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A disk-diffusion-based target identification platform for antibacterials (TIPA): an inducible assay for profiling MOAs of antibacterial compounds

  • Applied genetics and molecular biotechnology
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Abstract

One of the challenges in antibiotic lead discovery is the difficulty and time-consuming task of determining the mechanism of action (MOA) of antibacterial compounds. In this report, we describe the development and validation of a facile and inexpensive assay system utilizing disk diffusion of inhibitors on solid agar medium embedded with mixed pools of a comprehensive collection of Escherichia coli clones each containing a plasmid-borne inducible essential gene from E. coli. From individual clones, pilot small-scale (48 or 50 clones) assays, to full-scale target identification platform for antibacterials (TIPA) system, involving a variety of assay formats (liquid vs solid media, individual vs mix clones), we demonstrate that elevated resistance phenotypes of relevant cell clones were highly specific. In particular, the TIPA system was able to reveal cellular targets of several known antibacterial inhibitors: cerulenin, diazaborine, indolmycin, phosphomycin, and triclosan. Complementary to several existing MOA profiling schemes, the TIPA system offers a simple and low-cost method for elucidating the target proteins of antibacterial inhibitors, thus will facilitate discovery and development of novel antibacterial compounds to combat multidrug-resistant bacterial pathogens.

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Acknowledgments

Funding for this project has been provided by grants from the U.S. Department of Defense’s Army Research Office (W911NF-12-1-0059), NIH (S3GM083686), and CSUPERB (California State University Program for Education and Research in Biotechnology) (Entrepreneurial Joint Venture Matching grant) to H. H. Xu. I. Silva and M. S. Ward acknowledge scholarship support from the NIH Minority Biomedical Research Support-Research Initiative for Scientific Enhancement (MBRS-RISE) Program (5R25GM061331). Technical assistance from Walfre Martinez, Jere Wilson, Marlyn Rios, David Yang, Deisy Contreras, Peter Chong, Sonya Valentine, and Christopher Lam is greatly appreciated. We are grateful to Nara Institute of Science and Technology (Nara, Japan) for generously providing the E. coli essential gene overexpression clones.

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  1. Department of Biological Sciences, California State University, Los Angeles, 5151 State University Dr., Los Angeles, CA, 90032, USA

    Isba Silva, Lilian J. Real, Matthew S. Ward & H. Howard Xu

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  1. Isba Silva
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  2. Lilian J. Real
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  4. H. Howard Xu
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Correspondence to H. Howard Xu.

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Silva, I., Real, L.J., Ward, M.S. et al. A disk-diffusion-based target identification platform for antibacterials (TIPA): an inducible assay for profiling MOAs of antibacterial compounds. Appl Microbiol Biotechnol 98, 5551–5566 (2014). https://doi.org/10.1007/s00253-014-5623-9

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