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Contents

• Title: A novel FbFP-based biosensor toolbox for sensitive in vivo determination of intracellular pH

• Authors: Christian Rupprecht, Marcus Wingen, Janko Potzkeib, Thomas Gensch, Karl-Erich Jaeger, Thomas Drepper

• Institutions:

  • Institute of Molecular Enzyme Technology, Heinrich-Heine-University Düsseldorf

  • Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich

  • GO-Bio Projekt SenseUP, Forschungszentrum Jülich

  • Institute of Complex Systems ICS-4: Cellular Biophysics, Forschungszentrum Jülich

• Keywords: FRET biosensor, Flavin-binding fluorescent proteins (FbFP), Yellow fluorescent protein (YFP), Intracellular pH, Bacterial acid stress response

• Journal Info: Journal of Biotechnology 258 (2017) 25–32

Abstract

• Significance of Intracellular pH:

  • Critical for enzymatic conversion of metabolites and transport across cell membranes.

  • Changes in pH can cause cellular dysfunctions.

  • Monitoring pH helps elucidate dependencies in physiological and biotechnological processes.

• Genetically Encoded Biosensors:

  • Enable non-invasive, high-resolution pH determination.

• Fluorescence Biosensors for pH (FluBpH):

  • Constructed with FMN-binding fluorescent protein (EcFbFP) as donor and EYFP variants as pH-sensing acceptors.

  • Acids tolerate pKa ∼ 3.2.

  • Covers a physiologically relevant range with pKa values of 5.7, 6.1, and 7.5.

• Applications:

  • Evaluated using E. coli under acid stress conditions.

Introduction

• Importance of Intracellular pH:

  • Regulates metabolic activity and cell viability; significant changes can lead to protein misfolding or death.

  • Typical range for neutrophilic bacteria: pH 7.4–7.8.

• Acid Stress Response:

  • Alterations during acid or alkaline stress are well-documented.

  • Examples:

    • E. coli O157:H7 survives stomach's acid prior to colonization.

• Pathogens and Acid Resistance:

  • E. coli maintains cytoplasmic pH above 4.3.

  • Lactococcus lactis grows well at pH 6.5 and acidifies surrounding during glucose metabolism.

  • Helicobacter pylori thrives at low external pH, causing gastric ulcers.

Fluorescent Biosensors for pH Monitoring

• Two Categories of pH Sensors:

  1. Fluorescent Dyes:

     • Include SNARFs and BCECF; suitable for ranges pH 3.5 to 8.

     • Drawbacks: cytotoxicity, limited uptake, leakage.

  2. Genetically Encoded Fluorescent Protein-based Sensors:

     • Provide non-invasive targeting to specific cell types and compartments.

     • Ratiometric measurements based on pH-dependent fluorescence.

Donor and Acceptor Domains

• Donor Proteins:

  • CFP and ECFP with pKa around 6.4 and 5.6.

• Acceptor Variants:

  • Multiple EYFPs showing varied sensitivities and pKa values:

    • EYFP exhibits pKa of 7.1; EYFP-H148G pKa at 8.0.

    • Citrine known for low pKa (5.7) but more tolerant to halides.

Characterization of EcFbFP and EYFP Variants

• Stability Assessment:

  • EcFbFP shows stable emission from pH 4.0 to 9.0, confirmed individual pKa under different conditions.

  • EYFP variant pKa values affected by chloride concentration.

FluBpH Biosensor Development

• Design and Construct:

  • EcFbFP fusions with EYFP to create FluBpH series (5.7, 6.1, 7.5).

  • Ranges calibrated in physiological buffer assessing dynamic ranges.

• In Vivo and In Vitro Calibration:

  • Used with E. coli treatments to establish environments and validate pH sensor readings.

  • High correlation observed between in vivo and in vitro spectral responses.

Results and Analysis

• Acid Stress Monitoring with FluBpH:

  • Intracellular pH during various challenges recorded: typical values corresponded with expected physiological readings.

  • Support for mechanisms of acid resistance derived from FluBpH data.

  • FluBpH 5.7 proved effective under extreme acidic conditions, validating its non-invasive application for assessing survival/susceptibility in bacteria under pH stress.

Conclusions

• Development Impact:

  • FluBpH toolbox offers a robust method for intracellular pH measurement across a variety of conditions in diverse bacterial species, paving the way for further research in biotechnology and pathophysiology.

A Novel FbFP-based Biosensor Toolbox for Sensitive In Vivo Determination of Intracellular pH

Abstract

Intracellular pH is critical for metabolism and membrane transport, with significant changes leading to dysfunction. Genetically encoded biosensors, particularly Fluorescence Biosensors for pH (FluBpH) using EcFbFP and EYFP, allow non-invasive pH determination. Tested in E. coli under acid stress, the sensors cover pH ranges of 5.7, 6.1, and 7.5, effective for monitoring membrane transport.

Introduction

Intracellular pH regulates metabolic activity. Typical rates for neutrophilic bacteria are pH 7.4–7.8. Acid stress responses are well-studied, as seen in E. coli O157:H7 and Lactococcus lactis, with varying pH tolerances among pathogens.

Fluorescent Biosensors for pH Monitoring provide a promising approach to study the effects of pH on bacterial growth and metabolism, allowing for real-time tracking of changes in environmental conditions that can influence pathogenic behavior.

Two types: 1) Fluorescent Dyes (e.g., SNARFs, BCECF) limited by cytotoxicity; 2) Genetically Encoded Sensors providing ratiometric measurements for specific targeting.Donor Proteins: CFP and ECFP (pKa ~6.4 and 5.6)Acceptors: EYFP variants, with sensitivities and pKa values ranging from 5.7 to 8.0.

Development of FluBpH Biosensors

Design involved fusing EcFbFP with EYFP variants for sensor series (5.7, 6.1, 7.5). Correlation between in vivo and in vitro readings validated their effectiveness.

Results and Analysis

FluBpH sensors effectively tracked intracellular pH during acid stress, demonstrating their potential to assess bacterial survival under pH challenges.

Conclusions

The FluBpH toolbox provides a robust method for intracellular pH measurements, advancing research in biotechnology and pathophysiology.