scholarly journals CCCP experimental evolution ofEscherichia coliselects for mutations that increase EmrA activity or that downregulate other PMF-driven drug efflux pumps

2018 ◽  
Author(s):  
Jessie M. Griffith ◽  
Preston J. Basting ◽  
Katarina M. Bischof ◽  
Erintrude P. Wrona ◽  
Karina S. Kunka ◽  
...  
Keyword(s):  
Experimental Evolution ◽  
Efflux Pump ◽  
Enteric Bacteria ◽  
Efflux Pumps ◽  
Proton Motive Force ◽  
Motive Force ◽  
Drug Efflux ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
K 12

ABSTRACTExperimental evolution was conducted withEscherichia coliK-12 W3110 in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), an uncoupler of the proton motive force (PMF). Cultures were serially diluted daily 1:100 in broth medium containing 20-150 μM CCCP at pH 6.5 or at pH 8.0. After 1,000 generations, all populations showed 5- to 10-fold increase in CCCP resistance. Sequenced isolates showed mutations inemrABor in its negative repressormprA; the EmrAB-TolC multidrug efflux pump confers resistance to CCCP and nalidixic acid. Deletion ofemrAabolished the CCCP resistance of these strains. One CCCP-evolved isolate lackedemrAormprAmutations; this strain (C-B11-1) showed mutations in drug efflux regulatorscecR(ybiH) (upregulates drug pumps YbhG and YbhFSR) andgadE(upregulates drug pumpmdtEF). AcecR∷kanRdeletion conferred partial resistance to CCCP. A later evolved descendant of the C-B11 population showed mutations inybhR(MDR efflux). Another isolate showedacrB(MDR efflux pump). TheacrBisolate was sensitive to chloramphenicol and tetracycline, which are effluxed by AcrAB. Other mutant genes in CCCP-evolved strains includeadhE(alcohol dehydrogenase),rng(ribonuclease G), andcyaA(adenylate cyclase). Overall, experimental evolution revealed a CCCP fitness advantage for mutations increasing its own efflux via EmrA; and for mutations that may decrease proton-driven pumps that efflux other drugs not present (cecR, gadE, acrB, ybhR). These results are consistent with our previous report of drug sensitivity associated with evolved tolerance to a partial uncoupler (benzoate or salicylate).IMPORTANCEThe genetic responses of bacteria to depletion of proton motive force, and their effects on drug resistance, are poorly understood. Our evolution experiment reveals genetic mechanisms of adaptation to the PMF uncoupler CCCP, including selection for and against various multidrug efflux pumps. The results have implications for our understanding of the gut microbiome, which experiences high levels of organic acids that decrease PMF. Organic acid uncouplers may select against multidrug resistance in evolving populations of enteric bacteria.

scholarly journals Benzoate-TolerantEscherichia coliStrains from Experimental Evolution Lose the Gad Regulon, Multi-Drug Efflux Pumps, and Hydrogenases

2019 ◽  
Author(s):  
Jeremy P. Moore ◽  
Haofan Li ◽  
Morgan L. Engmann ◽  
Katarina M. Bischof ◽  
Karina S. Kunka ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Experimental Evolution ◽  
Efflux Pumps ◽  
Proton Motive Force ◽  
Motive Force ◽  
Signal Molecules ◽  
Drug Efflux ◽  
Drug Efflux Pumps ◽  
K 12

ABSTRACTBenzoate, a partial uncoupler of the proton motive force (PMF), selects for sensitivity to chloramphenicol and tetracycline inEscherichia coliK-12. Genetic mechanisms of increased benzoate tolerance and decreased drug resistance were analyzed in strains isolated from experimental evolution with benzoate. Transcriptomes showed reversal of benzoate-dependent regulation, including the Gad regulon for acid resistance and multi-drug resistance (MDR). Benzoate-evolved strains knocked down the nitrate reductasenarHJand the acid-consuming hydrogenase Hyd-3, but upregulated OmpF and other porins that admit nutrients and antibiotics. Mutations were found affecting many genes of aromatic catabolism such asfolDandadd. Several candidate genes from benzoate-evolved strains had a role in benzoate tolerance. Growth with benzoate or salicylate was increased by deletion of the Gad activatorariR, or by deletion of theslp-gadXacid fitness island encoding Gad regulators and the MDR pump MdtEF. Benzoate growth was also increased by deletion of MDR componentemrA, the RpoS post-transcriptional regulatorcspC, or the adenosine deaminaseadd. Growth in chloramphenicol with benzoate was decreased by a point mutation in the RNA polymerase alpha-subunitrpoA, which appeared in one chloramphenicol-sensitive benzoate-evolved strain. Growth in chloramphenicol was also decreased by deletion of Mar activatorrob, or ofrfaY(lipopolysaccharide biosynthesis). Hydrogenase Hyd-3 deletion increased benzoate tolerance. Overall, long-term culture in the presence of benzoate or salicylate favors loss of MDR efflux pumps and of hydrogenases that generate a futile cycle of PMF; and favors upregulation of large-hole porins that increase uptake of fermentable nutrients and of antibiotics.IMPORTANCEBenzoate is a common food preservative, and salicylate is the active form of aspirin. InE. coli, benzoate derivatives upregulate multiple regulons that export antibiotics and other toxic products, and downregulate large outer-membrane porins that allow antibiotic influx. But benzoate or salicylate exposure causes energy stress by depleting the proton motive force. In the absence of antibiotics, long-term benzoate exposure selects against energy-spending systems such as multi-drug efflux pumps and the proton-consuming hydrogenase. Selection favors upregulation of porins that admit fermentable substrates but also allow entry of antibiotics. Thus, evolution with benzoate requires a tradeoff for antibiotic sensitivity. Benzoate and salicylate are naturally occurring plant signal molecules that may influence the evolution of microbiomes in plants and in animal digestive tracts. Effects on hydrogenase activity may be relevant to the biotechnology of hydrogen production.

scholarly journals Experimental Evolution ofEscherichia coliK-12 in the Presence of Proton Motive Force (PMF) Uncoupler Carbonyl Cyanidem-Chlorophenylhydrazone Selects for Mutations Affecting PMF-Driven Drug Efflux Pumps

2018 ◽  
Vol 85 (5) ◽  
Author(s):  
Jessie M. Griffith ◽  
Preston J. Basting ◽  
Katarina M. Bischof ◽  
Erintrude P. Wrona ◽  
Karina S. Kunka ◽  
...  
Keyword(s):  
Experimental Evolution ◽  
Point Mutations ◽  
Proton Motive Force ◽  
Motive Force ◽  
Multidrug Efflux ◽  
Content Type ◽  
Fitness Advantage ◽  
Evolution Experiment ◽  
Drug Efflux Pumps ◽  
K 12

ABSTRACTExperimental evolution ofEscherichia coliK-12 with benzoate, a partial uncoupler of the proton motive force (PMF), selects for mutations that decrease antibiotic resistance. We conducted experimental evolution in the presence of carbonyl cyanidem-chlorophenylhydrazone (CCCP), a strong uncoupler. Cultures were serially diluted daily 1:100 in LBK medium containing 20 to 150 µM CCCP buffered at pH 6.5 or at pH 8.0. After 1,000 generations, the populations tolerated up to 150 µM CCCP. Sequenced isolates had mutations inmprA(emrR), which downregulates the EmrAB-TolC pump that exports CCCP. AmprA::kanRdeletion conferred growth at 60 μM CCCP, though not at the higher levels resisted by evolved strains (150 µM). SomemprAmutant strains also had point mutations affectingemrA, but deletion ofemrAabolished the CCCP resistance. Thus, CCCP-evolved isolates contained additional adaptations. One isolate lackedemrAormprAmutations but had mutations incecR(ybiH), whose product upregulates drug pumps YbhG and YbhFSR, and ingadE, which upregulates the multidrug pump MdtEF. AcecR::kanRdeletion conferred partial resistance to CCCP. Other multidrug efflux genes that had mutations includedybhRandacrAB. TheacrBisolate was sensitive to the AcrAB substrates chloramphenicol and tetracycline. Other mutant genes in CCCP-evolved strains includerng(RNase G) andcyaA(adenylate cyclase). Overall, experimental evolution revealed a CCCP-dependent fitness advantage for mutations increasing CCCP efflux via EmrA and for mutations that may deactivate proton-driven pumps for drugs not present (cecR,gadE,acrAB, andybhR). These results are consistent with our previous report of drug sensitivity associated with evolved benzoate tolerance.IMPORTANCEThe genetic responses of bacteria to depletion of proton motive force (PMF), and their effects on drug resistance, are poorly understood. PMF drives export of many antibiotics, but the energy cost may decrease fitness when antibiotics are absent. Our evolution experiment reveals genetic mechanisms of adaptation to the PMF uncoupler CCCP, including selection for increased CCCP efflux but also against the expression of PMF-driven pumps for drugs not present. The results have implications for our understanding of the gut microbiome, which experiences high levels of organic acids that decrease PMF.

scholarly journals A Broad-Specificity Multidrug Efflux Pump Requiring a Pair of Homologous SMR-Type Proteins

2000 ◽  
Vol 182 (8) ◽  
pp. 2311-2313 ◽  
Author(s):  
Donald L. Jack ◽  
Michael L. Storms ◽  
Jason H. Tchieu ◽  
Ian T. Paulsen ◽  
Milton H. Saier
Keyword(s):  
Escherichia Coli ◽  
Efflux Pump ◽  
Multidrug Resistant ◽  
Drug Efflux ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Resistant Phenotype ◽  
Small Multidrug Resistance ◽  
Drug Efflux Pumps ◽  
Broad Specificity

ABSTRACT The Bacillus subtilis genome encodes seven homologues of the small multidrug resistance (SMR) family of drug efflux pumps. Six of these homologues are paired in three distinct operons, and coexpression in Escherichia coli of one such operon,ykkCD, but not expression of either ykkC orykkD alone, gives rise to a broad specificity, multidrug-resistant phenotype including resistance to cationic, anionic, and neutral drugs.

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 Molecular Mechanism of MBX2319 Inhibition of Escherichia coli AcrB Multidrug Efflux Pump and Comparison with Other Inhibitors

2014 ◽  
Vol 58 (10) ◽  
pp. 6224-6234 ◽  
Author(s):  
Attilio V. Vargiu ◽  
Paolo Ruggerone ◽  
Timothy J. Opperman ◽  
Son T. Nguyen ◽  
Hiroshi Nikaido
Keyword(s):  
Efflux Pump ◽  
Efflux Pumps ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Efflux Pump Inhibitor ◽  
Content Type ◽  
X Ray Crystallography ◽  
Resistance Nodulation Division ◽  
Hydrophobic Portion ◽  
Dynamics Simulations

ABSTRACTEfflux pumps of the resistance nodulation division (RND) superfamily, such as AcrB, make a major contribution to multidrug resistance in Gram-negative bacteria. The development of inhibitors of the RND pumps would improve the efficacy of current and next-generation antibiotics. To date, however, only one inhibitor has been cocrystallized with AcrB. Thus,in silicostructure-based analysis is essential for elucidating the interaction between other inhibitors and the efflux pumps. In this work, we used computer docking and molecular dynamics simulations to study the interaction between AcrB and the compound MBX2319, a novel pyranopyridine efflux pump inhibitor with potent activity against RND efflux pumps ofEnterobacteriaceaespecies, as well as other known inhibitors (D13-9001, 1-[1-naphthylmethyl]-piperazine, and phenylalanylarginine-β-naphthylamide) and the binding of doxorubicin to the efflux-defective F610A variant of AcrB. We also analyzed the binding of a substrate, minocycline, for comparison. Our results show that MBX2319 binds very tightly to the lower part of the distal pocket in the B protomer of AcrB, strongly interacting with the phenylalanines lining the hydrophobic trap, where the hydrophobic portion of D13-9001 was found to bind by X-ray crystallography. Additionally, MBX2319 binds to AcrB in a manner that is similar to the way in which doxorubicin binds to the F610A variant of AcrB. In contrast, 1-(1-naphthylmethyl)-piperazine and phenylalanylarginine-β-naphthylamide appear to bind to somewhat different areas of the distal pocket in the B protomer of AcrB than does MBX2319. However, all inhibitors (except D13-9001) appear to distort the structure of the distal pocket, impairing the proper binding of substrates.

scholarly journals Inverted Regulation of Multidrug Efflux Pumps, Acid Resistance, and Porins in Benzoate-EvolvedEscherichia coliK-12

2019 ◽  
Vol 85 (16) ◽  
Author(s):  
Jeremy P. Moore ◽  
Haofan Li ◽  
Morgan L. Engmann ◽  
Katarina M. Bischof ◽  
Karina S. Kunka ◽  
...  
Keyword(s):  
Benzoic Acid ◽  
Experimental Evolution ◽  
Acid Resistance ◽  
Efflux Pumps ◽  
Proton Motive Force ◽  
Regulator Gene ◽  
Multidrug Efflux ◽  
Trade Off ◽  
Content Type ◽  
Multidrug Efflux Pumps

ABSTRACTBenzoic acid, a partial uncoupler of the proton motive force (PMF), selects for sensitivity to chloramphenicol and tetracycline during the experimental evolution ofEscherichia coliK-12. Transcriptomes ofE. coliisolates evolved with benzoate showed the reversal of benzoate-dependent regulation, including the downregulation of multidrug efflux pump genes, the gene for the Gad acid resistance regulon, the nitrate reductase genesnarHJ, and the gene for the acid-consuming hydrogenase Hyd-3. However, the benzoate-evolved strains had increased expression of OmpF and other large-hole porins that admit fermentable substrates and antibiotics. Candidate genes identified from benzoate-evolved strains were tested for their roles in benzoate tolerance and in chloramphenicol sensitivity. Benzoate or salicylate tolerance was increased by deletion of the Gad activatorariRor of the acid fitness island fromslpto the end of thegadXgene encoding Gad regulators and the multidrug pump genesmdtEF. Benzoate tolerance was also increased by deletion of multidrug component geneemrA, RpoS posttranscriptional regulator genecspC, adenosine deaminase geneadd, hydrogenase genehyc(Hyd-3), and the RNA chaperone/DNA-binding regulator genehfq. Chloramphenicol resistance was decreased by mutations in genes for global regulators, such as RNA polymerase alpha subunit generpoA, the Mar activator generob, andhfq. Deletion of lipopolysaccharide biosynthetic kinase generfaYdecreased the rate of growth in chloramphenicol. Isolates from experimental evolution with benzoate had many mutations affecting aromatic biosynthesis and catabolism, such asaroF(encoding tyrosine biosynthesis) andapt(encoding adenine phosphoribosyltransferase). Overall, benzoate or salicylate exposure selects for the loss of multidrug efflux pumps and of hydrogenases that generate a futile cycle of PMF and upregulates porins that admit fermentable nutrients and antibiotics.IMPORTANCEBenzoic acid is a common food preservative, and salicylic acid (2-hydroxybenzoic acid) is the active form of aspirin. At high concentrations, benzoic acid conducts a proton across the membrane, depleting the proton motive force. In the absence of antibiotics, benzoate exposure selects against proton-driven multidrug efflux pumps and upregulates porins that admit fermentable substrates but that also allow the entry of antibiotics. Thus, evolution with benzoate and related molecules, such as salicylates, requires a trade-off for antibiotic sensitivity, a trade-off that could help define a stable gut microbiome. Benzoate and salicylate are naturally occurring plant signal molecules that may modulate the microbiomes of plants and animal digestive tracts so as to favor fermenters and exclude drug-resistant pathogens.

scholarly journals Chlorpromazine and Amitriptyline Are Substrates and Inhibitors of the AcrB Multidrug Efflux Pump

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Elizabeth M. Grimsey ◽  
Chiara Fais ◽  
Robert L. Marshall ◽  
Vito Ricci ◽  
Maria Laura Ciusa ◽  
...  
Keyword(s):  
De Novo ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Inhibition Mechanism ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Content Type ◽  
Inhibitory Compounds ◽  
Serovar Typhimurium ◽  
Rnd Efflux Pumps

Efflux pumps of the resistance nodulation-cell division (RND) superfamily are major contributors to multidrug resistance for most of the Gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens. The development of inhibitors of these pumps would be highly desirable; however, several issues have thus far hindered all efforts at designing new efflux inhibitory compounds devoid of adverse effects. An alternative route to de novo design relies on the use of marketed drugs, for which side effects on human health have been already assessed. In this work, we provide experimental evidence that the antipsychotic drugs chlorpromazine and amitriptyline are inhibitors of the AcrB transporter, the engine of the major RND efflux pumps in Escherichia coli and Salmonella enterica serovar Typhimurium. Furthermore, in silico calculations have provided a molecular-level picture of the inhibition mechanism, allowing rationalization of experimental data and paving the way for similar studies with other classes of marketed compounds.

scholarly journals Berberine reverses multidrug resistance in Candida albicans by hijacking the drug efflux pump Mdr1p

2020 ◽  
Author(s):  
Yaojun Tong ◽  
Nuo Sun ◽  
Xiangming Wang ◽  
Qi Wei ◽  
Yu Zhang ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Multidrug Resistance ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Multidrug Resistant ◽  
Major Facilitator Superfamily ◽  
Drug Efflux ◽  
Multidrug Efflux Pump ◽  
Drug Efflux Pumps ◽  
Major Facilitator

AbstractClinical use of antimicrobials faces great challenges from the emergence of multidrug resistant (MDR) pathogens. The overexpression of drug efflux pumps is one of the major contributors to MDR. It is considered as a promising approach to overcome MDR by reversing the function of drug efflux pumps. In the life-threatening fungal pathogen Candida albicans, the major facilitator superfamily (MFS) transporter Mdr1p can excrete many structurally unrelated antifungals, leading to multidrug resistance. Here we report a counterintuitive case of reversing multidrug resistance in C. albicans by using a natural product berberine to hijack the overexpressed Mdr1p for its own importation. Moreover, we illustrate that the imported berberine accumulates in mitochondria, and compromises the mitochondrial function by impairing mitochondrial membrane potential and mitochondrial Complex I. It results in the selective elimination of Mdr1p overexpressed C. albicans cells. Furthermore, we show that berberine treatment can prolong the mean survival time (MST) of mice with a blood-borne dissemination of Mdr1p overexpressed multidrug resistant candidiasis. This study provided a potential direction of novel anti-MDR drug discovery by screening for multidrug efflux pump converters.

scholarly journals Mosaic Drug Efflux Gene Sequences from CommensalNeisseriaCan Lead to Low-Level Azithromycin Resistance Expressed byNeisseria gonorrhoeaeClinical Isolates

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
William M. Shafer
Keyword(s):  
Efflux Pump ◽  
Bioinformatic Analysis ◽  
Drug Efflux ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Content Type ◽  
Treatment Regimens ◽  
Resistance Markers ◽  
Low Levels ◽  
Azithromycin Resistance

ABSTRACTIn a previousmBioarticle, Wadsworth and colleagues (mBio 9:e01419-18, 2018, https://doi.org/10.1128/mBio.01419-18) describedNeisseria gonorrhoeaeisolates that express low levels of azithromycin (Azi) resistance. Whole-genome sequencing and bioinformatic analysis suggested that the isolates had acquired DNA from commensalNeisseriaspp. that caused numerous nucleotide changes in themtrlocus, which contains genes for a transcriptional repressor (MtrR) and three proteins (MtrC-MtrD-MtrE) that form a multidrug efflux pump known to export macrolides. Strong regions of linkage disequilibrium mapped to the overlappingmtrRandmtrCDEpromoters andmtrD.Genetic analyses revealed that these mosaic-like sequences increased transcription ofmtrCDEand MtrD function, respectively. These changes also had strong epistatic effects that collectively were responsible for decreased susceptibility to MtrCDE substrates, including Azi. The report emphasizes the importance of gene exchange among neisserial species and development of antibiotic resistance in gonococci, both of which have ramifications for detection of resistance markers and efficacy of antibiotic treatment regimens for gonorrhea.

scholarly journals Molecular Cloning and Characterization of an ABC Multidrug Efflux Pump, VcaM, in Non-O1 Vibrio cholerae

2003 ◽  
Vol 47 (8) ◽  
pp. 2413-2417 ◽  
Author(s):  
Nazmul Huda ◽  
Eun-Woo Lee ◽  
Jing Chen ◽  
Yuji Morita ◽  
Teruo Kuroda ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Vibrio Cholerae ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Atp Binding ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Hoechst 33342 ◽  
Hydrophobic Domain ◽  
Multidrug Efflux Pumps

ABSTRACT A gene responsible for multidrug resistance was cloned from the chromosomal DNA of non-O1 Vibrio cholerae NCTC 4716 by using as a host drug-hypersensitive Escherichia coli strain KAM32, which lacks major multidrug efflux pumps. E. coli cells transformed with the gene showed elevated levels of resistance to a number of structurally dissimilar drugs, such as tetracycline, norfloxacin, ciprofloxacin, doxorubicin, daunomycin, 4′,6-diamidino-2-phenylindole, and Hoechst 33342. We determined the nucleotide sequence and found one open reading frame. We designated the gene vcaM. The deduced product, VcaM, seems to be a polypeptide with 619 amino acid residues (69 kDa) that has a putative topology of six transmembrane segments in the N-terminal hydrophobic domain, followed by an ATP binding domain in the C-terminal hydrophilic region. The sequence of VcaM was shown to be similar to those of human multidrug resistance proteins P-glycoprotein MDR1 and lactococcal LmrA, which are driven by ATP. The efflux of Hoechst 33342 and doxorubicin from cells possessing VcaM was detected. The efflux activity was inhibited by reserpine and sodium o-vanadate, which are potent inhibitors of MDR1 and LmrA. Thus, we conclude that VcaM is a member of the family of multidrug efflux pumps of the ATP binding cassette type and the first experimentally proven example of a multidrug efflux pump of this family in gram-negative bacteria.

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