scholarly journals Pimozide Inhibits the AcrAB-TolC Efflux Pump in Escherichia coli

2013 ◽  
Vol 7 (1) ◽  
pp. 83-86 ◽  
Author(s):  
Jürgen A Bohnert ◽  
Sabine Schuster ◽  
Winfried V Kern
Keyword(s):  
Escherichia Coli ◽  
Ethidium Bromide ◽  
Binding Sites ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Nile Red ◽  
Neuroleptic Drug ◽  
Antimicrobial Compounds ◽  
Efflux Pump Inhibitors ◽  
Substrate Binding Sites

Efflux pump inhibitors (EPIs) are attractive compounds to reverse multidrug-resistance in clinically relevant bacterial pathogens. In this study we tested the ability of the neuroleptic drug pimozide to inhibit the Escherichia coli AcrAB-TolC efflux pump, whose overproduction confers resistance to various antimicrobial agents. A real-time Nile red efflux assay in the AcrAB – overproducing strain 3-AG100 revealed that pimozide was capable of full inhibition of this pump at a concentration of 100 µM, which is far below its intrinsic MIC (>1mM). However, MIC assay demonstrated very little effect of pimozide with regard to reduction in MICs of various antimicrobial compounds. Only oxacillin MICs were reduced twofold in the presence of pimozide at 100 and 200 µM. Since pimozide did considerably enhance accumulation of ethidium bromide in a fluorescence assay, ethidium bromide MIC assays in the presence and absence of this putative EPI were performed. They revealed that pimozide was able to reduce the MICs of ethidium bromide by 4-fold. In line with previous reports we suggest that the capability of EPIs to restore the susceptibility to antimicrobial agents can be highly substrate-specific due to different substrate binding sites.

scholarly journals Optimized Nile Red Efflux Assay of AcrAB-TolC Multidrug Efflux System Shows Competition between Substrates

2010 ◽  
Vol 54 (9) ◽  
pp. 3770-3775 ◽  
Author(s):  
Jürgen A. Bohnert ◽  
Brian Karamian ◽  
Hiroshi Nikaido
Keyword(s):  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Nile Red ◽  
Proton Conductor ◽  
Multidrug Efflux ◽  
Pump System ◽  
Tetraphenylphosphonium Chloride ◽  
Carbonyl Cyanide ◽  
Efflux Pump Inhibitors ◽  
Multidrug Efflux System

ABSTRACT AcrAB-TolC is the major constitutively expressed efflux pump system that provides resistance to a variety of antimicrobial agents and dyes in Escherichia coli. However, no systematically optimized real-time dye efflux assay has been published for the measurement of its activity and for detection of possible competition between substrates. Here, we report on the development of such an assay using a lipophilic dye, Nile Red. Energy-depleted cells were loaded with the dye in the presence of low (10 μM or less) concentrations of the proton conductor carbonyl cyanide m-chlorophenylhydrazone (CCCP). The CCCP was then removed, and efflux was triggered by energization with glucose. Various known efflux pump inhibitors and antimicrobials were checked for the ability to slow down Nile Red efflux, presumably through competition. Besides the known inhibitors Phe-Arg-β-naphthylamide and 1-naphthyl-methylpiperazine, several tetracyclic compounds (doxorubicin, minocycline, chlortetracycline, doxycycline, and tetracycline) and tetraphenylphosphonium chloride were found to interfere with dye efflux. This inhibition could not be explained by the depletion of proton motive force. None of the other tested antimicrobials, including macrolides, fluoroquinolones, and β-lactams, had any impact on Nile Red efflux, even at concentrations of up to 1 mM.

scholarly journals Human organic anion transporter MRP4 (ABCC4) is an efflux pump for the purine end metabolite urate with multiple allosteric substrate binding sites

2005 ◽  
Vol 288 (2) ◽  
pp. F327-F333 ◽  
Author(s):  
Rémon A. M. H. Van Aubel ◽  
Pascal H. E. Smeets ◽  
Jeroen J. M. W. van den Heuvel ◽  
Frans G. M. Russel
Keyword(s):  
Binding Sites ◽  
Efflux Pump ◽  
Substrate Binding ◽  
Organic Anion ◽  
Apical Cell ◽  
Cell Uptake ◽  
Hek293 Cells ◽  
Hill Coefficient ◽  
Anion Transporter ◽  
Substrate Binding Sites

The end product of human purine metabolism is urate, which is produced primarily in the liver and excreted by the kidney through a well-defined basolateral blood-to-cell uptake step. However, the apical cell-to-urine efflux mechanism is as yet unidentified. Here, we show that the renal apical organic anion efflux transporter human multidrug resistance protein 4 (MRP4), but not apical MRP2, mediates ATP-dependent urate transport via a positive cooperative mechanism ( Km of 1.5 ± 0.3 mM, Vmax of 47 ± 7 pmol·mg−1·min−1, and Hill coefficient of 1.7 ± 0.2). In HEK293 cells overexpressing MRP4, intracellular urate levels were lower than in control cells. Urate inhibited methotrexate transport (IC50 of 235 ± 8 μM) by MRP4, did not affect cAMP transport, whereas cGMP transport was stimulated. Urate shifted cGMP transport by MRP4 from positive cooperativity ( Km and Vmax value of 180 ± 20 μM and 58 ± 4 pmol·mg−1·min−1, respectively, Hill coefficient of 1.4 ± 0.1) to single binding site kinetics ( Km and Vmax value of 2.2 ± 0.9 mM and 280 ± 50 pmol·mg−1·min−1, respectively). Finally, MRP4 could transport urate simultaneously with cAMP or cGMP. We conclude that human MRP4 is a unidirectional efflux pump for urate with multiple allosteric substrate binding sites. We propose MRP4 as a candidate transporter for urinary urate excretion and suggest that MRP4 may also mediate hepatic export of urate into the circulation, because of its basolateral expression in the liver.

Characterization of Dual Substrate Binding Sites in the Homodimeric Structure of Escherichia coli mRNA Interferase MazF

2006 ◽  
Vol 357 (1) ◽  
pp. 139-150 ◽  
Author(s):  
Guang-Yao Li ◽  
Yonglong Zhang ◽  
Mitchell C.Y. Chan ◽  
Tapas K. Mal ◽  
Klaus P. Hoeflich ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Substrate Binding ◽  
Substrate Binding Sites ◽  
Dual Substrate

scholarly journals Identification of the Haemophilus influenzae tolC Gene by Susceptibility Profiles of Insertionally Inactivated Efflux Pump Mutants

2004 ◽  
Vol 48 (4) ◽  
pp. 1416-1418 ◽  
Author(s):  
Catherine M. Trepod ◽  
John E. Mott
Keyword(s):  
Escherichia Coli ◽  
Haemophilus Influenzae ◽  
Efflux Pump ◽  
Antimicrobial Compounds ◽  
The Third ◽  
Susceptibility Profiles ◽  
Isogenic Strains

ABSTRACT Isogenic strains containing insertional disruptions of 10 Haemophilus influenzae Rd genes were investigated for their effects on the susceptibility of the organism to various classes of antimicrobial compounds. MIC results show that HI1462, which encodes an Escherichia coli TolC homolog, is the third component of the H. influenzae AcrAB pump.

scholarly journals Rescued chlorhexidine activity by resveratrol against carbapenem-resistant Acinetobacter baumannii via down-regulation of AdeB efflux pump

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0243082
Author(s):  
Uthaibhorn Singkham-in ◽  
Paul G. Higgins ◽  
Dhammika Leshan Wannigama ◽  
Parichart Hongsing ◽  
Tanittha Chatsuwan
Keyword(s):  
Acinetobacter Baumannii ◽  
Ethidium Bromide ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Intracellular Accumulation ◽  
Efflux Pump Inhibitor ◽  
Carbapenem Resistant ◽  
Efflux Pump Inhibition ◽  
Synergistic Mechanism ◽  
Time Kill

The aim of this study was to determine the activity and synergistic mechanisms of resveratrol in combination with chlorhexidine against carbapenem-resistant Acinetobacter baumannii clinical isolates. The activity of resveratrol plus antimicrobial agents was determined by checkerboard and time-kill assay against carbapenem-resistant A. baumannii isolated from patients at the King Chulalongkorn Memorial Hospital, Bangkok, Thailand. Overexpression of efflux pumps that mediates chlorhexidine susceptibility was characterized by the ethidium bromide accumulation assay. The effect of resveratrol on the expression of efflux pump genes (adeB, adeJ, adeG abeS, and aceI) and the two-component regulators, adeR and adeS was determined by RT-qPCR. The combination of resveratrol and chlorhexidine resulted in strong synergistic and bactericidal activity against carbapenem-resistant A. baumannii. Up-regulation of adeB and aceI was induced by chlorhexidine. However, the addition of resveratrol increased chlorhexidine susceptibility with increased intracellular accumulation of ethidium bromide in A. baumannii indicating that resveratrol acts as an efflux pump inhibitor. Expression of adeB was significantly reduced in the combination of resveratrol with chlorhexidine indicating that resveratrol inhibits the AdeB efflux pump and restores chlorhexidine effect on A. baumannii. In conclusion, reduced adeB expression in A. baumannii was mediated by resveratrol suggesting that AdeB efflux pump inhibition contributes to the synergistic mechanism of resveratrol with chlorhexidine. Our finding highlights the potential importance of resveratrol in clinical applications.

scholarly journals Genetic and Enzymatic Experiments relating to the Quaternary Structure of ß-Galactosidase

1974 ◽  
Vol 27 (3) ◽  
pp. 321 ◽  
Author(s):  
J Langridge
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Quaternary Structure ◽  
Substrate Binding ◽  
Isolation And Characterization ◽  
Normal Enzyme ◽  
Substrate Binding Sites ◽  
Polypeptide Sequences

The quaternary structure of f:!-galactosidase, which consists of four identical subunits, has been studied by the isolation and characterization of appropriate mutants of Escherichia coli. Of 146 mutants examined, 19 were found to have enzymes with reduced subunit association. These altered enzymes are especially sensitive to inactivation by urea which, at concentrations that do not affect the normal enzyme, causes dissociation into subunits. Mapping with overlapping deletions showed that the mutations affecting quaternary structure are not distributed continuously, but occur in five or six groups within the gene. The mapping indicates that polypeptide sequences involved in subunit association and in substrate binding are contiguous. A model for f:!-galactosidase structure is suggested in which substrate binding sites are provided by the clefts formed between subunits when they associate.

Sign in / Sign up

Export Citation Format

Share Document