scholarly journals Functional Cloning and Characterization of the Multidrug Efflux Pumps NorM from Neisseria gonorrhoeae and YdhE from Escherichia coli

2008 ◽  
Vol 52 (9) ◽  
pp. 3052-3060 ◽  
Author(s):  
Feng Long ◽  
Corinne Rouquette-Loughlin ◽  
William M. Shafer ◽  
Edward W. Yu
Keyword(s):  
Escherichia Coli ◽  
Neisseria Gonorrhoeae ◽  
Ethidium Bromide ◽  
Antimicrobial Agents ◽  
Dissociation Constants ◽  
Efflux Pumps ◽  
Multidrug Efflux ◽  
Active Efflux ◽  
E Coli ◽  
Multidrug Efflux Pumps

ABSTRACT Active efflux of antimicrobial agents is one of the most important adapted strategies that bacteria use to defend against antimicrobial factors that are present in their environment. The NorM protein of Neisseria gonorrhoeae and the YdhE protein of Escherichia coli have been proposed to be multidrug efflux pumps that belong to the multidrug and toxic compound extrusion (MATE) family. In order to determine their antimicrobial export capabilities, we cloned, expressed, and purified these two efflux proteins and characterized their functions both in vivo and in vitro. E. coli strains expressing norM or ydhE showed elevated (twofold or greater) resistance to several antimicrobial agents, including fluoroquinolones, ethidium bromide, rhodamine 6G, acriflavine, crystal violet, berberine, doxorubicin, novobiocin, enoxacin, and tetraphenylphosphonium chloride. When they were expressed in E. coli, both transporters reduced the levels of ethidium bromide and norfloxacin accumulation through a mechanism requiring the proton motive force, and direct measurements of efflux confirmed that NorM behaves as an Na+-dependent transporter. The capacities of NorM and YdhE to recognize structurally divergent compounds were confirmed by steady-state fluorescence polarization assays, and the results revealed that these transporters bind to antimicrobials with dissociation constants in the micromolar region.

scholarly journals Expression of Pseudomonas aeruginosa Multidrug Efflux Pumps MexA-MexB-OprM and MexC-MexD-OprJ in a Multidrug-Sensitive Escherichia coli Strain

1998 ◽  
Vol 42 (1) ◽  
pp. 65-71 ◽  
Author(s):  
Ramakrishnan Srikumar ◽  
Tatiana Kon ◽  
Naomasa Gotoh ◽  
Keith Poole
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Crystal Violet ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Multidrug Efflux ◽  
Efflux System ◽  
E Coli ◽  
Multidrug Efflux Pumps

ABSTRACT The mexCD-oprJ and mexAB-oprM operons encode components of two distinct multidrug efflux pumps inPseudomonas aeruginosa. To assess the contribution of individual components to antibiotic resistance and substrate specificity, these operons and their component genes were cloned and expressed in Escherichia coli. Western immunoblotting confirmed expression of the P. aeruginosa efflux pump components in E. coli strains expressing and deficient in the endogenous multidrug efflux system (AcrAB), although only the ΔacrAB strain, KZM120, demonstrated increased resistance to antibiotics in the presence of the P. aeruginosa efflux genes. E. coli KZM120 expressing MexAB-OprM showed increased resistance to quinolones, chloramphenicol, erythromycin, azithromycin, sodium dodecyl sulfate (SDS), crystal violet, novobiocin, and, significantly, several β-lactams, which is reminiscent of the operation of this pump in P. aeruginosa. This confirmed previous suggestions that MexAB-OprM provides a direct contribution to β-lactam resistance via the efflux of this group of antibiotics. An increase in antibiotic resistance, however, was not observed when MexAB or OprM alone was expressed in KZM120. Thus, despite the fact that β-lactams act within the periplasm, OprM alone is insufficient to provide resistance to these agents. E. coli KZM120 expressing MexCD-OprJ also showed increased resistance to quinolones, chloramphenicol, macrolides, SDS, and crystal violet, though not to most β-lactams or novobiocin, again somewhat reminiscent of the antibiotic resistance profile of MexCD-OprJ-expressing strains ofP. aeruginosa. Surprisingly, E. coli KZM120 expressing MexCD alone also showed an increase in resistance to these agents, while an OprJ-expressing KZM120 failed to demonstrate any increase in antibiotic resistance. MexCD-mediated resistance, however, was absent in a tolC mutant of KZM120, indicating that MexCD functions in KZM120 in conjunction with TolC, the previously identified outer membrane component of the AcrAB-TolC efflux system. These data confirm that a tripartite efflux pump is necessary for the efflux of all substrate antibiotics and that the P. aeruginosa multidrug efflux pumps are functional and retain their substrate specificity in E. coli.

scholarly journals VmrA, a Member of a Novel Class of Na+-Coupled Multidrug Efflux Pumps from Vibrio parahaemolyticus

2002 ◽  
Vol 184 (2) ◽  
pp. 572-576 ◽  
Author(s):  
Jing Chen ◽  
Yuji Morita ◽  
M. Nazmul Huda ◽  
Teruo Kuroda ◽  
Tohru Mizushima ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ethidium Bromide ◽  
Vibrio Parahaemolyticus ◽  
Efflux Pumps ◽  
Efflux Transporters ◽  
Drug Transporter ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pumps ◽  
Tetraphenylphosphonium Chloride

ABSTRACT Gene vmrA, cloned from Vibrio parahaemolyticus, made Escherichia coli resistant to 4prime;,6-diamino-2-phenylindol, tetraphenylphosphonium chloride, acriflavine, and ethidium bromide. VmrA belongs to the DinF branch of MATE family efflux transporters. VmrA catalyzed acriflavine efflux and showed Na+/drug transporter activity because the addition of tetraphenylphosphonium to Na+-loaded cells caused Na+ efflux.

scholarly journals Impaired Efflux of the Siderophore Enterobactin Induces Envelope Stress in Escherichia coli

2019 ◽  
Author(s):  
Randi L. Guest ◽  
Emily A. Court ◽  
Jayne L. Waldon ◽  
Kiersten A. Schock ◽  
Tracy L. Raivio
Keyword(s):  
Escherichia Coli ◽  
Nadh Dehydrogenase ◽  
Enteric Bacteria ◽  
Efflux Pumps ◽  
Multidrug Efflux ◽  
Gram Negative ◽  
E Coli ◽  
Multidrug Efflux Pumps ◽  
Pathway Activation ◽  
Envelope Stress

AbstractThe Cpx response is one of several envelope stress responses that monitor and maintain the integrity of the gram-negative bacterial envelope. While several conditions that are known or predicted to generate misfolded inner membrane proteins activate the Cpx response, the molecular nature of the Cpx inducing cue is not yet known. Studies have demonstrated that mutation of multidrug efflux pumps activates the Cpx response in many gram-negative bacteria. In Vibrio cholerae, pathway activation is due to accumulation of the catechol siderophore vibriobactin. However, the mechanism by which the Cpx response is activated by mutation of efflux pumps in Escherichia coli remains unknown. Here we show that inhibition of efflux by deletion of tolC, the outer membrane channel of several multidrug efflux pumps, activates the Cpx response in E. coli as a result of impaired efflux of the siderophore enterobactin. Enterobactin accumulation in the tolC mutant reduces activity of the NADH oxidation arm of the aerobic respiratory chain. However, NADH dehydrogenase I, NADH dehydrogenase II, and cytochrome bo3 do not contribute to Cpx pathway activation in the E. coli tolC mutant. We show that the Cpx response down-regulates transcription of the enterobactin biosynthesis operon. These results suggest that the Cpx response promotes adaptation to envelope stress in enteric bacteria that are exposed to iron-limited environments, which are rich in envelope-damaging compounds and conditions.

scholarly journals Functional and Structural Roles of the Major Facilitator Superfamily Bacterial Multidrug Efflux Pumps

2020 ◽  
Vol 8 (2) ◽  
pp. 266 ◽  
Author(s):  
Sanath Kumar ◽  
Manjusha Lekshmi ◽  
Ammini Parvathi ◽  
Manisha Ojha ◽  
Nicholas Wenzel ◽  
...  
Keyword(s):  
Amino Acid ◽  
Antimicrobial Agents ◽  
Efflux Pumps ◽  
Multidrug Resistant ◽  
Amino Acid Sequences ◽  
Major Facilitator Superfamily ◽  
Multidrug Efflux ◽  
Ion Translocation ◽  
Multidrug Efflux Pumps ◽  
Major Facilitator

Pathogenic microorganisms that are multidrug-resistant can pose severe clinical and public health concerns. In particular, bacterial multidrug efflux transporters of the major facilitator superfamily constitute a notable group of drug resistance mechanisms primarily because multidrug-resistant pathogens can become refractory to antimicrobial agents, thus resulting in potentially untreatable bacterial infections. The major facilitator superfamily is composed of thousands of solute transporters that are related in terms of their phylogenetic relationships, primary amino acid sequences, two- and three-dimensional structures, modes of energization (passive and secondary active), and in their mechanisms of solute and ion translocation across the membrane. The major facilitator superfamily is also composed of numerous families and sub-families of homologous transporters that are conserved across all living taxa, from bacteria to humans. Members of this superfamily share several classes of highly conserved amino acid sequence motifs that play essential mechanistic roles during transport. The structural and functional importance of multidrug efflux pumps that belong to the major facilitator family and that are harbored by Gram-negative and -positive bacterial pathogens are considered here.

scholarly journals Functional Cloning and Characterization of a Multidrug Efflux Pump, MexHI-OpmD, from a Pseudomonas aeruginosa Mutant

2003 ◽  
Vol 47 (9) ◽  
pp. 2990-2992 ◽  
Author(s):  
Hiroshi Sekiya ◽  
Takehiko Mima ◽  
Yuji Morita ◽  
Teruo Kuroda ◽  
Tohru Mizushima ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Ethidium Bromide ◽  
Rhodamine 6G ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Multidrug Efflux ◽  
Chromosomal Dna ◽  
Multidrug Efflux Pump ◽  
Multidrug Efflux Pumps

ABSTRACT We isolated mutant YM644, which showed elevated resistance to norfloxacin, ethidium bromide, acriflavine, and rhodamine 6G, from Pseudomonas aeruginosa YM64, a strain that lacks four major multidrug efflux pumps. The genes responsible for the resistance were mexHI-opmD. Elevated ethidium extrusion was observed with cells of YM644 and YM64 harboring a plasmid carrying the genes. Disruption of the genes in the chromosomal DNA of YM644 made the cells sensitive to the drugs.

scholarly journals 1-benzyl-1,4-diazepane reduces the efflux of resistance-nodulation-cell division pumps in Escherichia coli

2020 ◽  
Vol 15 (11) ◽  
pp. 987-999
Author(s):  
Enrico Casalone ◽  
Tiziano Vignolini ◽  
Laura Braconi ◽  
Lucia Gardini ◽  
Marco Capitanio ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ethidium Bromide ◽  
Inhibitory Concentration ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Efflux Pump Inhibitor ◽  
E Coli ◽  
Microdilution Method ◽  
Photoactivated Localization Microscopy ◽  
Mixed Mechanism

Aim: To investigate the action mechanism of 1-benzyl-1,4-diazepane (1-BD) as efflux pump inhibitor (EPI) in Escherichia coli mutants: Δ acrAB or overexpressing AcrAB and AcrEF efflux pumps. Materials & methods: Effect of 1-BD on: antibiotic potentiation, by microdilution method; membrane functionality, by fluorimetric assays; ethidium bromide accumulation, by fluorometric real-time efflux assay; AcrB expression, by quantitative photoactivated localization microscopy. Results: 1-BD decreases the minimal inhibitory concentration of levofloxacin and other antibiotics and increase ethidium bromide accumulation in E. coli overexpressing efflux pumps but not in the Δ acrAB strain. 1-BD increases membranes permeability, without sensibly affecting inner membrane polarity and decreases acrAB transcription. Conclusion: 1-BD acts as an EPI in E. coli with a mixed mechanism, different from that of major reference EPIs.

scholarly journals Roles of Multidrug Efflux Pumps on the Biofilm Formation of Escherichia coli K-12

2011 ◽  
Vol 16 (2) ◽  
pp. 69-72 ◽  
Author(s):  
KAYO MATSUMURA ◽  
SOICHI FURUKAWA ◽  
HIROKAZU OGIHARA ◽  
YASUSHI MORINAGA
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Efflux Pumps ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pumps ◽  
K 12

scholarly journals In vitro synergy between Sodium Deoxycholate and Furazolidone against Enterobacteria via Inhibition of Multidrug Efflux Pumps

2019 ◽  
Author(s):  
Vuong Van Hung Le ◽  
Catrina Olivera ◽  
Julian Spagnuolo ◽  
Ieuan Davies ◽  
Jasna Rakonjac
Keyword(s):  
Synergistic Effect ◽  
Model Organism ◽  
Sodium Deoxycholate ◽  
Efflux Pumps ◽  
Bacterial Cells ◽  
Multidrug Efflux ◽  
Resistance Development ◽  
E Coli ◽  
Multidrug Efflux Pumps

AbstractAntimicrobial combinations have been proven to be a promising approach in the confrontation with multi-drug resistant bacterial pathogens, owing to enhancement of antibacterial efficacy, deceleration of resistance development rate and mitigation of side effects by lowering the doses of two drugs. In the present study, we report that combination of furazolidone (FZ) and other nitrofurans with a secondary bile salt, Sodium Deoxycholate (DOC), generates a profound synergistic effect on growth inhibition and lethality in enterobacteria, including Escherichia coli, Salmonella, Citrobacter gillenii and Klebsiella pneumoniae. Taking E. coli as the model organism to study the mechanism of DOC-FZ synergy, we found that the synergistic effect involves FZ-mediated inhibition of efflux pumps that normally remove DOC from bacterial cells. We further show that the FZ–mediated nitric oxide production contributes to the synergistic effect. This is to our knowledge the first report of nitrofuran-DOC synergy against Gram-negative bacteria.

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