Hypocrates is a genetically encoded fluorescent biosensor for (pseudo)hypohalous acids and their derivatives

The lack of tools to monitor the dynamics of (pseudo)hypohalous acids in live cells and tissues hinders a better understanding of inflammatory processes. Here we present a fluorescent genetically encoded biosensor, Hypocrates, for the visualization of (pseudo)hypohalous acids and their derivatives. Hypocrates consists of a circularly permuted yellow fluorescent protein integrated into the structure of the transcription repressor NemR from Escherichia coli. We show that Hypocrates is ratiometric, reversible, and responds to its analytes in the 106 M−1s−1 range. Solving the Hypocrates X-ray structure provided insights into its sensing mechanism, allowing determination of the spatial organization in this circularly permuted fluorescent protein-based redox probe. We exemplify its applicability by imaging hypohalous stress in bacteria phagocytosed by primary neutrophils. Finally, we demonstrate that Hypocrates can be utilized in combination with HyPerRed for the simultaneous visualization of (pseudo)hypohalous acids and hydrogen peroxide dynamics in a zebrafish tail fin injury model.


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March 2021

Data analysis
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Sample size
Data exclusions

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In vitro experiments: OriginPro 9.0 and GraphPad Prism8 for calculations. Crystal structure determination: XDS 0.6.5.5 for data processing, Phaser 2.7.16 in the Phenix gui for molecular replacement, Phenix.refinement 1.11.1_2575 for refinement, Coot 0. For in vitro experiments, no sample size calculation protocols were used. The minimum number of independent measurements was 3 for most assays since the used equipment provided sufficient level of data consistency as can be seen in the Source Data file. For some tests, in which the relationships among variables were investigated, the points were measured in single replicas; however, in such cases the desired accuracy was achieved due to sufficient number of steps. Some experiments were performed in a single replica (e.g. the registration of CD spectra or fluorescence excitation spectra in bacteria) since pronounced qualitative effects were investigated. These experiments were never used to construct any quantitative conclusions, and their aim was to visually demonstrate the main trends in the behavior of the proteins. For HeLa Kyoto experiments, no sample size calculation protocols were used. The minimum number of independent experiments was 1 (usually, 3) and the minimum number of cells per measurement was 25 or more. We presume that such sample sizes are sufficient, because we observed relatively low variability between individual cells as can be seen in the Source Data file. Generally, the selected sample sizes are common in the field. For experiments with neutrophils, no sample size calculation protocols were used. In each case, the number of independent measurements was 3 and the total number of cells per experiment was 35. As can be seen in the Source Data file, dispersion was relatively weak within a given condition. Generally, the selected sample sizes are common in the field. For Danio rerio experiments, no statistical methods were used to predetermine sample size. We always mounted 3 to 4 larvae per experiment for imaging and we reproduced the experiment 3 or 4 times. Some larvae are out of focus and cannot be used for imaging. As shown in the Source Data file, dispersion was weak within a given condition, and difference was statistically significant between conditions.
For in vitro experiments, no data were excluded. For HeLa Kyoto experiments, no data were excluded. For experiments with neutrophils, no data were excluded. For Danio rerio experiments, no data were excluded. For imaging, embryos were mounted in agarose. Animals out of focus were discarded.
In vitro experiments were mostly performed in duplicates or triplicates. All replication attempts were successful and provided matching results. Moreover, many experiments were independently performed in two different laboratories and still provided a good level of data consistency.
HeLa Kyoto experiments were mostly performed in triplicates. All replication attempts were successful and provided matching results. Experiments with neutrophils were performed in triplicates. All replication attempts were successful and provided matching results. For Danio rerio experiments, all the findings were replicated. For the Hypocrates sensor, the data correspond to 4 independent experiments performed within 2 months of work. We checked with ANOVA followed by Tukey's post hoc test whether independent experiments gave significantly different results, which was never the case. Consequently, independent experiments were pooled. For the HyPerRed sensor, 3 independent experiments were performed, and treated as above.
No randomization was required for in vitro experiments. Since tested aliquots were taken from the concentrated protein solution which was Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Note that full information on the approval of the study protocol must also be provided in the manuscript.
obtained according to a fixed protocol, we can assume that the in vitro samples were randomly allocated. In HeLa Kyoto experiments, the imaged cells were plated from the same culture in the same conditions; therefore, no randomization was required. The cell line we use is isogenic and consequently cells were randomly allocated. In each experiment with neutrophils, the imaged cells were taken from the total mixture of cells collected from all the volunteers at the beginning of the day and sensors were tested in parallel with controls; therefore, no randomization was required. Since the cells from all participants were present in each sample, we can assume that these cells were randomly allocated. For Danio rerio experiments, the animals were picked from a pool of animals. The strain we use is isogenic and consequently larvae were randomly allocated.
Blinding procedures were not required for in vitro experiments. In these tests, the responses of the proteins were measured by the optical equipment automatically. Since all data were processed according to the same protocol, any blinding procedures were not required. In HeLa Kyoto experiments, the responses of the sensors were measured by the optical equipment automatically. Since all images were processed according to the same protocol, any blinding procedures were not required.
In experiments with neutrophils, the responses of the sensors were measured by the optical equipment automatically. Since all images were processed according to the same protocol, any blinding procedures were not required. For Danio rerio experiments, the investigators were not blinded to group allocation during data collection which was conducted according to a standard protocol for all animals; however, all quantifications were performed in blind.
None of the cell lines have been authenticated.
HeLa Kyoto were tested negative for mycoplasma contamination.
None commonly misidentified lines were used.
No wild animals were used in this study.
No field-collected samples were used in this study.