A Ratiometric pH Sensor for Gram-Positive and Gram-Negative Bacteria

Area of Science:

• Microbiology
• Biochemistry
• Cell Biology

Background:

• Bacterial populations exhibit phenotypic heterogeneity, impacting their response to environmental stressors like antibiotics and immune system components.
• Understanding single-cell physiology is crucial for analyzing bacterial adaptation to environmental changes, particularly pH stress.
• Current methods for real-time cytoplasmic pH monitoring in bacteria are limited.

Purpose of the Study:

• To develop and validate a ratiometric fluorescent probe for sensing bacterial cytoplasmic pH.
• To assess the probe's utility in real-time monitoring of pH fluctuations and identifying pH-sensitive phenotypes.
• To evaluate the probe's application in studying bacterial interactions with immune cells.

Main Methods:

• Synthesis of hemicyanine dye-based ratiometric fluorescent probes.
• Uptake and validation of probes in Gram-positive (e.g., Staphylococcus epidermidis, MRSA) and Gram-negative (e.g., Escherichia coli) bacteria.
• Real-time monitoring of cytoplasmic pH changes in response to environmental conditions and during phagocytosis by immune cells.

Main Results:

• The fluorescent probes demonstrated rapid and reversible responses to cytoplasmic pH changes across a biologically relevant range.
• Probes were successfully validated for cytoplasmic pH sensing in multiple bacterial species, including clinically relevant strains like MRSA.
• The probes facilitated the identification of pH-sensitive bacterial phenotypes and monitored phagocytosis of virulent strains by immune cells.

Conclusions:

• The developed ratiometric fluorescent probes are a promising tool for analyzing bacterial cytoplasmic pH and phenotypic heterogeneity.
• These probes can provide insights into the physiological states of bacterial populations, including antibiotic-resistant and persistent strains.
• This technology offers a valuable approach for studying bacterial adaptation and host-pathogen interactions in real-time.