Fluorescent trimethoprim conjugate probes to assess drug accumulation in wild type and mutant Escherichia coli

Phetsang, Wanida, Pelingon, Ruby, Butler, Mark S., KC, Sanjaya, Pitt, Miranda, Kaeslin, Geraldine, Cooper, Matthew A. and Blaskovich, Mark A. T. (2016) Fluorescent trimethoprim conjugate probes to assess drug accumulation in wild type and mutant Escherichia coli. ACS Infectious Diseases, 2 10: 688-701. doi:10.1021/acsinfecdis.6b00080


Author Phetsang, Wanida
Pelingon, Ruby
Butler, Mark S.
KC, Sanjaya
Pitt, Miranda
Kaeslin, Geraldine
Cooper, Matthew A.
Blaskovich, Mark A. T.
Title Fluorescent trimethoprim conjugate probes to assess drug accumulation in wild type and mutant Escherichia coli
Formatted title
Fluorescent trimethoprim conjugate probes to assess drug accumulation in wild type and mutant Escherichia coli
Journal name ACS Infectious Diseases   Check publisher's open access policy
ISSN 2373-8227
Publication date 2016-01-01
Year available 2016
Sub-type Article (original research)
DOI 10.1021/acsinfecdis.6b00080
Open Access Status DOI
Volume 2
Issue 10
Start page 688
End page 701
Total pages 14
Place of publication Washington, DC United States
Publisher American Chemical Society
Language eng
Abstract Reduced susceptibility to antimicrobials in Gram-negative bacteria may result from multiple resistance mechanisms, including increased efflux pump activity or reduced porin protein expression. Up-regulation of the efflux pump system is closely associated with multidrug resistance (MDR). To help investigate the role of efflux pumps on compound accumulation, a fluorescence-based assay was developed using fluorescent derivatives of trimethoprim (TMP), a broad-spectrum synthetic antibiotic that inhibits an intracellular target, dihydrofolate reductase (DHFR). Novel fluorescent TMP probes inhibited eDHFR activity with comparable potency to TMP, but did not kill or inhibit growth of wild type Escherichia coli. However, bactericidal activity was observed against an efflux pump deficient E. coli mutant strain (Delta tolC). A simple and quick fluorescence assay was developed to measure cellular accumulation of the TMP probe using either fluorescence spectroscopy or flow cytometry, with validation by LC-MS/MS. This fluorescence assay may provide a simple method to assess efflux pump activity with standard laboratory equipment.
Formatted abstract
Reduced susceptibility to antimicrobials in Gram-negative bacteria may result from multiple resistance mechanisms, including increased efflux pump activity or reduced porin protein expression. Up-regulation of the efflux pump system is closely associated with multidrug resistance (MDR). To help investigate the role of efflux pumps on compound accumulation, a fluorescence-based assay was developed using fluorescent derivatives of trimethoprim (TMP), a broad-spectrum synthetic antibiotic that inhibits an intracellular target, dihydrofolate reductase (DHFR). Novel fluorescent TMP probes inhibited eDHFR activity with comparable potency to TMP, but did not kill or inhibit growth of wild type Escherichia coli. However, bactericidal activity was observed against an efflux pump deficient E. coli mutant strain (ΔtolC). A simple and quick fluorescence assay was developed to measure cellular accumulation of the TMP probe using either fluorescence spectroscopy or flow cytometry, with validation by LC-MS/MS. This fluorescence assay may provide a simple method to assess efflux pump activity with standard laboratory equipment.
Keyword Antibiotics
Cellular accumulation
Efflux pump
Fluorescent probes
Trimethoprim
Q-Index Code C1
Q-Index Status Provisional Code
Grant ID APP1059354
WT141107
LIEF LE130100078
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: HERDC Pre-Audit
Institute for Molecular Bioscience - Publications
 
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Created: Fri, 09 Sep 2016, 01:47:17 EST by Susan Allen on behalf of Institute for Molecular Bioscience