title: Resistance to AFN-1252 arises from missense mutations in Staphylococcus aureus enoyl-acyl carrier protein reductase (FabI).
abstract: AFN-1252 is a potent antibiotic against Staphylococcus aureus that targets the enoyl-acyl carrier protein reductase (FabI). A thorough screen for AFN-1252-resistant strains was undertaken to identify the spectrum of mechanisms for acquired resistance. A missense mutation in fabI predicted to encode FabI(M99T) was isolated 49 times, and a single isolate was predicted to encode FabI(Y147H). AFN-1252 only bound to the NADPH form of FabI, and the close interactions between the drug and Met-99 and Tyr-147 explained how the mutations would result in resistant enzymes. The clone expressing FabI(Y147H) had a pronounced growth defect that was rescued by exogenous fatty acid supplementation, and the purified protein had less than 5% of the enzymatic activity of FabI. FabI(Y147F) was also catalytically defective but retained its sensitivity to AFN-1252, illustrating the importance of the conserved Tyr-147 hydroxyl group in FabI function. The strains expressing FabI(M99T) exhibited normal growth, and the biochemical properties of the purified protein were indistinguishable from those of FabI. The AFN-1252 Ki(app) increased from 4 nm in FabI to 69 nm in FabI(M99T), accounting for the increased resistance of the corresponding mutant strain. The low activity of FabI(Y147H) precluded an accurate Ki measurement. The strain expressing FabI(Y147H) was also resistant to triclosan; however, the strain expressing FabI(M99T) was more susceptible. Strains with higher levels of AFN-1252 resistance were not obtained. The AFN-1252-resistant strains remained sensitive to submicromolar concentrations of AFN-1252, which blocked growth through inhibition of fatty acid biosynthesis at the FabI step.
authors.affiliation: From the Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee 38105.
authors.forename: Jiangwei
authors.initials: J
authors.lastname: Yao
authors.forename: John B
authors.initials: JB
authors.lastname: Maxwell
authors.forename: Charles O
authors.initials: CO
authors.lastname: Rock
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001572:Benzofurans / Q000494:pharmacology*; D001665:Binding Sites; D024881:Drug Resistance, Bacterial / Q000235:genetics; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary; D011753:Pyrones / Q000494:pharmacology*; D013211:Staphylococcus aureus / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*
chemical_list: C521531:API 1252; D000900:Anti-Bacterial Agents; D001572:Benzofurans; D011753:Pyrones; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)
keywords: Acyl Carrier Protein; Antibiotic Resistance; Antibiotics Action; Drug Resistance; Enoyl-ACP Reductase; FabI; Fatty Acid Synthase; Staphylococcus aureus