High-Throughput Flow Cytometry Screening of Multidrug Efflux Systems

Book Title

Bacterial Multidrug Exporters

Authors

Mark Haynes, University of New Mexico
Matthew Garcia, University of New Mexico
Ryan Peters, University of New Mexico
Anna Waller, University of New Mexico
Pietro Tedesco, National Research Council - Italy; University of Naples - Italy
Oleg Ursu, University of New Mexico
Cristian G. Bologa, University of New Mexico
Radleigh Santos, Torrey Pines Institute for Molecular StudiesFollow
Clemencia Pinilla, Torrey Pines Institute for Molecular Studies
Terry H. Wu, University of New Mexico
Julie A. Lovchik, University of New Mexico
Tudor I. Oprea, University of New Mexico
Larry A. Sklar, University of New Mexico
George P. Tegos, Harvard Medical School; Massachusetts General Hospital

Document Type

Book Chapter

ISBN

978-1-4939-7452-8

Publication Date

2018

Editors

Akihito Yamaguchi, Kunihiko Nishino

Keywords

Flow cytometry, RND efflux transporters, Antibiotic enhancement, Biological warfare bacterial agents, Burkholderia sp., Francisella sp.

Description

The resistance nodulation cell division (RND) family of proteins are inner membrane transporters that associate with periplasmic adaptor proteins and outer membrane porins to affect substrate transport from the cytosol and periplasm in Gram-negative bacteria. Various structurally diverse compounds are substrates of RND transporters. Along with their notable role in antibiotic resistance, these transporters are essential for niche colonization, quorum sensing, and virulence as well as for the removal of fatty acids and bile salts. As such, RNDs are an attractive target for antimicrobial development. However, while enhancing the utility of antibiotics with an RND inhibitor is an appealing concept, only a small core of chemotypes has been identified as efflux pump inhibitors (EPIs). Thus, our key objective is the development and validation of an efflux profiling and discovery strategy for RND model systems. Here we describe a flow cytometric dye accumulation assay that uses fluorescein diacetate (FDA) to interrogate the model Gram-negative pathogens Escherichia coli, Franscisella tularensis, and Burkholderia pseudomallei. Fluorochrome retention is increased in the presence of known efflux inhibitors and in RND deletion strains. The assay can be used in a high-throughput format to evaluate efflux of dye-substrate candidates and to screen chemical libraries for novel EPIs. Triaged compounds that inhibit efflux in pathogenic strains are tested for growth inhibition and antibiotic potentiation using microdilution culture plates in a select agent Biosafety Level-3 (BSL3) environment. This combined approach demonstrates the utility of flow cytometric analysis for efflux activity and provides a useful platform in which to characterize efflux in pathogenic Gram-negative bacteria. Screening small molecule libraries for novel EPI candidates offers the potential for the discovery of new classes of antibacterial compounds.

DOI

10.1007/978-1-4939-7454-2

Publisher

Humana Press

City

New York

Additional Information

Pages 293-318

Disciplines

Mathematics

NSUWorks Citation

Haynes, Mark; Matthew Garcia; Ryan Peters; Anna Waller; Pietro Tedesco; Oleg Ursu; Cristian G. Bologa; Radleigh Santos; Clemencia Pinilla; Terry H. Wu; Julie A. Lovchik; Tudor I. Oprea; Larry A. Sklar; and George P. Tegos. (2018). High-Throughput Flow Cytometry Screening of Multidrug Efflux Systems. In Akihito Yamaguchi, Kunihiko Nishino (Eds.), Bacterial Multidrug Exporters .

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