title: Detection and Quantification of Ribosome Inhibition by Aminoglycoside Antibiotics in Living Bacteria Using an Orthogonal Ribosome-Controlled Fluorescent Reporter.
abstract: The ribosome is the quintessential antibacterial drug target, with many structurally and mechanistically distinct classes of antibacterial agents acting by inhibiting ribosome function. Detecting and quantifying ribosome inhibition by small molecules and investigating their binding modes and mechanisms of action are critical to antibacterial drug discovery and development efforts. To develop a ribosome inhibition assay that is operationally simple, yet provides direct information on the drug target and the mechanism of action, we have developed engineered E. coli strains harboring an orthogonal ribosome-controlled green fluorescent protein (GFP) reporter that produce fluorescent signal when the orthogonal ribosome is inhibited. As a proof of concept, we demonstrate that these strains, when coexpressing homogeneous populations of aminoglycoside resistant ribosomes, act as sensitive and quantitative detectors of ribosome inhibition by a set of 12 structurally diverse aminoglycoside antibiotics. We suggest that this strategy can be extended to quantifying ribosome inhibition by other drug classes.
authors.affiliation: Department of Chemistry and Chemical Biology, ‡Department of Biology, and §Center for Biomedical Engineering, University of New Mexico , Albuquerque, New Mexico 87131-0001, United States.
authors.forename: Shijie
authors.initials: S
authors.lastname: Huang
authors.affiliation: Department of Chemistry and Chemical Biology, ‡Department of Biology, and §Center for Biomedical Engineering, University of New Mexico , Albuquerque, New Mexico 87131-0001, United States.
authors.forename: Xuechen
authors.initials: X
authors.lastname: Zhu
authors.affiliation: Department of Chemistry and Chemical Biology, ‡Department of Biology, and §Center for Biomedical Engineering, University of New Mexico , Albuquerque, New Mexico 87131-0001, United States.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Melançon
mesh_terms: D000617:Aminoglycosides / Q000737:chemistry / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology; D001681:Biological Assay; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000187:drug effects; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D015394:Molecular Structure; D012270:Ribosomes / Q000187:drug effects* / Q000378:metabolism
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins