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Biotechnology Letters

, Volume 40, Issue 2, pp 359–367 | Cite as

Evaluation of NfsA-like nitroreductases from Neisseria meningitidis and Bartonella henselae for enzyme-prodrug therapy, targeted cellular ablation, and dinitrotoluene bioremediation

  • Michelle H. Rich
  • Abigail V. Sharrock
  • Kelsi R. Hall
  • David F. Ackerley
  • Joanna K. MacKichan
Original Research Paper

Abstract

Objectives

To characterize the activities of two candidate nitroreductases, Neisseria meningitidis NfsA (NfsA_Nm) and Bartonella henselae (PnbA_Bh), with the nitro-prodrugs, CB1954 and metronidazole, and the environmental pollutants 2,4- and 2,6-dinitrotoluene.

Results

NfsA_Nm and PnbA_Bh were evaluated in Escherichia coli over-expression assays and as His6-tagged proteins in vitro. With the anti-cancer prodrug CB1954, both enzymes were more effective than the canonical O2-insensitive nitroreductase E. coli NfsB (NfsB_Ec), NfsA_Nm exhibiting comparable levels of activity to the leading nitroreductase candidate E. coli NfsA (NfsA_Ec). NfsA_Nm is also the first NfsA-family nitroreductase shown to produce a substantial proportion of 4-hydroxylamine end-product. NfsA_Nm and PnbA_Bh were again more efficient than NfsB_Ec at aerobic activation of metronidazole to a cytotoxic form, with NfsA_Nm appearing a promising candidate for improving zebrafish-targeted cell ablation models. NfsA_Nm was also more active than either NfsA_Ec or NfsB_Ec with 2,4- or 2,6-dinitrotoluene substrates, whereas PnbA_Bh was relatively inefficient with either substrate.

Conclusions

NfsA_Nm is a promising new nitroreductase candidate for several diverse biotechnological applications.

Keywords

CB1954 Dinitrotoluene Gene-directed enzyme-prodrug therapy Metronidazole NfsA Nitroreductase PnbA 

Notes

Acknowledgements

This work was partially supported by grants from the Lottery Health Research Fund and the Wellington Medical Research Fund (2014/234) to JKM, and the Royal Society of New Zealand Marsden Fund (VUW1502 to DFA). MHR, KRH and AVS were supported by Victoria University of Wellington PhD Scholarships, with MHR additionally supported by the Cancer Society of New Zealand and AVS by a Dick and Mary Earle Scholarship in Technology.

Supporting information

Supplementary Table 1—IC50 evaluation for E. coli strains expressing wild type nitroreductases.

Supplementary Fig. 1—Kinetics of purified nitroreductases with CB1954.

Supplementary Fig. 2—Kinetics of purified nitroreductases with metronidazole.

Supplementary Fig. 3—Kinetics of purified nitroreductases with 2,4-dinitrotoluene.

Supplementary Fig. 4—Kinetics of purified nitroreductases with 2,6-dinitrotoluene.

Supplementary material

10529_2017_2472_MOESM1_ESM.docx (913 kb)
Supplementary material 1 (DOCX 912 kb)

References

  1. Akiva E, Copp JN, Tokuriki N, Babbitt PC (2017) Evolutionary and molecular foundations of multiple contemporary functions of the nitroreductase superfamily. Proc Natl Acad Sci USA.  https://doi.org/10.1073/pnas.1706849114 PubMedPubMedCentralGoogle Scholar
  2. Aviv G et al (2014) A unique megaplasmid contributes to stress tolerance and pathogenicity of an emergent Salmonella enterica serovar Infantis strain. Environ Microbiol 16:977–994CrossRefPubMedGoogle Scholar
  3. Breitschwerdt EB (2014) Bartonellosis: one health perspectives for an emerging infectious disease. ILAR J 55:46–58CrossRefPubMedGoogle Scholar
  4. Copp JN, Williams EM, Rich MH, Patterson AV, Smaill JB, Ackerley DF (2014) Toward a high-throughput screening platform for directed evolution of enzymes that activate genotoxic prodrugs. Protein Eng Des Sel 27:399–403CrossRefPubMedGoogle Scholar
  5. Copp JN et al (2017) Engineering a multifunctional nitroreductase for improved activation of prodrugs and PET probes for cancer gene therapy. Cell Chem Biol 24:391–403CrossRefPubMedGoogle Scholar
  6. Curado S, Anderson RM, Jungblut B, Mumm J, Schroeter E, Stainier DY (2007) Conditional targeted cell ablation in zebrafish: a new tool for regeneration studies. Dev Dyn 236:1025–1035CrossRefPubMedGoogle Scholar
  7. Denny WA (2002) Nitroreductase-based GDEPT. Curr Pharm Des 8:1349–1361CrossRefPubMedGoogle Scholar
  8. Gabutti G, Stefanati A, Kuhdari P (2015) Epidemiology of Neisseria meningitidis infections: case distribution by age and relevance of carriage. J Prev Med Hyg 56:E116–120PubMedPubMedCentralGoogle Scholar
  9. Han S, Mukherji ST, Rice A, Hughes JB (2011) Determination of 2,4- and 2,6-dinitrotoluene biodegradation limits. Chemosphere 85:848–853CrossRefPubMedGoogle Scholar
  10. Heap JT et al (2014) Spores of Clostridium engineered for clinical efficacy and safety cause regression and cure of tumors in vivo. Oncotarget 5:1761–1769CrossRefPubMedPubMedCentralGoogle Scholar
  11. Helsby NA, Ferry DM, Patterson AV, Pullen SM, Wilson WR (2004) 2-Amino metabolites are key mediators of CB 1954 and SN 23862 bystander effects in nitroreductase GDEPT. Br J Cancer 90:1084–1092CrossRefPubMedPubMedCentralGoogle Scholar
  12. Johnson GR, Spain JC (2003) Evolution of catabolic pathways for synthetic compounds: bacterial pathways for degradation of 2,4-dinitrotoluene and nitrobenzene. Appl Microbiol Biotechnol 62:110–123CrossRefPubMedGoogle Scholar
  13. Knox RJ, Friedlos F, Sherwood RF, Melton RG, Anlezark GM (1992) The bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)–II. A comparison of an Escherichia coli nitroreductase and Walker DT diaphorase. Biochem Pharmacol 44:2297–2301CrossRefPubMedGoogle Scholar
  14. Lei B, Tu SC (1998) Mechanism of reduced flavin transfer from Vibrio harveyi NADPH-FMN oxidoreductase to luciferase. Biochemistry 37:14623–14629CrossRefPubMedGoogle Scholar
  15. Liochev SI, Hausladen A, Fridovich I (1999) Nitroreductase A is regulated as a member of the soxRS regulon of Escherichia coli. Proc Natl Acad Sci USA 96:3537–3539CrossRefPubMedPubMedCentralGoogle Scholar
  16. MacKichan JK, Gerns HL, Chen YT, Zhang P, Koehler JE (2008) A SacB mutagenesis strategy reveals that the Bartonella quintana variably expressed outer membrane proteins are required for bloodstream infection of the host. Infect Immun 76:788–795CrossRefPubMedGoogle Scholar
  17. Mathias JR, Zhang Z, Saxena MT, Mumm JS (2014) Enhanced cell-specific ablation in zebrafish using a triple mutant of Escherichia coli nitroreductase. Zebrafish 11:85–97CrossRefPubMedPubMedCentralGoogle Scholar
  18. Patel P et al (2009) A phase I/II clinical trial in localized prostate cancer of an adenovirus expressing nitroreductase with CB1954 [correction of CB1984]. Mol Ther 17:1292–1299CrossRefPubMedPubMedCentralGoogle Scholar
  19. Perez-Reinado E, Roldan MD, Castillo F, Moreno-Vivian C (2008) The NprA nitroreductase required for 2,4-dinitrophenol reduction in Rhodobacter capsulatus is a dihydropteridine reductase. Environ Microbiol 10:3174–3183CrossRefPubMedGoogle Scholar
  20. Prosser GA et al (2013) Creation and screening of a multi-family bacterial oxidoreductase library to discover novel nitroreductases that efficiently activate the bioreductive prodrugs CB1954 and PR-104A. Biochem Pharmacol 85:1091–1103CrossRefPubMedGoogle Scholar
  21. Pulliainen AT, Dehio C (2012) Persistence of Bartonella spp. stealth pathogens: from subclinical infections to vasoproliferative tumor formation. FEMS Microbiol Rev 36:563–599CrossRefPubMedGoogle Scholar
  22. Race PR, Lovering AL, White SA, Grove JI, Searle PF, Wrighton CW, Hyde EI (2007) Kinetic and structural characterisation of Escherichia coli nitroreductase mutants showing improved efficacy for the prodrug substrate CB1954. J Mol Biol 368:481–492CrossRefPubMedGoogle Scholar
  23. Ren X, MacKichan JK (2014) Disease-associated Neisseria meningitidis isolates inhibit wound repair in respiratory epithelial cells in a type IV pilus-independent manner. Infect Immun 82:5023–5034CrossRefPubMedPubMedCentralGoogle Scholar
  24. Roldan MD, Perez-Reinado E, Castillo F, Moreno-Vivian C (2008) Reduction of polynitroaromatic compounds: the bacterial nitroreductases. FEMS Microbiol Rev 32:474–500CrossRefPubMedGoogle Scholar
  25. Stephens DS (2009) Biology and pathogenesis of the evolutionarily successful, obligate human bacterium Neisseria meningitidis. Vaccine 27(Suppl 2):B71–77CrossRefPubMedPubMedCentralGoogle Scholar
  26. Vass SO, Jarrom D, Wilson WR, Hyde EI, Searle PF (2009) E. coli NfsA: an alternative nitroreductase for prodrug activation gene therapy in combination with CB1954. Br J Cancer 100:1903–1911CrossRefPubMedPubMedCentralGoogle Scholar
  27. White DT, Mumm JS (2013) The nitroreductase system of inducible targeted ablation facilitates cell-specific regenerative studies in zebrafish. Methods 62:232–240CrossRefPubMedPubMedCentralGoogle Scholar
  28. Williams EM et al (2015) Nitroreductase gene-directed enzyme prodrug therapy: insights and advances toward clinical utility. Biochem J 471:131–153CrossRefPubMedGoogle Scholar
  29. Zhang J, Kale V, Chen M (2015) Gene-directed enzyme prodrug therapy. AAPS J 17:102–110CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2017

Authors and Affiliations

  • Michelle H. Rich
    • 1
    • 2
  • Abigail V. Sharrock
    • 1
    • 2
  • Kelsi R. Hall
    • 1
    • 2
  • David F. Ackerley
    • 1
    • 2
  • Joanna K. MacKichan
    • 1
    • 2
  1. 1.School of Biological SciencesVictoria University of WellingtonWellingtonNew Zealand
  2. 2.Centre for BiodiscoveryVictoria University of WellingtonWellingtonNew Zealand

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