Biosensor for Multimodal Characterization of an Essential ABC Transporter for Next-Generation Antibiotic Research

Area of Science:

• Biochemistry
• Microbiology
• Biophysics

Background:

• Antibiotic resistance is a growing global health threat, necessitating the development of novel antimicrobial agents.
• The ATP-binding cassette (ABC) transporter MsbA is a critical survival protein in Gram-negative pathogenic bacteria and a key target for new antimicrobials.
• Supported lipid bilayers (SLBs) offer a versatile platform for studying membrane protein structure and function using various integrated techniques.

Purpose of the Study:

• To develop and validate a novel platform for monitoring the activity of the bacterial ABC transporter MsbA.
• To investigate the potential of electrochemical impedance spectroscopy (EIS) integrated with SLBs for detecting changes in MsbA function.
• To demonstrate the utility of this platform in evaluating MsbA activity and the effects of inhibitors.

Main Methods:

• Formation of supported lipid bilayers (SLBs) incorporating *Escherichia coli* MsbA.
• High-resolution microscopy techniques, including atomic force microscopy (AFM) and structured illumination microscopy (SIM), to assess SLB and protein integrity.
• Integration of SLBs on microelectrode arrays (MEAs) with poly(3,4-ethylenedioxy-thiophene) poly(styrene sulfonate) (PEDOT:PSS) for electrochemical measurements.
• Electrochemical impedance spectroscopy (EIS) to monitor ion flow through MsbA in response to ATP hydrolysis, correlated with biochemical assays.
• Evaluation of wild-type MsbA, MsbA mutants, and the inhibitor G907.

Main Results:

• Successful formation and characterization of SLBs containing functional *E. coli* MsbA.
• Demonstration that EIS can effectively monitor ion transport through MsbA in response to ATP.
• Correlation of EIS measurements with biochemical assays for MsbA-ATPase activity.
• Detection of altered MsbA activity in mutants and inhibition by G907 using the developed platform.

Conclusions:

• The integrated SLB-MEA platform provides a robust method for studying essential bacterial membrane transporters like MsbA.
• This approach enables the real-time monitoring of transporter activity and the assessment of potential drug inhibitors.
• The platform holds significant promise for accelerating the discovery and development of next-generation antimicrobials targeting essential microbial transporters.