Structure and Function of the Genomically-Encoded Fosfomycin Resistance Enzyme, FosB, from Staphylococcus Aureus

Abstract

The Gram-positive pathogen Staphylococcus aureus is a leading cause of global morbidity and mortality. Like many multi-drug resistant organisms, S. aureus contains antibiotic modifying enzymes that facilitate resistance to a multitude of antimicrobial compounds. FosB is a Mn2+-dependent fosfomycin-inactivating enzyme found in S. aureus that catalyzes nucleophillic addition of either L-cysteine (L-Cys) or bacillithiol (BSH) to the antibiotic resulting in a modified compound with no bacteriacidal properties. The three-dimensional x-ray crystal structure of FosBSa has been determined to a resolution of 1.15 A. Co-crystallization of FosBSa with either L-cys or BSH results in an unnatural disulfide bond between the exogenous thiol and the active site Cys9 of the enzyme. Two crystals of FosBSa contain Zn2+ in the active site, but subsequent kinetic analyses indicated that the enzyme is inhibited by Zn2+ for L-Cys transferase activity and only marginally active for BSH transferase activity. Fosfomycin-treated disk diffusion assays involving S. aureus Newman and the USA300 methicillin-resistant S. aureus (MRSA) demonstrate a marked increase in sensitivity of the organism to the antibiotic in either the BSH or FosB null strains, indicating that both are required for survival of the organism when treated with fosfomycin. This work identifies FosB as the sole fosfomycin-modifying pathway of S. aureus and establishes the enzyme as a potential therapeutic target for increased efficacy of the antibiotic against the pathogen.