Outer-Membrane Protease (OmpT) Based E. coli Sensing with Anionic Polythiophene and Unlabeled Peptide Substrate

Area of Science:

• Biochemistry and Molecular Biology
• Analytical Chemistry
• Environmental Microbiology

Background:

• Escherichia coli (E. coli) and Salmonella are significant causes of foodborne and waterborne illnesses.
• Rapid and sensitive detection methods are crucial for preventing outbreaks.
• Bacterial surface protease activity presents a potential target for detection strategies.

Purpose of the Study:

• To develop a simple, specific, and rapid assay for detecting E. coli in water.
• To utilize bacterial protease activity as a biomarker for E. coli detection.
• To investigate the use of polythiophene acetic acid (PTAA) and a peptide substrate for optical detection.

Main Methods:

• Development of an assay targeting the outer-membrane protease OmpT on E. coli.
• Utilizing polythiophene acetic acid (PTAA) as an optical reporter.
• Employing a specific peptide substrate (LL37FRRV) for OmpT, which interacts with PTAA to enhance fluorescence.
• Monitoring changes in PTAA fluorescence and conformation upon peptide cleavage by E. coli.

Main Results:

• The assay can detect E. coli at concentrations as low as 1 CFU/mL within 6 hours.
• Cleavage of the LL37FRRV peptide by E. coli's OmpT alters PTAA's fluorescence and conformation.
• Circular dichroism confirmed the formation of a helical PTAA-LL37FRRV construct and its disruption upon peptide cleavage.
• Investigation of LL37FRRV sequence variants provided insights into peptide-protease interactions.

Conclusions:

• A novel optical assay based on protease activity enables sensitive and specific detection of E. coli.
• The assay's reliance on peptide cleavage by E. coli offers a unique detection mechanism.
• This method holds promise for rapid, near-outbreak surveillance of E. coli in water samples.