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Development of an oxidative stress sensor in live bacteria using the optimized HyPer2 protein

  • B. Franco
  • Felipe Padilla-Vaca
  • Naurú Idalia Vargas-Maya
  • Luz Janeth Herrera-Gutiérrez
  • Josué Daniel Mora-Garduño
  • Margarita Daniela Tafoya-Ramírez
  • Itzel Páramo-Pérez
  • Fernando Anaya-Velázquez
  • Claudia Leticia Mendoza-Macías
Original Paper

Abstract

Oxidative stress is a key regulator in many cellular processes but also an important burden for living organisms. The source of oxidative damage usually is difficult to measure and assess with analytical tools or chemical indicators. One major limitation is to discriminate the presence of secondary oxidant molecules derived from the cellular metabolism after exposure to the oxidant or the scavenging capacity of reactive oxygen species by cells. Using a whole-cell reporter system based on an optimized HyPer2 protein for Escherichia coli expression, we demonstrate that, as previously shown for eukaryotic organisms, the effect at the transcriptional level of hydrogen peroxide can be monitored in vivo using flow cytometry of bacterial cells without the need of a direct analytical measurement. In this approach, we generated two different HyPer2 expression systems, one that is induced by IPTG and a second one that is induced by oxidative stress responsive promoters to control the expression of the HyPer2 protein and the exposure of higher H2O2 concentrations that has been shown to activate oxidative response genes. Both systems showed that the pathway that leads to the generation of H2O2 in vivo can be traced from H2O2 exposure. Our results indicate that hydrogen peroxide pulses can be readily detected in E. coli cells by a defined fluorescence signature that is H2O2 concentration-dependent. Our findings indicate that although less sensitive than purified protein or expressed in eukaryotic cells, HyPer2 is a good bacterial sensor for H2O2. As proof of concept, this system was used to trace the oxidative capacity of Toluidine Blue O showing that oxidative stress and redox imbalance is generated inside the cell. This system is expanding the repertoire of whole cell probes available for tracing cellular stress in bacteria.

Keywords

HyPer2 Hydrogen peroxide Whole cell reporter Flow cytometry 

Notes

Acknowledgements

The authors are grateful for the support by the institutional Grant from DAIP/Guanajuato University in the Convocatoria Institucional de Investigación Científica 2016–2017 to support this research. Authors are also grateful for the support by CONACyT/CIBIOGEM Grant No. 264456. Institutional support from the Grant: Apoyo Institucional para fortalecer la excelencia académica convenio 89/2016. The authors are grateful to Tannia Razo Soria for her skilled technical assistance.

Author contributions

BF and FP designed the study, received funding designed and the experiments. NIV-M, LJHG, JDM-G, MDT-R, IPP, and BF performed the experiments and prepared the figures. BF, FP, NIV-M, A-V and MM analyzed the data. BF and FP wrote the paper. A-V and MM revised the manuscript. All authors approved the final version of the manuscript.

Conflict of interest

The authors declare that they have no conflict or financial interest.

Supplementary material

10482_2018_1140_MOESM1_ESM.pptx (1 mb)
Supplementary material 1 (PPTX 1059 kb)
10482_2018_1140_MOESM2_ESM.docx (12 kb)
Supplementary material 2 (DOCX 12 kb)

References

  1. Alvarez AF, Malpica R, Contreras M, Escamilla E, Georgellis D (2010) Cytochrome d but not cytochrome o rescues the toluidine blue growth sensitivity of arc mutants of Escherichia coli. J Bacteriol 192(2):391–399CrossRefPubMedGoogle Scholar
  2. Belousov VV, Fradkov AF, Lukyanov KA, Staroverov DB, Shakhbazov KS, Terskikh AV, Lukyanov S (2006) Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat Methods 3(4):281–286CrossRefPubMedGoogle Scholar
  3. Betteridge DJ (2000) What is oxidative stress? Metabolism 49(2 Suppl 1):3–8CrossRefPubMedGoogle Scholar
  4. Bilan DS, Belousov VV (2016) HyPer family probes: state of the art. Antioxid Redox Signal 24(13):731–751CrossRefPubMedGoogle Scholar
  5. Boluki E, Kazemian H, Peeridogaheh H, Alikhani MY, Shahabi S, Beytollahi L, Ghorbanzadeh R (2017) Antimicrobial activity of photodynamic therapy in combination with colistin against a pan-drug resistant Acinetobacter baumannii isolated from burn patient. Photodiagnosis Photodyn Ther. 18:1–5CrossRefPubMedGoogle Scholar
  6. Brilkina AA, Peskova NN, Dudenkova VV, Gorokhova AA, Sokolova EA, Balalaeva IV (2018) Monitoring of hydrogen peroxide production under photodynamic treatment using protein sensor HyPer. J Photochem Photobiol B 178:296–301CrossRefPubMedGoogle Scholar
  7. Demple B, Halbrook J (1983) Inducible repair of oxidative DNA damage in E. coli. Nature (Lond) 304:466–468CrossRefGoogle Scholar
  8. González-Flecha B, Demple B (1994) Intracellular generation of superoxide as a by-product of Vibrio harveyi luciferase expressed in Escherichia coli. J Bacteriol 176(8):2293–2299CrossRefPubMedPubMedCentralGoogle Scholar
  9. González-Flecha B, Demple B (1995) Metabolic sources of hydrogen peroxide in aerobically growing Escherichia coli. J Biol Chem 270(23):13681–13687CrossRefPubMedGoogle Scholar
  10. González-Flecha B, Demple B (1997) Homeostatic regulation of intracellular hydrogen peroxide concentration in aerobically growing Escherichia coli. J Bacteriol 179(2):382–388CrossRefPubMedPubMedCentralGoogle Scholar
  11. González-Flecha B, Demple B (2000) Genetic responses to free radicals. Homeostasis and gene control. Ann NY Acad Sci 899:69–87CrossRefPubMedGoogle Scholar
  12. Gonzalez-Flecha B, Cutrin JC, Boveris A (1993) Time course and mechanism of oxidative stress and tissue damage in rat liver subjected to ischemia-reperfusion. J Clin Investig 91:456–464CrossRefPubMedGoogle Scholar
  13. Gravina F, Dobrzanski T, Olchanheski LR, Galvão CW, Reche PM, Pileggi SA, Azevedo RA, Sadowsky MJ, Pileggi M (2017) Metabolic Interference of sod gene mutations on catalase activity in Escherichia coli exposed to Gramoxone® (paraquat) herbicide. Ecotoxicol Environ Saf 139:89–96CrossRefPubMedGoogle Scholar
  14. Grenier F, Matteau D, Baby V, Rodrigue S (2014) Complete Genome Sequence of Escherichia coli BW25113. Genome Announc 2(5):e01038-14CrossRefPubMedPubMedCentralGoogle Scholar
  15. Li X, Imlay JA (2018) Improved measurements of scant hydrogen peroxide enable experiments that define its threshold of toxicity for Escherichia coli. Free Radic Biol Med 14(120):217–227CrossRefGoogle Scholar
  16. Lim JB, Barker KA, Huang BK, Sikes HD (2014) In-depth characterization of the fluorescent signal of HyPer, a probe for hydrogen peroxide, in bacteria exposed to external oxidative stress. J Microbiol Methods 106:33–39CrossRefPubMedGoogle Scholar
  17. Lindqvist A, Membrillo-Hernández J, Poole RK, Cook GM (2000) Roles of respiratory oxidases in protecting Escherichia coli K12 from oxidative stress. Antonie Van Leeuwenhoek 78(1):23–31CrossRefPubMedGoogle Scholar
  18. Lu C, Albano CR, Bentley WE, Rao G (2005) Quantitative and kinetic study of oxidative stress regulons using green fluorescent protein. Biotechnol Bioeng 89(5):574–587CrossRefPubMedGoogle Scholar
  19. Lushchak VI (2014) Free radicals, reactive oxygen species, oxidative stress and its classification. Chem Biol Interact 5(224):164–175CrossRefGoogle Scholar
  20. Marinho HS, Real C, Cyrne L, Soares H, Antunes F (2014) Hydrogen peroxide sensing, signaling and regulation of transcription factors. Redox Biol 23(2):535–562CrossRefGoogle Scholar
  21. Markvicheva KN, Bilan DS, Mishina NM, Gorokhovatsky AY, Vinokurov LM, Lukyanov S, Belousov VV (2011) A genetically encoded sensor for H2O2 with expanded dynamic range. Bioorg Med Chem 19(3):1079–1084CrossRefPubMedGoogle Scholar
  22. Padilla-Martínez F, Carrizosa-Villegas LA, Rangel-Serrano Á, Paramo-Pérez I, Mondragón-Jaimes V, Anaya-Velázquez F, Padilla-Vaca F, Franco B (2015) Cell damage detection using Escherichia coli reporter plasmids: fluorescent and colorimetric assays. Arch Microbiol 197(6):815–821CrossRefPubMedGoogle Scholar
  23. Prieto-Alamo MJ, Jurado J, Gallardo-Madueno R, Monje-Casas F, Holmgren A, Pueyo C (2000) Transcriptional regulation of glutaredoxin and thioredoxin pathways and related enzymes in response to oxidative stress. J Biol Chem 275(18):13398–13405CrossRefPubMedGoogle Scholar
  24. Rogers JK, Guzman CD, Taylor ND, Raman S, Anderson K, Church GM (2015) Synthetic biosensors for precise gene control and real-time monitoring of metabolites. Nucleic Acids Res 43(15):7648–7660CrossRefPubMedPubMedCentralGoogle Scholar
  25. Rogers JK, Taylor ND, Church GM (2016) Biosensor-based engineering of biosynthetic pathways. Curr Opin Biotechnol 42:84–91CrossRefPubMedGoogle Scholar
  26. Sies H (2015) Oxidative stress: a concept in redox biology and medicine. Redox Biol 4:180–183CrossRefPubMedPubMedCentralGoogle Scholar
  27. Sleight SC, Bartley BA, Lieviant JA, Sauro HM (2010) In-Fusion BioBrick assembly and re-engineering. Nucleic Acids Res 38(8):2624–2636CrossRefPubMedPubMedCentralGoogle Scholar
  28. Storz G, Imlay JA (1999) Oxidative stress. Curr Opin Microbiol 2(2):188–194CrossRefPubMedGoogle Scholar
  29. Wainwright M, Phoenix DA, Marland J, Wareing DR, Bolton FJ (1997) A study of photobactericidal activity in the phenothiazinium series. FEMS Immunol Med Microbiol 19(1):75–80CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  • B. Franco
    • 1
  • Felipe Padilla-Vaca
    • 1
  • Naurú Idalia Vargas-Maya
    • 1
  • Luz Janeth Herrera-Gutiérrez
    • 1
  • Josué Daniel Mora-Garduño
    • 1
  • Margarita Daniela Tafoya-Ramírez
    • 1
  • Itzel Páramo-Pérez
    • 1
  • Fernando Anaya-Velázquez
    • 1
  • Claudia Leticia Mendoza-Macías
    • 2
  1. 1.Departamento de Biología, División de Ciencias Naturales y ExactasUniversidad de GuanajuatoGuanajuatoMexico
  2. 2.Departamento de Farmacia, División de Ciencias Naturales y ExactasUniversidad de GuanajuatoGuanajuatoMexico

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