Development of an oxidative stress sensor in live bacteria using the optimized HyPer2 protein
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.
KeywordsHyPer2 Hydrogen peroxide Whole cell reporter Flow cytometry
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.
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.
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