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

Published version
Peer-reviewed

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Authors

Bali, Karan 
Guffick, Charlotte 
McCoy, Reece 
Kaminski, Clemens F  ORCID logo  https://orcid.org/0000-0002-5194-0962

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.

Description

Keywords

MsbA, PEDOT:PSS, atomic force microscopy, biosensor, electrochemical impedance spectroscopy, electrophysiology, structured illumination microscopy, supported lipid bilayer

Journal Title

ACS Appl Mater Interfaces

Conference Name

Journal ISSN

1944-8244
1944-8252

Volume Title

Publisher

American Chemical Society (ACS)
Sponsorship
Biotechnology and Biological Sciences Research Council (2114197)
Biotechnology and Biological Sciences Research Council (BB/R00224X/1)
Engineering and Physical Sciences Research Council (EP/H018301/1)
Wellcome Trust (089703/Z/09/Z)
Medical Research Council (MR/K015850/1)
Medical Research Council (MR/K02292X/1)
Engineering and Physical Sciences Research Council (EP/L015889/1)
Engineering and Physical Sciences Research Council (EP/S022953/1)
Engineering and Physical Sciences Research Council (2266415)
This research was funded by the Biotechnology and Biological Sciences Research Council (BBSRC) grant BB/R00224X/1 (to H.W.V.V). K.B. was funded by an Engineering and Physical Sciences Research Council (EPSRC)-Doctoral Training Partnership (DTP) PhD studentship (project 2266415). C.G. was funded by a BBSRC-DTP-Targeted PhD studentship (project 2114197). R.M. was funded by an EPSRC Cambridge Nanoscience and Nanotechnology (NanoDTC) PhD studentship (award EP/S022953/1). C.F.K. acknowledges funding from the EPSRC (EP/H018301/1 and EP/L015889/1), the Wellcome Trust (089703/Z/09/Z and 3-3249/Z/16/Z), the Medical Research Council (MR/K015850/1 and MR/K02292X/1), MedImmune, and Infinitus (China). I.M. acknowledges funding from the Royal Society (URF/R1/221795). R.M.O. acknowledges funding for this project by the Defense Advanced Research Projects Agency (DARPA) Army Research Office and accomplished under Cooperative Agreement W911NF-18-2-0152. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the official policies, either expressed or implied, of DARPA or the Army Research Office or the U.S. Government. The U.S. Government is authorized to reproduce and distribute reprints for Government purposes notwithstanding any copyright notation herein.
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