Detection and quantification of membrane-damaging antimicrobials using pHluorin2-based bacterial biosensors

Area of Science:

• Microbiology
• Biochemistry
• Molecular Biology

Background:

• Bacteriocins are antimicrobial peptides that disrupt bacterial membrane integrity and pH homeostasis.
• pHluorin2 is a fluorescent protein whose emission spectrum changes ratiometrically with pH.
• Monitoring intracellular pH changes in bacteria can indicate membrane damage.

Purpose of the Study:

• To develop and validate live bacterial biosensors for rapid detection of membrane-damaging antimicrobials.
• To utilize pHluorin2 fluorescence for real-time assessment of antimicrobial activity.
• To provide a faster alternative to conventional growth-dependent assays for bacteriocin characterization.

Main Methods:

• Constructed live bacterial biosensors expressing the pHluorin2 fluorescent protein.
• Measured changes in pHluorin2 fluorescence profiles in response to antimicrobial treatment.
• Utilized spectrophotometry, microscopy, and flow cytometry for fluorescence detection.
• Developed a protocol for freeze-dried, ready-to-use biosensor assay plates.

Main Results:

• pHluorin2-expressing bacteria exhibited rapid, measurable fluorescence changes upon treatment with membrane-disrupting compounds.
• The biosensor system detected antimicrobial activity orders of magnitude faster than traditional assays.
• The method proved effective for characterization, screening, and quantification of membrane-damaging agents.
• Freeze-dried biosensor bacteria maintained viability and functionality for assay plate preparation.

Conclusions:

• Live pHluorin2-expressing bacterial biosensors provide a highly efficient platform for studying membrane-damaging antimicrobials.
• This approach significantly accelerates the discovery and characterization of compounds like bacteriocins.
• The developed biosensor technology is versatile, applicable across various detection methods and high-throughput screening formats.