scholarly journals Extreme Acid Modulates Fitness Tradeoffs of Multidrug Efflux Pumps MdtEF-TolC and AcrAB-TolC in Escherichia coli K-12

2021 ◽  
Author(s):  
Samantha H. Schaffner ◽  
Abigail V. Lee ◽  
Minh T.N. Pham ◽  
Beimnet B. Kassaye ◽  
Haofan Li ◽  
...  
Keyword(s):  
Bile Acids ◽  
Efflux Pump ◽  
Growth Cycle ◽  
Acid Exposure ◽  
Fitness Cost ◽  
Multidrug Efflux ◽  
Fitness Advantage ◽  
Multidrug Efflux Pumps ◽  
Stomach Acid ◽  
Fitness Tradeoffs

Bacterial genomes encode various multidrug efflux pumps (MDR) whose specific conditions for fitness advantage are unknown. We show that the efflux pump MdtEF-TolC, in Escherichia coli , confers a fitness advantage during exposure to extreme acid (pH 2). Our flow cytometry method revealed pH-dependent fitness tradeoffs between bile acids (a major pump substrate) and salicylic acid, a membrane-permeant aromatic acid that induces a drug-resistance regulon but depletes proton motive force (PMF). The PMF drives MdtEF-TolC and related pumps such as AcrAB-TolC. Deletion of mdtE (with loss of pump MdtEF-TolC) increased the strain’s relative fitness during growth with or without salicylate or bile acids. However, when the growth cycle included a 2-h incubation at pH 2 (below the pH growth range), MdtEF-TolC conferred a fitness advantage. The fitness advantage required bile salts but was decreased by the presence of salicylate, whose uptake is amplified by acid. For comparison, AcrAB-TolC, the primary efflux pump for bile acids, conferred a PMF-dependent fitness advantage with or without acid exposure in the growth cycle. A different MDR pump, EmrAB-TolC, confered no selective benefit during growth in the presence of bile acids. Without bile acids, all three MDR pumps incurred a large fitness cost with salicylate when exposed at pH 2. These results are consistent with the increased uptake of salicylate at low pH. Overall, we showed that MdtEF-TolC is an MDR pump adapted for transient extreme-acid exposure; and that low pH amplifies the salicylate-dependent fitness cost for drug pumps. IMPORTANCE Antibiotics and other drugs that reach the gut must pass through stomach acid. Yet little is known of how extreme acid modulates the effect of drugs on gut bacteria. We find that extreme-acid exposure leads to a fitness advantage for a multidrug pump that otherwise incurs a fitness cost. At the same time, extreme acid amplifies the effect of salicylate selection against multidrug pumps. Thus, organic acids and stomach acid could play important roles in regulating multidrug resistance in the gut microbiome. Our flow cytometry assay provides a way to measure the fitness effects of extreme-acid exposure to various membrane-soluble organic acids including plant-derived nutrients and pharmaceutical agents. Therapeutic acids might be devised to control the prevalence of multidrug pumps in environmental and host-associated habitats.

scholarly journals Salicylate, Bile Acids and Extreme Acid Cause Fitness Tradeoffs for Multidrug Pumps in Escherichia coli K-12

2020 ◽  
Author(s):  
Samantha H. Schaffner ◽  
Abigail V. Lee ◽  
Minh T. N. Pham ◽  
Beimnet B. Kassaye ◽  
Haofan Li ◽  
...  
Keyword(s):  
Organic Acids ◽  
Bile Acids ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Fitness Cost ◽  
E Coli ◽  
Fitness Advantage ◽  
Multidrug Efflux Pumps ◽  
Fitness Tradeoffs ◽  
K 12

ABSTRACTThe aspirin derivative salicylate selects against bacterial multidrug efflux pumps of Escherichia coli K-12 such as MdtEF-TolC and EmrAB-TolC, and acid stress regulators such as GadE. Salicylate uptake is driven by the transmembrane pH gradient (ΔpH) and the proton motive force (PMF) which drives many efflux pumps. We used flow cytometry to measure the fitness tradeoffs of salicylate, bile acids, and extreme low pH for E. coli cultured with pump deletants. The AcrAB-TolC efflux pump conferred a fitness advantage in the presence of bile acids, an efflux substrate. Without bile acids, AcrA incurred a small fitness cost. The fitness advantage with bile acids was eliminated by the PMF uncoupler CCCP. The Gad acid fitness island encodes components of MdtEF-TolC (an acid-adapted efflux pump) as well as acid regulator GadE. The fitness advantage of E. coli cocultured with a Gad deletant (Δslp-gadX) was lost in the presence of salicylate. Salicylate caused an even larger fitness cost for GadE. MdtE incurred negative or neutral fitness under all media conditions, as did EmrA. But when the competition cycle included two hours at pH 2, MdtE conferred a fitness advantage. The MdtE advantage required the presence of bile acids. Thus, the MdtEF-TolC pump is useful to E. coli for transient extreme acid exposure comparable to passage through the acidic stomach. Salicylate selects against some multidrug efflux pumps, whereas bile acids selects for them; and these fitness tradeoffs are amplified by extreme acid.IMPORTANCEControl of drug resistance in gut microbial communities is a compelling problem for human health. Growth of gut bacteria is limited by host-produced acids such as bile acids, and may be modulated by plant-derived acids such as salicylic acid. Membrane-soluble organic acids can control bacterial growth by disrupting membranes, decreasing cell pH, and depleting PMF. Our flow cytometry assay measures the fitness effects of exposure to membrane-soluble organic acids, with growth cycles that may include a period of extreme acid. We find that extreme-acid exposure leads to a fitness advantage for a multidrug pump, MdtEF-TolC, which otherwise incurs a large fitness cost. Thus, organic acids and stomach acid may play important roles in controlling multidrug resistance in the gut microbiome. Therapeutic acids might be developed to limit the prevalence of multidrug resistance pumps in environmental and host-associated communities.

scholarly journals Insight into the AcrAB-TolC Complex Assembly Process Learned from Competition Studies

Antibiotics ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 830
Author(s):  
Prasangi Rajapaksha ◽  
Isoiza Ojo ◽  
Ling Yang ◽  
Ankit Pandeya ◽  
Thilini Abeywansha ◽  
...  
Keyword(s):  
Efflux Pump ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Assembly Process ◽  
Multidrug Efflux ◽  
E Coli ◽  
Multidrug Efflux Pumps ◽  
Negative Effect ◽  
Dynamic Assembly ◽  
Pump Assembly

The RND family efflux pump AcrAB-TolC in E. coli and its homologs in other Gram-negative bacteria are major players in conferring multidrug resistance to the cells. While the structure of the pump complex has been elucidated with ever-increasing resolution through crystallography and Cryo-EM efforts, the dynamic assembly process remains poorly understood. Here, we tested the effect of overexpressing functionally defective pump components in wild type E. coli cells to probe the pump assembly process. Incorporation of a defective component is expected to reduce the efflux efficiency of the complex, leading to the so called “dominant negative” effect. Being one of the most intensively studied bacterial multidrug efflux pumps, many AcrA and AcrB mutations have been reported that disrupt efflux through different mechanisms. We examined five groups of AcrB and AcrA mutants, defective in different aspects of assembly and substrate efflux. We found that none of them demonstrated the expected dominant negative effect, even when expressed at concentrations many folds higher than their genomic counterpart. The assembly of the AcrAB-TolC complex appears to have a proof-read mechanism that effectively eliminated the formation of futile pump complex.

scholarly journals Magnitude of Voriconazole Resistance in Clinical and Environmental Isolates of Aspergillus flavus and Investigation into the Role of Multidrug Efflux Pumps

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Raees A. Paul ◽  
Shivaprakash M. Rudramurthy ◽  
Manpreet Dhaliwal ◽  
Pankaj Singh ◽  
Anup K. Ghosh ◽  
...  
Keyword(s):  
Aspergillus Flavus ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Azole Resistance ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Environmental Isolates ◽  
Content Type ◽  
Multidrug Efflux Pumps

ABSTRACT The magnitude of azole resistance in Aspergillus flavus and its underlying mechanism is obscure. We evaluated the frequency of azole resistance in a collection of clinical (n = 121) and environmental isolates (n = 68) of A. flavus by the broth microdilution method. Six (5%) clinical isolates displayed voriconazole MIC greater than the epidemiological cutoff value. Two of these isolates with non-wild-type MIC were isolated from same patient and were genetically distinct, which was confirmed by amplified fragment length polymorphism analysis. Mutations associated with azole resistance were not present in the lanosterol 14-α demethylase coding genes (cyp51A, cyp51B, and cyp51C). Basal and voriconazole-induced expression of cyp51A homologs and various efflux pump genes was analyzed in three each of non-wild-type and wild-type isolates. All of the efflux pump genes screened showed low basal expression irrespective of the azole susceptibility of the isolate. However, the non-wild-type isolates demonstrated heterogeneous overexpression of many efflux pumps and the target enzyme coding genes in response to induction with voriconazole (1 μg/ml). The most distinctive observation was approximately 8- to 9-fold voriconazole-induced overexpression of an ortholog of the Candida albicans ATP binding cassette (ABC) multidrug efflux transporter, Cdr1, in two non-wild-type isolates compared to those in the reference strain A. flavus ATCC 204304 and other wild-type strains. Although the dominant marker of azole resistance in A. flavus is still elusive, the current study proposes the possible role of multidrug efflux pumps, especially that of Cdr1B overexpression, in contributing azole resistance in A. flavus.

scholarly journals Control of Biofilm Formation in Healthcare: Recent Advances Exploiting Quorum-Sensing Interference Strategies and Multidrug Efflux Pump Inhibitors

Materials ◽  
2018 ◽  
Vol 11 (9) ◽  
pp. 1676 ◽  
Author(s):  
Bindu Subhadra ◽  
Dong Kim ◽  
Kyungho Woo ◽  
Surya Surendran ◽  
Chul Choi
Keyword(s):  
Quorum Sensing ◽  
Biofilm Formation ◽  
Efflux Pump ◽  
Financial Burden ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Persistent Infections ◽  
Healthcare Settings ◽  
Recent Advances ◽  
Multidrug Efflux Pumps

Biofilm formation in healthcare is an issue of considerable concern, as it results in increased morbidity and mortality, imposing a significant financial burden on the healthcare system. Biofilms are highly resistant to conventional antimicrobial therapies and lead to persistent infections. Hence, there is a high demand for novel strategies other than conventional antibiotic therapies to control biofilm-based infections. There are two approaches which have been employed so far to control biofilm formation in healthcare settings: one is the development of biofilm inhibitors based on the understanding of the molecular mechanism of biofilm formation, and the other is to modify the biomaterials which are used in medical devices to prevent biofilm formation. This review will focus on the recent advances in anti-biofilm approaches by interrupting the quorum-sensing cellular communication system and the multidrug efflux pumps which play an important role in biofilm formation. Research efforts directed towards these promising strategies could eventually lead to the development of better anti-biofilm therapies than the conventional treatments.

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 Dehydrozingerone Exhibits Synergistic Antifungal Activities in Combination with Dodecanol against Budding Yeast via the Restriction of Multidrug Resistance

2018 ◽  
Vol 5 (02) ◽  
pp. e61-e67
Author(s):  
Chika Yamawaki ◽  
Yoshihiro Yamaguchi ◽  
Akira Ogita ◽  
Toshio Tanaka ◽  
Ken-ichi Fujita
Keyword(s):  
Drug Resistance ◽  
Antifungal Activity ◽  
Efflux Pump ◽  
Fungal Infections ◽  
Zingiber Officinale ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Yeast Saccharomyces Cerevisiae ◽  
Multidrug Transporters ◽  
Multidrug Efflux Pumps

AbstractDrug resistance in fungal infections has been a more frequent occurrence with the increasing number of immunocompromised patients. In efforts to overcome the problem of fungal drug resistance, we focused on the phenolic compound dehydrozingerone, which is isolated from Zingiber officinale. The effectiveness of this compound on the model yeast Saccharomyces cerevisiae has not been reported. In our study, dehydrozingerone showed a weak antifungal activity against the yeast, but demonstrated a synergistic effect in combination with dodecanol, which typically only restricts cell growth transiently. Efflux of rhodamine 6G through the multidrug efflux pumps was significantly restricted by dehydrozingerone. The transcription level of PDR5, encoding a primary multidrug efflux pump in S. cerevisiae, was enhanced with dodecanol treatment, whereas the level was reduced by dehydrozingerone. These results suggest that dehydrozingerone may be effective for potentiating antifungal activity of other drugs that are expelled from fungi by multidrug transporters like Pdr5p.

scholarly journals The Varied Role of Efflux Pumps of the MFS Family in the Interplay of Bacteria with Animal and Plant Cells

2019 ◽  
Vol 7 (9) ◽  
pp. 285 ◽  
Author(s):  
Pasqua ◽  
Grossi ◽  
Zennaro ◽  
Fanelli ◽  
Micheli ◽  
...  
Keyword(s):  
Regulatory Networks ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Major Facilitator Superfamily ◽  
Host Cells ◽  
Multidrug Efflux ◽  
Animal Cells ◽  
Multidrug Efflux Pumps ◽  
Wide Range ◽  
Major Facilitator

Efflux pumps represent an important and large group of transporter proteins found in all organisms. The importance of efflux pumps resides in their ability to extrude a wide range of antibiotics, resulting in the emergence of multidrug resistance in many bacteria. Besides antibiotics, multidrug efflux pumps can also extrude a large variety of compounds: Bacterial metabolites, plant-produced compounds, quorum-sensing molecules, and virulence factors. This versatility makes efflux pumps relevant players in interactions not only with other bacteria, but also with plant or animal cells. The multidrug efflux pumps belonging to the major facilitator superfamily (MFS) are widely distributed in microbial genomes and exhibit a large spectrum of substrate specificities. Multidrug MFS efflux pumps are present either as single-component transporters or as tripartite complexes. In this review, we will summarize how the multidrug MFS efflux pumps contribute to the interplay between bacteria and targeted host cells, with emphasis on their role in bacterial virulence, in the colonization of plant and animal host cells and in biofilm formation. We will also address the complexity of these interactions in the light of the underlying regulatory networks required for the effective activation of efflux pump genes.

scholarly journals Inhibitors of Bacterial Multidrug Efflux Pumps Potentiate Antimicrobial Photoinactivation

2008 ◽  
Vol 52 (9) ◽  
pp. 3202-3209 ◽  
Author(s):  
George P. Tegos ◽  
Kayo Masago ◽  
Fatima Aziz ◽  
Andrew Higginbotham ◽  
Frank R. Stermitz ◽  
...  
Keyword(s):  
Efflux Pump ◽  
Red Light ◽  
Antimicrobial Effect ◽  
Efflux Pumps ◽  
Photodynamic Inactivation ◽  
Multidrug Efflux ◽  
Efflux Pump Inhibitor ◽  
Multidrug Efflux Pumps ◽  
Resistance Nodulation Division ◽  
Major Facilitator

ABSTRACT Antimicrobial photodynamic inactivation (APDI) combines a nontoxic photoactivatable dye or photosensitizer (PS) with harmless visible light to generate singlet oxygen and reactive oxygen species that kill microbial cells. Cationic phenothiazinium dyes, such as toluidine blue O (TBO), are the only PS used clinically for APDI, and we recently reported that this class of PS are substrates of multidrug efflux pumps in both gram-positive and gram-negative bacteria. We now report that APDI can be significantly potentiated by combining the PS with an efflux pump inhibitor (EPI). Killing of Staphylococcus aureus mediated by TBO and red light is greatly increased by coincubation with known inhibitors of the major facilitator pump (NorA): the diphenyl urea INF271, reserpine, 5′-methoxyhydnocarpin, and the polyacylated neohesperidoside, ADH7. The potentiation effect is greatest in the case of S. aureus mutants that overexpress NorA and least in NorA null cells. Addition of the EPI before TBO has a bigger effect than addition of the EPI after TBO. Cellular uptake of TBO is increased by EPI. EPI increased photodynamic inactivation killing mediated by other phenothiazinium dyes, such as methylene blue and dimethylmethylene blue, but not that mediated by nonphenothiazinium PS, such as Rose Bengal and benzoporphyrin derivative. Killing of Pseudomonas aeruginosa mediated by TBO and light was also potentiated by the resistance nodulation division pump (MexAB-OprM) inhibitor phenylalanine-arginine beta-naphthylamide but to a lesser extent than for S. aureus. These data suggest that EPI could be used in combination with phenothiazinium salts and light to enhance their antimicrobial effect against localized infections.

scholarly journals Three Efflux Pumps Are Required To Provide Efficient Tolerance to Toluene in Pseudomonas putidaDOT-T1E

2001 ◽  
Vol 183 (13) ◽  
pp. 3967-3973 ◽  
Author(s):  
Antonia Rojas ◽  
Estrella Duque ◽  
Gilberto Mosqueda ◽  
Geir Golden ◽  
Ana Hurtado ◽  
...  
Keyword(s):  
Gas Phase ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Site Directed Mutagenesis ◽  
Multidrug Efflux ◽  
Triple Mutant ◽  
Multidrug Efflux Pumps ◽  
Mutant Strains ◽  
Resistance Nodulation Division ◽  
Identification And Characterization

ABSTRACT In Pseudomonas putida DOT-T1E multidrug efflux pumps of the resistance-nodulation-division family make a major contribution to solvent resistance. Two pumps have been identified: TtgABC, expressed constitutively, and TtgDEF, induced by aromatic hydrocarbons. A double mutant lacking both efflux pumps was able to survive a sudden toluene shock if and only if preinduced with small amounts of toluene supplied via the gas phase. In this article we report the identification and characterization in this strain of a third efflux pump, named TtgGHI. The ttgGHI genes form an operon that is expressed constitutively at high levels from a single promoter. In the presence of toluene the operon is expressed at an even higher level from two promoters, the constitutive one and a previously unreported one that is inducible and that partially overlaps the constitutive promoter. By site-directed mutagenesis we constructed a single ttgHmutant which was shown to be unable to survive sudden 0.3% (vol/vol) toluene shocks regardless of the preculture conditions. The mutation was transferred to single and double mutants to construct mutant strains in which two or all three pumps are knocked out. Survival analysis of induced and noninduced cells revealed that the TtgABC and TtgGHI pumps extruded toluene, styrene, m-xylene, ethylbenzene, and propylbenzene, whereas the TtgDEF pump removed only toluene and styrene. The triple mutant was hypersensitive to toluene, as shown by its inability to grow with toluene supplied via the vapor phase.

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.

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