategies bacteria use to evade antibiotic action. S-adenosylmethionine-dependent erythromycin resistance methyltransferases catalyze the methylation of 23S ribosomal RNA in bacteria, causing resistance to macrolides, lincosamides, and streptogramin type-B antibiotics. Given the diversity and number of identified variants of these enzymes, it is vital to devise ways of inhibiting their activity to rescue affected antibiotics. Here, we use computer-aided solvent mapping and virtual screening techniques to identify inhibitors of Erms displaying promising adjuvant properties. We further demonstrate that an E. coli model expressing a recombinant S. aureus ErmC (SaErmC) variant causes substantial resistance to representative macrolide and lincosamide antibiotics. Assessment of test compounds using this resistance model revealed candidates displaying promising adjuvant activity when combined with erythromycin or clindamycin. Antibiotic combinations with a principal candidate oxadiazole, JNAL-016, completely blocked SaErmC-mediated resistance against erythromycin, resulting in an antibiotic-sensitive phenotype in broth microdilution screening assays. This compound also suppressed ErmC activity, allowing erythromycin to regain its bactericidal properties when assessed in actively growing cultures using time-kill assays. JNAL-016 displayed a noncompetitive mode of inhibition against SaErmC activity in vitro and bound the purified enzyme with high affinity (Kd = 1.8 {+/-} 0.7 M) based on microscale thermophoresis data. Competition experiments suggested that JNAL-016 competes with SAM for its binding pocket on the enzyme, and this compound exhibited no toxicity against human embryonic kidney cells. These findings establish a practical strategy for targeting Erm-mediated resistance, which could lead to a viable adjuvant-based therapy against bacterial pathogens that weaponize variants of this class of methyltransferases.