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

Abstract

As the threat of antibiotic resistance increases, there is a particular focus on developing antimicrobials against pathogenic bacteria whose multidrug resistance is especially entrenched and concerning. One such target for novel antimicrobials is the ATP-binding cassette (ABC) transporter MsbA that is present in the plasma membrane of Gram-negative pathogenic bacteria where it is fundamental to the survival of these bacteria. Supported lipid bilayers (SLBs) are useful in monitoring membrane protein structure and function since they can be integrated with a variety of optical, biochemical, and electrochemical techniques. Here, we form SLBs containing Escherichia coli MsbA and use atomic force microscopy (AFM) and structured illumination microscopy (SIM) as high-resolution microscopy techniques to study the integrity of the SLBs and incorporated MsbA proteins. We then integrate these SLBs on microelectrode arrays (MEA) based on the conducting polymer poly(3,4-ethylenedioxy-thiophene) poly(styrene sulfonate) (PEDOT:PSS) using electrochemical impedance spectroscopy (EIS) to monitor ion flow through MsbA proteins in response to ATP hydrolysis. These EIS measurements can be correlated with the biochemical detection of MsbA-ATPase activity. To show the potential of this SLB approach, we observe not only the activity of wild-type MsbA but also the activity of two previously characterized mutants along with quinoline-based MsbA inhibitor G907 to show that EIS systems can detect changes in ABC transporter activity. Our work combines a multitude of techniques to thoroughly investigate MsbA in lipid bilayers as well as the effects of potential inhibitors of this protein. We envisage that this platform will facilitate the development of next-generation antimicrobials that inhibit MsbA or other essential membrane transporters in microorganisms.

In Vitro Models for Investigating Intestinal Host–Pathogen Interactions

“…[288] This has led to the fabrication of supported lipid bilayers on solid substrates like glass or silicon as well as on polymer cushions separating solid support and membrane. [289][290][291] The latter is especially promising for representing membranes in their native configuration due to preservation of the membrane's natural fluid character allowing for diffusion and dynamic rearrangement of lipid molecules and proteins, and prevention of transmembrane protein denaturation. [276] As such, conducting polymers are a useful subclass of polymer supports because they pro-vide these advantages while also being transparent and electrically conducting.…”

Multiparametric Sensing of Outer Membrane Vesicle-Derived Supported Lipid Bilayers Demonstrates the Specificity of Bacteriophage Interactions

“…A key part of the attraction of the SLB platform is its ability to be integrated with a range of measurement techniques. One such measurement technique is electrochemical impedance spectroscopy (EIS), used to probe the resistance and capacitance characteristics of the SLB . SLBs can be generated upon microelectrode arrays (MEAs), and moreover these MEAs can be functionalized with biocompatible conducting polymers, such as the polymer poly­(3,4-ethylenedioxythiophene) polystyrenesulfonate (PEDOT:PSS) to increase the device sensitivity.…”

“…One such measurement technique is electrochemical impedance spectroscopy (EIS), used to probe the resistance and capacitance characteristics of the SLB. 18 SLBs can be generated upon microelectrode arrays (MEAs), and moreover these MEAs can be functionalized with biocompatible conducting polymers, such as the polymer poly(3,4-ethylenedioxythiophene) polystyrenesulfonate (PEDOT:PSS) to increase the device sensitivity. PEDOT:PSS, owing to its ion-to-electron mixed conductivity and cushioned mechanical properties, is an ideal material for interfacing with biological materials (such as SLBs), leading to the emergence of organic bioelectronic devices.…”