Split fluorescent protein-mediated multimerization of cell wall binding domain for highly sensitive and selective...

Area of Science:

• Biotechnology
• Molecular Biology
• Biosensing

Background:

• Cell wall peptidoglycan binding domains (CBDs) on lytic enzymes facilitate specific bacterial binding.
• CBDs show potential as biorecognition molecules for non-destructive bacterial detection.
• Existing methods may lack sensitivity or speed for certain bacterial detection needs.

Purpose of the Study:

• To design and develop a novel self-complementing split fluorescent protein (FP) complex for enhanced bacterial detection.
• To create multimeric FP chains fused with specific CBDs ((FP-CBD)n) for improved signal amplification.
• To demonstrate the utility of these complexes for sensitive detection of Staphylococcus aureus and Bacillus anthracis.

Main Methods:

• Construction of multimeric split FP-CBD fusion proteins ((FP-CBD)n).
• Utilized flow cytometry to assess cell surface fluorescence.
• Employed surface plasmon resonance to determine binding affinity to peptidoglycan.
• Incorporated enzymatic detection modalities, such as horseradish peroxidase (HRP) coupling.

Main Results:

• Increased FP stoichiometry in (FP-CBD)n complexes led to enhanced cell surface fluorescence.
• Demonstrated nanomolar binding affinity to isolated peptidoglycan via surface plasmon resonance.
• HRP-coupled (FP-CBD)n complexes achieved catalytic amplification, reaching a limit of detection of 10^3 cells/droplet for S. aureus and B. anthracis within 15 minutes.

Conclusions:

• Multimeric split FP-CBD fusions serve as effective biorecognition molecules with amplified signals for bacterial biosensing.
• The modular design allows for functional versatility and multiplexed detection capabilities.
• This approach significantly enhances sensitivity and speed for detecting specific bacterial pathogens.