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 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 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 Structure and function of efflux pumps that confer resistance to drugs

2003 ◽  
Vol 376 (2) ◽  
pp. 313-338 ◽  
Author(s):  
M. Ines BORGES-WALMSLEY ◽  
Kenneth S. McKEEGAN ◽  
Adrian R. WALMSLEY
Keyword(s):  
Drug Resistance ◽  
Cancer Cells ◽  
Conformational Changes ◽  
Human Cancer ◽  
Efflux Pumps ◽  
Structure And Function ◽  
Drug Efflux ◽  
Diverse Range ◽  
Drug Efflux Pumps ◽  
And Function

Resistance to therapeutic drugs encompasses a diverse range of biological systems, which all have a human impact. From the relative simplicity of bacterial cells, fungi and protozoa to the complexity of human cancer cells, resistance has become problematic. Stated in its simplest terms, drug resistance decreases the chance of providing successful treatment against a plethora of diseases. Worryingly, it is a problem that is increasing, and consequently there is a pressing need to develop new and effective classes of drugs. This has provided a powerful stimulus in promoting research on drug resistance and, ultimately, it is hoped that this research will provide novel approaches that will allow the deliberate circumvention of well understood resistance mechanisms. A major mechanism of resistance in both microbes and cancer cells is the membrane protein-catalysed extrusion of drugs from the cell. Resistant cells exploit proton-driven antiporters and/or ATP-driven ABC (ATP-binding cassette) transporters to extrude cytotoxic drugs that usually enter the cell by passive diffusion. Although some of these drug efflux pumps transport specific substrates, many are transporters of multiple substrates. These multidrug pumps can often transport a variety of structurally unrelated hydrophobic compounds, ranging from dyes to lipids. If we are to nullify the effects of efflux-mediated drug resistance, we must first of all understand how these efflux pumps can accommodate a diverse range of compounds and, secondly, how conformational changes in these proteins are coupled to substrate translocation. These are key questions that must be addressed. In this review we report on the advances that have been made in understanding the structure and function of drug efflux pumps.

scholarly journals The Combined Treatment with Chemotherapeutic Agents and the Dualsteric Muscarinic Agonist Iper-8-Naphthalimide Affects Drug Resistance in Glioblastoma Stem Cells

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1877
Author(s):  
Claudia Guerriero ◽  
Carlo Matera ◽  
Donatella Del Bufalo ◽  
Marco De Amici ◽  
Luciano Conti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Drug Resistance ◽  
Efflux Pumps ◽  
Chemotherapeutic Agents ◽  
Drug Efflux ◽  
Low Doses ◽  
Conventional Chemotherapy ◽  
Glioblastoma Stem Cells ◽  
Chemotherapy Drugs ◽  
Drug Efflux Pumps

Background: Glioblastoma multiforme (GBM) is characterized by heterogeneous cell populations. Among these, the Glioblastoma Stem Cells (GSCs) fraction shares some similarities with Neural Stem Cells. GSCs exhibit enhanced resistance to conventional chemotherapy drugs. Our previous studies demonstrated that the activation of M2 muscarinic acetylcholine receptors (mAChRs) negatively modulates GSCs proliferation and survival. The aim of the present study was to analyze the ability of the M2 dualsteric agonist Iper-8-naphthalimide (N-8-Iper) to counteract GSCs drug resistance. Methods: Chemosensitivity to M2 dualsteric agonist N-8-Iper and chemotherapy drugs such as temozolomide, doxorubicin, or cisplatin was evaluated in vitro by MTT assay in two different GSC lines. Drug efflux pumps expression was evaluated by RT-PCR and qRT-PCR. Results: By using sub-toxic concentrations of N-8-Iper combined with the individual chemotherapeutic agents, we found that only low doses of the M2 agonist combined with doxorubicin or cisplatin or temozolomide were significantly able to counteract cell growth in both GSC lines. Moreover, we evaluated as the exposure to high and low doses of N-8-Iper downregulated the ATP-binding cassette (ABC) drug efflux pumps expression levels. Conclusions: Our results revealed the ability of the investigated M2 agonist to counteract drug resistance in two GSC lines, at least partially by downregulating the ABC drug efflux pumps expression. The combined effects of low doses of conventional chemotherapy and M2 agonists may thus represent a novel promising pharmacological approach to impair the GSC-drug resistance in the GBM therapy.

scholarly journals Verapamil Increases the Bioavailability and Efficacy of Bedaquiline but Not Clofazimine in a Murine Model of Tuberculosis

2017 ◽  
Vol 62 (1) ◽  
Author(s):  
Jian Xu ◽  
Rokeya Tasneen ◽  
Charles A. Peloquin ◽  
Deepak V. Almeida ◽  
Si-Yang Li ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Oral Bioavailability ◽  
Systemic Exposure ◽  
Efflux Pumps ◽  
Drug Efflux ◽  
Content Type ◽  
Acquired Drug Resistance ◽  
Drug Efflux Pumps

ABSTRACT Drug efflux pumps play important roles in intrinsic and acquired drug resistance. Verapamil, an efflux inhibitor that enhances the activity of bedaquiline, clofazimine, and other drugs against Mycobacterium tuberculosis, has been proposed as a potential adjunctive agent for treatment of tuberculosis (TB). However, the extent to which verapamil enhances in vivo efficacy by inhibiting bacterial efflux pumps versus inhibiting mammalian drug transporters to improve oral bioavailability has not been delineated. We found that verapamil potentiated the in vitro activity of bedaquiline and clofazimine against M. tuberculosis clinical isolates, including those harboring rv0678 mutations. Verapamil increased the efficacy of bedaquiline in a murine TB model by the same extent to which it increased systemic bedaquiline exposure. However, verapamil showed no effect on the oral bioavailability or efficacy of clofazimine in mice. The addition of verapamil increased the sterilizing activity of a regimen composed of bedaquiline, clofazimine, and pyrazinamide. These results confirm that verapamil has adjunctive activity in vivo, but they also demonstrate that the adjunctive effect is likely due to enhanced systemic exposure to companion drugs via effects on mammalian transporters, rather than inhibition of bacterial pumps. Therefore, there may be no advantage to administering verapamil versus increasing the doses of companion drugs.

The Role of NIR Fluorescence in MDR Cancer Treatment: From Targeted Imaging to Phototherapy

2020 ◽  
Vol 27 (33) ◽  
pp. 5510-5529
Author(s):  
Zengtao Wang ◽  
Qingqing Meng ◽  
Shaoshun Li
Keyword(s):  
Cancer Treatment ◽  
Near Infrared ◽  
Combined Therapy ◽  
Efflux Pumps ◽  
Cross Resistance ◽  
Great Promise ◽  
Drug Efflux ◽  
Nir Dyes ◽  
Nir Imaging ◽  
Drug Efflux Pumps

Background: Multidrug Resistance (MDR) is defined as a cross-resistance of cancer cells to various chemotherapeutics and has been demonstrated to correlate with drug efflux pumps. Visualization of drug efflux pumps is useful to pre-select patients who may be insensitive to chemotherapy, thus preventing patients from unnecessary treatment. Near-Infrared (NIR) imaging is an attractive approach to monitoring MDR due to its low tissue autofluorescence and deep tissue penetration. Molecular NIR imaging of MDR cancers requires stable probes targeting biomarkers with high specificity and affinity. Objective: This article aims to provide a concise review of novel NIR probes and their applications in MDR cancer treatment. Results: Recently, extensive research has been performed to develop novel NIR probes and several strategies display great promise. These strategies include chemical conjugation between NIR dyes and ligands targeting MDR-associated biomarkers, native NIR dyes with inherent targeting ability, activatable NIR probes as well as NIR dyes loaded nanoparticles. Moreover, NIR probes have been widely employed for photothermal and photodynamic therapy in cancer treatment, which combine with other modalities to overcome MDR. With the rapid advancing of nanotechnology, various nanoparticles are incorporated with NIR dyes to provide multifunctional platforms for controlled drug delivery and combined therapy to combat MDR. The construction of these probes for MDR cancers targeted NIR imaging and phototherapy will be discussed. Multimodal nanoscale platform which integrates MDR monitoring and combined therapy will also be encompassed. Conclusion: We believe these NIR probes project a promising approach for diagnosis and therapy of MDR cancers, thus holding great potential to reach clinical settings in cancer treatment.

scholarly journals Interdependence of multi-drug efflux pumps and quorum sensing systems in Pseudomonas aeruginosa

2014 ◽  
Vol 21 ◽  
pp. 92
Author(s):  
K. Ganguly ◽  
J.L. Phillips ◽  
M.S. Wren ◽  
P.E. Pardington ◽  
S. Gnanakaran ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Efflux Pumps ◽  
Drug Efflux ◽  
Sensing Systems ◽  
Drug Efflux Pumps
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