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High-frequency transposition for determining antibacterial mode of action

Nature Chemical Biology volume 7, pages 720–729 (2011)Cite this article

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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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Figure 1: Modulation of gene expression through Tnp mutagenesis.
Figure 2: Bacteriophage-mediated Tnp delivery and in situ drugR selection.
Figure 3: Tnp-mediated triclosan resistance in S. aureus COL.
Figure 4: Tnp-mediated CDFI resistance in S. aureus COL.
Figure 5: Tnp-mediated ciprofloxacin resistance in S. aureus RN4220.
Figure 6: Tnp-mediated indolmycin resistance in S. aureus COL.
Figure 7: Tnp-mediated antifolate resistance in S. aureus RN4220.

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References

  1. Theuretzbacher, U. Future antibiotics scenarios: is the tide starting to turn? Int. J. Antimicrob. Agents 34, 15–20 (2009).

    Article  CAS  PubMed  Google Scholar 

  2. Rice, L.B. The clinical consequences of antimicrobial resistance. Curr. Opin. Microbiol. 12, 476–481 (2009).

    Article  CAS  PubMed  Google Scholar 

  3. Brötz-Oesterhelt, H. & Sass, P. Postgenomic strategies in antibacterial drug discovery. Future Microbiol. 5, 1553–1579 (2010).

    Article  PubMed  Google Scholar 

  4. Boucher, H.W. et al. Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. Clin. Infect. Dis. 48, 1–12 (2009).

    Article  PubMed  Google Scholar 

  5. Mills, S.D. When will the genomics investment pay off for antibacterial discovery? Biochem. Pharmacol. 71, 1096–1102 (2006).

    Article  CAS  PubMed  Google Scholar 

  6. Alekshun, M.N. & Levy, S.B. Molecular mechanisms of antibacterial multidrug resistance. Cell 128, 1037–1050 (2007).

    Article  CAS  PubMed  Google Scholar 

  7. Bockstael, K. & Van Aerschot, A. Antimicrobial resistance in bacteria. Cent. Eur. J. Med. 4, 141–155 (2009).

    CAS  Google Scholar 

  8. Lange, R.P., Locher, H.H., Wyss, P.C. & Then, R.L. The targets of currently used antibacterial agents: lessons for drug discovery. Curr. Pharm. Des. 13, 3140–3154 (2007).

    Article  CAS  PubMed  Google Scholar 

  9. Silver, L.L. Challenges of antibacterial discovery. Clin. Microbiol. Rev. 24, 71–109 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Payne, D.J., Gwynn, M.N., Holmes, D.J. & Pompliano, D.L. Drugs for bad bugs: confronting the challenges of antibacterial discovery. Nat. Rev. Drug Discov. 6, 29–40 (2007).

    Article  CAS  Google Scholar 

  11. Breithaupt, H. The new antibiotics. Nat. Biotechnol. 17, 1165–1169 (1999).

    Article  CAS  PubMed  Google Scholar 

  12. Miesel, L., Greene, J. & Black, T.A. Genetic strategies for antibacterial drug discovery. Nat. Rev. Genet. 4, 442–456 (2003).

    Article  CAS  PubMed  Google Scholar 

  13. Payne, D.J., Gwynn, M.N., Holmes, D.J. & Rosenberg, M. Genomic approaches to antibacterial discovery. Methods Mol. Biol. 266, 231–259 (2004).

    CAS  PubMed  Google Scholar 

  14. Bordi, C. et al. In vitro mutagenesis of Bacillus subtilis by using a modified Tn7 transposon with an outward-facing inducible promoter. Appl. Environ. Microbiol. 74, 3419–3425 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Salipante, S.J., Barlow, M. & Hall, B.G. GeneHunter, a transposon tool for identification and isolation of cryptic antibiotic resistance genes. Antimicrob. Agents Chemother. 47, 3840–3845 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Carleton, S., Projan, S.J., Highlander, S.K., Moghazeh, S.M. & Novick, R.P. Control of pT181 replication II. Mutational analysis. EMBO J. 3, 2407–2414 (1984).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Xia, M., Lunsford, R.D., McDevitt, D. & Iordanescu, S. Rapid method for the identification of essential genes in Staphylococcus aureus. Plasmid 42, 144–149 (1999).

    Article  CAS  PubMed  Google Scholar 

  18. Novick, R.P., Edelman, I. & Lofdahl, S. Small Staphylococcus aureus plasmids are transduced as linear multimers that are formed and resolved by replicative processes. J. Mol. Biol. 192, 209–220 (1986).

    Article  CAS  PubMed  Google Scholar 

  19. Yansura, D.G. & Henner, D.J. Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis. Proc. Natl. Acad. Sci. USA 81, 439–443 (1984).

    Article  CAS  PubMed  Google Scholar 

  20. Luong, T.T., Ouyang, S., Bush, K. & Lee, C.Y. Type 1 capsule genes of Staphylococcus aureus are carried in a staphylococcal cassette chromosome genetic element. J. Bacteriol. 184, 3623–3629 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Anthony, R.M., Schuitema, A.R., Oskam, L. & Klatser, P.R. Direct detection of Staphylococcus aureus mRNA using a flow through microarray. J. Microbiol. Methods 60, 47–54 (2005).

    Article  CAS  PubMed  Google Scholar 

  22. Heath, R.J., Li, J., Roland, G.E. & Rock, C.O. Inhibition of the Staphylococcus aureus NADPH-dependent enoyl-acyl carrier protein reductase by triclosan and hexachlorophene. J. Biol. Chem. 275, 4654–4659 (2000).

    Article  CAS  PubMed  Google Scholar 

  23. Huber, J. et al. Chemical genetic identification of peptidoglycan inhibitors potentiating carbapenem activity against methicillin-resistant Staphylococcus aureus. Chem. Biol. 16, 837–848 (2009).

    Article  CAS  PubMed  Google Scholar 

  24. Ruiz, N. Bioinformatics identification of MurJ (MviN) as the peptidoglycan lipid II flippase in Escherichia coli. Proc. Natl. Acad. Sci. USA 105, 15553–15557 (2008).

    Article  CAS  PubMed  Google Scholar 

  25. Champoux, J.J. DNA topoisomerases: structure, function, and mechanism. Annu. Rev. Biochem. 70, 369–413 (2001).

    Article  CAS  PubMed  Google Scholar 

  26. Ng, E.Y., Trucksis, M. & Hooper, D.C. Quinolone resistance mutations in topoisomerase IV: relationship to the flqA locus and genetic evidence that topoisomerase IV is the primary target and DNA gyrase is the secondary target of fluoroquinolones in Staphylococcus aureus. Antimicrob. Agents Chemother. 40, 1881–1888 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Ng, E.Y., Trucksis, M. & Hooper, D.C. Quinolone resistance mediated by norA: physiologic characterization and relationship to flqB, a quinolone resistance locus on the Staphylococcus aureus chromosome. Antimicrob. Agents Chemother. 38, 1345–1355 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Ince, D. & Hooper, D.C. Quinolone resistance due to reduced target enzyme expression. J. Bacteriol. 185, 6883–6892 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Donald, R.G. et al. A Staphylococcus aureus fitness test platform for mechanism-based profiling of antibacterial compounds. Chem. Biol. 16, 826–836 (2009).

    Article  CAS  PubMed  Google Scholar 

  30. Hurdle, J.G., O'Neill, A.J. & Chopra, I. Anti-staphylococcal activity of indolmycin, a potential topical agent for control of staphylococcal infections. J. Antimicrob. Chemother. 54, 549–552 (2004).

    Article  CAS  PubMed  Google Scholar 

  31. Werner, R.G., Thorpe, L.F., Reuter, W. & Nierhaus, K.H. Indolmycin inhibits prokaryotic tryptophanyl-tRNA ligase. Eur. J. Biochem. 68, 1–3 (1976).

    Article  CAS  PubMed  Google Scholar 

  32. Cassels, R., Oliva, B. & Knowles, D. Occurrence of the regulatory nucleotides ppGpp and pppGpp following induction of the stringent response in staphylococci. J. Bacteriol. 177, 5161–5165 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Wolz, C., Geiger, T. & Goerke, C. The synthesis and function of the alarmone (p)ppGpp in firmicutes. Int. J. Med. Microbiol. 300, 142–147 (2010).

    Article  CAS  PubMed  Google Scholar 

  34. Brandenberger, M., Tschierske, M., Giachino, P., Wada, A. & Berger-Bachi, B. Inactivation of a novel three-cistronic operon tcaR-tcaA-tcaB increases teicoplanin resistance in Staphylococcus aureus. Biochim. Biophys. Acta 1523, 135–139 (2000).

    Article  CAS  PubMed  Google Scholar 

  35. Law, C.J., Maloney, P.C. & Wang, D.N. Ins and outs of major facilitator superfamily antiporters. Annu. Rev. Microbiol. 62, 289–305 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. de Crécy-Lagard, V., El, Y.B., de la Garza, R.D., Noiriel, A. & Hanson, A.D. Comparative genomics of bacterial and plant folate synthesis and salvage: predictions and validations. BMC Genomics 8, 245 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Bermingham, A. & Derrick, J.P. The folic acid biosynthesis pathway in bacteria: evaluation of potential for antibacterial drug discovery. Bioessays 24, 637–648 (2002).

    Article  CAS  PubMed  Google Scholar 

  38. Hawser, S., Lociuro, S. & Islam, K. Dihydrofolate reductase inhibitors as antibacterial agents. Biochem. Pharmacol. 71, 941–948 (2006).

    Article  CAS  PubMed  Google Scholar 

  39. Sammes, P.G. Sulfonamides and sulfones in Comprehensive Medicinal Chemistry (eds. Sammes, P.G. & Taylor, J.B.) 255–270 (Pergamon Press, Oxford, 1990).

  40. Saxild, H.H., Brunstedt, K., Nielsen, K.I., Jarmer, H. & Nygaard, P. Definition of the Bacillus subtilis PurR operator using genetic and bioinformatic tools and expansion of the PurR regulon with glyA, guaC, pbuG, xpt-pbuX, yqhZ-folD, and pbuO. J. Bacteriol. 183, 6175–6183 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Freiberg, C., Brunner, N., Macko, L. & Fischer, H.P. Discovering antibiotic efficacy biomarkers: toward mechanism-specific high content compound screening. Mol. Cell. Proteomics 5, 2326–2335 (2006).

    Article  CAS  PubMed  Google Scholar 

  42. Swedberg, G., Castensson, S. & Skold, O. Characterization of mutationally altered dihydropteroate synthase and its ability to form a sulfonamide-containing dihydrofolate analog. J. Bacteriol. 137, 129–136 (1979).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Lampe, D.J., Churchill, M.E. & Robertson, H.M. A purified mariner transposase is sufficient to mediate transposition in vitro. EMBO J. 15, 5470–5479 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Bae, T. et al. Staphylococcus aureus virulence genes identified by Bursa aurealis mutagenesis and nematode killing. Proc. Natl. Acad. Sci. USA 101, 12312–12317 (2004).

    Article  CAS  PubMed  Google Scholar 

  45. Chaudhuri, R.R. et al. Comprehensive identification of essential Staphylococcus aureus genes using transposon-mediated differential hybridisation (TMDH). BMC Genomics 10, 291 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Wright, G.D. The antibiotic resistome: the nexus of chemical and genetic diversity. Nat. Rev. Microbiol. 5, 175–186 (2007).

    Article  CAS  PubMed  Google Scholar 

  47. Meredith, T.C., Swoboda, J.G. & Walker, S. Late-stage polyribitol phosphate wall teichoic acid biosynthesis in Staphylococcus aureus. J. Bacteriol. 190, 3046–3056 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Pitcher, D.G., Saunders, N.A. & Owen, R.J. Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett. Appl. Microbiol. 8, 151–156 (1989).

    Article  CAS  Google Scholar 

  49. Ralser, M. et al. An efficient and economic enhancer mix for PCR. Biochem. Biophys. Res. Commun. 347, 747–751 (2006).

    Article  CAS  PubMed  Google Scholar 

  50. Livak, K.J. & Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408 (2001).

    Article  CAS  PubMed  Google Scholar 

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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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  1. Timothy C Meredith

    Present address: Current address: Infectious Diseases, Novartis Institute for Biomedical Research, Cambridge, Massachusetts, USA.,

Authors and Affiliations

  1. Infectious Diseases Division, Merck Frosst Center for Therapeutic Research, Kirkland, Quebec, Canada.,

    Hao Wang, David Claveau, John P Vaillancourt, Terry Roemer & Timothy C Meredith

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Contributions

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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Correspondence to Terry Roemer or Timothy C Meredith.

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Competing interests

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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