Abstract
Connecting bacterial growth inhibitors to molecular targets at the whole-cell level is a major impediment to antibacterial development. Herein we report the design of a highly efficient and versatile bacteriophage-based mariner transposon delivery system in Staphylococcus aureus for determining inhibitor mode of action. Using bacteriophage-mediated delivery of concatameric minitransposon cassettes, we generated nonclonal transposant libraries with genome-wide insertion-site coverage in either laboratory or methicillin-resistant strain backgrounds and screened for drug resistance in situ on a single agar plate in one step. A gradient of gene-target expression levels, along with a correspondingly diverse assortment of drug-resistant phenotypes, was achieved by fitting the transposon cassette with a suite of outward-facing promoters. Using a panel of antibiotics, we demonstrate the ability to unveil not only an inhibitor's molecular target but also its route of cellular entry, efflux susceptibility and other off-target resistance mechanisms.
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Acknowledgements
We are grateful to all of the scientists who worked at the Merck Frosst Center for Therapeutic Research for having provided a stimulating environment for infectious disease research.
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T.C.M. designed the system and experiments, T.C.M. and H.W. performed the experiments, D.C. conducted the real time-PCR experiments, and J.P.V. provided bioinformatics support and analysis. T.C.M., H.W. and T.R. analyzed the data. T.C.M. wrote the manuscript.
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H.W., D.C. J.P.V., T.R. and T.C.M. are present or past employees of Merck, as stated in the affiliations, and potentially own stock and/or hold stock options in the company.
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Wang, H., Claveau, D., Vaillancourt, J. et al. High-frequency transposition for determining antibacterial mode of action. Nat Chem Biol 7, 720–729 (2011). https://doi.org/10.1038/nchembio.643
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DOI: https://doi.org/10.1038/nchembio.643
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