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Sample records for active efflux pump

  1. Active efflux of fluoroquinolones in Mycobacterium smegmatis mediated by LfrA, a multidrug efflux pump.

    PubMed Central

    Liu, J; Takiff, H E; Nikaido, H

    1996-01-01

    The lfrA gene cloned from chromosomal DNA of quinolone-resistant Mycobacterium smegmatis mc2-552 conferred low-level resistance to fluoroquinolones when present on multicopy plasmids. Sequence analysis suggested that lfrA encodes a membrane efflux pump of the major facilitator family (H. E. Takiff, M. Cimino, M. C. Musso, T. Weisbrod, R. Martinez, M. B. Delgado, L Salazar, B. R. Bloom, and W. R. Jacbos, Jr., Proc. Natl. Acad. Sci. USA 93:362-366, 1996). In this work, we studied the role of LfrA in the accumulation of fluoroquinolones by M. smegmatis. The steady-state accumulation level of a hydrophilic quinolone, norfloxacin, by M. smegmatis harboring a plasmid carrying the lfrA gene was about 50% of that by the parent strain but was increased to the same level as that of the parent strain by addition of a proton conductor, carbonyl cyanide m-chorophenylhydrazone. Norfloxacin efflux mediated by LfrA was competed for strongly by ciprofloxacin but not by nalidixic acid. Furthermore, we showed that portions of norfloxacin accumulated by starved cells were pumped out upon reenergization of the cells, and the rates of this efflux showed evidence of saturation at higher intracellular concentrations of the drug. These results suggest that the LfrA polypeptide catalyzes the active efflux of several quinolones. PMID:8682782

  2. Ethidium Bromide MIC Screening for Enhanced Efflux Pump Gene Expression or Efflux Activity in Staphylococcus aureus▿

    PubMed Central

    Patel, Diixa; Kosmidis, Christos; Seo, Susan M.; Kaatz, Glenn W.

    2010-01-01

    Multidrug resistance efflux pumps contribute to antimicrobial and biocide resistance in Staphylococcus aureus. The detection of strains capable of efflux is time-consuming and labor-intensive using currently available techniques. A simple and inexpensive method to identify such strains is needed. Ethidium bromide is a substrate for all but one of the characterized S. aureus multidrug-resistant (MDR) efflux pumps (NorC), leading us to examine the utility of simple broth microtiter MIC determinations using this compound in identifying efflux-proficient strains. Quantitative reverse transcription-PCR identified the increased expression of one or more MDR efflux pump genes in 151/309 clinical strains (49%). Ethidium bromide MIC testing was insensitive (48%) but specific (92%) in identifying strains with gene overexpression, but it was highly sensitive (95%) and specific (99%) in identifying strains capable of ethidium efflux. The increased expression of norA with or without other genes was most commonly associated with efflux, and in the majority of cases that efflux was inhibited by reserpine. Ethidium bromide MIC testing is a simple and straightforward method to identify effluxing strains and can provide accurate predictions of efflux prevalence in large strain sets in a short period of time. PMID:20855743

  3. Goldenseal (Hydrastis canadensis L.) extracts synergistically enhance the antibacterial activity of berberine via efflux pump inhibition.

    PubMed

    Ettefagh, Keivan A; Burns, Johnna T; Junio, Hiyas A; Kaatz, Glenn W; Cech, Nadja B

    2011-05-01

    Goldenseal (Hydrastis canadensis L.) is used to combat inflammation and infection. Its antibacterial activity in vitRO has been attributed to its alkaloids, the most abundant of which is berberine. The goal of these studies was to compare the composition, antibacterial activity, and efflux pump inhibitory activity of ethanolic extracts prepared from roots and aerial portions of H. canadensis. Ethanolic extracts were prepared separately from roots and aerial portions of six H. canadensis plants. Extracts were analyzed for alkaloid concentration using LC-MS and tested for antimicrobial activity against Staphylococcus aureus (NCTC 8325-4) and for inhibition of ethidium bromide efflux. Synergistic antibacterial activity was observed between the aerial extract (FIC 0.375) and to a lesser extent the root extract (FIC 0.750) and berberine. The aerial extract inhibited ethidium bromide efflux from wild-type S. aureus but had no effect on the expulsion of this compound from an isogenic derivative deleted for norA. Our studies indicate that the roots of H. canadensis contain higher levels of alkaloids than the aerial portions, but the aerial portions synergize with berberine more significantly than the roots. Furthermore, extracts from the aerial portions of H. canadensis contain efflux pump inhibitors, while efflux pump inhibitory activity was not observed for the root extract. The three most abundant H. canadensis alkaloids, berberine, hydrastine, and canadine, are not responsible for the efflux pump inhibitory activity of the extracts from H. canadensis aerial portions. PMID:21157683

  4. NorA efflux pump inhibitory activity of coumarins from Mesua ferrea.

    PubMed

    Roy, Somendu K; Kumari, Neela; Pahwa, Sonika; Agrahari, Udai C; Bhutani, Kamlesh K; Jachak, Sanjay M; Nandanwar, Hemraj

    2013-10-01

    The purpose of this investigation was to study the modulator and efflux pump inhibitor activity of coumarins isolated from Mesua ferrea against clinical strains as well as NorA-over expressed strain of Staphylococcus aureus 1199B. Seven coumarins were tested for modulator activity using ethidium bromide (EtBr) as a substrate. Compounds 1, 4-7 modulated the MIC of EtBr by ≥ 2 fold against wild type clinical strains of S. aureus 1199 and S. aureus 1199B, whereas compounds 4-7 modulated the MIC of EtBr by ≥ 16 fold against MRSA 831. Compounds 1, 4-7 also reduced the MIC of norfloxacin by ≥ 8 fold against S. aureus 1199B, and 4-6 reduced the MIC of norfloxacin by ≥ 8 fold against MRSA 831 at half of their MICs. Inhibition of EtBr efflux by NorA-overproducing S. aureus 1199B and MRSA 831 confirmed the role of compounds 4-6 as NorA efflux pump inhibitors (EPI). Dose-dependent activity at sub-inhibitory concentration (6.25 μg/mL) suggested that compounds 4 and 5 are promising EPI compared to verapamil against 1199B and MRSA 831 strains. PMID:23892000

  5. Efflux Pump Blockers in Gram-Negative Bacteria: The New Generation of Hydantoin Based-Modulators to Improve Antibiotic Activity

    PubMed Central

    Otręebska-Machaj, Ewa; Chevalier, Jacqueline; Handzlik, Jadwiga; Szymańska, Ewa; Schabikowski, Jakub; Boyer, Gérard; Bolla, Jean-Michel; Kieć-Kononowicz, Katarzyna; Pagès, Jean-Marie; Alibert, Sandrine

    2016-01-01

    Multidrug resistant (MDR) bacteria are an increasing health problem with the shortage of new active antibiotic agents. Among effective mechanisms that contribute to the spread of MDR Gram-negative bacteria are drug efflux pumps that expel clinically important antibiotic classes out of the cell. Drug pumps are attractive targets to restore the susceptibility toward the expelled antibiotics by impairing their efflux activity. Arylhydantoin derivatives were investigated for their potentiation of activities of selected antibiotics described as efflux substrates in Enterobacter aerogenes expressing or not AcrAB pump. Several compounds increased the bacterial susceptibility toward nalidixic acid, chloramphenicol and sparfloxacin and were further pharmacomodulated to obtain a better activity against the AcrAB producing bacteria. PMID:27199950

  6. Biochemistry of Bacterial Multidrug Efflux Pumps

    PubMed Central

    Kumar, Sanath; Varela, Manuel F.

    2012-01-01

    Bacterial pathogens that are multi-drug resistant compromise the effectiveness of treatment when they are the causative agents of infectious disease. These multi-drug resistance mechanisms allow bacteria to survive in the presence of clinically useful antimicrobial agents, thus reducing the efficacy of chemotherapy towards infectious disease. Importantly, active multi-drug efflux is a major mechanism for bacterial pathogen drug resistance. Therefore, because of their overwhelming presence in bacterial pathogens, these active multi-drug efflux mechanisms remain a major area of intense study, so that ultimately measures may be discovered to inhibit these active multi-drug efflux pumps. PMID:22605991

  7. Identification of a multidrug efflux pump in Mycobacterium smegmatis.

    PubMed

    Bansal, Ankita; Mallik, Dhriti; Kar, Debasish; Ghosh, Anindya S

    2016-07-01

    Cell wall impermeability and active efflux of drugs are among the primary reasons for drug resistance in mycobacteria. Efflux pumps are tripartite membrane localized transport proteins that expel drug molecules outside the cells. Several of such efflux pumps are annotated in mycobacteria, but few have been characterized, like MSMEG_2991, a putative efflux pump permease of Mycobacterium smegmatis To substantiate this, we overexpressed MSMEG_2991 protein in Escherichia coli 2443. Expression of MSMEG_2991 elevated the resistance towards structurally unrelated groups of antibiotics. An active antibiotic efflux pump nature of MSMEG_2991 was revealed by assessing the acquisition of ciprofloxacin in the absence and presence of the efflux pump inhibitor, carbonyl cyanide m-chlorophenyl hydrazone, indicating the involvement of proton-motive force (pmf) during the efflux activity. MSMEG_2991 expression elevated biofilm formation in E. coli by 4-fold, keeping parity to some of the earlier reported efflux pumps. In silico analysis suggested the presence of 12 transmembrane helices in MSMEG_2991 resembling EmrD efflux pump of E. coli Based on in vivo and in silico analyses, MSMEG_2991 may be designated as a pmf-mediated multidrug efflux pump protein that expels diverse groups of antibiotics and might as well be involved in the biofilm enhancement. PMID:27190152

  8. Natural and Synthetic Polymers as Inhibitors of Drug Efflux Pumps

    PubMed Central

    2007-01-01

    Inhibition of efflux pumps is an emerging approach in cancer therapy and drug delivery. Since it has been discovered that polymeric pharmaceutical excipients such as Tweens® or Pluronics® can inhibit efflux pumps, various other polymers have been investigated regarding their potential efflux pump inhibitory activity. Among them are polysaccharides, polyethylene glycols and derivatives, amphiphilic block copolymers, dendrimers and thiolated polymers. In the current review article, natural and synthetic polymers that are capable of inhibiting efflux pumps as well as their application in cancer therapy and drug delivery are discussed. PMID:17896100

  9. How to Measure Export via Bacterial Multidrug Resistance Efflux Pumps.

    PubMed

    Blair, Jessica M A; Piddock, Laura J V

    2016-01-01

    Bacterial multidrug resistance (MDR) efflux pumps are an important mechanism of antibiotic resistance and are required for many pathogens to cause infection. They are also being harnessed to improve microbial biotechnological processes, including biofuel production. Therefore, scientists of many specialties must be able to accurately measure efflux activity. However, myriad methodologies have been described and the most appropriate method is not always clear. Within the scientific literature, many methods are misused or data arising are misinterpreted. The methods for measuring efflux activity can be split into two groups, (i) those that directly measure efflux and (ii) those that measure the intracellular accumulation of a substrate, which is then used to infer efflux activity. Here, we review the methods for measuring efflux and explore the most recent advances in this field, including single-cell or cell-free technologies and mass spectrometry, that are being used to provide more detailed information about efflux pump activity. PMID:27381291

  10. How to Measure Export via Bacterial Multidrug Resistance Efflux Pumps

    PubMed Central

    Blair, Jessica M. A.

    2016-01-01

    ABSTRACT Bacterial multidrug resistance (MDR) efflux pumps are an important mechanism of antibiotic resistance and are required for many pathogens to cause infection. They are also being harnessed to improve microbial biotechnological processes, including biofuel production. Therefore, scientists of many specialties must be able to accurately measure efflux activity. However, myriad methodologies have been described and the most appropriate method is not always clear. Within the scientific literature, many methods are misused or data arising are misinterpreted. The methods for measuring efflux activity can be split into two groups, (i) those that directly measure efflux and (ii) those that measure the intracellular accumulation of a substrate, which is then used to infer efflux activity. Here, we review the methods for measuring efflux and explore the most recent advances in this field, including single-cell or cell-free technologies and mass spectrometry, that are being used to provide more detailed information about efflux pump activity. PMID:27381291

  11. Antimicrobial and Efflux Pump Inhibitory Activity of Caffeoylquinic Acids from Artemisia absinthium against Gram-Positive Pathogenic Bacteria

    PubMed Central

    Fiamegos, Yiannis C.; Kastritis, Panagiotis L.; Exarchou, Vassiliki; Han, Haley; Bonvin, Alexandre M. J. J.; Vervoort, Jacques; Lewis, Kim; Hamblin, Michael R.; Tegos, George P.

    2011-01-01

    Background Traditional antibiotics are increasingly suffering from the emergence of multidrug resistance amongst pathogenic bacteria leading to a range of novel approaches to control microbial infections being investigated as potential alternative treatments. One plausible antimicrobial alternative could be the combination of conventional antimicrobial agents/antibiotics with small molecules which block multidrug efflux systems known as efflux pump inhibitors. Bioassay-driven purification and structural determination of compounds from plant sources have yielded a number of pump inhibitors which acted against gram positive bacteria. Methodology/Principal Findings In this study we report the identification and characterization of 4′,5′-O-dicaffeoylquinic acid (4′,5′-ODCQA) from Artemisia absinthium as a pump inhibitor with a potential of targeting efflux systems in a wide panel of Gram-positive human pathogenic bacteria. Separation and identification of phenolic compounds (chlorogenic acid, 3′,5′-ODCQA, 4′,5′-ODCQA) was based on hyphenated chromatographic techniques such as liquid chromatography with post column solid-phase extraction coupled with nuclear magnetic resonance spectroscopy and mass spectroscopy. Microbial susceptibility testing and potentiation of well know pump substrates revealed at least two active compounds; chlorogenic acid with weak antimicrobial activity and 4′,5′-ODCQA with pump inhibitory activity whereas 3′,5′-ODCQA was ineffective. These intitial findings were further validated with checkerboard, berberine accumulation efflux assays using efflux-related phenotypes and clinical isolates as well as molecular modeling methodology. Conclusions/Significance These techniques facilitated the direct analysis of the active components from plant extracts, as well as dramatically reduced the time needed to analyze the compounds, without the need for prior isolation. The calculated energetics of the docking poses supported the

  12. In vitro transport activity of the fully assembled MexAB-OprM efflux pump from Pseudomonas aeruginosa

    NASA Astrophysics Data System (ADS)

    Verchère, Alice; Dezi, Manuela; Adrien, Vladimir; Broutin, Isabelle; Picard, Martin

    2015-04-01

    Antibiotic resistance is a major public health issue and many bacteria responsible for human infections have now developed a variety of antibiotic resistance mechanisms. For instance, Pseudomonas aeruginosa, a disease-causing Gram-negative bacteria, is now resistant to almost every class of antibiotics. Much of this resistance is attributable to multidrug efflux pumps, which are tripartite membrane protein complexes that span both membranes and actively expel antibiotics. Here we report an in vitro procedure to monitor transport by the tripartite MexAB-OprM pump. By combining proteoliposomes containing the MexAB and OprM portions of the complex, we are able to assay energy-dependent substrate translocation in a system that mimics the dual-membrane architecture of Gram-negative bacteria. This assay facilitates the study of pump transport dynamics and could be used to screen pump inhibitors with potential clinical use in restoring therapeutic activity of old antibiotics.

  13. Structure-Activity Relationships of a Novel Pyranopyridine Series of Gram-negative Bacterial Efflux Pump Inhibitors

    PubMed Central

    Nguyen, Son T.; Kwasny, Steven M.; Ding, Xiaoyuan; Cardinale, Steven C.; McCarthy, Courtney T.; Kim, Hong-Suk; Nikaido, Hiroshi; Peet, Norton P.; Williams, John D.; Bowlin, Terry L.; Opperman, Timothy J.

    2015-01-01

    Recently we described a novel pyranopyridine inhibitor (MBX2319) of RND-type efflux pumps of the Enterobacteriaceae. MBX2319 (3,3-dimethyl-5-cyano-8-morpholino-6-(phenethylthio)-3,4-dihydro-1H-pyrano[3,4-c]pyridine) is structurally distinct from other known Gram-negative efflux pump inhibitors (EPIs), such as 1-(1-naphthylmethyl)-piperazine (NMP), phenylalanylarginine-β-naphthylamide (PAβN), D13-9001, and the pyridopyrimidine derivatives. Here, we report the synthesis and biological evaluation of 60 new analogs of MBX2319 that were designed to probe the structure activity relationships (SARs) of the pyranopyridine scaffold. The results of these studies produced a molecular activity map of the scaffold, which identifies regions that are critical to efflux inhibitory activities and those that can be modified to improve potency, metabolic stability and solubility. Several compounds, such as 22d–f, 22i and 22k, are significantly more effective than MBX2319 at potentiating the antibacterial activity of levofloxacin and piperacillin against Escherichia coli. PMID:25818767

  14. The "racemic approach" in the evaluation of the enantiomeric NorA efflux pump inhibition activity of 2-phenylquinoline derivatives.

    PubMed

    Carotti, Andrea; Ianni, Federica; Sabatini, Stefano; Di Michele, Alessandro; Sardella, Roccaldo; Kaatz, Glenn W; Lindner, Wolfgang; Cecchetti, Violetta; Natalini, Benedetto

    2016-09-10

    Among the mechanisms adopted by bacteria, efflux pumps (EPs) have been recognized as being significantly involved in contributing to resistance to commonly used antibacterial agents. However, little is known about their three-dimensional structures or the steric requirements for their inhibition. Lack of such knowledge includes NorA, one of the most studied Staphylococcus aureus EPs. In the present study, the use of two commercialized Cinchona alkaloid-based zwitterionic chiral stationary phases allowed the enantioseparation of four 2-((2-(4-propoxyphenyl)quinolin-4-yl)oxy)alkylamines 1-4 previously found to be potent S. aureus NorA efflux pump inhibitors when tested as racemates. In the identified optimal polar-ionic conditions (MeOH/THF/H2O-49/49/2 (v/v/v)+25mM formic acid, 12.5mM diethylamine), repeated consecutive injections of 1 allowed the isolation of sufficient amount of its enantiomers (2.6mg and 2.8mg, for (R)-1 and (S)-1, respectively) and then to evaluate their ability to inhibit the S. aureus NorA efflux pump. The biological evaluation highlighted the main contribution of the (R)-1 enantiomer to both the EtBr efflux inhibition and synergistic effect with against SA-1199B (norA+/A116E GrlA) respect to the racemate activity. The comparison between the experimental electronic circular dichroism and the time-dependent density functional theory calculations spectra of the two isolated enantiomeric fractions allowed for all compounds a clear and easy assignment of the enantiomeric elution order. PMID:27429367

  15. Efflux pump inhibitors: targeting mycobacterial efflux systems to enhance TB therapy.

    PubMed

    Pule, Caroline M; Sampson, Samantha L; Warren, Robin M; Black, Philippa A; van Helden, Paul D; Victor, Tommie C; Louw, Gail E

    2016-01-01

    The emergence of drug resistance continues to plague TB control, with a global increase in the prevalence of MDR-TB. This acts as a gateway to XDR-TB and thus emphasizes the urgency for drug development and optimal treatment options. Bedaquiline is the first new anti-TB drug approved by the FDA in 40 years and has been shown to be an effective treatment option for MDR Mycobacterium tuberculosis infection. Bedaquiline has also recently been included in clinical trials for new regimens with the aim of improving and shortening treatment periods. Alarmingly, efflux-mediated bedaquiline resistance, as well as efflux-mediated cross-resistance to clofazimine, has been identified in treatment failures. This mechanism of resistance results in efflux of a variety of anti-TB drugs from the bacterial cell, thereby decreasing the intracellular drug concentration. In doing so, the bacillus is able to render the antibiotic treatment ineffective. Recent studies have explored strategies to reverse the resistance phenotype conferred by efflux pump activation. It was observed that the addition of efflux pump inhibitors partially restored drug susceptibility in vitro and in vivo. This has significant clinical implications, especially in MDR-TB management where treatment options are extremely limited. This review aims to highlight the current efflux pump inhibitors effective against M. tuberculosis, the effect of efflux pump inhibitors on mycobacterial growth and the clinical promise of treatment with efflux pump inhibitors and standard anti-TB therapy. PMID:26472768

  16. First evidence for the presence of efflux pump in the earthworm Eisenia andrei.

    PubMed

    Hackenberger, Branimir K; Velki, Mirna; Stepić, Sandra; Hackenberger, Davorka K

    2012-01-01

    Efflux pumps are transport proteins involved in the extrusion of toxic substrates from cells to the external environment. Activities of efflux pumps have been found in many organisms, however such activity has not been evidenced in earthworms. Adult Eisenia andrei earthworms were exposed to efflux modulators - verapamil (a known inhibitor of efflux pump protein) and dexamethasone (a known inducer of efflux activity) - and the amount of absorbed fluorescent dye rhodamine B was measured. The results showed that verapamil inhibited efflux activity and decreased removal of rhodamine B, whereas dexamethasone induced efflux activity and increased removal of rhodamine B. This is the first evidence of the presence of efflux pump in earthworm Eisenia andrei. Since earthworms are often used as test organisms due to their sensitive reactions towards environmental influences, the discovery of efflux pump activity can contribute to the better understanding of toxicity of certain pollutants. PMID:22033226

  17. The Influence of Efflux Pump Inhibitors on the Activity of Non-Antibiotic NSAIDS against Gram-Negative Rods

    PubMed Central

    Laudy, Agnieszka E.; Mrowka, Agnieszka; Krajewska, Joanna; Tyski, Stefan

    2016-01-01

    Background Most patients with bacterial infections suffer from fever and various pains that require complex treatments with antibiotics, antipyretics, and analgaesics. The most common drugs used to relieve these symptoms are non-steroidal anti-inflammatory drugs (NSAIDs), which are not typically considered antibiotics. Here, we investigate the effects of NSAIDs on bacterial susceptibility to antibiotics and the modulation of bacterial efflux pumps. Methodology The activity of 12 NSAID active substances, paracetamol (acetaminophen), and eight relevant medicinal products was analyzed with or without pump inhibitors against 89 strains of Gram-negative rods by determining the MICs. Furthermore, the effects of NSAIDs on the susceptibility of clinical strains to antimicrobial agents with or without PAβN (Phe-Arg-β-naphtylamide) were measured. Results The MICs of diclofenac, mefenamic acid, ibuprofen, and naproxen, in the presence of PAβN, were significantly (≥4-fold) reduced, decreasing to 25–1600 mg/L, against the majority of the studied strains. In the case of acetylsalicylic acid only for 5 and 7 out of 12 strains of P. mirabilis and E. coli, respectively, a 4-fold increase in susceptibility in the presence of PAβN was observed. The presence of Aspirin resulted in a 4-fold increase in the MIC of ofloxacin against only two strains of E. coli among 48 tested clinical strains, which included species such as E. coli, K. pneumoniae, P. aeruginosa, and S. maltophilia. Besides, the medicinal products containing the following NSAIDs, diclofenac, mefenamic acid, ibuprofen, and naproxen, did not cause the decrease of clinical strains’ susceptibility to antibiotics. Conclusions The effects of PAβN on the susceptibility of bacteria to NSAIDs indicate that some NSAIDs are substrates for efflux pumps in Gram-negative rods. Morever, Aspirin probably induced efflux-mediated resistance to fluoroquinolones in a few E. coli strains. PMID:26771525

  18. Multidrug Efflux Pumps in the Genus Erwinia: Physiology and Regulation of Efflux Pump Gene Expression.

    PubMed

    Thekkiniath, J; Ravirala, R; San Francisco, M

    2016-01-01

    Plant pathogens belonging to the genus Erwinia cause diseases in several economically important plants. Plants respond to bacterial infection with a powerful chemical arsenal and signaling molecules to rid themselves of the microbes. Although our understanding of how Erwinia initiate infections in plants has become clear, a comprehensive understanding of how these bacteria rid themselves of noxious antimicrobial agents during the infection is important. Multidrug efflux pumps are key factors in bacterial resistance toward antibiotics by reducing the level of antimicrobial compounds in the bacterial cell. Erwinia induce the expression of efflux pump genes in response to plant-derived antimicrobials. The capability of Erwinia to co-opt plant defense signaling molecules such as salicylic acid to trigger multidrug efflux pumps might have developed to ensure bacterial survival in susceptible host plants. In this review, we discuss the developments in Erwinia efflux pumps, focusing in particular on efflux pump function and the regulation of efflux pump gene expression. PMID:27571694

  19. Efflux pump activity in fluoroquinolone and tetracycline resistant Salmonella and E. coli implicated in reduced susceptibility to household antimicrobial cleaning agents.

    PubMed

    Thorrold, C A; Letsoalo, M E; Dusé, A G; Marais, E

    2007-02-15

    It has been shown that the inappropriate use of antimicrobial household agents selects for organisms with resistance mechanisms (e.g. efflux pumps), which could lead to the development of antibiotic resistance. The reverse hypothesis, that antibiotic-resistant organisms become tolerant to other antibacterial agents (e.g. disinfectants) due to the action of efflux pumps, has however not been extensively examined. The objective of this study was to establish whether there is a link between antibiotic resistance in potential gastrointestinal pathogens and reduced sensitivity of these organisms to commonly used household antimicrobial agents. In this study, tetracycline and ofloxacin sensitive and resistant Escherichia coli (9 strains) and Salmonella spp. (8 strains) were isolated from poultry and clinical samples. In order to assess whether these bacteria had active efflux pumps, ethidium bromide accumulation assays were performed. Extrusion of the active components of three commercial household agents (triclosan, sodium salicylate, and ortho-phenylphenol) by efflux pumps was tested using spectrophotometric accumulation assays. In order to simulate the kitchen environment, in-use disinfectant testing using the commercial household agents was performed to determine changes in their efficacy due to antibiotic resistance. Active efflux pump activity and extrusion of all three active ingredients was observed only in the antibiotic resistant organisms. The antibiotic sensitive bacteria were also more susceptible than the resistant isolates to the household antimicrobial agents at concentrations below that recommended by the manufacturer. These resistant bacteria could potentially be selected for and result in hard to treat infections. PMID:17126442

  20. Antibiotic-potentiation activities of four Cameroonian dietary plants against multidrug-resistant Gram-negative bacteria expressing efflux pumps

    PubMed Central

    2014-01-01

    Background The continuous spread of multidrug-resistant (MDR) bacteria, partially due to efflux pumps drastically reduced the efficacy of the antibiotic armory, increasing the frequency of therapeutic failure. The search for new compounds to potentiate the efficacy of commonly used antibiotics is therefore important. The present study was designed to evaluate the ability of the methanol extracts of four Cameroonian dietary plants (Capsicum frutescens L. var. facilulatum, Brassica oleacera L. var. italica, Brassica oleacera L. var. butyris and Basilicum polystachyon (L.) Moench.) to improve the activity of commonly used antibiotics against MDR Gram-negative bacteria expressing active efflux pumps. Methods The qualitative phytochemical screening of the plant extracts was performed using standard methods whilst the antibacterial activity was performed by broth micro-dilution method. Results All the studied plant extracts revealed the presence of alkaloids, phenols, flavonoids, triterpenes and sterols. The minimal inhibitory concentrations (MIC) of the studied extracts ranged from 256-1024 μg/mL. Capsicum frutescens var. facilulatum extract displayed the largest spectrum of activity (73%) against the tested bacterial strains whilst the lower MIC value (256 μg/mL) was recorded with Basilicum polystachyon against E. aerogenes ATCC 13048 and P. stuartii ATCC 29916. In the presence of PAβN, the spectrum of activity of Brassica oleacera var. italica extract against bacteria strains increased (75%). The extracts from Brassica oleacera var. butyris, Brassica oleacera var. italica, Capsicum frutescens var. facilulatum and Basilicum polystachyon showed synergistic effects (FIC ≤ 0.5) against the studied bacteria, with an average of 75.3% of the tested antibiotics. Conclusion These results provide promising information for the potential use of the tested plants alone or in combination with some commonly used antibiotics in the fight against MDR Gram-negative bacteria

  1. A mass spectrometry-based assay for improved quantitative measurements of efflux pump inhibition.

    PubMed

    Brown, Adam R; Ettefagh, Keivan A; Todd, Daniel; Cole, Patrick S; Egan, Joseph M; Foil, Daniel H; Graf, Tyler N; Schindler, Bryan D; Kaatz, Glenn W; Cech, Nadja B

    2015-01-01

    Bacterial efflux pumps are active transport proteins responsible for resistance to selected biocides and antibiotics. It has been shown that production of efflux pumps is up-regulated in a number of highly pathogenic bacteria, including methicillin resistant Staphylococcus aureus. Thus, the identification of new bacterial efflux pump inhibitors is a topic of great interest. Existing assays to evaluate efflux pump inhibitory activity rely on fluorescence by an efflux pump substrate. When employing these assays to evaluate efflux pump inhibitory activity of plant extracts and some purified compounds, we observed severe optical interference that gave rise to false negative results. To circumvent this problem, a new mass spectrometry-based method was developed for the quantitative measurement of bacterial efflux pump inhibition. The assay was employed to evaluate efflux pump inhibitory activity of a crude extract of the botanical Hydrastis Canadensis, and to compare the efflux pump inhibitory activity of several pure flavonoids. The flavonoid quercetin, which appeared to be completely inactive with a fluorescence-based method, showed an IC50 value of 75 μg/mL with the new method. The other flavonoids evaluated (apigenin, kaempferol, rhamnetin, luteolin, myricetin), were also active, with IC50 values ranging from 19 μg/mL to 75 μg/mL. The assay described herein could be useful in future screening efforts to identify efflux pump inhibitors, particularly in situations where optical interference precludes the application of methods that rely on fluorescence. PMID:25961825

  2. A Mass Spectrometry-Based Assay for Improved Quantitative Measurements of Efflux Pump Inhibition

    PubMed Central

    Brown, Adam R.; Ettefagh, Keivan A.; Todd, Daniel; Cole, Patrick S.; Egan, Joseph M.; Foil, Daniel H.; Graf, Tyler N.; Schindler, Bryan D.; Kaatz, Glenn W.; Cech, Nadja B.

    2015-01-01

    Bacterial efflux pumps are active transport proteins responsible for resistance to selected biocides and antibiotics. It has been shown that production of efflux pumps is up-regulated in a number of highly pathogenic bacteria, including methicillin resistant Staphylococcus aureus. Thus, the identification of new bacterial efflux pump inhibitors is a topic of great interest. Existing assays to evaluate efflux pump inhibitory activity rely on fluorescence by an efflux pump substrate. When employing these assays to evaluate efflux pump inhibitory activity of plant extracts and some purified compounds, we observed severe optical interference that gave rise to false negative results. To circumvent this problem, a new mass spectrometry-based method was developed for the quantitative measurement of bacterial efflux pump inhibition. The assay was employed to evaluate efflux pump inhibitory activity of a crude extract of the botanical Hydrastis Canadensis, and to compare the efflux pump inhibitory activity of several pure flavonoids. The flavonoid quercetin, which appeared to be completely inactive with a fluorescence-based method, showed an IC50 value of 75 μg/mL with the new method. The other flavonoids evaluated (apigenin, kaempferol, rhamnetin, luteolin, myricetin), were also active, with IC50 values ranging from 19 μg/mL to 75 μg/mL. The assay described herein could be useful in future screening efforts to identify efflux pump inhibitors, particularly in situations where optical interference precludes the application of methods that rely on fluorescence. PMID:25961825

  3. Recent advances toward a molecular mechanism of efflux pump inhibition

    PubMed Central

    Opperman, Timothy J.; Nguyen, Son T.

    2015-01-01

    Multidrug resistance (MDR) in Gram-negative pathogens, such as the Enterobacteriaceae and Pseudomonas aeruginosa, poses a significant threat to our ability to effectively treat infections caused by these organisms. A major component in the development of the MDR phenotype in Gram-negative bacteria is overexpression of Resistance-Nodulation-Division (RND)-type efflux pumps, which actively pump antibacterial agents and biocides from the periplasm to the outside of the cell. Consequently, bacterial efflux pumps are an important target for developing novel antibacterial treatments. Potent efflux pump inhibitors (EPIs) could be used as adjunctive therapies that would increase the potency of existing antibiotics and decrease the emergence of MDR bacteria. Several potent inhibitors of RND-type efflux pump have been reported in the literature, and at least three of these EPI series were optimized in a pre-clinical development program. However, none of these compounds have been tested in the clinic. One of the major hurdles to the development of EPIs has been the lack of biochemical, computational, and structural methods that could be used to guide rational drug design. Here, we review recent reports that have advanced our understanding of the mechanism of action of several potent EPIs against RND-type pumps. PMID:25999939

  4. Potentiation of antibacterial activity of the MB-1 siderophore-monobactam conjugate using an efflux pump inhibitor.

    PubMed

    Tomaras, Andrew P; Crandon, Jared L; McPherson, Craig J; Nicolau, David P

    2015-04-01

    Preliminary enthusiasm over the encouraging spectrum and in vitro activities of siderophore conjugates, such as MB-1, was recently tempered by unexpected variability in in vivo efficacy. The need for these conjugates to compete for iron with endogenously produced siderophores has exposed a significant liability for this novel antibacterial strategy. Here, we have exploited dependence on efflux for siderophore secretion in Pseudomonas aeruginosa and provide evidence that efflux inhibition may circumvent this in vivo-relevant resistance liability. PMID:25605364

  5. Potentiation of Antibacterial Activity of the MB-1 Siderophore-Monobactam Conjugate Using an Efflux Pump Inhibitor

    PubMed Central

    Crandon, Jared L.; McPherson, Craig J.; Nicolau, David P.

    2015-01-01

    Preliminary enthusiasm over the encouraging spectrum and in vitro activities of siderophore conjugates, such as MB-1, was recently tempered by unexpected variability in in vivo efficacy. The need for these conjugates to compete for iron with endogenously produced siderophores has exposed a significant liability for this novel antibacterial strategy. Here, we have exploited dependence on efflux for siderophore secretion in Pseudomonas aeruginosa and provide evidence that efflux inhibition may circumvent this in vivo-relevant resistance liability. PMID:25605364

  6. An overview of bacterial efflux pumps and computational approaches to study efflux pump inhibitors.

    PubMed

    Jamshidi, Shirin; Sutton, J Mark; Rahman, Khondaker M

    2016-02-01

    Micro-organisms express a wide range of transmembrane pumps known as multidrug efflux pumps that improve the micro-organism's ability to survive in severe environments and contribute to resistance against antibiotic and antimicrobial agents. There is significant interest in developing efflux inhibitors as an adjunct to treatment with current and next generation of antibiotics. A greater understanding of drug recognition and transport by multidrug efflux pumps is needed to develop clinically useful inhibitors, given the breadth of molecules that can be effluxed by these systems. We summarize some structural and functional data that could provide insights into the inhibition of transport mechanisms of these intricate molecular nanomachines with a focus on the advances in computational approaches. PMID:26824720

  7. Development of efflux pump inhibitors in antituberculosis therapy.

    PubMed

    Song, Lele; Wu, Xueqiong

    2016-06-01

    Resistance and tolerance to antituberculosis (anti-TB) drugs, especially the first-line drugs, has become a serious problem in anti-TB therapy. Efflux of antimicrobial agents via bacterial efflux pumps is one of the main reasons for drug resistance. Efflux pump inhibitors (EPIs) bind to efflux pumps to inhibit drug efflux and thus enhance the drug effect and reduce drug resistance. Studies on EPIs targeting the efflux pumps of Mycobacterium tuberculosis (Mtb) help to understand Mtb resistance and to identify the potential drug target and are of significance in guiding the development of new anti-TB drugs and optimal combinations. Currently, there are many potential EPIs under study, but none of them has been used clinically for anti-TB therapy. In this article, we will provide an overview on the current development of EPIs targeting the efflux pumps of Mtb and discuss their potential clinical applications. PMID:27211826

  8. Multidrug efflux pumps of Gram-positive bacteria.

    PubMed

    Schindler, Bryan D; Kaatz, Glenn W

    2016-07-01

    Gram-positive organisms are responsible for some of the most serious of human infections. Resistance to front-line antimicrobial agents can complicate otherwise curative therapy. These organisms possess multiple drug resistance mechanisms, with drug efflux being a significant contributing factor. Efflux proteins belonging to all five transporter families are involved, and frequently can transport multiple structurally unrelated compounds resulting in a multidrug resistance (MDR) phenotype. In addition to clinically relevant antimicrobial agents, MDR efflux proteins can transport environmental biocides and disinfectants which may allow persistence in the healthcare environment and subsequent acquisition by patients or staff. Intensive research on MDR efflux proteins and the regulation of expression of their genes is ongoing, providing some insight into the mechanisms of multidrug recognition and transport. Inhibitors of many of these proteins have been identified, including drugs currently being used for other indications. Structural modifications guided by structure-activity studies have resulted in the identification of potent compounds. However, lack of broad-spectrum pump inhibition combined with potential toxicity has hampered progress. Further work is required to gain a detailed understanding of the multidrug recognition process, followed by application of this knowledge in the design of safer and more highly potent inhibitors. PMID:27449594

  9. The role of efflux pumps in macrolide resistance in Mycobacterium avium complex.

    PubMed

    Rodrigues, Liliana; Sampaio, Daniela; Couto, Isabel; Machado, Diana; Kern, Winfried V; Amaral, Leonard; Viveiros, Miguel

    2009-12-01

    Mycobacteriumavium complex (MAC) is clinically important since it can cause severe infections in acquired immune deficiency syndrome (AIDS) patients and other immunocompromised individuals. Use of the macrolides clarithromycin and azithromycin has improved the outcome of MAC infections, but therapeutic failure is still a major problem. In this work, we studied efflux pump activity in MAC clinical strains and evaluated the contribution of active efflux to macrolide resistance. Eighteen clinical strains isolated from AIDS patients were evaluated for macrolide resistance in the presence and absence of the efflux pump inhibitors (EPIs) thioridazine, chlorpromazine and verapamil. The efflux activity of these strains was then assessed by a semi-automated fluorometric method that detects extrusion of ethidium bromide (EtBr), a known efflux pump substrate. Resistance to clarithromycin was significantly reduced in the presence of thioridazine, chlorpromazine and verapamil. The same EPIs were effective in decreasing the efflux of EtBr from MAC cells. Moreover, increased retention of [(14)C]-erythromycin in the presence of these EPIs further demonstrated that active efflux contributes to MAC resistance to macrolides. This study demonstrates that efflux pumps play an important role in MAC resistance to antibiotics. PMID:19740629

  10. Clinically Relevant Chromosomally Encoded Multidrug Resistance Efflux Pumps in Bacteria

    PubMed Central

    Piddock, Laura J. V.

    2006-01-01

    Efflux pump genes and proteins are present in both antibiotic-susceptible and antibiotic-resistant bacteria. Pumps may be specific for one substrate or may transport a range of structurally dissimilar compounds (including antibiotics of multiple classes); such pumps can be associated with multiple drug (antibiotic) resistance (MDR). However, the clinical relevance of efflux-mediated resistance is species, drug, and infection dependent. This review focuses on chromosomally encoded pumps in bacteria that cause infections in humans. Recent structural data provide valuable insights into the mechanisms of drug transport. MDR efflux pumps contribute to antibiotic resistance in bacteria in several ways: (i) inherent resistance to an entire class of agents, (ii) inherent resistance to specific agents, and (iii) resistance conferred by overexpression of an efflux pump. Enhanced efflux can be mediated by mutations in (i) the local repressor gene, (ii) a global regulatory gene, (iii) the promoter region of the transporter gene, or (iv) insertion elements upstream of the transporter gene. Some data suggest that resistance nodulation division systems are important in pathogenicity and/or survival in a particular ecological niche. Inhibitors of various efflux pump systems have been described; typically these are plant alkaloids, but as yet no product has been marketed. PMID:16614254

  11. [Significance of efflux pumps in multidrug resistance of Gram-negative bacteria].

    PubMed

    Wiercińska, Olga; Chojecka, Agnieszka; Kanclerski, Krzysztof; Rőhm-Rodowald, Ewa; Jakimiak, Bożenna

    2015-01-01

    The phenomenon of multidrug. resistance of bacteria is a serious problem of modern medicine. This resistance largely is a consequence of abuse and improper use of antibacterial substances, especially antibiotics and chemotherapeutics in hospital settings. Multidrug resistance is caused by a number of interacting mechanisms of resistance. Recent studies have indicated that efflux pumps and systems of efflux pumps are an important determinant of this phenomenon. Contribute to this particular RND efflux systems of Gram-negative bacteria, which possess a wide range of substrates such as antibiotics, dyes, detergents, toxins and active substances of disinfectants and antiseptics. These transporters are usually encoded on bacterial chromosomes. Genes encoding efflux pumps' proteins may also be carried on plasmids and other mobile genetic elements. Such pumps are usually specific to a small group of substrates, but as an additional mechanism of resistance may contribute to the multidrug resistance. PMID:26084076

  12. Efflux Pump Control Alters Synthetic Gene Circuit Function.

    PubMed

    Diao, Junchen; Charlebois, Daniel A; Nevozhay, Dmitry; Bódi, Zoltán; Pál, Csaba; Balázsi, Gábor

    2016-07-15

    Synthetic biology aims to design new biological systems for predefined purposes, such as the controlled secretion of biofuels, pharmaceuticals, or other chemicals. Synthetic gene circuits regulating an efflux pump from the ATP-binding cassette (ABC) protein family could achieve this. However, ABC efflux pumps can also drive out intracellular inducer molecules that control the gene circuits. This will introduce an implicit feedback that could alter gene circuit function in ways that are poorly understood. Here, we used two synthetic gene circuits inducible by tetracycline family molecules to regulate the expression of a yeast ABC pump (Pdr5p) that pumps out the inducer. Pdr5p altered the dose-responses of the original gene circuits substantially in Saccharomyces cerevisiae. While one aspect of the change could be attributed to the efflux pumping function of Pdr5p, another aspect remained unexplained. Quantitative modeling indicated that reduced regulator gene expression in addition to efflux pump function could fully explain the altered dose-responses. These predictions were validated experimentally. Overall, we highlight how efflux pumps can alter gene circuit dynamics and demonstrate the utility of mathematical modeling in understanding synthetic gene circuit function in new circumstances. PMID:27111147

  13. Current Advances in Developing Inhibitors of Bacterial Multidrug Efflux Pumps.

    PubMed

    Mahmood, Hannah Y; Jamshidi, Shirin; Sutton, J Mark; Rahman, Khondaker M

    2016-01-01

    Antimicrobial resistance represents a significant challenge to future healthcare provision. An acronym ESKAPEE has been derived from the names of the organisms recognised as the major threats although there are a number of other organisms, notably Neisseria gonorrhoeae, that have become equally challenging to treat in the clinic. These pathogens are characterised by the ability to rapidly develop and/or acquire resistance mechanisms in response to exposure to different antimicrobial agents. A key part of the armoury of these pathogens is a series of efflux pumps, which effectively exclude or reduce the intracellular concentration of a large number of antibiotics, making the pathogens significantly more resistant. These efflux pumps are the topic of considerable interest, both from the perspective of basic understanding of efflux pump function, and its role in drug resistance but also as targets for the development of novel adjunct therapies. The necessity to overcome antimicrobial resistance has encouraged investigations into the characterisation of resistance-modifying efflux pump inhibitors to block the mechanisms of drug extrusion, thereby restoring antibacterial susceptibility and returning existing antibiotics into the clinic. A greater understanding of drug recognition and transport by multidrug efflux pumps is needed to develop clinically useful inhibitors, given the breadth of molecules that can be effluxed by these systems. This review discusses different bacterial EPIs originating from both natural source and chemical synthesis and examines the challenges to designing successful EPIs that can be useful against multidrug resistant bacteria. PMID:26947776

  14. Multidrug Efflux Pumps in Staphylococcus aureus: an Update

    PubMed Central

    Costa, Sofia Santos; Viveiros, Miguel; Amaral, Leonard; Couto, Isabel

    2013-01-01

    The emergence of infections caused by multi- or pan-resistant bacteria in the hospital or in the community settings is an increasing health concern. Albeit there is no single resistance mechanism behind multiresistance, multidrug efflux pumps, proteins that cells use to detoxify from noxious compounds, seem to play a key role in the emergence of these multidrug resistant (MDR) bacteria. During the last decades, experimental data has established their contribution to low level resistance to antimicrobials in bacteria and their potential role in the appearance of MDR phenotypes, by the extrusion of multiple, unrelated compounds. Recent studies suggest that efflux pumps may be used by the cell as a first-line defense mechanism, avoiding the drug to reach lethal concentrations, until a stable, more efficient alteration occurs, that allows survival in the presence of that agent. In this paper we review the current knowledge on MDR efflux pumps and their intricate regulatory network in Staphylococcus aureus, a major pathogen, responsible from mild to life-threatening infections. Particular emphasis will be given to the potential role that S. aureus MDR efflux pumps, either chromosomal or plasmid-encoded, have on resistance towards different antimicrobial agents and on the selection of drug - resistant strains. We will also discuss the many questions that still remain on the role of each specific efflux pump and the need to establish appropriate methodological approaches to address all these questions. PMID:23569469

  15. Expression of homologous RND efflux pump genes is dependent upon AcrB expression: implications for efflux and virulence inhibitor design

    PubMed Central

    Blair, Jessica M. A.; Smith, Helen E.; Ricci, Vito; Lawler, Amelia J.; Thompson, Louisa J.; Piddock, Laura J. V.

    2015-01-01

    Objectives Enterobacteriaceae have multiple efflux pumps that confer intrinsic resistance to antibiotics. AcrB mediates clinically relevant multidrug resistance and is required for virulence and biofilm formation, making it an attractive target for the design of inhibitors. The aim of this study was to assess the viability of single transporters as a target for efflux inhibition using Salmonella Typhimurium as the model pathogen. Methods The expression of resistance–nodulation–division (RND) efflux pump genes in response to the inactivation of single or multiple homologues was measured using real-time RT–PCR. Phenotypes of mutants were characterized by measuring antimicrobial susceptibility, dye accumulation and the ability to cause infection in vitro. Results The expression of all RND efflux pump genes was increased when single or multiple acr genes were inactivated, suggesting a feedback mechanism that activates the transcription of homologous efflux pump genes. When two or three acr genes were inactivated, the mutants had further reduced efflux, altered susceptibility to antimicrobials (including increased susceptibility to some, but conversely and counterintuitively, decreased susceptibility to some others) and were more attenuated in the tissue culture model than mutants lacking single pumps were. Conclusions These data indicate that it is critical to understand which pumps an inhibitor is active against and the effect of this on the expression of homologous systems. For some antimicrobials, an inhibitor with activity against multiple pumps will have a greater impact on susceptibility, but an unintended consequence of this may be decreased susceptibility to other drugs, such as aminoglycosides. PMID:25288678

  16. Overexpression of MexAB-OprM efflux pump in carbapenem-resistant Pseudomonas aeruginosa.

    PubMed

    Pan, Ya-Ping; Xu, Yuan-Hong; Wang, Zhong-Xin; Fang, Ya-Ping; Shen, Ji-Lu

    2016-08-01

    Efflux pump systems are one of the most important mechanisms conferring multidrug resistance in Pseudomonas aeruginosa. MexAB-OprM efflux pump is one of the largest multi-drug resistant efflux pumps with high-level expression, which is controlled by regulatory genes mexR, nalC, and nalD. This study investigated the role of efflux pump MexAB-OprM in 75 strains of carbapenem-resistant P. aeruginosa and evaluated the influence of point mutation of the regulatory genes. The minimum inhibitory concentrations of imipenem and meropenem, with or without MC207110, an efflux pump inhibitor, were determined by agar dilution method to select the positive strains for an overexpressed active efflux pump. Carba NP test and EDTA-disk synergy test were used for the detection of carbapenemase and metallo-β-lactamases, respectively. The gene mexA, responsible for the fusion protein structure, and the reference gene rpoD of the MexAB-OprM pump were amplified by real-time PCR. The quantity of relative mRNA expression was determined simultaneously. By PCR method, the efflux regulatory genes mexR, nalC, and nalD and outer membrane protein OprD2 were amplified for the strains showing overexpression of MexAB-OprM and subsequently analyzed by BLAST. Among the 75 P. aeruginosa strains, the prevalence of efflux pump-positive phenotype was 17.3 % (13/75). Carba NP test and EDTA-disk synergy test were all negative in the 13 strains. PCR assay results showed that ten strains overexpressed the MexAB-OprM efflux pump and were all positive for the regulatory genes mexR, nalC, and nalD. Sequence analysis indicated that of the ten isolates, nine had a mutation (Gly → Glu) at 71st amino acid position in NalC, and eight also had a mutation (Ser → Arg) at 209th position in NalC. Only one strain had a mutation (Thr → Ile) at the 158th amino acid position in NalD, whereas eight isolates had mutations in MexR. In conclusion, overexpression of efflux pump MexAB-OprM plays an important role in

  17. Mitochondria Influence CDR1 Efflux Pump Activity, Hog1-Mediated Oxidative Stress Pathway, Iron Homeostasis, and Ergosterol Levels in Candida albicans

    PubMed Central

    Thomas, Edwina; Roman, Elvira; Claypool, Steven; Manzoor, Nikhat; Pla, Jesús

    2013-01-01

    Mitochondrial dysfunction in Candida albicans is known to be associated with drug susceptibility, cell wall integrity, phospholipid homeostasis, and virulence. In this study, we deleted CaFZO1, a key component required during biogenesis of functional mitochondria. Cells with FZO1 deleted displayed fragmented mitochondria, mitochondrial genome loss, and reduced mitochondrial membrane potential and were rendered sensitive to azoles and peroxide. In order to understand the cellular response to dysfunctional mitochondria, genome-wide expression profiling of fzo1Δ/Δ cells was performed. Our results show that the increased susceptibility to azoles was likely due to reduced efflux activity of CDR efflux pumps, caused by the missorting of Cdr1p into the vacuole. In addition, fzo1Δ/Δ cells showed upregulation of genes involved in iron assimilation, in iron-sufficient conditions, characteristic of iron-starved cells. One of the consequent effects was downregulation of genes of the ergosterol biosynthesis pathway with a commensurate decrease in cellular ergosterol levels. We therefore connect deregulated iron metabolism to ergosterol biosynthesis pathway in response to dysfunctional mitochondria. Impaired activation of the Hog1 pathway in the mutant was the basis for increased susceptibility to peroxide and increase in reactive oxygen species, indicating the importance of functional mitochondria in controlling Hog1-mediated oxidative stress response. Mitochondrial phospholipid levels were also altered as indicated by an increase in phosphatidylserine and phosphatidylethanolamine and decrease in phosphatidylcholine in fzo1Δ/Δ cells. Collectively, these findings reinforce the connection between functional mitochondria and azole tolerance, oxidant-mediated stress, and iron homeostasis in C. albicans. PMID:23979757

  18. Identification of Acinetobacter baumannii Serum-Associated Antibiotic Efflux Pump Inhibitors

    PubMed Central

    Blanchard, Catlyn; Barnett, Pamela; Perlmutter, Jessamyn

    2014-01-01

    Adaptive antibiotic resistance is a newly described phenomenon by which Acinetobacter baumannii induces efflux pump activity in response to host-associated environmental cues that may, in part, account for antibiotic treatment failures against clinically defined susceptible strains. To that end, during adaptation to growth in human serum, the organism induces approximately 22 putative efflux-associated genes and displays efflux-mediated minocycline tolerance at antibiotic concentrations corresponding to patient serum levels. Here, we show that in addition to minocycline, growth in human serum elicits A. baumannii efflux-mediated tolerance to the antibiotics ciprofloxacin, meropenem, tetracycline, and tigecycline. Moreover, using a whole-cell high-throughput screen and secondary assays, we identified novel serum-associated antibiotic efflux inhibitors that potentiated the activities of antibiotics toward serum-grown A. baumannii. Two compounds, Acinetobacter baumannii efflux pump inhibitor 1 (ABEPI1) [(E)-4-((4-chlorobenzylidene)amino)benezenesulfonamide] and ABEPI2 [N-tert-butyl-2-(1-tert-butyltetrazol-5-yl)sulfanylacetamide], were shown to lead to minocycline accumulation within A. baumannii during serum growth and inhibit the efflux potential of the organism. While both compounds also inhibited the antibiotic efflux properties of the bacterial pathogen Pseudomonas aeruginosa, they did not display significant cytotoxicity toward human cells or mammalian Ca2+ channel inhibitory effects, suggesting that ABEPI1 and ABEPI2 represent promising structural scaffolds for the development of new classes of bacterial antibiotic efflux pump inhibitors that can be used to potentiate the activities of current and future antibiotics for the therapeutic intervention of Gram-negative bacterial infections. PMID:25114126

  19. Tripartite assembly of RND multidrug efflux pumps

    PubMed Central

    Daury, Laetitia; Orange, François; Taveau, Jean-Christophe; Verchère, Alice; Monlezun, Laura; Gounou, Céline; Marreddy, Ravi K. R.; Picard, Martin; Broutin, Isabelle; Pos, Klaas M.; Lambert, Olivier

    2016-01-01

    Tripartite multidrug efflux systems of Gram-negative bacteria are composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. They are assumed to form ducts inside the periplasm facilitating drug exit across the outer membrane. Here we present the reconstitution of native Pseudomonas aeruginosa MexAB–OprM and Escherichia coli AcrAB–TolC tripartite Resistance Nodulation and cell Division (RND) efflux systems in a lipid nanodisc system. Single-particle analysis by electron microscopy reveals the inner and outer membrane protein components linked together via the periplasmic adaptor protein. This intrinsic ability of the native components to self-assemble also leads to the formation of a stable interspecies AcrA–MexB–TolC complex suggesting a common mechanism of tripartite assembly. Projection structures of all three complexes emphasize the role of the periplasmic adaptor protein as part of the exit duct with no physical interaction between the inner and outer membrane components. PMID:26867482

  20. Tripartite assembly of RND multidrug efflux pumps

    NASA Astrophysics Data System (ADS)

    Daury, Laetitia; Orange, François; Taveau, Jean-Christophe; Verchère, Alice; Monlezun, Laura; Gounou, Céline; Marreddy, Ravi K. R.; Picard, Martin; Broutin, Isabelle; Pos, Klaas M.; Lambert, Olivier

    2016-02-01

    Tripartite multidrug efflux systems of Gram-negative bacteria are composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. They are assumed to form ducts inside the periplasm facilitating drug exit across the outer membrane. Here we present the reconstitution of native Pseudomonas aeruginosa MexAB-OprM and Escherichia coli AcrAB-TolC tripartite Resistance Nodulation and cell Division (RND) efflux systems in a lipid nanodisc system. Single-particle analysis by electron microscopy reveals the inner and outer membrane protein components linked together via the periplasmic adaptor protein. This intrinsic ability of the native components to self-assemble also leads to the formation of a stable interspecies AcrA-MexB-TolC complex suggesting a common mechanism of tripartite assembly. Projection structures of all three complexes emphasize the role of the periplasmic adaptor protein as part of the exit duct with no physical interaction between the inner and outer membrane components.

  1. Involvement of Antibiotic Efflux Machinery in Glutathione-Mediated Decreased Ciprofloxacin Activity in Escherichia coli.

    PubMed

    Goswami, Manish; Subramanian, Mahesh; Kumar, Ranjeet; Jass, Jana; Jawali, Narendra

    2016-07-01

    We have analyzed the contribution of different efflux components to glutathione-mediated abrogation of ciprofloxacin's activity in Escherichia coli and the underlying potential mechanism(s) behind this phenomenon. The results indicated that glutathione increased the total active efflux, thereby partially contributing to glutathione-mediated neutralization of ciprofloxacin's antibacterial action in E. coli However, the role of glutathione-mediated increased efflux becomes evident in the absence of a functional TolC-AcrAB efflux pump. PMID:27139480

  2. Engineering microbial biofuel tolerance and export using efflux pumps

    PubMed Central

    Dunlop, Mary J; Dossani, Zain Y; Szmidt, Heather L; Chu, Hou Cheng; Lee, Taek Soon; Keasling, Jay D; Hadi, Masood Z; Mukhopadhyay, Aindrila

    2011-01-01

    Many compounds being considered as candidates for advanced biofuels are toxic to microorganisms. This introduces an undesirable trade-off when engineering metabolic pathways for biofuel production because the engineered microbes must balance production against survival. Cellular export systems, such as efflux pumps, provide a direct mechanism for reducing biofuel toxicity. To identify novel biofuel pumps, we used bioinformatics to generate a list of all efflux pumps from sequenced bacterial genomes and prioritized a subset of targets for cloning. The resulting library of 43 pumps was heterologously expressed in Escherichia coli, where we tested it against seven representative biofuels. By using a competitive growth assay, we efficiently distinguished pumps that improved survival. For two of the fuels (n-butanol and isopentanol), none of the pumps improved tolerance. For all other fuels, we identified pumps that restored growth in the presence of biofuel. We then tested a beneficial pump directly in a production strain and demonstrated that it improved biofuel yields. Our findings introduce new tools for engineering production strains and utilize the increasingly large database of sequenced genomes. PMID:21556065

  3. A Simple Method for Assessment of MDR Bacteria for Over-Expressed Efflux Pumps.

    PubMed

    Martins, Marta; McCusker, Matthew P; Viveiros, Miguel; Couto, Isabel; Fanning, Séamus; Pagès, Jean-Marie; Amaral, Leonard

    2013-01-01

    It is known that bacteria showing a multi-drug resistance phenotype use several mechanisms to overcome the action of antibiotics. As a result, this phenotype can be a result of several mechanisms or a combination of thereof. The main mechanisms of antibiotic resistance are: mutations in target genes (such as DNA gyrase and topoisomerase IV); over-expression of efflux pumps; changes in the cell envelope; down regulation of membrane porins, and modified lipopolysaccharide component of the outer cell membrane (in the case of Gram-negative bacteria). In addition, adaptation to the environment, such as quorum sensing and biofilm formation can also contribute to bacterial persistence. Due to the rapid emergence and spread of bacterial isolates showing resistance to several classes of antibiotics, methods that can rapidly and efficiently identify isolates whose resistance is due to active efflux have been developed. However, there is still a need for faster and more accurate methodologies. Conventional methods that evaluate bacterial efflux pump activity in liquid systems are available. However, these methods usually use common efflux pump substrates, such as ethidium bromide or radioactive antibiotics and therefore, require specialized instrumentation, which is not available in all laboratories. In this review, we will report the results obtained with the Ethidium Bromide-agar Cartwheel method. This is an easy, instrument-free, agar based method that has been modified to afford the simultaneous evaluation of as many as twelve bacterial strains. Due to its simplicity it can be applied to large collections of bacteria to rapidly screen for multi-drug resistant isolates that show an over-expression of their efflux systems. The principle of the method is simple and relies on the ability of the bacteria to expel a fluorescent molecule that is substrate for most efflux pumps, ethidium bromide. In this approach, the higher the concentration of ethidium bromide required to

  4. MAPK1 of Leishmania donovani Modulates Antimony Susceptibility by Downregulating P-Glycoprotein Efflux Pumps

    PubMed Central

    Garg, Mansi

    2015-01-01

    Emergence of resistance to pentavalent antimonials has become a severe obstacle in the treatment of visceral leishmaniasis (VL) in the Indian subcontinent. Mitogen-activated protein kinases (MAPKs) are well-known mediators of signal transduction of eukaryotes, regulating important processes, like proliferation, differentiation, stress response, and apoptosis. In Leishmania, MAPK1 has been shown to be consistently downregulated in antimony-resistant field isolates, suggesting that it has a role in antimony resistance. The present work investigates the molecular mechanism of MAPK1 in antimony resistance in Leishmania donovani. The L. donovani MAPK1 (LdMAPK1) single-allele replacement mutants exhibited increased resistance to Sb(III) (5.57-fold) compared to wild-type promastigotes, while overexpressing parasites became much more susceptible to antimony. The LdMAPK1-mediated drug sensitivity was directly related to antimony-induced apoptotic death of the parasite, as was evidenced by a 4- to 5-fold decrease in cell death parameters in deletion mutants and a 2- to 3-fold increase in MAPK1-overexpressing cells. LdMAPK1-underexpressing parasites also exhibited increased P-glycoprotein (P-gp)-mediated efflux pump activity, while a significant decrease in pump activity was observed in overexpressing cells. This change in efflux pump activity was directly related to expression levels of P-gp in all cell lines. However, episomal complementation of the gene restored normal growth, drug sensitivity, P-gp expression, and efflux pump activity. The data indicate that LdMAPK1 negatively regulates the expression of P-glycoprotein-type efflux pumps in the parasite. The decrease in efflux pump activity with an increase in LdMAPK1 expression may result in increased antimony accumulation in the parasite, making it more vulnerable to the drug. PMID:25870075

  5. Action of cholecalciferol and alpha-tocopherol on Staphylococcus aureus efflux pumps.

    PubMed

    Tintino, Saulo R; Morais-Tintino, Cícera D; Campina, Fábia F; Pereira, Raimundo L; Costa, Maria do S; Braga, Maria Flaviana B M; Limaverde, Paulo W; Andrade, Jacqueline C; Siqueira-Junior, José P; Coutinho, Henrique Douglas Melo; Balbino, Valdir Q; Leal-Balbino, Tereza C; Ribeiro-Filho, Jaime; Quintans-Júnior, Lucindo J

    2016-01-01

    Alpha-tocopherol is one the most abundant and biologically active isoforms of vitamin E. This compound is a potent antioxidant and one of most studied isoforms of vitamin E. Vitamin D3 (cholecalciferol) is an important nutrient for calcium homeostasis and bone health, that has also been recognized as a potent modulator of the immune response. Methicillin-resistant Staphylococcus aureus (MRSA) is the most important causative agent of both nosocomial and community-acquired infections. The aim of this study was to evaluate the inhibitory effect of alpha-tocopherol and cholecalciferol on both S. aureus and multidrug resistant S. aureus efflux pumps. The RN4220 strain has the plasmid pUL5054 that is the carrier of gene that encodes the macrolide resistance protein (an efflux pump) MsrA; the IS-58 strain possesses the TetK tetracycline efflux protein in its genome and the 1199B strain resists to hydrophilic fluoroquinolones via a NorA-mediated mechanism. The antibacterial activity was evaluated by determining the Minimal Inhibitory Concentration (MIC) and a possible inhibition of efflux pumps was associated to a reduction of the MIC. In this work we observed that in the presence of the treatments there was a decrease in the MIC for the RN4220 and IS-58 strains, suggesting that the substances presented an inhibitory effect on the efflux pumps of these strains. Significant efforts have been done to identify efflux pump inhibitors (EPIs) from natural sources and, therefore, the antibacterial properties of cholecalciferol and alpha-tocopherol might be attributed to a direct effect on the bacterial cell depending on their amphipathic structure. PMID:27298617

  6. Action of cholecalciferol and alpha-tocopherol on Staphylococcus aureus efflux pumps

    PubMed Central

    Tintino, Saulo R.; Morais-Tintino, Cícera D.; Campina, Fábia F.; Pereira, Raimundo L.; Costa, Maria do S.; Braga, Maria Flaviana B.M.; Limaverde, Paulo W.; Andrade, Jacqueline C.; Siqueira-Junior, José P.; Coutinho, Henrique Douglas Melo; Balbino, Valdir Q.; Leal-Balbino, Tereza C.; Ribeiro-Filho, Jaime; Quintans-Júnior, Lucindo J.

    2016-01-01

    Alpha-tocopherol is one the most abundant and biologically active isoforms of vitamin E. This compound is a potent antioxidant and one of most studied isoforms of vitamin E. Vitamin D3 (cholecalciferol) is an important nutrient for calcium homeostasis and bone health, that has also been recognized as a potent modulator of the immune response. Methicillin-resistant Staphylococcus aureus (MRSA) is the most important causative agent of both nosocomial and community-acquired infections. The aim of this study was to evaluate the inhibitory effect of alpha-tocopherol and cholecalciferol on both S. aureus and multidrug resistant S. aureus efflux pumps. The RN4220 strain has the plasmid pUL5054 that is the carrier of gene that encodes the macrolide resistance protein (an efflux pump) MsrA; the IS-58 strain possesses the TetK tetracycline efflux protein in its genome and the 1199B strain resists to hydrophilic fluoroquinolones via a NorA-mediated mechanism. The antibacterial activity was evaluated by determining the Minimal Inhibitory Concentration (MIC) and a possible inhibition of efflux pumps was associated to a reduction of the MIC. In this work we observed that in the presence of the treatments there was a decrease in the MIC for the RN4220 and IS-58 strains, suggesting that the substances presented an inhibitory effect on the efflux pumps of these strains. Significant efforts have been done to identify efflux pump inhibitors (EPIs) from natural sources and, therefore, the antibacterial properties of cholecalciferol and alpha-tocopherol might be attributed to a direct effect on the bacterial cell depending on their amphipathic structure. PMID:27298617

  7. Characterization of MATE-Type Multidrug Efflux Pumps from Klebsiella pneumoniae MGH78578

    PubMed Central

    Ogawa, Wakano; Minato, Yusuke; Dodan, Hayata; Onishi, Motoyasu; Tsuchiya, Tomofusa; Kuroda, Teruo

    2015-01-01

    We previously described the cloning of genes related to drug resistance from Klebsiella pneumoniae MGH78578. Of these, we identified a putative gene encoding a MATE-type multidrug efflux pump, and named it ketM. Escherichia coli KAM32 possessing ketM on a plasmid showed increased minimum inhibitory concentrations for norfloxacin, ciprofloxacin, cefotaxime, acriflavine, Hoechst 33342, and 4',6-diamidino-2-phenyl indole (DAPI). The active efflux of DAPI was observed in E. coli KAM32 possessing ketM on a plasmid. The expression of mRNA for ketM was observed in K. pneumoniae cells, and we subsequently disrupted ketM in K. pneumoniae ATCC10031. However, no significant changes were observed in drug resistance levels between the parental strain ATCC10031 and ketM disruptant, SKYM. Therefore, we concluded that KetM was a multidrug efflux pump, that did not significantly contribute to intrinsic resistance to antimicrobial chemicals in K. pneumoniae. MATE-type transporters are considered to be secondary transporters; therefore, we investigated the coupling cations of KetM. DAPI efflux by KetM was observed when lactate was added to produce a proton motive force, indicating that KetM effluxed substrates using a proton motive force. However, the weak efflux of DAPI by KetM was also noted when NaCl was added to the assay mixture without lactate. This result suggests that KetM may utilize proton and sodium motive forces. PMID:25807080

  8. Alkylaminoquinolines inhibit the bacterial antibiotic efflux pump in multidrug-resistant clinical isolates.

    PubMed Central

    Malléa, Monique; Mahamoud, Abdallah; Chevalier, Jacqueline; Alibert-Franco, Sandrine; Brouant, Pierre; Barbe, Jacques; Pagès, Jean-Marie

    2003-01-01

    Over the last decade, MDR (multidrug resistance) has increased worldwide in microbial pathogens by efflux mechanisms, leading to treatment failures in human infections. Several Gram-negative bacteria efflux pumps have been described. These proteinaceous channels are capable of expelling structurally different drugs across the envelope and conferring antibiotic resistance in various bacterial pathogens. Combating antibiotic resistance is an urgency and the blocking of efflux pumps is an attractive response to the emergence of MDR phenotypes in infectious bacteria. In the present study, various alkylaminoquinolines were tested as potential inhibitors of drug transporters. We showed that alkylaminoquinolines are capable of restoring susceptibilities to structurally unrelated antibiotics in clinical isolates of MDR Gram-negative bacteria. Antibiotic efflux studies indicated that 7-nitro-8-methyl-4-[2'-(piperidino)ethyl]aminoquinoline acts as an inhibitor of the AcrAB-TolC efflux pump and restores a high level of intracellular drug concentration. Inhibitory activity of this alkylaminoquinoline is observed on clinical isolates showing different resistance phenotypes. PMID:12959639

  9. Efflux Pump, the Masked Side of ß-Lactam Resistance in Klebsiella pneumoniae Clinical Isolates

    PubMed Central

    Pages, Jean-Marie; Lavigne, Jean-Philippe; Leflon-Guibout, Véronique; Marcon, Estelle; Bert, Frédéric; Noussair, Latifa; Nicolas-Chanoine, Marie-Hélène

    2009-01-01

    Background β-lactamase production and porin decrease are the well-recognized mechanisms of acquired ß-lactam resistance in Klebsiella pneumoniae isolates. However, such mechanisms proved to be absent in K. pneumoniae isolates that are non susceptible to cefoxitin (FOX) and succeptible to amoxicillin+clavulanic acid in our hospital. Assessing the role of efflux pumps in this β-lactam phenotype was the aim of this study. Methodology/Findings MICs of 9 β-lactams, including cloxacillin (CLX), and other antibiotic families were tested alone and with an efflux pump inhibitor (EPI), then with both CLX (subinhibitory concentrations) and EPI against 11 unique bacteremia K. pneumoniae isolates displaying the unusual phenotype, and 2 ATCC strains. CLX and EPI-dose dependent effects were studied on 4 representatives strains. CLX MICs significantly decreased when tested with EPI. A similar phenomenon was observed with piperacillin+tazobactam whereas MICs of the other β-lactams significantly decreased only in the presence of both EPI and CLX. Thus, FOX MICs decreased 128 fold in the K. pneumoniae isolates but also16 fold in ATCC strain. Restoration of FOX activity was CLX dose-dependent suggesting a competitive relationship between CLX and the other β-lactams with regard to their efflux. For chloramphenicol, erythromycin and nalidixic acid whose resistance was also due to efflux, adding CLX to EPI did not increase their activity suggesting differences between the efflux process of these molecules and that of β-lactams. Conclusion This is the first study demonstrating that efflux mechanism plays a key role in the β-lactam susceptibility of clinical isolates of K. pneumoniae. Such data clearly evidence that the involvement of efflux pumps in ß-lactam resistance is specially underestimated in clinical isolates. PMID:19279676

  10. Chlorinated phenols control the expression of the multi-drug resistance efflux pump MexAB-OprM in Pseudomonas aeruginosa by activating NalC

    PubMed Central

    Ghosh, Sudeshna; Cremers, Claudia M.; Jakob, Ursula; Love, Nancy G.

    2011-01-01

    Summary NalC is a TetR type regulator that represses the multidrug efflux pump MexAB-OprM in Pseudomonas aeruginosa. Here we explain the mechanism of NalC mediated regulation of MexAB-OprM. We show that NalC non-covalently binds chlorinated phenols and chemicals containing chlorophenol sidechains such as triclosan. NalC-chlorinated phenol binding results in its dissociation from promoter DNA and up-regulation of NalC’s downstream targets, including the MexR antirepressor ArmR. ArmR up-regulation and MexR-ArmR complex formation have previously been shown to upregulate MexAB-OprM. In vivo mexB and armR expression analyses were used to corroborate in vitro NalC chlorinated phenol binding. We also show that the interaction between chlorinated phenols and NalC is reversible, such that removal of these chemicals restored NalC promoter DNA binding. Thus, the NalC-chlorinated phenol interaction is likely a pertinent physiological mechanism that P. aeruginosa uses to control expression of the MexAB-OprM efflux pump. PMID:21231970

  11. Expression of Efflux Pumps and Fatty Acid Activator One Genes in Azole Resistant Candida Glabrata Isolated From Immunocompromised Patients.

    PubMed

    Farahyar, Shirin; Zaini, Farideh; Kordbacheh, Parivash; Rezaie, Sassan; Falahati, Mehraban; Safara, Mahin; Raoofian, Reza; Hatami, Kamran; Mohebbi, Masoumeh; Heidari, Mansour

    2016-07-01

    Acquired azole resistance in opportunistic fungi causes severe clinical problems in immunosuppressed individuals. This study investigated the molecular mechanisms of azole resistance in clinical isolates of Candida glabrata. Six unmatched strains were obtained from an epidemiological survey of candidiasis in immunocompromised hosts that included azole and amphotericin B susceptible and azole resistant clinical isolates. Candida glabrata CBS 138 was used as reference strain. Antifungal susceptibility testing of clinical isolates was evaluated using Clinical and Laboratory Standards Institute (CLSI) methods. Complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) technology, semi-quantitative RT-PCR, and sequencing were employed for identification of potential genes involved in azole resistance. Candida glabrata Candida drug resistance 1 (CgCDR1) and Candida glabrata Candida drug resistance 2 (CgCDR2) genes, which encode for multidrug transporters, were found to be upregulated in azole-resistant isolates (≥2-fold). Fatty acid activator 1 (FAA1) gene, belonging to Acyl-CoA synthetases, showed expression in resistant isolates ≥2-fold that of the susceptible isolates and the reference strain. This study revealed overexpression of the CgCDR1, CgCDR2, and FAA1 genes affecting biological pathways, small hydrophobic compounds transport, and lipid metabolism in the resistant clinical C.glabrata isolates. PMID:27424018

  12. Engineering bacterial efflux pumps for solar-powered bioremediation of surface waters.

    PubMed

    Kapoor, Vikram; Wendell, David

    2013-05-01

    Antibiotics are difficult to selectively remove from surface waters by present treatment methods. Bacterial efflux pumps have evolved the ability to discriminately expel antibiotics and other noxious agents via proton and ATP driven pathways. Here, we describe light-dependent removal of antibiotics by engineering the bacterial efflux pump AcrB into a proteovesicle system. We have created a chimeric protein with the requisite proton motive force by coupling AcrB to the light-driven proton pump Delta-rhodopsin (dR) via a glycophorin A transmembrane domain. This creates a solar powered protein material capable of selectively capturing antibiotics from bulk solutions. Using environmental water and direct sunlight, our AcrB-dR vesicles removed almost twice as much antibiotic as the treatment standard, activated carbon. Altogether, the AcrB-dR system provides an effective means of extracting antibiotics from surface waters as well as potential antibiotic recovery through vesicle solubilization. PMID:23581993

  13. The ins and outs of RND efflux pumps in Escherichia coli

    PubMed Central

    Anes, João; McCusker, Matthew P.; Fanning, Séamus; Martins, Marta

    2015-01-01

    Infectious diseases remain one of the principal causes of morbidity and mortality in the world. Relevant authorities including the WHO and CDC have expressed serious concern regarding the continued increase in the development of multidrug resistance among bacteria. They have also reaffirmed the urgent need for investment in the discovery and development of new antibiotics and therapeutic approaches to treat multidrug resistant (MDR) bacteria. The extensive use of antimicrobial compounds in diverse environments, including farming and healthcare, has been identified as one of the main causes for the emergence of MDR bacteria. Induced selective pressure has led bacteria to develop new strategies of defense against these chemicals. Bacteria can accomplish this by several mechanisms, including enzymatic inactivation of the target compound; decreased cell permeability; target protection and/or overproduction; altered target site/enzyme and increased efflux due to over-expression of efflux pumps. Efflux pumps can be specific for a single substrate or can confer resistance to multiple antimicrobials by facilitating the extrusion of a broad range of compounds including antibiotics, heavy metals, biocides and others, from the bacterial cell. To overcome antimicrobial resistance caused by active efflux, efforts are required to better understand the fundamentals of drug efflux mechanisms. There is also a need to elucidate how these mechanisms are regulated and how they respond upon exposure to antimicrobials. Understanding these will allow the development of combined therapies using efflux inhibitors together with antibiotics to act on Gram-negative bacteria, such as the emerging globally disseminated MDR pathogen Escherichia coli ST131 (O25:H4). This review will summarize the current knowledge on resistance-nodulation-cell division efflux mechanisms in E. coli, a bacteria responsible for community and hospital-acquired infections, as well as foodborne outbreaks worldwide

  14. Efflux pump inhibitors (EPIs) as new antimicrobial agents against Pseudomonas aeruginosa

    PubMed Central

    Askoura, Momen; Mottawea, Walid; Abujamel, Turki; Taher, Ibrahim

    2011-01-01

    Pseudomonas aeruginosa is an opportunistic human pathogen and one of the leading causes of nosocomial infections worldwide. The difficulty in treatment of pseudomonas infections arises from being multidrug resistant (MDR) and exhibits resistance to most antimicrobial agents due to the expression of different mechanisms overcoming their effects. Of these resistance mechanisms, the active efflux pumps in Pseudomonas aeruginosa that belong to the resistance nodulation division (RND) plays a very important role in extruding the antibiotics outside the bacterial cells providing a protective means against their antibacterial activity. Beside its role against the antimicrobial agents, these pumps can extrude biocides, detergents, and other metabolic inhibitors. It is clear that efflux pumps can be targets for new antimicrobial agents. Peptidomimetic compounds such as phenylalanine arginyl β-naphthylamide (PAβN) have been introduced as efflux pump inhibitors (EPIs); their mechanism of action is through competitive inhibition with antibiotics on the efflux pump resulting in increased intracellular concentration of antibiotic, hence, restoring its antibacterial activity. The advantage of EPIs is the difficulty to develop bacterial resistance against them, but the disadvantage is their toxic property hindering their clinical application. The structure activity relationship of these compounds showed other derivatives from PAβN that are higher in their activity with higher solubility in biological fluids and decreased toxicity level. This raises further questions on how can we compact Pseudomonas infections. Of particular importance, the recent resurgence in the use of older antibiotics such as polymyxins and probably applying stricter control measures in order to prevent their spread in clinical sittings. PMID:21594004

  15. Focus on the Outer Membrane Factor OprM, the Forgotten Player from Efflux Pumps Assemblies.

    PubMed

    Phan, Gilles; Picard, Martin; Broutin, Isabelle

    2015-01-01

    Antibiotics have been used extensively during several decades and we are now facing the emergence of multidrug resistant strains. It has become a major public concern, urging the need to discover new strategies to combat them. Among the different ways used by bacteria to resist antibiotics, the active efflux is one of the main mechanisms. In Gram-negative bacteria the efflux pumps are comprised of three components forming a long edifice crossing the complete cell wall from the inside to the outside of the cell. Blocking these pumps would permit the restoration of the effectiveness of the current antibiotherapy which is why it is important to increase our knowledge on the different proteins involved in these complexes. A tremendous number of experiments have been performed on the inner membrane protein AcrB from Escherichia coli and, to a lesser extent, the protein partners forming the AcrAB-TolC pump, but less information is available concerning the efflux pumps from other virulent Gram-negative bacteria. The present review will focus on the OprM outer membrane protein from the MexAB-OprM pump of Pseudomonas aeruginosa, highlighting similarities and differences compare to the archetypal AcrAB-TolC in terms of structure, function, and assembly properties. PMID:27025640

  16. Focus on the Outer Membrane Factor OprM, the Forgotten Player from Efflux Pumps Assemblies

    PubMed Central

    Phan, Gilles; Picard, Martin; Broutin, Isabelle

    2015-01-01

    Antibiotics have been used extensively during several decades and we are now facing the emergence of multidrug resistant strains. It has become a major public concern, urging the need to discover new strategies to combat them. Among the different ways used by bacteria to resist antibiotics, the active efflux is one of the main mechanisms. In Gram-negative bacteria the efflux pumps are comprised of three components forming a long edifice crossing the complete cell wall from the inside to the outside of the cell. Blocking these pumps would permit the restoration of the effectiveness of the current antibiotherapy which is why it is important to increase our knowledge on the different proteins involved in these complexes. A tremendous number of experiments have been performed on the inner membrane protein AcrB from Escherichia coli and, to a lesser extent, the protein partners forming the AcrAB-TolC pump, but less information is available concerning the efflux pumps from other virulent Gram-negative bacteria. The present review will focus on the OprM outer membrane protein from the MexAB-OprM pump of Pseudomonas aeruginosa, highlighting similarities and differences compare to the archetypal AcrAB-TolC in terms of structure, function, and assembly properties. PMID:27025640

  17. The Role of Efflux Pumps in Schistosoma mansoni Praziquantel Resistant Phenotype

    PubMed Central

    Armada, Ana; Belo, Silvana; Carrilho, Emanuel; Viveiros, Miguel; Afonso, Ana

    2015-01-01

    Background Schistosomiasis is a neglected disease caused by a trematode of the genus Schistosoma that is second only to malaria in public health significance in Africa, South America, and Asia. Praziquantel (PZQ) is the drug of choice to treat this disease due to its high cure rates and no significant side effects. However, in the last years increasingly cases of tolerance to PZQ have been reported, which has caused growing concerns regarding the emergency of resistance to this drug. Methodology/Principal Findings Here we describe the selection of a parasitic strain that has a stable resistance phenotype to PZQ. It has been reported that drug resistance in helminths might involve efflux pumps such as members of ATP-binding cassette transport proteins, including P-glycoprotein and multidrug resistance-associated protein families. Here we evaluate the role of efflux pumps in Schistosoma mansoni resistance to PZQ, by comparing the efflux pumps activity in susceptible and resistant strains. The evaluation of the efflux activity was performed by an ethidium bromide accumulation assay in presence and absence of Verapamil. The role of efflux pumps in resistance to PZQ was further investigated comparing the response of susceptible and resistant parasites in the absence and presence of different doses of Verapamil, in an ex vivo assay, and these results were further reinforced through the comparison of the expression levels of SmMDR2 RNA by RT-PCR. Conclusions/Significance This work strongly suggests the involvement of Pgp-like transporters SMDR2 in Praziquantel drug resistance in S. mansoni. Low doses of Verapamil successfully reverted drug resistance. Our results might give an indication that a combination therapy with PZQ and natural or synthetic Pgp modulators can be an effective strategy for the treatment of confirmed cases of resistance to PZQ in S. mansoni. PMID:26445012

  18. Pharmacophore generation of 2-substituted benzothiazoles as AdeABC efflux pump inhibitors in A. baumannii.

    PubMed

    Yilmaz, S; Altinkanat-Gelmez, G; Bolelli, K; Guneser-Merdan, D; Over-Hasdemir, M U; Yildiz, I; Aki-Yalcin, E; Yalcin, I

    2014-01-01

    RND family efflux pumps are important for multidrug resistance in Gram-negative bacteria. To date no efflux pump inhibitors for clinical use have been found, so developing the specific inhibitors of this pump system will be beneficial for the treatment of infections caused by these multidrug-resistant pathogens. A set of BSN-coded 2-substituted benzothiazoles were tested alone and in combination with ciprofloxacin (CIP) against the RND family efflux pump AdeABC overexpressor Acinetobacter baumannii SbMox-2 strain. The results indicated that the BSN compounds did not have antimicrobial activity when tested alone. However, if they were applied in combination with CIP, it was observed that the antibiotic had antimicrobial activity against the tested pathogen, possessing a minimum inhibitory concentration value that could be utilized in clinical treatment. A 3D-common features pharmacophore model was applied by using the HipHop method and the generated pharmacophore hypothesis revealed that the hydrogen bond acceptor property of nitrogen in the thiazole ring and the oxygen of the amide substituted at the second position of the benzothiazole ring system were significant for binding to the target protein. Moreover, three hydrophobic aromatic features were found to be essential for inhibitory activity. PMID:24905472

  19. Evaluation of the tannic acid inhibitory effect against the NorA efflux pump of Staphylococcus aureus.

    PubMed

    Tintino, Saulo R; Oliveira-Tintino, Cícera D M; Campina, Fábia F; Silva, Raimundo L P; Costa, Maria do S; Menezes, Irwin R A; Calixto-Júnior, João T; Siqueira-Junior, José P; Coutinho, Henrique D M; Leal-Balbino, Tereza C; Balbino, Valdir Q

    2016-08-01

    During the early periods of antibiotic usage, bacterial infections were considered tamed. However, widespread antibiotic use has promoted the emergence of antibiotic-resistant pathogens, including multidrug resistant strains. Active efflux is a mechanism for bacterial resistance to inhibitory substances, known simply as drug efflux pumps. The bacterium Staphylococcus aureus is an important pathogenic bacterium responsible for an array of infections. The NorA efflux pump has been shown to be responsible for moderate fluoroquinolone resistance of S. aureus. The inhibition of the efflux pump was assayed using a sub-inhibitory concentration of standard efflux pump inhibitors and tannic acid (MIC/8), where its capacity to decrease the MIC of Ethidium bromide (EtBr) and antibiotics due to the possible inhibitory effect of these substances was observed. The MICs of EtBr and antibiotics were significantly reduced in the presence of tannic acid, indicating the inhibitory effect of this agent against the efflux pumps of both strains causing a three-fold reduction of the MIC when compared with the control. These results indicate the possible usage of tannic acid as an adjuvant in antibiotic therapy against multidrug resistant bacteria (MDR). PMID:27057677

  20. Role of Efflux Pumps and Intracellular Thiols in Natural Antimony Resistant Isolates of Leishmania donovani

    PubMed Central

    Rai, Smita; Bhaskar; Goel, Sudhir K.; Nath Dwivedi, Upendra; Sundar, Shyam; Goyal, Neena

    2013-01-01

    Background In view of the recent upsurge in the phenomenon of therapeutic failure, drug resistance in Leishmania, developed under natural field conditions, has become a great concern yet little understood. Accordingly, the study of determinants of antimony resistance is urgently warranted. Efflux transporters have been reported in Leishmania but their role in clinical resistance is still unknown. The present study was designed to elucidate the mechanism of natural antimony resistance in L. donovani field isolates by analyzing the functionality of efflux pump(s) and expression profiles of known genes involved in transport and thiol based redox metabolism Methodology/Principal Findings We selected 7 clinical isolates (2 sensitive and 5 resistant) in addition to laboratory sensitive reference and SbIII resistant mutant strains for the present study. Functional characterization using flow cytometry identified efflux pumps that transported substrates of both P-gp and MRPA and were inhibited by the calmodulin antagonist trifluoperazine. For the first time, verapamil sensitive efflux pumps for rhodamine 123 were observed in L. donovani that were differentially active in resistant isolates. RT-PCR confirmed the over-expression of MRPA in isolates with high resistance index only. Resistant isolates also exhibited consistent down regulation of AQP1 and elevated intracellular thiol levels which were accompanied with increased expression of ODC and TR genes. Interestingly, γ-GCS is not implicated in clinical resistance in L. donovani isolates. Conclusions/Significance Here we demonstrate for the first time, the role of P-gp type plasma membrane efflux transporter(s) in antimony resistance in L. donovani field isolates. Further, decreased levels of AQP1 and elevated thiols levels have emerged as biomarkers for clinical resistance. PMID:24069359

  1. Inhibitors of Bacterial Multidrug Efflux Pumps Potentiate Antimicrobial Photoinactivation▿

    PubMed Central

    Tegos, George P.; Masago, Kayo; Aziz, Fatima; Higginbotham, Andrew; Stermitz, Frank R.; Hamblin, Michael R.

    2008-01-01

    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. PMID:18474586

  2. Sarothrin from Alkanna orientalis is an antimicrobial agent and efflux pump inhibitor

    PubMed Central

    Bame, Jessica R.; Graf, Tyler N.; Junio, Hiyas A.; Bussey, R. Owen; Jarmusch, Scott A.; El-Elimat, Tamam; Falkinham, Joseph O.; Oberlies, Nicholas H.; Cech, Richard A.; Cech, Nadja B.

    2015-01-01

    An Alkanna orientalis leaf and flower extract inhibited the growth of Staphylococcus aureus, a pathogen that causes an estimated 478,000 hospitalizations in the US annually. Bioassay-guided fractionation of A. orientalis resulted in isolation of the flavonoid sarothrin (5,7,4′-trihydroxy-3,6,8-trimethoxyflavone), which inhibited the growth of Mycobacterium smegmatis (MIC 75 μM) and S. aureus (MIC >800 μM), and possessed efflux pump inhibitory activity. This is the first report of antimicrobial or efflux pump inhibitory activity of sarothrin, and of its presence in A. orientalis. Our findings suggest that the effectiveness of A. orientalis extracts is due to a combination of multiple constituents, including sarothrin. PMID:23468310

  3. Chalcone inhibitors of the NorA efflux pump in Staphylococcus aureus whole cells and enriched everted membrane vesicles.

    PubMed

    Holler, Jes Gitz; Slotved, Hans-Christian; Mølgaard, Per; Olsen, Carl Erik; Christensen, Søren Brøgger

    2012-07-15

    A library of 117 chalcones was screened for efflux pump inhibitory (EPI) activity against NorA mediated ethidium bromide efflux. Five of the chalcones (5-7, 9, and 10) were active and two chalcones (9 and 10) were equipotent to reserpine with IC(50)-values of 9.0 and 7.7 μM, respectively. Twenty chalcones were subsequently proved to be inhibitors of the NorA efflux pump in everted membrane vesicles. Compounds 5, 7, and 9 synergistically increased the effect of ciprofloxacin on Staphylococcus aureus. Our results suggest that chalcones might be developed into drugs for overcoming multidrug resistance based on efflux transporters of microorganisms. PMID:22682300

  4. Low-Level Resistance of Staphylococcus aureus to Thrombin-Induced Platelet Microbicidal Protein 1 In Vitro Associated with qacA Gene Carriage Is Independent of Multidrug Efflux Pump Activity

    PubMed Central

    Bayer, A. S.; Kupferwasser, L. I.; Brown, M. H.; Skurray, R. A.; Grkovic, S.; Jones, T.; Mukhopadhay, K.; Yeaman, M. R.

    2006-01-01

    Thrombin-induced platelet microbial protein 1 (tPMP-1), a cationic antimicrobial polypeptide released from thrombin-stimulated rabbit platelets, targets the Staphylococcus aureus cytoplasmic membrane to initiate its microbicidal effects. In vitro resistance to tPMP-1 correlates with survival advantages in vivo. In S. aureus, the plasmid-carried qacA gene encodes a multidrug transporter, conferring resistance to organic cations (e.g., ethidium [Et]) via proton motive force (PMF)-energized export. We previously showed that qacA also confers a tPMP-1-resistant (tPMP-1r) phenotype in vitro. The current study evaluated whether (i) transporters encoded by the qacB and qacC multidrug resistance genes also confer tPMP-1r and (ii) tPMP-1r mediated by qacA is dependent on efflux pump activity. In contrast to tPMP-1r qacA-bearing strains, the parental strain and its isogenic qacB- and qacC-containing strains were tPMP-1 susceptible (tPMP-1s). Efflux pump inhibition by cyanide m-chlorophenylhydrazone abrogated Etr, but not tPMP-1r, in the qacA-bearing strain. In synergy assays, exposure of the qacA-bearing strain to tPMP-1 did not affect the susceptibility of Et (ruling out Et-tPMP-1 cotransport). The following cytoplasmic membrane parameters did not differ significantly between the qacA-bearing and parental strains: contents of the major phospholipids; asymmetric distributions of the positively charged species, lysyl-phosphotidylglycerol; fatty acid composition; and relative surface charge. Of note, the qacA-bearing strain exhibited greater membrane fluidity than that of the parental, qacB-, or qacC-bearing strain. In conclusion, among these families of efflux pumps, only the multidrug transporter encoded by qacA conferred a tPMP-1r phenotype. These data suggest that qacA-encoded tPMP-1r results from the impact of a specific transporter upon membrane structure or function unrelated to PMF-dependent peptide efflux. PMID:16801425

  5. Novel epitopes identified from efflux pumps of Mycobacterium tuberculosis could induce cytotoxic T lymphocyte response

    PubMed Central

    Zhai, Ming-xia; Chen, Fei; Zhao, Yuan-yuan; Wu, Ya-hong; Li, Guo-dong; Qi, Yuan-ming

    2015-01-01

    Overcoming drug-resistance is one of the major challenges to control tuberculosis (TB). The up-regulation of efflux pumps is one common mechanism that leads to drug-resistance. Therefore, immunotherapy targeting these efflux pump antigens could be promising strategy to be combined with current chemotherapy. Considering that CD8+ cytotoxic T lymphocytes (CTLs) induced by antigenic peptides (epitopes) could elicit HLA-restricted anti-TB immune response, efflux pumps from classical ABC family (Mycobacterium tuberculosis, Mtb) were chosen as target antigens to identify CTL epitopes. HLA-A2 restricted candidate peptides from Rv2937, Rv2686c and Rv2687c of Mycobacterium tuberculosis were predicted, synthesized and tested. Five peptides could induce IFN-γ release and cytotoxic activity in PBMCs from HLA-A2+ PPD+ donors. Results from HLA-A2/Kb transgenic mice immunization assay suggested that four peptides Rv2937-p168, Rv2937-p266, Rv2686c-p151, and Rv2686c-p181 could induce significant CTL response in vivo. These results suggested that these novel epitopes could be used as immunotherapy candidates to TB drug-resistance. PMID:26417538

  6. Multidrug Efflux Pumps from Enterobacteriaceae, Vibrio cholerae and Staphylococcus aureus Bacterial Food Pathogens

    PubMed Central

    Andersen, Jody L.; He, Gui-Xin; Kakarla, Prathusha; KC, Ranjana; Kumar, Sanath; Lakra, Wazir Singh; Mukherjee, Mun Mun; Ranaweera, Indrika; Shrestha, Ugina; Tran, Thuy; Varela, Manuel F.

    2015-01-01

    Foodborne illnesses caused by bacterial microorganisms are common worldwide and constitute a serious public health concern. In particular, microorganisms belonging to the Enterobacteriaceae and Vibrionaceae families of Gram-negative bacteria, and to the Staphylococcus genus of Gram-positive bacteria are important causative agents of food poisoning and infection in the gastrointestinal tract of humans. Recently, variants of these bacteria have developed resistance to medically important chemotherapeutic agents. Multidrug resistant Escherichia coli, Salmonella enterica, Vibrio cholerae, Enterobacter spp., and Staphylococcus aureus are becoming increasingly recalcitrant to clinical treatment in human patients. Of the various bacterial resistance mechanisms against antimicrobial agents, multidrug efflux pumps comprise a major cause of multiple drug resistance. These multidrug efflux pump systems reside in the biological membrane of the bacteria and actively extrude antimicrobial agents from bacterial cells. This review article summarizes the evolution of these bacterial drug efflux pump systems from a molecular biological standpoint and provides a framework for future work aimed at reducing the conditions that foster dissemination of these multidrug resistant causative agents through human populations. PMID:25635914

  7. The pseudo-atomic structure of an RND-type tripartite multidrug efflux pump.

    PubMed

    Du, Dijun; Voss, Jarrod; Wang, Zhao; Chiu, Wah; Luisi, Ben F

    2015-09-01

    Microorganisms encode several classes of transmembrane molecular pumps that can expel a wide range of chemically distinct toxic substances. These machines contribute to the capacity of the organisms to withstand harsh environments, and they help to confer resistance against clinical antimicrobial agents. In Gram-negative bacteria, some of the pumps comprise tripartite assemblies that actively transport drugs and other harmful compounds across the cell envelope. We describe recent structural and functional data that have provided insights into the architecture and transport mechanism of the AcrA-AcrB-TolC pump of Escherichia coli. This multidrug efflux pump is powered by proton electrochemical gradients through the activity of AcrB, a member of the resistance/nodulation/cell division (RND) transporter family. Crystallographic data reveal how the small protein AcrZ binds to AcrB in a concave surface of the transmembrane domain, and we discuss how this interaction may affect the efflux activities of the transporter. PMID:25803077

  8. Enhancement of antibiotic activity by efflux inhibitors against multidrug resistant Mycobacterium tuberculosis clinical isolates from Brazil

    PubMed Central

    Coelho, Tatiane; Machado, Diana; Couto, Isabel; Maschmann, Raquel; Ramos, Daniela; von Groll, Andrea; Rossetti, Maria L.; Silva, Pedro A.; Viveiros, Miguel

    2015-01-01

    Drug resistant tuberculosis continues to increase and new approaches for its treatment are necessary. The identification of M. tuberculosis clinical isolates presenting efflux as part of their resistant phenotype has a major impact in tuberculosis treatment. In this work, we used a checkerboard procedure combined with the tetrazolium microplate-based assay (TEMA) to study single combinations between antituberculosis drugs and efflux inhibitors (EIs) against multidrug resistant M. tuberculosis clinical isolates using the fully susceptible strain H37Rv as reference. Efflux activity was studied on a real-time basis by a fluorometric method that uses ethidium bromide as efflux substrate. Quantification of efflux pump genes mRNA transcriptional levels were performed by RT-qPCR. The fractional inhibitory concentrations (FIC) indicated synergistic activity for the interactions between isoniazid, rifampicin, amikacin, ofloxacin, and ethidium bromide plus the EIs verapamil, thioridazine and chlorpromazine. The FICs ranged from 0.25, indicating a four-fold reduction on the MICs, to 0.015, 64-fold reduction. The detection of active efflux by real-time fluorometry showed that all strains presented intrinsic efflux activity that contributes to the overall resistance which can be inhibited in the presence of the EIs. The quantification of the mRNA levels of the most important efflux pump genes on these strains shows that they are intrinsically predisposed to expel toxic compounds as the exposure to subinhibitory concentrations of antibiotics were not necessary to increase the pump mRNA levels when compared with the non-exposed counterpart. The results obtained in this study confirm that the intrinsic efflux activity contributes to the overall resistance in multidrug resistant clinical isolates of M. tuberculosis and that the inhibition of efflux pumps by the EIs can enhance the clinical effect of antibiotics that are their substrates. PMID:25972842

  9. Structure of the AcrAB-TolC multidrug efflux pump.

    PubMed

    Du, Dijun; Wang, Zhao; James, Nathan R; Voss, Jarrod E; Klimont, Ewa; Ohene-Agyei, Thelma; Venter, Henrietta; Chiu, Wah; Luisi, Ben F

    2014-05-22

    The capacity of numerous bacterial species to tolerate antibiotics and other toxic compounds arises in part from the activity of energy-dependent transporters. In Gram-negative bacteria, many of these transporters form multicomponent 'pumps' that span both inner and outer membranes and are driven energetically by a primary or secondary transporter component. A model system for such a pump is the acridine resistance complex of Escherichia coli. This pump assembly comprises the outer-membrane channel TolC, the secondary transporter AcrB located in the inner membrane, and the periplasmic AcrA, which bridges these two integral membrane proteins. The AcrAB-TolC efflux pump is able to transport vectorially a diverse array of compounds with little chemical similarity, thus conferring resistance to a broad spectrum of antibiotics. Homologous complexes are found in many Gram-negative species, including in animal and plant pathogens. Crystal structures are available for the individual components of the pump and have provided insights into substrate recognition, energy coupling and the transduction of conformational changes associated with the transport process. However, how the subunits are organized in the pump, their stoichiometry and the details of their interactions are not known. Here we present the pseudo-atomic structure of a complete multidrug efflux pump in complex with a modulatory protein partner from E. coli. The model defines the quaternary organization of the pump, identifies key domain interactions, and suggests a cooperative process for channel assembly and opening. These findings illuminate the basis for drug resistance in numerous pathogenic bacterial species. PMID:24747401

  10. Genotypic and phenotypic detection of efflux pump in Rhodococcus equi

    PubMed Central

    Gressler, Letícia Trevisan; de Vargas, Agueda Castagna; da Costa, Mateus Matiuzzi; Pötter, Luciana; da Silveira, Bibiana Petri; Sangioni, Luis Antônio; de Avila Botton, Sônia

    2014-01-01

    The req_39680 gene, associated to a putative efflux system, was detected in 60% (54/90) of R. equi isolates by PCR. The phenotypic expression of efflux mechanism was verified in 20% of the isolates using ethidium bromide. For the first time, the expression of efflux mechanism was demonstrated in R. equi. PMID:25242956

  11. Modulation of Bacterial Multidrug Resistance Efflux Pumps of the Major Facilitator Superfamily

    PubMed Central

    Kumar, Sanath; Mukherjee, Mun Mun; Varela, Manuel F.

    2013-01-01

    Bacterial infections pose a serious public health concern, especially when an infectious disease has a multidrug resistant causative agent. Such multidrug resistant bacteria can compromise the clinical utility of major chemotherapeutic antimicrobial agents. Drug and multidrug resistant bacteria harbor several distinct molecular mechanisms for resistance. Bacterial antimicrobial agent efflux pumps represent a major mechanism of clinical resistance. The major facilitator superfamily (MFS) is one of the largest groups of solute transporters to date and includes a significant number of bacterial drug and multidrug efflux pumps. We review recent work on the modulation of multidrug efflux pumps, paying special attention to those transporters belonging primarily to the MFS. PMID:25750934

  12. Multidrug Efflux Pumps Attenuate the Effect of MGMT Inhibitors.

    PubMed

    Tomaszowski, Karl-Heinz; Schirrmacher, Ralf; Kaina, Bernd

    2015-11-01

    Various mechanisms of drug resistance attenuate the effectiveness of cancer therapeutics, including drug transport and DNA repair. The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) is a key factor determining the resistance against alkylating anticancer drugs inducing the genotoxic DNA lesions O(6)-methylguanine and O(6)-chloroethylguanine, and MGMT inactivation or depletion renders cells more susceptible to treatment with methylating and chloroethylating agents. Highly specific and efficient inhibitors of the repair protein MGMT were designed, including O(6)-benzylguanine (O(6)BG) and O(6)-(4-bromothenyl)guanine (O(6)BTG) that are nontoxic on their own. Unfortunately, these inhibitors do not select between MGMT in normal and cancer cells, causing nontarget effects in the healthy tissue. Therefore, a targeting strategy for MGMT inhibitors is required. Here, we used O(6)BG and O(6)BTG conjugated to β-d-glucose (O(6)BG-Glu and O(6)BTG-Glu, respectively) in order to selectively inhibit MGMT in tumors, harnessing their high demand for glucose. Both glucose conjugates efficiently inhibited MGMT in several cancer cell lines, but with different extents of sensitization to DNA alkylating agents, with lomustine being more effective than temozolomide. We further show that the glucose conjugates are subject to ATP-binding cassette (ABC) transporter mediated efflux, involving P-glycoprotein, MRP1, and BCRP, which impacts the efficiency of MGMT inhibition. Surprisingly, also O(6)BG and O(6)BTG were subject to an active transport out of the cell. We also show that pharmacological inhibition of efflux transporters increases the induction of cell death following treatment with these MGMT inhibitors and temozolomide. We conclude that strategies of attenuating the efflux by ABC transporters are required for achieving successful MGMT targeting. PMID:26379107

  13. Characterization of a Novel Pyranopyridine Inhibitor of the AcrAB Efflux Pump of Escherichia coli

    PubMed Central

    Kwasny, Steven M.; Kim, Hong-Suk; Nguyen, Son T.; Houseweart, Chad; D'Souza, Sanjay; Walker, Graham C.; Peet, Norton P.; Nikaido, Hiroshi; Bowlin, Terry L.

    2014-01-01

    Members of the resistance-nodulation-division (RND) family of efflux pumps, such as AcrAB-TolC of Escherichia coli, play major roles in multidrug resistance (MDR) in Gram-negative bacteria. A strategy for combating MDR is to develop efflux pump inhibitors (EPIs) for use in combination with an antibacterial agent. Here, we describe MBX2319, a novel pyranopyridine EPI with potent activity against RND efflux pumps of the Enterobacteriaceae. MBX2319 decreased the MICs of ciprofloxacin (CIP), levofloxacin, and piperacillin versus E. coli AB1157 by 2-, 4-, and 8-fold, respectively, but did not exhibit antibacterial activity alone and was not active against AcrAB-TolC-deficient strains. MBX2319 (3.13 μM) in combination with 0.016 μg/ml CIP (minimally bactericidal) decreased the viability (CFU/ml) of E. coli AB1157 by 10,000-fold after 4 h of exposure, in comparison with 0.016 μg/ml CIP alone. In contrast, phenyl-arginine-β-naphthylamide (PAβN), a known EPI, did not increase the bactericidal activity of 0.016 μg/ml CIP at concentrations as high as 100 μM. MBX2319 increased intracellular accumulation of the fluorescent dye Hoechst 33342 in wild-type but not AcrAB-TolC-deficient strains and did not perturb the transmembrane proton gradient. MBX2319 was broadly active against Enterobacteriaceae species and Pseudomonas aeruginosa. MBX2319 is a potent EPI with possible utility as an adjunctive therapeutic agent for the treatment of infections caused by Gram-negative pathogens. PMID:24247144

  14. Structure of the AcrAB-TolC multidrug efflux pump

    PubMed Central

    Du, Dijun; Wang, Zhao; James, Nathan R.; Voss, Jarrod E.; Klimont, Ewa; Ohene-Agyei, Thelma; Venter, Henrietta; Chiu, Wah; Luisi, Ben F.

    2015-01-01

    The capacity of numerous bacterial species to tolerate antibiotics and other toxic compounds arises in part from the activity of energy-dependent transporters. In Gram-negative bacteria, many of these transporters form multicomponent ‘pumps’ that span both inner and outer membranes and are driven energetically by a primary or secondary transporter component1-7. A model system for such a pump is the acridine resistance complex of Escherichia coli1. This pump assembly comprises the outer-membrane channel TolC, the secondary transporter AcrB located in the inner membrane, and the periplasmic AcrA, which bridges these two integral membrane proteins. The AcrAB-TolC efflux pump is able to vectorially transport a diverse array of compounds with little chemical similarity, and accordingly confers resistance to a broad spectrum of antibiotics. Homologous complexes are found in many Gram-negative species, including pathogens of animals and plants. Crystal structures are available for the individual pump components2-7 and these have provided insights into substrate recognition, energy coupling and the transduction of conformational changes associated with the transport process. How the subunits are organised in the pump, their stoichiometry and the details of their interactions are not known and are under debate. In this manuscript, we present the pseudoatomic structure of a complete multidrug efflux pump in complex with a modulatory protein partner8. The model defines the quaternary organization of the pump, identifies key domain interactions, and suggests a cooperative process for channel assembly and opening. These findings illuminate the basis for drug resistance in numerous pathogenic bacterial species. PMID:24747401

  15. Rifaximin resistance in Escherichia coli associated with inflammatory bowel disease correlates with prior rifaximin use, mutations in rpoB, and activity of Phe-Arg-β-naphthylamide-inhibitable efflux pumps.

    PubMed

    Kothary, Vishesh; Scherl, Ellen J; Bosworth, Brian; Jiang, Zhi-Dong; Dupont, Herbert L; Harel, Josee; Simpson, Kenneth W; Dogan, Belgin

    2013-02-01

    Escherichia coli is implicated in the pathogenesis of inflammatory bowel disease (IBD). Rifaximin, a nonabsorbable derivative of rifampin effective against E. coli, improves symptoms in mild-to-moderate IBD. However, rifaximin resistance can develop in a single step in vitro. We examined the prevalence and mechanisms of rifaximin resistance in 62 strains of E. coli isolated from the ileal mucosa of 50 patients (19 with ileal Crohn's disease [L1+L3], 6 with colonic Crohn's disease [L2], 13 with ulcerative colitis [UC], 4 with symptomatic non-IBD diagnoses [NI], and 8 healthy [H]). Resistance (MIC > 1,024 mg/liter) was present in 12/48 IBD-associated ileal E. coli strains. Resistance correlated with prior rifaximin treatment (P < 0.00000001) but not with the presence of ileal inflammation (P = 0.73) or E. coli phylogroup. Mutations in a 1,057-bp region of rpoB, which encodes the bacterial target of rifaximin, were identified in 10/12 resistant strains versus 0/50 sensitive strains (P < 0.000000001) and consisted of seven amino acid substitutions. The efflux pump inhibitor Phe-Arg-β-naphthylamide (PAβN) lowered the MIC of 9/12 resistant strains 8- to 128-fold. Resistance was stable in the absence of rifaximin in 10/12 resistant strains after 30 passages. We conclude that IBD-associated ileal E. coli frequently manifest resistance to rifaximin that correlates with prior rifaximin use, amino acid substitutions in rpoB, and activity of PAβN-inhibitable efflux pumps, but not with the presence of ileal inflammation or E. coli phylogroup. These findings have significant implications for treatment trials targeting IBD-associated E. coli. PMID:23183443

  16. Rifaximin Resistance in Escherichia coli Associated with Inflammatory Bowel Disease Correlates with Prior Rifaximin Use, Mutations in rpoB, and Activity of Phe-Arg-β-Naphthylamide-Inhibitable Efflux Pumps

    PubMed Central

    Kothary, Vishesh; Scherl, Ellen J.; Bosworth, Brian; Jiang, Zhi-Dong; DuPont, Herbert L.; Harel, Josee

    2013-01-01

    Escherichia coli is implicated in the pathogenesis of inflammatory bowel disease (IBD). Rifaximin, a nonabsorbable derivative of rifampin effective against E. coli, improves symptoms in mild-to-moderate IBD. However, rifaximin resistance can develop in a single step in vitro. We examined the prevalence and mechanisms of rifaximin resistance in 62 strains of E. coli isolated from the ileal mucosa of 50 patients (19 with ileal Crohn's disease [L1+L3], 6 with colonic Crohn's disease [L2], 13 with ulcerative colitis [UC], 4 with symptomatic non-IBD diagnoses [NI], and 8 healthy [H]). Resistance (MIC > 1,024 mg/liter) was present in 12/48 IBD-associated ileal E. coli strains. Resistance correlated with prior rifaximin treatment (P < 0.00000001) but not with the presence of ileal inflammation (P = 0.73) or E. coli phylogroup. Mutations in a 1,057-bp region of rpoB, which encodes the bacterial target of rifaximin, were identified in 10/12 resistant strains versus 0/50 sensitive strains (P < 0.000000001) and consisted of seven amino acid substitutions. The efflux pump inhibitor Phe-Arg-β-naphthylamide (PAβN) lowered the MIC of 9/12 resistant strains 8- to 128-fold. Resistance was stable in the absence of rifaximin in 10/12 resistant strains after 30 passages. We conclude that IBD-associated ileal E. coli frequently manifest resistance to rifaximin that correlates with prior rifaximin use, amino acid substitutions in rpoB, and activity of PAβN-inhibitable efflux pumps, but not with the presence of ileal inflammation or E. coli phylogroup. These findings have significant implications for treatment trials targeting IBD-associated E. coli. PMID:23183443

  17. The efflux pump SmeDEF contributes to trimethoprim-sulfamethoxazole resistance in Stenotrophomonas maltophilia.

    PubMed

    Sánchez, María Blanca; Martínez, José Luis

    2015-07-01

    Trimethoprim-sulfamethoxazole (co-trimoxazole) is one of the antimicrobials of choice for the treatment of Stenotrophomonas maltophilia infections. The analysis of mutants either lacking or overexpressing the efflux pump SmeDEF shows that this efflux pump contributes to intrinsic and acquired co-trimoxazole resistance in S. maltophilia. Since SmeDEF can extrude a variety of antibiotics, selection with such antimicrobials, including quinolones, might also select for S. maltophilia co-trimoxazole resistance. PMID:25918144

  18. Multidrug efflux pumps as main players in intrinsic and acquired resistance to antimicrobials.

    PubMed

    Hernando-Amado, Sara; Blanco, Paula; Alcalde-Rico, Manuel; Corona, Fernando; Reales-Calderón, Jose A; Sánchez, María B; Martínez, José L

    2016-09-01

    Multidrug efflux pumps constitute a group of transporters that are ubiquitously found in any organism. In addition to other functions with relevance for the cell physiology, efflux pumps contribute to the resistance to compounds used for treating different diseases, including resistance to anticancer drugs, antibiotics or antifungal compounds. In the case of antimicrobials, efflux pumps are major players in both intrinsic and acquired resistance to drugs currently in use for the treatment of infectious diseases. One important aspect not fully explored of efflux pumps consists on the identification of effectors able to induce their expression. Indeed, whereas the analysis of clinical isolates have shown that mutants overexpressing these resistance elements are frequently found, less is known on the conditions that may trigger expression of efflux pumps, hence leading to transient induction of resistance in vivo, a situation that is barely detectable using classical susceptibility tests. In the current article we review the structure and mechanisms of regulation of the expression of bacterial and fungal efflux pumps, with a particular focus in those for which a role in clinically relevant resistance has been reported. PMID:27620952

  19. Broad Specificity Efflux pumps and Their Role in Multidrug Resistance of Gram Negative Bacteria

    PubMed Central

    Nikaido, Hiroshi; Pagès, Jean-Marie

    2013-01-01

    Antibiotic resistance mechanisms reported in Gram-negative bacteria are producing a worldwide health problem. The continuous dissemination of «multi-drug resistant» (MDR) bacteria drastically reduces the efficacy of our antibiotic “arsenal” and consequently increases the frequency of therapeutic failure. In MDR bacteria, the over-expression of efflux pumps that expel structurally-unrelated drugs contributes to the reduced susceptibility by decreasing the intracellular concentration of antibiotics. During the last decade, several clinical data indicate an increasing involvement of efflux pumps in the emergence and dissemination of resistant Gram-negative bacteria. It is necessary to clearly define the molecular, functional and genetic bases of the efflux pump in order to understand the translocation of antibiotic molecules through the efflux transporter. The recent investigation on the efflux pump AcrB at its structural and physiological level, including the identification of drug affinity sites and kinetic parameters for various antibiotics, may open the way to rationally develop an improved new generation of antibacterial agents as well as efflux inhibitors in order to efficiently combat efflux-based resistance mechanisms. PMID:21707670

  20. Doxorubicin induces drug efflux pumps in Candida albicans.

    PubMed

    Kofla, Grzegorz; Turner, Vincent; Schulz, Bettina; Storch, Ulrike; Froelich, Daniela; Rognon, Bénédicte; Coste, Alix T; Sanglard, Dominique; Ruhnke, Markus

    2011-02-01

    Candida albicans is one of the most important opportunistic fungal pathogens. It can cause serious fungal diseases in immunocompromised patients, including those with cancer. Treatment failures due to the emergence of drug-resistant C. albicans strains have become a serious clinical problem. Resistance incidents were often mediated by fungal efflux pumps which are closely related to the human ABC transporter P-glycoprotein (P-gp). P-gp is often overexpressed in cancer cells and confers resistance to many cytotoxic drugs. We examined whether cytotoxic drugs commonly used for cancer treatment (doxorubicin and cyclophosphamide) could alter the expression of genes responsible for the development of fluconazole resistance in Candida cells in the way they can influence homologous genes in cancer cell lines. ABC transporters (CDR1 and CDR2) and other resistance genes (MDR1 and ERG11) were tested by real-time PCR for their expression in C. albicans cells at the mRNA level after induction by antineoplastic drugs. The results were confirmed by a lacZ gene reporter system and verified at the protein level using GFP and immunoblotting. We showed that doxorubicin is a potent inducer of CDR1/CDR2 expression in C. albicans at both the mRNA and protein level and thus causes an increase in fluconazole MIC values. However, cyclophosphamide, which is not a substrate of human P-gp, did not induce ABC transporter expression in C. albicans. Neither doxorubicin nor cyclophosphamide could influence the expression of the other resistance genes (MDR1 and ERG11). The induction of CDR1/CDR2 by doxorubicin in C. albicans and the resulting alteration of antifungal susceptibility might be of clinical relevance for the antifungal treatment of Candida infections occurring after anticancer chemotherapy with doxorubicin. PMID:20818920

  1. Tigecycline Susceptibility and the Role of Efflux Pumps in Tigecycline Resistance in KPC-Producing Klebsiella pneumoniae

    PubMed Central

    Chen, Qiong; Ruan, Zhi; Hua, Xiaoting; Zhou, Hua; Yu, Yunsong

    2015-01-01

    KPC-producing Klebsiella pneumoniae isolates have emerged as important pathogens of nosocomial infections, and tigecycline is one of the antibiotics recommended for severe infections caused by KPC-producing K. pneumoniae. To identify the susceptibility profile of KPC-producing K. pneumoniae to tigecycline and investigate the role of efflux pumps in tigecycline resistance, a total of 215 KPC-producing K. pneumoniae isolates were collected. The minimum inhibitory concentration (MIC) of tigecycline was determined by standard broth microdilution tests. Isolates showing resistance to tigecycline underwent susceptibility test with efflux pump inhibitors. Expression levels of efflux pump genes (acrB and oqxB) and their regulators (ramA, marA, soxS and rarA) were examined by real-time PCR, and the correlation between tigecycline MICs and gene expression levels were analysed. Our results show that the tigecycline resistance rate in these isolates was 11.2%. Exposure of the tigecycline-resistant isolates to the efflux pump inhibitor NMP resulted in an obvious decrease in MICs and restored susceptibility to tigecycline in 91.7% of the isolates. A statistically significant association between acrB expression and tigecycline MICs was observed, and overexpression of ramA was found in three tigecycline-resistant isolates, further analysis confirmed ramR mutations existed in these isolates. Transformation of one mutant with wild-type ramR restored susceptibility to tigecycline and repressed overexpression of ramA and acrB. These data indicate that efflux pump AcrAB, which can be up-regulated by ramR mutations and subsequent ramA activation, contributed to tigecycline resistance in K. pneumoniae clinical isolates. PMID:25734903

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

    PubMed Central

    Vargiu, Attilio V.; Ruggerone, Paolo; Opperman, Timothy J.; Nguyen, Son T.

    2014-01-01

    Efflux 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 silico structure-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 of Enterobacteriaceae species, 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. PMID:25114133

  3. The MexJK Efflux Pump of Pseudomonas aeruginosa Requires OprM for Antibiotic Efflux but Not for Efflux of Triclosan

    PubMed Central

    Chuanchuen, Rungtip; Narasaki, Craig T.; Schweizer, Herbert P.

    2002-01-01

    Using the biocide triclosan as a selective agent, several triclosan-resistant mutants of a susceptible Pseudomonas aeruginosa strain were isolated. Cloning and characterization of a DNA fragment conferring triclosan resistance from one of these mutants revealed a hitherto uncharacterized efflux system of the resistance nodulation cell division (RND) family, which was named MexJK and which is encoded by the mexJK operon. Expression of this operon is negatively regulated by the product of mexL, a gene located upstream of and transcribed divergently from mexJK. The triclosan-resistant mutant contained a single nucleotide change in mexL, which caused an amino acid change in the putative helix-turn-helix domain of MexL. The MexL protein belongs to the TetR family of repressor proteins. The MexJK system effluxed tetracycline and erythromycin but only in the presence of the outer membrane protein channel OprM; OprJ and OprN did not function with MexJK. Triclosan efflux required neither of the outer membrane protein channels tested but necessitated the MexJ membrane fusion protein and the MexK inner membrane RND transporter. The results presented in this study suggest that MexJK may function as a two-component RND pump for triclosan efflux but must associate with OprM to form a tripartite antibiotic efflux system. Furthermore, the results confirm that triclosan is an excellent tool for the study of RND multidrug efflux systems and that this popular biocide therefore readily selects mutants which are cross-resistant with antibiotics. PMID:12193619

  4. Virtual screening for novel Staphylococcus Aureus NorA efflux pump inhibitors from natural products.

    PubMed

    Thai, Khac-Minh; Ngo, Trieu-Du; Phan, Thien-Vy; Tran, Thanh-Dao; Nguyen, Ngoc-Vinh; Nguyen, Thien-Hai; Le, Minh-Tri

    2015-01-01

    NorA is a member of the Major Facilitator Superfamily (MFS) drug efflux pumps that have been shown to mediate antibiotic resistance in Staphylococcus aureus (SA). In this study, QSAR analysis, virtual screening and molecular docking were implemented in an effort to discover novel SA NorA efflux pump inhibitors. Originally, a set of 47 structurally diverse compounds compiled from the literature was used to develop linear QSAR models and another set of 15 different compounds were chosen for extra validation. The final model which was estimated by statistical values for the full data set (n = 45, Q(2) = 0.80, RMSE = 0.20) and for the external test set (n = 15, R(2) = 0.60, |res|max = 0.75, |res|min = 0.02) was applied on the collection of 182 flavonoides and the traditional Chinese medicine (TCM) database to screen for novel NorA inhibitors. Finally, 33 lead compounds that met the Lipinski's rules of five/three and had good predicted pIC50 values from in silico screening process were employed to analyze the binding ability by docking studies on NorA homology model in place of its unavailable crystal structures at two active sites, the central channel and the Walker B. PMID:25181985

  5. An Inducible Fusaric Acid Tripartite Efflux Pump Contributes to the Fusaric Acid Resistance in Stenotrophomonas maltophilia

    PubMed Central

    Hu, Rouh-Mei; Liao, Sih-Ting; Huang, Chiang-Ching; Huang, Yi-Wei; Yang, Tsuey-Ching

    2012-01-01

    Background Fusaric acid (5-butylpicolinic acid), a mycotoxin, is noxious to some microorganisms. Stenotrophomonas maltophilia displays an intrinsic resistance to fusaric acid. This study aims to elucidate the mechanism responsible for the intrinsic fusaric acid resistance in S. maltophilia. Methodology A putative fusaric acid resistance-involved regulon fuaR-fuaABC was identified by the survey of the whole genome sequence of S. maltophilia K279a. The fuaABC operon was verified by reverse transcriptase-PCR. The contribution of the fuaABC operon to the antimicrobial resistance was evaluated by comparing the antimicrobials susceptibility between the wild-type strain and fuaABC knock-out mutant. The regulatory role of fuaR in the expression of the fuaABC operon was assessed by promoter transcription fusion assay. Results The fuaABC operon was inducibly expressed by fusaric acid and the inducibility was fuaR dependent. FuaR functioned as a repressor of the fuaABC operon in absence of a fusaric acid inducer and as an activator in its presence. Overexpression of the fuaABC operon contributed to the fusaric acid resistance. Significance A novel tripartite fusaric acid efflux pump, FuaABC, was identified in this study. Distinct from the formally classification, the FuaABC may constitute a new type of subfamily of the tripartite efflux pump. PMID:23236431

  6. Resistance-nodulation-division efflux pump acrAB is modulated by florfenicol and contributes to drug resistance in the fish pathogen Piscirickettsia salmonis.

    PubMed

    Sandoval, Rodrigo; Oliver, Cristian; Valdivia, Sharin; Valenzuela, Karla; Haro, Ronie E; Sánchez, Patricio; Olavarría, Víctor H; Valenzuela, Paulina; Avendaño-Herrera, Rubén; Romero, Alex; Cárcamo, Juan G; Figueroa, Jaime E; Yáñez, Alejandro J

    2016-06-01

    Piscirickettsia salmonis is a fastidious intracellular pathogen responsible for high mortality rates in farmed salmonids, with serious economic consequences for the Chilean aquaculture industry. Oxytetracycline and florfenicol are the most frequently used antibiotics against P. salmonis, but routine use could contribute to drug resistance. This study identified differentiated florfenicol susceptibilities in two P. salmonis strains, LF-89 and AUSTRAL-005. The less susceptible isolate, AUSTRAL-005, also showed a high ethidium bromide efflux rate, indicating a higher activity of general efflux pump genes than LF-89. The P. salmonis genome presented resistance nodulation division (RND) family members, a family containing typical multidrug resistance-related efflux pumps in Gram-negative bacteria. Additionally, efflux pump acrAB genes were overexpressed in AUSTRAL-005 following exposure to the tolerated maximal concentration of florfenicol, in contrast to LF-89. These results indicate that tolerated maximum concentrations of florfenicol can modulate RND gene expression and increase efflux pump activity. We propose that the acrAB efflux pump is essential for P. salmonis survival at critical florfenicol concentrations and for the generation of antibiotic-resistant bacterial strains. PMID:27190287

  7. In vitro activity of levofloxacin against planktonic and biofilm Stenotrophomonas maltophilia lifestyles under conditions relevant to pulmonary infection in cystic fibrosis, and relationship with SmeDEF multidrug efflux pump expression.

    PubMed

    Pompilio, Arianna; Crocetta, Valentina; Verginelli, Fabio; Bonaventura, Giovanni Di

    2016-07-01

    The activity of levofloxacin against planktonic and biofilm Stenotrophomonas maltophilia cells and the role played by the multidrug efflux pump SmeDEF were evaluated under conditions relevant to the cystic fibrosis (CF) lung. MIC, MBC and MBEC of levofloxacin were assessed, against five CF strains, under 'standard' (CLSI-recommended) and 'CF-like' (pH 6.8, 5% CO2, in a synthetic CF sputum) conditions. Levofloxacin was tested against biofilms at concentrations (10, 50 and 100 μg mL(-1)) corresponding to achievable serum levels and sputum levels by aerosolisation. smeD expression was evaluated, under both conditions, in planktonic and biofilm cells by RT-PCR. The bactericidal effect of levofloxacin was decreased, in three out of five strains tested, under 'CF-like' conditions (MBC: 2-4 vs 8-16 μg mL(-1), under 'standard' and 'CF-like' conditions, respectively). Biofilm was intrinsically resistant to levofloxacin, regardless of conditions tested (MBECs ≥ 100 μg mL(-1) for all strains). Only under 'CF-like' conditions, smeD expression increased during planktonic-to-biofilm transition, and in biofilm cells compared to stationary planktonic cells. Our findings confirmed that S. maltophilia biofilm is intrinsically resistant to therapeutic concentrations of levofloxacin. Under conditions relevant to CF, smeD overexpression could contribute to levofloxacin resistance. Further studies are warranted to define the clinical relevance of our findings. PMID:27242375

  8. Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins

    PubMed Central

    Mealman, Tiffany D.; McEvoy, Megan M.; Blackburn, Ninian J.

    2014-01-01

    Copper is an essential nutrient for all aerobic organisms but is toxic in excess. At the host–pathogen interface, macrophages respond to bacterial infection by copper-dependent killing mechanisms, whereas the invading bacteria are thought to counter with an up-regulation of copper transporters and efflux pumps. The tripartite efflux pump CusCBA and its metallochaperone CusF are vital to the detoxification of copper and silver ions in the periplasm of Escherichia coli. However, the mechanism of efflux by this complex, which requires the activation of the inner membrane pump CusA, is poorly understood. Here, we use selenomethionine (SeM) active site labels in a series of biological X-ray absorption studies at the selenium, copper, and silver edges to establish a “switch” role for the membrane fusion protein CusB. We determine that metal-bound CusB is required for activation of cuprous ion transfer from CusF directly to a site in the CusA antiporter, showing for the first time (to our knowledge) the in vitro activation of the Cus efflux pump. This metal-binding site of CusA is unlike that observed in the crystal structures of the CusA protein and is composed of one oxygen and two sulfur ligands. Our results suggest that metal transfer occurs between CusF and apo-CusB, and that, when metal-loaded, CusB plays a role in the regulation of metal ion transfer from CusF to CusA in the periplasm. PMID:25313055

  9. Efflux Pump Overexpression in Multiple-Antibiotic-Resistant Mutants of Bacteroides fragilis

    PubMed Central

    Pumbwe, Lilian; Glass, Daniel; Wexler, Hannah M.

    2006-01-01

    Multidrug-resistant mutants of a wild-type Bacteroides fragilis strain (strain ADB77) and a quadruple resistance nodulation division family efflux pump deletion mutant (ADB77 ΔbmeB1 ΔbmeB3 ΔbmeB12 ΔbmeB15) were selected with antimicrobials. Ampicillin, doripenem, imipenem, levofloxacin, and metronidazole selected for mutants from both strains; cefoxitin selected for mutants from strain ADB77 only; and sodium dodecyl sulfate selected mutants from ADB77ΔbmeB1 ΔbmeB3 ΔbmeB12 ΔbmeB15 only. The mutants overexpressed one or more efflux pumps. PMID:16940115

  10. Clonal relatedness is a predictor of spontaneous multidrug efflux pump gene overexpression in Staphylococcus aureus.

    PubMed

    Schindler, Bryan D; Jacinto, Pauline L; Buensalido, Joseph Adrian L; Seo, Susan M; Kaatz, Glenn W

    2015-05-01

    Increased expression of genes encoding multidrug resistance efflux pumps (MDR-EPs) contributes to antimicrobial agent and biocide resistance in Staphylococcus aureus. Previously identified associations between norA overexpression and spa type t002 meticillin-resistant S. aureus (MRSA), and a similar yet weaker association between mepA overexpression and type t008 meticillin-susceptible S. aureus (MSSA), in clinical isolates are suggestive of clonal dissemination. It is also possible that related strains are prone to mutations resulting in overexpression of specific MDR-EP genes. Exposure of non-MDR-EP-overexpressing clinical isolates to biocides and dyes can select for MDR-EP-overexpressing mutants. spa types t002 and t008 isolates are predominated by multilocus sequencing typing sequence types (STs) 5 and 8, respectively. In this study, non-MDR-EP gene-overexpressing clinical isolates (MRSA and MSSA) representing ST5 and ST8 were subjected to single exposures of ethidium bromide (EtBr) to select for EtBr-resistant mutants. Measurements of active EtBr transport among mutants were used to demonstrate an efflux-proficient phenotype. Using quantitative reverse-transcription PCR, it was found that EtBr-resistant mutants of ST5 and ST8 parental strains predominantly overexpressed mepA (100%) and mdeA (83%), respectively, regardless of meticillin sensitivity. Associations between clonal lineage and MDR-EP gene overexpression differed from those previously observed and suggest the latter is due to clonal spread of efflux-proficient strains. The predilection of in vitro-selected mutants of related strains to overexpress the same MDR-EP gene indicates the presence of a consistent mutational process. PMID:25548027

  11. Adaptive drug resistance mediated by root-nodulation-cell division efflux pumps.

    PubMed

    Daniels, C; Ramos, J L

    2009-01-01

    Bacterial resistance to antibiotics is a major therapeutic problem. Bacteria use the same mechanisms for developing resistance to antibiotics as they do for developing resistance to biocide compounds present in some cleaning and personal care products. Root-nodulation-cell division (RND) family efflux pumps are a common means of multidrug resistance, and induction of their expression can explain the observed cross-resistance found between antibiotics and biocides in laboratory strains. Hence, there is a relationship between the active chemicals used in household products, organic solvents and antibiotics. The widespread use of biocide-containing modern-day household products may promote the development of microbial resistance and, in particular, cross-resistance to antibiotics. PMID:19220351

  12. Functionally Cloned pdrM from Streptococcus pneumoniae Encodes a Na+ Coupled Multidrug Efflux Pump

    PubMed Central

    Hashimoto, Kohei; Ogawa, Wakano; Nishioka, Toshihiro; Tsuchiya, Tomofusa; Kuroda, Teruo

    2013-01-01

    Multidrug efflux pumps play an important role as a self-defense system in bacteria. Bacterial multidrug efflux pumps are classified into five families based on structure and coupling energy: resistance−nodulation−cell division (RND), small multidrug resistance (SMR), major facilitator (MF), ATP binding cassette (ABC), and multidrug and toxic compounds extrusion (MATE). We cloned a gene encoding a MATE-type multidrug efflux pump from Streptococcus pneumoniae R6, and designated it pdrM. PdrM showed sequence similarity with NorM from Vibrio parahaemolyticus, YdhE from Escherichia coli, and other bacterial MATE-type multidrug efflux pumps. Heterologous expression of PdrM let to elevated resistance to several antibacterial agents, norfloxacin, acriflavine, and 4′,6-diamidino-2-phenylindole (DAPI) in E. coli KAM32 cells. PdrM effluxes acriflavine and DAPI in a Na+- or Li+-dependent manner. Moreover, Na+ efflux via PdrM was observed when acriflavine was added to Na+-loaded cells expressing pdrM. Therefore, we conclude that PdrM is a Na+/drug antiporter in S. pneumoniae. In addition to pdrM, we found another two genes, spr1756 and spr1877,that met the criteria of MATE-type by searching the S. pneumoniae genome database. However, cloned spr1756 and spr1877 did not elevate the MIC of any of the investigated drugs. mRNA expression of spr1756, spr1877, and pdrM was detected in S. pneumoniae R6 under laboratory growth conditions. Therefore, spr1756 and spr1877 are supposed to play physiological roles in this growth condition, but they may be unrelated to drug resistance. PMID:23555691

  13. The SmeYZ efflux pump of Stenotrophomonas maltophilia contributes to drug resistance, virulence-related characteristics, and virulence in mice.

    PubMed

    Lin, Yi-Tsung; Huang, Yi-Wei; Chen, Shiang-Jiuun; Chang, Chia-Wei; Yang, Tsuey-Ching

    2015-07-01

    The resistance-nodulation-division (RND)-type efflux pump is one of the causes of the multidrug resistance of Stenotrophomonas maltophilia. The roles of the RND-type efflux pump in physiological functions and virulence, in addition to antibiotic extrusion, have attracted much attention. In this study, the contributions of the constitutively expressed SmeYZ efflux pump to drug resistance, virulence-related characteristics, and virulence were evaluated. S. maltophilia KJ is a clinical isolate of multidrug resistance. The smeYZ isogenic deletion mutant, KJΔYZ, was constructed by a gene replacement strategy. The antimicrobial susceptibility, virulence-related physiological characteristics, susceptibility to human serum and neutrophils, and in vivo virulence between KJ and KJΔYZ were comparatively assessed. The SmeYZ efflux pump contributed resistance to aminoglycosides and trimethoprim-sulfamethoxazole. Inactivation of smeYZ resulted in attenuation of oxidative stress susceptibility, swimming, flagella formation, biofilm formation, and secreted protease activity. Furthermore, loss of SmeYZ increased susceptibility to human serum and neutrophils and decreased in vivo virulence in a murine model. These findings suggest the possibility of attenuation of the resistance and virulence of S. maltophilia with inhibitors of the SmeYZ efflux pump. PMID:25918140

  14. The efflux pump inhibitor timcodar improves the potency of antimycobacterial agents.

    PubMed

    Grossman, Trudy H; Shoen, Carolyn M; Jones, Steven M; Jones, Peter L; Cynamon, Michael H; Locher, Christopher P

    2015-03-01

    Previous studies indicated that inhibition of efflux pumps augments tuberculosis therapy. In this study, we used timcodar (formerly VX-853) to determine if this efflux pump inhibitor could increase the potency of antituberculosis (anti-TB) drugs against Mycobacterium tuberculosis in in vitro and in vivo combination studies. When used alone, timcodar weakly inhibited M. tuberculosis growth in broth culture (MIC, 19 μg/ml); however, it demonstrated synergism in drug combination studies with rifampin, bedaquiline, and clofazimine but not with other anti-TB agents. When M. tuberculosis was cultured in host macrophage cells, timcodar had about a 10-fold increase (50% inhibitory concentration, 1.9 μg/ml) in the growth inhibition of M. tuberculosis and demonstrated synergy with rifampin, moxifloxacin, and bedaquiline. In a mouse model of tuberculosis lung infection, timcodar potentiated the efficacies of rifampin and isoniazid, conferring 1.0 and 0.4 log10 reductions in bacterial burden in lung, respectively, compared to the efficacy of each drug alone. Furthermore, timcodar reduced the likelihood of a relapse infection when evaluated in a mouse model of long-term, chronic infection with treatment with a combination of rifampin, isoniazid, and timcodar. Although timcodar had no effect on the pharmacokinetics of rifampin in plasma and lung, it did increase the plasma exposure of bedaquiline. These data suggest that the antimycobacterial drug-potentiating activity of timcodar is complex and drug dependent and involves both bacterial and host-targeted mechanisms. Further study of the improvement of the potency of antimycobacterial drugs and drug candidates when used in combination with timcodar is warranted. PMID:25534740

  15. In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

    PubMed Central

    Verchère, Alice; Dezi, Manuela; Broutin, Isabelle; Picard, Martin

    2014-01-01

    There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution of efflux pumps from the Gram-negative bacteria Pseudomonas aeruginosa in a biomimetic environment that allows for an accurate investigation of their activity of transport. Three prerequisites are fulfilled: compartmentation in a lipidic environment, use of a relevant index for transport, and generation of a proton gradient. The membrane protein transporter is reconstituted into liposomes together with bacteriorhodopsin, a light-activated proton pump that generates a proton gradient that is robust as well as reversible and tunable. The activity of the protein is deduced from the pH variations occurring within the liposome, using pyranin, a pH-dependent fluorescent probe. We describe a step-by-step procedure where membrane protein purification, liposome formation, protein reconstitution, and transport analysis are addressed. Although they were specifically designed for an RND transporter, the described methods could potentially be adapted for use with any other membrane protein transporter energized by a proton gradient. PMID:24638061

  16. Premature Termination of MexR Leads to Overexpression of MexAB-OprM Efflux Pump in Pseudomonas aeruginosa in a Tertiary Referral Hospital in India

    PubMed Central

    2016-01-01

    Objectives The present study was undertaken to investigate the mutations that are present in mexR gene of multidrug resistant (MDR) isolates of Pseudomonas aeruginosa collected from a tertiary referral hospital of north east India. Methods 76 MDR clinical isolates of P. aeruginosa were obtained from the patients who were admitted to or attended the clinics of Silchar medical college and hospital. They were screened phenotypically for the presence of efflux pump activity by an inhibitor based method. Acquired resistance mechanisms were detected by multiplex PCR. Real time PCR was performed to study the expression of mexA gene of MexAB-OprM efflux pump in isolates with increase efflux pump activity. mexR gene of the isolates with overexpressed MexAB-OprM efflux pump was amplified, sequenced and analysed. Results Out of 76 MDR isolates, 24 were found to exhibit efflux pump activity phenotypically against ciprofloxacin and meropenem. Acquired resistance mechanisms were absent in 11 of them and among those isolates, 8 of them overexpressed MexAB-OprM. All the 8 isolates possessed mutation in mexR gene. 11 transversions, 4 transitions, 2 deletion mutations and 2 insertion mutations were found in all the isolates. However, the most significant observation was the formation of a termination codon at 35th position which resulted in the termination of the polypeptide and leads to overexpression of the MexAB-OprM efflux pump. Conclusions This study highlighted emergence of a novel mutation which is probably associated with multi drug resistance. Therefore, further investigations and actions are needed to prevent or at least hold back the expansion and emergence of newer mutations in nosocomial pathogens which may compromise future treatment options. PMID:26866484

  17. LmrS is a multidrug efflux pump of the major facilitator superfamily from Staphylococcus aureus.

    PubMed

    Floyd, Jody L; Smith, Kenneth P; Kumar, Sanath H; Floyd, Jared T; Varela, Manuel F

    2010-12-01

    A multidrug efflux pump designated LmrS (lincomycin resistance protein of Staphylococcus aureus), belonging to the major facilitator superfamily (MFS) of transporters, was cloned, and the role of LmrS in antimicrobial efflux was evaluated. The highest relative increase in MIC, 16-fold, was observed for linezolid and tetraphenylphosphonium chloride (TPCL), followed by an 8-fold increase for sodium dodecyl sulfate (SDS), trimethoprim, and chloramphenicol. LmrS has 14 predicted membrane-spanning domains and is homologous to putative lincomycin resistance proteins of Bacillus spp., Lactobacillus spp., and Listeria spp. PMID:20855745

  18. Structural basis for the blockade of MATE multidrug efflux pumps

    DOE PAGESBeta

    Radchenko, Martha; Symersky, Jindrich; Nie, Rongxin; Lu, Min

    2015-08-06

    Multidrug and toxic compound extrusion (MATE) transporters underpin multidrug resistance by using the H+ or Na+ electrochemical gradient to extrude different drugs across cell membranes. MATE transporters can be further parsed into the DinF, NorM and eukaryotic subfamilies based on their amino-acid sequence similarity. Here we report the 3.0 Å resolution X-ray structures of a protonation-mimetic mutant of an H+-coupled DinF transporter, as well as of an H+-coupled DinF and a Na+-coupled NorM transporters in complexes with verapamil, a small-molecule pharmaceutical that inhibits MATE-mediated multidrug extrusion. Combining structure-inspired mutational and functional studies, we confirm the biological relevance of our crystalmore » structures, reveal the mechanistic differences among MATE transporters, and suggest how verapamil inhibits MATE-mediated multidrug efflux. Our findings offer insights into how MATE transporters extrude chemically and structurally dissimilar drugs and could inform the design of new strategies for tackling multidrug resistance.« less

  19. Structural basis for the blockade of MATE multidrug efflux pumps

    SciTech Connect

    Radchenko, Martha; Symersky, Jindrich; Nie, Rongxin; Lu, Min

    2015-08-06

    Multidrug and toxic compound extrusion (MATE) transporters underpin multidrug resistance by using the H+ or Na+ electrochemical gradient to extrude different drugs across cell membranes. MATE transporters can be further parsed into the DinF, NorM and eukaryotic subfamilies based on their amino-acid sequence similarity. Here we report the 3.0 Å resolution X-ray structures of a protonation-mimetic mutant of an H+-coupled DinF transporter, as well as of an H+-coupled DinF and a Na+-coupled NorM transporters in complexes with verapamil, a small-molecule pharmaceutical that inhibits MATE-mediated multidrug extrusion. Combining structure-inspired mutational and functional studies, we confirm the biological relevance of our crystal structures, reveal the mechanistic differences among MATE transporters, and suggest how verapamil inhibits MATE-mediated multidrug efflux. Our findings offer insights into how MATE transporters extrude chemically and structurally dissimilar drugs and could inform the design of new strategies for tackling multidrug resistance.

  20. Structural basis for the blockade of MATE multidrug efflux pumps

    NASA Astrophysics Data System (ADS)

    Radchenko, Martha; Symersky, Jindrich; Nie, Rongxin; Lu, Min

    2015-08-01

    Multidrug and toxic compound extrusion (MATE) transporters underpin multidrug resistance by using the H+ or Na+ electrochemical gradient to extrude different drugs across cell membranes. MATE transporters can be further parsed into the DinF, NorM and eukaryotic subfamilies based on their amino-acid sequence similarity. Here we report the 3.0 Å resolution X-ray structures of a protonation-mimetic mutant of an H+-coupled DinF transporter, as well as of an H+-coupled DinF and a Na+-coupled NorM transporters in complexes with verapamil, a small-molecule pharmaceutical that inhibits MATE-mediated multidrug extrusion. Combining structure-inspired mutational and functional studies, we confirm the biological relevance of our crystal structures, reveal the mechanistic differences among MATE transporters, and suggest how verapamil inhibits MATE-mediated multidrug efflux. Our findings offer insights into how MATE transporters extrude chemically and structurally dissimilar drugs and could inform the design of new strategies for tackling multidrug resistance.

  1. Structural basis for the blockade of MATE multidrug efflux pumps

    PubMed Central

    Radchenko, Martha; Symersky, Jindrich; Nie, Rongxin; Lu, Min

    2015-01-01

    Multidrug and toxic compound extrusion (MATE) transporters underpin multidrug resistance by using the H+ or Na+ electrochemical gradient to extrude different drugs across cell membranes. MATE transporters can be further parsed into the DinF, NorM and eukaryotic subfamilies based on their amino-acid sequence similarity. Here we report the 3.0 Å resolution X-ray structures of a protonation-mimetic mutant of an H+-coupled DinF transporter, as well as of an H+-coupled DinF and a Na+-coupled NorM transporters in complexes with verapamil, a small-molecule pharmaceutical that inhibits MATE-mediated multidrug extrusion. Combining structure-inspired mutational and functional studies, we confirm the biological relevance of our crystal structures, reveal the mechanistic differences among MATE transporters, and suggest how verapamil inhibits MATE-mediated multidrug efflux. Our findings offer insights into how MATE transporters extrude chemically and structurally dissimilar drugs and could inform the design of new strategies for tackling multidrug resistance. PMID:26246409

  2. Interaction of Ocular Hypotensive Agents (PGF2α Analogs—Bimatoprost, Latanoprost, and Travoprost) With MDR Efflux Pumps on the Rabbit Cornea

    PubMed Central

    Mitra, Ashim K.

    2009-01-01

    Purpose: The objectives of this work were (i) to screen ocular hypotensive prostaglandin (PGF2α) analogs—bimatoprost, latanoprost, and travoprost as well as their free acid forms—for interaction with efflux pumps on the cornea and (ii) to assess the modulation of efflux upon co-administration of these prostaglandin analogs. Methods: Cultured rabbit primary corneal epithelial cells (rPCEC) were employed as an in vitro model for rabbit cornea. Transporter-specific interaction studies were carried out using Madin-Darby canine kidney (MDCK) cells overexpressing MDR1, MRP1, MRP2, MRP5, and BCRP. Freshly excised rabbit cornea was used as an ex vivo model to determine transcorneal permeability. Results: Cellular accumulation studies clearly showed that all prostaglandin analogs and their free acid forms are substrates of MRP1, MRP2, and MRP5. Bimatoprost was the only prostaglandin analog in this study to interact with P-gp. In addition, none of these molecules showed any affinity for BCRP. Ki values of these prostaglandin analogs obtained from dose-dependent inhibition of erythromycin efflux in rPCEC showed bimatoprost (82.54 μM) and travoprost (94.77 μM) to have similar but higher affinity to efflux pumps than latanoprost (163.20 μM). Ex vivo studies showed that the permeation of these molecules across cornea was significantly elevated in the presence of specific efflux modulators. Finally, both in vitro and ex vivo experiments demonstrated that the efflux of these prostaglandin analogs could be modulated by co-administering them together. Conclusion: Bimatoprost, latanoprost, travoprost, and their free acid forms are substrates of multiple drug efflux pumps on the cornea. Co-administration of these molecules together is a viable strategy to overcome efflux, which could simultaneously elicit a synergistic pharmacological effect, since these molecules have been shown to activate different receptor population for the reduction of intraocular pressure (IOP). PMID

  3. Structure of the periplasmic adaptor protein from a major facilitator superfamily (MFS) multidrug efflux pump

    PubMed Central

    Hinchliffe, Philip; Greene, Nicholas P.; Paterson, Neil G.; Crow, Allister; Hughes, Colin; Koronakis, Vassilis

    2014-01-01

    Periplasmic adaptor proteins are key components of bacterial tripartite efflux pumps. The 2.85 Å resolution structure of an MFS (major facilitator superfamily) pump adaptor, Aquifex aeolicus EmrA, shows linearly arranged α-helical coiled-coil, lipoyl, and β-barrel domains, but lacks the fourth membrane-proximal domain shown in other pumps to interact with the inner membrane transporter. The adaptor α-hairpin, which binds outer membrane TolC, is exceptionally long at 127 Å, and the β-barrel contains a conserved disordered loop. The structure extends the view of adaptors as flexible, modular components that mediate diverse pump assembly, and suggests that in MFS tripartite pumps a hexamer of adaptors could provide a periplasmic seal. PMID:24996185

  4. Structure of the periplasmic adaptor protein from a major facilitator superfamily (MFS) multidrug efflux pump.

    PubMed

    Hinchliffe, Philip; Greene, Nicholas P; Paterson, Neil G; Crow, Allister; Hughes, Colin; Koronakis, Vassilis

    2014-08-25

    Periplasmic adaptor proteins are key components of bacterial tripartite efflux pumps. The 2.85 Å resolution structure of an MFS (major facilitator superfamily) pump adaptor, Aquifex aeolicus EmrA, shows linearly arranged α-helical coiled-coil, lipoyl, and β-barrel domains, but lacks the fourth membrane-proximal domain shown in other pumps to interact with the inner membrane transporter. The adaptor α-hairpin, which binds outer membrane TolC, is exceptionally long at 127 Å, and the β-barrel contains a conserved disordered loop. The structure extends the view of adaptors as flexible, modular components that mediate diverse pump assembly, and suggests that in MFS tripartite pumps a hexamer of adaptors could provide a periplasmic seal. PMID:24996185

  5. Effects of chlorophyll-derived efflux pump inhibitor pheophorbide a and pyropheophorbide a on growth and macrolide antibiotic resistance of indicator and anaerobic swine manure bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Natural plant compounds, such as the chlorophyll a catabolites pheophorbide a (php) and pyropheophorbide a (pyp), are potentially active in the gastrointestinal tracts and wastes of livestock as antimicrobial resistance-modifying agents through inhibition of bacterial efflux pumps. To investigate w...

  6. From phenothiazine to 3-phenyl-1,4-benzothiazine derivatives as inhibitors of the Staphylococcus aureus NorA multidrug efflux pump.

    PubMed

    Sabatini, Stefano; Kaatz, Glenn W; Rossolini, Gian Maria; Brandini, David; Fravolini, Arnaldo

    2008-07-24

    Overexpression of efflux pumps is an important mechanism by which bacteria evade effects of substrate antimicrobial agents and inhibition of such pumps is a promising strategy to circumvent this resistance mechanism. NorA is a Staphylococcus aureus multidrug efflux pump, the activity of which confers decreased susceptibility to many structurally unrelated agents, including fluoroquinolones, resulting in a multidrug resistant (MDR) phenotype. In this work, a series of 1,4-benzothiazine derivatives were designed and synthesized as a minimized structural template of phenothiazine MDR efflux pump inhibitors (EPIs) in an effort to identify more potent S. aureus NorA EPIs. Almost all derivatives evaluated showed good activity in combination with ciprofloxacin against S. aureus ATCC 25923; some were capable of completely restoring ciprofloxacin activity in a norA-overexpressing strain (SA-K2378). Compounds 6k and 7j displayed good activity against SA-1199B, a strain that also overexpresses norA, in an ethidium bromide (EtBr) efflux inhibition assay. PMID:18578473

  7. Use of a yeast-based membrane protein expression technology to overexpress drug resistance efflux pumps.

    PubMed

    Lamping, Erwin; Cannon, Richard D

    2010-01-01

    Azole antifungal drugs are used widely to treat people with oral fungal infections. Unfortunately, fungi can develop resistance to these drugs. This resistance can be due to the overexpression or mutation of cytochrome P450 14alpha-lanosterol demethylase, also known as ERG11 or CYP51, and/or the overexpression of membrane-located multidrug efflux pumps. We have developed a heterologous membrane protein expression system that can be used to study the structure and function of these proteins in the non-pathogenic, genetically stable, and versatile eukaryotic model organism, Saccharomyces cerevisiae. In this chapter we describe the techniques used to express the Candida albicans efflux pump Cdr1p in S. cerevisiae. PMID:20717788

  8. All0809/8/7 is a DevBCA-like ABC-type efflux pump required for diazotrophic growth in Anabaena sp. PCC 7120.

    PubMed

    Staron, Peter; Maldener, Iris

    2012-10-01

    Efflux pumps export a wide variety of proteinaceous and non-proteinaceous substrates across the Gram-negative cell wall. For the filamentous cyanobacterium Anabaena sp. strain PCC 7120, the ATP-driven glycolipid efflux pump DevBCA-TolC has been shown to be crucial for the differentiation of N(2)-fixing heterocysts from photosynthetically active vegetative cells. In this study, a homologous system was described. All0809/8/7-TolC form a typical ATP-driven efflux pump as shown by surface plasmon resonance. This putative exporter is also involved in diazotrophic growth of Anabaena sp. PCC 7120. A mutant in all0809 encoding the periplasmic membrane fusion protein of the pump was not able to grow without combined nitrogen. Although heterocysts of this mutant were not distinguishable from those of the wild-type in light and electron micrographs, they were impaired in providing the microoxic environment necessary for N(2) fixation. RT-PCR of all0809 transcripts and localization studies on All0807-GFP revealed that All0809/8/7 was initially downregulated during heterocyst maturation and upregulated at later stages of heterocyst formation in all cells of the filament. A substrate of the efflux pump could not be identified in ATP hydrolysis assays. We discuss a role for All0809/8/7-TolC in maintaining the continuous periplasm and how this would be of special importance for heterocyst differentiation. PMID:22859614

  9. Structure and function of efflux pumps that confer resistance to drugs.

    PubMed Central

    Borges-Walmsley, M Ines; McKeegan, Kenneth S; Walmsley, Adrian R

    2003-01-01

    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. PMID:13678421

  10. Amino acid amides of piperic acid (PA) and 4-ethylpiperic acid (EPA) as NorA efflux pump inhibitors of Staphylococcus aureus.

    PubMed

    Wani, Naiem Ahmad; Singh, Samsher; Farooq, Saleem; Shankar, Sudha; Koul, Surrinder; Khan, Inshad Ali; Rai, Rajkishor

    2016-09-01

    A total of eighteen piperic acid (PA) and 4-ethylpiperic acid (EPA) amides (C1-C18) with α-, β- and γ-amino acids were synthesized, characterized and evaluated for their efflux pump inhibitory activity against ciprofloxacin resistant Staphylococcus aureus. The amides were screened against NorA overexpressing S. aureus SA-1199B and wild type S. aureus SA-1199 using ethidium bromide as NorA efflux pump substrate. EPI C6 was found to be most potent and reduced the MIC of ciprofloxacin by 16 fold followed by C18 which showed 4 fold reduction of MIC. Ethidium bromide efflux inhibition and accumulation assay proved these compounds as NorA inhibitors. PMID:27503686

  11. Evolution from a natural flavones nucleus to obtain 2-(4-Propoxyphenyl)quinoline derivatives as potent inhibitors of the S. aureus NorA efflux pump.

    PubMed

    Sabatini, Stefano; Gosetto, Francesca; Manfroni, Giuseppe; Tabarrini, Oriana; Kaatz, Glenn W; Patel, Diixa; Cecchetti, Violetta

    2011-08-25

    Overexpression of efflux pumps is an important mechanism by which bacteria evade the effects of substrate antimicrobial agents. Inhibition of such pumps is a promising strategy to circumvent this resistance mechanism. NorA is a Staphylococcus aureus efflux pump that confers reduced susceptibility to many structurally unrelated agents, including fluoroquinolones, resulting in a multidrug resistant phenotype. In this work, a series of 2-phenyl-4(1H)-quinolone and 2-phenyl-4-hydroxyquinoline derivatives, obtained by modifying the flavone nucleus of known efflux pump inhibitors (EPIs), were synthesized in an effort to identify more potent S. aureus NorA EPIs. The 2-phenyl-4-hydroxyquinoline derivatives 28f and 29f display potent EPI activity against SA-1199B, a strain that overexpresses norA, in an ethidium bromide efflux inhibition assay. The same compounds, in combination with ciprofloxacin, were able to completely restore its antibacterial activity against both S. aureus SA-K2378 and SA-1199B, norA-overexpressing strains. PMID:21751791

  12. Enhanced Efflux Activity Facilitates Drug Tolerance in Dormant Bacterial Cells

    PubMed Central

    Pu, Yingying; Zhao, Zhilun; Li, Yingxing; Zou, Jin; Ma, Qi; Zhao, Yanna; Ke, Yuehua; Zhu, Yun; Chen, Huiyi; Baker, Matthew A.B.; Ge, Hao; Sun, Yujie; Xie, Xiaoliang Sunney; Bai, Fan

    2016-01-01

    Summary Natural variations in gene expression provide a mechanism for multiple phenotypes to arise in an isogenic bacterial population. In particular, a sub-group termed persisters show high tolerance to antibiotics. Previously, their formation has been attributed to cell dormancy. Here we demonstrate that bacterial persisters, under β-lactam antibiotic treatment, show less cytoplasmic drug accumulation as a result of enhanced efflux activity. Consistently, a number of multi-drug efflux genes, particularly the central component TolC, show higher expression in persisters. Time-lapse imaging and mutagenesis studies further establish a positive correlation between tolC expression and bacterial persistence. The key role of efflux systems, among multiple biological pathways involved in persister formation, indicates that persisters implement a positive defense against antibiotics prior to a passive defense via dormancy. Finally, efflux inhibitors and antibiotics together effectively attenuate persister formation, suggesting a combination strategy to target drug tolerance. PMID:27105118

  13. Contribution of the Ade Resistance-Nodulation-Cell Division-Type Efflux Pumps to Fitness and Pathogenesis of Acinetobacter baumannii

    PubMed Central

    Yoon, Eun-Jeong; Balloy, Viviane; Fiette, Laurence; Chignard, Michel; Courvalin, Patrice

    2016-01-01

    ABSTRACT Overexpression of chromosomal resistance-nodulation-cell division (RND)-type efflux systems with broad substrate specificity contributes to multidrug resistance (MDR) in Acinetobacter baumannii. We have shown that modulation of expression of the structural genes for the efflux systems AdeABC and AdeIJK confers MDR and results in numerous alterations of membrane-associated cellular functions, in particular biofilm formation. However, the contribution of these RND pumps to cell fitness and virulence has not yet been studied. The biological cost of an antibiotic resistance mechanism is a key parameter in determining its stability and dissemination. From an entirely sequenced susceptible clinical isolate, we have generated a set of isogenic derivatives having single point mutations resulting in overexpression of each efflux system or with every pump deleted by allelic replacement. We found that overproduction of the pumps results in a significant decrease in fitness of the bacterial host when measured by competition experiments in vitro. Fitness and virulence were also evaluated in vivo both in systemic and pulmonary infection models in immunocompetent mice. A diminished competitiveness of the AdeABC-overexpressing mutant was observed only after intraperitoneal inoculation, but not after intranasal inoculation, the latter mimicking the most frequent type of human A. baumannii infection. However, in mice infected intranasally, this mutant was more virulent and stimulated an enhanced neutrophil activation in the lungs. Altogether, these data account for the observation that adeABC overexpression is common in MDR A. baumannii frequently found in ventilator-associated pneumonia. PMID:27247231

  14. Reversal of efflux mediated antifungal resistance underlies synergistic activity of two monoterpenes with fluconazole.

    PubMed

    Ahmad, Aijaz; Khan, Amber; Manzoor, Nikhat

    2013-01-23

    Thymol (THY) and carvacrol (CARV), the principal chemical components of thyme oil have long been known for their wide use in medicine due to antimicrobial and disinfectant properties. This study, however, draws attention to a possible synergistic antifungal effect of these monoterpenes with azole antimycotic-fluconazole. Resistance to azoles in Candida albicans involves over-expression of efflux-pump genes MDR1, CDR1, CDR2 or mutations and over-expression of target gene ERG11. The inhibition of drug efflux pumps is considered a feasible strategy to overcome clinical antifungal resistance. To put forward this approach, we investigated the combination effects of these monoterpenes and FLC against 38 clinically obtained FLC-sensitive, and eleven FLC-resistant Candida isolates. Synergism was observed with combinations of THY-FLC and CARV-FLC evaluated by checkerboard microdilution method and nature of the interactions was calculated by FICI. In addition, antifungal activity was assessed using agar-diffusion and time-kill curves. The drug efflux activity was determined using two dyes, Rhodamine6G (R6G) and fluorescent Hoechst 33342. No significant differences were observed in dye uptakes between FLC-susceptible and resistant isolates, incubated in glucose free buffer. However, a significantly higher efflux was recorded in FLC-resistant isolates when glucose was added. Both monoterpenes inhibited efflux by 70-90%, showing their high potency to block drug transporter pumps. Significant differences, in the expression levels of CDR1 and MDR1, induced by monoterpenes revealed reversal of FLC-resistance. The selectively fungicidal characteristics and ability to restore FLC susceptibility in resistant isolates signify a promising candidature of THY and CARV as antifungal agents in combinational treatments for candidiasis. PMID:23111348

  15. Adaptive and Mutational Resistance: Role of Porins and Efflux Pumps in Drug Resistance

    PubMed Central

    Fernández, Lucía

    2012-01-01

    Summary: The substantial use of antibiotics in the clinic, combined with a dearth of new antibiotic classes, has led to a gradual increase in the resistance of bacterial pathogens to these compounds. Among the various mechanisms by which bacteria endure the action of antibiotics, those affecting influx and efflux are of particular importance, as they limit the interaction of the drug with its intracellular targets and, consequently, its deleterious effects on the cell. This review evaluates the impact of porins and efflux pumps on two major types of resistance, namely, mutational and adaptive types of resistance, both of which are regarded as key phenomena in the global rise of antibiotic resistance among pathogenic microorganisms. In particular, we explain how adaptive and mutational events can dramatically influence the outcome of antibiotic therapy by altering the mechanisms of influx and efflux of antibiotics. The identification of porins and pumps as major resistance markers has opened new possibilities for the development of novel therapeutic strategies directed specifically against these mechanisms. PMID:23034325

  16. Protoporphyrin (PPIX) efflux by the MacAB-TolC pump in Escherichia coli.

    PubMed

    Turlin, Evelyne; Heuck, Gesine; Simões Brandão, Maria Inês; Szili, Noémie; Mellin, J R; Lange, Norbert; Wandersman, Cécile

    2014-12-01

    In most organisms, heme biosynthesis is strictly controlled so as to avoid heme and heme precursor accumulation, which is toxic. Escherichia coli regulates heme biosynthesis by a feedback loop involving heme-induced proteolytic cleavage of HemA, glutamyl-tRNA reductase, which is the first enzyme in the heme biosynthetic pathway. We show here that heme homeostasis can be disrupted by overproduction of YfeX, a cytoplasmic protein that captures iron from heme that we named deferrochelatase. We also show that it is disrupted by iron chelation, which reduces the intracellular iron concentration necessary for loading iron into protoporphyrin IX (PPIX, the immediate heme precursor). In both cases, we established that there is an increased PPIX concentration and we demonstrate that this compound is expelled by the MacAB-TolC pump, an efflux pump involved in E. coli and Salmonella for macrolide efflux. The E. coli macAB and tolC mutants accumulate PPIX and are sensitive to photo-inactivation. The MacAB-TolC pump is required for Salmonella typhimurium survival in macrophages. We propose that PPIX is an endogenous substrate of the MacAB-TolC pump in E. coli and S. typhimurium and that this compound is produced inside bacteria when natural heme homeostasis is disrupted by iron shortage, as happens when bacteria invade the mammalian host. PMID:25257218

  17. Marinobacter adhaerens HP15 harbors two CzcCBA efflux pumps involved in zinc detoxification.

    PubMed

    Stahl, Antje; Pletzer, Daniel; Mehmood, Amna; Ullrich, Matthias S

    2015-09-01

    Several members of the ubiquitously found γ-proteobacterial genus Marinobacter were described or assumed to inhabit marine environments naturally enriched in heavy metals. However, direct studies that describe the ability of this genus to occupy such environments have not been conducted. To cope with heavy metal stress, bacteria possess specific efflux pumps as tools for detoxification, among which the CzcCBA type efflux system is one representative. Previous studies showed that this system plays an important role in resistance towards cadmium, zinc, and cobalt. Up to now, no study had focused on characterization of Czc pumps in Marinobacter sp. or other marine prokaryotes. Herein, we elucidated the function of two CzcCBA pumps encoded by Marinobacter adhaerens HP15's genome during exposure to cadmium, zinc, and cobalt. Single and double knock-out mutants lacking the corresponding two czcCBA operons were generated and analyzed in terms of their resistance profiles. Both operons appeared to be important for zinc resistance but had no role in tolerance towards cadmium or cobalt. One of the mutations was genetically complemented thereby restoring the wild type phenotype. In accordance with the resistance pattern, expression of the genes coding for both CzcCBA pumps was induced by zinc but neither by cadmium nor cobalt. PMID:26122890

  18. Synthesis and Biological Evaluation of Pentacyclic Strychnos Alkaloids as Selective Modulators of the ABCC10 (MRP7) Efflux Pump

    PubMed Central

    2015-01-01

    The selective modulation of ATP-binding cassette (ABC) efflux pumps overexpressed in multidrug resistant cancers (MDR) and attendant resensitization to chemotherapeutic agents represent a promising strategy for treating cancer. We have synthesized four novel pentacyclic Strychnos alkaloids alstolucines B (2), F (3), and A (5) and N-demethylalstogucine (4), in addition to known Strychnos alkaloid echitamidine (16), and we evaluated compounds 1–5 in biochemical assays with ABCC10 and P-glycoprotein (P-gp). Alstolucines B (2) and F (3) inhibited ABCC10 ATPase activity at 12.5 μM without affecting P-gp function; moreover, they resensitized ABCC10-transfected cell lines to paclitaxel at 10 μM. Altogether, the alstolucines represent promising lead candidates in the development of modulators of ABCC10 for MDR cancers overexpressing this pump. PMID:25419978

  19. A data-driven approach to modeling the tripartite structure of multidrug resistance efflux pumps.

    PubMed

    Phillips, Joshua L; Gnanakaran, S

    2015-01-01

    Many bacterial pathogens are becoming increasingly resistant to antibiotic treatments, and a detailed understanding of the molecular basis of antibiotic resistance is critical for the development of next-generation approaches for combating bacterial infections. Studies focusing on pathogens have revealed the profile of resistance in these organisms to be due primarily to the presence of multidrug resistance efflux pumps: tripartite protein complexes which span the periplasm bridging the inner and outer membranes of Gram-negative bacteria. An atomic-level resolution tripartite structure remains imperative to advancing our understanding of the molecular mechanisms of pump function using both theoretical and experimental approaches. We develop a fast and consistent method for constructing tripartite structures which leverages existing data-driven models and provide molecular modeling approaches for constructing tripartite structures of multidrug resistance efflux pumps. Our modeling studies reveal that conformational changes in the inner membrane component responsible for drug translocation have limited impact on the conformations of the other pump components, and that two distinct models derived from conflicting experimental data are both consistent with all currently available measurements. Additionally, we investigate putative drug translocation pathways via geometric simulations based on the available crystal structures of the inner membrane pump component, AcrB, bound to two drugs which occupy distinct binding sites: doxorubicin and linezolid. These simulations suggest that smaller drugs may enter the pump through a channel from the cytoplasmic leaflet of the inner membrane, while both smaller and larger drug molecules may enter through a vestibule accessible from the periplasm. PMID:24957790

  20. Pgp efflux pump decreases the cytostatic effect of CENP-E inhibitor GSK923295.

    PubMed

    Tcherniuk, Sergey O; Oleinikov, Andrew V

    2015-05-28

    Human kinesin CENP-E is an attractive target for cancer chemotherapy. The allosteric CENP-E inhibitor GSK923295 was proposed as a promising anticancer compound with potent cytostatic effect. In our work, we have analyzed the influence of the Pgp efflux pump on the cytostatic effect of GSK923295. We have demonstrated that multidrug resistant MESSA Dx5 cells overexpressing Pgp are 70-80 times more resistant to GSK923295 than their parental counterpart MESSA cells. Addition of 20 µM verapamil restored the drug sensibility of MESSA Dx5 cells. Combinations of GSK923295 with verapamil showed nearly additive effects in MESSA and synergistic effects in MESSA Dx5 cells. Our results demonstrate that tumors possessing Pgp could be more resistant to GSK923295, and that overexpression of Pgp can decrease the therapeutic effect of this drug. Development of structural analogs of GSK923295 which would not be a substrate of the Pgp efflux pump or addition of Pgp pump inhibitors can significantly improve the cytostatic effect of this drug. PMID:25725449

  1. In vivo evaluation of anionic thiolated polymers as oral delivery systems for efflux pump inhibition.

    PubMed

    Palmberger, Thomas F; Laffleur, Flavia; Greindl, Melanie; Bernkop-Schnürch, Andreas

    2015-08-01

    Recently, the cationic polymer thiolated chitosan has been reported to modulate drug absorption by inhibition of intestinal efflux pumps. The objective of this study was to evaluate in vitro and in vivo whether thiolated anionic biopolymers also show an efflux pump inhibitory effect in order to improve intestinal transcellular drug uptake. Therefore, three thiomers have been synthesized due covalent attachment of cysteine to various polymer backbones: pectin-cysteine (pect-cys), carboxymethylcellulose-cysteine (CMC-cys) and alginate-cysteine (alg-cys). In vitro, the permeation enhancing properties of these thiomers and their corresponding unmodified polymers have been evaluated on rat small intestine in Ussing-type chambers, using sulforhodamine 101 (SR-101) as MRP2 model substrate. In comparison to buffer only, SR-101 transport in presence of pect-cys, CMC-cys and alg-cys was improved 1.5-fold, 1.8-fold and 3.0-fold, respectively. Due to the comparatively best in vitro performance of thiolated alginate, it has been chosen for in vivo studies: a SR-101 solution containing 4% (w/v) alg-cys led to an AUC0 ≥ 12 of SR-101 of 109 ng ml(-1)h in rats representing a 3.8-fold improvement in comparison to a SR-101 buffer solution. Unmodified alginate improved the AUC0 ≥ 12 of SR-101 by a factor of 1.9. These findings suggest thiolated alginate as promising auxiliary agent for drugs being anionic efflux pump substrates, since the oral bioavailability of a MRP2 substrate could be significantly improved. PMID:26095915

  2. A novel nanoparticle formulation overcomes multiple types of membrane efflux pumps in human breast cancer cells.

    PubMed

    Prasad, Preethy; Cheng, Ji; Shuhendler, Adam; Rauth, Andrew M; Wu, Xiao Yu

    2012-04-01

    Multidrug resistance (MDR) in cancer cells can involve overexpression of different types of membrane drug efflux pumps and other drug resistance mechanisms. Hence, inhibition of one resistance mechanism may not be therapeutically effective. Previously we demonstrated a new polymer lipid hybrid nanoparticle (PLN) system was able to circumvent drug resistance of P-glycoprotein (P-gp) overexpressing breast cancer cells. The objectives of the present study were 2-fold: (1) to evaluate the ability of the PLN system to overcome two other membrane efflux pumps-multidrug resistance protein 1 (MRP1+) and breast cancer resistance protein (BCRP+) overexpressed on human breast cancer cell lines MCF7 VP (MRP1+) and MCF7 MX (BCRP+); and (2) to evaluate possible synergistic effects of doxorubicin (Dox)-mitomycin C (MMC) in these cell lines. These objectives were accomplished by measuring in vitro cellular uptake, intracellular trafficking, and cytotoxicity (using a clonogenic assay and median effect analysis), of Dox, MMC, or Dox-MMC co-loaded PLN. Treatment of MDR cells with PLN encapsulating single anticancer agents significantly enhanced cell kill compared to free Dox or MMC solutions. Dox-MMC co-loaded PLN were 20-30-folds more effective in killing MDR cells than free drugs. Co-encapsulated Dox-MMC was more effective in killing MDR cells than single agent-encapsulated PLN. Microscopic images showed perinuclear localization of fluorescently labelled PLN in all cell lines. These results are consistent with our previous results for P-gp overexpressing breast cancer cells suggesting the PLN system can overcome multiple types of membrane efflux pumps increasing the cytotoxicity of Dox-MMC at significantly lower doses than free drugs. PMID:25786718

  3. Enhancing tolerance to short-chain alcohols by engineering the Escherichia coli AcrB efflux pump to secrete the non-native substrate n-butanol.

    PubMed

    Fisher, Michael A; Boyarskiy, Sergey; Yamada, Masaki R; Kong, Niwen; Bauer, Stefan; Tullman-Ercek, Danielle

    2014-01-17

    The microbial conversion of sugars to fuels is a promising technology, but the byproducts of biomass pretreatment processes and the fuels themselves are often toxic at industrially relevant levels. One promising solution to these problems is to engineer efflux pumps to secrete fuels and inhibitory chemicals from the cell, increasing microbial tolerance and enabling higher fuel titer. Toward that end, we used a directed evolution strategy to generate variants of the Escherichia coli AcrB efflux pump that act on the non-native substrate n-butanol, enhancing growth rates of E. coli in the presence of this biofuel by up to 25%. Furthermore, these variants confer improved tolerance to isobutanol and straight-chain alcohols up to n-heptanol. Single amino acid changes in AcrB responsible for this phenotype were identified. We have also shown that both the chemical and genetic inactivation of pump activity eliminate the tolerance conferred by AcrB pump variants, supporting our assertion that the variants secrete the non-native substrates. This strategy can be applied to create an array of efflux pumps that modulate the intracellular concentrations of small molecules of interest to microbial fuel and chemical production. PMID:23991711

  4. Effects of Efflux Pump Inhibitors on Colistin Resistance in Multidrug-Resistant Gram-Negative Bacteria.

    PubMed

    Ni, Wentao; Li, Yanjun; Guan, Jie; Zhao, Jin; Cui, Junchang; Wang, Rui; Liu, Youning

    2016-05-01

    We tested the effects of various putative efflux pump inhibitors on colistin resistance in multidrug-resistant Gram-negative bacteria. Addition of 10 mg/liter cyanide 3-chlorophenylhydrazone (CCCP) to the test medium could significantly decrease the MICs of colistin-resistant strains. Time-kill assays showed CCCP could reverse colistin resistance and inhibit the regrowth of the resistant subpopulation, especially in Acinetobacter baumannii and Stenotrophomonas maltophilia These results suggest colistin resistance in Gram-negative bacteria can be suppressed and reversed by CCCP. PMID:26953203

  5. Expression of Multidrug Resistance Efflux Pump Gene norA Is Iron Responsive in Staphylococcus aureus

    PubMed Central

    Deng, Xin; Sun, Fei; Ji, Quanjiang; Liang, Haihua; Missiakas, Dominique; Lan, Lefu

    2012-01-01

    Staphylococcus aureus utilizes efflux transporter NorA to pump out a wide range of structurally dissimilar drugs, conferring low-level multidrug resistance. The regulation of norA expression has yet to be fully understood although past studies have revealed that this gene is under the control of the global transcriptional regulator MgrA and the two-component system ArlRS. To identify additional regulators of norA, we screened a transposon library in strain Newman expressing the transcriptional fusion norA-lacZ for altered β-galactosidase activity. We identify a transposon insertion in fhuB, a gene that encodes a ferric hydroxamate uptake system permease, and propose that the norA transcription is iron responsive. In agreement with this observation, addition of FeCl3 repressed the induction of norA-lacZ, suggesting that bacterial iron uptake plays an important role in regulating norA transcription. In addition, a fur (ferric uptake regulator) deletion exhibited compromised norA transcription and reduced resistance to quinolone compared to the wild-type strain, indicating that fur functions as a positive regulator of norA. A putative Fur box identified in the promoter region of norA was confirmed by electrophoretic mobility shift and DNase I footprint assays. Finally, by employing a siderophore secretion assay, we reveal that NorA may contribute to the export of siderophores. Collectively, our experiments uncover some novel interactions between cellular iron level and norA regulation in S. aureus. PMID:22267518

  6. AdeIJK, a Resistance-Nodulation-Cell Division Pump Effluxing Multiple Antibiotics in Acinetobacter baumannii▿

    PubMed Central

    Damier-Piolle, Laurence; Magnet, Sophie; Brémont, Sylvie; Lambert, Thierry; Courvalin, Patrice

    2008-01-01

    We have identified a second resistance-nodulation-cell division (RND)-type efflux pump, AdeIJK, in clinical isolate Acinetobacter baumannii BM4454. The adeI, adeJ, and adeK genes encode, respectively, the membrane fusion, RND, and outer membrane components of the pump. AdeJ belongs to the AcrB protein family (57% identity with AcrB from Escherichia coli). mRNA analysis by Northern blotting and reverse transcription-PCR indicated that the genes were cotranscribed. Overexpression of the cloned adeIJK operon was toxic in both E. coli and Acinetobacter. The adeIJK genes were detected in all of the 60 strains of A. baumannii tested. The two latter observations suggest that the AdeIJK complex might contribute to intrinsic but not to acquired antibiotic resistance in Acinetobacter. To characterize the substrate specificity of the pump, we have constructed derivatives of BM4454 in which adeIJK (strain BM4579), adeABC (strain BM4561), or both groups of genes (strain BM4652) were inactivated by deletion-insertion. Determination of the antibiotic susceptibility of these strains and of BM4652 and BM4579, in which the adeIJK operon was provided in trans, indicated that the AdeIJK pump contributes to resistance to β-lactams, chloramphenicol, tetracycline, erythromycin, lincosamides, fluoroquinolones, fusidic acid, novobiocin, rifampin, trimethoprim, acridine, safranin, pyronine, and sodium dodecyl sulfate. The chemical structure of these molecules suggests that amphiphilic compounds are the preferred substrates. The AdeABC and AdeIJK efflux systems contributed in a more than additive fashion to tigecycline resistance. PMID:18086852

  7. Disulphide cross linked pullulan based cationic polymer for improved gene delivery and efflux pump inhibition.

    PubMed

    S, Priya S; R, Rekha M

    2016-10-01

    Multidrug resistance is a hurdle to successful cancer chemotherapy. Over expression of P-glycoprotein (P-gp) is a prime contributing factor for drug resistance. In this study, a disulphide cross-linked pullulan-based cationic polymer (PPSS) was synthesized to act simultaneously as gene delivery vehicle and efflux pump inhibitor. The PPSS nanoplexes were of size <200nm with the zeta potential of +15 to +20mV. The cytotoxicity studies using C6 and L929 cells showed that PPSS polymers are non-toxic even at high polymer concentrations. The PPSS/pDNA nanoplex showed superior uptake in confocal microscopy with 97% uptake by flow cytometry. The efficacy of efflux pump inhibition by the PPSS nanoplex was established by the enhanced intracellular retention of DOX. The enhanced cell death by p53/PPSS/DOX nanoplexes was attributed to the synergistic effect of P-gp inhibition and p53 transfection efficiency. Therefore, this multifunctional polymeric system may have significant promise for therapeutic application against cancer drug resistance. PMID:27459414

  8. Importance of multidrug efflux pumps in the antimicrobial resistance property of clinical multidrug-resistant isolates of Neisseria gonorrhoeae.

    PubMed

    Golparian, Daniel; Shafer, William M; Ohnishi, Makoto; Unemo, Magnus

    2014-06-01

    The contribution of drug efflux pumps in clinical isolates of Neisseria gonorrhoeae that express extensively drug-resistant or multidrug-resistant phenotypes has heretofore not been examined. Accordingly, we assessed the effect on antimicrobial resistance of loss of the three gonococcal efflux pumps associated with a known capacity to export antimicrobials (MtrC-MtrD-MtrE, MacA-MacB, and NorM) in such clinical isolates. We report that the MIC of several antimicrobials, including seven previously and currently recommended for treatment was significantly impacted. PMID:24733458

  9. Role of the Tet38 Efflux Pump in Staphylococcus aureus Internalization and Survival in Epithelial Cells

    PubMed Central

    Truong-Bolduc, Q. C.; Bolduc, G. R.; Medeiros, H.; Vyas, J. M.; Wang, Y.

    2015-01-01

    We previously identified the protein Tet38 as a chromosomally encoded efflux pump of Staphylococcus aureus that confers resistance to tetracycline and certain unsaturated fatty acids. Tet38 also contributes to mouse skin colonization. In this study, we discovered a novel regulator of tet38, named tetracycline regulator 21 (TetR21), that bound specifically to the tet38 promoter and repressed pump expression. A ΔtetR21 mutant showed a 5-fold increase in tet38 transcripts and an 8-fold increase in resistance to tetracycline and fatty acids. The global regulator MgrA bound to the tetR21 promoter and indirectly repressed the expression of tet38. To further assess the full role of Tet38 in S. aureus adaptability, we tested its effect on host cell invasion using A549 (lung) and HMEC-1 (heart) cell lines. We used S. aureus RN6390, its Δtet38, ΔtetR21, and ΔmgrA mutants, and a Δtet38 ΔtetR21 double mutant. After 2 h of contact, the Δtet38 mutant was internalized in 6-fold-lower numbers than RN6390 in A549 and HMEC-1 cells, and the ΔtetR21 mutant was internalized in 2-fold-higher numbers than RN6390. A slight increase of 1.5-fold in internalization was found for the ΔmgrA mutant. The growth patterns of RN6390 and the ΔmgrA and ΔtetR21 mutants within A549 cells were similar, while no growth was observed for the Δtet38 mutant. These data indicate that the Tet38 efflux pump is regulated by TetR21 and contributes to the ability of S. aureus to internalize and replicate within epithelial cells. PMID:26324534

  10. Structure-function analysis of the ATP-driven glycolipid efflux pump DevBCA reveals complex organization with TolC/HgdD.

    PubMed

    Staron, Peter; Forchhammer, Karl; Maldener, Iris

    2014-01-31

    In Gram-negative bacteria, trans-envelope efflux pumps have periplasmic membrane fusion proteins (MFPs) as essential components. MFPs act as mediators between outer membrane factors (OMFs) and inner membrane factors (IMFs). In this study, structure-function relations of the ATP-driven glycolipid efflux pump DevBCA-TolC/HgdD from the cyanobacterium Anabaena sp. PCC 7120 were analyzed. The binding of the MFP DevB to the OMF TolC absolutely required the respective tip-regions. The interaction of DevB with the IMF DevAC mainly involved the β-barrel and the lipoyl domain. Efficient binding to DevAC and TolC, substrate recognition and export activity by DevAC were dependent on stable DevB hexamers. PMID:24361095

  11. Structures of Gate Loop Variants of the AcrB Drug Efflux Pump Bound by Erythromycin Substrate

    PubMed Central

    Ababou, Abdessamad; Koronakis, Vassilis

    2016-01-01

    Gram-negative bacteria such as E. coli use tripartite efflux pumps such as AcrAB-TolC to expel antibiotics and noxious compounds. A key feature of the inner membrane transporter component, AcrB, is a short stretch of residues known as the gate/switch loop that divides the proximal and distal substrate binding pockets. Amino acid substitutions of the gate loop are known to decrease antibiotic resistance conferred by AcrB. Here we present two new AcrB gate loop variants, the first stripped of its bulky side chains, and a second in which the gate loop is removed entirely. By determining the crystal structures of the variant AcrB proteins in the presence and absence of erythromycin and assessing their ability to confer erythromycin tolerance, we demonstrate that the gate loop is important for AcrB export activity but is not required for erythromycin binding. PMID:27403665

  12. Efflux Pump Inhibitor Phenylalanine-Arginine Β-Naphthylamide Effect on the Minimum Inhibitory Concentration of Imipenem in Acinetobacter baumannii Strains Isolated From Hospitalized Patients in Shahid Motahari Burn Hospital, Tehran, Iran

    PubMed Central

    Gholami, Mehrdad; Hashemi, Ali; Hakemi-Vala, Mojdeh; Goudarzi, Hossein; Hallajzadeh, Masoumeh

    2015-01-01

    Background: Acinetobacter baumannii has emerged as a highly troublesome pathogen and a leading cause of mortality and morbidity among hospitalized burn patients. Objectives: The aims of this study were to determine the frequency of the AdeABC genes and the role of the efflux pump (s) in the imipenem resistance of A. baumannii strains isolated from burn patients. Materials and Methods: This study was conducted on 60 A. baumannii isolates collected from 240 wound samples of burn patients admitted to the Burn Unit of Shahid Motahari Burn hospital, Tehran, Iran. Antibiotic susceptibility tests were performed using the Kirby-Bauer disc diffusion and broth microdilution according to the clinical and laboratory standards institute (CLSI) guidelines. The activity of the efflux pump was evaluated using the efflux pump inhibitor, the phenylalanine-arginine Β-naphthylamide (PAΒN). The AdeABC genes were detected by polymerase chain reaction (PCR) and sequencing. Results: In this study, 100% of the isolates were resistant to cefotaxime, ceftazidime, ceftriaxone, ciprofloxacin, cefepime, piperacillin, meropenem, co-trimoxazole, and piperacillin/tazobactam; 56 (94%) to gentamicin; 50 (81%) to amikacin; 58 (97%) to imipenem; and 45 (76%) to tetracycline. Additionally,all the isolates were susceptible to colistin. The susceptibility of the strains to imipenem was highly increased in the presence of the efflux pump inhibitor such that for 58 (96.6%) of the isolates, the PAΒN reduced the minimum inhibitory concentrations (MIC) by 4- to 64-fold. The adeA and adeB genes were detected in 60 (100%) of the isolates, and the adeC gene was present in 51 (85%). Conclusions: The efflux pump may play a role in antibiotic resistance in A. baumannii isolates. The ability of A. baumannii isolates to acquire drug resistance by the efflux pump mechanism is a concern. Thus, new strategies are required in order to eliminate the efflux transport activity from resistant A. baumannii isolates causing

  13. Effect of six fluoroquinolones on the expression of four efflux pumps in the multidrug resistant Escherichia coli isolates.

    PubMed

    Liu, Haixia; Liu, Xiaoqiang; Li, Yinqian; Hao, Caiju

    2015-07-01

    In this study, a total of 78 Escherichia coli clinical isolates were isolated from canines diagnosed with urinary tract infections. 23/78 isolates (29.5 %) showed multidrug resistance (MDR) phenotype, including the isolates both susceptible to fluoroquinolones (FQs) (FQ(S)-MDR, n = 12) and resistant to FQs (FQ(R)-MDR, n = 11). For these MDR isolates, mutations within quinolone-resistance determining region of gyrA and parC were determined by PCR amplification and DNA sequencing. The relative quantification of emrE, acrB, macB, and mdfA genes expression in MDR isolates was determined by quantitative real-time PCR before and after exposure to the FQs (10 µg/ml). The results showed that a temporary exposure to FQs could lead to various degrees of up or down-regulation on the expression of four efflux pumps in MDR isolates depending on the resistant phenotype and the activities of the FQs. Generally, the FQ(R)-MDR isolates showed more obvious changes in average expression levels of these transporters versus the FQ(S)-MDR isolates, with a largest increase in emrE, and followed by acrB, while the expression of macB and mdfA did not change as radically. Meanwhile, there is a reverse relationship between the expression changes and the activities of the FQs tested. The expression was higher in the isolates exposed to enrofloxacin, ciprofloxacin, and orbifloxacin, and followed by the marbofloxacin, gatifloxacin, and pradofloxacin, and the average expression levels of some efflux pumps even decreased as the isolates were exposed to gatifloxacin or pradofloxacin. PMID:25854862

  14. Assembly and Channel Opening of Outer Membrane Protein in Tripartite Drug Efflux Pumps of Gram-negative Bacteria*

    PubMed Central

    Xu, Yongbin; Moeller, Arne; Jun, So-Young; Le, Minho; Yoon, Bo-Young; Kim, Jin-Sik; Lee, Kangseok; Ha, Nam-Chul

    2012-01-01

    Gram-negative bacteria are capable of expelling diverse xenobiotic substances from within the cell by use of three-component efflux pumps in which the energy-activated inner membrane transporter is connected to the outer membrane channel protein via the membrane fusion protein. In this work, we describe the crystal structure of the membrane fusion protein MexA from the Pseudomonas aeruginosa MexAB-OprM pump in the hexameric ring arrangement. Electron microscopy study on the chimeric complex of MexA and the outer membrane protein OprM reveals that MexA makes a tip-to-tip interaction with OprM, which suggests a docking model for MexA and OprM. This docking model agrees well with genetic results and depicts detailed interactions. Opening of the OprM channel is accompanied by the simultaneous exposure of a protein structure resembling a six-bladed cogwheel, which intermeshes with the complementary cogwheel structure in the MexA hexamer. Taken together, we suggest an assembly and channel opening model for the MexAB-OprM pump. This study provides a better understanding of multidrug resistance in Gram-negative bacteria. PMID:22308040

  15. Transcriptional Analysis of MexAB-OprM Efflux Pumps System of Pseudomonas aeruginosa and Its Role in Carbapenem Resistance in a Tertiary Referral Hospital in India

    PubMed Central

    2015-01-01

    Carbapenem resistance presents severe threat to the treatment of multidrug resistant Pseudomonas aeruginosa infections. The study was undertaken to investigate the role of efflux pumps in conferring meropenem resistance and effect of single dose exposure of meropenem on transcription level of mexA gene in clinical isolates of P. aeruginosa from a tertiary referral hospital of India. Further, in this investigation an effort was made to assess whether different components of MexAB-OprM operon expresses in the same manner and the extent of contributions of those components in meropenem resistance in its natural host (P. aeruginosa) and in a heterologous host (E. coli). Out of 83 meropenem nonsusceptible isolates, 22 isolates were found to possess efflux pump activity phenotypically. Modified hodge test and multiplex PCR confirmed the absence of carbapenemase genes in those isolates. All of them were of multidrug resistant phenotype and were resistant to all the carbepenem drug tested. MexAB-OprM efflux pump was found to be overexpressed in all the study isolates. It could be observed that single dose exposure meropenem could give rise to trivial increase in transcription of mexA gene. Different constructs of MexAB-OprM (mexR-mexA-mexB-OprM; mexA-mexB-OprM; mexA-mexB) could be expressed in both its natural (P. aeruginosa PAO1) and heterologous host (E. coli JM107) but transcription level of mexA gene varied in both the hosts before and after single dose exposure of meropenem. Different components of the operon failed to enhance meropenem resistance in E. coli JM107 and P. aeruginosa PAO1. This study could prove that MexAB-OprM efflux pump can significantly contribute to meropenem resistance in hospital isolates of P. aeruginosa where an acquired resistant mechanism is absent. Thus, equal importance should be given for diagnosis of intrinsic resistance mechanism so as to minimize treatment failure. As meropenem could not enhance mexA transcriptions significantly, there

  16. Crystal Structure of Escherichia coli CusC the Outer Membrane Component of a Heavy Metal Efflux Pump

    SciTech Connect

    R Kulathila; R Kulathila; M Indic; B van den Berg

    2011-12-31

    While copper has essential functions as an enzymatic co-factor, excess copper ions are toxic for cells, necessitating mechanisms for regulating its levels. The cusCBFA operon of E. coli encodes a four-component efflux pump dedicated to the extrusion of Cu(I) and Ag(I) ions. We have solved the X-ray crystal structure of CusC, the outer membrane component of the Cus heavy metal efflux pump, to 2.3 {angstrom} resolution. The structure has the largest extracellular opening of any outer membrane factor (OMF) protein and suggests, for the first time, the presence of a tri-acylated N-terminal lipid anchor. The CusC protein does not have any obvious features that would make it specific for metal ions, suggesting that the narrow substrate specificity of the pump is provided by other components of the pump, most likely by the inner membrane component CusA.

  17. Substrate path in the AcrB multidrug efflux pump of Escherichia coli

    PubMed Central

    Husain, Fasahath; Nikaido, Hiroshi

    2010-01-01

    A major tripartite multidrug efflux pump of Escherichia coli, AcrAB-TolC, confers resistance to a wide variety of compounds. The drug molecule is captured by AcrB probably from the periplasm or the periplasm/inner membrane interface, and is passed through AcrB and then TolC to the medium. Currently there exist numerous crystallographic and mutation data concerning the regions of AcrB and its homologs that may interact with substrates. Starting with these data, we devised fluorescence assays in whole cells to determine the entire substrate path through AcrB. We tested 48 residues in AcrB along the predicted substrate path and 25 gave positive results, based on the covalent labeling of cysteine residues by a lipophilic dye-maleimide and the blocking of Nile Red efflux by covalent labeling with bulky maleimide reagents. These residues are all located in the periplasmic domain, in regions we designate as the lower part of the large external cleft, the cleft itself, the crystallographically defined binding pocket, and the gate between the pocket and the funnel. Our observations suggest that the substrate is captured in the lower cleft region of AcrB, then transported through the binding pocket, the gate, and finally to the AcrB funnel that connects AcrB to TolC. PMID:20804453

  18. Dipeptide Prodrug Approach to Evade Efflux Pumps and CYP3A4 Metabolism of Lopinavir

    PubMed Central

    Patel, Mitesh; Sheng, Ye; Mandava, Nanda K.; Pal, Dhananjay; Mitra, Ashim K.

    2014-01-01

    Oral absorption of lopinavir (LPV) is limited due to P-glycoprotein (P-gp) and multidrug resistance-associated protein2 (MRP2) mediated efflux by intestinal epithelial cells. Moreover, LPV is extensively metabolized by CYP3A4 enzymes. In the present study, dipeptide prodrug approach was employed to circumvent efflux pumps (P-gp and MRP2) and CYP3A4 mediated metabolism of LPV. Valine-isoleucine-LPV (Val-Ile-LPV) was synthesized and identified by LCMS and NMR techniques. The extent of LPV and Val-Ile-LPV interactions with P-gp and MRP2 was studied by uptake and transport studies across MDCK-MDR1 and MDCK-MRP2 cells. To determine the metabolic stability, time and concentration dependent degradation study was performed in liver microsomes. Val-Ile-LPV exhibited significantly higher aqueous solubility relative to LPV. This prodrug generated higher stability under acidic pH. Val-Ile-LPV demonstrated significantly lower affinity towards P-gp and MRP2 relative to LPV. Transepithelial transport of Val-Ile-LPV was significantly higher in the absorptive direction (apical to basolateral) relative to LPV. Importantly, Val-Ile-LPV was recognized as an excellent substrate by peptide transporter. Moreover, Val-Ile-LPV displayed significantly higher metabolic stability relative to LPV. Results obtained from this study suggested that dipeptide prodrug approach is a viable option to elevate systemic levels of LPV following oral administration PMID:25261710

  19. The human multidrug resistance-associated protein MRP is a plasma membrane drug-efflux pump.

    PubMed Central

    Zaman, G J; Flens, M J; van Leusden, M R; de Haas, M; Mülder, H S; Lankelma, J; Pinedo, H M; Scheper, R J; Baas, F; Broxterman, H J

    1994-01-01

    The multidrug-resistance associated protein MRP is a 180- to 195-kDa membrane protein associated with resistance of human tumor cells to cytotoxic drugs. We have investigated how MRP confers drug resistance in SW-1573 human lung carcinoma cells by generating a subline stably transfected with an expression vector containing MRP cDNA. MRP-overexpressing SW-1573 cells are resistant to doxorubicin, daunorubicin, vincristine, VP-16, colchicine, and rhodamine 123, but not to 4'-(9-acridinylamino)methanesulfon-m-anisidide or taxol. The intracellular accumulation of drug (daunorubicin, vincristine, and VP-16) is decreased and the efflux of drug (daunorubicin) is increased in the transfectant. The decreased accumulation of daunorubicin is abolished by permeabilization of the plasma membrane with digitonin, showing that MRP can lower the intracellular daunorubicin level against a concentration gradient. Anti-MRP antisera predominantly stain the plasma membrane of MRP-overexpressing cells. We conclude that MRP is a plasma membrane drug-efflux pump. Images PMID:7916458

  20. Azole resistance in Candida spp. isolated from Catú Lake, Ceará, Brazil: an efflux-pump-mediated mechanism

    PubMed Central

    Brilhante, Raimunda S.N.; Paiva, Manoel A.N.; Sampaio, Célia M.S.; Castelo-Branco, Débora S.C.M.; Teixeira, Carlos E.C.; de Alencar, Lucas P.; Bandeira, Tereza J.P.G.; Monteiro, André J.; Cordeiro, Rossana A.; Pereira-Neto, Waldemiro A.; Sidrim, José J.C.; Moreira, José L.B.; Rocha, Marcos F.G.

    2016-01-01

    Since, there is no study reporting the mechanism of azole resistance among yeasts isolated from aquatic environments; the present study aims to investigate the occurrence of antifungal resistance among yeasts isolated from an aquatic environment, and assess the efflux-pump activity of the azole-resistant strains to better understand the mechanism of resistance for this group of drugs. For this purpose, monthly water and sediment samples were collected from Catú Lake, Ceará, Brazil, from March 2011 to February 2012. The obtained yeasts were identified based on morphological and biochemical characteristics. Of the 46 isolates, 37 were Candida spp., 4 were Trichosporon asahii, 3 were Cryptococcus laurentii, 1 Rhodotorula mucilaginosa, and 1 was Kodamaea ohmeri. These isolates were subjected to broth microdilution assay with amphotericin B, itraconazole, and fluconazole, according to the methodology standardized by the Clinical and Laboratory Standards Institute (CLSI). The minimum inhibitory concentrations (MICs) of amphotericin B, itraconazole, and fluconazole were 0.03125–2 μg/mL, 0.0625 to ≥16 μg/mL, and 0.5 to ≥64 μg/mL, respectively, and 13 resistant azole-resistant Candida isolates were detected. A reduction in the azole MICs leading to the phenotypical reversal of the azole resistance was observed upon addition of efflux-pump inhibitors. These findings suggest that the azole resistance among environmental Candida spp. is most likely associated with the overexpression of efflux-pumps. PMID:26887224

  1. Hyper Accumulation of Arsenic in Mutants of Ochrobactrum tritici Silenced for Arsenite Efflux Pumps

    PubMed Central

    Piedade, Ana Paula; Morais, Paula V.

    2015-01-01

    Ochrobactrum tritici SCII24T is a highly As-resistant bacterium, with two previously described arsenic resistance operons, ars1 and ars2. Among a large number of genes, these operons contain the arsB and Acr3 genes that encode the arsenite efflux pumps responsible for arsenic resistance. Exploring the genome of O. tritici SCII24T, an additional putative operon (ars3) was identified and revealed the presence of the Acr3_2 gene that encodes for an arsenite efflux protein but which came to prove to not be required for full As resistance. The genes encoding for arsenite efflux pumps, identified in this strain, were inactivated to develop microbial accumulators of arsenic as new tools for bioremediation. Six different mutants were produced, studied and three were more useful as biotools. O. tritici wild type and the Acr3-mutants showed the highest resistance to As(III), being able to grow up to 50 mM of arsenite. On the other hand, arsB-mutants were not able to grow at concentrations higher than 1 mM As(III), and were the most As(III) sensitive mutants. In the presence of 1 mM As(III), the strain with arsB and Acr3_1 mutated showed the highest intracellular arsenic concentration (up to 17 ng(As)/mg protein), while in assays with 5 mM As(III), the single arsB-mutant was able to accumulate the highest concentration of arsenic (up to 10 ng(As)/mg protein). Therefore, arsB is the main gene responsible for arsenite resistance in O. tritici. However, both genes arsB and Acr3_1 play a crucial role in the resistance mechanism, depending on the arsenite concentration in the medium. In conclusion, at moderate arsenite concentrations, the double arsB- and Acr3_1-mutant exhibited a great ability to accumulate arsenite and can be seen as a promising bioremediation tool for environmental arsenic detoxification. PMID:26132104

  2. Brain Efflux Index To Investigate the Influence of Active Efflux on Brain Distribution of Pemetrexed and Methotrexate

    PubMed Central

    Li, Li; Agarwal, Sagar

    2013-01-01

    Antifolates, in particular methotrexate (MTX), have been widely used in the treatment of primary and secondary tumors of the central nervous system (CNS). Pemetrexed (PMX) is a novel antifolate that also exhibits potent antitumor activity against CNS malignancies. Studies have shown that brain distribution of both antifolates is significantly restricted, possible due to active efflux transport at the blood-brain barrier (BBB). This study characterizes the brain-to-blood transport of PMX and MTX and examines the role of several efflux transporters in brain distribution of the antifolates by use of the intracerebral microinjection technique (brain efflux index). The results from this study show that both PMX and MTX undergo saturable efflux transport across the BBB, with elimination half-lives of approximately 39 minutes and 29 minutes, respectively. Of the various efflux transporters this study investigated, multidrug resistance-associated protein 2 (Mrp2) does not play an important role in the brain distribution of the two antifolate drugs. Interestingly, breast-cancer resistance protein (Bcrp) makes a significant contribution to the brain elimination of MTX but not PMX. In addition, the brain-to-blood transport of both antifolates was inhibited by probenecid and benzylpenicillin, suggesting the involvement of organic anion transporters in the efflux of these compounds from the brain, with organic anion transporter 3 (Oat3) being a possibility. Our results suggest that one of the underlying mechanisms behind the limited brain distribution of PMX and MTX is active efflux transport processes at the BBB, including a benzylpenicillin-sensitive transport system and/or the active transporter Bcrp. PMID:23297298

  3. P-glycoprotein in adriamycin-resistant cells functions as an efflux pump for benzopyrene, a chemical carcinogen

    SciTech Connect

    Chao Yeh, G.; Poore, C.M.; Lopaczynska, J.; Phang, J.M. )

    1991-03-15

    The physiological function of multidrug resistant gene (MDR 1) coded P-glycoprotein 170 (P-gp) in normal tissues remains unknown. The authors propose that P-gp functions as an efflux pump in normal tissues for benzopyrene and other xenobiotic substances. To examine their hypothesis the authors used a series of MDR human breast cancer MCF-7 cells with increasing degrees of drug resistance, expression of MDR and levels of P-gp. First, they found the IC{sub 50} for benzopyrene is linearly correlated with the levels of P-gp at different stages of adriamycin resistant MCF-7 cells. Using P-gp ({sup 3}H)azidopine labeling as a measurement of P-gp they found benzopyrene competes for labeling of P-gp. Finally, they directly measured cellular efflux of benzopyrene with adherent cell laser cytometry and found that resistant cells expressing high levels of P-gp showed rapid efflux of benzopyrene. By contrast, drug sensitive wild type cells with undetectable P-gp showed negligible efflux. They conclude that P-gp can function as an efflux pump for benzopyrene and suggest that P-gp may be a cellular mechanism for resistance to carcinogens.

  4. A linkage between SmeIJK efflux pump, cell envelope integrity, and σE-mediated envelope stress response in Stenotrophomonas maltophilia.

    PubMed

    Huang, Yi-Wei; Liou, Rung-Shiuan; Lin, Yi-Tsung; Huang, Hsin-Hui; Yang, Tsuey-Ching

    2014-01-01

    Resistance nodulation division (RND) efflux pumps, such as the SmeIJK pump of Stenotrophomonas maltophilia, are known to contribute to the multidrug resistance in Gram-negative bacteria. However, some RND pumps are constitutively expressed even though no antimicrobial stresses occur, implying that there should be some physical implications for these RND pumps. In this study, the role of SmeIJK in antimicrobials resistance, envelope integrity, and σE-mediated envelope stress response (ESR) of S. maltophilia was assessed. SmeIJK was involved in the intrinsic resistance of S. maltophilia KJ to aminoglycosides and leucomycin. Compared with the wild-type KJ, the smeIJK deletion mutant exhibited growth retardation in the MH medium, an increased sensitivity to membrane-damaging agents (MDAs), as well as activation of an σE-mediated ESR. Moreover, the expression of smeIJK was further induced by sub-lethal concentrations of MDAs or surfactants in an σE-dependent manner. These data collectively suggested an alternative physiological role of smeIJK in cell envelope integrity maintenance and σE-mediated ESR beyond the efflux of antibiotics. Because of the necessity of the physiological role of SmeIJK in protecting S. maltophilia from the envelope stress, smeIJK is constitutively expressed, which, in turn, contributes the intrinsic resistance to aminoglycoside and leucomycin. This is the first demonstration of the linkage among RND-type efflux pump, cell envelope integrity, and σE-mediated ESR in S. maltophilia. PMID:25390933

  5. A Linkage between SmeIJK Efflux Pump, Cell Envelope Integrity, and σE-Mediated Envelope Stress Response in Stenotrophomonas maltophilia

    PubMed Central

    Huang, Yi-Wei; Liou, Rung-Shiuan; Lin, Yi-Tsung; Huang, Hsin-Hui; Yang, Tsuey-Ching

    2014-01-01

    Resistance nodulation division (RND) efflux pumps, such as the SmeIJK pump of Stenotrophomonas maltophilia, are known to contribute to the multidrug resistance in Gram-negative bacteria. However, some RND pumps are constitutively expressed even though no antimicrobial stresses occur, implying that there should be some physical implications for these RND pumps. In this study, the role of SmeIJK in antimicrobials resistance, envelope integrity, and σE-mediated envelope stress response (ESR) of S. maltophilia was assessed. SmeIJK was involved in the intrinsic resistance of S. maltophilia KJ to aminoglycosides and leucomycin. Compared with the wild-type KJ, the smeIJK deletion mutant exhibited growth retardation in the MH medium, an increased sensitivity to membrane-damaging agents (MDAs), as well as activation of an σE-mediated ESR. Moreover, the expression of smeIJK was further induced by sub-lethal concentrations of MDAs or surfactants in an σE-dependent manner. These data collectively suggested an alternative physiological role of smeIJK in cell envelope integrity maintenance and σE-mediated ESR beyond the efflux of antibiotics. Because of the necessity of the physiological role of SmeIJK in protecting S. maltophilia from the envelope stress, smeIJK is constitutively expressed, which, in turn, contributes the intrinsic resistance to aminoglycoside and leucomycin. This is the first demonstration of the linkage among RND-type efflux pump, cell envelope integrity, and σE-mediated ESR in S. maltophilia. PMID:25390933

  6. Functional cloning and characterization of the multidrug efflux pumps NorM from Neisseria gonorrhoeae and YdhE from Escherichia coli.

    PubMed

    Long, Feng; Rouquette-Loughlin, Corinne; Shafer, William M; Yu, Edward W

    2008-09-01

    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. PMID:18591276

  7. Development of a novel antimicrobial screening system targeting the pyoverdine-mediated iron acquisition system and xenobiotic efflux pumps.

    PubMed

    Sato, Kazuki; Ushioda, Kenichi; Akiba, Keiji; Matsumoto, Yoshimi; Maseda, Hideaki; Ando, Tasuke; Isogai, Emiko; Nakae, Taiji; Yoneyama, Hiroshi

    2015-01-01

    The iron acquisition systems in Pseudomonas aeruginosa are inducible in response to low-iron conditions and important for growth of this organism under iron limitation. OprM is the essential outer membrane subunit of the MexAB-OprM xenobiotic efflux pump. We designed and constructed a new model antimicrobial screening system targeting both the iron-uptake system and xenobiotic efflux pumps. The oprM gene was placed immediately downstream of the ferri-pyoverdine receptor gene, fpvA, in the host lacking chromosomal oprM and the expression of oprM was monitored by an antibiotic susceptibility test under iron depleted and replete conditions. The recombinant cells showed wild-type susceptibility to pump substrate antibiotics, e.g., aztreonam, under iron limitation and became supersusceptible to them under iron repletion, suggesting that expression of oprM is under control of the iron acquisition system. Upon screening of a chemical library comprising 2952 compounds using this strain, a compound-ethyl 2-(1-acetylpiperidine-4-carboxamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate-was found to enhance the efficacy of aztreonam under iron limitation, suggesting that the compound inhibits either the iron acquisition system or the MexAB-OprM efflux pump. This compound was subsequently found to inhibit the growth of wild-type cells in the presence of sublethal amounts of aztreonam, regardless of the presence or absence of dipyridyl, an iron-chelator. The compound was eventually identified to block the function of the MexAB-OprM efflux pump, showing the validity of this new method. PMID:25939068

  8. Contribution of Efflux Pumps, Porins, and β-Lactamases to Multidrug Resistance in Clinical Isolates of Acinetobacter baumannii

    PubMed Central

    Rumbo, C.; Gato, E.; López, M.; Ruiz de Alegría, C.; Fernández-Cuenca, F.; Martínez-Martínez, L.; Vila, J.; Pachón, J.; Cisneros, J. M.; Rodríguez-Baño, J.; Pascual, A.

    2013-01-01

    We investigated the mechanisms of resistance to carbapenems, aminoglycosides, glycylcyclines, tetracyclines, and quinolones in 90 multiresistant clinical strains of Acinetobacter baumannii isolated from two genetically unrelated A. baumannii clones: clone PFGE-ROC-1 (53 strains producing the OXA-58 β-lactamase enzyme and 18 strains with the OXA-24 β-lactamase) and clone PFGE-HUI-1 (19 strains susceptible to carbapenems). We used real-time reverse transcriptase PCR to correlate antimicrobial resistance (MICs) with expression of genes encoding chromosomal β-lactamases (AmpC and OXA-51), porins (OmpA, CarO, Omp33, Dcap-like, OprB, Omp25, OprC, OprD, and OmpW), and proteins integral to six efflux systems (AdeABC, AdeIJK, AdeFGH, CraA, AbeM, and AmvA). Overexpression of the AdeABC system (level of expression relative to that by A. baumannii ATCC 17978, 30- to 45-fold) was significantly associated with resistance to tigecycline, minocycline, and gentamicin and other biological functions. However, hyperexpression of the AdeIJK efflux pump (level of expression relative to that by A. baumannii ATCC 17978, 8- to 10-fold) was significantly associated only with resistance to tigecycline and minocycline (to which the TetB efflux system also contributed). TetB and TetA(39) efflux pumps were detected in clinical strains and were associated with resistance to tetracyclines and doxycycline. The absence of the AdeABC system and the lack of expression of other mechanisms suggest that tigecycline-resistant strains of the PFGE-HUI-1 clone may be associated with a novel resistance-nodulation-cell efflux pump (decreased MICs in the presence of the inhibitor Phe-Arg β-naphthylamide dihydrochloride) and the TetA(39) system. PMID:23939894

  9. Contribution of efflux pumps, porins, and β-lactamases to multidrug resistance in clinical isolates of Acinetobacter baumannii.

    PubMed

    Rumbo, C; Gato, E; López, M; Ruiz de Alegría, C; Fernández-Cuenca, F; Martínez-Martínez, L; Vila, J; Pachón, J; Cisneros, J M; Rodríguez-Baño, J; Pascual, A; Bou, G; Tomás, M

    2013-11-01

    We investigated the mechanisms of resistance to carbapenems, aminoglycosides, glycylcyclines, tetracyclines, and quinolones in 90 multiresistant clinical strains of Acinetobacter baumannii isolated from two genetically unrelated A. baumannii clones: clone PFGE-ROC-1 (53 strains producing the OXA-58 β-lactamase enzyme and 18 strains with the OXA-24 β-lactamase) and clone PFGE-HUI-1 (19 strains susceptible to carbapenems). We used real-time reverse transcriptase PCR to correlate antimicrobial resistance (MICs) with expression of genes encoding chromosomal β-lactamases (AmpC and OXA-51), porins (OmpA, CarO, Omp33, Dcap-like, OprB, Omp25, OprC, OprD, and OmpW), and proteins integral to six efflux systems (AdeABC, AdeIJK, AdeFGH, CraA, AbeM, and AmvA). Overexpression of the AdeABC system (level of expression relative to that by A. baumannii ATCC 17978, 30- to 45-fold) was significantly associated with resistance to tigecycline, minocycline, and gentamicin and other biological functions. However, hyperexpression of the AdeIJK efflux pump (level of expression relative to that by A. baumannii ATCC 17978, 8- to 10-fold) was significantly associated only with resistance to tigecycline and minocycline (to which the TetB efflux system also contributed). TetB and TetA(39) efflux pumps were detected in clinical strains and were associated with resistance to tetracyclines and doxycycline. The absence of the AdeABC system and the lack of expression of other mechanisms suggest that tigecycline-resistant strains of the PFGE-HUI-1 clone may be associated with a novel resistance-nodulation-cell efflux pump (decreased MICs in the presence of the inhibitor Phe-Arg β-naphthylamide dihydrochloride) and the TetA(39) system. PMID:23939894

  10. Carpobrotus edulis methanol extract inhibits the MDR efflux pumps, enhances killing of phagocytosed S. aureus and promotes immune modulation.

    PubMed

    Ordway, Diane; Hohmann, Judit; Viveiros, Miguel; Viveiros, Antonio; Molnar, Joseph; Leandro, Clara; Arroz, Maria Jorge; Gracio, Maria Amelia; Amaral, Leonard

    2003-05-01

    Although alkaloids from the family Aizoaceae have anticancer activity, species of this family have received little attention. Because these alkaloids also exhibit properties normally associated with compounds that have activity at the level of the plasma membrane, a methanol extract of Carpobrotus edulis, a common plant found along the Portuguese coast, was studied for properties normally associated with plasma membrane active compounds. The results of this study show that the extract is non-toxic at concentrations that inhibit a verapamil sensitive efflux pump of L5178 mouse T cell lymphoma cell line thereby rendering these multi-drug resistant cells susceptible to anticancer drugs. These non-toxic concentrations also prime THP-1 human monocyte-derived macrophages to kill ingested Staphylococcus aureus and to promote the release of lymphokines associated with cellular immune functions. The extract also induces the proliferation of THP-1 cells within 1 day of exposure to quantities normally associated with phytohaemagglutinin. The potential role of the compound(s) isolated from this plant in cancer biology is intriguing and is currently under investigation. It is supposed that the resistance modifier and immunomodulatory effect of this plant extract can be exploited in the experimental chemotherapy of cancer and bacterial or viral infections. PMID:12748989

  11. Efflux pump inhibitor potentiates antimicrobial photodynamic inactivation of Enterococcus faecalis biofilm.

    PubMed

    Kishen, Anil; Upadya, Megha; Tegos, George P; Hamblin, Michael R

    2010-01-01

    Microbial biofilm architecture contains numerous protective features, including extracellular polymeric material that render biofilms impermeable to conventional antimicrobial agents. This study evaluated the efficacy of antimicrobial photodynamic inactivation (aPDI) of Enterococcus faecalis biofilms. The ability of a cationic, phenothiazinium photosensitizer, methylene blue (MB) and an anionic, xanthene photosensitizer, rose bengal (RB) to inactivate biofilms of E. faecalis (OG1RF and FA 2-2) and disrupt the biofilm structure was evaluated. Bacterial cells were tested as planktonic suspensions, intact biofilms and biofilm-derived suspensions obtained by the mechanical disruption of biofilms. The role of a specific microbial efflux pump inhibitor (EPI), verapamil hydrochloride in the MB-mediated aPDI of E. faecalis biofilms was also investigated. The results showed that E. faecalis biofilms exhibited significantly higher resistance to aPDI when compared with E. faecalis in suspension (P < 0.001). aPDI with cationic MB produced superior inactivation of E. faecalis strains in a biofilm along with significant destruction of biofilm structure when compared with anionic RB (P < 0.05). The ability to inactivate biofilm bacteria was further enhanced when the EPI was used with MB (P < 0.001). These experiments demonstrated the advantage of a cationic phenothiazinium photosensitizer combined with an EPI to inactivate biofilm bacteria and disrupt biofilm structure. PMID:20860692

  12. Structure and function of Neisseria gonorrhoeae MtrF illuminates a class of antimetabolite efflux pumps.

    PubMed

    Su, Chih-Chia; Bolla, Jani Reddy; Kumar, Nitin; Radhakrishnan, Abhijith; Long, Feng; Delmar, Jared A; Chou, Tsung-Han; Rajashankar, Kanagalaghatta R; Shafer, William M; Yu, Edward W

    2015-04-01

    Neisseria gonorrhoeae is an obligate human pathogen and the causative agent of the sexually transmitted disease gonorrhea. The control of this disease has been compromised by the increasing proportion of infections due to antibiotic-resistant strains, which are growing at an alarming rate. N. gonorrhoeae MtrF is an integral membrane protein that belongs to the AbgT family of transporters for which no structural information is available. Here, we describe the crystal structure of MtrF, revealing a dimeric molecule with architecture distinct from all other families of transporters. MtrF is a bowl-shaped dimer with a solvent-filled basin extending from the cytoplasm to halfway across the membrane bilayer. Each subunit of the transporter contains nine transmembrane helices and two hairpins, posing a plausible pathway for substrate transport. A combination of the crystal structure and biochemical functional assays suggests that MtrF is an antibiotic efflux pump mediating bacterial resistance to sulfonamide antimetabolite drugs. PMID:25818299

  13. Vulvovaginal candidiasis: species distribution, fluconazole resistance and drug efflux pump gene overexpression.

    PubMed

    Zhang, Jie-Yu; Liu, Jin-Hui; Liu, Fa-Di; Xia, Yan-Hua; Wang, Jing; Liu, Xi; Zhang, Zhi-Qin; Zhu, Na; Yan-Yan; Ying, Ying; Huang, Xiao-Tian

    2014-10-01

    The increasing incidence of vulvovaginal candidiasis (VVC) and the emergence of fluconazole resistance are an indisputable fact. However, little information is available regarding the correlation between fluconazole resistance in vaginal Candida albicans and the expression of drug efflux pump genes. In this study, we investigated the species distribution, fluconazole susceptibility profiles and the mechanisms of fluconazole resistance in Candida strains. In total, 785 clinical Candida isolates were collected from patients with VVC. C. albicans was the most frequently isolated species(n = 529) followed by C. glabrata (n = 164) and C. krusei (n = 57). Of all Candida isolates, 4.7% were resistant to fluconazole. We randomly selected 18 fluconazole resistant isolates of C. albicans to evaluate the expression of CDR1, CDR2, MDR1 and FLU1 genes. Compared with fluconazole-susceptible C. albicans isolates, CDR1 gene expression displayed 3.16-fold relative increase, which was statistically significant. CDR2, MDR1 and FLU1 overexpression was observed in several fluconazole-resistant C. albicans isolates, but statistical significance was not achieved. These results demonstrate a high frequency of non-albicans species (32.6%); however, C. albicans is the most common Candida species implicated in vaginitis, and this strain displays considerable fluconazole resistance. Meanwhile, our study further indicates that fluconazole resistance in C. albicans may correlate with CDR1 gene overexpression. PMID:24962255

  14. Efflux pump-deficient mutants as a platform to search for microbes that produce antibiotics.

    PubMed

    Molina-Santiago, Carlos; Udaondo, Zulema; Daddaoua, Abdelali; Roca, Amalia; Martín, Jesús; Pérez-Victoria, Ignacio; Reyes, Fernando; Ramos, Juan-Luis

    2015-07-01

    Pseudomonas putida DOT-T1E-18 is a strain deficient in the major antibiotic efflux pump (TtgABC) that exhibits an overall increased susceptibility to a wide range of drugs when compared with the wild-type strain. We used this strain as a platform to search for microbes able to produce antibiotics that inhibit growth. A collection of 2400 isolates from soil, sediments and water was generated and a drop assay developed to identify, via growth inhibition halos, strains that prevent the growth of DOT-T1E-18 on solid Luria-Bertani plates. In this study, 35 different isolates that produced known and unknown antibiotics were identified. The most potent inhibitor of DOT-T1E-18 growth was an isolate named 250J that, through multi-locus sequence analysis, was identified as a Pseudomonas sp. strain. Culture supernatants of 250J contain four different xantholysins that prevent growth of Gram-positive bacteria, Gram-negative and fungi. Two of the xantholysins were produced in higher concentrations and purified. Xantholysin A was effective against Bacillus, Lysinibacillus and Rhodococcus strains, and the effect against these microbes was enhanced when used in combination with other antibiotics such as ampicillin, gentamicin and kanamycin. Xantholysin C was also efficient against Gram-positive bacteria and showed an interesting antimicrobial effect against Pseudomonas strains, and a synergistic inhibitory effect with ampicillin, chloramphenicol and gentamicin. PMID:26059350

  15. Expression of multidrug resistance efflux pump genes in clinical and environmental isolates of Staphylococcus aureus.

    PubMed

    Kosmidis, Christos; Schindler, Bryan D; Jacinto, Pauline L; Patel, Diixa; Bains, Karanpreet; Seo, Susan M; Kaatz, Glenn W

    2012-09-01

    Increased expression of multidrug resistance efflux pump (MDR-EP) genes in clinical isolates of Staphylococcus aureus occurs frequently, but its temporal and geographic variability is unknown. Such strains may contaminate the hospital environment, posing an infection control problem. Nearly 700 clinical isolates from different geographic locales as well as 91 environmental isolates recovered from two Detroit hospitals were studied. Ethidium bromide (EtBr) minimum inhibitory concentration (MIC), quantitative expression of all characterised chromosomal MDR-EP genes, and the presence of qacA/B and smr were determined for all strains. In addition, for norA- and/or mepA-overexpressing strains, the spa type was established. MDR-EP gene overexpression varied temporally and geographically, and overexpressing strains were present in the hospital environment. Increased expression of norA was associated with meticillin resistance and spa type t002, a rare type among control strains, consistent with widespread dissemination of a norA-overexpressing, meticillin-resistant S. aureus (MRSA) clone. Clonal spread also played a role for spa type t008, mepA-overexpressing, meticillin-susceptible strains. An EtBr MIC of ≤12.5 μg/mL was highly specific (>90%) in identifying strains lacking MDR-EP gene overexpression. PMID:22766161

  16. Efflux pump-deficient mutants as a platform to search for microbes that produce antibiotics

    PubMed Central

    Molina-Santiago, Carlos; Udaondo, Zulema; Daddaoua, Abdelali; Roca, Amalia; Martín, Jesús; Pérez-Victoria, Ignacio; Reyes, Fernando; Ramos, Juan-Luis

    2015-01-01

    Pseudomonas putida DOT-T1E-18 is a strain deficient in the major antibiotic efflux pump (TtgABC) that exhibits an overall increased susceptibility to a wide range of drugs when compared with the wild-type strain. We used this strain as a platform to search for microbes able to produce antibiotics that inhibit growth. A collection of 2400 isolates from soil, sediments and water was generated and a drop assay developed to identify, via growth inhibition halos, strains that prevent the growth of DOT-T1E-18 on solid Luria–Bertani plates. In this study, 35 different isolates that produced known and unknown antibiotics were identified. The most potent inhibitor of DOT-T1E-18 growth was an isolate named 250J that, through multi-locus sequence analysis, was identified as a Pseudomonas sp. strain. Culture supernatants of 250J contain four different xantholysins that prevent growth of Gram-positive bacteria, Gram-negative and fungi. Two of the xantholysins were produced in higher concentrations and purified. Xantholysin A was effective against Bacillus, Lysinibacillus and Rhodococcus strains, and the effect against these microbes was enhanced when used in combination with other antibiotics such as ampicillin, gentamicin and kanamycin. Xantholysin C was also efficient against Gram-positive bacteria and showed an interesting antimicrobial effect against Pseudomonas strains, and a synergistic inhibitory effect with ampicillin, chloramphenicol and gentamicin. PMID:26059350

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

    PubMed Central

    Sekiya, Hiroshi; Mima, Takehiko; Morita, Yuji; Kuroda, Teruo; Mizushima, Tohru; Tsuchiya, Tomofusa

    2003-01-01

    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. PMID:12937010

  18. Re-evolution of the 2-phenylquinolines: ligand-based design, synthesis, and biological evaluation of a potent new class of Staphylococcus aureus NorA efflux pump inhibitors to combat antimicrobial resistance.

    PubMed

    Sabatini, Stefano; Gosetto, Francesca; Iraci, Nunzio; Barreca, Maria Letizia; Massari, Serena; Sancineto, Luca; Manfroni, Giuseppe; Tabarrini, Oriana; Dimovska, Mirjana; Kaatz, Glenn W; Cecchetti, Violetta

    2013-06-27

    Overexpression of efflux pumps is an important mechanism by which bacteria evade the effects of antimicrobial agents that are substrates. NorA is a Staphylococcus aureus efflux pump that confers reduced susceptibility to many structurally unrelated agents, including fluoroquinolones, biocides, and dyes, resulting in a multidrug resistant (MDR) phenotype. In this work, a series of 2-phenylquinoline derivatives was designed by means of ligand-based pharmacophore modeling in an attempt to identify improved S. aureus NorA efflux pump inhibitors (EPIs). Most of the 2-phenylquinoline derivatives displayed potent EPI activity against the norA overexpressing strain SA-1199B. The antibacterial activity of ciprofloxacin, when used in combination with some of the synthesized compounds, was completely restored in SA-1199B and SA-K2378, a strain overexpressing norA from a multicopy plasmid. Compounds 3m and 3q also showed potent synergistic activity with the ethidium bromide dye in a strain overexpressing the MepA MDR efflux pump. PMID:23710549

  19. Pyrazolo[4,3-c][1,2]benzothiazines 5,5-dioxide: a promising new class of Staphylococcus aureus NorA efflux pump inhibitors.

    PubMed

    Sabatini, Stefano; Gosetto, Francesca; Serritella, Serena; Manfroni, Giuseppe; Tabarrini, Oriana; Iraci, Nunzio; Brincat, Jean Pierre; Carosati, Emanuele; Villarini, Milena; Kaatz, Glenn W; Cecchetti, Violetta

    2012-04-12

    The increasing resistance to antibacterials commonly employed in the clinic and the growth of multidrug resistant strains suggest that the development of new therapeutic approaches should be of primary concern. In this context, EPIs may restore life to old drugs. In the present work, the EPI activity of the COX-2 inhibitor celecoxib was confirmed and a new class of pyrazolo[4,3-c][1,2]benzothiazine 5,5-dioxide analogues acting as inhibitors of the Staphylococcus aureus NorA multidrug efflux pump was identified. PMID:22432682

  20. The in-vitro antimicrobial effect of non-antibiotics and putative inhibitors of efflux pumps on Pseudomonas aeruginosa and Staphylococcus aureus.

    PubMed

    Hendricks, O; Butterworth, T S; Kristiansen, J E

    2003-09-01

    The anti-microbial activity of six non-antibiotics (one amino-ethylchloride, three phenothiazines, two tricyclic antidepressives) were tested on 20 clinical isolates of Pseudomonas aeruginosa, one clinical isolate of Klebsiella pneumoniae, 2 ATTC strains and 14 clinical isolates of Staphylococccus aureus, using the plate dilution method. The effects on P. aeruginosa were independent of antibiotic resistance pattern and the species Stenotrophomonas maltophilia was found to be the most susceptible to the non-antibiotics, with MIC values as low as 20 mg/l for some of the substances. The 16 S. aureus strains tested were all particularly susceptible to the anti-microbial effects of the putative inhibitors of efflux pumps thioridazine and trifluoperazine with MIC values of < or =16 mg/l independently of the methicillin resistance profile of the strains. Because phenothiazines are well known to inhibit efflux pumps our results may indicate the existence of such pumps. Current works in progress are attempts at reversing the antibiotic resistance of selected bacterial strains using specific non-antibiotics and their stereo-chemical isomers. PMID:13678831

  1. Expression of the CDR1 efflux pump in clinical Candida albicans isolates is controlled by a negative regulatory element

    SciTech Connect

    Gaur, Naseem Akhtar; Manoharlal, Raman; Saini, Preeti; Prasad, Tulika; Mukhopadhyay, Gauranga; Hoefer, Milan; Morschhaeuser, Joachim; Prasad, Rajendra . E-mail: rp47@hotmail.com

    2005-06-24

    Resistance to azole antifungal drugs in clinical isolates of the human fungal pathogen Candida albicans is often caused by constitutive overexpression of the CDR1 gene, which encodes a multidrug efflux pump of the ABC transporter superfamily. To understand the relevance of a recently identified negative regulatory element (NRE) in the CDR1 promoter for the control of CDR1 expression in the clinical scenario, we investigated the effect of mutation or deletion of the NRE on CDR1 expression in two matched pairs of azole-sensitive and resistant clinical isolates of C. albicans. Expression of GFP or lacZ reporter genes from the wild type CDR1 promoter was much higher in the azole-resistant C. albicans isolates than in the azole-susceptible isolates, reflecting the known differences in CDR1 expression in these strains. Deletion or mutation of the NRE resulted in enhanced reporter gene expression in azole-sensitive strains, but did not further increase the already high CDR1 promoter activity in the azole-resistant strains. In agreement with these findings, electrophoretic mobility shift assays showed a reduced binding to the NRE of nuclear extracts from the resistant C. albicans isolates as compared with extracts from the sensitive isolates. These results demonstrate that the NRE is involved in maintaining CDR1 expression at basal levels and that this repression is overcome in azole-resistant clinical C. albicans isolates, resulting in constitutive CDR1 overexpression and concomitant drug resistance.

  2. Catch me if you can: a biotinylated proteoliposome affinity assay for the investigation of assembly of the MexA-MexB-OprM efflux pump from Pseudomonas aeruginosa

    PubMed Central

    Ntsogo Enguéné, Véronique Yvette; Verchère, Alice; Phan, Gilles; Broutin, Isabelle; Picard, Martin

    2015-01-01

    Efflux pumps are membrane transporters that actively extrude various substrates, leading to multidrug resistance (MDR). In this study, we have designed a new test that allows investigating the assembly of the MexA-MexB-OprM efflux pump from the Gram negative bacteria Pseudomonas aeruginosa. The method relies on the streptavidin-mediated pull-down of OprM proteoliposomes upon interaction with MexAB proteoliposomes containing a biotin function carried by lipids. We give clear evidence for the importance of MexA in promoting and stabilizing the assembly of the MexAB-OprM complex. In addition, we have investigated the effect of the role of the lipid anchor of MexA as well as the role of the proton motive force on the assembly and disassembly of the efflux pump. The assay presented here allows for an accurate investigation of the assembly with only tens of microgram of protein and could be adapted to 96 wells plates. Hence, this work provides a basis for the medium-high screening of efflux pump inhibitors (EPIs). PMID:26082762

  3. Catch me if you can: a biotinylated proteoliposome affinity assay for the investigation of assembly of the MexA-MexB-OprM efflux pump from Pseudomonas aeruginosa.

    PubMed

    Ntsogo Enguéné, Véronique Yvette; Verchère, Alice; Phan, Gilles; Broutin, Isabelle; Picard, Martin

    2015-01-01

    Efflux pumps are membrane transporters that actively extrude various substrates, leading to multidrug resistance (MDR). In this study, we have designed a new test that allows investigating the assembly of the MexA-MexB-OprM efflux pump from the Gram negative bacteria Pseudomonas aeruginosa. The method relies on the streptavidin-mediated pull-down of OprM proteoliposomes upon interaction with MexAB proteoliposomes containing a biotin function carried by lipids. We give clear evidence for the importance of MexA in promoting and stabilizing the assembly of the MexAB-OprM complex. In addition, we have investigated the effect of the role of the lipid anchor of MexA as well as the role of the proton motive force on the assembly and disassembly of the efflux pump. The assay presented here allows for an accurate investigation of the assembly with only tens of microgram of protein and could be adapted to 96 wells plates. Hence, this work provides a basis for the medium-high screening of efflux pump inhibitors (EPIs). PMID:26082762

  4. Discovery of 4-acetyl-3-(4-fluorophenyl)-1-(p-tolyl)-5-methylpyrrole as a dual inhibitor of human P-glycoprotein and Staphylococcus aureus Nor A efflux pump.

    PubMed

    Bharate, Jaideep B; Singh, Samsher; Wani, Abubakar; Sharma, Sadhana; Joshi, Prashant; Khan, Inshad A; Kumar, Ajay; Vishwakarma, Ram A; Bharate, Sandip B

    2015-05-21

    Polysubstituted pyrrole natural products, lamellarins, are known to overcome multi-drug resistance in cancer via the inhibition of p-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) efflux pumps. Herein, a series of simplified polysubstituted pyrroles, prepared via a one-pot domino protocol, were screened for P-gp inhibition in P-gp overexpressing human adenocarcinoma LS-180 cells using a rhodamine 123 efflux assay. Several compounds showed the significant inhibition of P-gp at 50 μM, as indicated by increase in the intracellular accumulation of Rh123 in LS-180 cells. Furthermore, pyrrole 5i decreased the efflux of digoxin, a FDA approved P-gp substrate in MDCK-MDR1 cells with an IC50 of 11.2 μM. In in vivo studies, following the oral administration of a P-gp substrate drug, rifampicin, along with compound , the Cmax and AUC0-∞ of rifampicin was enhanced by 31% and 46%, respectively. All the compounds were then screened for their ability to potentiate ciprofloxacin activity via the inhibition of Staphylococcus aureus Nor A efflux pump. Pyrrole showed the significant inhibition of S. aureus Nor A efflux pump with 8- and 4-fold reductions in the MIC of ciprofloxacin at 50 and 6.25 μM, respectively. The molecular docking studies of compound with the human P-gp and S. aureus Nor A efflux pump identified its plausible binding site and key interactions. Thus, the results presented herein strongly indicate the potential of this scaffold for its use as multi-drug resistance reversal agent or bioavailability enhancer. PMID:25865846

  5. Molecular basis for inhibition of AcrB multidrug efflux pump by novel and powerful pyranopyridine derivatives.

    PubMed

    Sjuts, Hanno; Vargiu, Attilio V; Kwasny, Steven M; Nguyen, Son T; Kim, Hong-Suk; Ding, Xiaoyuan; Ornik, Alina R; Ruggerone, Paolo; Bowlin, Terry L; Nikaido, Hiroshi; Pos, Klaas M; Opperman, Timothy J

    2016-03-29

    TheEscherichia coliAcrAB-TolC efflux pump is the archetype of the resistance nodulation cell division (RND) exporters from Gram-negative bacteria. Overexpression of RND-type efflux pumps is a major factor in multidrug resistance (MDR), which makes these pumps important antibacterial drug discovery targets. We have recently developed novel pyranopyridine-based inhibitors of AcrB, which are orders of magnitude more powerful than the previously known inhibitors. However, further development of such inhibitors has been hindered by the lack of structural information for rational drug design. Although only the soluble, periplasmic part of AcrB binds and exports the ligands, the presence of the membrane-embedded domain in AcrB and its polyspecific binding behavior have made cocrystallization with drugs challenging. To overcome this obstacle, we have engineered and produced a soluble version of AcrB [AcrB periplasmic domain (AcrBper)], which is highly congruent in structure with the periplasmic part of the full-length protein, and is capable of binding substrates and potent inhibitors. Here, we describe the molecular basis for pyranopyridine-based inhibition of AcrB using a combination of cellular, X-ray crystallographic, and molecular dynamics (MD) simulations studies. The pyranopyridines bind within a phenylalanine-rich cage that branches from the deep binding pocket of AcrB, where they form extensive hydrophobic interactions. Moreover, the increasing potency of improved inhibitors correlates with the formation of a delicate protein- and water-mediated hydrogen bond network. These detailed insights provide a molecular platform for the development of novel combinational therapies using efflux pump inhibitors for combating multidrug resistant Gram-negative pathogens. PMID:26976576

  6. Upregulation of the Saccharomyces cerevisiae efflux pump Tpo1 rescues an Imp2 transcription factor-deficient mutant from bleomycin toxicity.

    PubMed

    Berra, Siham; Ayachi, Sami; Ramotar, Dindial

    2014-07-01

    Yeast mutants lacking the transcriptional co-activator Imp2 are hypersensitive to the anticancer drug bleomycin, although the gene targets involved in this process remain elusive. A search for multicopy suppressors that rescue the imp2Δ mutant from bleomycin toxicity revealed the transcriptional activator Yap1, which can turn on many target genes such as transporters involved in regulating drug resistance. We show that YAP1 overexpression stimulated the expression of the TPO1 gene encoding a polyamine efflux pump, and that Yap1 failed to rescue the imp2Δ mutant from bleomycin toxicity in the absence of the TPO1 gene. Moreover, TPO1 overexpression, and not the related transporter gene QDR3, conferred upon the tpo1Δ imp2Δ double mutant parental resistance to bleomycin. We conclude that YAP1 overexpression rescues the imp2Δ mutant from bleomycin toxicity by triggering Tpo1 expression to expel the drug. Our data provide the first evidence that bleomycin could be a substrate for the Tpo1 efflux pump. PMID:24599794

  7. Regulation of the MtrC-MtrD-MtrE efflux-pump system modulates the in vivo fitness of Neisseria gonorrhoeae.

    PubMed

    Warner, Douglas M; Folster, Jason P; Shafer, William M; Jerse, Ann E

    2007-12-15

    The Neisseria gonorrhoeae MtrC-MtrD-MtrE multidrug-resistance efflux pump expels macrolide antibiotics, penicillin, and antimicrobial effectors of the innate defense. Mutation of the mtrR locus, which encodes a transcriptional repressor of the mtrCDE operon, increases gonococcal resistance to these agents. Here we report that, in a mouse infection model, an mtrR mutant is more fit than the wild-type bacteria. Consistent with derepression of the mtrCDE operon as the primary reason for the fitness benefit, an mtrR,mtrE double mutant and an mtrE mutant showed no difference in survival phenotype. Gonococcal mutants deficient in MtrA, an activator of the mtrCDE operon, exhibited significantly reduced fitness in vivo, and mtrA mutants with spontaneous compensatory mtrR mutations were selected during infection. These results confirm the importance of the MtrC-MtrD-MtrE efflux-pump system during experimental gonococcal genital-tract infection and also illustrate an antibiotic-resistance mechanism that is accompanied by a fitness benefit rather than a fitness cost. PMID:18190261

  8. Impact of anatase and rutile titanium dioxide nanoparticles on uptake carriers and efflux pumps in Caco-2 gut epithelial cells

    NASA Astrophysics Data System (ADS)

    Dorier, M.; Brun, E.; Veronesi, G.; Barreau, F.; Pernet-Gallay, K.; Desvergne, C.; Rabilloud, T.; Carapito, C.; Herlin-Boime, N.; Carrière, M.

    2015-04-01

    TiO2 microparticles are widely used in food products, where they are added as a white food colouring agent. This food additive contains a significant amount of nanoscale particles; still the impact of TiO2 nanoparticles (TiO2-NPs) on gut cells is poorly documented. Our study aimed at evaluating the impact of rutile and anatase TiO2-NPs on the main functions of enterocytes, i.e. nutrient absorption driven by solute-liquid carriers (SLC transporters) and protection against other xenobiotics driven by efflux pumps from the ATP-binding cassette (ABC) family. We show that acute exposure of Caco-2 cells to both anatase (12 nm) and rutile (20 nm) TiO2-NPs induce early upregulation of a battery of efflux pumps and nutrient transporters. In addition they cause overproduction of reactive oxygen species and misbalance redox repair systems, without inducing cell mortality or DNA damage. Taken together, these data suggest that TiO2-NPs may increase the functionality of gut epithelial cells, particularly their property to form a protective barrier against exogenous toxicants and to absorb nutrients.TiO2 microparticles are widely used in food products, where they are added as a white food colouring agent. This food additive contains a significant amount of nanoscale particles; still the impact of TiO2 nanoparticles (TiO2-NPs) on gut cells is poorly documented. Our study aimed at evaluating the impact of rutile and anatase TiO2-NPs on the main functions of enterocytes, i.e. nutrient absorption driven by solute-liquid carriers (SLC transporters) and protection against other xenobiotics driven by efflux pumps from the ATP-binding cassette (ABC) family. We show that acute exposure of Caco-2 cells to both anatase (12 nm) and rutile (20 nm) TiO2-NPs induce early upregulation of a battery of efflux pumps and nutrient transporters. In addition they cause overproduction of reactive oxygen species and misbalance redox repair systems, without inducing cell mortality or DNA damage. Taken

  9. Hoechst 33342 Is a Hidden “Janus” amongst Substrates for the Multidrug Efflux Pump LmrP

    PubMed Central

    Neuberger, Arthur; van Veen, Hendrik W.

    2015-01-01

    Multidrug transporters mediate the active extrusion of antibiotics and toxic ions from the cell. This reaction is thought to be based on a switch of the transporter between two conformational states, one in which the interior substrate binding cavity is available for substrate binding at the inside of the cell, and another in which the cavity is exposed to the outside of the cell to enable substrate release. Consistent with this model, cysteine cross-linking studies with the Major Facilitator Superfamily drug/proton antiporter LmrP from Lactococcus lactis demonstrated binding of transported benzalkonium to LmrP in its inward-facing state. The fluorescent dye Hoechst 33342 is a substrate for many multidrug transporters and is extruded by efflux pumps in microbial and mammalian cells. Surprisingly, and in contrast to other multidrug transporters, LmrP was found to actively accumulate, rather than extrude, Hoechst 33342 in lactococcal cells. Consistent with this observation, LmrP expression was associated with cellular sensitivity, rather than resistance to Hoechst 33342. Thus, we discovered a hidden “Janus” amongst LmrP substrates that is translocated in reverse direction across the membrane by binding to outward-facing LmrP followed by release from inward-facing LmrP. These findings are in agreement with distance measurements by electron paramagnetic resonance in which Hoechst 33342 binding was found to stabilize LmrP in its outward-facing conformation. Our data have important implications for the use of multidrug exporters in selective targeting of “Hoechst 33342-like” drugs to cells and tissues in which these transporters are expressed. PMID:26540112

  10. Use of a combined effect model approach for discriminating between ABCB1- and ABCC1-type efflux activities in native bivalve gill tissue.

    PubMed

    Faria, Melissa; Pavlichenko, Vasiliy; Burkhardt-Medicke, Kathleen; Soares, Amadeu M V M; Altenburger, Rolf; Barata, Carlos; Luckenbach, Till

    2016-04-15

    Aquatic organisms, such as bivalves, employ ATP binding cassette (ABC) transporters for efflux of potentially toxic chemicals. Anthropogenic water contaminants can, as chemosensitizers, disrupt efflux transporter function enabling other, putatively toxic compounds to enter the organism. Applying rapid amplification of cDNA ends (RACE) PCR we identified complete cDNAs encoding ABCB1- and ABCC1-type transporter homologs from zebra mussel providing the molecular basis for expression of both transporter types in zebra mussel gills. Further, efflux activities of both transporter types in gills were indicated with dye accumulation assays where efflux of the dye calcein-am was sensitive to both ABCB1- (reversin 205, verapamil) and ABCC1- (MK571) type specific inhibitors. The assumption that different inhibitors targeted different efflux pump types was confirmed when comparing measured effects of binary inhibitor compound mixtures in dye accumulation assays with predictions from mixture effect models. Effects by the MK571/reversin 205 mixture corresponded better with independent action, whereas reversin 205/verapamil joint effects were better predicted by the concentration addition model indicating different and equal targets, respectively. The binary mixture approach was further applied to identify the efflux pump type targeted by environmentally relevant chemosensitizing compounds. Pentachlorophenol and musk ketone, which were selected after a pre-screen of twelve compounds that previously had been identified as chemosensitizers, showed mixture effects that corresponded better with concentration addition when combined with reversine 205 but with independent action predictions when combined with MK571 indicating targeting of an ABCB1-type efflux pump by these compounds. PMID:26929997

  11. Antibiotic resistance and multidrug-resistant efflux pumps expression in lactic acid bacteria isolated from pozol, a nonalcoholic Mayan maize fermented beverage.

    PubMed

    Wacher-Rodarte, Maria Del Carmen; Trejo-Muñúzuri, Tanya Paulina; Montiel-Aguirre, Jesús Fernando; Drago-Serrano, Maria Elisa; Gutiérrez-Lucas, Raúl L; Castañeda-Sánchez, Jorge Ismael; Sainz-Espuñes, Teresita

    2016-05-01

    Pozol is a handcrafted nonalcoholic Mayan beverage produced by the spontaneous fermentation of maize dough by lactic acid bacteria. Lactic acid bacteria (LAB) are carriers of chromosomal encoded multidrug-resistant efflux pumps genes that can be transferred to pathogens and/or confer resistance to compounds released during the fermentation process causing food spoiling. The aim of this study was to evaluate the antibiotic sensibility and the transcriptional expression of ABC-type efflux pumps in LAB isolated from pozol that contributes to multidrug resistance. Analysis of LAB and Staphylococcus (S.) aureus ATCC 29213 and ATCC 6538 control strains to antibiotic susceptibility, minimal inhibitory concentration (MIC), and minimal bactericidal concentration (MBC) to ethidium bromide were based in "standard methods" whereas the ethidium bromide efflux assay was done by fluorometric assay. Transcriptional expression of efflux pumps was analyzed by RT-PCR. LAB showed antibiotic multiresistance profiles, moreover, Lactococcus (L.) lactis and Lactobacillus (L.) plantarum displayed higher ethidium bromide efflux phenotype than S. aureus control strains. Ethidium bromide resistance and ethidium bromide efflux phenotypes were unrelated with the overexpression of lmrD in L. lactics, or the underexpression of lmrA in L. plantarum and norA in S. aureus. These findings suggest that, moreover, the analyzed efflux pumps genes, other unknown redundant mechanisms may underlie the antibiotic resistance and the ethidium bromide efflux phenotype in L. lactis and L. plantarum. Phenotypic and molecular drug multiresistance assessment in LAB may improve a better selection of the fermentation starter cultures used in pozol, and to control the antibiotic resistance widespread and food spoiling for health safety. PMID:27247772

  12. Multidrug efflux pumps: the structures of prokaryotic ATP-binding cassette transporter efflux pumps and implications for our understanding of eukaryotic P-glycoproteins and homologues.

    PubMed

    Kerr, Ian D; Jones, Peter M; George, Anthony M

    2010-02-01

    One of the Holy Grails of ATP-binding cassette transporter research is a structural understanding of drug binding and transport in a eukaryotic multidrug resistance pump. These transporters are front-line mediators of drug resistance in cancers and represent an important therapeutic target in future chemotherapy. Although there has been intensive biochemical research into the human multidrug pumps, their 3D structure at atomic resolution remains unknown. The recent determination of the structure of a mouse P-glycoprotein at subatomic resolution is complemented by structures for a number of prokaryotic homologues. These structures have provided advances into our knowledge of the ATP-binding cassette exporter structure and mechanism, and have provided the template data for a number of homology modelling studies designed to reconcile biochemical data on these clinically important proteins. PMID:19961540

  13. Novel Piperazine Arylideneimidazolones Inhibit the AcrAB-TolC Pump in Escherichia coli and Simultaneously Act as Fluorescent Membrane Probes in a Combined Real-Time Influx and Efflux Assay.

    PubMed

    Bohnert, Jürgen A; Schuster, Sabine; Kern, Winfried V; Karcz, Tadeusz; Olejarz, Agnieszka; Kaczor, Aneta; Handzlik, Jadwiga; Kieć-Kononowicz, Katarzyna

    2016-04-01

    In this study, we tested five compounds belonging to a novel series of piperazine arylideneimidazolones for the ability to inhibit the AcrAB-TolC efflux pump. The biphenylmethylene derivative (BM-19) and the fluorenylmethylene derivative (BM-38) were found to possess the strongest efflux pump inhibitor (EPI) activities in the AcrAB-TolC-overproducingEscherichia colistrain 3-AG100, whereas BM-9, BM-27, and BM-36 had no activity at concentrations of up to 50 μM in a Nile red efflux assay. MIC microdilution assays demonstrated that BM-19 at 1/4 MIC (intrinsic MIC, 200 μM) was able to reduce the MICs of levofloxacin, oxacillin, linezolid, and clarithromycin 8-fold. BM-38 at 1/4 MIC (intrinsic MIC, 100 μM) was able to reduce only the MICs of oxacillin and linezolid (2-fold). Both compounds markedly reduced the MIC of rifampin (BM-19, 32-fold; and BM-38, 4-fold), which is suggestive of permeabilization of the outer membrane as an additional mechanism of action. Nitrocefin hydrolysis assays demonstrated that in addition to their EPI activity, both compounds were in fact weak permeabilizers of the outer membrane. Moreover, it was found that BM-19, BM-27, BM-36, and BM-38 acted as near-infrared-emitting fluorescent membrane probes, which allowed for their use in a combined influx and efflux assay and thus for tracking of the transport of an EPI across the outer membrane by an efflux pump in real time. The EPIs BM-38 and BM-19 displayed the most rapid influx of all compounds, whereas BM-27, which did not act as an EPI, showed the slowest influx. PMID:26824939

  14. Hyperinduction of host beta interferon by a Listeria monocytogenes strain naturally overexpressing the multidrug efflux pump MdrT.

    PubMed

    Schwartz, Kierstyn T; Carleton, Joshua D; Quillin, Sarah J; Rollins, Stuart D; Portnoy, Daniel A; Leber, Jess H

    2012-04-01

    Many pathogens regulate or modify their immune-stimulating ligands to avoid detection by their infected hosts. Listeria monocytogenes, a facultative intracellular bacterial pathogen, interacts with multiple components of mammalian innate immunity during its infection cycle. During replication within the cytosol of infected cells, L. monocytogenes utilizes two multidrug efflux pumps, MdrM and MdrT, to secrete the small nucleic acid second messenger cyclic-di-AMP (c-di-AMP). Host recognition of c-di-AMP triggers the production of type I interferons, including beta interferon (IFN-β), which, surprisingly, promote L. monocytogenes virulence. In this study, we have examined the capacity of multiple laboratory and clinical isolates of L. monocytogenes to stimulate host production of IFN-β. We have identified the L. monocytogenes strain LO28 as able to hyperinduce IFN-β production in infected cells ∼30-fold more than the common laboratory clone L. monocytogenes strain 10403S. Genomic analyses determined that LO28 contains a naturally occurring loss-of-function allele of the transcriptional regulator BrtA and correspondingly derepresses expression of MdrT. Surprisingly, while derepression of MdrT resulted in hyperstimulation of IFN-β, it results in significant attenuation in multiple mouse models of infection. While type I interferons may promote L. monocytogenes virulence, this study demonstrates that unregulated expression of the c-di-AMP-secreting efflux pump MdrT significantly restricts virulence in vivo by an unknown mechanism. PMID:22290148

  15. Insertion sequence disruption of adeR and ciprofloxacin resistance caused by efflux pumps and gyrA and parC mutations in Acinetobacter baumannii.

    PubMed

    Lopes, B S; Amyes, S G B

    2013-02-01

    Acinetobacter baumannii is a pathogenic bacterium responsible for a wide range of infections in immunocompromised patients. This study examined the role of insertional inactivation of the adeR gene and its effect on adeABC gene expression along with characterisation of the gyrA and parC mutations involved in ciprofloxacin resistance in three A. baumannii clinical isolates and their derivatives. Primers designed for the detection of adeSRABC detected the presence of ISAba16, which disrupted the adeR gene in strain Ab12M, and ISAba1, which disrupted the same gene in strains Ab18 and Ab209. A second copy of ISAba1 was detected upstream of the adeA gene in Ab209 leading to AdeABC pump expression. AdeIJK pump expression was seen in all of the isolates but was not as significant as AdeABC expression. Minimum inhibitory concentrations of ciprofloxacin were ≥256 mg/L for all of the isolates and a decrease of ≥8-fold was seen following addition of the efflux pump inhibitor 1-(1-naphthylmethyl)-piperazine. Fluorometric analysis also demonstrated active efflux, with upregulation of adeIJK and some genes of the adeABC operon in some strains. Sequencing of the quinolone resistance-determining region of the gyrA and parC genes revealed a Ser83→Leu change in the gyrA gene and a novel change of Ser80→Trp in the parC gene of Ab12, Ab12M and Ab209; in Ab18 there was a Ser80→Leu change in parC. This study shows the multifactorial contribution of different mechanisms in A. baumannii leading to ciprofloxacin resistance. PMID:23217848

  16. MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 7: highly soluble and in vivo active quaternary ammonium analogue D13-9001, a potential preclinical candidate.

    PubMed

    Yoshida, Ken-Ichi; Nakayama, Kiyoshi; Ohtsuka, Masami; Kuru, Noriko; Yokomizo, Yoshihiro; Sakamoto, Atsunobu; Takemura, Makoto; Hoshino, Kazuki; Kanda, Hiroko; Nitanai, Hironobu; Namba, Kenji; Yoshida, Kumi; Imamura, Yuichiro; Zhang, Jason Z; Lee, Ving J; Watkins, William J

    2007-11-15

    A series of 4-oxo-4H-pyrido[1,2-a]pyrimidine derivatives, substituted at the 2-position with piperidines bearing quaternary ammonium salt side chains, were synthesized and evaluated for their ability to potentiate the activity of the fluoroquinolone levofloxacin (LVFX) and the beta-lactam aztreonam (AZT) in Pseudomonas aeruginosa. Attachment of the charged entity using an N-ethylcarbamoyloxy linker led to the discovery of the highly soluble compound 22 (D13-9001), which maintained good potency in vitro and displayed excellent activity in vivo in a rat pneumonia model of P. aeruginosa. PMID:17869116

  17. Interaction Mediated by the Putative Tip Regions of MdsA and MdsC in the Formation of a Salmonella-Specific Tripartite Efflux Pump

    PubMed Central

    Song, Saemee; Hwang, Soonhye; Lee, Seunghwa; Ha, Nam-Chul; Lee, Kangseok

    2014-01-01

    To survive in the presence of a wide range of toxic compounds, gram-negative bacteria expel such compounds via tripartite efflux pumps that span both the inner and outer membranes. The Salmonella-specific MdsAB pump consists of MdsB, a resistance-nodulation-division (RND)-type inner membrane transporter (IMT) that requires the membrane fusion protein (MFP) MdsA, and an outer membrane protein (OMP; MdsC or TolC) to form a tripartite efflux complex. In this study, we investigated the role of the putative tip regions of MdsA and its OMPs, MdsC and TolC, in the formation of a functional MdsAB-mediated efflux pump. Comparative analysis indicated that although sequence homologies of MdsA and MdsC with other MFPs and OMPs, respectively, are extremely low, key residues in the putative tip regions of these proteins are well conserved. Mutagenesis studies on these conserved sites demonstrated their importance for the physical and functional interactions required to form an MdsAB-mediated pump. Our studies suggest that, despite differences in the primary amino acid sequences and functions of various OMPs and MFPs, interactions mediated by the conserved tip regions of OMP and MFP are required for the formation of functional tripartite efflux pumps in gram-negative bacteria. PMID:24960027

  18. Mutagenesis and Modeling To Predict Structural and Functional Characteristics of the Staphylococcus aureus MepA Multidrug Efflux Pump

    PubMed Central

    Schindler, Bryan D.; Patel, Diixa; Seo, Susan M.

    2013-01-01

    MepA is a multidrug and toxin extrusion (MATE) family protein and the only MATE protein encoded within the Staphylococcus aureus genome. Structural data for MATE proteins are limited to a single high-resolution example, NorM of Vibrio cholerae. Substitution mutations were created in MepA using gradient plates containing both a substrate and reserpine as an efflux pump inhibitor. Site-directed mutagenesis of plasmid-based mepA was used to reproduce these mutations, as well as unique or low-frequency mutations identified in mepA-overexpressing clinical strains, and to mutagenize conserved acidic residues. The effect of these changes on protein function was quantitated in a norA-disrupted host strain by susceptibility testing with and without inhibitors and by determining the proficiency of ethidium efflux. Up-function substitutions clustered in the carboxy half of MepA, near the cytoplasmic face of the protein. Repeated application of the same gradient plate conditions frequently reproduced identical substitution mutations, suggesting that individual residues are required for interaction with specific substrates. Acidic residues critical to protein function were identified in helices 4 and 5. In silico modeling revealed an outward-facing molecule, with helices 1, 2, 4, 7, 8, and 10 having contact with a central cavity that may represent a substrate translocation pathway. Functionally important residues within this cavity included S81, A161, M291, and A302. These data provide a critical starting point for understanding how MATE multidrug efflux proteins function and will be useful in refining crystallographic data when they are available. PMID:23175649

  19. Clinical Strains of Pseudomonas aeruginosa Overproducing MexAB-OprM and MexXY Efflux Pumps Simultaneously

    PubMed Central

    Llanes, Catherine; Hocquet, Didier; Vogne, Christelle; Benali-Baitich, Dounia; Neuwirth, Catherine; Plésiat, Patrick

    2004-01-01

    Simultaneous overexpression of the MexAB-OprM and MexXY efflux systems was demonstrated by real-time reverse transcription-PCR and immunoblotting experiments for 12 multiresistant clinical isolates of Pseudomonas aeruginosa. DNA sequencing analysis showed that nine of these strains (named agrZ mutants) harbored mutations in mexZ, the product of which downregulates the expression of the mexXY operon. In addition, 8 of the 12 strains exhibited mutations in genes known to control transcription of the mexAB-oprM operon. Four of them were nalB mutants with alterations in the repressor gene mexR, three of them appeared to be nalC mutants deficient in gene PA3721 and overexpressing gene PA3720, and one strain was a nalB nalC double mutant. For MexAB-OprM as well as for MexXY, no clear correlation could be established between (i) the types of mutations, (ii) the expression level of mexA or mexX, and (iii) resistance to effluxed antibiotics. Finally, three isolates, named agrW mutants, overproduced MexXY and had an intact mexZ gene, and four strains overproduced MexAB-OprM and had intact mexR and PA3721 genes (nalD mutants). These data show that clinical isolates are able to broaden their drug resistance profiles by coexpressing two Mex efflux pumps and suggest the existence of additional regulators for MexAB-OprM and MexXY. PMID:15105137

  20. An H+-Coupled Multidrug Efflux Pump, PmpM, a Member of the MATE Family of Transporters, from Pseudomonas aeruginosa

    PubMed Central

    He, Gui-Xin; Kuroda, Teruo; Mima, Takehiko; Morita, Yuji; Mizushima, Tohru; Tsuchiya, Tomofusa

    2004-01-01

    We cloned the gene PA1361 (we designated the gene pmpM), which seemed to encode a multidrug efflux pump belonging to the MATE family, of Pseudomonas aeruginosa by the PCR method using the drug-hypersensitive Escherichia coli KAM32 strain as a host. Cells of E. coli possessing the pmpM gene showed elevated resistance to several antimicrobial agents. We observed energy-dependent efflux of ethidium from cells possessing the pmpM gene. We found that PmpM is an H+-drug antiporter, and this finding is the first reported case of an H+-coupled efflux pump in the MATE family. Disruption and reintroduction of the pmpM gene in P. aeruginosa revealed that PmpM is functional and that benzalkonium chloride, fluoroquinolones, ethidium bromide, acriflavine, and tetraphenylphosphonium chloride are substrates for PmpM in this microorganism. PMID:14679249

  1. Gallic acid-based indanone derivative interacts synergistically with tetracycline by inhibiting efflux pump in multidrug resistant E. coli.

    PubMed

    Dwivedi, Gaurav Raj; Tiwari, Nimisha; Singh, Aastha; Kumar, Akhil; Roy, Sudeep; Negi, Arvind Singh; Pal, Anirban; Chanda, Debabrata; Sharma, Ashok; Darokar, Mahendra P

    2016-03-01

    The purpose of the present study was to study the synergy potential of gallic acid-based derivatives in combination with conventional antibiotics using multidrug resistant cultures of Escherichia coli. Gallic acid-based derivatives significantly reduced the MIC of tetracycline against multidrug resistant clinical isolate of E. coli. The best representative, 3-(3',4,'5'-trimethoxyphenyl)-4,5,6-trimethoxyindanone-1, an indanone derivative of gallic acid, was observed to inhibit ethidium bromide efflux and ATPase which was also supported by in silico docking. This derivative extended the post-antibiotic effect and decreased the mutation prevention concentration of tetracycline. This derivative in combination with TET was able to reduce the concentration of TNFα up to 18-fold in Swiss albino mice. This derivative was nontoxic and well tolerated up to 300 mg/kg dose in subacute oral toxicity study in mice. This is the first report of gallic acid-based indanone derivative as drug resistance reversal agent acting through ATP-dependent efflux pump inhibition. PMID:26658982

  2. Effects of chlorophyll-derived efflux pump inhibitor pheophorbide a and pyropheophorbide a on erythromycin resistance of Staphylococcus aureus, Enterococcus faecalis, Salmonella Typhimurium and Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The purpose of this study was to validate the hypothesis that pheophorbide a and pyropheophorbide a reduce erythromycin resistance of reference strains of facultative anaerobic bacteria with multidrug or macrolide efflux pumps, as indicative of their effect on bacteria indigenous to anaerobic swine ...

  3. Crystal structures of OprN and OprJ, outer membrane factors of multidrug tripartite efflux pumps of Pseudomonas aeruginosa.

    PubMed

    Yonehara, Ryo; Yamashita, Eiki; Nakagawa, Atsushi

    2016-06-01

    The genome of Pseudomonas aeruginosa encodes tripartite efflux pumps that extrude functionally and structurally dissimilar antibiotics from the bacterial cell. MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM are the main tripartite efflux pumps responsible for multidrug resistance in P. aeruginosa. The outer membrane factors OprN, OprJ, and OprM are essential components of functional tripartite efflux pumps. To elucidate the structural basis of multidrug resistance, we determined the crystal structures of OprN and OprJ. These structures revealed several features, including tri-acylation of the N-terminal cysteine, a small pore in the β-barrel domain, and a tightly sealed gate in the α-barrel domain. Despite the overall similarity of OprN, OprJ, and OprM, a comparison of their structures and electrostatic distributions revealed subtle differences at the periplasmic end of the α-barrel domain. These results suggested that the overall structures of these outer membrane factors are specifically optimized for particular tripartite efflux pumps. Proteins 2016; 84:759-769. © 2016 Wiley Periodicals, Inc. PMID:26914226

  4. Multidrug efflux transporter activity in sea urchin embryos:Does localization provide a diffusive advantage?

    NASA Astrophysics Data System (ADS)

    Song, Xianfeng; Setayeshgar, Sima; Cole, Bryan; Hamdoun, Amro; Epel, David

    2008-03-01

    Experiments have shown upregulation of multidrug efflux transporter activity approximately 30 min after fertilization in the sea urchin embryo [1]. These ATP-hydrolyzing transporter proteins pump moderately hydrophobic molecules out of the cell and represent the cell's first line of defense againstexogenous toxins. It has also been shown that transporters are moved in vesicles along microfilaments and localized to tips of microvilli prior to activation. We have constructed a geometrically realistic model of the embryo, including microvilli, to explore the functional role of this localization in the efficient elimination of toxins from the standpoint of diffusion. We compute diffusion of toxins in extracellular, membrane and intracellular spaces coupled with transporter activity, using experimentally derived values for physical parameters. For transporters uniformly distributed along microvilli and tip-localized transporters we compare regions in parameter space where each distribution provides diffusive advantage, and comment on the physically expected conditions. [1] A. M. Hamdoun, G. N. Cherr, T. A. Roepke and D. Epel, Developmental Biology 276 452 (2004).

  5. Identification and properties of plasma membrane azole efflux pumps from the pathogenic fungi Cryptococcus gattii and Cryptococcus neoformans

    PubMed Central

    Basso, Luiz R.; Gast, Charles E.; Bruzual, Igor; Wong, Brian

    2015-01-01

    Objectives Cryptococcus gattii from the North American Northwest (NW) have higher azole MICs than do non-NW C. gattii or Cryptococcus neoformans. Since mechanisms of azole resistance in C. gattii are not known, we identified C. gattii and C. neoformans plasma membrane azole efflux pumps and characterized their properties. Methods The C. gattii R265 genome was searched for orthologues of known fungal azole efflux genes, expression of candidate genes was assessed by RT–PCR and the expressed genes' cDNAs were cloned and expressed in Saccharomyces cerevisiae. Azole MICs and intracellular [3H]fluconazole were measured in C. gattii and C. neoformans and in S. cerevisiae expressing each cDNA of interest, as was [3H]fluconazole uptake by post-Golgi vesicles (PGVs) isolated from S. cerevisiae sec6-4 mutants expressing each cDNA of interest. Results Intracellular [3H]fluconazole concentrations were inversely correlated with fluconazole MICs only in 25 NW C. gattii strains. S. cerevisiae expressing three C. gattii cDNAs (encoded by orthologues of C. neoformans AFR1 and MDR1 and the previously unstudied gene AFR2) and their C. neoformans counterparts had higher azole MICs and lower intracellular [3H]fluconazole concentrations than did empty-vector controls. PGVs from S. cerevisiae expressing all six Cryptococcus cDNAs also accumulated more [3H]fluconazole than did controls, and [3H]fluconazole transport by all six transporters of interest was ATP dependent and was inhibited by excess unlabelled fluconazole, voriconazole, itraconazole and posaconazole. Conclusions We conclude that C. gattii and C. neoformans AFR1, MDR1 and AFR2 encode ABC transporters that pump multiple azoles out of S. cerevisiae cells, thereby causing azole resistance. PMID:25630649

  6. Biofilm Formation Caused by Clinical Acinetobacter baumannii Isolates Is Associated with Overexpression of the AdeFGH Efflux Pump

    PubMed Central

    He, Xinlong; Lu, Feng; Yuan, Fenglai; Jiang, Donglin; Zhao, Peng; Zhu, Jie; Cheng, Huali

    2015-01-01

    Chronic wound infections are associated with biofilm formation, which in turn has been correlated with drug resistance. However, the mechanism by which bacteria form biofilms in clinical environments is not clearly understood. This study was designed to investigate the biofilm formation potency of Acinetobacter baumannii and the potential association of biofilm formation with genes encoding efflux pumps, quorum-sensing regulators, and outer membrane proteins. A total of 48 clinically isolated A. baumannii strains, identified by enterobacterial repetitive intergenic consensus (ERIC)-PCR as types A-II, A-III, and A-IV, were analyzed. Three representative strains, which were designated A. baumannii ABR2, ABR11, and ABS17, were used to evaluate antimicrobial susceptibility, biofilm inducibility, and gene transcription (abaI, adeB, adeG, adeJ, carO, and ompA). A significant increase in the MICs of different classes of antibiotics was observed in the biofilm cells. The formation of a biofilm was significantly induced in all the representative strains exposed to levofloxacin. The levels of gene transcription varied between bacterial genotypes, antibiotics, and antibiotic concentrations. The upregulation of adeG correlated with biofilm induction. The consistent upregulation of adeG and abaI was detected in A-III-type A. baumannii in response to levofloxacin and meropenem (1/8 to 1/2× the MIC), conditions which resulted in the greatest extent of biofilm induction. This study demonstrates a potential role of the AdeFGH efflux pump in the synthesis and transport of autoinducer molecules during biofilm formation, suggesting a link between low-dose antimicrobial therapy and a high risk of biofilm infections caused by A. baumannii. This study provides useful information for the development of antibiofilm strategies. PMID:26033730

  7. Evolution and dissemination of OqxAB-like efflux pumps, an emerging quinolone resistance determinant among members of Enterobacteriaceae.

    PubMed

    Wong, Marcus Ho Yin; Chan, Edward Wai Chi; Chen, Sheng

    2015-01-01

    The OqxAB efflux pump, a plasmid-mediated quinolone resistance (PMQR) determinant, has become increasingly prevalent among members of Enterobacteriaceae over the past decade. To investigate the evolution and dissemination routes of the oqxAB operon, we assessed the prevalence of oqxAB-like elements among various Gram-negative bacterial species and analyzed the genotypic and phenotypic characteristics of organisms harboring such elements. With a comprehensive genotyping approach, a chromosome-based oqxAB operon was detectable in all Klebsiella pneumoniae strains tested, including organisms isolated before the year 1984. Sequence and phylogenetic analyses confirmed that the oqxAB operon in K. pneumoniae isolates was genetically closest to their plasmid-borne counterparts recoverable only from Escherichia coli and Salmonella isolates collected from the year 2003 onward. Chromosomal elements with much lower sequence homology were also found among the Enterobacter spp. but not other Gram-negative species. Contrary to the quinolone resistance phenotypes which were consistently observable among organisms with oqxAB-harboring plasmids, chromosomal oqxAB elements generally did not confer quinolone resistance, except for K. pneumoniae strains, which exhibited a typical oqxAB-mediated phenotype characterized by cross-resistance to olaquindox, chloramphenicol, and the quinolones. Gene expression analysis illustrated that such phenotypes were due to elevated expression of the chromosomal oqxAB operon. Furthermore, transposition of the oqxAB operon from the bacterial chromosome to plasmids was found to result in a >80-fold increase in the level of expression of the OqxAB pump, confirming its status as the first constitutively expressed efflux system located in bacterial mobile elements. PMID:25801572

  8. Integration Host Factor is required for FarR repression of the farAB-encoded efflux pump of Neisseria gonorrhoeae.

    PubMed

    Lee, Eun-Hee; Hill, Stuart A; Napier, Ruth; Shafer, William M

    2006-06-01

    The farAB operon encodes an efflux pump system that mediates the resistance of Neisseria gonorrhoeae to antimicrobial long-chain fatty acids. We previously observed that expression of farAB is negatively regulated by the FarR repressor. In this study, we examined the molecular mechanism by which FarR represses expression of farAB. DNase I footprinting analysis, coupled with a deletion analysis of the farAB promoter region, indicated that FarR binds to three sites (termed sites A, B and C) within the DNA sequence upstream of farA; genetic analysis revealed, however, that site B is not required for FarR repression of farAB. This repression also required the presence of Integration Host Factor (IHF), which was found to bind to sequences located between FarR binding sites A and C. We determined that IHF binding to the farAB promoter region could inhibit transcription in vitro and that such binding induced a bending of the target DNA, which we propose to be important in regulating this operon. IHF binding to the promoter region was found to stabilize the binding of FarR to its binding sites A and C and as a consequence, enhanced repression of farAB expression mediated by FarR. We propose a model in which expression of the farAB-encoded efflux pump in N. gonorrhoeae is modulated by the DNA binding activities of FarR and IHF. PMID:16796676

  9. Inducible Expression of a Resistance-Nodulation-Division-Type Efflux Pump in Staphylococcus aureus Provides Resistance to Linoleic and Arachidonic Acids

    PubMed Central

    Alnaseri, Heba; Arsic, Benjamin; Schneider, James E. T.; Kaiser, Julienne C.; Scinocca, Zachariah C.; Heinrichs, David E.

    2015-01-01

    ABSTRACT Although Staphylococcus aureus is exposed to antimicrobial fatty acids on the skin, in nasal secretions, and in abscesses, a specific mechanism of inducible resistance to this important facet of innate immunity has not been identified. Here, we have sequenced the genome of S. aureus USA300 variants selected for their ability to grow at an elevated concentration of linoleic acid. The fatty acid-resistant clone FAR7 had a single nucleotide polymorphism resulting in an H121Y substitution in an uncharacterized transcriptional regulator belonging to the AcrR family, which was divergently transcribed from a gene encoding a member of the resistance-nodulation-division superfamily of multidrug efflux pumps. We named these genes farR and farE, for regulator and effector of fatty acid resistance, respectively. Several lines of evidence indicated that FarE promotes efflux of antimicrobial fatty acids and is regulated by FarR. First, expression of farE was strongly induced by arachidonic and linoleic acids in an farR-dependent manner. Second, an H121Y substitution in FarR resulted in increased expression of farE and was alone sufficient to promote increased resistance of S. aureus to linoleic acid. Third, inactivation of farE resulted in a significant reduction in the inducible resistance of S. aureus to the bactericidal activity of 100 μM linoleic acid, increased accumulation of [14C]linoleic acid by growing cells, and severely impaired growth in the presence of nonbactericidal concentrations of linoleic acid. Cumulatively, these findings represent the first description of a specific mechanism of inducible resistance to antimicrobial fatty acids in a Gram-positive pathogen. IMPORTANCE Staphylococcus aureus colonizes approximately 25% of humans and is a leading cause of human infectious morbidity and mortality. To persist on human hosts, S. aureus must have intrinsic defense mechanisms to cope with antimicrobial fatty acids, which comprise an important component of

  10. Differential susceptibility to carbapenems due to the AdeABC efflux pump among nosocomial outbreak isolates of Acinetobacter baumannii in a Chinese hospital.

    PubMed

    Huang, Lei; Sun, Liying; Xu, Guobing; Xia, Tiean

    2008-11-01

    A nosocomial outbreak of carbapenem-resistant Acinetobacter baumannii occurred from February to November 2004 in the surgical intensive care unit (SICU) and the pediatric intensive care unit (PICU) of our hospital. Two separate clones prevailed, the antimicrobial susceptibility profiles of which were different. The PICU isolates produced OXA-23 oxacillinase, whereas no carbapenemases were detected from SICU isolates. No obvious outer membrane protein change was seen. Efflux pump phenotype was detected from SICU isolates. Efflux pump-encoding gene adeB was positive in these isolates and negative in PICU isolates. Through further study, we found that AdeABC efflux system gene contents were common in the SICU isolates. Then we compared the expression level of adeB in resistant and susceptible isolates using quantitative real-time polymerase chain reaction and found that increased expression of AdeABC efflux pump may play an important role in reduced meropenem susceptibility among A. baumannii in the SICU of our hospital. PMID:18687557

  11. Association of hp1181 and hp1184 Genes With the Active Efflux Phenotype in Multidrug-Resistant Isolates of Helicobacter pylori

    PubMed Central

    Falsafi, Tahereh; Ehsani, Azadeh; Attaran, Bahareh; Niknam, Vahid

    2016-01-01

    Background During the last decades the rate of multidrug resistance among clinical Helicobacter pylori isolates has increased. Active pumping out of the drugs may be an important mechanism for multidrug resistance in H. pylori strains. Objectives The aim of this study was to evaluate the association of two H. pylori efflux-genes, hp1181 and hp1184 with the active-efflux phenotype in MDR clinical-strains of H. pylori. Materials and Methods Minimal inhibitory concentration (MIC) and drug accumulation for β-lactames, Tetracycline (TET), Erythromycin (ERY), Metronidazole (MTZ), Ciprofloxacin (CIP) and Ethidium Bromide (EtBr) was performed in the presence and absence of carbonyl cyanide M-Chlorophenyl Hydrazone (CCCP). Presence of hp1181 and hp1184 genes was detected by the polymerase chain reaction (PCR). RT-PCR was performed to compare expression of efflux genes by MDR strains, demonstrating active efflux with the strains without active efflux. Results Two- to four-fold decrease in minimum inhibitory concentration (MIC) and two-fold increase in accumulation were observed for EtBr in the presence of CCCP for 67% (8) of 12 MDR strains. With CCCP, two- to four-fold decrease in MIC and 1.4- to 1.8-fold increase in the accumulation of β-lactames, TET, CIP and MTZ were obtained for 42% (5) of the MDR strains. Six, five and three of the 12 MDR strains amplified hp1184, hp1181, and both of them, respectively. The RT-PCR product for expression of hp1181 by MDR strains was approximately 100 bp shorter than that of the 26695 susceptible standard strain. Conclusions Expression of the genes hp1184 and hp1181 are associated with the specific active efflux of EtBr and non-related antibiotics, respectively. For displaying these phenotypes, a post-transcriptional regulation step may be required. PMID:27303615

  12. Inactivation of SmeSyRy Two-Component Regulatory System Inversely Regulates the Expression of SmeYZ and SmeDEF Efflux Pumps in Stenotrophomonas maltophilia

    PubMed Central

    Lin, Yi-Tsung; Ning, Hsiao-Chen; Yang, Tsuey-Ching

    2016-01-01

    SmeYZ efflux pump is a critical pump responsible for aminoglycosides resistance, virulence-related characteristics (oxidative stress susceptibility, motility, and secreted protease activity), and virulence in Stenotrophomonas maltophilia. However, the regulatory circuit involved in SmeYZ expression is little known. A two-component regulatory system (TCS), smeRySy, transcribed divergently from the smeYZ operon is the first candidate to be considered. To assess the role of SmeRySy in smeYZ expression, the smeRySy isogenic deletion mutant, KJΔRSy, was constructed by gene replacement strategy. Inactivation of smeSyRy correlated with a higher susceptibility to aminoglycosides concomitant with an increased resistance to chloramphenicol, ciprofloxacin, tetracycline, and macrolides. To elucidate the underlying mechanism responsible for the antimicrobials susceptibility profiles, the SmeRySy regulon was firstly revealed by transcriptome analysis and further confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and promoter transcription fusion constructs assay. The results demonstrate that inactivation of smeRySy decreased the expression of SmeYZ pump and increased the expression of SmeDEF pump, which underlies the ΔsmeSyRy-mediated antimicrobials susceptibility profile. To elucidate the cognate relationship between SmeSy and SmeRy, a single mutant, KJΔRy, was constructed and the complementation assay of KJΔRSy with smeRy were performed. The results support that SmeSy-SmeRy TCS is responsible for the regulation of smeYZ operon; whereas SmeSy may be cognate with another unidentified response regulator for the regulation of smeDEF operon. The impact of inverse expression of SmeYZ and SmeDEF pumps on physiological functions was evaluated by mutants construction, H2O2 susceptibility test, swimming, and secreted protease activity assay. The increased expression of SmeDEF pump in KJΔRSy may compensate, to some extents, the SmeYZ downexpression

  13. Regulation of retinoid mediated cholesterol efflux involves liver X receptor activation in mouse macrophages.

    PubMed

    Manna, Pulak R; Sennoune, Souad R; Martinez-Zaguilan, Raul; Slominski, Andrzej T; Pruitt, Kevin

    2015-08-14

    Removal of cholesterol from macrophage-derived foam cells is a critical step to the prevention of atherosclerotic lesions. We have recently demonstrated the functional importance of retinoids in the regulation of the steroidogenic acute regulatory (StAR) protein that predominantly mediates the intramitochondrial transport of cholesterol in target tissues. In the present study, treatment of mouse macrophages with retinoids, particularly all-trans retinoic acid (atRA) and 9-cis RA, resulted in increases in cholesterol efflux to apolipoprotein AI (Apo-A1). Activation of the PKA pathway by a cAMP analog, (Bu)2cAMP, markedly augmented retinoid mediated cholesterol efflux. Macrophages overexpressing hormone-sensitive lipase increased the hydrolysis of cholesteryl esters and concomitantly enhanced the efficacy of retinoic acid receptor and liver X receptor (LXR) ligands on StAR and ATP-binding cassette transporter A1 (ABCA1) protein levels. RAs elevated StAR promoter activity in macrophages, and an increase in StAR levels augmented cholesterol efflux to Apo-A1, suggesting retinoid-mediated efflux of cholesterol involves enhanced oxysterol production. Further studies revealed that retinoids activate the LXR regulated genes, sterol receptor-element binding protein-1c and ABCA1. These findings provide insights into the regulatory events in which retinoid signaling effectively enhances macrophage cholesterol efflux and indicate that retinoid therapy may have important implications in limiting and/or regressing atherosclerotic cardiovascular disease. PMID:26119689

  14. Molecular basis of polyspecificity of the Small Multidrug Resistance Efflux Pump AbeS from Acinetobacter baumannii.

    PubMed

    Lytvynenko, Iryna; Brill, Shlomo; Oswald, Christine; Pos, Klaas M

    2016-02-13

    Secondary multidrug efflux transporters play a key role in the bacterial resistance phenotype. One of the major questions concerns the polyspecific recognition of substrates by these efflux pumps. To understand the molecular basis of this promiscuous recognition, we compared the substrate specificity of the well-studied Escherichia coli small multidrug resistance protein EmrE with that of the poorly studied Acinetobacter baumannii homologue AbeS. The latter drug/H(+) antiporter is a 109-amino-acid membrane protein with predicted four transmembrane helices. It effectively confers resistance toward ethidium, acriflavine and benzalkonium in an E. coli ΔemrEΔmdfA background. Purified AbeS and the substrate-specific hyperactive variant A16G bind tetraphenylphosphonium with nanomolar affinity and exhibit electrogenic transport of 1-methyl-4-phenylpyridinium after reconstitution into liposomes. A16G hyperactivity was apparent toward acriflavine and ethidium, resulting in 7- to 10-fold higher normalized IC50 values, respectively, but not toward substrates 1-methyl-4-phenylpyridinium and benzalkonium. Substitution of Y3 and A42 with Ala or Ser, respectively, also displayed a substrate-dependent phenotype, as these variants were strongly affected in their properties to confer resistance against acriflavine and ethidium, but not against benzalkonium. The size and planarity of the conjugated aromatic moieties appear to be a critical and subtle criterion for substrate recognition by these transporters. Rather moderate changes in the property of side chains postulated to be part of the substrate binding site result in a large phenotypical difference. These observations provide indications for the molecular basis of specificity within the binding pocket of polyspecific transporters. PMID:26707198

  15. Evidence for an Efflux Pump Mediating Multiple Antibiotic Resistance in Salmonella enterica Serovar Typhimurium

    PubMed Central

    Piddock, Laura J. V.; White, David G.; Gensberg, Karl; Pumbwe, Lilian; Griggs, Deborah J.

    2000-01-01

    The mechanism of multiple antibiotic resistance in six isolates of Salmonella enterica serovar Typhimurium recovered from a patient treated with ciprofloxacin was studied to investigate the role of efflux in the resistance phenotype. Compared to the patient's pretherapy isolate (L3), five of six isolates accumulated less ciprofloxacin, three of six isolates accumulated less chloramphenicol, and all six accumulated less tetracycline. The accumulation of one or more antibiotics was increased by carbonyl cyanide m-chlorophenylhydrazone to concentrations similar to those accumulated by L3 for all isolates except one, in which accumulation of all three agents remained approximately half that of L3. All isolates had the published wild-type sequences of marO and marR. No increased expression of marA, tolC, or soxS was observed by Northern blotting; however, three isolates showed increased expression of acrB, which was confirmed by quantitative competitive reverse transcription-PCR. However, there were no mutations within acrR or the promoter region of acrAB in any of the isolates. PMID:11036033

  16. Ferroportin and Exocytoplasmic Ferroxidase Activity Are Required for Brain Microvascular Endothelial Cell Iron Efflux*

    PubMed Central

    McCarthy, Ryan C.; Kosman, Daniel J.

    2013-01-01

    The mechanism(s) of iron flux across the brain microvasculature endothelial cells (BMVEC) of the blood-brain barrier remains unknown. Although both hephaestin (Hp) and the ferrous iron permease ferroportin (Fpn) have been identified in BMVEC, their roles in iron efflux have not been examined. Using a human BMVEC line (hBMVEC), we have demonstrated that these proteins are required for iron efflux from these cells. Expression of both Hp and Fpn protein was confirmed in hBMVEC by immunoblot and indirect immunofluorescence; we show that hBMVEC express soluble ceruloplasmin (Cp) transcript as well. Depletion of endogenous Hp and Cp via copper chelation leads to the reduction of hBMVEC Fpn protein levels as well as a complete inhibition of 59Fe efflux. Both hBMVEC Fpn protein and 59Fe efflux activity are restored upon incubation with 6.6 nm soluble plasma Cp. These results are independent of the source of cell iron, whether delivered as transferrin- or non-transferrin-bound 59Fe. Our results demonstrate that iron efflux from hBMVEC Fpn requires the action of an exocytoplasmic ferroxidase, which can be either endogenous Hp or extracellular Cp. PMID:23640881

  17. A Resistance-Nodulation-Cell Division Family Xenobiotic Efflux Pump in an Obligate Anaerobe, Porphyromonas gingivalis

    PubMed Central

    Ikeda, Takeshi; Yoshimura, Fuminobu

    2002-01-01

    Porphyromonas gingivalis, a gram-negative obligate anaerobe, contains two homologs of an Escherichia coli resistance-nodulation-cell division-type multidrug exporter gene, acrB, in putative operons, together with homologs of membrane fusion protein gene acrA and outer membrane channel gene tolC. MIC determination and accumulation assays with mutants with disruptions of one or more genes showed that one cluster, named xepCAB, pumped out multiple agents including rifampin, puromycin, and ethidium bromide. PMID:12234854

  18. Coupling of remote alternating-access transport mechanisms for protons and substrates in the multidrug efflux pump AcrB

    PubMed Central

    Eicher, Thomas; Seeger, Markus A; Anselmi, Claudio; Zhou, Wenchang; Brandstätter, Lorenz; Verrey, François; Diederichs, Kay; Faraldo-Gómez, José D; Pos, Klaas M

    2014-01-01

    Membrane transporters of the RND superfamily confer multidrug resistance to pathogenic bacteria, and are essential for cholesterol metabolism and embryonic development in humans. We use high-resolution X-ray crystallography and computational methods to delineate the mechanism of the homotrimeric RND-type proton/drug antiporter AcrB, the active component of the major efflux system AcrAB-TolC in Escherichia coli, and one most complex and intriguing membrane transporters known to date. Analysis of wildtype AcrB and four functionally-inactive variants reveals an unprecedented mechanism that involves two remote alternating-access conformational cycles within each protomer, namely one for protons in the transmembrane region and another for drugs in the periplasmic domain, 50 Å apart. Each of these cycles entails two distinct types of collective motions of two structural repeats, coupled by flanking α-helices that project from the membrane. Moreover, we rationalize how the cross-talk among protomers across the trimerization interface might lead to a more kinetically efficient efflux system. DOI: http://dx.doi.org/10.7554/eLife.03145.001 PMID:25248080

  19. Carbapenem Activities against Pseudomonas aeruginosa: Respective Contributions of OprD and Efflux Systems

    PubMed Central

    Köhler, Thilo; Michea-Hamzehpour, Mehri; Epp, Simone F.; Pechere, Jean-Claude

    1999-01-01

    While meropenem MICs were strongly influenced by the presence or absence of the MexAB-OprM efflux pump in both OprD-proficient and -deficient strain backgrounds, MICs of imipenem and of ER-35786 remained unchanged, demonstrating that meropenem is a substrate of MexAB-OprM but not imipenem and ER-35786. In vitro, all three carbapenems selected loss of OprD as a first mechanism of resistance. However, in an OprD-deficient background, meropenem was able to select MexAB-OprM overproducers as a secondary resistance mechanism, while ER-35786 selected a mutant cross-resistant to sparfloxacin and cefpirome. PMID:9925552

  20. Multidrug Efflux Systems in Microaerobic and Anaerobic Bacteria

    PubMed Central

    Xu, Zeling; Yan, Aixin

    2015-01-01

    Active drug efflux constitutes an important mechanism of antibiotic and multidrug resistance in bacteria. Understanding the distribution, expression, and physiological functions of multidrug efflux pumps, especially under physiologically and clinically relevant conditions of the pathogens, is the key to combat drug resistance. In animal hosts, most wounded, infected and inflamed tissues display low oxygen tensions. In this article, we summarize research development on multidrug efflux pumps in the medicinally relevant microaerobic and anaerobic pathogens and their implications in the effort to combat drug-resistant infections. PMID:27025630

  1. Genome Sequence of a Clinical Strain of Acinetobacter baumannii Belonging to the ST79/PFGE-HUI-1 Clone Lacking the AdeABC (Resistance-Nodulation-Cell Division-Type) Efflux Pump.

    PubMed

    López, M; Álvarez-Fraga, L; Gato, E; Blasco, L; Poza, M; Fernández-García, L; Bou, G; Tomás, M

    2016-01-01

    Increased expression of chromosomal genes for resistance-nodulation-cell division-type efflux systems plays a major role in the multidrug resistance of Acinetobacter baumannii Little is known about the genetic characteristics of clinical strains of Acinetobacter baumannii lacking the AdeABC pump. In this study, we sequenced the genome of clinical strain Ab421 GEIH-2010 (belonging to clone ST79/PFGE-HUI-1 from the GEIH-REIPI Ab. 2010 project) which lacks this efflux pump. PMID:27609928

  2. Characterization and regulation of the Resistance-Nodulation-Cell Division-type multidrug efflux pumps MdtABC and MdtUVW from the fire blight pathogen Erwinia amylovora

    PubMed Central

    2014-01-01

    Background The Gram-negative bacterium Erwinia amylovora is the causal agent of the devastating disease fire blight in rosaceous plants such as apple, pear, quince, raspberry, and cotoneaster. In order to survive and multiply in a host, microbes must be able to circumvent the toxic effects of antimicrobial plant compounds, such as flavonoids and tannins. E. amylovora uses multidrug efflux transporters that recognize and actively export toxic compounds out of the cells. Here, two heterotrimeric resistance-nodulation-cell division (RND)-type multidrug efflux pumps, MdtABC and MdtUVW, from E. amylovora were identified. These RND systems are unusual in that they contain two different RND proteins forming a functional pump. Results To find the substrate specificities of the two efflux systems, we overexpressed the transporters in a hypersensitive mutant lacking the major RND pump AcrB. Both transporters mediated resistance to several flavonoids, fusidic acid and novobiocin. Additionally, MdtABC mediated resistance towards josamycin, bile salts and silver nitrate, and MdtUVW towards clotrimazole. The ability of the mdtABC- and mdtUVW-deficient mutants to multiply in apple rootstock was reduced. Quantitative RT-PCR analyses revealed that the expression of the transporter genes was induced during infection of apple rootstock. The polyphenolic plant compound tannin, as well as the heavy metal salt tungstate was found to induce the expression of mdtABC. Finally, the expression of the mdtABC genes was shown to be regulated by BaeR, the response regulator of the two-component system BaeSR, a cell envelope stress response system that controls the adaptive responses to changes in the environment. Conclusions The expression of MdtABC and MdtUVW is induced during growth of E. amylovora in planta. We identified the plant polyphenol tannin as inducer of mdtABC expression. The reduced ability of the mdtABC- and mdtUVW-deficient mutants to multiply in apple rootstock suggests that the

  3. Detection of AdeABC efflux pump genes in tetracycline-resistant Acinetobacter baumannii isolates from burn and ventilator-associated pneumonia patients

    PubMed Central

    Beheshti, Maryam; Talebi, Malihe; Ardebili, Abdollah; Bahador, Abbas; Lari, Abdolaziz Rastegar

    2014-01-01

    Purpose: Acinetobacter baumannii is the most prevalent nosocomial pathogen which have been emerged in the past three decades worldwide. The aim of this study was to assess the distribution of the AdeABC efflux pump genes, associated with tetracycline resistance in Acinetobacter baumannii isolates collected from burn infection and Ventilator Associated Pneumonia (VAP). Materials and Methods: Ninety-eight A. baumannii isolates were collected from two different hospitals in Tehran, Iran. Tetracycline susceptibility testing was performed by disk diffusion and agar dilution methods according to the CLSI guidelines. The presence of adeSR, adeB, drug efflux system genes in resistant isolates was assessed by polymerase chain reaction (PCR). Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as a chemical inhibitor agent to assess the contribution of AdeABC efflux pump in tetracycline resistance isolates. Results: Approximately 48% (47 out of 98) of isolates showed resistance to tetracycline which 14 (14.2%) isolates were corresponded to burn infection and the remaining 33 (33.8%) strains were isolated from VAP. All tetracycline resistant isolates have AdeABC in PCR assay. The reduction of tetracycline MICs by using 50 μg/ml CCCP were as follows: in 18 isolates 2-4 fold reduction in MICs, 26 isolates showed 8 fold reduction,1 isolate showed 16 fold, 1 isolate showed 32 fold and the remaining 1 isolate showed 128 fold reduction in MICs. Conclusion: The results showed significant correlation between tetracycline resistance and AdeABC efflux pump genes in resistant A. baumannii isolates. PMID:25400404

  4. Effect of iron on expression of efflux pump (adeABC) and quorum sensing (luxI, luxR) genes in clinical isolates of Acinetobacter baumannii.

    PubMed

    Modarresi, Farzan; Azizi, Omid; Shakibaie, Mohammad Reza; Motamedifar, Mohammad; Valibeigi, Behnaz; Mansouri, Shahla

    2015-11-01

    Resistance-nodulation-division efflux system (RND) adeABC contributes to intrinsic resistance to various drug classes in Acinetobacter baumannii. Similarly, quorum sensing (QS) plays an important role in the biofilm formation and pathogenicity of this bacterium. The aims of this study were to evaluate the influence of iron limitation on the expression of efflux pump (adeABC) genes and QS (luxI, luxR) system by relative quantitative real-time polymerase chain reaction (qRT-PCR). In addition, DNA sequence and phylogenetic relatedness of biofilm-associated protein (Bap) gene was also investigated. Sixty-five multidrug-resistant isolates of A. baumannii were recovered from ICU patients of three hospitals in Kerman, Iran. The isolates were highly resistant to at least 11 antibiotics (MIC ≥64 μg/mL); however, 87% and 89% were susceptible to colistin and tigecycline, respectively (MIC 0.05 μg/mL) (p ≤ 0.05). We detected the presence of RND efflux pump, QS, and bap genes with the frequencies of 92% (adeA), 61.5% (adeB), 84.6% (adeC), 80% (luxI), 61% (luxR), and 66% (bap), respectively. qRT-PCR analysis showed that in some isolates, expression of both adeABC and luxI/R was increased more than fourfold in the presence of low iron (20 μm), suggesting the additional regulatory role of iron on both efflux pump and QS system. Alignment and phylogenetic analysis on the strong biofilm forming isolates confirmed that the fragments amplified were indeed part of bap gene and deduced sequence was similar to A. baumannii K9B410. PMID:26350174

  5. Regulation of the AcrAB multidrug efflux pump in Salmonella enterica serovar Typhimurium in response to indole and paraquat.

    PubMed

    Nikaido, Eiji; Shirosaka, Ikue; Yamaguchi, Akihito; Nishino, Kunihiko

    2011-03-01

    Salmonella enterica serovar Typhimurium has at least nine multidrug efflux pumps. Among these, AcrAB is constitutively expressed and is the most efficient, playing a role in both drug resistance and virulence. The acrAB locus is induced by indole, Escherichia coli-conditioned medium, and bile salts. This induction is dependent on RamA through the binding sequence in the upstream region of acrA that binds RamA. In the present study, we made a detailed investigation of the ramA and acrAB induction mechanisms in Salmonella in response to indole, a biological oxidant for bacteria. We found that acrAB and ramA induction in response to indole is dependent on RamR. However, the cysteine residues of RamR do not play a role in the induction of ramA in response to indole, and the oxidative effect of indole is therefore not related to ramA induction via RamR. Furthermore, we showed that paraquat, a superoxide generator, induces acrAB but not ramA. We further discovered that the mechanism of acrAB induction in response to paraquat is dependent on SoxS. The data indicate that there are at least two independent induction pathways for acrAB in response to extracellular signals such as indole and paraquat. We propose that Salmonella utilizes these regulators for acrAB induction in response to extracellular signals in order to adapt itself to environmental conditions. PMID:21148208

  6. Thiomers: Influence of molecular mass and thiol group content of poly(acrylic acid) on efflux pump inhibition.

    PubMed

    Grabovac, Vjera; Laffleur, Flavia; Bernkop-Schnürch, Andreas

    2015-09-30

    The aim of the present study was to investigate the influence of molecular mass and thiol group content of poly(acrylic acid)-cysteine conjugates on the permeation of sulforhodamine 101 and penicillin G. acting as substrates for multidrug resistance-associated protein 2 efflux pump. Poly(acrylic acids) of 2 kDa, 100 kDa, 250 kDa, 450 kDa and 3000 kDa were conjugated with cysteine. The thiol group content of all these polymers was in the range from 343.3 ± 48.4 μmol/g to 450.3 ± 76.1 μmol/g. Transport studies were performed on rat small intestine mounted in Ussing-type chambers. Since 250 kDa poly(acrylic acid) showed the highest permeation enhancing effect, additionally thiolated 250 kDa polyacrylates displaying 157.2 μmol/g, 223.0 ± 18.1 and 355.9 μmol/g thiol groups were synthesized in order to investigate the influence of thiol group content on the permeation enhancement. The permeation of sulforhodamine was 3.93- and 3.85-fold improved using 250 kDa poly(acrylic acid)-cysteine conjugate exhibiting 355.9 ± 39.5 μmol/g and 223.0 ± 18.1 μmol/g thiol groups. Using the same conjugates the permeation of penicillin G was 1.70- and 1.59-fold improved, respectively. The study demonstrates that thiolated poly(acrylic acid) inhibits Mrp2 mediated transport and that the extent of inhibition depends on the molecular mass and degree of thiolation of the polymer. PMID:26238816

  7. Nek7 is an essential mediator of NLRP3 activation downstream of potassium efflux

    PubMed Central

    He, Yuan; Zeng, Melody Y.; Yang, Dahai; Motro, Benny; Núñez, Gabriel

    2016-01-01

    Inflammasomes are intracellular protein complexes that drive the activation of inflammatory caspases1. To date, four inflammasomes involving NLRP1, NLRP3, NLRC4 and AIM2 have been described that recruit the common adaptor ASC to activate caspase-1, leading to the secretion of mature IL-1β and IL-182,3. The NLRP3 inflammasome has been implicated in the pathogenesis of several acquired inflammatory diseases4,5 as well as Cryopyrin-associated periodic fever syndromes (CAPS) caused by inherited NLRP3 mutations6,7. Potassium efflux is a common step that is essential for NLRP3 inflammasome activation induced by multiple stimuli8,9. Despite extensive investigation, the molecular mechanism leading to NLRP3 activation in response to potassium efflux remains unknown. We report here the identification of Nek7, a member of the family of mammalian NIMA-related kinases (Neks)10, as an NLRP3-binding protein that acts downstream of potassium efflux to regulate NLRP3 oligomerization and activation. In the absence of Nek7, caspase-1 activation and IL-1β release were abrogated in response to signals that activate NLRP3, but not NLRC4 or AIM2 inflammasome. NLRP3-activating stimuli promoted the NLRP3-Nek7 interaction in a process dependent on potassium efflux. NLRP3 associated with the catalytic domain of Nek7, but the catalytic activity of Nek7 was dispensable for activation of the NLRP3 inflammasome. Activated macrophages formed a high-molecular-mass NLRP3-Nek7 complex, which along with ASC oligomerization and ASC speck formation were abrogated in the absence of Nek7. Nek7 was required for macrophages harboring the CAPS-associated NLRP3R258W activating mutation to activate caspase-1. Mouse chimeras reconstituted with wild-type, Nek7−/− or Nlrp3−/− hematopoietic cells revealed that Nek7 was required for NLRP3 inflammasome activation in vivo. These studies demonstrate that Nek7 is an essential protein that acts downstream of potassium efflux to mediate NLRP3 inflammasome

  8. NEK7 is an essential mediator of NLRP3 activation downstream of potassium efflux.

    PubMed

    He, Yuan; Zeng, Melody Y; Yang, Dahai; Motro, Benny; Núñez, Gabriel

    2016-02-18

    Inflammasomes are intracellular protein complexes that drive the activation of inflammatory caspases. So far, four inflammasomes involving NLRP1, NLRP3, NLRC4 and AIM2 have been described that recruit the common adaptor protein ASC to activate caspase-1, leading to the secretion of mature IL-1β and IL-18 proteins. The NLRP3 inflammasome has been implicated in the pathogenesis of several acquired inflammatory diseases as well as cryopyrin-associated periodic fever syndromes (CAPS) caused by inherited NLRP3 mutations. Potassium efflux is a common step that is essential for NLRP3 inflammasome activation induced by many stimuli. Despite extensive investigation, the molecular mechanism leading to NLRP3 activation in response to potassium efflux remains unknown. Here we report the identification of NEK7, a member of the family of mammalian NIMA-related kinases (NEK proteins), as an NLRP3-binding protein that acts downstream of potassium efflux to regulate NLRP3 oligomerization and activation. In the absence of NEK7, caspase-1 activation and IL-1β release were abrogated in response to signals that activate NLRP3, but not NLRC4 or AIM2 inflammasomes. NLRP3-activating stimuli promoted the NLRP3-NEK7 interaction in a process that was dependent on potassium efflux. NLRP3 associated with the catalytic domain of NEK7, but the catalytic activity of NEK7 was shown to be dispensable for activation of the NLRP3 inflammasome. Activated macrophages formed a high-molecular-mass NLRP3-NEK7 complex, which, along with ASC oligomerization and ASC speck formation, was abrogated in the absence of NEK7. NEK7 was required for macrophages containing the CAPS-associated NLRP3(R258W) activating mutation to activate caspase-1. Mouse chimaeras reconstituted with wild-type, Nek7(-/-) or Nlrp3(-/-) haematopoietic cells showed that NEK7 was required for NLRP3 inflammasome activation in vivo. These studies demonstrate that NEK7 is an essential protein that acts downstream of potassium efflux to

  9. Correlation between AcrB trimer association affinity and efflux activity.

    PubMed

    Ye, Cui; Wang, Zhaoshuai; Lu, Wei; Zhong, Meng; Chai, Qian; Wei, Yinan

    2014-06-17

    The majority of membrane proteins function as oligomers. However, it remains largely unclear how the oligomer stability of protein complexes correlates with their function. Understanding the relationship between oligomer stability and activity is essential to protein research and to virtually all cellular processes that depend on the function of protein complexes. Proteins make lasting or transient interactions as they perform their functions. Obligate oligomeric proteins exist and function exclusively at a specific oligomeric state. Although oligomerization is clearly critical for such proteins to function, a direct correlation between oligomer affinity and biological activity has not yet been reported. Here, we used an obligate trimeric membrane transporter protein, AcrB, as a model to investigate the correlation between its relative trimer affinity and efflux activity. AcrB is a component of the major multidrug efflux system in Escherichia coli. We created six AcrB constructs with mutations at the transmembrane intersubunit interface, and we determined their activities using both a drug susceptibility assay and an ethidium bromide accumulation assay. The relative trimer affinities of these mutants in detergent micelles were obtained using blue native polyacrylamide gel electrophoresis. A correlation between the relative trimer affinity and substrate efflux activity was observed, in which a threshold trimer stability was required to maintain efflux activity. The trimer affinity of the wild-type protein was approximately 3 kcal/mol more stable than the threshold value. Once the threshold was reached, an additional increase of stability in the range observed had no observable effect on protein activity. PMID:24854514

  10. Metabolomics Analysis Reveals the Participation of Efflux Pumps and Ornithine in the Response of Pseudomonas putida DOT-T1E Cells to Challenge with Propranolol

    PubMed Central

    Sayqal, Ali; Xu, Yun; Trivedi, Drupad K.; AlMasoud, Najla; Ellis, David I.; Rattray, Nicholas J. W.; Goodacre, Royston

    2016-01-01

    Efflux pumps are critically important membrane components that play a crucial role in strain tolerance in Pseudomonas putida to antibiotics and aromatic hydrocarbons that result in these toxicants being expelled from the bacteria. Here, the effect of propranolol on P. putida was examined by sudden addition of 0.2, 0.4 and 0.6 mg mL-1 of this β-blocker to several strains of P. putida, including the wild type DOT-T1E and the efflux pump knockout mutants DOT-T1E-PS28 and DOT-T1E-18. Bacterial viability measurements reveal that the efflux pump TtgABC plays a more important role than the TtgGHI pump in strain tolerance to propranolol. Mid-infrared (MIR) spectroscopy was then used as a rapid, high-throughput screening tool to investigate any phenotypic changes resulting from exposure to varying levels of propranolol. Multivariate statistical analysis of these MIR data revealed gradient trends in resultant ordination scores plots, which were related to the concentration of propranolol. MIR illustrated phenotypic changes associated with the presence of this drug within the cell that could be assigned to significant changes that occurred within the bacterial protein components. To complement this phenotypic fingerprinting approach metabolic profiling was performed using gas chromatography mass spectrometry (GC-MS) to identify metabolites of interest during the growth of bacteria following toxic perturbation with the same concentration levels of propranolol. Metabolic profiling revealed that ornithine, which was only produced by P. putida cells in the presence of propranolol, presents itself as a major metabolic feature that has important functions in propranolol stress tolerance mechanisms within this highly significant and environmentally relevant species of bacteria. PMID:27331395

  11. The CydDC ABC transporter of Escherichia coli: new roles for a reductant efflux pump.

    PubMed

    Shepherd, Mark

    2015-10-01

    The CydDC complex of Escherichia coli is a heterodimeric ATP-binding cassette (ABC) transporter that exports cysteine and glutathione to the periplasm. These reductants are thought to modulate periplasmic redox poise, impacting upon the disulfide folding of periplasmic and secreted proteins involved in bacterial virulence. Diminished CydDC activity abolishes the assembly of functional bd-type respiratory oxidases and perturbs haem ligation during the assembly of c-type cytochromes. The focus herein is upon a newly-discovered interaction of the CydDC complex with a haem cofactor; haem has recently been shown to modulate CydDC activity and structural modelling reveals a potential haem-binding site on the periplasmic surface of the complex. These findings have important implications for future investigations into the potential roles for the CydDC-bound haem in redox sensing and tolerance to nitric oxide (NO). PMID:26517902

  12. Overcoming Multidrug Resistance in Candida albicans: Macrocyclic Diterpenes from Euphorbia Species as Potent Inhibitors of Drug Efflux Pumps.

    PubMed

    Nim, Shweta; Mónico, Andreia; Rawal, Manpreet Kaur; Duarte, Noélia; Prasad, Rajendra; Di Pietro, Attilio; Ferreira, Maria-José U

    2016-08-01

    Thirteen macrocyclic diterpenes (1-13) of the jatrophane and lathyrane types, either isolated from Euphorbia species or obtained by chemical derivatization, were evaluated for their ability to inhibit the drug efflux activity of Candida albicans CaCdr1p and CaMdr1p multidrug transporters overexpressed in a Saccharomyces cerevisiae strain. Their inhibitory potential was assessed through a functional assay of Nile Red accumulation monitored by flow cytometry. A chemosensitization assay, using the checkerboard method, was also performed with the active compounds in order to evaluate their type of interaction with fluconazole.In the transport assay, most compounds were found to inhibit both transporters, most likely as non-substrates, as shown by relative resistance indices close to unity. In contrast, the jatrophanes euphopubescenol (10) and euphomelliferene A (11) were selective for CaMdr1p and CaCdr1p, respectively. Moreover, when used in combination with fluconazole, compounds 12 and 13 displayed strong synergistic interactions (FICI = 0.071) against the yeast strain overexpressing CaMdr1p, decreasing the MIC80 of the antifungal agent 13-fold. Both compounds were also able to reduce the effective concentration of this antifungal agent by 4- to 8-fold against an azole-resistant clinical isolate of C. albicans (F5). PMID:27145238

  13. Tolerance of Listeria monocytogenes to Quaternary Ammonium Sanitizers Is Mediated by a Novel Efflux Pump Encoded by emrE.

    PubMed

    Kovacevic, Jovana; Ziegler, Jennifer; Wałecka-Zacharska, Ewa; Reimer, Aleisha; Kitts, David D; Gilmour, Matthew W

    2016-02-01

    A novel genomic island (LGI1) was discovered in Listeria monocytogenes isolates responsible for the deadliest listeriosis outbreak in Canada, in 2008. To investigate the functional role of LGI1, the outbreak strain 08-5578 was exposed to food chain-relevant stresses, and the expression of 16 LGI1 genes was measured. LGI1 genes with putative efflux (L. monocytogenes emrE [emrELm]), regulatory (lmo1851), and adhesion (sel1) functions were deleted, and the mutants were exposed to acid (HCl), cold (4°C), salt (10 to 20% NaCl), and quaternary ammonium-based sanitizers (QACs). Deletion of lmo1851 had no effect on the L. monocytogenes stress response, and deletion of sel1 did not influence Caco-2 and HeLa cell adherence/invasion, whereas deletion of emrE resulted in increased susceptibility to QACs (P < 0.05) but had no effect on the MICs of gentamicin, chloramphenicol, ciprofloxacin, erythromycin, tetracycline, acriflavine, and triclosan. In the presence of the QAC benzalkonium chloride (BAC; 5 μg/ml), 14/16 LGI1 genes were induced, and lmo1861 (putative repressor gene) was constitutively expressed at 4 °C, 37 °C, and 52 °C and in the presence of UV exposure (0 to 30 min). Following 1 h of exposure to BAC (10 μg/ml), upregulation of emrE (49.6-fold), lmo1851 (2.3-fold), lmo1861 (82.4-fold), and sigB (4.1-fold) occurred. Reserpine visibly suppressed the growth of the ΔemrELm strain, indicating that QAC tolerance is due at least partially to efflux activity. These data suggest that a minimal function of LGI1 is to increase the tolerance of L. monocytogenes to QACs via emrELm. Since QACs are commonly used in the food industry, there is a concern that L. monocytogenes strains possessing emrE will have an increased ability to survive this stress and thus to persist in food processing environments. PMID:26590290

  14. Tolerance of Listeria monocytogenes to Quaternary Ammonium Sanitizers Is Mediated by a Novel Efflux Pump Encoded by emrE

    PubMed Central

    Ziegler, Jennifer; Wałecka-Zacharska, Ewa; Reimer, Aleisha; Kitts, David D.; Gilmour, Matthew W.

    2015-01-01

    A novel genomic island (LGI1) was discovered in Listeria monocytogenes isolates responsible for the deadliest listeriosis outbreak in Canada, in 2008. To investigate the functional role of LGI1, the outbreak strain 08-5578 was exposed to food chain-relevant stresses, and the expression of 16 LGI1 genes was measured. LGI1 genes with putative efflux (L. monocytogenes emrE [emrELm]), regulatory (lmo1851), and adhesion (sel1) functions were deleted, and the mutants were exposed to acid (HCl), cold (4°C), salt (10 to 20% NaCl), and quaternary ammonium-based sanitizers (QACs). Deletion of lmo1851 had no effect on the L. monocytogenes stress response, and deletion of sel1 did not influence Caco-2 and HeLa cell adherence/invasion, whereas deletion of emrE resulted in increased susceptibility to QACs (P < 0.05) but had no effect on the MICs of gentamicin, chloramphenicol, ciprofloxacin, erythromycin, tetracycline, acriflavine, and triclosan. In the presence of the QAC benzalkonium chloride (BAC; 5 μg/ml), 14/16 LGI1 genes were induced, and lmo1861 (putative repressor gene) was constitutively expressed at 4°C, 37°C, and 52°C and in the presence of UV exposure (0 to 30 min). Following 1 h of exposure to BAC (10 μg/ml), upregulation of emrE (49.6-fold), lmo1851 (2.3-fold), lmo1861 (82.4-fold), and sigB (4.1-fold) occurred. Reserpine visibly suppressed the growth of the ΔemrELm strain, indicating that QAC tolerance is due at least partially to efflux activity. These data suggest that a minimal function of LGI1 is to increase the tolerance of L. monocytogenes to QACs via emrELm. Since QACs are commonly used in the food industry, there is a concern that L. monocytogenes strains possessing emrE will have an increased ability to survive this stress and thus to persist in food processing environments. PMID:26590290

  15. FarR regulates the farAB-encoded efflux pump of Neisseria gonorrhoeae via an MtrR regulatory mechanism.

    PubMed

    Lee, E-H; Rouquette-Loughlin, C; Folster, J P; Shafer, W M

    2003-12-01

    The farAB operon of Neisseria gonorrhoeae encodes an efflux pump which mediates gonococcal resistance to antibacterial fatty acids. It was previously observed that expression of the farAB operon was positively regulated by MtrR, which is a repressor of the mtrCDE-encoded efflux pump system (E.-H. Lee and W. M. Shafer, Mol. Microbiol. 33:839-845, 1999). This regulation was believed to be indirect since MtrR did not bind to the farAB promoter. In this study, computer analysis of the gonococcal genome sequence database, lacZ reporter fusions, and gel mobility shift assays were used to elucidate the regulatory mechanism by which expression of the farAB operon is modulated by MtrR in gonococci. We identified a regulatory protein belonging to the MarR family of transcriptional repressors and found that it negatively controls expression of farAB by directly binding to the farAB promoter. We designated this regulator FarR to signify its role in regulating the farAB operon. We found that MtrR binds to the farR promoter, thereby repressing farR expression. Hence, MtrR regulates farAB in a positive fashion by modulating farR expression. This MtrR regulatory cascade seems to play an important role in adjusting levels of the FarAB and MtrCDE efflux pumps to prevent their excess expression in gonococci. PMID:14645274

  16. Auxin efflux carrier activity and auxin accumulation regulate cell division and polarity in tobacco cells.

    PubMed

    Petrásek, Jan; Elckner, Miroslav; Morris, David A; Zazímalová, Eva

    2002-12-01

    Division and growth of most types of in vitro-cultured plant cells require an external source of auxin. In such cultures, the ratio of external to internal auxin concentration is crucial for the regulation of the phases of the standard growth cycle. In this report the internal concentration of auxin in suspension-cultured cells of Nicotiana tabacum L., strain VBI-0, was manipulated either (i) by increasing 10-fold the normal concentration of 1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid in the external medium; or (ii) by addition 1-N-naphthylphthalamic acid (NPA; an inhibitor of auxin efflux and of auxin efflux carrier traffic). Both treatments delayed the onset of cell division for 6-7 days without loss of cell viability. In both cases, cell division activity subsequently resumed coincident with a reduction in the ability of cells to accumulate [(3)H]NAA from an external medium. Following renewed cell division, a significant proportion of the NPA-treated cells but not those grown at high auxin concentration, exhibited changes in the orientation of new cell divisions and loss of polarity. We conclude that cell division, but not cell elongation, is prevented when the internal auxin concentration rises above a critical threshold value and that the directed traffic of auxin efflux carriers to the plasma membrane may regulate the orientation of cell divisions. PMID:12447544

  17. Self-Pumping Active Gel

    NASA Astrophysics Data System (ADS)

    Wu, Kun-Ta; Hishamunda, Jean Bernard; Fraden, Seth; Dogic, Zvonimir

    Isotropic active gels are the network which is consist of cross-linked building blocks and the structure of which changes randomly and isotropically with time. Dogic et. al. show that pairs of anti-parallel microtubules form extensile bundles, which merge, extend, and buckle. In an unconfined system, the dynamics of these bundles causes spontaneous turbulent-like flow driven by motion of microscopic molecular motors. We found that confining these active gels in a millimeter sized toroids causes a transition into a new dynamical state characterized by circulation currents persisting for hours until ATP is depleted. We show how toroid dimensions impact the properties of self-organized circular currents, how directions of circulation can be designed by engineering ratchet-shaped boundaries, and how circulations of connected toroids can be either synchronized or antisynchronized. Furthermore, we demonstrate that the flow rate in the circulation is independent of curvature and length of flow path. The flow rate persists for centimeters without decay, disregarding conventional pipe flow resistance. Such findings pave the path to self-pumping pipe transport and performing physical work with biological system.

  18. Induction of mexCD-oprJ operon for a multidrug efflux pump by disinfectants in wild-type Pseudomonas aeruginosa PAO1.

    PubMed

    Morita, Yuji; Murata, Takeshi; Mima, Takehiko; Shiota, Sumiko; Kuroda, Teruo; Mizushima, Tohru; Gotoh, Naomasa; Nishino, Takeshi; Tsuchiya, Tomofusa

    2003-04-01

    Induction of the MexCD-OprJ multidrug efflux pump was investigated in wild-type Pseudomonas aeruginosa PAO1. MexCD-OprJ was induced by clinically important disinfectants such as benzalkonium chloride and chlorhexidine gluconate, and by some cytotoxic agents such as tetraphenylphosphonium chloride, ethidium bromide and rhodamine 6G. MexCD-OprJ was not induced by norfloxacin, tetracycline, chloramphenicol, streptomycin, erythromycin or carbenicillin, although they are substrates for the pump. Cells of PAO1 showed increased resistance to norfloxacin when grown in the presence of the inducers of the mexCD-oprJ operon mentioned above. These results indicate that MexCD-OprJ plays an important role in intrinsic multidrug resistance in wild-type P. aeruginosa in hospitals where disinfectants are used frequently. PMID:12654738

  19. K+ efflux agonists induce NLRP3 inflammasome activation independently of Ca2+ signaling1

    PubMed Central

    Katsnelson, Michael A.; Rucker, L. Graham; Russo, Hana M.; Dubyak, George R.

    2015-01-01

    Perturbation of intracellular ion homeostasis is a major cellular stress signal for activation of NLRP3 inflammasome signaling that results in caspase-1 mediated production of IL-1β and pyroptosis. However, the relative contributions of decreased cytosolic [K+] versus increased cytosolic [Ca2+] remain disputed and incompletely defined. We investigated roles for elevated cytosolic [Ca2+] in NLRP3 activation and downstream inflammasome signaling responses in primary murine dendritic cells and macrophages in response to two canonical NLRP3 agonists (ATP and nigericin) that facilitate primary K+ efflux by mechanistically distinct pathways or the lysosome-destabilizing agonist Leu-Leu-O-methyl ester (LLME). The study provides three major findings relevant to this unresolved area of NLRP3 regulation. First, increased cytosolic [Ca2+] was neither a necessary nor sufficient signal for the NLRP3 inflammasome cascade during activation by endogenous ATP-gated P2X7 receptor channels, the exogenous bacterial ionophore nigericin, or the lysosomotropic agent LLME. Second, agonists for three Ca2+-mobilizing G protein-coupled receptors (formyl peptide receptor/FPR; P2Y2 purinergic receptor/P2Y2R; calcium-sensing receptor/CaSR) expressed in murine dendritic cells were ineffective as activators of rapidly induced NLRP3 signaling when directly compared to the K+ efflux agonists. Third, the intracellular Ca2+ buffer, BAPTA, and the channel blocker, 2-aminoethoxydiphenyl borate (2-APB), widely used reagents for disruption of Ca2+-dependent signaling pathways, strongly suppressed nigericin-induced NLRP3 inflammasome signaling via mechanisms dissociated from their canonical or expected effects on Ca2+ homeostasis. The results indicate that the ability of K+ efflux agonists to activate NLRP3 inflammasome signaling can be dissociated from changes in cytosolic [Ca2+] as a necessary or sufficient signal. PMID:25762778

  20. RND-Type Efflux Pumps in Multidrug-Resistant Clinical Isolates of Acinetobacter baumannii: Major Role for AdeABC Overexpression and AdeRS Mutations

    PubMed Central

    Yoon, Eun-Jeong; Courvalin, Patrice

    2013-01-01

    Increased expression of chromosomal genes for resistance-nodulation-cell division (RND)-type efflux systems plays a major role in the multidrug resistance (MDR) of Acinetobacter baumannii. However, the relative contributions of the three most prevalent pumps, AdeABC, AdeFGH, and AdeIJK, have not been evaluated in clinical settings. We have screened 14 MDR clinical isolates shown to be distinct on the basis of multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) for the presence and overexpression of the three Ade efflux systems and analyzed the sequences of the regulators AdeRS, a two-component system, for AdeABC and AdeL, a LysR-type regulator, for AdeFGH. Gene adeB was detected in 13 of 14 isolates, and adeG and the intrinsic adeJ gene were detected in all strains. Significant overexpression of adeB was observed in 10 strains, whereas only 7 had moderately increased levels of expression of AdeFGH, and none overexpressed AdeIJK. Thirteen strains had reduced susceptibility to tigecycline, but there was no correlation between tigecycline MICs and the levels of AdeABC expression, suggesting the presence of other mechanisms for tigecycline resistance. No mutations were found in the highly conserved LysR regulator of the nine strains expressing AdeFGH. In contrast, functional mutations were found in conserved domains of AdeRS in all the strains that overexpressed AdeABC with two mutational hot spots, one in AdeS near histidine 149 suggesting convergent evolution and the other in the DNA binding domain of AdeR compatible with horizontal gene transfer. This report outlines the high incidence of AdeABC efflux pump overexpression in MDR A. baumannii as a result of a variety of single mutations in the corresponding two-component regulatory system. PMID:23587960

  1. RND-type efflux pumps in multidrug-resistant clinical isolates of Acinetobacter baumannii: major role for AdeABC overexpression and AdeRS mutations.

    PubMed

    Yoon, Eun-Jeong; Courvalin, Patrice; Grillot-Courvalin, Catherine

    2013-07-01

    Increased expression of chromosomal genes for resistance-nodulation-cell division (RND)-type efflux systems plays a major role in the multidrug resistance (MDR) of Acinetobacter baumannii. However, the relative contributions of the three most prevalent pumps, AdeABC, AdeFGH, and AdeIJK, have not been evaluated in clinical settings. We have screened 14 MDR clinical isolates shown to be distinct on the basis of multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) for the presence and overexpression of the three Ade efflux systems and analyzed the sequences of the regulators AdeRS, a two-component system, for AdeABC and AdeL, a LysR-type regulator, for AdeFGH. Gene adeB was detected in 13 of 14 isolates, and adeG and the intrinsic adeJ gene were detected in all strains. Significant overexpression of adeB was observed in 10 strains, whereas only 7 had moderately increased levels of expression of AdeFGH, and none overexpressed AdeIJK. Thirteen strains had reduced susceptibility to tigecycline, but there was no correlation between tigecycline MICs and the levels of AdeABC expression, suggesting the presence of other mechanisms for tigecycline resistance. No mutations were found in the highly conserved LysR regulator of the nine strains expressing AdeFGH. In contrast, functional mutations were found in conserved domains of AdeRS in all the strains that overexpressed AdeABC with two mutational hot spots, one in AdeS near histidine 149 suggesting convergent evolution and the other in the DNA binding domain of AdeR compatible with horizontal gene transfer. This report outlines the high incidence of AdeABC efflux pump overexpression in MDR A. baumannii as a result of a variety of single mutations in the corresponding two-component regulatory system. PMID:23587960

  2. Microbicides Alter the Expression and Function of RND-Type Efflux Pump AdeABC in Biofilm-Associated Cells of Acinetobacter baumannii Clinical Isolates.

    PubMed

    Krishnamoorthy, Suvarna; Shah, Bhavikkumar P; Lee, Hiu Ham; Martinez, Luis R

    2016-01-01

    Acinetobacter baumannii is a Gram-negative bacterium that causes nosocomial infections worldwide. This microbe's propensity to form biofilms allows it to persist and to survive on clinical abiotic surfaces for long periods. In fact, A. baumannii biofilm formation and its multidrug-resistant nature severely compromise our capacity to care for patients in hospital environments. In contrast, microbicides such as cetrimide (CT) and chlorhexidine (CHX) play important roles in the prevention and treatment of infections. We assessed the efficacy of CT and CHX, either alone or in combination, in eradicating A. baumannii biofilms formed by clinical isolates, by using stainless steel washers to mimic hard abiotic surfaces found in hospital settings. We demonstrated that increasing amounts of each microbicide, alone or in combination, were able to damage and to reduce the viability of A. baumannii biofilms efficaciously. Interestingly, the adeB gene of the resistance-nodulation-cell division (RND) family is predominantly associated with acquired resistance to antimicrobials in A. baumannii. We showed that CT and CHX adversely modified the expression and function of the RND-type efflux pump AdeABC in biofilm-associated A. baumannii cells. Furthermore, we established that these microbicides decreased the negative charges on A. baumannii cell membranes, causing dysregulation of the efflux pump and leading to cell death. Our findings suggest that CT and CHX, alone or in combination, can be used efficaciously for eradication of A. baumannii from hospital surfaces, in order to reduce infections caused by this nosocomial agent. PMID:26459900

  3. Vertical integration of thermally activated heat pumps

    SciTech Connect

    Chen, F.C.

    1985-01-01

    Many thermally activated heat pump systems are being developed along technology lines, such as, engine-driven and absorption heat pumps. Their thermal performances are temperature dependent. Based on the temperature-dependent behavior of heat pump cycle performance and the energy cascading idea, the concept of vertically integrating various thermally activated heat pump technologies to maximize resources utilization is explored. Based on a preliminary analysis, it is found that integrating a desiccant dehumidification subsystem to an engine-driven heat pump could improve its cooling performance by 36% and integrating an ejector to it could improve its cooling performance by 20%. The added advantage of an ejector-coupled engine-driven heat pump is its system simplicity which should result in equipment cost savings.

  4. Polyamines cause plasma membrane depolarization, activate Ca2+-, and modulate H+-ATPase pump activity in pea roots.

    PubMed

    Pottosin, Igor; Velarde-Buendía, Ana María; Bose, Jayakumar; Fuglsang, Anja T; Shabala, Sergey

    2014-06-01

    Polyamines regulate a variety of cation and K(+) channels, but their potential effects on cation-transporting ATPases are underexplored. In this work, noninvasive microelectrode ion flux estimation and conventional microelectrode techniques were applied to study the effects of polyamines on Ca(2+) and H(+) transport and membrane potential in pea roots. Externally applied spermine or putrescine (1mM) equally activated eosin yellow (EY)-sensitive Ca(2+) pumping across the root epidermis and caused net H(+) influx or efflux. Proton influx induced by spermine was suppressed by EY, supporting the mechanism in which Ca(2+) pump imports 2 H(+) per each exported Ca(2+). Suppression of the Ca(2+) pump by EY diminished putrescine-induced net H(+) efflux instead of increasing it. Thus, activities of Ca(2+) and H(+) pumps were coupled, likely due to the H(+)-pump inhibition by intracellular Ca(2+). Additionally, spermine but not putrescine caused a direct inhibition of H(+) pumping in isolated plasma membrane vesicles. Spermine, spermidine, and putrescine (1mM) induced membrane depolarization by 70, 50, and 35 mV, respectively. Spermine-induced depolarization was abolished by cation transport blocker Gd(3+), was insensitive to anion channels' blocker niflumate, and was dependent on external Ca(2+). Further analysis showed that uptake of polyamines but not polyamine-induced cationic (K(+)+Ca(2+)+H(+)) fluxes were a main cause of membrane depolarization. Polyamine increase is a common component of plant stress responses. Activation of Ca(2+) efflux by polyamines and contrasting effects of polyamines on net H(+) fluxes and membrane potential can contribute to Ca(2+) signalling and modulate a variety of transport processes across the plasma membrane under stress. PMID:24723394

  5. A Requirement of TolC and MDR Efflux Pumps for Acid Adaptation and GadAB Induction in Escherichia coli

    PubMed Central

    Tatsumi, Ryoko; Rosner, Judah L.; Wachi, Masaaki; Slonczewski, Joan L.

    2011-01-01

    Background The TolC outer membrane channel is a key component of several multidrug resistance (MDR) efflux pumps driven by H+ transport in Escherichia coli. While tolC expression is under the regulation of the EvgA-Gad acid resistance regulon, the role of TolC in growth at low pH and extreme-acid survival is unknown. Methods and Principal Findings TolC was required for extreme-acid survival (pH 2) of strain W3110 grown aerobically to stationary phase. A tolC deletion decreased extreme-acid survival (acid resistance) of aerated pH 7.0-grown cells by 105-fold and of pH 5.5-grown cells by 10-fold. The requirement was specific for acid resistance since a tolC defect had no effect on aerobic survival in extreme base (pH 10). TolC was required for expression of glutamate decarboxylase (GadA, GadB), a key component of glutamate-dependent acid resistance (Gad). TolC was also required for maximal exponential growth of E. coli K-12 W3110, in LBK medium buffered at pH 4.5–6.0, but not at pH 6.5–8.5. The TolC growth requirement in moderate acid was independent of Gad. TolC-associated pump components EmrB and MdtB contributed to survival in extreme acid (pH 2), but were not required for growth at pH 5. A mutant lacking the known TolC-associated efflux pumps (acrB, acrD, emrB, emrY, macB, mdtC, mdtF, acrEF) showed no growth defect at acidic pH and a relatively small decrease in extreme-acid survival when pre-grown at pH 5.5. Conclusions TolC and proton-driven MDR efflux pump components EmrB and MdtB contribute to E. coli survival in extreme acid and TolC is required for maximal growth rates below pH 6.5. The TolC enhancement of extreme-acid survival includes Gad induction, but TolC-dependent growth rates below pH 6.5 do not involve Gad. That MDR resistance can enhance growth and survival in acid is an important consideration for enteric organisms passing through the acidic stomach. PMID:21541325

  6. Tunicamycin Depresses P-Glycoprotein Glycosylation Without an Effect on Its Membrane Localization and Drug Efflux Activity in L1210 Cells

    PubMed Central

    Šereš, Mário; Cholujová, Dana; Bubenčíkova, Tatiana; Breier, Albert; Sulová, Zdenka

    2011-01-01

    P-glycoprotein (P-gp), also known as ABCB1, is a member of the ABC transporter family of proteins. P-gp is an ATP-dependent drug efflux pump that is localized to the plasma membrane of mammalian cells and confers multidrug resistance in neoplastic cells. P-gp is a 140-kDa polypeptide that is glycosylated to a final molecular weight of 170 kDa. Our experimental model used two variants of L1210 cells in which overexpression of P-gp was achieved: either by adaptation of parental cells (S) to vincristine (R) or by transfection with the human gene encoding P-gp (T). R and T cells were found to differ from S cells in transglycosylation reactions in our recent studies. The effects of tunicamycin on glycosylation, drug efflux activity and cellular localization of P-gp in R and T cells were examined in the present study. Treatment with tunicamycin caused less concentration-dependent cellular damage to R and T cells compared with S cells. Tunicamycin inhibited P-gp N-glycosylation in both of the P-gp-positive cells. However, tunicamycin treatment did not alter either the P-gp cellular localization to the plasma membrane or the P-gp transport activity. The present paper brings evidence that independently on the mode of P-gp expression (selection with drugs or transfection with a gene encoding P-gp) in L1210 cells, tunicamycin induces inhibition of N-glycosylation of this protein, without altering its function as plasma membrane drug efflux pump. PMID:22174631

  7. Expression of the AcrAB Components of the AcrAB-TolC Multidrug Efflux Pump of Yersinia enterocolitica Is Subject to Dual Regulation by OmpR

    PubMed Central

    Raczkowska, Adrianna; Trzos, Joanna; Lewandowska, Olga; Nieckarz, Marta; Brzostek, Katarzyna

    2015-01-01

    OmpR is a transcriptional regulator implicated in the control of various cellular processes and functions in Enterobacteriaceae. This study was undertaken to identify genes comprising the OmpR regulon in the human gastrointestinal pathogen Yersinia enterocolitica. Derivatives of an ompR-negative strain with random transposon insertions creating transcriptional fusions with the reporter gene lacZ were isolated. These were supplied with the wild-type ompR allele in trans and then screened for OmpR-dependent changes in β-galactosidase activity. Using this strategy, five insertions in genes/operons positively regulated by OmpR and two insertions in genes negatively regulated by this protein were identified. Genetic analysis of one of these fusion strains revealed that the gene acrR, encoding transcriptional repressor AcrR is negatively regulated by OmpR. Differential analysis of membrane proteins by SDS-PAGE followed by mass spectrometry identified the protein AcrB, a component of the AcrAB-TolC multidrug efflux pump, as being positively regulated by OmpR. Analysis of the activity of the acrR and acrAB promoters using gfp fusions confirmed their OmpR-dependent repression and activation, respectively. The identification of putative OmpR-binding sites and electrophoretic mobility shift assays confirmed that this regulator binds specifically to both promoter regions with different affinity. Examination of the activity of the acrR and acrAB promoters after the exposure of cells to different chemicals showed that bile salts can act as an OmpR-independent inducer. Taken together, our findings suggest that OmpR positively controls the expression of the AcrAB-TolC efflux pump involved in the adaptive response of Y. enterocolitica O:9 to different chemical stressors, thus conferring an advantage in particular ecological niches. PMID:25893523

  8. Expression of the AcrAB Components of the AcrAB-TolC Multidrug Efflux Pump of Yersinia enterocolitica Is Subject to Dual Regulation by OmpR.

    PubMed

    Raczkowska, Adrianna; Trzos, Joanna; Lewandowska, Olga; Nieckarz, Marta; Brzostek, Katarzyna

    2015-01-01

    OmpR is a transcriptional regulator implicated in the control of various cellular processes and functions in Enterobacteriaceae. This study was undertaken to identify genes comprising the OmpR regulon in the human gastrointestinal pathogen Yersinia enterocolitica. Derivatives of an ompR-negative strain with random transposon insertions creating transcriptional fusions with the reporter gene lacZ were isolated. These were supplied with the wild-type ompR allele in trans and then screened for OmpR-dependent changes in β-galactosidase activity. Using this strategy, five insertions in genes/operons positively regulated by OmpR and two insertions in genes negatively regulated by this protein were identified. Genetic analysis of one of these fusion strains revealed that the gene acrR, encoding transcriptional repressor AcrR is negatively regulated by OmpR. Differential analysis of membrane proteins by SDS-PAGE followed by mass spectrometry identified the protein AcrB, a component of the AcrAB-TolC multidrug efflux pump, as being positively regulated by OmpR. Analysis of the activity of the acrR and acrAB promoters using gfp fusions confirmed their OmpR-dependent repression and activation, respectively. The identification of putative OmpR-binding sites and electrophoretic mobility shift assays confirmed that this regulator binds specifically to both promoter regions with different affinity. Examination of the activity of the acrR and acrAB promoters after the exposure of cells to different chemicals showed that bile salts can act as an OmpR-independent inducer. Taken together, our findings suggest that OmpR positively controls the expression of the AcrAB-TolC efflux pump involved in the adaptive response of Y. enterocolitica O:9 to different chemical stressors, thus conferring an advantage in particular ecological niches. PMID:25893523

  9. Inhibition of topoisomerase I activity and efflux drug transporters' expression by xanthohumol. from hops.

    PubMed

    Lee, Sung Ho; Kim, Hyun Jung; Lee, Jung Sun; Lee, Ik-Soo; Kang, Bok Yun

    2007-11-01

    Xanthohumol (XN) and its related compounds were evaluated for their cytotoxicity against four different human cancer cell lines, A549 (lung), SK-OV-3 (ovarian), SK-MEL-2 (melanoma), and HCT-15 (colon) using a sulforhodamine B assay. XN showed the most active cytotoxicity against the human cancer cell lines. Isoxanthohumol, 8-prenylnaringenin, and xanthohumol 4'-O-beta-D-glucopyranoside showed comparable cytotoxicity and (2S)-5-methoxy-8-prenylnaringenin 7-O-beta-D-glucopyranoside was the least cytotoxic compound. The anticancer properties of XN, the most active cytotoxic compound, were further investigated. XN showed an inhibitory effect on the activity of DNA topoisomerase I (topo I), which was measured from the relaxation of supercoiled DNA. The inhibition of topo I by XN might explain the cytotoxicity against the human cancer cell lines. Moreover, the expression of the drug efflux genes was investigated to predict the drug resistance. XN clearly decreased the mRNA levels of ABCB1 (MDR1), ABCC1 (MRP1), ABCC2 (MRP2), and ABCC3 (MRP3). These results suggest that XN has anticancer properties by inhibiting the topo I activity and it might be used in conjunction with other anticancer chemotherapeutic agents to reduce the drug resistance inhibiting the efflux drug transporters. PMID:18087812

  10. Effects of aging on agonist-activated sup 86 Rb efflux in arteries of Fischer 344 rats

    SciTech Connect

    Cox, R.H.; Tulenko, T.N. )

    1989-08-01

    Segments of thoracic aorta (DTA), tail artery (TA), and mesenteric artery branches (MAB) were obtained from male Fischer 344 rats at ages of 1, 2, 6, 12, 24, and 30 mo and were used to determine the effects of aging on agonist-activated {sup 86}Rb (and {sup 42}K) efflux. At all three arterial sites, basal efflux decreased during development (1-6 mo), but no further changes were observed with aging (6-30 mo). The initial efflux response to 10 microM norepinephrine (NE) in the presence of 1 microM propranolol exhibited either no change (DTA) or an increase (TA and MAB) during development (1-6 mo), but all three sites showed a large decrease during aging (6-30 mo). Changes in the steady-state response to NE paralleled changes in the basal efflux at all ages and arterial sites. The initial efflux response to 75 mM K+-physiological salt solution (PSS) for the DTA in the presence of 1 microM phentolamine and 1 microM propranolol decreased during development followed by an increase during aging, whereas for the TA and MAB, there were no significant changes with age. The steady-state efflux response to K+ decreased during development at all three sites but was increased only for the DTA during aging. The steady-state efflux response to K+ was not altered for the TA and MAB during aging. Efflux responses using {sup 42}K were qualitatively similar, but rate constants were quantitatively larger than those with {sup 86}Rb at all three arterial sites and at all ages.

  11. Bacterial multidrug efflux transporters.

    PubMed

    Delmar, Jared A; Su, Chih-Chia; Yu, Edward W

    2014-01-01

    Infections caused by bacteria are a leading cause of death worldwide. Although antibiotics remain a key clinical therapy, their effectiveness has been severely compromised by the development of drug resistance in bacterial pathogens. Multidrug efflux transporters--a common and powerful resistance mechanism--are capable of extruding a number of structurally unrelated antimicrobials from the bacterial cell, including antibiotics and toxic heavy metal ions, facilitating their survival in noxious environments. Transporters of the resistance-nodulation-cell division (RND) superfamily typically assemble as tripartite efflux complexes spanning the inner and outer membranes of the cell envelope. In Escherichia coli, the CusCFBA complex, which mediates resistance to copper(I) and silver(I) ions, is the only known RND transporter specific to heavy metals. Here, we describe the current knowledge of individual pump components of the Cus system, a paradigm for efflux machinery, and speculate on how RND pumps assemble to fight diverse antimicrobials. PMID:24702006

  12. Comparison of effects of cromakalim and pinacidil on mechanical activity and 86Rb efflux in dog coronary arteries

    SciTech Connect

    Masuzawa, K.; Asano, M.; Matsuda, T.; Imaizumi, Y.; Watanabe, M. )

    1990-05-01

    Effects of two K+ channel openers, cromakalim and pinacidil, on mechanical activity and on 86Rb efflux were compared in strips of dog coronary arteries. Cromakalim and pinacidil produced the relaxation in 20.9 mM K(+)-contracted strips with a pD2 of 6.53 and 5.95, respectively. In 65.9 mM K(+)-contracted strips, high concentrations of pinacidil, but not cromakalim, produced relaxation. Ca+(+)-induced contractions in 80 mM K(+)-depolarized strips were also inhibited by pinacidil but not by cromakalim. Glibenclamide, a blocker of ATP-regulated K+ (KATP) channels, competitively antagonized the relaxant responses to cromakalim with a pA2 value of 7.62. However, the antagonism by glibenclamide of the relaxant responses to pinacidil was not a typical competitive type, suggesting the contribution of other effects than the KATP channel opening activity to the relaxant effects of pinacidil. In resting strips preloaded with 86Rb, cromakalim and pinacidil increased the basal 86Rb efflux in a dose-dependent manner. The increase in the 86Rb efflux induced by cromakalim was greater than that by pinacidil. When the effects of cromakalim and pinacidil on the 86Rb efflux were determined in the 20.9 or 65.9 mM K(+)-contracted strips, both drugs increased the 86Rb efflux. Under the same conditions nifedipine, a Ca(+)+ channel blocker, produced the relaxation that is accompanied by the decrease in 86Rb efflux. The increase in the 86Rb efflux induced by cromakalim was much greater than that by pinacidil.

  13. Efflux systems in bacterial pathogens: an opportunity for therapeutic intervention? An industry view.

    PubMed

    Lynch, A Simon

    2006-03-30

    The efflux systems of bacteria protect cells from antibiotics and biocides by actively transporting compounds out of the cytoplasm and/or periplasm and thereby limit their steady-state accumulation at their site(s) of action. The impact of efflux systems on the efficacy of antibiotics used in human medicine and animal husbandry is becoming increasingly apparent from the characterization of drug-resistant strains with altered drug efflux properties. In most instances, efflux-mediated antibiotic resistance arises from mutational events that result in their elevated expression and, in the case of efflux pumps with broad substrate specificity, can confer multi-drug resistance (MDR) to structurally unrelated antibiotics. Knowledge of the role of efflux systems in conferring antibiotic resistance has now been successfully exploited in the pharmaceutical industry and contributed, in part, to the development of new members of the macrolide and tetracycline classes of antibiotics that circumvent the efflux-based resistance mechanisms that have limited the clinical utility of their progenitors. The therapeutic utility of compounds that inhibit bacterial drug efflux pumps and therein potentiate the activity of a co-administered antibiotic agent remains to be validated in the clinical setting, but the approach holds promise for the future in improving the efficacy and/or extending the clinical utility of existing antibiotics. This review discusses the potential of further exploiting the knowledge of efflux-mediated antibiotic resistance in bacteria toward the discovery and development of new chemotherapeutic agents. PMID:16290174

  14. The Pseudomonas aeruginosa efflux pump MexGHI-OpmD transports a natural phenazine that controls gene expression and biofilm development.

    PubMed

    Sakhtah, Hassan; Koyama, Leslie; Zhang, Yihan; Morales, Diana K; Fields, Blanche L; Price-Whelan, Alexa; Hogan, Deborah A; Shepard, Kenneth; Dietrich, Lars E P

    2016-06-21

    Redox-cycling compounds, including endogenously produced phenazine antibiotics, induce expression of the efflux pump MexGHI-OpmD in the opportunistic pathogen Pseudomonas aeruginosa Previous studies of P. aeruginosa virulence, physiology, and biofilm development have focused on the blue phenazine pyocyanin and the yellow phenazine-1-carboxylic acid (PCA). In P. aeruginosa phenazine biosynthesis, conversion of PCA to pyocyanin is presumed to proceed through the intermediate 5-methylphenazine-1-carboxylate (5-Me-PCA), a reactive compound that has eluded detection in most laboratory samples. Here, we apply electrochemical methods to directly detect 5-Me-PCA and find that it is transported by MexGHI-OpmD in P. aeruginosa strain PA14 planktonic and biofilm cells. We also show that 5-Me-PCA is sufficient to fully induce MexGHI-OpmD expression and that it is required for wild-type colony biofilm morphogenesis. These physiological effects are consistent with the high redox potential of 5-Me-PCA, which distinguishes it from other well-studied P. aeruginosa phenazines. Our observations highlight the importance of this compound, which was previously overlooked due to the challenges associated with its detection, in the context of P. aeruginosa gene expression and multicellular behavior. This study constitutes a unique demonstration of efflux-based self-resistance, controlled by a simple circuit, in a Gram-negative pathogen. PMID:27274079

  15. Characterization of AcrD, a Resistance-Nodulation-Cell Division-type multidrug efflux pump from the fire blight pathogen Erwinia amylovora

    PubMed Central

    2014-01-01

    Background Multidrug efflux pumps are membrane translocases that have the ability to extrude a variety of structurally unrelated compounds from the cell. AcrD, a resistance-nodulation-cell division (RND) transporter, was shown to be involved in efflux of highly hydrophilic aminoglycosides and a limited number of amphiphilic compounds in E. coli. Here, a homologue of AcrD in the plant pathogen and causal agent of fire blight disease Erwinia amylovora was identified. Results The substrate specificity of AcrD was studied by overexpression of the corresponding gene from a high-copy plasmid in E. amylovora Ea1189-3, which is hypersensitive to many drugs due to a deficiency of the major multidrug pump AcrB. AcrD mediated resistance to several amphiphilic compounds including clotrimazole and luteolin, two compounds hitherto not described as substrates of AcrD in enterobacteria. However, AcrD was not able to expel aminoglycosides. An acrD mutant exhibited full virulence on apple rootstock and immature pear fruits. RT-PCR analysis revealed an induction of acrD expression in infected apple tissue but not on pear fruits. Moreover, a direct binding of BaeR, the response regulator of the two-component regulatory system BaeSR, to the acrD promoter was observed as has already been shown in other enterobacteria. Conclusions AcrD from E. amylovora is involved in resistance to a limited number of amphiphilic compounds, but in contrast to AcrD of E. coli, it is not involved in resistance to aminoglycosides. The expression of acrD was up-regulated by addition of the substrates deoxycholate, naringenin, tetracycline and zinc. AcrD appears to be regulated by the BaeSR two-component system, an envelope stress signal transduction pathway. PMID:24443882

  16. Characterization and distribution of drug resistance associated β-lactamase, membrane porin and efflux pump genes in MDR A. baumannii isolated from Zhenjiang, China

    PubMed Central

    Yang, Huijian; Huang, Lan; Barnie, Prince Amoah; Su, Zhaoliang; Mi, Zuhuang; Chen, Jianguo; Aparna, Vasudevan; Kumar, Dinesh; Xu, Huaxi

    2015-01-01

    Background: Acinetobacter baumannii (A. baumannii), especially the multidrug resistant A. baumannii (MDR-AB) is becoming a common opportunistic pathogen in hospital, and constitutes significant public health threats. This study aimed at investigating the relationship between drug resistance with expression of class A-D β-lactamase genes, mutation in membrane porin and over-expression of efflux pump genes among A. baumannii isolated from Zhengjiang, China. Methods: Antibiotic susceptibility assays were performed using Kirby-Bauer disc diffusion method. PCR was used to detect β-lactamase genes and carO, oprD, adeR, adeS. Real-time PCR was used to assess the mRNA expression level of efflux pump gene adeB. The software of DNAMAN was applied to assemble oprD and carO sequences, and the sequences were compared with those retrieved from GenBank (http://www.ncbi.nlm.nih.gov/). Results: 27 isolates (61.4%) in this study were MDR-AB, in which five β-lactamases including TEM, CTX-M-2, ADC, OXA-23 and OXA-51 were found, and the positive rate was 96.3% (26), 14.8% (4), 92.6% (25), 88.9% (24) and 92.6% (25), respectively. In addition, the expression level of adeB mRNA was significantly increased in MDR-AB, it might due to adeR mutation. Some mutations were also found in carO and oprD. Conclusion: MDR-AB showed high relationship with β-lactamase, mutation in membrane porin and overexpression of adeB, which may directly relates to the mutation in regulating gene adeR. PMID:26629028

  17. Altered agonist-activated sup 86 Rb+ efflux from arteries in canine renal hypertension

    SciTech Connect

    Cox, R.H.; Bagshaw, R.J. )

    1989-07-01

    Basal rate constants for {sup 86}Rb+ efflux from renal arteries of renal hypertensive dogs were lower than those of control animals whereas no differences were found for coronary arteries. Norepinephrine produced parallel increases in efflux rate constants for hypertensive and control renal arteries, but serotonin produced smaller responses in hypertensive compared to control coronary arteries.

  18. Modeling the tripartite drug efflux pump archetype: structural and functional studies of the macromolecular constituents reveal more than their names imply.

    PubMed

    Elkins, C A; Beenken, K E

    2005-12-01

    It is a remarkable age in molecular biology when one can argue that our current understanding of a process is influenced as much by structural studies as it is by genetic and physiological manipulations. This statement is particularly poignant with membrane proteins for which structural knowledge has been long impeded by the inability to easily obtain crystal structures in a lipid matrix. Thus, several high-resolution structures of the components comprising tripartite multidrug efflux pumps from Escherichia coli and Pseudomonas aeruginosa are now available and were received with much acclaim over ever-evolving crystal structures of soluble, aqueous proteins. These structures, in conjunction with functional mutagenesis studies, have provided insight into substrate capture and binding domains and redefined the potential interactions between individual pump constituents. However, correct assembly of the components is still a matter of debate as is the functional contribution of each to the translocation of drug substrates over long distances spanning the Gram-negative cell envelope. PMID:16433187

  19. [Investigation of the effect of efflux pump inhibitors to MIC values of ciprofloxacin in clinical isolates of Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii and Staphylococcus aureus].

    PubMed

    Cetinkaya, Ebru; Coban, Ahmet Yilmaz; Durupinar, Belma

    2008-10-01

    The aim of this study was to investigate the effects of efflux pump inhibitors on the minimal inhibitory concentration (MIC) values of ciprofloxacin (CIP) in fluoroquinolone-resistant 42 Pseudomonas aeruginosa (n= 42), Escherichia coil (n= 97), Acinetobacter baumannii (n= 58) and Staphylococcus aureus (n= 80) strains isolated from clinical specimens. For this purpose phenylalanyl-arginyl-beta-naphthylamide (PA beta N) was used for P. aeruginosa, E. coli, A. baumannii and reserpine for S. aureus isolates as pump inhibitors. Fluoroquinolone resistance of the clinical isolates were determined by VITEK2 Compact (BioMerieux, France) automated system and confirmed with standard broth microdilution method. For the investigation of the effects of inhibitor agents, the MIC values were also determined in the presence of 25 microg/ml and 100 microg/ml PA beta N and 20 microg/ml reserpine. In the presence of 25 mg/l PA beta N, 61.9% of CIP resistant P. aeruginosa strains converted to susceptible ones, while this rate was 73.8% in the presence of 100 mg/l PA beta N. In A. baumannii clinical isolates, 8.6% and 15.5% of CIP-resistant strains have become susceptible in the presence of 25 mg/l and 100 mg/l PA beta N, respectively. Similarly the MIC values for CIP have decreased > or = 4 folds in 42.2%, and > or = 2 folds in 30.9% of E. coli isolates, in the presence of 25 mg/l PA beta N, however, there was no change in MICs of 26.9% of E. coli strains. The MIC values have also been lowered for > or = 4 folds in 83.6%, and two folds in 13.4% of E. coli strains by the use of 100 mg/l PA beta N concentration, however, no decrease in MIC values was detected in 3% of the isolates. 20 mg/l of reserpine have caused a decrease of > or = 4 folds in 8.75%, and two folds in 33.75% of S. aureus isolates, while there was no change in MIC values of 57.5% of S. aureus strains. Our results showed that PA beta N causes significant reduction in MIC values for CIP in the clinical isolates of P

  20. PV water pumping: NEOS Corporation recent PV water pumping activities

    SciTech Connect

    Lane, C.

    1995-11-01

    NEOS Corporation has been very active in PV-powered water pumping, particularly with respect to electric utilities. Most of the recent activity has been through the Photovoltaic Services Network (PSN). The PSN is an independent, not-for-profit organization comprised of all types of electric utilities: rural electric coops, public power districts, investor-owned utilities, and power marketing agencies. The PSN`s mission is to work pro-actively to promote utility involvement in PV through education and training. PV information is distributed by the PSN in three primary forms: (1) consultation with PSN technical service representatives: (2) literature generated by the PSN; and (3) literature published by other organizations. The PSN can also provide assistance to members in developing PV customer service programs. The PSN`s product support activities include consolidation of information on existing packaged PV systems and facilitation of the development of new PV product packages that meet utility-defined specifications for cost performance, and reliability. The PSN`s initial product support efforts will be focused on commercially available packaged PV systems for a variety of off-grid applications. In parallel with this effort, if no products exist that meet the PSN`s functional specifications, the PSN will initiate the second phase of product development support process by encouraging the development of new packaged systems. Through these services and product support activities, the PSN anticipates engaging all segments for the PV industry, thus providing benefits to PV systems suppliers as well as local PV service contractors.This paper describes field testing of pv power systems for water pumping.

  1. The effects of the putative potassium channel activator WAY-120,491 on 86Rb efflux from the rabbit aorta

    SciTech Connect

    Lodge, N.J.; Cohen, R.B.; Havens, C.N.; Colatsky, T.J. )

    1991-02-01

    WAY-120,491 ((-)-(3S-trans)-2-(3,4-dihydro-3-hydroxy-2,2-dimethyl-6-(trifluoromet hox y)- 2H-1-benzopyran-4-yl)-2,3-dihydro-1H-isoindol-1-one) is a novel antihypertensive agent. We have investigated the effects of this compound on contractile force and 86Rb efflux, using the rabbit aorta, in order to assess its K channel activator properties. K channel blockers and ionic conditions thought to modulate specific K channel types have been used to provide insight into the K channel(s) affected by this compound. WAY-120,491 evoked relaxation of precontracted rabbit aortic rings and increased the rate of 86Rb efflux from strips of rabbit aorta; both effects occurring in a concentration-dependent manner. The WAY-120,491 (1 microM)-induced 86Rb efflux was inhibited by tetraethylammonium (IC50 = 0.38 mM), indicating that the increased efflux was mediated by K channels. Glyburide completely blocked the WAY-120,491 (1 microM)-evoked 86Rb efflux with 50% block occurring at a concentration of 0.48 microM. Glyburide also antagonized the WAY-120,491-induced relaxation of aortic rings. Omission of Ca from the solution bathing the aorta did not inhibit the WAY-120,491 induced 86Rb efflux but rather caused an augmentation of the response. It is concluded that WAY-120,491 may be classified as a K channel opener. Furthermore, the K channel upon which WAY-120,491 acts exhibits some characteristics normally associated with the ATP regulated K channel although the involvement of other K channel types has not been ruled out.

  2. Efflux-Mediated Antifungal Drug Resistance†

    PubMed Central

    Cannon, Richard D.; Lamping, Erwin; Holmes, Ann R.; Niimi, Kyoko; Baret, Philippe V.; Keniya, Mikhail V.; Tanabe, Koichi; Niimi, Masakazu; Goffeau, Andre; Monk, Brian C.

    2009-01-01

    Summary: Fungi cause serious infections in the immunocompromised and debilitated, and the incidence of invasive mycoses has increased significantly over the last 3 decades. Slow diagnosis and the relatively few classes of antifungal drugs result in high attributable mortality for systemic fungal infections. Azole antifungals are commonly used for fungal infections, but azole resistance can be a problem for some patient groups. High-level, clinically significant azole resistance usually involves overexpression of plasma membrane efflux pumps belonging to the ATP-binding cassette (ABC) or the major facilitator superfamily class of transporters. The heterologous expression of efflux pumps in model systems, such Saccharomyces cerevisiae, has enabled the functional analysis of efflux pumps from a variety of fungi. Phylogenetic analysis of the ABC pleiotropic drug resistance family has provided a new view of the evolution of this important class of efflux pumps. There are several ways in which the clinical significance of efflux-mediated antifungal drug resistance can be mitigated. Alternative antifungal drugs, such as the echinocandins, that are not efflux pump substrates provide one option. Potential therapeutic approaches that could overcome azole resistance include targeting efflux pump transcriptional regulators and fungal stress response pathways, blockade of energy supply, and direct inhibition of efflux pumps. PMID:19366916

  3. Evidence of a Substrate-Discriminating Entrance Channel in the Lower Porter Domain of the Multidrug Resistance Efflux Pump AcrB.

    PubMed

    Schuster, Sabine; Vavra, Martina; Kern, Winfried V

    2016-07-01

    Efflux pumps of the resistance nodulation cell division (RND) transporter family, such as AcrB of Escherichia coli, play an important role in the development of multidrug resistance, but the molecular basis for their substrate promiscuity is not yet completely understood. From a collection of highly clarithromycin-resistant AcrB periplasmic domain mutants derived from in vitro random mutagenesis, we identified variants with an unusually altered drug resistance pattern characterized by increased susceptibility to many drugs of lower molecular weight, including fluoroquinolones, tetracyclines, and oxazolidinones, but unchanged or increased resistance to drugs of higher molecular weight, including macrolides. Sequencing of 14 such "divergent resistance" phenotype mutants and 15 control mutants showed that this unusual phenotype was associated with mutations at residues I38 and I671 predominantly to phenylalanine and threonine, respectively, both conferring a similar susceptibility pattern. Reconstructed I38F and I671T single mutants as well as an engineered I38F I671T double mutant with proved efflux competence revealed an equivalent phenotype with enhanced or unchanged resistance to many large AcrB substrates but increased susceptibility to several lower-molecular-weight drugs known to bind within the distal binding pocket. The two isoleucines located in close vicinity to each other in the lower porter domain of AcrB beneath the bottom of the proximal binding pocket may be part of a preferential small-drug entrance pathway that is compromised by the mutations. This finding supports recent indications of distinct entrance channels used by compounds with different physicochemical properties, of which molecular size appears to play a prominent role. PMID:27161641

  4. Bioluminescent imaging of ABCG2 efflux activity at the blood-placenta barrier.

    PubMed

    Kumar, Jeyan S; Wei, Bih-Rong; Madigan, James P; Simpson, R Mark; Hall, Matthew D; Gottesman, Michael M

    2016-01-01

    Physiologic barriers such as the blood placenta barrier (BPB) and the blood brain barrier protect the underlying parenchyma from pathogens and toxins. ATP-binding cassette (ABC) transporters are transmembrane proteins found at these barriers, and function to efflux xenobiotics and maintain chemical homeostasis. Despite the plethora of ex vivo and in vitro data showing the function and expression of ABC transporters, no imaging modality exists to study ABC transporter activity in vivo at the BPB. In the present study, we show that in vitro models of the placenta possess ABCG2 activity and can specifically transport D-luciferin, the endogenous substrate of firefly luciferase. To test ABCG2 transport activity at the BPB, we devised a breeding strategy to generate a bioluminescent pregnant mouse model to demonstrate transporter function in vivo. We found that coadministering the ABCG2 inhibitors Ko143 and gefitinib with D-luciferin increased bioluminescent signal from fetuses and placentae, whereas the control P-gp inhibitor DCPQ had no effect. We believe that our bioluminescent pregnant mouse model will facilitate greater understanding of the BPB and ABCG2 activity in health and disease. PMID:26853103

  5. Bioluminescent imaging of ABCG2 efflux activity at the blood-placenta barrier

    PubMed Central

    Kumar, Jeyan S.; Wei, Bih-Rong; Madigan, James P.; Simpson, R. Mark; Hall, Matthew D.; Gottesman, Michael M.

    2016-01-01

    Physiologic barriers such as the blood placenta barrier (BPB) and the blood brain barrier protect the underlying parenchyma from pathogens and toxins. ATP-binding cassette (ABC) transporters are transmembrane proteins found at these barriers, and function to efflux xenobiotics and maintain chemical homeostasis. Despite the plethora of ex vivo and in vitro data showing the function and expression of ABC transporters, no imaging modality exists to study ABC transporter activity in vivo at the BPB. In the present study, we show that in vitro models of the placenta possess ABCG2 activity and can specifically transport D-luciferin, the endogenous substrate of firefly luciferase. To test ABCG2 transport activity at the BPB, we devised a breeding strategy to generate a bioluminescent pregnant mouse model to demonstrate transporter function in vivo. We found that coadministering the ABCG2 inhibitors Ko143 and gefitinib with D-luciferin increased bioluminescent signal from fetuses and placentae, whereas the control P-gp inhibitor DCPQ had no effect. We believe that our bioluminescent pregnant mouse model will facilitate greater understanding of the BPB and ABCG2 activity in health and disease. PMID:26853103

  6. Resistance-nodulation-cell division-type efflux pump involved in aminoglycoside resistance in Acinetobacter baumannii strain BM4454.

    PubMed

    Magnet, S; Courvalin, P; Lambert, T

    2001-12-01

    Multidrug-resistant strain Acinetobacter baumannii BM4454 was isolated from a patient with a urinary tract infection. The adeB gene, which encodes a resistance-nodulation-cell division (RND) protein, was detected in this strain by PCR with two degenerate oligodeoxynucleotides. Insertional inactivation of adeB in BM4454, which generated BM4454-1, showed that the corresponding protein was responsible for aminoglycoside resistance and was involved in the level of susceptibility to other drugs including fluoroquinolones, tetracyclines, chloramphenicol, erythromycin, trimethoprim, and ethidium bromide. Study of ethidium bromide accumulation in BM4454 and BM4454-1, in the presence or in the absence of carbonyl cyanide m-chlorophenylhydrazone, demonstrated that AdeB was responsible for the decrease in intracellular ethidium bromide levels in a proton motive force-dependent manner. The adeB gene was part of a cluster that included adeA and adeC which encodes proteins homologous to membrane fusion and outer membrane proteins of RND-type three-component efflux systems, respectively. The products of two upstream open reading frames encoding a putative two-component regulatory system might be involved in the regulation of expression of the adeABC gene cluster. PMID:11709311

  7. Resistance-Nodulation-Cell Division-Type Efflux Pump Involved in Aminoglycoside Resistance in Acinetobacter baumannii Strain BM4454

    PubMed Central

    Magnet, Sophie; Courvalin, Patrice; Lambert, Thierry

    2001-01-01

    Multidrug-resistant strain Acinetobacter baumannii BM4454 was isolated from a patient with a urinary tract infection. The adeB gene, which encodes a resistance-nodulation-cell division (RND) protein, was detected in this strain by PCR with two degenerate oligodeoxynucleotides. Insertional inactivation of adeB in BM4454, which generated BM4454-1, showed that the corresponding protein was responsible for aminoglycoside resistance and was involved in the level of susceptibility to other drugs including fluoroquinolones, tetracyclines, chloramphenicol, erythromycin, trimethoprim, and ethidium bromide. Study of ethidium bromide accumulation in BM4454 and BM4454-1, in the presence or in the absence of carbonyl cyanide m-chlorophenylhydrazone, demonstrated that AdeB was responsible for the decrease in intracellular ethidium bromide levels in a proton motive force-dependent manner. The adeB gene was part of a cluster that included adeA and adeC which encodes proteins homologous to membrane fusion and outer membrane proteins of RND-type three-component efflux systems, respectively. The products of two upstream open reading frames encoding a putative two-component regulatory system might be involved in the regulation of expression of the adeABC gene cluster. PMID:11709311

  8. KpnEF, a New Member of the Klebsiella pneumoniae Cell Envelope Stress Response Regulon, Is an SMR-Type Efflux Pump Involved in Broad-Spectrum Antimicrobial Resistance

    PubMed Central

    Rajamohan, Govindan

    2013-01-01

    Klebsiella pneumoniae has been frequently associated with nosocomial infections. Efflux systems are ubiquitous transporters that also function in drug resistance. Genome analysis of K. pneumoniae strain NTUH-K2044 revealed the presence of ∼15 putative drug efflux systems. We discuss here for the first time the characterization of a putative SMR-type efflux pump, an ebrAB homolog (denoted here as kpnEF) with respect to Klebsiella physiology and the multidrug-resistant phenotype. Analysis of hypermucoviscosity revealed direct involvement of kpnEF in capsule synthesis. The ΔkpnEF mutant displayed higher sensitivity to hyperosmotic (∼2.8-fold) and high bile (∼4.0-fold) concentrations. Mutation in kpnEF resulted in increased susceptibility to cefepime, ceftriaxone, colistin, erythromycin, rifampin, tetracycline, and streptomycin; mutated strains changed from being resistant to being susceptible, and the resistance was restored upon complementation. The ΔkpnEF mutant displayed enhanced sensitivity toward structurally related compounds such as sodium dodecyl sulfate, deoxycholate, and dyes, including clinically relevant disinfectants such as benzalkonium chloride, chlorhexidine, and triclosan. The prevalence of kpnEF in clinical strains broadens the diversity of antibiotic resistance in K. pneumoniae. Experimental evidence of CpxR binding to the efflux pump promoter and quantification of its expression in a cpxAR mutant background demonstrated kpnEF to be a member of the Cpx regulon. This study helps to elucidate the unprecedented biological functions of the SMR-type efflux pump in Klebsiella spp. PMID:23836167

  9. Ion Channel Blockers as Antimicrobial Agents, Efflux Inhibitors, and Enhancers of Macrophage Killing Activity against Drug Resistant Mycobacterium tuberculosis

    PubMed Central

    Perdigão, João; Couto, Isabel; Portugal, Isabel; Martins, Marta; Amaral, Leonard; Anes, Elsa; Viveiros, Miguel

    2016-01-01

    Given the ability of M. tuberculosis to survive as an intracellular pathogen and its propensity to develop resistance to the existing antituberculosis drugs, its treatment requires new approaches. Here the antimycobacterial properties of verapamil, thioridazine, chlorpromazine, flupenthixol and haloperidol were investigated against a panel of drug resistant M. tuberculosis strains, both in vitro and on human-infected macrophages. These compounds are efflux inhibitors that share among them the characteristic of being ion channel blockers. In vitro, all compounds exhibited synergistic inhibitory activities when combined with isoniazid and rifampicin, and were able to inhibit active efflux, demonstrating their role as efflux inhibitors. Gene expression analysis showed that M. tuberculosis efflux genes were overexpressed in response to antibiotic exposure, in vitro and within macrophages, irrespective of their resistance pattern. These compounds displayed a rapid and high killing activity against M. tuberculosis, associated with a decrease in intracellular ATP levels demonstrating that the bactericidal action of the ion channel blockers against M. tuberculosis clinical strains is associated with their interference with energy metabolism. The compounds led to a decrease in the intracellular mycobacterial load by increasing phagosome acidification and activating lysosomal hydrolases. The results presented in this study enable us to propose the following mechanism of action for these compounds: a) in the bacteria, the compounds generate a cascade of events involving the inhibition of the respiratory chain complexes and energy production for efflux activity. Indirectly, this reduce the resistance level to antituberculosis drugs potentiating their activity; b) on the host cell, the treatment with the ion channel blockers increases phagosome acidification and induces the expression of phagosomal hydrolases, leading to bacterial growth restriction irrespective of their

  10. Ion Channel Blockers as Antimicrobial Agents, Efflux Inhibitors, and Enhancers of Macrophage Killing Activity against Drug Resistant Mycobacterium tuberculosis.

    PubMed

    Machado, Diana; Pires, David; Perdigão, João; Couto, Isabel; Portugal, Isabel; Martins, Marta; Amaral, Leonard; Anes, Elsa; Viveiros, Miguel

    2016-01-01

    Given the ability of M. tuberculosis to survive as an intracellular pathogen and its propensity to develop resistance to the existing antituberculosis drugs, its treatment requires new approaches. Here the antimycobacterial properties of verapamil, thioridazine, chlorpromazine, flupenthixol and haloperidol were investigated against a panel of drug resistant M. tuberculosis strains, both in vitro and on human-infected macrophages. These compounds are efflux inhibitors that share among them the characteristic of being ion channel blockers. In vitro, all compounds exhibited synergistic inhibitory activities when combined with isoniazid and rifampicin, and were able to inhibit active efflux, demonstrating their role as efflux inhibitors. Gene expression analysis showed that M. tuberculosis efflux genes were overexpressed in response to antibiotic exposure, in vitro and within macrophages, irrespective of their resistance pattern. These compounds displayed a rapid and high killing activity against M. tuberculosis, associated with a decrease in intracellular ATP levels demonstrating that the bactericidal action of the ion channel blockers against M. tuberculosis clinical strains is associated with their interference with energy metabolism. The compounds led to a decrease in the intracellular mycobacterial load by increasing phagosome acidification and activating lysosomal hydrolases. The results presented in this study enable us to propose the following mechanism of action for these compounds: a) in the bacteria, the compounds generate a cascade of events involving the inhibition of the respiratory chain complexes and energy production for efflux activity. Indirectly, this reduce the resistance level to antituberculosis drugs potentiating their activity; b) on the host cell, the treatment with the ion channel blockers increases phagosome acidification and induces the expression of phagosomal hydrolases, leading to bacterial growth restriction irrespective of their

  11. Optical pumping of generalized laser active materials.

    PubMed

    Fry, F H

    1967-11-01

    Results are presented of a computer-based study on the rate of excitation in the active cores of two types of optically pumped lasers as a function of a number of parameters of the active core. The absorption bands of the active materials are generated by Lorentzian and Gaussian functions. The excitation rate of the active core is proportional to the width of the absorption band at all depths of penetration. The plots of excitation rate as a function of frequency show curves similar to line reversal spectra and emphasize the importance of excitation some distance from the center of the absorption band in the slab model. In the cylindrical model, this wing pumping is even more important due to focusing. The effect of refractive index on the excitation rate is also described. PMID:20062337

  12. Rapid activation of catalase followed by citrate efflux effectively improves aluminum tolerance in the roots of chick pea (Cicer arietinum).

    PubMed

    Sharma, Manorma; Sharma, Vinay; Tripathi, Bhumi Nath

    2016-05-01

    The present study demonstrates the comparative response of two contrasting genotypes (aluminum (Al) tolerant and Al sensitive) of chick pea (Cicer arietinum) against Al stress. The Al-tolerant genotype (RSG 974) showed lesser inhibition of root growth as well as lower oxidative damages, measured in terms of the accumulation of H2O2 and lipid peroxidation compared to the Al-sensitive genotype (RSG 945). The accumulation of Al by roots of both genotypes was almost equal at 96 and 144 h after Al treatment; however, it was higher in Al-tolerant than Al-sensitive genotype at 48 h after Al treatment. Further, the Al-mediated induction of superoxide dismutase (SOD) activity was significantly higher in Al-tolerant than Al-sensitive genotype. Ascorbate peroxidase (APX) activity was almost similar in both genotypes. Al treatment promptly activated catalase activity in Al-tolerant genotype, and it was remarkably higher than that of Al-sensitive genotype. As another important Al detoxification mechanism, citrate efflux was almost equal in both genotypes except at 1000 μM Al treatment for 96 and 144 h. Further, citrate carrier and anion channel inhibitor experiment confirmed the contribution of citrate efflux in conferring Al tolerance in Al-tolerant genotype. Based on the available data, the present study concludes that rapid activation of catalase (also SOD) activity followed by citrate efflux effectively improves Al tolerance in chick pea. PMID:26615604

  13. Role of OXA-23 and AdeABC efflux pump for acquiring carbapenem resistance in an Acinetobacter baumannii strain carrying the blaOXA-66 gene.

    PubMed

    Lee, Yangsoon; Yum, Jong Hwa; Kim, Chang-Ki; Yong, Dongeun; Jeon, Eun Hee; Jeong, Seok Hoon; Ahn, Jee Young; Lee, Kyungwon

    2010-01-01

    This study was performed to determine the mechanisms for acquiring carbapenem resistance in six clinical isolates of Acinetobacter baumannii. All isolates showed similar SmaI-macrorestriction patterns with less than 3 band differences by PFGE. The isolates showed a high level resistance (>32 mg/L) to both imipenem and meropenem by Etest. Phe-Arg-beta-naphthylamide lowered the MICs of carbapenems. Real-time PCR experiments showed that expression levels of the adeB gene in the six A. baumannii isolates were 10- to 40-times higher than those of imipenem-susceptible strains. Direct sequencing of PCR products showed that all isolates carried the bla(OXA-23) gene, which was preceded by ISAba1. The bla(OXA-23) probe hybridized with approximately 500-kb I-CeuI chromosomal fragments, but not with a plasmid. These findings suggest that overexpression of the AdeABC efflux pump as well as chromosome-borne OXA-23 may play a role in acquiring carbapenem resistance in our A. baumannii isolates. PMID:20124329

  14. Structural and biochemical characterization of MepR, a multidrug binding transcription regulator of the Staphylococcus aureus multidrug efflux pump MepA

    PubMed Central

    Kumaraswami, Muthiah; Schuman, Jason T.; Seo, Susan M.; Kaatz, Glenn W.; Brennan, Richard G.

    2009-01-01

    MepR is a multidrug binding transcription regulator that represses expression of the Staphylococcus aureus multidrug efflux pump gene, mepA, as well as its own gene. MepR is induced by multiple cationic toxins, which are also substrates of MepA. In order to understand the gene regulatory and drug-binding mechanisms of MepR, we carried out biochemical, in vivo and structural studies. The 2.40 Å resolution structure of drug-free MepR reveals the most open MarR family protein conformation to date, which will require a huge conformational change to bind cognate DNA. DNA-binding data show that MepR uses a dual regulatory binding mode as the repressor binds the mepA operator as a dimer of dimers, but binds the mepR operator as a single dimer. Alignment of the six half sites reveals the consensus MepR binding site, 5'-GTTAGAT-3'. ‘Drug’ binding studies show that MepR binds to ethidium and DAPI with comparable affinities (Kd = 2.6 and 4.5 μM, respectively), but with significantly lower affinity to the larger rhodamine 6G (Kd = 62.6 μM). Mapping clinically relevant or in vitro selected MepR mutants onto the MepR structure suggests that their defective repressor phenotypes are due to structural and allosteric defects. PMID:19129225

  15. Structures of the TetR-like simocyclinone efflux pump repressor, SimR, and the mechanism of ligand-mediated derepression.

    PubMed

    Le, Tung B K; Stevenson, Clare E M; Fiedler, Hans-Peter; Maxwell, Anthony; Lawson, David M; Buttner, Mark J

    2011-04-22

    Simocyclinone D8 (SD8), a potent DNA gyrase inhibitor made by Streptomyces antibioticus, is exported from the producing organism by the SimX efflux pump. The expression of simX is under the control of SimR, a member of the TetR family of transcriptional regulators. SimR represses simX transcription by binding to operators in the intergenic region between simR and simX. Previously, we have shown that the mature antibiotic SD8 or its biosynthetic intermediate, simocyclinone C4, can dissociate SimR from its operators, leading to derepression of simX and export of SD8 from the cell. This provides a mechanism that couples the biosynthesis of the antibiotic to its export. Here, we report the crystal structures of SimR alone and in complex with either SD8 or simocyclinone C4. The ligand-binding pocket is unusual compared to those of other characterized TetR-family transcriptional regulators: the structures show an extensive ligand-binding pocket spanning both monomers in the functional dimeric unit, with the aminocoumarin moiety of SD8 buried in the protein core, while the angucyclic polyketide moiety is partially exposed to bulk solvent. Through comparisons of the structures, we postulate a derepression mechanism for SimR that invokes rigid-body motions of the subunits relative to one another, coupled with a putative locking mechanism to restrict further conformational change. PMID:21354180

  16. Both STAT3 activation and cholesterol efflux contribute to the anti-inflammatory effect of apoA-I/ABCA1 interaction in macrophages.

    PubMed

    Tang, Chongren; Houston, Barbara A; Storey, Carl; LeBoeuf, Renee C

    2016-05-01

    ABCA1 exports excess cholesterol from cells to apoA-I and is essential for HDL synthesis. Genetic studies have shown that ABCA1 protects against cardiovascular disease. We have previously shown that the interaction of apoA-I with ABCA1 activates signaling molecule Janus kinase 2 (JAK2), which optimizes the cholesterol efflux activity of ABCA1. ABCA1-mediated activation of JAK2 also activates signal transducer and activator of transcription 3 (STAT3), which significantly attenuates proinflammatory cytokine expression in macrophages. To determine the mechanisms of the anti-inflammatory effects of apoA-I/ABCA1 interaction, we identified two special ABCA1 mutants, one with normal STAT3-activating capacity but lacking cholesterol efflux ability and the other with normal cholesterol efflux ability but lacking STAT3-activating capacity. We showed that activation of STAT3 by the interaction of apoA-I/ABCA1 without cholesterol efflux could significantly decrease proinflammatory cytokine expression in macrophages. Mechanistic studies showed that the anti-inflammatory effect of the apoA-I/ABCA1/STAT3 pathway is suppressor of cytokine signaling 3 dependent. Moreover, we showed that apoA-I/ABCA1-mediated cholesterol efflux without STAT3 activation can also reduce proinflammatory cytokine expression in macrophages. These findings suggest that the interaction of apoA-I/ABCA1 activates cholesterol efflux and STAT3 branch pathways to synergistically suppress inflammation in macrophages. PMID:26989082

  17. Motesanib (AMG706), a potent multikinase inhibitor, antagonizes multidrug resistance by inhibiting the efflux activity of the ABCB1

    PubMed Central

    Wang, Yi-Jun; Kathawala, Rishil J.; Zhang, Yun-Kai; Patel, Atish; Kumar, Priyank; Shukla, Suneet; Fung, King Leung; Ambudkar, Suresh V.; Talele, Tanaji T.; Chen, Zhe-Sheng

    2014-01-01

    Cancer cells often become resistant to chemotherapy through a phenomenon known as multidrug resistance (MDR). Several factors are responsible for the development of MDR, preeminent among them being the accelerated drug efflux mediated by overexpression of ATP binding cassette (ABC) transporters. Some small molecule tyrosine kinase inhibitors (TKIs) were recently reported to modulate the activity of ABC transporters. Therefore, the purpose of this study was to determine if motesanib, a multikinase inhibitor, could reverse ABCB1-mediated MDR. The results showed that motesanib significantly sensitized both ABCB1-transfected and drug-selected cell lines overexpressing this transporter to its substrate anticancer drugs. Motesanib significantly increased the accumulation of [3H]-paclitaxel in ABCB1 overexpressing cells by blocking the efflux function of ABCB1 transporter. In contrast, no significant change in the expression levels and localization pattern of ABCB1 was observed when ABCB1 overexpressing cells were exposed to 3 µM motesanib for 72 h. Moreover, motesanib stimulated the ATPase activity of ABCB1 in a concentration-dependent manner, indicating a direct interaction with the transporter. Consistent with these findings, the docking studies indicated favorable binding of motesanib within the transmembrane region of homology modeled human ABCB1. Here, we report for the first time, motesanib, at clinically achievable plasma concentrations, antagonizes MDR by inhibiting the efflux activity of the ABCB1 transporter. These findings may be useful for cancer combination therapy with TKIs in the clinic. PMID:24937702

  18. Motesanib (AMG706), a potent multikinase inhibitor, antagonizes multidrug resistance by inhibiting the efflux activity of the ABCB1.

    PubMed

    Wang, Yi-Jun; Kathawala, Rishil J; Zhang, Yun-Kai; Patel, Atish; Kumar, Priyank; Shukla, Suneet; Fung, King Leung; Ambudkar, Suresh V; Talele, Tanaji T; Chen, Zhe-Sheng

    2014-08-15

    Cancer cells often become resistant to chemotherapy through a phenomenon known as multidrug resistance (MDR). Several factors are responsible for the development of MDR, preeminent among them being the accelerated drug efflux mediated by overexpression of ATP binding cassette (ABC) transporters. Some small molecule tyrosine kinase inhibitors (TKIs) were recently reported to modulate the activity of ABC transporters. Therefore, the purpose of this study was to determine if motesanib, a multikinase inhibitor, could reverse ABCB1-mediated MDR. The results showed that motesanib significantly sensitized both ABCB1-transfected and drug-selected cell lines overexpressing this transporter to its substrate anticancer drugs. Motesanib significantly increased the accumulation of [(3)H]-paclitaxel in ABCB1 overexpressing cells by blocking the efflux function of ABCB1 transporter. In contrast, no significant change in the expression levels and localization pattern of ABCB1 was observed when ABCB1 overexpressing cells were exposed to 3μM motesanib for 72h. Moreover, motesanib stimulated the ATPase activity of ABCB1 in a concentration-dependent manner, indicating a direct interaction with the transporter. Consistent with these findings, the docking studies indicated favorable binding of motesanib within the transmembrane region of homology modeled human ABCB1. Here, we report for the first time, motesanib, at clinically achievable plasma concentrations, antagonizes MDR by inhibiting the efflux activity of the ABCB1 transporter. These findings may be useful for cancer combination therapy with TKIs in the clinic. PMID:24937702

  19. Quercetin increases macrophage cholesterol efflux to inhibit foam cell formation through activating PPARγ-ABCA1 pathway

    PubMed Central

    Sun, Liqiang; Li, En; Wang, Feng; Wang, Tao; Qin, Zhiping; Niu, Shaohui; Qiu, Chunguang

    2015-01-01

    The accumulation of cholesterol in macrophages could induce the formation of foam cells and increase the risk of developing atherosclerosis. We wonder if quercetin, one of flavonoids with anti-inflammation functions in different cell types, could elevate the development of foam cells formation in atherosclerosis. We treated foam cells derived from oxLDL induced THP-1 cells with quercetin, and evaluated the foam cells formation, cholesterol content and apoptosis of the cells. We found that quercetin induced the expression of ABCA1 in differentiated THP-1 cells, and increased the cholesterol efflux from THP-1 cell derived foam cells. Eventually, cholesterol level and the formation of foam cell derived from THP-1 cells decreased after quercetin treatment. In addition, quercetin activated PPARγ-LXRα pathway to upregulate ABCA1 expression through increasing protein level of PPARγ and its transcriptional activity. Inhibition of PPARγ activity by siRNA knockdown or the addition of chemical inhibitor, GW9662, abolished quercetin induced ABCA1 expression and cholesterol efflux in THP-1 derived macrophages. Our data demonstrated that quercetin increased cholesterol efflux from macrophages through upregulating the expressions of PPARγ and ABCA1. Taken together, increasing uptake of quercetin or quercetin-rich foods would be an effective way to lower the risk of atherosclerosis. PMID:26617799

  20. Efflux-Mediated Drug Resistance in Bacteria: an Update

    PubMed Central

    Li, Xian-Zhi; Nikaido, Hiroshi

    2010-01-01

    Drug efflux pumps play a key role in drug resistance and also serve other functions in bacteria. There has been a growing list of multidrug and drug-specific efflux pumps characterized from bacteria of human, animal, plant and environmental origins. These pumps are mostly encoded on the chromosome although they can also be plasmid-encoded. A previous article (Li X-Z and Nikaido H, Drugs, 2004; 64[2]: 159–204) had provided a comprehensive review regarding efflux-mediated drug resistance in bacteria. In the past five years, significant progress has been achieved in further understanding of drug resistance-related efflux transporters and this review focuses on the latest studies in this field since 2003. This has been demonstrated in multiple aspects that include but are not limited to: further molecular and biochemical characterization of the known drug efflux pumps and identification of novel drug efflux pumps; structural elucidation of the transport mechanisms of drug transporters; regulatory mechanisms of drug efflux pumps; determining the role of the drug efflux pumps in other functions such as stress responses, virulence and cell communication; and development of efflux pump inhibitors. Overall, the multifaceted implications of drug efflux transporters warrant novel strategies to combat multidrug resistance in bacteria. PMID:19678712

  1. Prevalence of Genes of OXA-23 Carbapenemase and AdeABC Efflux Pump Associated with Multidrug Resistance of Acinetobacter baumannii Isolates in the ICU of a Comprehensive Hospital of Northwestern China

    PubMed Central

    Jia, Wei; Li, Caiyun; Zhang, Haiyun; Li, Gang; Liu, Xiaoming; Wei, Jun

    2015-01-01

    The objective of this study was to explore the molecular epidemiology and the genetic support of clinical multidrug resistant (MDR) Acinetobacter baumannii (A. baumannii) isolates in an ICU ward of a comprehensive hospital. A total of 102 non-duplicate drug-resistant A. baumannii isolates were identified and 93 (91.1%) of them were MDR strains. Molecular analysis demonstrated that carbapenemase genes blaOXA-23 and blaOXA-51 were presented in all 93 MDR isolates (100%), but other carbapenemase genes, including blaOXA-24, blaOXA-58, blaIMP-1, blaIMP-4, blaSIM, and blaVIM genes were completely absent in all isolates. In addition, genes of AdeABC efflux system were detected in 88.2% (90/102) isolates. Interestingly, an addition to efflux pump inhibitor, reserpine could significantly enhance the susceptibility of MDR isolates to moxifloxacin, cefotaxime, and imipenem (p < 0.01). Clonal relationship analysis further grouped these clinical drug-resistant isolates into nine clusters, and the MDR strains were mainly in clusters A, B, C, and D, which include 16, 13, 25, and 15 isolates, respectively. This study demonstrated that clinical isolates carrying carbapenemase-encoding genes blaOXA-23 and AdeABC efflux pump genes are the main prevalent MDR A. baumannii, and the co-expression of oxacillinase and efflux pump proteins are thus considered to be the important reason for the prevalence of this organism in the ICU of this hospital. PMID:26308027

  2. Metal-induced conformational changes in ZneB suggest an active role of membrane fusion proteins in efflux resistance systems.

    PubMed

    De Angelis, Fabien; Lee, John K; O'Connell, Joseph D; Miercke, Larry J W; Verschueren, Koen H; Srinivasan, Vasundara; Bauvois, Cédric; Govaerts, Cédric; Robbins, Rebecca A; Ruysschaert, Jean-Marie; Stroud, Robert M; Vandenbussche, Guy

    2010-06-15

    Resistance nodulation cell division (RND)-based efflux complexes mediate multidrug and heavy-metal resistance in many Gram-negative bacteria. Efflux of toxic compounds is driven by membrane proton/substrate antiporters (RND protein) in the plasma membrane, linked by a membrane fusion protein (MFP) to an outer-membrane protein. The three-component complex forms an efflux system that spans the entire cell envelope. The MFP is required for the assembly of this complex and is proposed to play an important active role in substrate efflux. To better understand the role of MFPs in RND-driven efflux systems, we chose ZneB, the MFP component of the ZneCAB heavy-metal efflux system from Cupriavidus metallidurans CH34. ZneB is shown to be highly specific for Zn(2+) alone. The crystal structure of ZneB to 2.8 A resolution defines the basis for metal ion binding in the coordination site at a flexible interface between the beta-barrel and membrane proximal domains. The conformational differences observed between the crystal structures of metal-bound and apo forms are monitored in solution by spectroscopy and chromatography. The structural rearrangements between the two states suggest an active role in substrate efflux through metal binding and release. PMID:20534468

  3. ACTIVE EFFLUX OF ORGANIC SOLVENTS BY PSEUDOMONAS PUTIDA S12 IS INDUCED BY SOLVENTS

    EPA Science Inventory

    Induction of the membrane-associated organic solvent efflux system SrpABC of Pseudomonas putida S12 was examined by cloning a 312-bp DNA fragment, containing the srp promoter, in the broad-host-range reporter vector pKRZ-1. Compounds that are capable of inducing expression of the...

  4. The binding of triclosan to SmeT, the repressor of the multidrug efflux pump SmeDEF, induces antibiotic resistance in Stenotrophomonas maltophilia.

    PubMed

    Hernández, Alvaro; Ruiz, Federico M; Romero, Antonio; Martínez, José L

    2011-06-01

    The wide utilization of biocides poses a concern on the impact of these compounds on natural bacterial populations. Furthermore, it has been demonstrated that biocides can select, at least in laboratory experiments, antibiotic resistant bacteria. This situation has raised concerns, not just on scientists and clinicians, but also on regulatory agencies, which are demanding studies on the impact that the utilization of biocides may have on the development on resistance and consequently on the treatment of infectious diseases and on human health. In the present article, we explored the possibility that the widely used biocide triclosan might induce antibiotic resistance using as a model the opportunistic pathogen Stenotrophomonas maltophilia. Biochemical, functional and structural studies were performed, focusing on SmeDEF, the most relevant antibiotic- and triclosan-removing multidrug efflux pump of S. maltophilia. Expression of smeDEF is regulated by the repressor SmeT. Triclosan released SmeT from its operator and induces the expression of smeDEF, thus reducing the susceptibility of S. maltophilia to antibiotics in the presence of the biocide. The structure of SmeT bound to triclosan is described. Two molecules of triclosan were found to bind to one subunit of the SmeT homodimer. The binding of the biocide stabilizes the N terminal domain of both subunits in a conformation unable to bind DNA. To our knowledge this is the first crystal structure obtained for a transcriptional regulator bound to triclosan. This work provides the molecular basis for understanding the mechanisms allowing the induction of phenotypic resistance to antibiotics by triclosan. PMID:21738470

  5. Two host-induced Ralstonia solanacearum genes, acrA and dinF, encode multidrug efflux pumps and contribute to bacterial wilt virulence.

    PubMed

    Brown, Darby G; Swanson, Jill K; Allen, Caitilyn

    2007-05-01

    Multidrug efflux pumps (MDRs) are hypothesized to protect pathogenic bacteria from toxic host defense compounds. We created mutations in the Ralstonia solanacearum acrA and dinF genes, which encode putative MDRs in the broad-host-range plant pathogen. Both mutations reduced the ability of R. solanacearum to grow in the presence of various toxic compounds, including antibiotics, phytoalexins, and detergents. Both acrAB and dinF mutants were significantly less virulent on the tomato plant than the wild-type strain. Complementation restored near-wild-type levels of virulence to both mutants. Addition of either dinF or acrAB to Escherichia coli MDR mutants KAM3 and KAM32 restored the resistance of these strains to several toxins, demonstrating that the R. solanacearum genes can function heterologously to complement known MDR mutations. Toxic and DNA-damaging compounds induced expression of acrA and dinF, as did growth in both susceptible and resistant tomato plants. Carbon limitation also increased expression of acrA and dinF, while the stress-related sigma factor RpoS was required at a high cell density (>10(7) CFU/ml) to obtain wild-type levels of acrA expression both in minimal medium and in planta. The type III secretion system regulator HrpB negatively regulated dinF expression in culture at high cell densities. Together, these results show that acrAB and dinF encode MDRs in R. solanacearum and that they contribute to the overall aggressiveness of this phytopathogen, probably by protecting the bacterium from the toxic effects of host antimicrobial compounds. PMID:17337552

  6. Co-treatment with grapefruit juice inhibits while chronic administration activates intestinal P-glycoprotein-mediated drug efflux.

    PubMed

    Panchagnula, R; Bansal, T; Varma, M V S; Kaul, C L

    2005-12-01

    P-Glycoprotein (P-gp) mediated efflux is recognized as a significant biochemical barrier affecting oral absorption for a number of drugs. Various conflicting reports have been published regarding the effects of grapefruit juice (GFJ) on P-gp-mediated drug efflux, in which GFJ has been shown both to inhibit and activate it. Hence, the present study adopted a two-way approach, involving both co-treatment and chronic administration. Bi-directional transport of paclitaxel (PCL) was carried out in the absence and presence of GFJ extract, in rat everted ileum sac. Further, the effect of chronic administration of GFJ to rats was characterized by permeability studies with indinavir (INDI). Co-treatment of GFJ extract at 100% concentration reduced the asymmetric transport of PCL (efflux ratio = 20.8) by increasing absorptive (A --> B) transport by 921% and reducing secretory (B --> A) transport by 41%. Further, GFJ showed a concentration dependent effect on PCL permeability. Imipramine, a passive permeability marker with absorptive permeability of 15.33 +/- 4.26 x 10(-6) cm/s showed no asymmetric transport and also no significant (P < 0.05) change in permeability in the presence of GFJ. Chronic administration of GFJ resulted in a significant decrease in absorptive transport of indinavir, which was even greater than that produced by rifampicin pretreatment. No change in permeability of propranolol, a passive permeability marker, was observed. Further, the decrease in absorptive transport of INDI was reversed by the P-gp inhibitor verapamil. In conclusion, GFJ extract inhibited P-gp-mediated efflux in co-treatment, whereas chronic administration led to increased levels of P-gp expression, thus having a profound effect on intestinal absorption and GFJ-drug interactions in vivo. PMID:16398269

  7. Substrate-Dependent Activation of the Vibrio cholerae vexAB RND Efflux System Requires vexR

    PubMed Central

    Taylor, Dawn L.; Ante, Vanessa M.; Bina, X. Renee; Howard, Mondraya F.; Bina, James E.

    2015-01-01

    Vibrio cholerae encodes six resistance-nodulation-division (RND) efflux systems which function in antimicrobial resistance, virulence factor production, and intestinal colonization. Among the six RND efflux systems, VexAB exhibited broad substrate specificity and played a predominant role in intrinsic antimicrobial resistance. The VexAB system was encoded in an apparent three gene operon that included vexR; which encodes an uncharacterized TetR family regulator. In this work we examined the role of vexR in vexRAB expression. We found that VexR bound to the vexRAB promoter and vexR deletion resulted in decreased vexRAB expression and increased susceptibility to VexAB antimicrobial substrates. Substrate-dependent induction of vexRAB was dependent on vexR and episomal vexR expression provided a growth advantage in the presence of the VexAB substrate deoxycholate. The expression of vexRAB increased, in a vexR-dependent manner, in response to the loss of RND efflux activity. This suggested that VexAB may function to export intracellular metabolites. Support for this hypothesis was provided by data showing that vexRAB was upregulated in several metabolic mutants including tryptophan biosynthetic mutants that were predicted to accumulate indole. In addition, vexRAB was found to be upregulated in response to exogenous indole and to contribute to indole resistance. The collective results indicate that vexR is required for vexRAB expression in response to VexAB substrates and that the VexAB RND efflux system modulates the intracellular levels of metabolites that could otherwise accumulate to toxic levels. PMID:25695834

  8. Substrate-dependent activation of the Vibrio cholerae vexAB RND efflux system requires vexR.

    PubMed

    Taylor, Dawn L; Ante, Vanessa M; Bina, X Renee; Howard, Mondraya F; Bina, James E

    2015-01-01

    Vibrio cholerae encodes six resistance-nodulation-division (RND) efflux systems which function in antimicrobial resistance, virulence factor production, and intestinal colonization. Among the six RND efflux systems, VexAB exhibited broad substrate specificity and played a predominant role in intrinsic antimicrobial resistance. The VexAB system was encoded in an apparent three gene operon that included vexR; which encodes an uncharacterized TetR family regulator. In this work we examined the role of vexR in vexRAB expression. We found that VexR bound to the vexRAB promoter and vexR deletion resulted in decreased vexRAB expression and increased susceptibility to VexAB antimicrobial substrates. Substrate-dependent induction of vexRAB was dependent on vexR and episomal vexR expression provided a growth advantage in the presence of the VexAB substrate deoxycholate. The expression of vexRAB increased, in a vexR-dependent manner, in response to the loss of RND efflux activity. This suggested that VexAB may function to export intracellular metabolites. Support for this hypothesis was provided by data showing that vexRAB was upregulated in several metabolic mutants including tryptophan biosynthetic mutants that were predicted to accumulate indole. In addition, vexRAB was found to be upregulated in response to exogenous indole and to contribute to indole resistance. The collective results indicate that vexR is required for vexRAB expression in response to VexAB substrates and that the VexAB RND efflux system modulates the intracellular levels of metabolites that could otherwise accumulate to toxic levels. PMID:25695834

  9. Fluid transport by the cornea endothelium is dependent on buffering lactic acid efflux.

    PubMed

    Li, Shimin; Kim, Edward; Bonanno, Joseph A

    2016-07-01

    Maintenance of corneal hydration is dependent on the active transport properties of the corneal endothelium. We tested the hypothesis that lactic acid efflux, facilitated by buffering, is a component of the endothelial fluid pump. Rabbit corneas were perfused with bicarbonate-rich (BR) or bicarbonate-free (BF) Ringer of varying buffering power, while corneal thickness was measured. Perfusate was collected and analyzed for lactate efflux. In BF with no added HEPES, the maximal corneal swelling rate was 30.0 ± 4.1 μm/h compared with 5.2 ± 0.9 μm/h in BR. Corneal swelling decreased directly with [HEPES], such that with 60 mM HEPES corneas swelled at 7.5 ± 1.6 μm/h. Perfusate [lactate] increased directly with [HEPES]. Similarly, reducing the [HCO3 (-)] increased corneal swelling and decreased lactate efflux. Corneal swelling was inversely related to Ringer buffering power (β), whereas lactate efflux was directly related to β. Ouabain (100 μM) produced maximal swelling and reduction in lactate efflux, whereas carbonic anhydrase inhibition and an monocarboxylic acid transporter 1 inhibitor produced intermediate swelling and decreases in lactate efflux. Conversely, 10 μM adenosine reduced the swelling rate to 4.2 ± 0.8 μm/h and increased lactate efflux by 25%. We found a strong inverse relation between corneal swelling and lactate efflux (r = 0.98, P < 0.0001). Introducing lactate in the Ringer transiently increased corneal thickness, reaching a steady state (0 ± 0.6 μm/h) within 90 min. We conclude that corneal endothelial function does not have an absolute requirement for bicarbonate; rather it requires a perfusing solution with high buffering power. This facilitates lactic acid efflux, which is directly linked to water efflux, indicating that lactate flux is a component of the corneal endothelial pump. PMID:27225657

  10. Dendritic cells phenotype fitting under hypoxia or lipopolysaccharide; adenosine 5'-triphosphate-binding cassette transporters far beyond an efflux pump.

    PubMed

    Lloberas, N; Rama, I; Llaudó, I; Torras, J; Cerezo, G; Cassis, L; Franquesa, M; Merino, A; Benitez-Ribas, D; Cruzado, J M; Herrero-Fresneda, I; Bestard, O; Grinyó, J M

    2013-06-01

    -transporters emerge as potential targets in immunosuppressive therapies interfering with DCs maturation, thereby abrogating innate immune response when it is activated after ischaemia. PMID:23600833

  11. The Heterodimeric ABC Transporter EfrCD Mediates Multidrug Efflux in Enterococcus faecalis

    PubMed Central

    Hürlimann, Lea M.; Corradi, Valentina; Hohl, Michael; Bloemberg, Guido V.; Tieleman, D. Peter

    2016-01-01

    Nosocomial infections with Enterococcus faecalis are an emerging health problem. However, drug efflux pumps contributing to intrinsic drug resistance are poorly studied in this Gram-positive pathogen. In this study, we functionally investigated seven heterodimeric ABC transporters of E. faecalis that are annotated as drug efflux pumps. Deletion of ef0789-ef0790 on the chromosome of E. faecalis resulted in increased susceptibility to daunorubicin, doxorubicin, ethidium, and Hoechst 33342, and the corresponding transporter was named EfrCD. Unexpectedly, the previously described heterodimeric multidrug ABC transporter EfrAB contributes marginally to drug efflux in the endogenous context of E. faecalis. In contrast, heterologous expression in Lactococcus lactis revealed that EfrAB, EfrCD, and the product of ef2226-ef2227 (EfrEF) mediate the efflux of fluorescent substrates and confer resistance to multiple dyes and drugs, including fluoroquinolones. Four of seven transporters failed to exhibit drug efflux activity for the set of drugs and dyes tested, even upon overexpression in L. lactis. Since all seven transporters were purified as heterodimers after overexpression in L. lactis, a lack of drug efflux activity is not attributed to poor expression or protein aggregation. Reconstitution of the purified multidrug transporters EfrAB, EfrCD, and EfrEF in proteoliposomes revealed functional coupling between ATP hydrolysis and drug binding. Our analysis creates an experimental basis for the accurate prediction of drug efflux transporters and indicates that many annotated multidrug efflux pumps might be incapable of drug transport and thus might fulfill other physiological functions in the cell. PMID:27381387

  12. The Heterodimeric ABC Transporter EfrCD Mediates Multidrug Efflux in Enterococcus faecalis.

    PubMed

    Hürlimann, Lea M; Corradi, Valentina; Hohl, Michael; Bloemberg, Guido V; Tieleman, D Peter; Seeger, Markus A

    2016-09-01

    Nosocomial infections with Enterococcus faecalis are an emerging health problem. However, drug efflux pumps contributing to intrinsic drug resistance are poorly studied in this Gram-positive pathogen. In this study, we functionally investigated seven heterodimeric ABC transporters of E. faecalis that are annotated as drug efflux pumps. Deletion of ef0789-ef0790 on the chromosome of E. faecalis resulted in increased susceptibility to daunorubicin, doxorubicin, ethidium, and Hoechst 33342, and the corresponding transporter was named EfrCD. Unexpectedly, the previously described heterodimeric multidrug ABC transporter EfrAB contributes marginally to drug efflux in the endogenous context of E. faecalis In contrast, heterologous expression in Lactococcus lactis revealed that EfrAB, EfrCD, and the product of ef2226-ef2227 (EfrEF) mediate the efflux of fluorescent substrates and confer resistance to multiple dyes and drugs, including fluoroquinolones. Four of seven transporters failed to exhibit drug efflux activity for the set of drugs and dyes tested, even upon overexpression in L. lactis Since all seven transporters were purified as heterodimers after overexpression in L. lactis, a lack of drug efflux activity is not attributed to poor expression or protein aggregation. Reconstitution of the purified multidrug transporters EfrAB, EfrCD, and EfrEF in proteoliposomes revealed functional coupling between ATP hydrolysis and drug binding. Our analysis creates an experimental basis for the accurate prediction of drug efflux transporters and indicates that many annotated multidrug efflux pumps might be incapable of drug transport and thus might fulfill other physiological functions in the cell. PMID:27381387

  13. Ventral Tegmental Area Neurotensin Signaling Links the Lateral Hypothalamus to Locomotor Activity and Striatal Dopamine Efflux in Male Mice

    PubMed Central

    Patterson, Christa M.; Wong, Jenny-Marie T.; Leinninger, Gina M.; Allison, Margaret B.; Mabrouk, Omar S.; Kasper, Chelsea L.; Gonzalez, Ian E.; Mackenzie, Alexander; Jones, Justin C.

    2015-01-01

    Projections from the lateral hypothalamic area (LHA) innervate components of the mesolimbic dopamine (MLDA) system, including the ventral tegmental area (VTA) and nucleus accumbens (NAc), to modulate motivation appropriately for physiologic state. Neurotensin (NT)-containing LHA neurons respond to multiple homeostatic challenges and project to the VTA, suggesting that these neurons could link such signals to MLDA function. Indeed, we found that pharmacogenetic activation of LHA NT neurons promoted prolonged DA-dependent locomotor activity and NAc DA efflux, suggesting the importance of VTA neurotransmitter release by LHA NT neurons for the control of MLDA function. Using a microdialysis-mass spectrometry technique that we developed to detect endogenous NT in extracellular fluid in the mouse brain, we found that activation of LHA NT cells acutely increased the extracellular concentration of NT (a known activator of VTA DA cells) in the VTA. In contrast to the prolonged elevation of extracellular NAc DA, however, VTA NT concentrations rapidly returned to baseline. Intra-VTA infusion of NT receptor antagonist abrogated the ability of LHA NT cells to increase extracellular DA in the NAc, demonstrating that VTA NT promotes NAc DA release. Thus, transient LHA-derived NT release in the VTA couples LHA signaling to prolonged changes in DA efflux and MLDA function. PMID:25734363

  14. V-ATPase Proton Pumping Activity Is Required for Adult Zebrafish Appendage Regeneration

    PubMed Central

    Monteiro, Joana; Aires, Rita; Becker, Jörg D.; Jacinto, António; Certal, Ana C.; Rodríguez-León, Joaquín

    2014-01-01

    The activity of ion channels and transporters generates ion-specific fluxes that encode electrical and/or chemical signals with biological significance. Even though it is long known that some of those signals are crucial for regeneration, only in recent years the corresponding molecular sources started to be identified using mainly invertebrate or larval vertebrate models. We used adult zebrafish caudal fin as a model to investigate which and how ion transporters affect regeneration in an adult vertebrate model. Through the combined use of biophysical and molecular approaches, we show that V-ATPase activity contributes to a regeneration-specific H+ ef`flux. The onset and intensity of both V-ATPase expression and H+ efflux correlate with the different regeneration rate along the proximal-distal axis. Moreover, we show that V-ATPase inhibition impairs regeneration in adult vertebrate. Notably, the activity of this H+ pump is necessary for aldh1a2 and mkp3 expression, blastema cell proliferation and fin innervation. To the best of our knowledge, this is the first report on the role of V-ATPase during adult vertebrate regeneration. PMID:24671205

  15. SLC30A10 Is a Cell Surface-Localized Manganese Efflux Transporter, and Parkinsonism-Causing Mutations Block Its Intracellular Trafficking and Efflux Activity

    PubMed Central

    Leyva-Illades, Dinorah; Chen, Pan; Zogzas, Charles E.; Hutchens, Steven; Mercado, Jonathan M.; Swaim, Caleb D.; Morrisett, Richard A.; Bowman, Aaron B.

    2014-01-01

    Manganese (Mn) is an essential metal, but elevated cellular levels are toxic and may lead to the development of an irreversible parkinsonian-like syndrome that has no treatment. Mn-induced parkinsonism generally occurs as a result of exposure to elevated Mn levels in occupational or environmental settings. Additionally, patients with compromised liver function attributable to diseases, such as cirrhosis, fail to excrete Mn and may develop Mn-induced parkinsonism in the absence of exposure to elevated Mn. Recently, a new form of familial parkinsonism was reported to occur as a result of mutations in SLC30A10. The cellular function of SLC30A10 and the mechanisms by which mutations in this protein cause parkinsonism are unclear. Here, using a combination of mechanistic and functional studies in cell culture, Caenorhabditis elegans, and primary midbrain neurons, we show that SLC30A10 is a cell surface-localized Mn efflux transporter that reduces cellular Mn levels and protects against Mn-induced toxicity. Importantly, mutations in SLC30A10 that cause familial parkinsonism blocked the ability of the transporter to traffic to the cell surface and to mediate Mn efflux. Although expression of disease-causing SLC30A10 mutations were not deleterious by themselves, neurons and worms expressing these mutants exhibited enhanced sensitivity to Mn toxicity. Our results provide novel insights into the mechanisms involved in the onset of a familial form of parkinsonism and highlight the possibility of using enhanced Mn efflux as a therapeutic strategy for the potential management of Mn-induced parkinsonism, including that occurring as a result of mutations in SLC30A10. PMID:25319704

  16. Accelerometer based calf muscle pump activity monitoring.

    PubMed

    O'Donovan, Karol J; O'Keeffe, Derek T; Grace, Pierce A; Lyons, Gerard M

    2005-10-01

    Long distance travel is associated with increased risk of deep vein thrombosis (DVT). There is an increased risk of travel related DVT in passengers with a predisposition to thrombosis. Assisting blood circulation in the lower limb will reduce the risk of DVT. Leg exercises are recommended as a DVT preventative measure while flying but this fails to account for a passenger who is distracted by in flight entertainment or who falls asleep for an extended period. A method for monitoring calf muscle pump activity using accelerometers has been developed and evaluated. The proposed technique could be used to alert the traveller that there is a need to exercise their calf muscle, thus reducing the risk of DVT. PMID:16139770

  17. Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID

    PubMed Central

    Zourelidou, Melina; Absmanner, Birgit; Weller, Benjamin; Barbosa, Inês CR; Willige, Björn C; Fastner, Astrid; Streit, Verena; Port, Sarah A; Colcombet, Jean; de la Fuente van Bentem, Sergio; Hirt, Heribert; Kuster, Bernhard; Schulze, Waltraud X; Hammes, Ulrich Z; Schwechheimer, Claus

    2014-01-01

    The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the—in many cells—asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant. DOI: http://dx.doi.org/10.7554/eLife.02860.001 PMID:24948515

  18. Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID.

    PubMed

    Zourelidou, Melina; Absmanner, Birgit; Weller, Benjamin; Barbosa, Inês C R; Willige, Björn C; Fastner, Astrid; Streit, Verena; Port, Sarah A; Colcombet, Jean; de la Fuente van Bentem, Sergio; Hirt, Heribert; Kuster, Bernhard; Schulze, Waltraud X; Hammes, Ulrich Z; Schwechheimer, Claus

    2014-01-01

    The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the--in many cells--asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant. PMID:24948515

  19. Isolation of Escherichia coli Strains with AcrAB–TolC Efflux Pump-Associated Intermediate Interpretation or Resistance to Fluoroquinolone, Chloramphenicol and Aminopenicillin from Dogs Admitted to a University Veterinary Hospital

    PubMed Central

    SATO, Toyotaka; YOKOTA, Shin-ichi; ICHIHASHI, Risa; MIYAUCHI, Tomoka; OKUBO, Torahiko; USUI, Masaru; FUJII, Nobuhiro; TAMURA, Yutaka

    2014-01-01

    ABSTRACT Understanding the prevalence of antimicrobial-resistance and the relationship between emergence of resistant bacteria and clinical treatment can facilitate design of effective treatment strategies. We here examined antimicrobial susceptibilities of Escherichia coli isolated from dogs admitted to a university hospital (University hospital) and companion animal clinics (Community clinics) in the same city and investigated underlying multidrug-resistance mechanisms. The prevalence of E. coli with intermediate and resistant interpretations to ampicillin (AMP), enrofloxacin (ENR) and chloramphenicol (CHL) was higher in the University hospital than in the Community clinics cases. Use of antimicrobials, including fluoroquinolone, was also significantly higher in the University hospital than in the Community clinics cases. Upon isolation using ENR-supplemented agar plates, all ENR-resistant isolates had 3–4 nucleotide mutations that accompanied by amino acid substitutions in the quinolone-resistance-determining regions of gyrA, parC and parE, and 94.7% of all isolates derived from the University hospital showed AMP and/or CHL resistance and possessed blaTEM and/or catA1. The average mRNA expression levels of acrA, acrB and tolC and the prevalence of organic solvent tolerance, in isolates derived from ENR-supplemented agar plates were significantly higher in the University hospital than in the Community clinics isolates. Thus, E. coli derived from the University hospital cases more often showed concomitant decreased susceptibilities to aminopenicillins, fluoroquinolones and CHL than did those derived from the Community clinics; this was related to an active AcrAB–TolC efflux pump, in addition to acquisition of specific resistance genes and genetic mutations. PMID:24646457

  20. Expression of the RND-type efflux pump AdeABC in Acinetobacter baumannii is regulated by the AdeRS two-component system.

    PubMed

    Marchand, Isabelle; Damier-Piolle, Laurence; Courvalin, Patrice; Lambert, Thierry

    2004-09-01

    The AdeABC pump of Acinetobacter baumannii BM4454, which confers resistance to various antibiotic classes including aminoglycosides, is composed of the AdeA, AdeB, and AdeC proteins; AdeB is a member of the RND superfamily. The adeA, adeB, and adeC genes are contiguous and adjacent to adeS and adeR, which are transcribed in the opposite direction and which specify proteins homologous to sensors and regulators of two-component systems, respectively (S. Magnet, P. Courvalin, and T. Lambert, Antimicrob. Agents Chemother. 45:3375-3380, 2001). Analysis by Northern hybridization indicated that the three genes were cotranscribed, although mRNAs corresponding to adeAB and adeC were also present. Cotranscription of the two regulatory genes was demonstrated by reverse transcription-PCR. Inactivation of adeS led to aminoglycoside susceptibility. Transcripts corresponding to adeAB were not detected in susceptible A. baumannii CIP 70-10 but were present in spontaneous gentamicin-resistant mutants obtained in vitro. Analysis of these mutants revealed the substitutions Thr153-->Met in AdeS downstream from the putative His-149 site of autophosphorylation, which is presumably responsible for the loss of phosphorylase activity by the sensor, and Pro116-->Leu in AdeR at the first residue of the alpha(5) helix of the receiver domain, which is involved in interactions that control the output domain of response regulators. These mutations led to constitutive expression of the pump and, thus, to antibiotic resistance. These data indicate that the AdeABC pump is cryptic in wild A. baumannii due to stringent control by the AdeRS two-component system. PMID:15328088

  1. Expression of the RND-Type Efflux Pump AdeABC in Acinetobacter baumannii Is Regulated by the AdeRS Two-Component System

    PubMed Central

    Marchand, Isabelle; Damier-Piolle, Laurence; Courvalin, Patrice; Lambert, Thierry

    2004-01-01

    The AdeABC pump of Acinetobacter baumannii BM4454, which confers resistance to various antibiotic classes including aminoglycosides, is composed of the AdeA, AdeB, and AdeC proteins; AdeB is a member of the RND superfamily. The adeA, adeB, and adeC genes are contiguous and adjacent to adeS and adeR, which are transcribed in the opposite direction and which specify proteins homologous to sensors and regulators of two-component systems, respectively (S. Magnet, P. Courvalin, and T. Lambert, Antimicrob. Agents Chemother. 45:3375-3380, 2001). Analysis by Northern hybridization indicated that the three genes were cotranscribed, although mRNAs corresponding to adeAB and adeC were also present. Cotranscription of the two regulatory genes was demonstrated by reverse transcription-PCR. Inactivation of adeS led to aminoglycoside susceptibility. Transcripts corresponding to adeAB were not detected in susceptible A. baumannii CIP 70-10 but were present in spontaneous gentamicin-resistant mutants obtained in vitro. Analysis of these mutants revealed the substitutions Thr153→Met in AdeS downstream from the putative His-149 site of autophosphorylation, which is presumably responsible for the loss of phosphorylase activity by the sensor, and Pro116→Leu in AdeR at the first residue of the α5 helix of the receiver domain, which is involved in interactions that control the output domain of response regulators. These mutations led to constitutive expression of the pump and, thus, to antibiotic resistance. These data indicate that the AdeABC pump is cryptic in wild A. baumannii due to stringent control by the AdeRS two-component system. PMID:15328088

  2. Antitumor activity of 7-aminocarboxycoumarin derivatives, a new class of potent inhibitors of lactate influx but not efflux.

    PubMed

    Draoui, Nihed; Schicke, Olivier; Seront, Emmanuel; Bouzin, Caroline; Sonveaux, Pierre; Riant, Olivier; Feron, Olivier

    2014-06-01

    High lactate concentration in tumors is associated with bad prognosis. Lactate is released by glycolytic cells in tumors and recaptured by oxidative cancer cells to feed the tricarboxylic acid (TCA) cycle after conversion into pyruvate. Monocarboxylate transporters (MCT) mediate these fluxes of proton-linked lactate and represent attractive targets to interrupt lactate shuttle and to inhibit tumor growth. Here, we investigated the properties of 7-aminocarboxycoumarins (7ACC) developed to selectively interfere with lactate fluxes in the lactate-rich tumor microenvironment. The pharmacologic properties of two compounds of this family, including their effects on lactate influx and efflux and antitumor activity, were investigated using human cancer cell lines and mouse xenograft models. Contrary to the reference MCT1 inhibitor AR-C155858, 7ACC unexpectedly inhibited lactate influx but not efflux in tumor cells expressing MCT1 and MCT4 transporters. 7ACC delayed the growth of cervix SiHa tumors, colorectal HCT116 tumors, and orthoptopic MCF-7 breast tumors. MCT target engagement was confirmed by the lack of activity of 7ACC on bladder UM-UC-3 carcinoma that does not express functional MCT. 7ACC also inhibited SiHa tumor relapse after treatment with cisplatin. Finally, we found that contrary to AR-C155858, 7ACC did not prevent the cell entry of the substrate-mimetic drug 3-bromopyruvate (3BP) through MCT1, and contributed to the inhibition of tumor relapse after 3BP treatment. In conclusion, our results indicate that 7ACC selectively affects a single part of the MCT symporter translocation cycle, leading to strict inhibition of lactate influx. This singular activity is associated with antitumor effects less prone to resistance and side effects. PMID:24672058

  3. Inhibition by islet-activating protein, pertussis toxin, of P2-purinergic receptor-mediated iodide efflux and phosphoinositide turnover in FRTL-5 cells

    SciTech Connect

    Okajima, F.; Sho, K.; Kondo, Y.

    1988-08-01

    Exposure of FRTL-5 thyroid cells to ATP (1 microM to 1 mM) resulted in the stimulation of I- efflux in association with the induction of inositol trisphosphate production and intracellular Ca2+ mobilization. Nonhydrolyzable ATP derivatives, ADP and GTP, were also as effective in magnitude as ATP, whereas neither AMP nor adenosine exerted significant effect on I- efflux, suggesting a P2-purinergic receptor-mediated activation of I- efflux. Treatment of the cells with the islet-activating protein (IAP) pertussis toxin, which ADP-ribosylated a 41,000 mol wt membrane protein, effectively suppressed the phosphoinositide response to ATP in addition to ATP-dependent I- efflux at agonist concentrations below 10 microM. In contrast, the I- efflux stimulated by TSH, A23187, or phorbol myristate acetate was insusceptible to IAP. The IAP substrate, probably GTP-binding protein, is hence proposed to mediate the activation of P2-purinergic receptor-linked phospholipase-C in FRTL-5 cells. However, the responses to ATP, its nonhydrolyzable derivatives, or ADP at the higher agonist concentrations, especially above 100 microM, were only partially inhibited by IAP, even though the IAP substrate was totally ADP ribosylated by the toxin. The responses to GTP in the whole concentration range tested were not influenced by IAP treatment. Thus, signals arising from the P2-receptor might be transduced to phospholipase-C by two different pathways, i.e. IAP-sensitive and insensitive ones, and result in the stimulation of I- efflux.

  4. Methane efflux from marine sediments in passive and active margins: Estimations from bioenergetic reaction-transport simulations

    NASA Astrophysics Data System (ADS)

    Dale, A. W.; Van Cappellen, P.; Aguilera, D. R.; Regnier, P.

    2008-01-01

    A simplified version of a kinetic-bioenergetic reaction model for anaerobic oxidation of methane (AOM) in marine sediments [Dale, A.W., Regnier, P., Van Cappellen, P., 2006. Bioenergetic controls on anaerobic oxidation of methane (AOM) in coastal marine sediments: a theoretical analysis. Am. J. Sci. 306, 246-294.] is used to assess the impact of transport processes on biomass distributions, AOM rates and methane release fluxes from the sea floor. The model explicitly represents the functional microbial groups and the kinetic and bioenergetic limitations of the microbial metabolic pathways involved in AOM. Model simulations illustrate the dominant control exerted by the transport regime on the activity and abundance of AOM communities. Upward fluid flow at active seep systems restricts AOM to a narrow subsurface reaction zone and sustains high rates of methane oxidation. In contrast, pore-water transport dominated by molecular diffusion leads to deeper and broader zones of AOM, characterized by much lower rates and biomasses. Under steady-state conditions, less than 1% of the upward dissolved methane flux reaches the water column, irrespective of the transport regime. However, a sudden increase in the advective flux of dissolved methane, for example as a result of the destabilization of methane hydrates, causes a transient efflux of methane from the sediment. The benthic efflux of dissolved methane is due to the slow growth kinetics of the AOM community and lasts on the order of 60 years. This time window is likely too short to allow for a significant escape of pore-water methane following a large scale gas hydrate dissolution event such as the one that may have accompanied the Paleocene/Eocene Thermal Maximum (PETM).

  5. Dissociation between the induction of potassium efflux and cytostatic activity of polyene macrolides in mammalian cells.

    PubMed Central

    Malewicz, B; Jenkin, H M; Borowski, E

    1980-01-01

    The paper contains data on the induction of K+ efflux and viability of baby hamster kidney (BHK-21) cells after their treatment with macrolide antibiotics inducing specific pores in membrane. New water-soluble semisynthetic derivatives of amphotericin B and aureofacin (N-glycosyl and trimethylammonium methyl ester derivatives) as well as the parent compounds were used to compare the concentration of antibiotics inducing permeabilizing and cytostatic effects. We found that a two- to eight-times-higher concentration of polyene antibiotic was required to observe a cytostatic effect than for release of 50% of the cellular potassium (K50 concentration) from BHK-21 cells. These differences were larger for water-soluble derivatives than for the parent compounds. The amount of intracellular potassium in treated cells incubated under optimal growth conditions was higher than in cells which had been further washed with K+-free maintenance medium. The membrane permeability changes induced by low concentrations of specific polyenes were observed to be reversible. BHK-21 cells were able to repair polyene-induced membrane permeability within 3 to 12 h under optimal growth conditions, after cell treatment with K50 concentration of specific macrolide antibiotics. The repair phenomenon is postulated as an explanation for the dissociation observed between permeabilizing and cytostatic effect of specific polyenes in BHK-21 cells. PMID:7396461

  6. Possibilities of increasing the pumping efficiency of solid active medium laser generators by optimizing the pumping cavity profile

    NASA Astrophysics Data System (ADS)

    Dontu, O.; Ganatsios, S.; Alexandrescu, N.

    2008-03-01

    The paper presents some design elements concerning the optical pumping cavities of the laser generators with active solid medium, as well as the way of increasing their performance. We start from the fact that the laser cavity is a closed optical system, where the active laser medium and the pumping source are conjugated, in order to achieve a maximum concentration of the light flux of the pumping source towards the active medium. We discuss the simple elliptical section laser pumping cavities (with one pumping lamp) and triple elliptical (with three lamps), also presenting a series on calculus nomograms, very useful to those who design the laser generation optical pumping cavities.

  7. Intra-annual dynamics of stem CO2 efflux in relation to cambial activity and xylem development in Pinus cembra

    PubMed Central

    GRUBER, A.; WIESER, G.; OBERHUBER, W.

    2010-01-01

    Summary The relationship between stem CO2 efflux (ES), cambial activity and xylem production in Pinus cembra was determined at the timberline (1950 m a.s.l.) of the Central Austrian Alps, throughout one year. ES was measured continuously from June 2006 to August 2007 using an infrared gas-analysis system. Cambial activity and xylem production was determined by repeated microcore sampling of the developing tree ring and radial increment was monitored using automated point dendrometers. Aside of temperature, the number of living tracheids and cambial cells was predominantly responsible for ES: ES normalized to 10°C (ES10) was significantly correlated to number of living cells throughout the year (r2 = 0,574; p < 0,001). However, elevated ES and missing correlation between ES10 and xylem production was detected during cambial reactivation in April and during transition from active phase to rest, which occurred in August and lasted until early September. Results of this study indicate that (i) during seasonal variations in cambial activity non-linearity between ES and xylem production occurs and (ii) elevated metabolic activity during transition stages in the cambial activity-dormancy cycle influence the carbon budget of Pinus cembra. Daily radial stem increment was primarily influenced by the number of enlarging cells and was not correlated to ES. PMID:19203979

  8. Robust passive and active efflux of cellular cholesterol to a designer functional mimic of high density lipoprotein

    PubMed Central

    Luthi, Andrea J.; Lyssenko, Nicholas N.; Quach, Duyen; McMahon, Kaylin M.; Millar, John S.; Vickers, Kasey C.; Rader, Daniel J.; Phillips, Michael C.; Mirkin, Chad A.; Thaxton, C. Shad

    2015-01-01

    The ability of HDL to support macrophage cholesterol efflux is an integral part of its atheroprotective action. Augmenting this ability, especially when HDL cholesterol efflux capacity from macrophages is poor, represents a promising therapeutic strategy. One approach to enhancing macrophage cholesterol efflux is infusing blood with HDL mimics. Previously, we reported the synthesis of a functional mimic of HDL (fmHDL) that consists of a gold nanoparticle template, a phospholipid bilayer, and apo A-I. In this work, we characterize the ability of fmHDL to support the well-established pathways of cellular cholesterol efflux from model cell lines and primary macrophages. fmHDL received cell cholesterol by unmediated (aqueous) and ABCG1- and scavenger receptor class B type I (SR-BI)-mediated diffusion. Furthermore, the fmHDL holoparticle accepted cholesterol and phospholipid by the ABCA1 pathway. These results demonstrate that fmHDL supports all the cholesterol efflux pathways available to native HDL and thus, represents a promising infusible therapeutic for enhancing macrophage cholesterol efflux. fmHDL accepts cholesterol from cells by all known pathways of cholesterol efflux: unmediated, ABCG1- and SR-BI-mediated diffusion, and through ABCA1. PMID:25652088

  9. Robust passive and active efflux of cellular cholesterol to a designer functional mimic of high density lipoprotein.

    PubMed

    Luthi, Andrea J; Lyssenko, Nicholas N; Quach, Duyen; McMahon, Kaylin M; Millar, John S; Vickers, Kasey C; Rader, Daniel J; Phillips, Michael C; Mirkin, Chad A; Thaxton, C Shad

    2015-05-01

    The ability of HDL to support macrophage cholesterol efflux is an integral part of its atheroprotective action. Augmenting this ability, especially when HDL cholesterol efflux capacity from macrophages is poor, represents a promising therapeutic strategy. One approach to enhancing macrophage cholesterol efflux is infusing blood with HDL mimics. Previously, we reported the synthesis of a functional mimic of HDL (fmHDL) that consists of a gold nanoparticle template, a phospholipid bilayer, and apo A-I. In this work, we characterize the ability of fmHDL to support the well-established pathways of cellular cholesterol efflux from model cell lines and primary macrophages. fmHDL received cell cholesterol by unmediated (aqueous) and ABCG1- and scavenger receptor class B type I (SR-BI)-mediated diffusion. Furthermore, the fmHDL holoparticle accepted cholesterol and phospholipid by the ABCA1 pathway. These results demonstrate that fmHDL supports all the cholesterol efflux pathways available to native HDL and thus, represents a promising infusible therapeutic for enhancing macrophage cholesterol efflux. fmHDL accepts cholesterol from cells by all known pathways of cholesterol efflux: unmediated, ABCG1- and SR-BI-mediated diffusion, and through ABCA1. PMID:25652088

  10. The α-barrel tip region of Escherichia coli TolC homologs of Vibrio vulnificus interacts with the MacA protein to form the functional macrolide-specific efflux pump MacAB-TolC.

    PubMed

    Lee, Minho; Kim, Hyun-Lee; Song, Saemee; Joo, Minju; Lee, Seunghwa; Kim, Daeyoung; Hahn, Yoonsoo; Ha, Nam-Chul; Lee, Kangseok

    2013-04-01

    TolC and its homologous family of proteins are outer membrane factors that are essential for exporting small molecules and toxins across the outer membrane in Gram-negative bacteria. Two open reading frames in the Vibrio vulnificus genome that encode proteins homologous to Escherichia coli TolC, designated TolCV1 and TolCV2, have 51.3% and 29.6% amino acid identity to TolC, respectively. In this study, we show that TolCV1 and TolCV2 functionally and physically interacted with the membrane fusion protein, MacA, a component of the macrolide-specific MacAB-TolC pump of E. coli. We further show that the conserved residues located at the aperture tip region of the α-hairpin of TolCV1 and TolCV2 played an essential role in the formation of the functional MacAB-TolC pump using site-directed mutational analyses. Our findings suggest that these outer membrane factors have conserved tip-to-tip interaction with the MacA membrane fusion protein for action of the drug efflux pump in Gram-negative bacteria. PMID:23625214

  11. United States Department of Energy Thermally Activated Heat Pump Program

    SciTech Connect

    Fiskum, R.J.; Adcock, P.W.; DeVault, R.C.

    1996-06-01

    The US Department of Energy (DOE) is working with partners from the gas heating and cooling industry to improve energy efficiency using advance absorption technologies, to eliminate chlorofluorocarbons (CFCs) and hydrochlorofluorocarbons (HCFCs), to reduce global warming through more efficient combustion of natural gas, and to impact electric peak demand of air conditioning. To assist industry in developing these gas heating and cooling absorption technologies, the US DOE sponsors the Thermally Activated Heat Pump Program. It is divided into five key activities, addressing residential gas absorption heat pumps, large commercial chillers, advanced absorption fluids, computer-aided design, and advanced ``Hi-Cool`` heat pumps.

  12. Homologs of the Acinetobacter baumannii AceI Transporter Represent a New Family of Bacterial Multidrug Efflux Systems

    PubMed Central

    Liu, Qi; Henderson, Peter J. F.

    2015-01-01

    ABSTRACT Multidrug efflux systems are a major cause of resistance to antimicrobials in bacteria, including those pathogenic to humans, animals, and plants. These proteins are ubiquitous in these pathogens, and five families of bacterial multidrug efflux systems have been identified to date. By using transcriptomic and biochemical analyses, we recently identified the novel AceI (Acinetobacter chlorhexidine efflux) protein from Acinetobacter baumannii that conferred resistance to the biocide chlorhexidine, via an active efflux mechanism. Proteins homologous to AceI are encoded in the genomes of many other bacterial species and are particularly prominent within proteobacterial lineages. In this study, we expressed 23 homologs of AceI and examined their resistance and/or transport profiles. MIC analyses demonstrated that, like AceI, many of the homologs conferred resistance to chlorhexidine. Many of the AceI homologs conferred resistance to additional biocides, including benzalkonium, dequalinium, proflavine, and acriflavine. We conducted fluorimetric transport assays using the AceI homolog from Vibrio parahaemolyticus and confirmed that resistance to both proflavine and acriflavine was mediated by an active efflux mechanism. These results show that this group of AceI homologs represent a new family of bacterial multidrug efflux pumps, which we have designated the proteobacterial antimicrobial compound efflux (PACE) family of transport proteins. PMID:25670776

  13. Efflux as a glutaraldehyde resistance mechanism in Pseudomonas fluorescens and Pseudomonas aeruginosa biofilms.

    PubMed

    Vikram, Amit; Bomberger, Jennifer M; Bibby, Kyle J

    2015-01-01

    A major challenge in microbial biofilm control is biocide resistance. Phenotypic adaptations and physical protective effects have been historically thought to be the primary mechanisms for glutaraldehyde resistance in bacterial biofilms. Recent studies indicate the presence of genetic mechanisms for glutaraldehyde resistance, but very little is known about the contributory genetic factors. Here, we demonstrate that efflux pumps contribute to glutaraldehyde resistance in Pseudomonas fluorescens and Pseudomonas aeruginosa biofilms. The RNA-seq data show that efflux pumps and phosphonate degradation, lipid biosynthesis, and polyamine biosynthesis metabolic pathways were induced upon glutaraldehyde exposure. Furthermore, chemical inhibition of efflux pumps potentiates glutaraldehyde activity, suggesting that efflux activity contributes to glutaraldehyde resistance. Additionally, induction of known modulators of biofilm formation, including phosphonate degradation, lipid biosynthesis, and polyamine biosynthesis, may contribute to biofilm resistance and resilience. Fundamental understanding of the genetic mechanism of biocide resistance is critical for the optimization of biocide use and development of novel disinfection strategies. Our results reveal genetic components involved in glutaraldehyde resistance and a potential strategy for improved control of biofilms. PMID:25824217

  14. Efflux as a Glutaraldehyde Resistance Mechanism in Pseudomonas fluorescens and Pseudomonas aeruginosa Biofilms

    PubMed Central

    Vikram, Amit; Bomberger, Jennifer M.

    2015-01-01

    A major challenge in microbial biofilm control is biocide resistance. Phenotypic adaptations and physical protective effects have been historically thought to be the primary mechanisms for glutaraldehyde resistance in bacterial biofilms. Recent studies indicate the presence of genetic mechanisms for glutaraldehyde resistance, but very little is known about the contributory genetic factors. Here, we demonstrate that efflux pumps contribute to glutaraldehyde resistance in Pseudomonas fluorescens and Pseudomonas aeruginosa biofilms. The RNA-seq data show that efflux pumps and phosphonate degradation, lipid biosynthesis, and polyamine biosynthesis metabolic pathways were induced upon glutaraldehyde exposure. Furthermore, chemical inhibition of efflux pumps potentiates glutaraldehyde activity, suggesting that efflux activity contributes to glutaraldehyde resistance. Additionally, induction of known modulators of biofilm formation, including phosphonate degradation, lipid biosynthesis, and polyamine biosynthesis, may contribute to biofilm resistance and resilience. Fundamental understanding of the genetic mechanism of biocide resistance is critical for the optimization of biocide use and development of novel disinfection strategies. Our results reveal genetic components involved in glutaraldehyde resistance and a potential strategy for improved control of biofilms. PMID:25824217

  15. AcrS/EnvR Represses Expression of the acrAB Multidrug Efflux Genes in Escherichia coli▿

    PubMed Central

    Hirakawa, Hidetada; Takumi-Kobayashi, Asuka; Theisen, Ulrike; Hirata, Takahiro; Nishino, Kunihiko; Yamaguchi, Akihito

    2008-01-01

    The acrS regulatory gene is located upstream of the acrEF multidrug efflux system genes. However, the roles of AcrS in regulation of drug efflux pumps have not been clearly understood. Here we show that AcrS represses other multidrug efflux genes, acrAB, which encode a major efflux system in Escherichia coli. PMID:18567659

  16. Tigecycline Efflux as a Mechanism for Nonsusceptibility in Acinetobacter baumannii.

    PubMed

    Peleg, Anton Y; Adams, Jennifer; Paterson, David L

    2007-06-01

    Tigecycline has an extended spectrum of in vitro antimicrobial activities, including that against multidrug-resistant Acinetobacter. After identifying bloodstream isolates of Acinetobacter with reduced susceptibilities to tigecycline, we performed a study to assess tigecycline efflux mediated by the resistance-nodulation-division-type transporter AdeABC. After exposure of two tigecycline-nonsusceptible isolates to the efflux pump inhibitor phenyl-arginine-beta-naphthylamide (PABN), a fourfold reduction in the tigecycline MIC was observed. Both tigecycline-susceptible and -nonsusceptible isolates were found to carry the gene coding for the transmembrane component of the AdeABC pump, adeB, and the two-component regulatory system comprising adeS and adeR. Previously unreported point mutations were identified in the regulatory system in tigecycline-nonsusceptible isolates. Real-time PCR identified 40-fold and 54-fold increases in adeB expression in the two tigecycline-nonsusceptible isolates compared to that in a tigecycline-susceptible isolate. In vitro exposure of a tigecycline-susceptible clinical strain to tigecycline caused a rapid rise in the MIC of tigecycline from 2 microg/ml to 24 microg/ml, which was reversible with PABN. A 25-fold increase in adeB expression was observed in a comparison between this tigecycline-susceptible isolate and its isogenic tigecycline-nonsusceptible mutant. These results indicate that an efflux-based mechanism plays a role in reduced tigecycline susceptibility in Acinetobacter. PMID:17420217

  17. Tigecycline Efflux as a Mechanism for Nonsusceptibility in Acinetobacter baumannii▿

    PubMed Central

    Peleg, Anton Y.; Adams, Jennifer; Paterson, David L.

    2007-01-01

    Tigecycline has an extended spectrum of in vitro antimicrobial activities, including that against multidrug-resistant Acinetobacter. After identifying bloodstream isolates of Acinetobacter with reduced susceptibilities to tigecycline, we performed a study to assess tigecycline efflux mediated by the resistance-nodulation-division-type transporter AdeABC. After exposure of two tigecycline-nonsusceptible isolates to the efflux pump inhibitor phenyl-arginine-β-naphthylamide (PABN), a fourfold reduction in the tigecycline MIC was observed. Both tigecycline-susceptible and -nonsusceptible isolates were found to carry the gene coding for the transmembrane component of the AdeABC pump, adeB, and the two-component regulatory system comprising adeS and adeR. Previously unreported point mutations were identified in the regulatory system in tigecycline-nonsusceptible isolates. Real-time PCR identified 40-fold and 54-fold increases in adeB expression in the two tigecycline-nonsusceptible isolates compared to that in a tigecycline-susceptible isolate. In vitro exposure of a tigecycline-susceptible clinical strain to tigecycline caused a rapid rise in the MIC of tigecycline from 2 μg/ml to 24 μg/ml, which was reversible with PABN. A 25-fold increase in adeB expression was observed in a comparison between this tigecycline-susceptible isolate and its isogenic tigecycline-nonsusceptible mutant. These results indicate that an efflux-based mechanism plays a role in reduced tigecycline susceptibility in Acinetobacter. PMID:17420217

  18. Role of outer membrane barrier in efflux-mediated tetracycline resistance of Escherichia coli.

    PubMed Central

    Thanassi, D G; Suh, G S; Nikaido, H

    1995-01-01

    Accumulation of tetracycline in Escherichia coli was studied to determine its permeation pathway and to provide a basis for understanding efflux-mediated resistance. Passage of tetracycline across the outer membrane appeared to occur preferentially via the porin OmpF, with tetracycline in its magnesium-bound form. Rapid efflux of magnesium-chelated tetracycline from the periplasm was observed. In E. coli cells that do not contain exogenous tetracycline resistance genes, the steady-state level of tetracycline accumulation was decreased when porins were absent or when the fraction of Mg(2+)-chelated tetracycline was small. This is best explained by assuming the presence of a low-level endogenous active efflux system that bypasses the outer membrane barrier. When influx of tetracycline is slowed, this efflux is able to reduce the accumulation of tetracycline in the cytoplasm. In contrast, we found no evidence of a special outer membrane bypass mechanism for high-level efflux via the Tet protein, which is an inner membrane efflux pump coded for by exogenous tetA genes. Fractionation and equilibrium density gradient centrifugation experiments showed that the Tet protein is not localized to regions of inner and outer membrane adhesion. Furthermore, a high concentration of tetracycline was found in the compartment that rapidly equilibrated with the medium, most probably the periplasm, of Tet-containing E. coli cells, and the level of tetracycline accumulation in Tet-containing cells was not diminished by the mutational loss of the OmpF porin. These results suggest that the Tet protein, in contrast to the endogenous efflux system(s), pumps magnesium-chelated tetracycline into the periplasm. A quantitative model of tetracycline fluxes in E. coli cells of various types is presented. PMID:7860612

  19. Cysteines Introduced into Extracellular Loops 1 and 4 of Human P-Glycoprotein That Are Close Only in the Open Conformation Spontaneously Form a Disulfide Bond That Inhibits Drug Efflux and ATPase Activity*

    PubMed Central

    Loo, Tip W.; Clarke, David M.

    2014-01-01

    P-glycoprotein (P-gp) is an ATP-binding cassette drug pump that protects us from toxic compounds and confers multidrug resistance. The protein is organized into two halves. The halves contain a transmembrane domain (TMD) with six transmembrane segments and a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the TMD1/TMD2 and NBD1/NBD2 interfaces, respectively. ATP-dependent drug efflux involves changes between the open inward-facing (NBDs apart, extracellular loops (ECLs) close together) and the closed outward-facing (NBDs close together, ECLs apart) conformations. It is controversial, however, whether the open conformation only exists transiently in intact cells because of the presence of high levels of ATP. To test for the presence of an open conformation in intact cells, reporter cysteines were placed in extracellular loops 1 (A80C, N half) and 4 (R741C, C half). The rationale was that cysteines A80C/R741C would only come close enough to form a disulfide bond in an open conformation (6.9 Å apart) because they are separated widely (30.4 Å apart) in the closed conformation. It was observed that the mutant A80C/R741C cross-linked spontaneously (>90%) when expressed in cells. In contrast to previous reports showing that trapping P-gp in a closed conformation highly activated ATPase activity, here we show that A80C/R741C cross-linking inhibited ATPase activity and drug efflux. Both activities were restored when the cross-linked mutant was treated with a thiol-reducing agent. The results show that an open conformation can be readily detected in cells and that cross-linking of cysteines placed in ECLs 1 and 4 inhibits activity. PMID:25053414

  20. Properties of AdeABC and AdeIJK Efflux Systems of Acinetobacter baumannii Compared with Those of the AcrAB-TolC System of Escherichia coli

    PubMed Central

    Sugawara, Etsuko

    2014-01-01

    Acinetobacter baumannii contains RND-family efflux systems AdeABC and AdeIJK, which pump out a wide range of antimicrobial compounds, as judged from the MIC changes occurring upon deletion of the responsible genes. However, these studies may miss changes because of the high backgrounds generated by the remaining pumps and by β-lactamases, and it is unclear how the activities of these pumps compare quantitatively with those of the well-studied AcrAB-TolC system of Escherichia coli. We expressed adeABC and adeIJK of A. baumannii, as well as E. coli acrAB, in an E. coli host from which acrAB was deleted. The A. baumannii pumps were functional in E. coli, and the MIC changes that were observed largely confirmed the substrate range already reported, with important differences. Thus, the AdeABC system pumped out all β-lactams, an activity that was often missed in deletion studies. When the expression level of the pump genes was adjusted to a similar level for a comparison with AcrAB-TolC, we found that both A. baumannii efflux systems pumped out a wide range of compounds, but AdeABC was less effective than AcrAB-TolC in the extrusion of lipophilic β-lactams, novobiocin, and ethidium bromide, although it was more effective at tetracycline efflux. AdeIJK was remarkably more effective than a similar level of AcrAB-TolC in the efflux of β-lactams, novobiocin, and ethidium bromide, although it was less so in the efflux of erythromycin. These results thus allow us to compare these efflux systems on a quantitative basis, if we can assume that the heterologous systems are fully functional in the E. coli host. PMID:25246403

  1. Bacterial multi-drug efflux transporters

    PubMed Central

    Delmar, Jared A.; Su, Chih-Chia; Yu, Edward W.

    2016-01-01

    Infections caused by bacteria remain a leading cause of death worldwide. While antibiotics remain a key clinical therapy, their effectiveness has been severely compromised by the development of drug resistance in these pathogens. A common and powerful resistance mechanism, multi-drug efflux transporters are capable of extruding a number of structurally unrelated antimicrobials from the bacterial cell, including antibiotics and toxic heavy metal ions, facilitating their survival in noxious environments. Those transporters belonging to the resistance-nodulation-cell division (RND) superfamily typically assemble as tripartite efflux complexes, spanning the inner and outer membranes of the cell envelope. In Escherichia coli, the CusCFBA complex, which mediates resistance to copper(I) and silver(I) ions, is the only known RND transporter with a specificity for heavy metals. Herein, we describe the current knowledge of individual pump components of the Cus system, a paradigm for efflux machinery, and speculate on how RND pumps assemble to fight diverse antimicrobials. PMID:24702006

  2. Rheumatoid Arthritis Disease Activity Is Determinant for ABCB1 and ABCG2 Drug-Efflux Transporters Function

    PubMed Central

    Atisha-Fregoso, Yemil; Lima, Guadalupe; Pascual-Ramos, Virginia; Baños-Peláez, Miguel; Fragoso-Loyo, Hilda; Jakez-Ocampo, Juan; Contreras-Yáñez, Irazú; Llorente, Luis

    2016-01-01

    Objective To compare drug efflux function of ABCB1 and ABCG2 transporters in rheumatoid arthritis (RA) patients with active disease and in remission. Methods Twenty two active RA patients (DAS28 ≥3.2) and 22 patients in remission (DAS28<2.6) were selected from an early RA clinic. All patients were evaluated at study inclusion and six months later. ABCB1 and ABCG2 functional activity was measured in peripheral lymphocytes by flow cytometry. The percentage of cells able to extrude substrates for ABCB1 and ABCG2 was recorded. Results Active patients had higher ABCB1 and ABCG2 activity compared with patients in remission (median [interquartile range]): 3.9% (1.4–22.2) vs (1.3% (0.6–3.2), p = 0.003 and 3.9% (1.1–13.3) vs 0.9% (0.5–1.9) p = 0.006 respectively. Both transporters correlated with disease activity assessed by DAS28, rho = 0.45, p = 0.002 and rho = 0.47, p = 0.001 respectively. Correlation was observed between the time from the beginning of treatment and transporter activity: rho = 0.34, p = 0.025 for ABCB1 and rho = 0.35, p = 0.018 for ABCG2. The linear regression model showed that DAS28 and the time from the onset of treatment are predictors of ABCB1 and ABCG2 functional activity, even after adjustment for treatment. After six months we calculated the correlation between change in DAS28 and change in the functional activity in both transporters and found a moderate and significant correlation for ABCG2 (rho = 0.28, p = 0.04) and a non-significant correlation for ABCB1 (rho = 0.22, p = 0.11). Conclusions Patients with active RA have an increased function of ABCB1 and ABCG2, and disease activity is the main determinant of this phenomena. PMID:27442114

  3. Impairment of the ABCA1 and SR-BI-mediated cholesterol efflux pathways and HDL anti-inflammatory activity in Alzheimer's disease.

    PubMed

    Khalil, Abdelouahed; Berrougui, Hicham; Pawelec, Graham; Fulop, Tamas

    2012-01-01

    The aim of our study was to investigate the effect of Alzheimer's disease (AD) on the cholesterol efflux capacity and anti-inflammatory activity of HDL. HDL and apoA-I were isolated from 20 healthy subjects and from 39 AD patients. Our results showed that serum- and HDL-mediated cholesterol efflux is significantly impaired in AD patients. This impairment of serum and HDL cholesterol efflux capacity was significantly inversely correlated to the AD severity as evaluated by MMSE scores. Results obtained from SR-BI-enriched Fu5AH and ABCA1-enriched J774 cells revealed that AD impaired the interaction of HDL and apoA-I with both the ABCA1 transporter and SR-BI receptor. Purified apoA-I from AD patients also failed to remove free excess cholesterol from ABCA1-enriched J774 macrophages. Interestingly, the decrease in plasma α-tocopherol content and the increase in MDA formation and HDL relative electrophoretic mobility indicated that AD patients had higher levels of oxidative stress. The anti-inflammatory activity of HDL was also significantly lower in AD patients as measured by the level of ICAM-1 expression. In conclusion, our study provides evidence for the first time that the functionality of HDL is impaired in AD and that this alteration might be caused by AD-associated oxidative stress and inflammation. PMID:22178419

  4. Export pumps in Epulopiscium fishelsoni, the symbiotic giant gut bacterium in Acanthurus nigrofuscus

    NASA Astrophysics Data System (ADS)

    Bresler, V.; Fishelson, L.

    2006-04-01

    Activity of the potential antixenobiotic efflux pumps of Epulopiscium fishelsoni (epulos), the symbiotic giant gut bacterium of the algivorous surgeonfish Acanthurus nigrofuscus, was studied in vivo using various specific substrates and microfluorometry. Kinetic and inhibitor analyses revealed the following vital efflux activities: (1) verapamil-sensitive efflux of amphiphilic cationic compounds rhodamine B, Hoechst 33342, and ethidium bromide; (2) verapamil-sensitive efflux of hydrophobic neutral fluorescein diacetate; (3) verapamil-insensitive efflux of hydrophilic anionic fluorescein; and (4) verapamil-insensitive efflux of glutathione- S-bimane. Cytosolic enzymes, nonspecific esterase and glutathione S-transferase, were shown to participate in xenobiotic metabolism. The results suggest that the activity of the potential efflux pump in epulos are similar to those described in other bacteria but are kinetically characterized by an unusually high transport rate, probably mediated by hyperplasia of the plasma membrane. Further studies of the export pumps in epulos may unmask their evolutionary adaptation to a xenobiotic-rich host gut content.

  5. Structural Elements in the Transmembrane and Cytoplasmic Domains of the Metal Transporter SLC30A10 Are Required for Its Manganese Efflux Activity.

    PubMed

    Zogzas, Charles E; Aschner, Michael; Mukhopadhyay, Somshuvra

    2016-07-29

    Homozygous mutations in SLC30A10 lead to the development of familial manganese-induced parkinsonism. We previously demonstrated that SLC30A10 is a cell surface-localized manganese efflux transporter, and parkinsonism-causing mutations block its trafficking and efflux activity. Interestingly, other transporters in the SLC30 family mediate zinc efflux. Determining the mechanisms that allow SLC30A10 to transport manganese, which are unclear, is essential to understand its role in parkinsonism. Here, we generated a predicted structure of SLC30A10, based on the structure of the bacterial zinc transporter YiiP, and performed functional studies. In YiiP, side chains of residues Asp-45 and Asp-49 in the second and His-153 and Asp-157 in the fifth transmembrane segments coordinate zinc and are required for transport. In SLC30A10, the corresponding residues are Asn-43 and Asp-47 in the second and His-244 and Asp-248 in the fifth transmembrane segments. Surprisingly, although alanine substitution of Asp-248 abolished manganese efflux, that of Asn-43 and Asp-47 did not. Instead, side chains of charged or polar residues adjacent to Asp-248 in the first (Glu-25) or fourth (Asn-127) transmembrane segments were required. Further analyses revealed that residues His-333 and His-350 in the cytoplasmic C-terminal domain were required for full activity. However, the C-terminal domain failed to transfer manganese transport capability to a related zinc transporter. Overall, our results indicate that residues in the transmembrane and C-terminal domains together confer optimal manganese transport capability to SLC30A10 and suggest that the mechanism of ion coordination in the transmembrane domain of SLC30A10 may be substantially different from that in YiiP/other SLC30 proteins. PMID:27307044

  6. Pathways of Arsenic Uptake and Efflux

    PubMed Central

    Yang, Hung-Chi; Fu, Hsueh-Liang; Lin, Yung-Feng; Rosen, Barry P.

    2015-01-01

    Arsenic is the most prevalent environmental toxic substance and ranks first on the U.S. Environmental Protection Agency’s Superfund List. Arsenic is a carcinogen and a causative agent of numerous human diseases. Paradoxically arsenic is used as a chemotherapeutic agent for treatment of acute promyelocytic leukemia. Inorganic arsenic has two biological important oxidation states: As(V) (arsenate) and As(III) (arsenite). Arsenic uptake is adventitious because the arsenate and arsenite are chemically similar to required nutrients. Arsenate resembles phosphate and is a competitive inhibitor of many phosphate-utilizing enzymes. Arsenate is taken up by phosphate transport systems. In contrast, at physiological pH, the form of arsenite is As(OH)3, which resembles organic molecules such as glycerol. Consequently, arsenite is taken into cells by aquaglyceroporin channels. Arsenic efflux systems are found in nearly every organism and evolved to rid cells of this toxic metalloid. These efflux systems include members of the multidrug resistance protein family and the bacterial exchangers Acr3 and ArsB. ArsB can also be a subunit of the ArsAB As(III)-translocating ATPase, an ATP-driven efflux pump. The ArsD metallochaperone binds cytosolic As(III) and transfers it to the ArsA subunit of the efflux pump. Knowledge of the pathways and transporters for arsenic uptake and efflux is essential for understanding its toxicity and carcinogenicity and for rational design of cancer chemotherapeutic drugs. PMID:23046656

  7. PUMPS

    DOEpatents

    Thornton, J.D.

    1959-03-24

    A pump is described for conveving liquids, particure it is not advisable he apparatus. The to be submerged in the liquid to be pumped, a conduit extending from the high-velocity nozzle of the injector,and means for applying a pulsating prcesure to the surface of the liquid in the conduit, whereby the surface oscillates between positions in the conduit. During the positive half- cycle of an applied pulse liquid is forced through the high velocity nozzle or jet of the injector and operates in the manner of the well known water injector and pumps liquid from the main intake to the outlet of the injector. During the negative half-cycle of the pulse liquid flows in reverse through the jet but no reverse pumping action takes place.

  8. BBA, a Synthetic Derivative of 23-hydroxybutulinic Acid, Reverses Multidrug Resistance by Inhibiting the Efflux Activity of MRP7 (ABCC10)

    PubMed Central

    Chen, Jun-Jiang; Patel, Atish; Sodani, Kamlesh; Xiao, Zhi-Jie; Tiwari, Amit K.; Zhang, Dong-Mei; Li, Ying-Jie; Yang, Dong-Hua; Ye, Wen-Cai; Chen, Si-Dong; Chen, Zhe-Sheng

    2013-01-01

    Natural products are frequently used for adjuvant chemotherapy in cancer treatment. 23-O-(1,4'-bipiperidine-1-carbonyl) betulinic acid (BBA) is a synthetic derivative of 23-hydroxybutulinic acid (23-HBA), which is a natural pentacyclic triterpene and the major active constituent of the root of Pulsatillachinensis. We previously reported that BBA could reverse P-glycoprotein (P-gp/ABCB1)-mediated multidrug resistance (MDR). In the present study, we investigated whether BBA has the potential to reverse multidrug resistance protein 7 (MRP7/ABCC10)-mediated MDR. We found that BBA concentration-dependently enhanced the sensitivity of MRP7-transfected HEK293 cells to paclitaxel, docetaxel and vinblastine. Accumulation and efflux experiments demonstrated that BBA increased the intracellular accumulation of [3H]-paclitaxel by inhibiting the efflux of [3H]-paclitaxel from HEK293/MRP7 cells. In addition, immunoblotting and immunofluorescence analyses indicated no significant alteration of MRP7 protein expression and localization in plasma membranes after treatment with BBA. These results demonstrate that BBA reverses MRP7-mediated MDR through blocking the drug efflux function of MRP7 without affecting the intracellular ATP levels. Our findings suggest that BBA has the potential to be used in combination with conventional chemotherapeutic agents to augment the response to chemotherapy. PMID:24069321

  9. Identification and characterization of the emhABC efflux system for polycyclic aromatic hydrocarbons in Pseudomonas fluorescens cLP6a.

    PubMed

    Hearn, Elizabeth M; Dennis, Jonathan J; Gray, Murray R; Foght, Julia M

    2003-11-01

    The hydrocarbon-degrading environmental isolate Pseudomonas fluorescens LP6a possesses an active efflux mechanism for the polycyclic aromatic hydrocarbons phenanthrene, anthracene, and fluoranthene but not for naphthalene or toluene. PCR was used to detect efflux pump genes belonging to the resistance-nodulation-cell division (RND) superfamily in a plasmid-cured derivative, P. fluorescens cLP6a, which is unable to metabolize hydrocarbons. One RND pump, whose gene was identified in P. fluorescens cLP6a and was designated emhB, showed homology to the multidrug and solvent efflux pumps in Pseudomonas aeruginosa and Pseudomonas putida. The emhB gene is located in a gene cluster with the emhA and emhC genes, which encode the membrane fusion protein and outer membrane protein components of the efflux system, respectively. Disruption of emhB by insertion of an antibiotic resistance cassette demonstrated that the corresponding gene product was responsible for the efflux of polycyclic aromatic hydrocarbons. The emhB gene disruption did not affect the resistance of P. fluorescens cLP6a to tetracycline, erythromycin, trimethoprim, or streptomycin, but it did decrease resistance to chloramphenicol and nalidixic acid, indicating that the EmhABC system also functions in the efflux of these compounds and has an unusual selectivity. Phenanthrene efflux was observed in P. aeruginosa, P. putida, and Burkholderia cepacia but not in Azotobacter vinelandii. Polycyclic aromatic hydrocarbons represent a new class of nontoxic, highly hydrophobic compounds that are substrates of RND efflux systems, and the EmhABC system in P. fluorescens cLP6a has a narrow substrate range for these hydrocarbons and certain antibiotics. PMID:14563857

  10. Population pharmacokinetic modelling of non-linear brain distribution of morphine: influence of active saturable influx and P-glycoprotein mediated efflux

    PubMed Central

    Groenendaal, D; Freijer, J; de Mik, D; Bouw, M R; Danhof, M; de Lange, E C M

    2007-01-01

    Background and purpose: Biophase equilibration must be considered to gain insight into the mechanisms underlying the pharmacokinetic-pharmacodynamic (PK-PD) correlations of opioids. The objective was to characterise in a quantitative manner the non-linear distribution kinetics of morphine in brain. Experimental approach: Male rats received a 10-min infusion of 4 mg kg−1 of morphine, combined with a continuous infusion of the P-glycoprotein (Pgp) inhibitor GF120918 or vehicle, or 40 mg kg−1 morphine alone. Unbound extracellular fluid (ECF) concentrations obtained by intracerebral microdialysis and total blood concentrations were analysed using a population modelling approach. Key results: Blood pharmacokinetics of morphine was best described with a three-compartment model and was not influenced by GF120918. Non-linear distribution kinetics in brain ECF was observed with increasing dose. A one compartment distribution model was developed, with separate expressions for passive diffusion, active saturable influx and active efflux by Pgp. The passive diffusion rate constant was 0.0014 min−1. The active efflux rate constant decreased from 0.0195 min−1 to 0.0113 min−1 in the presence of GF120918. The active influx was insensitive to GF120918 and had a maximum transport (Nmax/Vecf) of 0.66 ng min−1 ml−1 and was saturated at low concentrations of morphine (C50=9.9 ng ml−1). Conclusions and implications: Brain distribution of morphine is determined by three factors: limited passive diffusion; active efflux, reduced by 42% by Pgp inhibition; low capacity active uptake. This implies blood concentration-dependency and sensitivity to drug-drug interactions. These factors should be taken into account in further investigations on PK-PD correlations of morphine. PMID:17471182

  11. Identification and molecular characterization of an efflux system involved in Pseudomonas putida S12 multidrug resistance.

    PubMed

    Kieboom, J; de Bont, J

    2001-01-01

    The authors previously described srpABC, an operon involved in proton-dependent solvent efflux in the solvent-tolerant Pseudomonas putida S12. Recently, it was shown that organic solvents and not antibiotics induce this operon. In the present study, the authors characterize a new efflux pump, designated ArpABC, on the basis of two isolated chloramphenicol-sensitive transposon mutants. The arpABC operon is involved in the active efflux of multiple antibiotics, such as tetracycline, chloramphenicol, carbenicillin, streptomycin, erythromycin and novobiocin. The deduced amino acid sequences encoded by the three genes involved show a striking resemblance to proteins of the resistance/nodulation/cell division family, which are involved in both organic solvent and multiple drug efflux. These findings demonstrate that ArpABC is highly homologous to the MepABC and TtgABC efflux systems for organic solvents and multiple antibiotics. However, ArpABC does not contribute to organic solvent tolerance in P. putida S12 but is solely involved in multidrug resistance. PMID:11160799

  12. Microbial Efflux Systems and Inhibitors: Approaches to Drug Discovery and the Challenge of Clinical Implementation

    PubMed Central

    Kourtesi, Christina; Ball, Anthony R; Huang, Ying-Ying; Jachak, Sanjay M; Vera, D Mariano A; Khondkar, Proma; Gibbons, Simon; Hamblin, Michael R; Tegos, George P

    2013-01-01

    Conventional antimicrobials are increasingly ineffective due to the emergence of multidrug-resistance among pathogenic microorganisms. The need to overcome these deficiencies has triggered exploration for novel and unconventional approaches to controlling microbial infections. Multidrug efflux systems (MES) have been a profound obstacle in the successful deployment of antimicrobials. The discovery of small molecule efflux system blockers has been an active and rapidly expanding research discipline. A major theme in this platform involves efflux pump inhibitors (EPIs) from natural sources. The discovery methodologies and the available number of natural EPI-chemotypes are increasing. Advances in our understanding of microbial physiology have shed light on a series of pathways and phenotypes where the role of efflux systems is pivotal. Complementing existing antimicrobial discovery platforms such as photodynamic therapy (PDT) with efflux inhibition is a subject under investigation. This core information is a stepping stone in the challenge of highlighting an effective drug development path for EPIs since the puzzle of clinical implementation remains unsolved. This review summarizes advances in the path of EPI discovery, discusses potential avenues of EPI implementation and development, and underlines the need for highly informative and comprehensive translational approaches. PMID:23569468

  13. Reversal of efflux of an anticancer drug in human drug-resistant breast cancer cells by inhibition of protein kinase Cα (PKCα) activity.

    PubMed

    Kim, Chan Woo; Asai, Daisuke; Kang, Jeong-Hun; Kishimura, Akihiro; Mori, Takeshi; Katayama, Yoshiki

    2016-02-01

    P-glycoprotein (Pgp) is a 170-kDa transmembrane protein that mediates the efflux of anticancer drugs from cells. Pgp overexpression has a distinct role in cells exhibiting multidrug resistance (MDR). We examined reversal of drug resistance in human MDR breast cancer cells by inhibition of protein kinase Cα (PKCα) activity, which is associated with Pgp-mediated efflux of anticancer drugs. PKCα activity was confirmed by measurement of phosphorylation levels of a PKCα-specific peptide substrate (FKKQGSFAKKK-NH2), showing relatively higher basal activity in drug-resistant MCF-7/ADR cells (84 %) than that in drug-sensitive MCF-7 cells (63 %). PKCα activity was effectively suppressed by the PKC inhibitor, Ro-31-7549, and reversal of intracellular accumulation of doxorubicin was observed by inhibition of PKCα activity in MCF-7/ADR cells compared with their intrinsic drug resistance. Importantly, increased accumulation of doxorubicin could enhance the therapeutic efficacy of doxorubicin in MDR cells significantly. These results suggest a potential for overcoming MDR via inhibition of PKCα activity with conventional anticancer drugs. PMID:26323260

  14. An Arg-Gly-Asp peptide stimulates Ca2+ efflux from osteoclast precursors through a novel mechanism

    NASA Technical Reports Server (NTRS)

    Yamakawa, K.; Duncan, R.; Hruska, K. A.

    1994-01-01

    We examined the effect of a peptide containing the Arg-Gly-Asp (RGD) sequence on 45Ca2+ efflux from osteoclast precursors. 45Ca(2+)-loaded osteoclast precursors were treated with GRGDSP (170 microM) for 10 min after 30 min of basal perfusion with a bicarbonate-containing buffer. GRGDSP significantly increased fractional efflux of Ca2+ from treated cells compared with vehicle-treated cells (P < 0.01) or cells treated with up to 200 micrograms/ml of a control peptide containing GRGESP. The effect of RGD was sustained for 15 min after the peptide was removed from the perfusate, but control levels of Ca2+ efflux returned by 1 h. The Ca2+ efflux effect of GRGDSP was most likely due to activation of the plasma membrane Ca(2+)-adenosinetriphosphatase (Ca(2+)-ATPase) pump, as indicated by its inhibition with vanadate and a calmodulin antagonist, N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide, and the absence of an effect of Na+/Ca2+ exchange inhibition. An inhibitor of cyclic nucleotide-dependent protein kinases, N-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide (0.1 mM), failed to inhibit GRGDSP-stimulated Ca2+ efflux. However, genistein and herbimycin A, inhibitors of protein-tyrosine kinases, blocked Ca2+ efflux stimulated by GRGDSP. The results indicate that RGD sequences of matrix proteins may stimulate Ca2+ efflux from osteoclasts through activation of protein-tyrosine kinases and suggest that GRGDSP-stimulated Ca2+ efflux is mediated via the plasma membrane Ca(2+)-ATPase.

  15. Pseudoatomic Structure of the Tripartite Multidrug Efflux Pump AcrAB-TolC Reveals the Intermeshing Cogwheel-like Interaction between AcrA and TolC.

    PubMed

    Jeong, Hyeongseop; Kim, Jin-Sik; Song, Saemee; Shigematsu, Hideki; Yokoyama, Takeshi; Hyun, Jaekyung; Ha, Nam-Chul

    2016-02-01

    The resistance-nodulation-division type tripartite pump AcrAB-TolC and its homologs are responsible for multidrug resistance in Gram-negative bacteria by expelling a wide variety of toxic substrates. The three essential components, AcrA, AcrB, and TolC, must function in concert with each respective binding partner within the complex. In this study, we report an 8.2-Å resolution cryo-electron microscopy (cryo-EM) 3D reconstruction of the complex that consists of an AcrAB fusion protein and a chimeric TolC protein. The pseudoatomic structure derived from the cryo-EM reconstruction clearly demonstrates a model only compatible with the adaptor bridging mechanism, wherein the funnel-like AcrA hexamer forms an intermeshing cogwheel-like interaction with the α-barrel tip region of TolC. These observations provide a structural milestone for understanding multidrug resistance in pathogenic Gram-negative bacteria, and may also lead to the design of new antibacterial drugs. PMID:26777412

  16. Snow-melting season CO2 efflux along the trans-Alaska pipeline

    NASA Astrophysics Data System (ADS)

    Kim, Y.; Nakai, T.

    2011-12-01

    This research was conducted to estimate CO2 effluxes in exposed and snow-covered soils along the trans-Alaska pipeline (ca. 660 km) during snow-melting seasons of April 2010 and April-May 2011. In-situ CO2 efflux was measured with a dynamic chamber system that consisted of a chamber (22 cm in diameter and 6 cm high), pump, NDIR (CO2 analyzer), and a laptop computer. Soil temperature and snow depth were measured with a portable thermocouple and from snow pit-wall. The difference in snow-melting season CO2 efflux was remarkably showed in exposed and snow-covered soils of boreal forest and tundra, suggesting the distinctly latitudinal CO2 efflux gradient. Mean CO2 efflux was 0.88±0.51 and 2.4±3.4 gCO2-C/m2/day in soil temperature of -1.8±4.0 and -1.1±3.4 °C during the snow-melting period of 2010 and 2011, respectively. When the snow was disappeared, mean CO2 efflux was 1.3±0.3 and 5.4±3.7 gCO2-C/m2/day for 2010 and 2011; on the other hand, when the seasonal covered snow was melting, mean CO2 efflux was 0.2±0.2 and 0.3±0.3 gCO2-C/m2/day for both years. However, the coastal site near Arctic sea was not still melted, showing much lower CO2 efflux was 0.02±0.02 and 0.08±0.12 gCO2-C/m2/day in soil temperature of -12.4±2.2 and -12.9±3.4 °C for 2010 and 2011, respectively. A relationship between mean CO2 efflux at each site and mean soil temperature at 5 cm below the surface along the trans-Alaska pipeline is a good exponential, which the equation is as follows: CO2 efflux = 885×exp(0.335×Ts) (R2=0.86; p<0.001) and CO2 efflux = 888×exp(0.337×Ts) (R2=0.92; p<0.001) for 2010 and 2011, respectively. CO2 efflux in a white spruce forest during the snow-thawing season was measured in four directions from the bottom stem, suggesting that distinct differences of CO2 efflux between the exposed soil and the snow-covered soil in the four directions. This may be due to the fast decomposition of soil organic carbon and/or active root respiration in the exposed soil

  17. Controlling epileptiform activity with organic electronic ion pumps.

    PubMed

    Williamson, Adam; Rivnay, Jonathan; Kergoat, Loïg; Jonsson, Amanda; Inal, Sahika; Uguz, Ilke; Ferro, Marc; Ivanov, Anton; Sjöström, Theresia Arbring; Simon, Daniel T; Berggren, Magnus; Malliaras, George G; Bernard, Christophe

    2015-05-27

    In treating epilepsy, the ideal solution is to act at a seizure's onset, but only in the affected regions of the brain. Here, an organic electronic ion pump is demonstrated, which directly delivers on-demand pure molecules to specific brain regions. State-of-the-art organic devices and classical pharmacology are combined to control pathological activity in vitro, and the results are verified with electrophysiological recordings. PMID:25866154

  18. Control of Cl− Efflux in Chara corallina by Cytosolic pH, Free Ca2+, and Phosphorylation Indicates a Role of Plasma Membrane Anion Channels in Cytosolic pH Regulation1

    PubMed Central

    Johannes, Eva; Crofts, Alan; Sanders, Dale

    1998-01-01

    Enhanced Cl− efflux during acidosis in plants is thought to play a role in cytosolic pH (pHc) homeostasis by short-circuiting the current produced by the electrogenic H+ pump, thereby facilitating enhanced H+ efflux from the cytosol. Using an intracellular perfusion technique, which enables experimental control of medium composition at the cytosolic surface of the plasma membrane of charophyte algae (Chara corallina), we show that lowered pHc activates Cl− efflux via two mechanisms. The first is a direct effect of pHc on Cl− efflux; the second mechanism comprises a pHc-induced increase in affinity for cytosolic free Ca2+ ([Ca2+]c), which also activates Cl− efflux. Cl− efflux was controlled by phosphorylation/dephosphorylation events, which override the responses to both pHc and [Ca2+]c. Whereas phosphorylation (perfusion with the catalytic subunit of protein kinase A in the presence of ATP) resulted in a complete inhibition of Cl− efflux, dephosphorylation (perfusion with alkaline phosphatase) arrested Cl− efflux at 60% of the maximal level in a manner that was both pHc and [Ca2+]c independent. These findings imply that plasma membrane anion channels play a central role in pHc regulation in plants, in addition to their established roles in turgor/volume regulation and signal transduction. PMID:9733536

  19. Steady-state heating of active fibres under optical pumping

    SciTech Connect

    Gainov, V V; Shaidullin, R I; Ryabushkin, Oleg A

    2011-07-31

    We have measured the temperature in the core of rare-earth-doped optical fibres under lasing conditions at high optical pump powers using a fibre Mach - Zehnder interferometer and probe light of wavelength far away from the absorption bands of the active ions. From the observed heating kinetics of the active medium, the heat transfer coefficient on the polymer cladding - air interface has been estimated. The temperature of the active medium is shown to depend on the thermal and optical properties of the polymer cladding. (fiber and integrated optics)

  20. Neutrophil IL-1β processing induced by pneumolysin is mediated by the NLRP3/ASC inflammasome and caspase-1 activation, and is dependent on K+ efflux

    PubMed Central

    Karmakar, Mausita; Katsnelson, Michael; Malak, Hesham A.; Greene, Neil G.; Howell, Scott J.; Hise, Amy G.; Camilli, Andrew; Kadioglu, Aras; Dubyak, George R.; Pearlman, Eric

    2014-01-01

    Although neutrophils are the most abundant cells in acute infection and inflammation, relatively little attention has been paid to their role in inflammasome formation and IL-1β processing. In the current study, we investigated the mechanism by which neutrophils process IL-1β in response to Streptococcus pneumoniae. Using a murine model of S. pneumoniae corneal infection, we demonstrated a requirement for IL-1β in bacterial clearance, and showed that NLRP3, ASC and caspase-1 are essential for IL-1β production and bacterial killing in the cornea. Neutrophils in infected corneas had multiple specks with enzymatically active caspase-1 (FLICA-660+), and bone marrow neutrophils stimulated with heat killed S. pneumoniae (signal 1) and pneumolysin (signal 2) exhibited multiple specks after staining with FLICA-660, NLRP3 or ASC. High molecular weight ASC complexes were also detected, consistent with oligomer formation. Pneumolysin induced K+ efflux in neutrophils, and blocking K+ efflux inhibited caspase-1 activation and IL-1β processing; however, neutrophils did not undergo pyroptosis, indicating that K+ efflux and IL-1β processing is not a consequence of cell death. There was also no role for lysosomal destabilization or neutrophil elastase in pneumolysin mediated IL-1β processing in neutrophils. Together, these findings demonstrate an essential role for neutrophil derived IL-1β in S. pneumoniae infection, and elucidate the role of the NLRP3 inflammasome in neutrophil cleavage and secretion of mature IL-1β. Given the ubiquitous presence of neutrophils in acute bacterial and fungal infections, these findings will have implications for other microbial diseases. PMID:25609842

  1. Effects of turbulent pumping on stellar activity cycles

    NASA Astrophysics Data System (ADS)

    Do Cao, O.; Brun, A. S.

    2011-12-01

    Stellar magnetic activity of solar like stars is thought to be due to an internal dynamo. While the Sun has been the subject of intense research for refining dynamo models, observations of magnetic cyclic activity in solar type stars have become more and more available, opening a new path to understand the underlying physics behind stellar cycles. For instance, it is key to understand how stellar rotation rate influences magnetic cycle period P_cyc. Recent numerical simulations of advection-dominated Babcock Leighton models have demonstrated that it is difficult to explain this observed trend given a) the strong influence of the cycle period to the meridional circulation amplitude and b) the fact that 3D models indicate that meridional flows become weaker as the rotation rate increases. In this paper, we introduce the turbulent pumping mechanism as another advective process capable also of transporting the magnetic fields. We found that this model is now able to reproduce the observations under the assumption that this effect increases as \\Omega2. The turbulent pumping becomes indeed another major player able to circumvent the meridional circulation. However, for high rotation rates (\\Omega ≃ 5 \\Omega_⊙), its effects dominate those of the meridional circulation, entering a new class of regime dominated by the advection of turbulent pumping and thus leading to a cyclic activity qualitatively different from that of the Sun.

  2. Peptide mediators of cholesterol efflux

    DOEpatents

    Bielicki, John K.; Johansson, Jan

    2013-04-09

    The present invention provides a family of non-naturally occurring polypeptides having cholesterol efflux activity that parallels that of full-length apolipoproteins (e.g., Apo AI and Apo E), and having high selectivity for ABAC1 that parallels that of full-length apolipoproteins. The invention also provides compositions comprising such polypeptides, methods of identifying, screening and synthesizing such polypeptides, and methods of treating, preventing or diagnosing diseases and disorders associated with dyslipidemia, hypercholesterolemia and inflammation.

  3. The Peptide Toxin Amylosin of Bacillus amyloliquefaciens from Moisture-Damaged Buildings Is Immunotoxic, Induces Potassium Efflux from Mammalian Cells, and Has Antimicrobial Activity

    PubMed Central

    Teplova, Vera V.; Andersson, Maria A.; Mikkola, Raimo; Kankkunen, Päivi; Matikainen, Sampsa; Gahmberg, Carl G.; Andersson, Leif C.; Salkinoja-Salonen, Mirja

    2015-01-01

    Amylosin, a heat-stable channel-forming non-ribosomally synthesized peptide toxin produced by strains of Bacillus amyloliquefaciens isolated from moisture-damaged buildings, is shown in this paper to have immunotoxic and cytotoxic effects on human cells as well as antagonistic effects on microbes. Human macrophages exposed to 50 ng of amylosin ml−1 secreted high levels of cytokines interleukin-1β (IL-1β) and IL-18 within 2 h, indicating activation of the NLRP3 inflammasome, an integral part of the innate immune system. At the same exposure level, expression of IL-1β and IL-18 mRNA increased. Amylosin caused dose-dependent potassium ion efflux from all tested mammalian cells (human monocytes and keratinocytes and porcine sperm cells) at 1 to 2 μM exposure. Amylosin also inhibited the motility of porcine sperm cells and depolarized the mitochondria of human keratinocytes. Amylosin may thus trigger the activation of the NLRP3 inflammasome and subsequently cytokine release by causing potassium efflux from exposed cells. The results of this study indicate that exposure to amylosin activates the innate immune system, which could offer an explanation for the inflammatory symptoms experienced by occupants of moisture-damaged buildings. In addition, the amylosin-producing B. amyloliquefaciens inhibited the growth of both prokaryotic and eukaryotic indoor microbes, and purified amylosin also had an antimicrobial effect. These antimicrobial effects could make amylosin producers dominant and therefore significant causal agents of health problems in some moisture-damaged sites. PMID:25681192

  4. Synthesis of α,β-Unsaturated Carbonyl-Based Compounds, Oxime and Oxime Ether Analogs as Potential Anticancer Agents for Overcoming Cancer Multidrug Resistance by Modulation of Efflux Pumps in Tumor Cells.

    PubMed

    Qin, Hua-Li; Leng, Jing; Zhang, Cheng-Pan; Jantan, Ibrahim; Amjad, Muhammad Wahab; Sher, Muhammad; Naeem-Ul-Hassan, Muhammad; Hussain, Muhammad Ajaz; Bukhari, Syed Nasir Abbas

    2016-04-14

    Sixty-nine novel α,β-unsaturated carbonyl based compounds, including cyclohexanone, tetralone, oxime, and oxime ether analogs, were synthesized. The antiproliferative activity determined by using seven different human cancer cell lines provided a structure-activity relationship. Compound 8ag exhibited high antiproliferative activity against Panc-1, PaCa-2, A-549, and PC-3 cell lines, with IC50 value of 0.02 μM, comparable to the positive control Erlotinib. The ten most active antiproliferative compounds were assessed for mechanistic effects on BRAF(V600E), EGFR TK kinases, and tubulin polymerization, and were investigated in vitro to reverse efflux-mediated resistance developed by cancer cells. Compound 8af exhibited the most potent BRAF(V600E) inhibitory activity with an IC50 value of 0.9 μM. Oxime analog 7o displayed the most potent EGFR TK inhibitory activity with an IC50 of 0.07 μM, which was analogous to the positive control. Some analogs including 7f, 8af, and 8ag showed a dual role as anticancer and MDR reversal agents. PMID:27010345

  5. Deformation of partially pumped active mirrors for high average-power diode-pumped solid-state lasers.

    PubMed

    Albach, Daniel; LeTouzé, Geoffroy; Chanteloup, Jean-Christophe

    2011-04-25

    We discuss the deformation of a partially pumped active mirror amplifier as a free standing disk, as implemented in several laser systems. We rely on the Lucia laser project to experimentally evaluate the analytical and numerical deformation models. PMID:21643092

  6. TLR2/MyD88/NF-κB Pathway, Reactive Oxygen Species, Potassium Efflux Activates NLRP3/ASC Inflammasome during Respiratory Syncytial Virus Infection

    PubMed Central

    Mgbemena, Victoria; Tsai, Su-Yu; Chang, Te-Hung; Berton, Michael T.; Morris, Ian R.; Allen, Irving C.; Ting, Jenny P.-Y.; Bose, Santanu

    2012-01-01

    Human respiratory syncytial virus (RSV) constitute highly pathogenic virus that cause severe respiratory diseases in newborn, children, elderly and immuno-compromised individuals. Airway inflammation is a critical regulator of disease outcome in RSV infected hosts. Although “controlled” inflammation is required for virus clearance, aberrant and exaggerated inflammation during RSV infection results in development of inflammatory diseases like pneumonia and bronchiolitis. Interleukin-1β (IL-1β) plays an important role in inflammation by orchestrating the pro-inflammatory response. IL-1β is synthesized as an immature pro-IL-1β form. It is cleaved by activated caspase-1 to yield mature IL-1β that is secreted extracellularly. Activation of caspase-1 is mediated by a multi-protein complex known as the inflammasome. Although RSV infection results in IL-1β release, the mechanism is unknown. Here in, we have characterized the mechanism of IL-1β secretion following RSV infection. Our study revealed that NLRP3/ASC inflammasome activation is crucial for IL-1β production during RSV infection. Further studies illustrated that prior to inflammasome formation; the “first signal” constitutes activation of toll-like receptor-2 (TLR2)/MyD88/NF-κB pathway. TLR2/MyD88/NF-κB signaling is required for pro-IL-1β and NLRP3 gene expression during RSV infection. Following expression of these genes, two “second signals” are essential for triggering inflammasome activation. Intracellular reactive oxygen species (ROS) and potassium (K+) efflux due to stimulation of ATP-sensitive ion channel promote inflammasome activation following RSV infection. Thus, our studies have underscored the requirement of TLR2/MyD88/NF-κB pathway (first signal) and ROS/potassium efflux (second signal) for NLRP3/ASC inflammasome formation, leading to caspase-1 activation and subsequent IL-1β release during RSV infection. PMID:22295065

  7. IP/sub 3/ stimulates CA/sup + +/ efflux from fusogenic carrot protoplasts

    SciTech Connect

    Rincon, M.; Boss, W.F.

    1986-04-01

    Polyphosphoinositide breakdown plays an important role in signal transduction in animal cells (Berridge and Irvine, 1984, Nature, 312:315). Upon stimulation, phospholipase C hydrolyzes phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate (IP/sub 3/) and diacylglycerol both of which act as cellular second messengers. IP/sub 3/ mobilizes Ca/sup + +/ from internal stores, hence the cytosolic free Ca/sup + +/ concentration increases and those physiological activities regulated by Ca/sup + +/ are stimulated. To test if plant cells also responded to IP/sub 3/, Ca/sup + +/ efflux studies were done with fusogenic carrot protoplasts released in EGTA. The protoplasts were preloaded with /sup 45/Ca/sup + +/ placed in a Ca/sup + +/-free medium, and efflux determined as /sup 45/Ca/sup + +/ loss from the protoplasts. IP/sub 3/ (10-20..mu..M) caused enhanced /sup 45/Ca/sup + +/ efflux and the response was sustained for at least 15 min. In plants, as in animals, the observed IP/sub 3/-enhanced /sup 45/Ca/sup + +/ efflux suggested that IP/sub 3/ released Ca/sup + +/ from internal stores, and the increased free cytosolic Ca/sup + +/ activated Ca/sup + +/ pumping mechanisms which restored the Ca/sup + +/ concentration in the cytosol to the normal level.

  8. Cross-Resistance between Triclosan and Antibiotics in Pseudomonas aeruginosa Is Mediated by Multidrug Efflux Pumps: Exposure of a Susceptible Mutant Strain to Triclosan Selects nfxB Mutants Overexpressing MexCD-OprJ

    PubMed Central

    Chuanchuen, Rungtip; Beinlich, Kerry; Hoang, Tung T.; Becher, Anna; Karkhoff-Schweizer, RoxAnn R.; Schweizer, Herbert P.

    2001-01-01

    Triclosan is an antiseptic frequently added to items as diverse as soaps, lotions, toothpaste, and many commonly used household fabrics and plastics. Although wild-type Pseudomonas aeruginosa expresses the triclosan target enoyl-acyl carrier protein reductase, it is triclosan resistant due to expression of the MexAB-OprM efflux system. Exposure of a susceptible Δ(mexAB-oprM) strain to triclosan selected multidrug-resistant bacteria at high frequencies. These bacteria hyperexpressed the MexCD-OprJ efflux system due to mutations in its regulatory gene, nfxB. The MICs of several drugs for these mutants were increased up to 500-fold, including the MIC of ciprofloxacin, which was increased 94-fold. Whereas the MexEF-OprN efflux system also participated in triclosan efflux, this antimicrobial was not a substrate for MexXY-OprM. PMID:11158736

  9. Cross-resistance between triclosan and antibiotics in Pseudomonas aeruginosa is mediated by multidrug efflux pumps: exposure of a susceptible mutant strain to triclosan selects nfxB mutants overexpressing MexCD-OprJ.

    PubMed

    Chuanchuen, R; Beinlich, K; Hoang, T T; Becher, A; Karkhoff-Schweizer, R R; Schweizer, H P

    2001-02-01

    Triclosan is an antiseptic frequently added to items as diverse as soaps, lotions, toothpaste, and many commonly used household fabrics and plastics. Although wild-type Pseudomonas aeruginosa expresses the triclosan target enoyl-acyl carrier protein reductase, it is triclosan resistant due to expression of the MexAB-OprM efflux system. Exposure of a susceptible Delta(mexAB-oprM) strain to triclosan selected multidrug-resistant bacteria at high frequencies. These bacteria hyperexpressed the MexCD-OprJ efflux system due to mutations in its regulatory gene, nfxB. The MICs of several drugs for these mutants were increased up to 500-fold, including the MIC of ciprofloxacin, which was increased 94-fold. Whereas the MexEF-OprN efflux system also participated in triclosan efflux, this antimicrobial was not a substrate for MexXY-OprM. PMID:11158736

  10. Recent NRC research activities addressing valve and pump issues

    SciTech Connect

    Morrison, D.L.

    1996-12-01

    The mission of the U.S. Nuclear Regulatory Commission (NRC) is to ensure the safe design, construction, and operation of commercial nuclear power plants and other facilities in the U.S.A. One of the main roles that the Office of Nuclear Regulatory Research (RES) plays in achieving the NRC mission is to plan, recommend, and implement research programs that address safety and technical issues deemed important by the NRC. The results of the research activities provide the bases for developing NRC positions or decisions on these issues. Also, RES performs confirmatory research for developing the basis to evaluate industry responses and positions on various regulatory requirements. This presentation summarizes some recent RES supported research activities that have addressed safety and technical issues related to valves and pumps. These activities include the efforts on determining valve and motor-operator responses under dynamic loads and pressure locking events, evaluation of monitoring equipment, and methods for detecting and trending aging of check valves and pumps. The role that RES is expected to play in future years to fulfill the NRC mission is also discussed.

  11. Dendritic cells phenotype fitting under hypoxia or lipopolysaccharide; adenosine 5′-triphosphate-binding cassette transporters far beyond an efflux pump

    PubMed Central

    Lloberas, N; Rama, I; Llaudó, I; Torras, J; Cerezo, G; Cassis, L; Franquesa, M; Merino, A; Benitez-Ribas, D; Cruzado, J M; Herrero-Fresneda, I; Bestard, O; Grinyó, J M

    2013-01-01

    -transporters emerge as potential targets in immunosuppressive therapies interfering with DCs maturation, thereby abrogating innate immune response when it is activated after ischaemia. PMID:23600833

  12. Impairment of blood-cerebrospinal fluid barrier properties by retrovirus-activated T lymphocytes: reduction in cerebrospinal fluid-to-blood efflux of prostaglandin E2.

    PubMed

    Khuth, Seng Thuon; Strazielle, Nathalie; Giraudon, Pascale; Belin, Marie-Françoise; Ghersi-Egea, Jean-François

    2005-09-01

    The choroid plexus epithelium forms the interface between the blood and the CSF. In conjunction with the tight junctions restricting the paracellular pathway, polarized specific transport systems in the choroidal epithelium allow a fine regulation of CSF-borne biologically active mediators. The highly vascularized stroma delimited by the choroidal epithelium can be a reservoir for retrovirus-infected or activated immune cells. In this work, new insight in the implication of the blood-CSF barrier in neuroinfectious and inflammatory diseases is provided by using a differentiated cellular model of the choroidal epithelium, exposed to infected T lymphocytes. We demonstrate that T cells activated by a retroviral infection, but not non-infected cells, reduce the transporter-mediated CSF-to-blood efflux of organic anions, in particular that of the potent pro-inflammatory prostaglandin PGE2, via the release of soluble factors. A moderate alteration of the paracellular permeability also occurs. We identified the viral protein Tax, oxygenated free radicals, matrix-metalloproteinases and pro-inflammatory cytokines as active molecules released during the exposure of the epithelium to infected T cells. Among them, tumour necrosis factor and interleukin 1 are directly involved in the mechanism underlying the decrease in some choroidal organic anion efflux. Given the strong involvement of CSF-borne PGE2 in sickness behaviour syndrome, these data suggest that the blood-CSF barrier plays an important role in the pathophysiology of neuroinflammation and neuroinfection, via changes in the transport processes controlling the CSF biodisposition of PGE2. PMID:16026393

  13. Commensal Bacteria-Induced Inflammasome Activation in Mouse and Human Macrophages Is Dependent on Potassium Efflux but Does Not Require Phagocytosis or Bacterial Viability.

    PubMed

    Chen, Kejie; Shanmugam, Nanda Kumar N; Pazos, Michael A; Hurley, Bryan P; Cherayil, Bobby J

    2016-01-01

    Gut commensal bacteria contribute to the pathogenesis of inflammatory bowel disease, in part by activating the inflammasome and inducing secretion of interleukin-1ß (IL-1ß). Although much has been learned about inflammasome activation by bacterial pathogens, little is known about how commensals carry out this process. Accordingly, we investigated the mechanism of inflammasome activation by representative commensal bacteria, the Gram-positive Bifidobacterium longum subspecies infantis and the Gram-negative Bacteroides fragilis. B. infantis and B. fragilis induced IL-1ß secretion by primary mouse bone marrow-derived macrophages after overnight incubation. IL-1ß secretion also occurred in response to heat-killed bacteria and was only partly reduced when phagocytosis was inhibited with cytochalasin D. Similar results were obtained with a wild-type immortalized mouse macrophage cell line but neither B. infantis nor B. fragilis induced IL-1ß secretion in a mouse macrophage line lacking the nucleotide-binding/leucine-rich repeat pyrin domain containing 3 (NLRP3) inflammasome. IL-1ß secretion in response to B. infantis and B. fragilis was significantly reduced when the wild-type macrophage line was treated with inhibitors of potassium efflux, either increased extracellular potassium concentrations or the channel blocker ruthenium red. Both live and heat-killed B. infantis and B. fragilis also induced IL-1ß secretion by human macrophages (differentiated THP-1 cells or primary monocyte-derived macrophages) after 4 hours of infection, and the secretion was inhibited by raised extracellular potassium and ruthenium red but not by cytochalasin D. Taken together, our findings indicate that the commensal bacteria B. infantis and B. fragilis activate the NLRP3 inflammasome in both mouse and human macrophages by a mechanism that involves potassium efflux and that does not require bacterial viability or phagocytosis. PMID:27505062

  14. Commensal Bacteria-Induced Inflammasome Activation in Mouse and Human Macrophages Is Dependent on Potassium Efflux but Does Not Require Phagocytosis or Bacterial Viability

    PubMed Central

    Chen, Kejie; Shanmugam, Nanda Kumar N.; Pazos, Michael A.; Hurley, Bryan P.; Cherayil, Bobby J.

    2016-01-01

    Gut commensal bacteria contribute to the pathogenesis of inflammatory bowel disease, in part by activating the inflammasome and inducing secretion of interleukin-1ß (IL-1ß). Although much has been learned about inflammasome activation by bacterial pathogens, little is known about how commensals carry out this process. Accordingly, we investigated the mechanism of inflammasome activation by representative commensal bacteria, the Gram-positive Bifidobacterium longum subspecies infantis and the Gram-negative Bacteroides fragilis. B. infantis and B. fragilis induced IL-1ß secretion by primary mouse bone marrow-derived macrophages after overnight incubation. IL-1ß secretion also occurred in response to heat-killed bacteria and was only partly reduced when phagocytosis was inhibited with cytochalasin D. Similar results were obtained with a wild-type immortalized mouse macrophage cell line but neither B. infantis nor B. fragilis induced IL-1ß secretion in a mouse macrophage line lacking the nucleotide-binding/leucine-rich repeat pyrin domain containing 3 (NLRP3) inflammasome. IL-1ß secretion in response to B. infantis and B. fragilis was significantly reduced when the wild-type macrophage line was treated with inhibitors of potassium efflux, either increased extracellular potassium concentrations or the channel blocker ruthenium red. Both live and heat-killed B. infantis and B. fragilis also induced IL-1ß secretion by human macrophages (differentiated THP-1 cells or primary monocyte-derived macrophages) after 4 hours of infection, and the secretion was inhibited by raised extracellular potassium and ruthenium red but not by cytochalasin D. Taken together, our findings indicate that the commensal bacteria B. infantis and B. fragilis activate the NLRP3 inflammasome in both mouse and human macrophages by a mechanism that involves potassium efflux and that does not require bacterial viability or phagocytosis. PMID:27505062

  15. Lack of efflux mediated quinolone resistance in Salmonella enterica serovars Typhi and Paratyphi A.

    PubMed

    Baucheron, Sylvie; Monchaux, Isabelle; Le Hello, Simon; Weill, François-Xavier; Cloeckaert, Axel

    2014-01-01

    Salmonella enterica serovars Typhi and Paratyphi A isolates from human patients in France displaying different levels of resistance to quinolones or fluoroquinolones were studied for resistance mechanisms to these antimicrobial agents. All resistant isolates carried either single or multiple target gene mutations (i.e., in gyrA, gyrB, or parC) correlating with the resistance levels observed. Active efflux, through upregulation of multipartite efflux systems, has also been previously reported as contributing mechanism for other serovars. Therefore, we investigated also the occurrence of non-target gene mutations in regulatory regions affecting efflux pump expression. However, no mutation was detected in these regions in both Typhi and Paratyphi isolates of this study. Besides, no overexpression of the major efflux systems was observed for these isolates. Nevertheless, a large deletion of 2334 bp was identified in the acrS-acrE region of all S. Typhi strains but which did not affect the resistance phenotype. As being specific to S. Typhi, this deletion could be used for specific molecular detection purposes. In conclusion, the different levels of quinolone or FQ resistance in both S. Typhi and S. Paratyphi A seem to rely only on target modifications. PMID:24478769

  16. Buckling of Dielectric Elastomeric Plates for Electrically Active Microfludic Pumps

    NASA Astrophysics Data System (ADS)

    Holmes, Douglas; Tavakol, Behrouz; Bozlar, Michael; Froehlicher, Guillaume; Stone, Howard; Aksay, Ilhan

    2013-11-01

    Fluid flow can be directed and controlled by a variety of mechanisms within industrial and biological environments. Advances in microfluidic technology have required innovative ways to control fluid flow on a small scale, and the ability to actively control fluid flow within microfluidic devices is crucial for advancements in nanofluidics, biomedical fluidic devices, and digital microfluidics. In this work, we present a means for microfluidic control via the electrical actuation of thin, flexible valves within microfluidic channels. These structures consist of a dielectric elastomer confined between two compliant electrodes that can be actively and reversibly buckle out of plane to pump fluids from an applied voltage. The out-of-plane deformation can be quantified using two parameters: net change in surface area and the shape of deformation. Change in surface area depends on the voltage, while the deformation shape, which significantly affects the flow rate, is a function of voltage, and the pressure and volume of the chambers on each side of the thin plate. The use of solid electrodes enables a robust and reversible pumping mechanism that will have will enable advancements in rapid microfluidic diagnostics, adaptive materials, and artificial muscles.

  17. An alkylaminoquinazoline restores antibiotic activity in Gram-negative resistant isolates.

    PubMed

    Mahamoud, Abdallah; Chevalier, Jacqueline; Baitiche, Milad; Adam, Elissavet; Pagès, Jean-Marie

    2011-02-01

    To date, various bacterial drug efflux pump inhibitors (EPIs) have been described. They exhibit variability in their activity spectrum with respect to antibiotic structural class and bacterial species. Among the various 4-alkylaminoquinazoline derivatives synthesized and studied in this work, one molecule, 1167, increased the susceptibility of important human-pathogenic, resistant, Gram-negative bacteria towards different antibiotic classes. This 4-(3-morpholinopropylamino)-quinazoline induced an increase in the activity of chloramphenicol, nalidixic acid, norfloxacin and sparfloxacin, which are substrates of the AcrAB-TolC and MexAB-OprM efflux pumps that act in these multidrug-resistant isolates. In addition, 1167 increased the intracellular concentration of chloramphenicol in efflux pump-overproducing strains. The rate of restoration depended on the structure of the antibiotic, suggesting that different sites in the efflux pumps may be involved. A molecule exhibiting a morpholine functional group and a propyl extension of the side chain was more active. PMID:21071494

  18. Zidovudine and ursodeoxycholic acid conjugation: design of a new prodrug potentially able to bypass the active efflux transport systems of the central nervous system.

    PubMed

    Dalpiaz, Alessandro; Paganetto, Guglielmo; Pavan, Barbara; Fogagnolo, Marco; Medici, Alessandro; Beggiato, Sarah; Perrone, Daniela

    2012-04-01

    We have synthesized a new prodrug obtained by the 5'-ester conjugation of zidovudine (AZT), an antiviral agent substrate of active efflux transport systems (AET), with ursodeoxycholic acid (UDCA), a bile acid able to permeate into the central nervous system (CNS). We have demonstrated, by HPLC analysis, that UDCA-AZT is quickly hydrolyzed in rat plasma and whole blood (half-life <10 s). The same compound was hydrolyzed with slower rates in human plasma (half-life =7.53 ± 0.44 h) and whole blood (half-life =3.71 ± 0.16 h), allowing to control the AZT release. UDCA-AZT appeared hydrolyzed also in rat brain (half-life = 7.24 ± 0.45 min) and liver homogenates (half-life = 2.70 ± 0.14 min). In the aim to study the permeation properties of the UDCA-AZT across physiological barriers, we have used an established human retinal pigment epithelium (HRPE) cell line to obtain a polarized cell monolayer showing epithelial features. The bidirectional permeation of 30 μM AZT across this monolayer was regulated by apparent permeability coefficients (P(E)) higher from the apical to basolateral compartments (P(E) = 209 ± 4 × 10⁻⁵ cm/min) than in the opposite way (P(E) = 133 ± 8 × 10⁻⁵ cm/min), in conformity with the in vivo behavior of AZT, actively effluxed from the CNS. The influx (P(E) = 39.1 ± 1.2 × 10⁻⁵ cm/min) and efflux (P(E) = 31.3 ± 3.6 × 10⁻⁵ cm/min) permeability coefficients of 30 μM UDCA-AZT were instead the same, suggesting the ability of the prodrug to avoid the AET systems and, potentially, to allow its accumulation in the CNS. The relatively low P(E) values of UDCA-AZT were associated with a partial hydrolysis during its permeation across the cell monolayer. PMID:22356133

  19. A Fluorescent Microplate Assay Quantifies Bacterial Efflux and Demonstrates Two Distinct Compound Binding Sites in AcrB

    PubMed Central

    Ferrari, Annette; Rijnbrand, R.; Erwin, Alice L.

    2015-01-01

    A direct assay of efflux by Escherichia coli AcrAB-TolC and related multidrug pumps would have great value in discovery of new Gram-negative antibiotics. The current understanding of how efflux is affected by the chemical structure and physical properties of molecules is extremely limited, derived from antibacterial data for compounds that inhibit growth of wild-type E. coli. We adapted a previously described fluorescent efflux assay to a 96-well microplate format that measured the ability of test compounds to compete for efflux with Nile Red (an environment-sensitive fluor), independent of antibacterial activity. We show that Nile Red and the lipid-sensitive probe DiBAC4-(3) [bis-(1,3-dibutylbarbituric acid)-trimethine oxonol] can quantify efflux competition in E. coli. We extend the previous findings that the tetracyclines compete with Nile Red and show that DiBAC4-(3) competes with macrolides. The extent of the competition shows a modest correlation with the effect of the acrB deletion on MICs within the compound sets for both dyes. Crystallographic studies identified at least two substrate binding sites in AcrB, the proximal and distal pockets. High-molecular-mass substrates bound the proximal pocket, while low-mass substrates occupied the distal pocket. As DiBAC4-(3) competes with macrolides but not with Nile Red, we propose that DiBAC4-(3) binds the proximal pocket and Nile Red likely binds the distal site. In conclusion, competition with fluorescent probes can be used to study the efflux process for diverse chemical structures and may provide information as to the site of binding and, in some cases, enable rank-ordering a series of related compounds by efflux. PMID:25645845

  20. Alternative to antibiotics against Pseudomonas aeruginosa: Effects of Glycyrrhiza glabra on membrane permeability and inhibition of efflux activity and biofilm formation in Pseudomonas aeruginosa and its in vitro time-kill activity.

    PubMed

    Chakotiya, Ankita Singh; Tanwar, Ankit; Narula, Alka; Sharma, Rakesh Kumar

    2016-09-01

    The multi-drug resistance offered by Pseudomonas aeruginosa to antibiotics can be attributed towards its propensity to develop biofilm, modification in cell membrane and to efflux antibacterial drugs. The present study explored the activity of Glycyrrhiza glabra and one of its pure compounds, glycyrrhizic acid against P. aeruginosa and their mechanism of action in terms of the effect on membrane permeability, efflux activity, and biofilm formation were determined. Minimum inhibitory concentrations were determined by using broth dilution technique. The minimum bactericidal concentrations were assessed on agar plate. The MIC of the extract and glycyrrhizic acid was found to be 200 and 100 μg ml(-1), respectively. The MBC was found to be 800 and 400 μg ml(-1) in the case of extract and glycyrrhizic acid, respectively. Time -dependent killing efficacy was also estimated. Flowcytometric analysis with staining methods was used to determine the effect of extract and glycyrrhizic acid at 2 × MIC on different physiological parameters and compared it with the standard (antibiotic). The growth of P. aeruginosa was significantly inhibited by extract and the pure compound. The herbal extract and the glycyrrhic acid were also found to effective in targeting the physiological parameters of the bacteria that involve cell membrane permeabilization, efflux activity, and biofilm formation. This study reports the antipseudomonal action of Glycyrrhiza glabra and one of its compound and provides insight into their mode of action. PMID:27392698

  1. Calf pump activity influencing venous hemodynamics in the lower extremity.

    PubMed

    Recek, Cestmir

    2013-03-01

    Calf muscle pump is the motive force enhancing return of venous blood from the lower extremity to the heart. It causes displacement of venous blood in both vertical and horizontal directions, generates ambulatory pressure gradient between thigh and lower leg veins, and bidirectional streaming within calf perforators. Ambulatory pressure gradient triggers venous reflux in incompetent veins, which induces ambulatory venous hypertension in the lower leg and foot. Bidirectional flow in calf perforators enables quick pressure equalization between deep and superficial veins of the lower leg; the outward (into the superficial veins) oriented component of the bidirectional flow taking place during calf muscle contraction is no pathological reflux but a physiological centripetal flow streaming via great saphenous vein into the femoral vein. Calf perforators are communicating channels between both systems making them conjoined vessels; they are not involved in the generation of pathological hemodynamic situations, nor do they cause ambulatory venous hypertension. The real cause why recurrences develop has not as yet been cleared. Pressure gradient arising during calf pump activity between the femoral vein and the saphenous remnant after abolition of saphenous reflux triggers biophysical and biochemical events, which might induce recurrence. Thus, abolition of saphenous reflux removes the hemodynamic disturbance, but at the same time it generates precondition for reflux recurrence and for the comeback of the previous pathological situation; this chain of events has been called hemodynamic paradox. PMID:24436580

  2. Staphylococcus aureus MnhF Mediates Cholate Efflux and Facilitates Survival under Human Colonic Conditions

    PubMed Central

    Sannasiddappa, Thippeswamy H.; Hood, Graham A.; Hanson, Kevan J.; Costabile, Adele; Gibson, Glenn R.

    2015-01-01

    Resistance to the innate defenses of the intestine is crucial for the survival and carriage of Staphylococcus aureus, a common colonizer of the human gut. Bile salts produced by the liver and secreted into the intestines are one such group of molecules with potent antimicrobial activity. The mechanisms by which S. aureus is able to resist such defenses in order to colonize and survive in the human gut are unknown. Here we show that mnhF confers resistance to bile salts, which can be abrogated by efflux pump inhibitors. MnhF mediates the efflux of radiolabeled cholic acid both in S. aureus and when heterologously expressed in Escherichia coli, rendering them resistant. Deletion of mnhF attenuated the survival of S. aureus in an anaerobic three-stage continuous-culture model of the human colon (gut model), which represents different anatomical areas of the large intestine. PMID:25824834

  3. Conditioning of Roots with Hypoxia Increases Aluminum and Acid Stress Tolerance by Mitigating Activation of K+ Efflux Channels by ROS in Barley: Insights into Cross-Tolerance Mechanisms.

    PubMed

    Ma, Yanling; Zhu, Min; Shabala, Lana; Zhou, Meixue; Shabala, Sergey

    2016-01-01

    Aluminum (Al) is prevalent in soils, but Al toxicity is manifested only under acid conditions. It causes severe damages to the root system. Short-term waterlogging stress can occur simultaneously with Al toxicity in areas with high rainfall or an inappropriate irrigation pattern. Barley (Hordeum vulgare L.) is one of the most Al-sensitive small-grained cereals. In this work, we have investigated effects of short-term treatments with hypoxia and phenolic acid (two major constraints in waterlogged soils) on root sensitivity to low-pH and Al stresses. We showed that hypoxia-primed roots maintained higher cell viability when exposed to low-pH/Al stress, in both elongation and mature root zones, and possessed superior ability to retain K(+) in response to low-pH/Al stresses. These priming effects were not related to higher H(+)-ATPase activity and better membrane potential maintenance, and could not be explained by the increased expression levels of HvHAK1, which mediates high-affinity K(+) uptake in roots. Instead, hypoxia-conditioned roots were significantly less sensitive to H2O2 treatment, indicated by the 10-fold reduction in the magnitude of K(+) efflux changes. This suggested that roots pre-treated with hypoxia desensitized reactive oxygen species (ROS)-inducible K(+) efflux channels in root epidermis, most probably via enhanced antioxidative capacity. A possible role for Ca(2+) in stress-induced ROS signaling pathways is also discussed. Overall, our results report, for the first time, the phenomenon of cross-protection between hypoxia and low-pH/Al stresses, and causally link it to the cell's ability to maintain cytosolic K(+) homeostasis. PMID:26581863

  4. Dielectric elastomer laminates for active membrane pump applications

    NASA Astrophysics Data System (ADS)

    Pope, Kimberly; Tews, Alyson; Frecker, Mary I.; Mockensturm, Eric; Goulbourne, Nakhiah C.; Snyder, Alan J.

    2004-07-01

    Previous research has demonstrated promise for the use of dielectric elastomer (DE) films in diaphragm pump applications. Because the films tend to be quite thin, single layers operate at very low pressures. To make this technology suitable for practical applications, the films may be organized into laminates which will operate at increased pressures. Radially stretched circular diaphragms of two materials were tested: 3M VHB 4905 polyacrylate and spin-cast Nusil CF19-2186 silicone. The diaphragms were stacked, each layer sharing an electrode with the adjacent layer. The stack was mounted on a sealed chamber and energized at varied electric fields while regulated pressure was applied to the interior chamber, displacing the diaphragm. The pressure-volume properties of the stacks were recorded for each activation state.

  5. Aromatic inhibitors derived from ammonia-pretreated lignocellulose hinder bacterial ethanologenesis by activating regulatory circuits controlling inhibitor efflux and detoxification

    PubMed Central

    Keating, David H.; Zhang, Yaoping; Ong, Irene M.; McIlwain, Sean; Morales, Eduardo H.; Grass, Jeffrey A.; Tremaine, Mary; Bothfeld, William; Higbee, Alan; Ulbrich, Arne; Balloon, Allison J.; Westphall, Michael S.; Aldrich, Josh; Lipton, Mary S.; Kim, Joonhoon; Moskvin, Oleg V.; Bukhman, Yury V.; Coon, Joshua J.; Kiley, Patricia J.; Bates, Donna M.; Landick, Robert

    2014-01-01

    Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a key barrier to the economically viable deployment of lignocellulosic biofuels. A chief contributor to this barrier is the impact on microbial processes and energy metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates, phenolic amides (for ammonia-pretreated biomass), phenolic aldehydes, and furfurals. To understand the bacterial pathways induced by inhibitors present in ammonia-pretreated biomass hydrolysates, which are less well studied than acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics of ammonia-pretreated corn stover hydrolysate (ACSH). To determine regulatory responses to the inhibitors normally present in ACSH, we measured transcript and protein levels in an Escherichia coli ethanologen using RNA-seq and quantitative proteomics during fermentation to ethanol of synthetic hydrolysates containing or lacking the inhibitors. Our study identified four major regulators mediating these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of these regulons was correlated with a reduced rate of ethanol production, buildup of pyruvate, depletion of ATP and NAD(P)H, and an inhibition of xylose conversion. The aromatic aldehyde inhibitor 5-hydroxymethylfurfural appeared to be reduced to its alcohol form by the ethanologen during fermentation, whereas phenolic acid and amide inhibitors were not metabolized. Together, our findings establish that the major regulatory responses to lignocellulose-derived inhibitors are mediated by transcriptional rather than translational regulators, suggest that energy consumed for inhibitor efflux and detoxification may limit biofuel production, and identify a network of regulators for future synthetic biology efforts. PMID:25177315

  6. Mycorrhiza-induced lower oxidative burst is related with higher antioxidant enzyme activities, net H2O2 effluxes, and Ca2+ influxes in trifoliate orange roots under drought stress.

    PubMed

    Zou, Ying-Ning; Huang, Yong-Ming; Wu, Qiang-Sheng; He, Xin-Hua

    2015-02-01

    Mechanisms of arbuscular mycorrhiza (AM)-induced lower oxidative burst of host plants under drought stress (DS) are not elucidated. A noninvasive microtest technology (NMT) was used to investigate the effects of Funneliformis mosseae on net fluxes of root hydrogen peroxide (H2O2) and calcium ions (Ca2+) in 5-month-old Poncirus trifoliata, in combination with catalase (CAT) and superoxide dismutase (SOD) activities as well as tissue superoxide radical (O2•-) and H2O2 concentrations under DS and well-watered (WW) conditions. A 2-month DS (55% maximum water holding capacity of growth substrates) significantly inhibited AM fungal root colonization, while AM symbiosis significantly increased plant biomass production, irrespective of water status. F. mosseae inoculation generally increased SOD and CAT activity but decreased O2•- and H2O2 concentrations in leaves and roots under WW and DS. Compared with non-AM seedlings, roots of AM seedlings had significantly higher net H2O2 effluxes and net Ca2+ influxes, especially in the meristem zone, but lower net H2O2 efflux in the elongation zone. Net Ca2+ influxes into roots were significantly positively correlated with root net H2O2 effluxes but negatively with root H2O2 concentrations. Results from this study suggest that AM-induced lower oxidative burst is related with higher antioxidant enzyme activities, root net H2O2 effluxes, and Ca2+ influxes under WW and DS. PMID:25085218

  7. Nrf2 activation ameliorates cytotoxic effects of arsenic trioxide in acute promyelocytic leukemia cells through increased glutathione levels and arsenic efflux from cells.

    PubMed

    Nishimoto, Shoichi; Suzuki, Toshihiro; Koike, Shin; Yuan, Bo; Takagi, Norio; Ogasawara, Yuki

    2016-08-15

    Carnosic acid (CA), a phenolic diterpene isolated from Rosmarinus officinalis, has been shown to activate nuclear transcription factor E2-related factor 2 (Nrf2), which plays a central role in cytoprotective responses to oxidative and electrophilic stress. Recently, the Nrf2-Kelch ECH associating protein 1 (Keap1) pathway has been associated with cancer drug resistance attributable to modulation of the expression and activation of antioxidant and detoxification enzymes. However, the exact mechanisms by which Nrf2 activation results in chemoresistance are insufficiently understood to date. This study investigated the mechanisms by which the cytotoxic effects of arsenic trioxide (ATO), an anticancer drug, were decreased in acute promyelocytic leukemia cells treated with CA, a typical activator of Nrf2 used to stimulate the Nrf2/Keap1 system. Our findings suggest that arsenic is non-enzymatically incorporated into NB4 cells and forms complexes that are dependent on intracellular glutathione (GSH) concentrations. In addition, the arsenic complexes are recognized as substrates by multidrug resistance proteins and subsequently excreted from the cells. Therefore, Nrf2-associated activation of the GSH biosynthetic pathway, followed by increased levels of intracellular GSH, are key mechanisms underlying accelerated arsenic efflux and attenuation of the cytotoxic effects of ATO. PMID:27317373

  8. Genomic potential for arsenic efflux and methylation varies among global Prochlorococcus populations.

    PubMed

    Saunders, Jaclyn K; Rocap, Gabrielle

    2016-01-01

    The globally significant picocyanobacterium Prochlorococcus is the main primary producer in oligotrophic subtropical gyres. When phosphate concentrations are very low in the marine environment, the mol:mol availability of phosphate relative to the chemically similar arsenate molecule is reduced, potentially resulting in increased cellular arsenic exposure. To mediate accidental arsenate uptake, some Prochlorococcus isolates contain genes encoding a full or partial efflux detoxification pathway, consisting of an arsenate reductase (arsC), an arsenite-specific efflux pump (acr3) and an arsenic-related repressive regulator (arsR). This efflux pathway was the only previously known arsenic detox pathway in Prochlorococcus. We have identified an additional putative arsenic mediation strategy in Prochlorococcus driven by the enzyme arsenite S-adenosylmethionine methyltransferase (ArsM) which can convert inorganic arsenic into more innocuous organic forms and appears to be a more widespread mode of detoxification. We used a phylogenetically informed approach to identify Prochlorococcus linked arsenic genes from both pathways in the Global Ocean Sampling survey. The putative arsenic methylation pathway is nearly ubiquitously present in global Prochlorococcus populations. In contrast, the complete efflux pathway is only maintained in populations which experience extremely low PO4:AsO4, such as regions in the tropical and subtropical Atlantic. Thus, environmental exposure to arsenic appears to select for maintenance of the efflux detoxification pathway in Prochlorococcus. The differential distribution of these two pathways has implications for global arsenic cycling, as their associated end products, arsenite or organoarsenicals, have differing biochemical activities and residence times. PMID:26151644

  9. Correlation of Ciprofloxacin Resistance with the AdeABC Efflux System in Acinetobacter baumannii Clinical Isolates

    PubMed Central

    Ardebili, Abdollah; Talebi, Malihe

    2014-01-01

    Background Acinetobacter baumannii is one of the most important pathogens capable of colonization in burn patients, leading to drug-resistant wound infections. This study evaluated the distribution of the AdeABC efflux system genes and their relationship to ciprofloxacin resistance in A. baumannii isolates collected from burn patients. Methods A total of 68 A. baumannii clinical strains were isolated from patients hospitalized in Motahari Burns Center in Tehran, Iran. Ciprofloxacin susceptibility was tested by the disk diffusion and agar dilution methods. PCR amplification of the adeRS-adeB drug efflux genes was performed for all resistant and susceptible isolates. To assess the role of the drug efflux pump in ciprofloxacin susceptibility, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as an efflux pump inhibitor (EPI). Results Approximately 95.6% of the Acinetobacter isolates were resistant to ciprofloxacin, with minimum inhibitory concentration (MIC) values ranging from 4 to ≥128 µg/mL. The susceptibility of 86.1% of the resistant isolates increased by factors of 2 to 64 in the presence of CCCP. All resistant isolates were positive for the adeRS-adeB genes, and 73.2% of them had mutations in the AdeRS regulatory system. Conclusions The results showed that AdeABC genes are common in A. baumannii, which might be associated with ciprofloxacin non-susceptibility, as indicated by the observed linkage to the presence of the genes essential for the activity of the AdeABC, several single mutations occurring in the adeRS regulatory system, and an increase of ciprofloxacin susceptibility in the presence of a CCCP EPI. PMID:25368818

  10. Calcium efflux activity of plasma membrane Ca2+ ATPase-4 (PMCA4) mediates cell cycle progression in vascular smooth muscle cells.

    PubMed

    Afroze, Talat; Yang, Ge; Khoshbin, Amir; Tanwir, Mansoor; Tabish, Taha; Momen, Abdul; Husain, Mansoor

    2014-03-01

    We explored the role played by plasma membrane calcium ATPase-4 (PMCA4) and its alternative splice variants in the cell cycle of vascular smooth muscle cells (VSMC). A novel variant (PMCA4e) was discovered. Quantitative real-time-PCR-quantified PMCA4 splice variant proportions differed in specific organs. The PMCA4a:4b ratio in uninjured carotid arteries (∼1:1) was significantly reduced by wire denudation injury (to ∼1:3) by modulation of alternative splicing, as confirmed by novel antibodies against PMCA4a/e and PMCA4b. Laser capture microdissection localized this shift to the media and adventitia. Primary carotid VSMC from PMCA4 knock-out (P4KO) mice showed impaired [(3)H]thymidine incorporation and G1 phase arrest as compared with wild type (P4WT). Electroporation of expression constructs encoding PMCA4a, PMCA4b, and a PMCA4b mutant lacking PDZ binding rescued this phenotype of P4KO cells, whereas a mutant with only 10% of normal Ca(2+) efflux activity could not. Microarray of early G1-synchronized VSMC showed 39-fold higher Rgs16 (NFAT (nuclear factor of activated T-cells) target; MAPK inhibitor) and 69-fold higher Decorin (G1 arrest marker) expression in P4KO versus P4WT. Validation by Western blot also revealed decreased levels of Cyclin D1 and NFATc3 in P4KO. Microarrays of P4KO VSMC rescued by PMCA4a or PMCA4b expression showed reversal of perturbed Rgs16, Decorin, and NFATc3 expression levels. However, PMCA4a rescue caused a 44-fold reduction in AP-2β, a known anti-proliferative transcription factor, whereas PMCA4b rescue resulted in a 50-fold reduction in p15 (Cyclin D1/Cdk4 inhibitor). We conclude that Ca(2+) efflux activity of PMCA4 underlies G1 progression in VSMC and that PMCA4a and PMCA4b differentially regulate specific downstream mediators. PMID:24448801

  11. Energy Metabolism and Drug Efflux in Mycobacterium tuberculosis

    PubMed Central

    Black, Philippa A.; Warren, Robin M.; Louw, Gail E.; van Helden, Paul D.; Victor, Thomas C.

    2014-01-01

    The inherent drug susceptibility of microorganisms is determined by multiple factors, including growth state, the rate of drug diffusion into and out of the cell, and the intrinsic vulnerability of drug targets with regard to the corresponding antimicrobial agent. Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains a significant source of global morbidity and mortality, further exacerbated by its ability to readily evolve drug resistance. It is well accepted that drug resistance in M. tuberculosis is driven by the acquisition of chromosomal mutations in genes encoding drug targets/promoter regions; however, a comprehensive description of the molecular mechanisms that fuel drug resistance in the clinical setting is currently lacking. In this context, there is a growing body of evidence suggesting that active extrusion of drugs from the cell is critical for drug tolerance. M. tuberculosis encodes representatives of a diverse range of multidrug transporters, many of which are dependent on the proton motive force (PMF) or the availability of ATP. This suggests that energy metabolism and ATP production through the PMF, which is established by the electron transport chain (ETC), are critical in determining the drug susceptibility of M. tuberculosis. In this review, we detail advances in the study of the mycobacterial ETC and highlight drugs that target various components of the ETC. We provide an overview of some of the efflux pumps present in M. tuberculosis and their association, if any, with drug transport and concomitant effects on drug resistance. The implications of inhibiting drug extrusion, through the use of efflux pump inhibitors, are also discussed. PMID:24614376

  12. Active magnetic bearings: As applied to centrifugal pumps

    NASA Technical Reports Server (NTRS)

    Nelik, Lev; Cooper, Paul; Jones, Graham; Galecki, Dennis; Pinckney, Frank; Kirk, Gordon

    1992-01-01

    Application of magnetic bearings to boiler feed pumps presents various attractive features, such as longer bearing life, lower maintenance costs, and improved operability through control of the rotordynamics. Magnetic bearings were fitted to an eight-stage, 600 hp boiler feed pump, which generates 2600 ft of heat at 680 gpm and 3560 rpm. In addition to the varied and severe operating environment in steady state operation of this pump in a power plant, it is also subjected to transient loads during frequent starts and stops. These loads can now be measured by the in-built instrumentation of the magnetic bearings. Following site installation, a follow-up bearing tune-up was performed, and pump transient response testing was conducted. The bearing response was completely satisfactory, ensuring trouble-free pump operation even in the range of reduced load. The experience gained so far through design and testing proves feasibility of magnetic bearings for boiler feed pumps, which sets the stage for application of even higher energy centrifugal pumps equipped with magnetic bearings.

  13. K+ efflux is the Common Trigger of NLRP3 inflammasome Activation by Bacterial Toxins and Particulate Matter

    PubMed Central

    Muñoz-Planillo, Raúl; Kuffa, Peter; Martínez-Colón, Giovanny; Smith, Brenna L.; Rajendiran, Thekkelnaycke M.; Núñez, Gabriel

    2013-01-01

    Summary The NLRP3 inflammasome is an important component of the innate immune system. However, its mechanism of activation remains largely unknown. We show that NLRP3 activators including bacterial pore-forming toxins, nigericin, ATP and particulate matter caused mitochondrial perturbation or the opening of a large membrane pore; but this was not required for NLRP3 activation. Furthermore, reactive oxygen species generation or a change in cell volume was not necessary for NLRP3 activation. Instead, the only common activity induced by all NLRP3 agonists was the permeation of the cell membrane to K+ and Na+. Notably, reduction of the intracellular K+ concentration was sufficient to activate NLRP3 whereas an increase in intracellular Na+ modulated, but was not strictly required for inflammasome activation. These results provide a unifying model for the activation of the NLRP3 inflammasome in which a drop in cytosolic K+ is the common step that is necessary and sufficient for caspase-1 activation. PMID:23809161

  14. NLRP3 inflammasome signaling is activated by low-level lysosome disruption but inhibited by extensive lysosome disruption: roles for K+ efflux and Ca2+ influx.

    PubMed

    Katsnelson, Michael A; Lozada-Soto, Kristen M; Russo, Hana M; Miller, Barbara A; Dubyak, George R

    2016-07-01

    Nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3 (NLRP3) is a cytosolic protein that nucleates assembly of inflammasome signaling platforms, which facilitate caspase-1-mediated IL-1β release and other inflammatory responses in myeloid leukocytes. NLRP3 inflammasomes are assembled in response to multiple pathogen- or environmental stress-induced changes in basic cell physiology, including the destabilization of lysosome integrity and activation of K(+)-permeable channels/transporters in the plasma membrane (PM). However, the quantitative relationships between lysosome membrane permeabilization (LMP), induction of increased PM K(+) permeability, and activation of NLRP3 signaling are incompletely characterized. We used Leu-Leu-O-methyl ester (LLME), a soluble lysosomotropic agent, to quantitatively track the kinetics and extent of LMP in relation to NLRP3 inflammasome signaling responses (ASC oligomerization, caspase-1 activation, IL-1β release) and PM cation fluxes in murine bone marrow-derived dendritic cells (BMDCs). Treatment of BMDCs with submillimolar (≤1 mM) LLME induced slower and partial increases in LMP that correlated with robust NLRP3 inflammasome activation and K(+) efflux. In contrast, supramillimolar (≥2 mM) LLME elicited extremely rapid and complete collapse of lysosome integrity that was correlated with suppression of inflammasome signaling. Supramillimolar LLME also induced dominant negative effects on inflammasome activation by the canonical NLRP3 agonist nigericin; this inhibition correlated with an increase in NLRP3 ubiquitination. LMP elicited rapid BMDC death by both inflammasome-dependent pyroptosis and inflammasome-independent necrosis. LMP also triggered Ca(2+) influx, which attenuated LLME-stimulated NLRP3 inflammasome signaling but potentiated LLME-induced necrosis. Taken together, these studies reveal a previously unappreciated signaling network that defines the coupling between LMP, changes

  15. Multidrug Pump Inhibitors Uncover Remarkable Activity of Plant Antimicrobials

    PubMed Central

    Tegos, George; Stermitz, Frank R.; Lomovskaya, Olga; Lewis, Kim

    2002-01-01

    Plant antimicrobials are not used as systemic antibiotics at present. The main reason for this is their low level of activity, especially against gram-negative bacteria. The reported MIC is often in the range of 100 to 1,000 μg/ml, orders of magnitude higher than those of common broad-spectrum antibiotics from bacteria or fungi. Major plant pathogens belong to the gram-negative bacteria, which makes the low level of activity of plant antimicrobials against this group of microorganisms puzzling. Gram-negative bacteria have an effective permeability barrier, comprised of the outer membrane, which restricts the penetration of amphipathic compounds, and multidrug resistance pumps (MDRs), which extrude toxins across this barrier. It is possible that the apparent ineffectiveness of plant antimicrobials is largely due to the permeability barrier. We tested this hypothesis in the present study by applying a combination of MDR mutants and MDR inhibitors. A panel of plant antimicrobials was tested by using a set of bacteria representing the main groups of plant pathogens. The human pathogens Pseudomonas aeruginosa, Escherichia coli, and Salmonella enterica serovar Typhimurium were also tested. The results show that the activities of the majority of plant antimicrobials were considerably greater against the gram-positive bacteria Staphylococcus aureus and Bacillus megaterium and that disabling of the MDRs in gram-negative species leads to a striking increase in antimicrobial activity. Thus, the activity of rhein, the principal antimicrobial from rhubarb, was potentiated 100- to 2,000-fold (depending on the bacterial species) by disabling the MDRs. Comparable potentiation of activity was observed with plumbagin, resveratrol, gossypol, coumestrol, and berberine. Direct measurement of the uptake of berberine, a model plant antimicrobial, confirmed that disabling of the MDRs strongly increases the level of penetration of berberine into the cells of gram-negative bacteria. These

  16. Nitrogen monoxide (NO)-mediated iron release from cells is linked to NO-induced glutathione efflux via multidrug resistance-associated protein 1

    PubMed Central

    Watts, Ralph N.; Hawkins, Clare; Ponka, Prem; Richardson, Des R.

    2006-01-01

    Nitrogen monoxide (NO) plays a role in the cytotoxic mechanisms of activated macrophages against tumor cells by inducing iron (Fe) release. We have shown that NO-mediated Fe efflux from cells required glutathione (GSH), and we have hypothesized that a GS–Fe–NO complex was released. Hence, we studied the role of the GSH-conjugate transporter multidrug resistance-associated protein 1 (MRP1) in NO-mediated Fe efflux. MCF7-VP cells overexpressing MRP1 exhibited a 3- to 4-fold increase in NO-mediated 59Fe and GSH efflux compared with WT cells (MCF7-WT) over 4 h. Similar results were found for other MRP1-overexpressing cell types but not those expressing another drug efflux pump, P-glycoprotein. NO-mediated 59Fe and GSH efflux were temperature- and energy-dependent and were significantly decreased by the GSH-depleting agent and MRP1 transport inhibitor l-buthionine-[S,R]-sulfoximine. Other MRP1 inhibitors, MK571, probenecid, and difloxacin, significantly inhibited NO-mediated 59Fe release. EPR spectroscopy demonstrated the dinitrosyl-dithiol-Fe complex (DNIC) peak in NO-treated cells was increased by MRP1 inhibitors, indicating inhibited DNIC transport from cells. The extent of DNIC accumulation correlated with the ability of MRP1 inhibitors to prevent NO-mediated 59Fe efflux. MCF7-VP cells were more sensitive than MCF7-WT cells to growth inhibition by effects of NO, which was potentiated by l-buthionine-[S,R]-sulfoximine. These data indicate the importance of GSH in NO-mediated inhibition of proliferation. Collectively, NO stimulates Fe and GSH efflux from cells via MRP1. Active transport of NO by MRP1 overcomes diffusion that is inefficient and nontargeted, which has broad ramifications for understanding NO biology. PMID:16679408

  17. The Arabidopsis AtPP2CA Protein Phosphatase Inhibits the GORK K+ Efflux Channel and Exerts a Dominant Suppressive Effect on Phosphomimetic-activating Mutations.

    PubMed

    Lefoulon, Cécile; Boeglin, Martin; Moreau, Bertrand; Véry, Anne-Aliénor; Szponarski, Wojciech; Dauzat, Myriam; Michard, Erwan; Gaillard, Isabelle; Chérel, Isabelle

    2016-03-18

    The regulation of the GORK (Guard Cell Outward Rectifying) Shaker channel mediating a massive K(+) efflux in Arabidopsis guard cells by the phosphatase AtPP2CA was investigated. Unlike the gork mutant, the atpp2ca mutants displayed a phenotype of reduced transpiration. We found that AtPP2CA interacts physically with GORK and inhibits GORK activity in Xenopus oocytes. Several amino acid substitutions in the AtPP2CA active site, including the dominant interfering G145D mutation, disrupted the GORK-AtPP2CA interaction, meaning that the native conformation of the AtPP2CA active site is required for the GORK-AtPP2CA interaction. Furthermore, two serines in the GORK ankyrin domain that mimic phosphorylation (Ser to Glu) or dephosphorylation (Ser to Ala) were mutated. Mutations mimicking phosphorylation led to a significant increase in GORK activity, whereas mutations mimicking dephosphorylation had no effect on GORK. In Xenopus oocytes, the interaction of AtPP2CA with "phosphorylated" or "dephosphorylated" GORK systematically led to inhibition of the channel to the same baseline level. Single-channel recordings indicated that the GORK S722E mutation increases the open probability of the channel in the absence, but not in the presence, of AtPP2CA. The dephosphorylation-independent inactivation mechanism of GORK by AtPP2CA is discussed in relation with well known conformational changes in animal Shaker-like channels that lead to channel opening and closing. In plants, PP2C activity would control the stomatal aperture by regulating both GORK and SLAC1, the two main channels required for stomatal closure. PMID:26801610

  18. The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

    PubMed Central

    Plésiat, Patrick

    2015-01-01

    SUMMARY The global emergence of multidrug-resistant Gram-negative bacteria is a growing threat to antibiotic therapy. The chromosomally encoded drug efflux mechanisms that are ubiquitous in these bacteria greatly contribute to antibiotic resistance and present a major challenge for antibiotic development. Multidrug pumps, particularly those represented by the clinically relevant AcrAB-TolC and Mex pumps of the resistance-nodulation-division (RND) superfamily, not only mediate intrinsic and acquired multidrug resistance (MDR) but also are involved in other functions, including the bacterial stress response and pathogenicity. Additionally, efflux pumps interact synergistically with other resistance mechanisms (e.g., with the outer membrane permeability barrier) to increase resistance levels. Since the discovery of RND pumps in the early 1990s, remarkable scientific and technological advances have allowed for an in-depth understanding of the structural and biochemical basis, substrate profiles, molecular regulation, and inhibition of MDR pumps. However, the development of clinically useful efflux pump inhibitors and/or new antibiotics that can bypass pump effects continues to be a challenge. Plasmid-borne efflux pump genes (including those for RND pumps) have increasingly been identified. This article highlights the recent progress obtained for organisms of clinical significance, together with methodological considerations for the characterization of MDR pumps. PMID:25788514

  19. Hypoxic Stress Induced by Hydralazine Leads to a Loss of Blood-Brain Barrier Integrity and an Increase in Efflux Transporter Activity

    PubMed Central

    Chatard, Morgane; Puech, Clémentine

    2016-01-01

    Understanding cellular and molecular mechanisms induced by hypoxic stress is crucial to reduce blood-brain barrier (BBB) disruption in some neurological diseases. Since the brain is a complex organ, it makes the interpretation of in vivo data difficult, so BBB studies are often investigated using in vitro models. However, the investigation of hypoxia in cellular pathways is complex with physical hypoxia because HIF-1α (factor induced by hypoxia) has a short half-life. We had set up an innovative and original method of induction of hypoxic stress by hydralazine that was more reproducible, which allowed us to study its impact on an in vitro BBB model. Our results showed that hydralazine, a mimetic agent of the hypoxia pathway, had the same effect as physical hypoxia, with few cytotoxicity effects on our cells. Hypoxic stress led to an increase of BBB permeability which corresponded to an opening of our BBB model. Study of tight junction proteins revealed that this hypoxic stress decreased ZO-1 but not occludin expression. In contrast, cells established a defence mechanism by increasing expression and activity of their efflux transporters (Pgp and MRP-1). This induction method of hypoxic stress by hydralazine is simple, reproducible, controllable and suitable to understand the cellular and molecular mechanisms involved by hypoxia on the BBB. PMID:27337093

  20. Activation of GPR55 Receptors Exacerbates oxLDL-Induced Lipid Accumulation and Inflammatory Responses, while Reducing Cholesterol Efflux from Human Macrophages

    PubMed Central

    Lanuti, Mirko; Talamonti, Emanuela; Maccarrone, Mauro; Chiurchiù, Valerio

    2015-01-01

    The G protein-coupled receptor GPR55 has been proposed as a new cannabinoid receptor associated with bone remodelling, nervous system excitability, vascular homeostasis as well as in several pathophysiological conditions including obesity and cancer. However, its physiological role and underlying mechanism remain unclear. In the present work, we demonstrate for the first time its presence in human macrophages and its increased expression in ox-LDL-induced foam cells. In addition, pharmacological activation of GPR55 by its selective agonist O-1602 increased CD36- and SRB-I-mediated lipid accumulation and blocked cholesterol efflux by downregulating ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, as well as enhanced cytokine- and pro-metalloprotease-9 (pro-MMP-9)-induced proinflammatory responses in foam cells. Treatment with cannabidiol, a selective antagonist of GPR55, counteracted these pro-atherogenic and proinflammatory O-1602-mediated effects. Our data suggest that GPR55 could play deleterious role in ox-LDL-induced foam cells and could be a novel pharmacological target to manage atherosclerosis and other related cardiovascular diseases. PMID:25970609

  1. Regulation of P-glycoprotein efflux activity by Z-guggulsterone of Commiphora mukul at the blood-brain barrier.

    PubMed

    Xu, Hong-Bin; Yu, Jing; Xu, Lu-Zhong; Fu, Jun

    2016-04-15

    The present study was to investigate whether Z-guggulsterone had the regulatory effect on the activity and expression of P-glycoprotein in rat brain microvessel endothelial cells (rBMECs) and in rat brain. Inorganic phosphate liberation assay, high performance liquid chromatography, and western blot analysis were performed to assess the P-glycoprotein ATPase activity, the accumulation of NaF and rhodamine 123, and P-glycoprotein and MRP1 expression. The results showed that Z-guggulsterone (0-100 μM) significantly enhanced basal P-glycoprotein ATPase activity in a concentration-dependent manner. Tetrandrine (0.1, 0.3, 1 μM) or cyclosporine A (0.1, 0.3, 1 μM) had non-competitively inhibitory manner on Z-guggulsterone-stimulated P-glycoprotein ATPase activity, suggesting that Z-guggulsterone might have unique binding site or regulating site on P-glycoprotein. However, Z-guggulsterone (30, 100 μM) had almost no influence on MRP1 expression in rBMECs. Further results revealed that Z-guggulsterone (50mg/kg) significantly increased the accumulation of rhodamine 123 by down-regulating P-glycoprotein expression in rat brain, as compared with control (P<0.05). Our studies suggested that Z-guggulsterone potentially inhibited the activity and expression of P-glycoprotein in rBMECs and in rat brain. PMID:27000241

  2. The quorum-sensing molecule farnesol is a modulator of drug efflux mediated by ABC multidrug transporters and synergizes with drugs in Candida albicans.

    PubMed

    Sharma, Monika; Prasad, Rajendra

    2011-10-01

    Overexpression of the CaCDR1-encoded multidrug efflux pump protein CaCdr1p (Candida drug resistance protein 1), belonging to the ATP binding cassette (ABC) superfamily of transporters, is one of the most prominent contributors of multidrug resistance (MDR) in Candida albicans. Thus, blocking or modulating the function of the drug efflux pumps represents an attractive approach in combating MDR. In the present study, we provide first evidence that the quorum-sensing molecule farnesol (FAR) is a specific modulator of efflux mediated by ABC multidrug transporters, such as CaCdr1p and CaCdr2p of C. albicans and ScPdr5p of Saccharomyces cerevisiae. Interestingly, FAR did not modulate the efflux mediated by the multidrug extrusion pump protein CaMdr1p, belonging to the major facilitator superfamily (MFS). Kinetic data revealed that FAR competitively inhibited rhodamine 6G efflux in CaCdr1p-overexpressing cells, with a simultaneous increase in an apparent K(m) without affecting the V(max) values and the ATPase activity. We also observed that when used in combination, FAR at a nontoxic concentration synergized with the drugs at their respective nonlethal concentrations, as was evident from their <0.5 fractional inhibitory concentration index (FICI) values and from the drop of 14- to 64-fold in the MIC(80) values in the wild-type strain and in azole-resistant clinical isolates of C. albicans. Our biochemical experiments revealed that the synergistic interaction of FAR with the drugs led to reactive oxygen species accumulation, which triggered early apoptosis, and that both could be partly reversed by the addition of an antioxidant. Collectively, FAR modulates drug extrusion mediated exclusively by ABC proteins and is synergistic to fluconazole (FLC), ketoconazole (KTC), miconazole (MCZ), and amphotericin (AMB). PMID:21768514

  3. CO2 efflux from subterranean nests of ant communities in a seasonal tropical forest, Thailand

    PubMed Central

    Hasin, Sasitorn; Ohashi, Mizue; Yamada, Akinori; Hashimoto, Yoshiaki; Tasen, Wattanachai; Kume, Tomonori; Yamane, Seiki

    2014-01-01

    Many ant species construct subterranean nests. The presence of their nests may explain soil respiration “hot spots”, an important factor in the high CO2 efflux from tropical forests. However, no studies have directly measured CO2 efflux from ant nests. We established 61 experimental plots containing 13 subterranean ant species to evaluate the CO2 efflux from subterranean ant nests in a tropical seasonal forest, Thailand. We examined differences in nest CO2 efflux among ant species. We determined the effects of environmental factors on nest CO2 efflux and calculated an index of nest structure. The mean CO2 efflux from nests was significantly higher than those from the surrounding soil in the wet and dry seasons. The CO2 efflux was species-specific, showing significant differences among the 13 ant species. The soil moisture content significantly affected nest CO2 efflux, but there was no clear relationship between nest CO2 efflux and nest soil temperature. The diameter of the nest entrance hole affected CO2 efflux. However, there was no significant difference in CO2 efflux rates between single-hole and multiple-hole nests. Our results suggest that in a tropical forest ecosystem the increase in CO2 efflux from subterranean ant nests is caused by species-specific activity of ants, the nest soil environment, and nest structure. PMID:25505521

  4. Studies of cryocooler based cryosorption pump with activated carbon panels operating at 11K

    NASA Astrophysics Data System (ADS)

    Kasthurirengan, S.; Behera, Upendra; Gangradey, Ranjana; Udgata, Swarup; Krishnamoorthy, V.

    2012-11-01

    Cryosorption pump is the only solution for pumping helium and hydrogen in fusion reactors. It is chosen because it offers highest pumping speed as well as the only suitable pump for the harsh environments in a tokamak. Towards the development of such cryosorption pumps, the optimal choice of the right activated carbon panels is essential. In order to characterize the performance of the panels with indigenously developed activated carbon, a cryocooler based cryosorption pump with scaled down sizes of panels is experimented. The results are compared with the commercial cryopanel used in a CTI cryosorption (model: Cryotorr 7) pump. The cryopanel is mounted on the cold head of the second stage GM cryocooler which cools the cryopanel down to 11K with first stage reaching about ~50K. With no heat load, cryopump gives the ultimate vacuum of 2.1E-7 mbar. The pumping speed of different gases such as nitrogen, argon, hydrogen, helium are tested both on indigenous and commercial cryopanel. These studies serve as a bench mark towards the development of better cryopanels to be cooled by liquid helium for use with tokamak.

  5. Continuous monitoring of soil gas efflux with Forced Diffusion (FD) chamber technique in a tundra ecosystem, Alaska

    NASA Astrophysics Data System (ADS)

    Kim, Y.; Park, S. J.; Lee, B. Y.

    2015-12-01

    Continuous measurements of soil carbon dioxide (CO2) efflux provide essential information about the soil carbon budget in response to an abruptly changing climate at Arctic and Subarctic scales. The Forced Diffusion (FD) chamber technique has a gas permeable membrane, which passively regulates the mixing of atmosphere and soil air in the chamber, in place of the active pumping system inside a regular dynamics efflux chamber system (Risk et al., 2011). Here the system has been modified the sampling routine to eliminate the problem of sensor drift. After that, we deployed the FD chamber system in a tundra ecosystem over the discontinuous permafrost regime of Council, Alaska. The representative understory plants are tussock (17 %), lichen (32 %), and moss (51 %), within a 40 נ40 m plot at an interval of five meters (81 points total) for efflux-measurement by dynamic chamber. The FD chamber monitored soil CO2 efflux from moss, lichen, and tussock regimes at an interval of 30 min during the growing season of 2015. As the results, mean soil CO2 effluxes in sphagnum moss, lichen, and tussock were 1.98 ± 1.10 (coefficient of variance: 55.8 %), 3.34 ± 0.84 (CV: 25.0 %), and 5.32 ± 1.48 (CV: 27.8 %) gCO2/m2/d, respectively. The difference between the 30-min efflux interval and the average efflux of three 10-min intervals is not significant for sphagnum (n = 196), lichen (n = 918), and tussock (n = 918) under a 95 % confidence level. The deploying interval was then set to 30 min and synchronized with eddy covariance tower data. During the deployment period of 2015, soil CO2 efflux over moss, lichen, and tussock using the FD chamber system were 44 ± 24, 73 ± 18, and 117 ± 33 gCO2/m2/period, respectively. Using the dynamic chamber, mean ecosystem respiration (Re) ranges for moss, lichen, and tussock were 2.2-2.6, 1.8-2.0, and 3.3-3.6 gCO2/m2/d, respectively, during June and July of 2015. These techniques provide the representativeness of spatiotemporal variation of soil

  6. Uncoupling of attenuated myo-(3H)inositol uptake and dysfunction in Na(+)-K(+)-ATPase pumping activity in hypergalactosemic cultured bovine lens epithelial cells

    SciTech Connect

    Cammarata, P.R.; Tse, D.; Yorio, T. )

    1991-06-01

    Attenuation of both the active transport of myo-inositol and Na(+)-K(+)-ATPase pumping activity has been implicated in the onset of sugar cataract and other diabetic complications in cell culture and animal models of the disease. Cultured bovine lens epithelial cells (BLECs) maintained in galactose-free Eagle's minimal essential medium (MEM) or 40 mM galactose with and without sorbinil for up to 5 days were examined to determine the temporal effects of hypergalactosemia on Na(+)-K(+)-ATPase and myo-inositol uptake. The Na(+)-K(+)-ATPase pumping activity after 5 days of continuous exposure to galactose did not change, as demonstrated by 86Rb uptake. The uptake of myo-(3H)inositol was lowered after 20 h of incubation in galactose and remained below that of the control throughout the 5-day exposure period. The coadministration of sorbinil to the galactose medium normalized the myo-(3H)inositol uptake. No significant difference in the rates of passive efflux of myo-(3H)inositol or 86Rb from preloaded galactose-treated and control cultures was observed. Culture-media reversal studies were also carried out to determine whether the galactose-induced dysfunction in myo-inositol uptake could be corrected. BLECs were incubated in galactose for 5 days, then changed to galactose-free physiological medium with and without sorbinil for a 1-day recovery period. myo-Inositol uptake was reduced to 34% of control after 6 days of continuous exposure to galactose. Within 24 h of media reversal, myo-inositol uptake returned to or exceeded control values in BLECs switched to either MEM or MEM with sorbinil.2+ reversible and occurred independently of changes in Na(+)-K(+)-ATPase pumping activity in cultured lens epithelium, indicating that the two parameters are not strictly associated and that the deficit in myo-inositol uptake occurs rapidly during hypergalactosemia.

  7. Cation Activation of the Basolateral Sodium-Potassium Pump in Turtle Colon

    PubMed Central

    Halm, D R; Dawson, D C

    1983-01-01

    The current generated by electrogenic sodium-potassium exchange at the basolateral membrane of the turtle colon can be measured directly in tissues that have been treated with serosal barium (to block the basolateral potassium conductance) and mucosal amphotericin B (to reduce the cation selectivity of the apical membrane). We studied the activation of this pump current by mucosal sodium and serosal potassium, rubidium, cesium, and ammonium. The kinetics of sodium activation were consistent with binding to three independent sites on the cytoplasmic side of the pump. The pump was not activated by cellular lithium ions. The kinetics of serosal cation activation were consistent with binding to two independent sites with the selectivity Rb > K > Cs > NH4. The properties and kinetics of the basolateral Na/K pump in the turtle colon are at least qualitatively similar to those ofthe well-characterized Na/K-ATPase of the human red blood cell . PMID:24244010

  8. Induction of Expression and Functional Activity of P-glycoprotein Efflux Transporter by Bioactive Plant Natural Products

    PubMed Central

    Abuznait, Alaa H.; Qosa, Hisham; O’Connell, Nicholas D.; Akbarian-Tefaghi, Jessica; Sylvester, Paul W.; El Sayed, Khalid A.; Kaddoumi, Amal

    2011-01-01

    The effect of bioactive plant natural products on the expression and functional activity of P-glycoprotein (P-gp) is poorly understood. Interactions of bioactive plant-based food and dietary supplements with P-gp can cause significant alteration of pharmacokinetic properties of P-gp substrate drugs when used in combination. This can augment toxicity and/or interfere with the drug’s therapeutic outcomes. This study investigated the effects of diverse commonly used plant natural products on the expression and activity of P-gp in human adenocarcinoma cells (LS-180). These natural products included the tobacco cembranoid (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (cembratriene), the palm oil-derived γ-tocotrienol, the extra-virgin olive oil-derived secoiridoid oleocanthal, and the triterpene acid asiatic acid derived from Melaleuca ericifolia and abundant in several other common plant dietary supplements. Treatment with 25 μM of cembratriene, oleocanthal, γ-tocotrienol, or asiatic acid showed 2.3-3.0-fold increase in P-gp expression as demonstrated by Western blotting. These results were consistent with those obtained by quantitative analysis of fluorescent micrographs for P-gp. Accumulation studies demonstrated 31-38% decrease in rhodamine 123 intracellular levels when LS-180 cells were treated with the investigated compounds as a result of P-gp induction. Bioactive natural products can up-regulate the P-gp expression and functionality, which may induce herb/food-drug interactions when concomitantly used with P-gp substrate drugs. PMID:21851848

  9. Interference study of a diode-pumped Nd : GGG active disk

    SciTech Connect

    Bufetova, G A; Nikolaev, D A; Trikshev, A I; Tsvetkov, V B; Shcherbakov, Ivan A

    2011-08-31

    We present the results of interference studies of a diode-pumped active disk element made of a gadolinium - gallium - garnet (GGG:Nd) crystal. The disk is cut perpendicular to the crystallographic axis [001], along which the pump beam propagates. With absorbing pump radiation, a thermal lens, which has been investigated by the interference methods using linearly polarised probe radiation, is formed in the disk. At the absorbed pump power up to 12 W, the interference pattern near the disk surface is a system of concentric rings. With increasing absorbed pump power up to 24 W, the outer rings transform into hexagons. It is shown that such an interference pattern in the garnet crystal is caused by thermally induced birefringence. (lasers)

  10. Proton pumping ATPase mediated fungicidal activity of two essential oil components.

    PubMed

    Bhatia, Rimple; Shreaz, Sheikh; Khan, Neelofar; Muralidhar, Sumathi; Basir, Seemi F; Manzoor, Nikhat; Khan, Luqman A

    2012-10-01

    This work evaluates the antifungal activity of two essential oil components against 28 clinical isolates (17 sensitive, 11 resistant) and 3 standard laboratory strains of Candida. Growth of the organisms was significantly effected in both solid and liquid media at different test compound concentrations. The minimum inhibitory concentrations (MICs) of Isoeugenol (compound 1) against 31 strains of Candida ranged 100-250 μg/ml and those of o -methoxy cinnamaldehyde (compound 2) ranged 200-500 μg/ml, respectively. Insight studies to mechanism suggested that these compounds exert antifungal activity by targeting H(+)-ATPase located in the membranes of pathogenic Candida species. At their respective MIC(90) average inhibition of H(+)-efflux for standard, clinical and resistant isolates caused by compound 1 and compound 2 was 70%, 74%, 82% and 42%, 42% and 43%. Respective inhibition of H(+)-efflux by fluconazole (5 μg/ml) was 94%, 92% and 10%. Inhibition of H(+)-ATPase leads to intracellular acidification and cell death. SEM analysis of Candida cells showed cell membrane breakage and alterations in morphology. Haemolytic activity on human erythrocytes was studied to exclude the possibility of further associated cytotoxicity. PMID:22143929

  11. Inhibition of the active lymph pump by flow in rat mesenteric lymphatics and thoracic duct

    NASA Technical Reports Server (NTRS)

    Gashev, Anatoliy A.; Davis, Michael J.; Zawieja, David C.; Delp, M. D. (Principal Investigator)

    2002-01-01

    There are only a few reports of the influence of imposed flow on an active lymph pump under conditions of controlled intraluminal pressure. Thus, the mechanisms are not clearly defined. Rat mesenteric lymphatics and thoracic ducts were isolated, cannulated and pressurized. Input and output pressures were adjusted to impose various flows. Lymphatic systolic and diastolic diameters were measured and used to determine contraction frequency and pump flow indices. Imposed flow inhibited the active lymph pump in both mesenteric lymphatics and in the thoracic duct. The active pump of the thoracic duct appeared more sensitive to flow than did the active pump of the mesenteric lymphatics. Imposed flow reduced the frequency and amplitude of the contractions and accordingly the active pump flow. Flow-induced inhibition of the active lymph pump followed two temporal patterns. The first pattern was a rapidly developing inhibition of contraction frequency. Upon imposition of flow, the contraction frequency immediately fell and then partially recovered over time during continued flow. This effect was dependent on the magnitude of imposed flow, but did not depend on the direction of flow. The effect also depended upon the rate of change in the direction of flow. The second pattern was a slowly developing reduction of the amplitude of the lymphatic contractions, which increased over time during continued flow. The inhibition of contraction amplitude was dependent on the direction of the imposed flow, but independent of the magnitude of flow. Nitric oxide was partly but not completely responsible for the influence of flow on the mesenteric lymph pump. Exposure to NO mimicked the effects of flow, and inhibition of the NO synthase by N (G)-monomethyl-L-arginine attenuated but did not completely abolish the effects of flow.

  12. Potent and selective mediators of cholesterol efflux

    DOEpatents

    Bielicki, John K; Johansson, Jan

    2015-03-24

    The present invention provides a family of non-naturally occurring polypeptides having cholesterol efflux activity that parallels that of full-length apolipoproteins (e.g., Apo AI and Apo E), and having high selectivity for ABAC1 that parallels that of full-length apolipoproteins. The invention also provides compositions comprising such polypeptides, methods of identifying, screening and synthesizing such polypeptides, and methods of treating, preventing or diagnosing diseases and disorders associated with dyslipidemia, hypercholesterolemia and inflammation.

  13. The activation of the sodium pump in pig red blood cells by internal and external cations.

    PubMed

    Brand, S C; Whittam, R

    1985-05-30

    A study has been made with pig red blood cells of the activation of the sodium pump by internal and external cations. Cell Na and K concentrations were altered using a PCMBS cation loading procedure. The procedure was characterised for resultant ionic conditions, maintenance of ATP levels and fragility. The activation of the sodium pump by external K was measured in cells suspended in choline (Na-free) solutions. External Cs was used as a substitute for K and elicited lower rates of pump activity. Both the Vmax and apparent Km for 42K influx and 134Cs influx increased as internal Na concentration was raised (within the non-saturating range). Vmax/apparent Km ratios for cation influx were constant. Raising external Cs concentration exerted a similar influence on pump activation by internal Na: both the maximum pump velocity and the apparent Na-site dissociation constant (K'Na) increased. The results provide evidence for a transmembrane connection between cation binding sites on opposite faces of the membrane and are consistent with a consecutive model for the sodium pump in pig red blood cells. PMID:2581622

  14. Trimerized apolipoprotein A-I (TripA) forms lipoproteins, activates lecithin: cholesterol acyltransferase, elicits lipid efflux, and is transported through aortic endothelial cells.

    PubMed

    Ohnsorg, Pascale M; Mary, Jean-Luc; Rohrer, Lucia; Pech, Michael; Fingerle, Jürgen; von Eckardstein, Arnold

    2011-12-01

    Apolipoprotein A-I (apoA-I) exerts many potentially anti-atherogenic properties and is therefore attractive for prevention and therapy of coronary heart disease. Since induction of apoA-I production by small molecules has turned out as difficult, application of exogenous apoA-I is pursued as an alternative therapeutic option. To counteract fast renal filtration of apoA-I, a trimeric high-molecular weight variant of apoA-I (TripA) was produced by recombinant technology. We compared TripA and apoA-I for important properties in reverse cholesterol transport. Reconstituted high-density lipoproteins (rHDL) containing TripA or apoA-I together with palmitoyl-2-oleyl-phosphatidylcholine (POPC) differed slightly by size. Compared to apoA-I, TripA activated lecithin:cholesterol acyltransferase (LCAT) with similar maximal velocity but concentration leading to half maximal velocity was slightly reduced (K(m)=2.1±0.3μg/mL vs. 0.59±0.06μg/mL). Both in the lipid-free form and as part of rHDL, TripA elicited cholesterol efflux from THP1-derived macrophages with similar kinetic parameters and response to liver-X-receptor activation as apoA-I. Lipid-free TripA is bound and transported by aortic endothelial cells through mechanisms which are competed by apoA-I and TripA and inhibited by knock-down of ATP-binding cassette transporter (ABC) A1. Pre-formed TripA/POPC particles were bound and transported by endothelial cells through mechanisms which are competed by excess native HDL as well as reconstituted HDL containing either apoA-I or TripA and which involve ABCG1 and scavenger receptor B1 (SR-BI). In conclusion, apoA-I and TripA show similar in vitro properties which are important for reverse cholesterol transport. These findings are important for further development of TripA as an anti-atherosclerotic drug. PMID:21930241

  15. Candida Efflux ATPases and Antiporters in Clinical Drug Resistance.

    PubMed

    Prasad, Rajendra; Rawal, Manpreet Kaur; Shah, Abdul Haseeb

    2016-01-01

    An enhanced expression of genes encoding ATP binding cassette (ABC) and major facilitator superfamily (MFS) transport proteins are known to contribute to the development of tolerance to antifungals in pathogenic yeasts. For example, the azole resistant (AR) clinical isolates of the opportunistic human fungal pathogen Candida albicans show an overexpression of CDR1 and/or CaMDR1 belonging to ABC and MFS, superfamilies, respectively. The reduced accumulation (due to rapid efflux) of drugs in AR isolates confirms the role of efflux pump proteins in the development of drug tolerance. Considering the importance of major multidrug transporters, the focus of recent research has been to understand the structure and function of these proteins which could help to design inhibitors/modulators of these pump proteins. This chapter focuses on some aspects of the structure and function of yeast transporter proteins particularly in relation to MDR in Candida. PMID:26721282

  16. The multi-xenobiotic resistance (MXR) efflux activity in hemocytes of Mytilus edulis is mediated by an ATP binding cassette transporter of class C (ABCC) principally inducible in eosinophilic granulocytes.

    PubMed

    Rioult, Damien; Pasquier, Jennifer; Boulangé-Lecomte, Céline; Poret, Agnès; Abbas, Imane; Marin, Matthieu; Minier, Christophe; Le Foll, Frank

    2014-08-01

    In marine and estuarine species, immunotoxic and/or immunomodulatory mechanisms are the crossroad of interactions between xenobiotics, microorganisms and physicochemical variations of the environment. In mussels, immunity relies exclusively on innate responses carried out by cells collectively called hemocytes and found in the open hemolymphatic circulatory system of these organisms. However, hemocytes do not form a homogenous population of immune cells since distinct subtypes of mussel blood cells can be distinguished by cytochemistry, flow cytometry or cell motility analysis. Previous studies have also shown that these cells are able to efflux xenobiotics by means of ATP binding cassette (ABC) transporter activities conferring a multixenobiotic resistance (MXR) phenotype. ABC transporters corresponding to vertebrate class B/P-glycoprotein (P-gp) and to class C/multidrug resistance related protein (MRP) are characterized in Mytilidae. Herein, we have investigated the relative contributions of ABCB- and ABCC-mediated efflux within the different hemocyte subpopulations of Mytilus edulis mussels, collected from areas differentially impacted by chemical contaminants in Normandy (France). RT-PCR analyses provide evidence for the presence of ABCB and ABCC transporters transcripts in hemocytes. Immunodetection of ABCB/P-gp with the monoclonal antibody UIC2 in living hemocytes revealed that expression was restricted to granular structures of spread cells. Efflux transporter activities, with calcein-AM as fluorescent probe, were measured by combining flow cytometry to accurate Coulter cell size measurements in order to get a cell-volume normalized fluorescence concentration. In these conditions, basal fluorescence levels were higher in hemocytes originating from Yport (control site) than in cells collected from the harbor of Le Havre, where mussels are more exposed to with persistent pollutants. By using specific ABCB/P-gp (verapamil, PSC833, zosuquidar) and ABCC/MRP (MK

  17. Negative relationships between erythrocyte Ca-pump activity and lead levels in mothers and newborns.

    PubMed

    Campagna, D; Huel, G; Hellier, G; Girard, F; Sahuquillo, J; Fagot-Campagna, A; Godin, J; Blot, P

    2000-12-01

    Lead poisoning induces hematological, gastrointestinal and neurological dysfunctions. One of the potential mechanisms is the inhibition of calcium-pump (Ca-pump), a transport protein. We investigated the effects of an environmental low lead exposure on Ca-pump activity in 247 mothers and their newborns. Maternal and cord blood, and newborn and mother hair, were sampled at delivery. Geometric means for mother and cord blood lead (Pb-B), and for mother and newborn hair lead (Pb-H), were 6.3 and 4.8 microg/dl, and 1.7 and 1.1 microg/g. Means for mother and cord basal Ca-pump activities were 2,442 and 2,675 nM/mg/hr. Mother enzymatic activity was negatively related to her Pb-B and Pb-H and to the cord Pb-B and newborn Pb-H levels. Newborn enzymatic activity was negatively related to his Pb-H level only. Adjustment for gestational age, child's sex, mother's age at delivery, alcohol, coffee and tea consumption, and smoking habits during pregnancy did not modify these relationships. Our findings support the hypothesis that lead toxicity could be in part mediated by a reduction of Ca-pump activity. This effect could be observed at low environmental exposure, in mothers and newborns. PMID:11191638

  18. Kinetics and stoichiometry of coupled Na efflux and Ca influx (Na/Ca exchange) in barnacle muscle cells.

    PubMed

    Rasgado-Flores, H; Santiago, E M; Blaustein, M P

    1989-06-01

    Coupled Na+ exit/Ca2+ entry (Na/Ca exchange operating in the Ca2+ influx mode) was studied in giant barnacle muscle cells by measuring 22Na+ efflux and 45Ca2+ influx in internally perfused, ATP-fueled cells in which the Na+ pump was poisoned by 0.1 mM ouabain. Internal free Ca2+, [Ca2+]i, was controlled with a Ca-EGTA buffering system containing 8 mM EGTA and varying amounts of Ca2+. Ca2+ sequestration in internal stores was inhibited with caffeine and a mitochondrial uncoupler (FCCP). To maximize conditions for Ca2+ influx mode Na/Ca exchange, and to eliminate tracer Na/Na exchange, all of the external Na+ in the standard Na+ sea water (NaSW) was replaced by Tris or Li+ (Tris-SW or LiSW, respectively). In both Na-free solutions an external Ca2+ (Cao)-dependent Na+ efflux was observed when [Ca2+]i was increased above 10(-8) M; this efflux was half-maximally activated by [Ca2+]i = 0.3 microM (LiSW) to 0.7 microM (Tris-SW). The Cao-dependent Na+ efflux was half-maximally activated by [Ca2+]o = 2.0 mM in LiSW and 7.2 mM in Tris-SW; at saturating [Ca2+]o, [Ca2+]i, and [Na+]i the maximal (calculated) Cao-dependent Na+ efflux was approximately 75 pmol#cm2.s. This efflux was inhibited by external Na+ and La3+ with IC50's of approximately 125 and 0.4 mM, respectively. A Nai-dependent Ca2+ influx was also observed in Tris-SW. This Ca2+ influx also required [Ca2+]i greater than 10(-8) M. Internal Ca2+ activated a Nai-independent Ca2+ influx from LiSW (tracer Ca/Ca exchange), but in Tris-SW virtually all of the Cai-activated Ca2+ influx was Nai-dependent (Na/Ca exchange). Half-maximal activation was observed with [Na+]i = 30 mM. The fact that internal Ca2+ activates both a Cao-dependent Na+ efflux and a Nai-dependent Ca2+ influx in Tris-SW implies that these two fluxes are coupled; the activating (intracellular) Ca2+ does not appear to be transported by the exchanger. The maximal (calculated) Nai-dependent Ca2+ influx was -25 pmol/cm2.s. At various [Na+]i between 6 and 106 m

  19. Natriuretic peptides stimulate the cardiac sodium pump via NPR-C-coupled NOS activation.

    PubMed

    William, M; Hamilton, E J; Garcia, A; Bundgaard, H; Chia, K K M; Figtree, G A; Rasmussen, H H

    2008-04-01

    Natriuretic peptides (NPs) and their receptors (NPRs) are expressed in the heart, but their effects on myocyte function are poorly understood. Because NPRs are coupled to synthesis of cGMP, an activator of the sarcolemmal Na(+)-K(+) pump, we examined whether atrial natriuretic peptide (ANP) regulates the pump. We voltage clamped rabbit ventricular myocytes and identified electrogenic Na(+)-K(+) pump current (arising from the 3:2 Na(+):K(+) exchange and normalized for membrane capacitance) as the shift in membrane current induced by 100 micromol/l ouabain. Ten nanomoles per liter ANP stimulated the Na(+)-K(+) pump when the intracellular compartment was perfused with pipette solutions containing 10 mmol/l Na(+) but had no effect when the pump was at near maximal activation with 80 mmol/l Na(+) in the pipette solution. Stimulation was abolished by inhibition of cGMP-activated protein kinase with KT-5823, nitric oxide (NO)-activated guanylyl cyclase with 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), or NO synthase with N(G)-nitro-L-arginine methyl ester (L-NAME). Since synthesis of cGMP by NPR-A and NPR-B is not NO dependent or ODQ sensitive, we exposed myocytes to AP-811, a highly selective ligand for the NPR-C "clearance" receptor. It abolished ANP-induced pump stimulation. Conversely, the selective NPR-C agonist ANP(4-23) reproduced stimulation. The stimulation was blocked by l-NAME. To examine NO production in response to ANP(4-23), we loaded myocytes with the NO-sensitive fluorescent dye diacetylated diaminofluorescein-2 and examined them by confocal microscopy. ANP(4-23) induced a significant increase in fluorescence, which was abolished by L-NAME. We conclude that NPs stimulate the Na(+)-K(+) pump via an NPR-C and NO-dependent pathway. PMID:18272821

  20. Case report: A novel apolipoprotein A-I missense mutation apoA-I (Arg149Ser)Boston associated with decreased lecithin-cholesterol acyltransferase activation and cellular cholesterol efflux.

    PubMed

    Anthanont, Pimjai; Asztalos, Bela F; Polisecki, Eliana; Zachariah, Benoy; Schaefer, Ernst J

    2015-01-01

    We report a novel heterozygous apolipoprotein A-I (apoA-I) missense mutation (c.517C>A, p.Arg149Ser, designated as apoA-IBoston) in a 67-year-old woman and her 2 sons, who had mean serum high-density lipoprotein (HDL) cholesterol, apoA-I, and apoA-I in very large α-1 HDL that were 10%, 35%, and 16% of normal, respectively (all P < .05). The percentage of HDL cholesterol in the esterified form was also significantly (P < .05) reduced to 52% of control values. Cholesteryl ester tranfer protein (CETP) activity was normal. The mean global, adenosine triphosphate (ATP)-binding cassette transporter A1 and scavenger receptor B type I-mediated cellular cholesterol efflux capacity in apoB-depleted serum from affected family members were 41%, 37%, 47%, 54%, and 48% of control values, respectively (all P < .05). lecithin-cholesterol acyltransferase (LCAT) activity in plasma was 71% of controls, whereas in the cell-based assay, it was 73% of control values (P < .05). The data indicate that this novel apoA-I missense is associated with markedly decreased levels of HDL cholesterol and very large α-1 HDL, as well as decreased serum cellular cholesterol efflux and LCAT activity, but not with premature coronary heart disease, similar to other apoA-I mutations that have been associated with decreased LCAT activity. PMID:26073399

  1. Cholesterol efflux is LXRα isoform-dependent in human macrophages

    PubMed Central

    2014-01-01

    Background The nuclear receptor liver X receptor (LXR) has two isoforms: LXRα and LXRβ. LXR activation promotes cholesterol efflux in macrophages, but the relative importance of each LXR isoform in mediating cholesterol efflux remains elusive. Methods We evaluated the ability of different doses of LXRs agonist T0901317 to affect cholesterol efflux in human macrophages and its relationship with mRNA and protein levels of several well-characterized proteins involved in cholesterol efflux, including ABCA1, ABCG1, SR-BI, LXRβ and LXRα, using quantitative real-time PCR, Western blotting, and siRNA techniques. Results Here we show that LXRα rather than LXRβ sustains baseline cholesterol efflux in human blood-derived macrophages. Treatment of human macrophages with a non-isoform-specific LXR agonist T0901317 substantially increased HDL- and apoA-I-mediated cholesterol efflux, which was associated with increased mRNA and protein expression levels of ABCA1, ABCG1, SR-BI, LXRα and LXRβ. The siRNA- mediated silencing of LXRα, but not LXRβ significantly reduced the protein levels of ABCA1,ABCG1, and SR-BI as wellas HDL- and ApoA1-mediated cholesterol in human macrophages. Conclusions These findings imply that LXRα- rather than LXRβ- specific agonists may promote reverse cholesterol transport in humans. PMID:24996838

  2. HCO3-stimulated Cl efflux in the gulf toadfish acclimated to sea water.

    PubMed

    Kormanik, G A; Evans, D H

    1979-04-01

    Unidirectional efflux of Cl was examined in the Gulf toadfish, Opsanus beta, in artificial seawater solutions with modified concentrations of Cl and HCO3. Removal of Cl HCO3 reduced Cl efflux. Addition of HCO3 at typical seawater concentrations stimulated Cl efflux, independent of changes in the transepithelial potential. This active, HCO3-stimulated Cl efflux is saturable, with a Km of 2.4 mM, typical of the concentration of HCO3 found in sea water, and independent of external pH. Active extrusion of Cl offsets the net diffusional and oral gain of Cl faced by O. beta in sea water. PMID:469477

  3. Efflux transporter engineering markedly improves amorphadiene production in Escherichia coli.

    PubMed

    Zhang, Congqiang; Chen, Xixian; Stephanopoulos, Gregory; Too, Heng-Phon

    2016-08-01

    Metabolic engineering aims at altering cellular metabolism to produce valuable products at high yields and titers. Achieving high titers and productivity can be challenging if final products are largely accumulated intracellularly. A potential solution to this problem is to facilitate the export of these substances from cells by membrane transporters. Amorphadiene, the precursor of antimalarial drug artemisinin, is known to be secreted from Escherichia coli overexpressing the biosynthetic pathway. In order to assess the involvement of various endogenous efflux pumps in amorphadiene transport, the effects of single gene deletion of 16 known multidrug-resistant membrane efflux transporters were examined. The outer membrane protein TolC was found to be intimately involved in amorphadiene efflux. The overexpression of tolC together with ABC family transporters (macAB) or MFS family transporters (emrAB or emrKY) enhanced amorphadiene titer by more than threefold. In addition, the overexpression of transporters in the lipopolysaccharide transport system (msbA, lptD, lptCABFG) was found to improve amorphadiene production. As efflux transporters often have a wide range of substrate specificity, the multiple families of transporters were co-expressed and synergistic benefits were observed in amorphadiene production. This strategy of screening and then rationally engineering transporters can be used to improve the production of other valuable compounds in E. coli. Biotechnol. Bioeng. 2016;113: 1755-1763. © 2016 Wiley Periodicals, Inc. PMID:26804325

  4. Sodium pump molecular activity and membrane lipid composition in two disparate ectotherms, and comparison with endotherms.

    PubMed

    Turner, Nigel; Hulbert, A J; Else, Paul L

    2005-02-01

    Previous research has shown that the lower sodium pump molecular activity observed in tissues of ectotherms compared to endotherms, is largely related to the lower levels of polyunsaturates and higher levels of monounsaturates found in the cell membranes of ectotherms. Marine-based ectotherms, however, have very polyunsaturated membranes, and in the current study, we measured molecular activity and membrane lipid composition in tissues of two disparate ectothermic species, the octopus (Octopus vulgaris) and the bearded dragon lizard (Pogona vitticeps), to determine whether the high level of membrane polyunsaturation generally observed in marine-based ectotherms is associated with an increased sodium pump molecular activity relative to other ectotherms. Phospholipids from all tissues of the octopus were highly polyunsaturated and contained high concentrations of the omega-3 polyunsaturate, docosahexaenoic acid (22:6 (n-3)). In contrast, phospholipids from bearded dragon tissues contained higher proportions of monounsaturates and lower proportions of polyunsaturates. Sodium pump molecular activity was only moderately elevated in tissues of the octopus compared to the bearded dragon, despite the much greater level of polyunsaturation in octopus membranes. When the current data were combined with data for the ectothermic cane toad, a significant (P = 0.003) correlation was observed between sodium pump molecular activity and the content of 22:6 (n-3) in the surrounding membrane. These results are discussed in relation to recent work which shows a similar relationship in endotherms. PMID:15726386

  5. Nanostructured lipid-carrageenan hybrid carriers (NLCCs) for controlled delivery of mitoxantrone hydrochloride to enhance anticancer activity bypassing the BCRP-mediated efflux.

    PubMed

    Ling, Guixia; Zhang, Tianhong; Zhang, Peng; Sun, Jin; He, Zhonggui

    2016-08-01

    Novel nanostructured lipid-carrageenan hybrid carriers (NLCCs) were exploited for controlled delivery of water soluble chemotherapeutic agent mitoxantrone hydrochloride (MTO) with high loading capacity, sustained release property, and potential for improving oral bioavailability and antitumor efficacy. By introducing the negative polymer of carrageenan, MTO was highly incorporated into NLCCs with encapsulation efficiency of 95.8% by electrostatic interaction. In vivo pharmacokinetics of MTO solution (MTO-Sol) and MTO-NLCCs in rats demonstrated that the apparent bioavailability of MTO-NLCCs was increased to approximate 3.5-fold compared to that of MTO-Sol. The cytotoxicity investigations by MTT method indicated that NLCCs could significantly enhanced the antitumor efficacy against resistant MCF-7/MX cells. The relative cellular association of MTO-NLCCs was 9.2-fold higher than that of MTO-Sol in breast cancer resistance protein (BCRP) over-expressing MCF-7/MX cells, implying that BCRP-mediated drug efflux was diminished by the introduction of NLCCs. The endocytosis inhibition study implied that the NLCCs entered the MCF-7/MX cells by clathrin-mediated endocytosis process, which can bypass the efflux of MTO mediated by BCRP. The new developed NLCCs provide an effective strategy for oral delivery of water-soluble MTO with improved encapsulation efficiency, oral bioavailability, and cytotoxicity against resistant breast cancer cells. PMID:26754913

  6. Effect of optical pumping on the refractive index and temperature in the core of active fibre

    SciTech Connect

    Gainov, V V; Ryabushkin, Oleg A

    2011-09-30

    This paper examines the refractive index change (RIC) induced in the core of Yb{sup 3+}-doped active silica fibres by pulsed pumping. RIC kinetic measurements with a Mach - Zehnder interferometer make it possible to separately assess the contributions of the electronic and thermal mechanisms to the RIC and evaluate temperature nonuniformities in the fibre.

  7. Electrogenic active proton pump in Rana esculenta skin and its role in sodium ion transport.

    PubMed Central

    Ehrenfeld, J; Garcia-Romeu, F; Harvey, B J

    1985-01-01

    Kinetic and electrophysiological studies were carried out in the in vitro Rana esculenta skin, bathed in dilute sodium solution, to characterize the proton pump and coupling between sodium absorption (JNa+n) and proton excretion (JH+n). JNa+n and JH+n were both dependent on transepithelial potential (psi ms); hyperpolarizing the skin decreased JNa+n and increased JH+n; depolarization produced the opposite effects. Amiloride (5 X 10(-5) M) at a clamped psi ms of +50 mV inhibited JNa+n without affecting JH+n. Variations of psi ms or pH had identical effects on JH+n. Ethoxzolamide inhibited JH+n and simultaneously increased psi ms by 15-30 mV. These changes were accompanied by depolarization of the apical membrane potential psi mc from -47 to -25 mV and an increase in apical membrane resistance of 30%; no significant effects on basolateral membrane potential (psi cs) and resistance (Rb) nor on shunt resistance (Rj) were observed. The proton pump appears to be localized at the apical membrane. The proton pump was also inhibited by deoxygenation, oligomycin, dicyclohexylcarbodiimide and vanadate (100, 78, 83 and 100% inhibition respectively). The variations of JH+n and of the measured electrical currents were significantly correlated. These findings are supportive evidence of a primary active proton pump, electrogenic and strictly linked to aerobic metabolism. The current-voltage (I-V) relation of the proton pump was obtained as the difference in the I-V curves of the apical membrane extracted before and after proton-pump inhibition by ethoxzolamide during amiloride block of sodium transport. The proton-pump current (IP) was best described by a saturable exponential function of psi mc. Maximal pump current (ImaxP) was calculated to be 200 nequiv h-1 cm-2 at a psi mc of +50 mV and the pump reversal potential ERP was -130 mV. The effect of ethoxzolamide to depolarize psi mc was dependent on the relation between psi mc and ERP. Maximal induced depolarization occurred at a

  8. Evaluation of a large capacity heat pump concept for active cooling of hypersonic aircraft structure

    NASA Technical Reports Server (NTRS)

    Pagel, L. L.; Herring, R. L.

    1978-01-01

    Results of engineering analyses assessing the conceptual feasibility of a large capacity heat pump for enhancing active cooling of hypersonic aircraft structure are presented. A unique heat pump arrangement which permits cooling the structure of a Mach 6 transport to aluminum temperatures without the aid of thermal shielding is described. The selected concept is compatible with the use of conventional refrigerants, with Freon R-11 selected as the preferred refrigerant. Condenser temperatures were limited to levels compatible with the use of conventional refrigerants by incorporating a unique multipass condenser design, which extracts mechanical energy from the hydrogen fuel, prior to each subsequent pass through the condenser. Results show that it is technically feasible to use a large capacity heat pump in lieu of external shielding. Additional analyses are required to optimally apply this concept.

  9. Insulin regulation of Na/K pump activity in rat hepatoma cells

    SciTech Connect

    Gelehrter, T.D.; Shreve, P.D.; Dilworth, V.M.

    1984-05-01

    Insulin rapidly increases Na/K pump activity in HTC rat hepatoma cells in tissue culture, as measured by the ouabain-sensitive influx of the potassium analogue 86Rb+. Increased influx is observed within minutes and is maximal (70% above control) within 1-2 h. The effect appears to be mediated by the insulin receptors, as: the concentration dependence on insulin is identical to that for insulin induction of tyrosine aminotransferase and stimulation of 2-aminoisobutyric acid transport, proinsulin is 6% as potent as insulin, and the effect is blocked by anti-receptor antibodies. The early stimulation of potassium influx is not blocked by cycloheximide and is not associated with an increased number of pump sites as measured by /sup 3/H-ouabain binding. The insulin effect is blocked by amiloride, which blocks sodium influx, and is mimicked by the sodium ionophore monensin, which increases sodium influx and intracellular accumulation. Insulin also rapidly increases the initial rate of /sup 22/Na+ influx, suggesting that insulin may enhance Na/K pump activity, in part, by increasing intracellular sodium concentration. Incubation of HTC cells with insulin for 24 h causes complete unresponsiveness to the insulin induction of transaminase and stimulation of amino acid transport, a phenomenon mediated by postbinding mechanisms. In contrast, similar incubation with insulin does not cause unresponsiveness to the insulin stimulation of Na/K pump activity. Therefore, the site of regulation of responsiveness to insulin must be distal to, or separate from, those events causing stimulation of ion fluxes.

  10. Heavy metal transport by the CusCFBA efflux system.

    PubMed

    Delmar, Jared A; Su, Chih-Chia; Yu, Edward W

    2015-11-01

    It is widely accepted that the increased use of antibiotics has resulted in bacteria with developed resistance to such treatments. These organisms are capable of forming multi-protein structures that bridge both the inner and outer membrane to expel diverse toxic compounds directly from the cell. Proteins of the resistance nodulation cell division (RND) superfamily typically assemble as tripartite efflux pumps, composed of an inner membrane transporter, a periplasmic membrane fusion protein, and an outer membrane factor channel protein. These machines are the most powerful antimicrobial efflux machinery available to bacteria. In Escherichia coli, the CusCFBA complex is the only known RND transporter with a specificity for heavy metals, detoxifying both Cu(+) and Ag(+) ions. In this review, we discuss the known structural information for the CusCFBA proteins, with an emphasis on their assembly, interaction, and the relationship between structure and function. PMID:26258953

  11. Role for c-jun N-terminal kinase in treatment-refractory acute myeloid leukemia (AML): signaling to multidrug-efflux and hyperproliferation.

    PubMed

    Cripe, L D; Gelfanov, V M; Smith, E A; Spigel, D R; Phillips, C A; Gabig, T G; Jung, S-H; Fyffe, J; Hartman, A D; Kneebone, P; Mercola, D; Burgess, G S; Boswell, H S

    2002-05-01

    A relationship was proved between constitutive activity of leukemic cell c-jun-N-terminal kinase (JNK) and treatment failure in AML. Specifically, early treatment failure was predicted by the presence of constitutive JNK activity. The mechanistic origins of this association was sought. A multidrug resistant leukemic cell line, HL-60/ADR, characterized by hyperexpression of c-jun and JNK activity, was transfected with a mutant c-jun vector, whose substrate N-terminal c-jun serines were mutated. Down-regulated expression occurred of c-jun/AP-1-dependent genes, catalase and glutathione-S-transferase (GST) pi, which participate in cellular homeostasis to oxidative stress and xenobiotic exposure. MRP-efflux was abrogated in HL-60/ADR cells with dominant-negative c-jun, perhaps because MRP1 protein expression was also lost. Heightened sensitivity to daunorubicin resulted in cells subjected to this change. Biochemical analysis in 67 primary adult AML samples established a statistical correlation between cellular expression of c-jun and JNK activity, JNK activity with hyperleukocytosis at presentation of disease, and with exuberant MRP efflux. These findings reflect the survival role for c-jun/AP-1 and its regulatory kinase previously demonstrated for yeast in homeostatic response to oxidative stress and in operation of ATP-binding cassette efflux pumps, and may support evolutionary conservation of such function. Thus, JNK and c-jun may be salient drug targets in multidrug resistant AML. PMID:11986940

  12. Kinetic analysis of receptor-controlled tracer efflux from sealed membrane fragments

    PubMed Central

    Bernhardt, Julius; Neumann, Eberhard

    1978-01-01

    A detailed kinetic analysis is presented for activator-receptor-mediated efflux of tracer substances from vesicular membrane systems in general and from sealed fragments of excitable membranes in particular. Rate constants and amplitudes, as the primary measurable quantities of the efflux kinetics, are expressed in terms of fundamental properties of vesicular membrane systems containing receptors of chemical gating systems. The experimental determination and theoretical analysis of single contributions to a complex receptor-controlled efflux has been treated for the acetylcholine receptor system; also the effect of “pharmacological densensitization” on efflux is explicitly formulated. The dependence of the measured efflux parameters on the concentration of activators can be used to derive the kinetic and thermodynamic constants for receptor activation and inactivation processes; a general kinetic scheme and two limiting cases are analyzed. The efflux of 22Na from “excitable microsacs” of Torpedo marmorata is treated as an example, and the power of the rigorous analytical method is demonstrated. In particular, the analysis of efflux amplitudes from only a few data points offers an alternative to the longer lasting measurements for obtaining efflux curves when a safety factor is involved, as in the case of tracer ions like 22Na. PMID:16592553

  13. MexXY multidrug efflux system of Pseudomonas aeruginosa

    PubMed Central

    Morita, Yuji; Tomida, Junko; Kawamura, Yoshiaki

    2012-01-01

    Anti-pseudomonas aminoglycosides, such as amikacin and tobramycin, are used in the treatment of Pseudomonas aeruginosa infections. However, their use is linked to the development of resistance. During the last decade, the MexXY multidrug efflux system has been comprehensively studied, and numerous reports of laboratory and clinical isolates have been published. This system has been increasingly recognized as one of the primary determinants of aminoglycoside resistance in P. aeruginosa. In P. aeruginosa cystic fibrosis isolates, upregulation of the pump is considered the most common mechanism of aminoglycoside resistance. Non-fermentative Gram-negative pathogens possessing very close MexXY orthologs such as Achromobacter xylosoxidans and various Burkholderia species (e.g., Burkholderia pseudomallei and B. cepacia complexes), but not B. gladioli, are intrinsically resistant to aminoglycosides. Here, we summarize the properties (e.g., discovery, mechanism, gene expression, clinical significance) of the P. aeruginosa MexXY pump and other aminoglycoside efflux pumps such as AcrD of Escherichia coli, AmrAB-OprA of B. pseudomallei, and AdeABC of Acinetobacter baumannii. MexXY inducibility of the PA5471 gene product, which is dependent on ribosome inhibition or oxidative stress, is noteworthy. Moreover, the discovery of the cognate outer membrane component (OprA) of MexXY in the multidrug-resistant clinical isolate PA7, serotype O12 deserves special attention. PMID:23233851

  14. Distribution and expression of the Ade multidrug efflux systems in Acinetobacter baumannii clinical isolates.

    PubMed

    Pagdepanichkit, Sirawit; Tribuddharat, Chanwit; Chuanchuen, Rungtip

    2016-09-01

    One hundred Acinetobacter baumannii clinical isolates were examined for inhibitory effect of reserpine and carbonyl cyanide m-chlorophenylhydrazone (CCCP) on the antimicrobial susceptibility and expression of 4 resistant-nodulation-cell division (RND)-type multidrug efflux systems, including AdeABC, AdeDE, AdeIJK, and AdeFGH, using RT-PCR. Ten A. baumannii isolates expressing AdeABC, AdeIJK, or AdeFGH were randomly selected for determination of transcription level and regulatory mutations. While all the isolates were resistant to multiple drugs, the reserpine and CCCP experiment showed that the multidrug resistance phenotype in most A. baumannii isolates was associated with efflux pumps. Most isolates expressed at least one of the RND-type efflux pumps tested (97%). AdeIJK expression was most common (97%), but none of the isolates produced AdeDE. Fifty-two percent of the A. baumannii isolates simultaneously produced up to 3 RND-type efflux systems (i.e., AdeABC, AdeFGH, and AdeIJK). No good correlation between the expression of RND-type efflux pumps and the type of antimicrobial resistance was observed. Overexpression of AdeABC, AdeIJK, and AdeFGH was not always related to the presence of mutations in their corresponding regulatory genes. This study highlights (i) the universal presence of the RND-type efflux pumps with variable levels of expression level among the A. baumannii in this collection and (ii) the complexity of their regulation of expression. PMID:27332787

  15. Efflux-Mediated Antibiotic Resistance in Acinetobacter spp. ▿

    PubMed Central

    Coyne, Sébastien; Courvalin, Patrice; Périchon, Bruno

    2011-01-01

    Among Acinetobacter spp., A. baumannii is the most frequently implicated in nosocomial infections, in particular in intensive care units. It was initially thought that multidrug resistance (MDR) in this species was due mainly to horizontal acquisition of resistance genes. However, it has recently become obvious that increased expression of chromosomal genes for efflux systems plays a major role in MDR. Among the five superfamilies of pumps, resistance-nodulation-division (RND) systems are the most prevalent in multiply resistant A. baumannii. RND pumps typically exhibit a wide substrate range that can include antibiotics, dyes, biocides, detergents, and antiseptics. Overexpression of AdeABC, secondary to mutations in the adeRS genes encoding a two-component regulatory system, constitutes a major mechanism of multiresistance in A. baumannii. AdeIJK, intrinsic to this species, is responsible for natural resistance, but since overexpression above a certain threshold is toxic for the host, its contribution to acquired resistance is minimal. The recently described AdeFGH, probably regulated by a LysR-type transcriptional regulator, also confers multidrug resistance when overexpressed. Non-RND efflux systems, such as CraA, AmvA, AbeM, and AbeS, have also been characterized for A. baumannii, as have AdeXYZ and AdeDE for other Acinetobacter spp. Finally, acquired narrow-spectrum efflux pumps, such as the major facilitator superfamily (MFS) members TetA, TetB, CmlA, and FloR and the small multidrug resistance (SMR) member QacE in Acinetobacter spp., have been detected and are mainly encoded by mobile genetic elements. PMID:21173183

  16. Some effects of way-120,491 on electrical and mechanical activity and on sup 42 K/ sup 85 Rb efflux in rat blood vessels

    SciTech Connect

    Edwards, G.; Weston, A.H. ); Oshiro, G. )

    1990-02-26

    The effects of WAY-120,491 (-)-(3A,4R-trans)-2-(2,3-dihydro-3-hydroxy-2,2-dimethyl-6-(trifluoromethoxy)-2H-1-benzopyran-4-yl)-2,3-dihydro-1H-isoindol-1-one have been examined in vitro using portal veins and aortic segments from male Sprague-Dawley rats. In aorta, WAY-120,491 (3.3-33nM) produced a dose-dependent relaxation of 20mM KC1 contractions, an effect antagonized by glibenclamide. WAY-120,491 had no effect on tension induced by 80mM CK1. When segments of aorta were loaded with {sup 86}Rb and {sup 42}K, WAY-120,491 (330nM-33{mu}M) increased the efflux of both isotopes, an effect antagonized by glibenclamide. In portal vein, WAY 120-491 (3.3-330nM) inhibited spontaneous tension development by abolishing electrical multispike complexes. In tissues loaded with {sup 86}Rb, WAY-120,491 (330nM-33{mu}M) increased the rate of {sup 86}Rb exchange. It is concluded that these effects of WAY-120,491 in rat blood vessels are consistent with the opening of plasmalemmal K-channels and that WAY-120,491 belongs to the pharmacological grouping known as the K-channel openers.

  17. Novel Resistance-Nodulation-Cell Division Efflux System AdeDE in Acinetobacter Genomic DNA Group 3

    PubMed Central

    Chau, Sze-Lok; Chu, Yiu-Wai; Houang, Elizabeth T. S.

    2004-01-01

    Resistance-nodulation-cell division type efflux pump AdeDE was identified in acinetobacters belonging to genomic DNA group 3. Inactivation of adeE showed that it may be responsible for reduced susceptibility to amikacin, ceftazidime, chloramphenicol, ciprofloxacin, erythromycin, ethidium bromide, meropenem, rifampin, and tetracycline. However, unlike what was found for other similar efflux systems, the open reading frame for the outer membrane component was not found downstream of the adeDE gene cluster. PMID:15388479

  18. Cooperation between prokaryotic (Lde) and eukaryotic (MRP) efflux transporters in J774 macrophages infected with Listeria monocytogenes: studies with ciprofloxacin and moxifloxacin.

    PubMed

    Lismond, Ann; Tulkens, Paul M; Mingeot-Leclercq, Marie-Paule; Courvalin, Patrice; Van Bambeke, Françoise

    2008-09-01

    Antibiotic efflux is observed in both eukaryotic and prokaryotic cells, modulating accumulation and resistance. The present study examines whether eukaryotic and prokaryotic fluoroquinolone transporters can cooperate in the context of an intracellular infection. We have used (i) J774 macrophages (comparing a ciprofloxacin-resistant cell line overexpressing an MRP-like transporter with wild-type cells with basal expression), (ii) Listeria monocytogenes (comparing a clinical isolate [CLIP21369] displaying ciprofloxacin resistance associated with overexpression of the Lde efflux system with a wild-type strain [EGD]), (iii) ciprofloxacin (substrate of both Lde and MRP) and moxifloxacin (nonsubstrate), and (iv) probenecid and reserpine (preferential inhibitors of MRP and Lde, respectively). The ciprofloxacin MICs for EGD were unaffected by reserpine, while those for CLIP21369 were decreased approximately fourfold (and made similar to those of EGD). Neither probenecid nor reserpine affected the moxifloxacin MICs against EGD or CLIP21369. In dose-response studies (0.01x to 100x MIC) in broth, reserpine fully restored the susceptibility of CLIP21369 to ciprofloxacin (no effect on EGD) but did not influence the activity of moxifloxacin. In studies with intracellular bacteria, reserpine, probenecid, and their combination increased the activity of ciprofloxacin in wild-type and ciprofloxacin-resistant macrophages in parallel with an increase in ciprofloxacin accumulation in macrophages for EGD and an increase in accumulation and decrease in MIC (in broth) for CLIP21369. Moxifloxacin accumulation and intracellular activity were consistently not affected by the inhibitors. A bacterial efflux pump may thus actively cooperate with a eukaryotic efflux transporter to reduce the activity of a common substrate (ciprofloxacin) toward an intracellular bacterial target. PMID:18573933

  19. Regulation of pumping function of the heart in developing body under changing regimens of motor activity.

    PubMed

    Vafina, E Z; Abzalov, R A; Abzalov, N I; Nikitin, A S; Gulyakov, A A

    2014-06-01

    We analyzed parameters of the pumping function of the heart in rats subjected to enhanced motor activity after a preliminary 70-day hypokinesia under conditions of α- and β-adrenergic receptor stimulation with norepinephrine followed by blockade of β-adrenergic receptor with propranolol (obsidian) and α1-adrenergic receptors with doxazosin. After norepinephrine administration, the HR and cardiac output were higher in rats with enhanced physical activity after preliminary hypokinesia than in rats with low physical activity. After propranolol administration, stroke volume and cardiac output in 100-day-old rats with limited activity were lower, and HR higher was than in rats with enhanced physical activity after preliminary 70-day hypokinesia. After administration of doxazosin, rats with limited motor activity demonstrated more pronounced changes in HR than rats with enhanced physical activity after preliminary 70-day hypokinesia. PMID:24970234

  20. Design and study of the efflux function of the EGFP fused MexAB-OprM membrane transporter in Pseudomonas aeruginosa using fluorescence spectroscopy.

    PubMed

    Ding, Feng; Lee, Kerry J; Vahedi-Faridi, Ardeschir; Yoneyama, Hiroshi; Osgood, Christopher J; Xu, Xiao-Hong Nancy

    2014-06-21

    block the translocation pore or restrict the movement of the individual pump domains, which may lead to partially restricted efflux activity. PMID:24781334

  1. An actively mode-locked Ho: YAG solid laser pumped by a Tm: YLF laser

    NASA Astrophysics Data System (ADS)

    Yao, B. Q.; Cui, Z.; Wang, J.; Duan, X. M.; Dai, T. Y.; Du, Y. Q.; Yuan, J. H.; Liu, W.

    2015-02-01

    A continuous wave mode-locked (CWML) Ho: YAG laser based on an acousto-optic modulator (AOM) pumped by a 1.9 μm Tm: YLF laser is demonstrated. This is the first time a report on an active CWML Ho: YAG laser has been published. A maximum output power of 1.04 W at 2097.25 nm in the CWML regime is obtained at a pump power of 13.2 W, corresponding to a slope efficiency of 13.3%. The mode-locked pulse repetition frequency is 82.76 MHz and the single pulse energy is 12.57 nJ. The mode-locked pulse width is 102 ps measured through a no-background second harmonic autocorrelation with KTP as the nonlinear crystal. Furthermore, the M2 factor is calculated to be 1.146.

  2. MBE growth of active regions for electrically pumped, cw-operating GaSb-based VCSELs

    NASA Astrophysics Data System (ADS)

    Kashani-Shirazi, K.; Bachmann, A.; Boehm, G.; Ziegler, S.; Amann, M.-C.

    2009-03-01

    Electrically pumped, cw-operating, single-mode GaSb-based VCSELs are attractive light sources for trace-gas sensing systems using tunable diode laser absorption spectroscopy (TDLAS) [A. Vicet, D.A. Yarekha, A. Pérona, Y. Rouillard, S. Gaillard, Spectrochimica Acta Part A 58 (2002) 2405-2412]. Only recently, the first electrically pumped (EP) devices emitting at 2.325 μm in cw-mode at room temperature have been reported [A. Bachmann, T. Lim, K. Kashani-Shirazi, O. Dier, C. Lauer, M.-C. Amann, Electronics Letters 44(3) (2008) 202-203]. The fabrication of these devices employs the molecular beam epitaxy (MBE) growth of GaSb/AlAsSb-distributed Bragg mirrors, a multi-quantum-well active region made of AlGaAsSb/InGaAsSb and an InAsSb/GaSb-buried-tunnel junction. As VCSELs are usually driven under high injection rates, an optimum electrical design of active regions is essential for high-performance devices. In this paper we present an enhanced simulation of current flow in the active region under operation conditions. The calculation includes carrier transport by drift, diffusion and tunneling. We discuss different design criteria and material compositions for active regions. Active regions with various barrier materials were incorporated into edge emitter samples to evaluate their performance. Aluminum-containing barriers show better internal efficiency compared to active regions with GaSb as the barrier material.

  3. Effects of Efflux Transporter Genes on Susceptibility of Escherichia coli to Tigecycline (GAR-936)

    PubMed Central

    Hirata, Takahiro; Saito, Asami; Nishino, Kunihiko; Tamura, Norihisa; Yamaguchi, Akihito

    2004-01-01

    The activity of tigecycline, 9-(t-butylglycylamido)-minocycline, against Escherichia coli KAM3 (acrB) strains harboring plasmids encoding various tetracycline-specific efflux transporter genes, tet(B), tet(C), and tet(K), and multidrug transporter genes, acrAB, acrEF, and bcr, was examined. Tigecycline showed potent activity against all three Tet-expressing, tetracycline-resistant strains, with the MICs for the strains being equal to that for the host strain. In the Tet(B)-containing vesicle study, tigecycline did not significantly inhibit tetracycline efflux-coupled proton translocation and at 10 μM did not cause proton translocation. This suggests that tigecycline is not recognized by the Tet efflux transporter at a low concentration; therefore, it exhibits significant antibacterial activity. These properties can explain its potent activity against bacteria with a Tet efflux resistance determinant. Tigecycline induced the Tet(B) protein approximately four times more efficiently than tetracycline, as determined by Western blotting, indicating that it is at least recognized by a TetR repressor. The MICs for multidrug efflux proteins AcrAB and AcrEF were increased fourfold. Tigecycline inhibited active ethidium bromide efflux from intact E. coli cells overproducing AcrAB. Therefore, tigecycline is a possible substrate of AcrAB and its close homolog, AcrEF, which are resistance-modulation-division-type multicomponent efflux transporters. PMID:15155219

  4. Directed evolution of an E. coli inner membrane transporter for improved efflux of biofuel molecules

    PubMed Central

    2013-01-01

    Background The depletion of fossil fuels and the rising need to meet global energy demands have led to a growing interest in microbial biofuel synthesis, particularly in Escherichia coli, due to its tractable characteristics. Besides engineering more efficient metabolic pathways for synthesizing biofuels, efforts to improve production yield by engineering efflux systems to overcome toxicity problems is also crucial. This study aims to enhance hydrocarbon efflux capability in E. coli by engineering a native inner membrane transporter, AcrB, using the directed evolution approach. Results We developed a selection platform based on competitive growth using a toxic substrate surrogate, which allowed rapid selection of AcrB variants showing enhanced efflux of linear and cyclic fuel molecule candidates, n-octane and α-pinene. Two mutants exhibiting increased efflux efficiency for n-octane and α-pinene by up to 47% and 400%, respectively, were isolated. Single-site mutants based on the mutations found in the isolated variants were synthesized and the amino acid substitutions N189H, T678S, Q737L and M844L were identified to have conferred improvement in efflux efficiency. The locations of beneficial mutations in AcrB suggest their contributions in widening the substrate channel, altering the dynamics of substrate efflux and promoting the assembly of AcrB with the outer membrane channel protein TolC for more efficient substrate export. It is interesting to note that three of the four beneficial mutations were located relatively distant from the known substrate channels, thus exemplifying the advantage of directed evolution over rational design. Conclusions Using directed evolution, we have isolated AcrB mutants with improved efflux efficiency for n-octane and α-pinene. The utilization of such optimized native efflux pumps will increase productivity of biofuels synthesis and alleviate toxicity and difficulties in production scale-up in current microbial platforms. PMID

  5. Alterations in cardiac sarcolemmal Ca/sup 2 +/ pump activity during diabetes mellitus

    SciTech Connect

    Heyliger, C.E.; Prakash, A.; McNeill, J.

    1987-03-01

    Diabetes mellitus is frequently associated with a primary cardiomyopathy. The mechanisms responsible for this heart disease are not clear, but an alteration in myocardial Ca/sup 2 +/ transport is believed to be involved in its development. Even though sarcolemma plays a crucial role in cellular Ca/sup 2 +/ transport, little appears to be known about its Ca/sup 2 +/ transporting capability in the diabetic myocardium. In this regard, the authors have examined the status of the cardiac sarcolemmal Ca/sup 2 +/ pump during diabetes mellitus. Purified sarcolemmal membranes were isolated from male Wistar diabetic rat hearts 8 wk after streptozotocin injection. Ca/sup 2 +/ pump activity assessed by measuring its Ca/sup 2 +/-stimulated adenosine triphosphatase and Ca/sup 2 +/-uptake ability in the absence and presence of calmodulin was significantly depressed in the diabetic myocardium relative to controls. These results did not appear to have been influenced by the minimal sarcoplasmic reticular and mitochondrial contamination of this membrane preparation. Hence, it appears that the sarcolemmal Ca/sup 2 +/ pump is defective in the diabetic myocardium and may be involved in the altered Ca/sup 2 +/ transport of the heart during diabetes mellitus.

  6. Metal Fluoride Inhibition of a P-type H+ Pump: STABILIZATION OF THE PHOSPHOENZYME INTERMEDIATE CONTRIBUTES TO POST-TRANSLATIONAL PUMP ACTIVATION.

    PubMed

    Pedersen, Jesper Torbøl; Falhof, Janus; Ekberg, Kira; Buch-Pedersen, Morten Jeppe; Palmgren, Michael

    2015-08-14

    The plasma membrane H(+)-ATPase is a P-type ATPase responsible for establishing electrochemical gradients across the plasma membrane in fungi and plants. This essential proton pump exists in two activity states: an autoinhibited basal state with a low turnover rate and a low H(+)/ATP coupling ratio and an activated state in which ATP hydrolysis is tightly coupled to proton transport. Here we characterize metal fluorides as inhibitors of the fungal enzyme in both states. In contrast to findings for other P-type ATPases, inhibition of the plasma membrane H(+)-ATPase by metal fluorides was partly reversible, and the stability of the inhibition varied with the activation state. Thus, the stability of the ATPase inhibitor complex decreased significantly when the pump transitioned from the activated to the basal state, particularly when using beryllium fluoride, which mimics the bound phosphate in the E2P conformational state. Taken together, our results indicate that the phosphate bond of the phosphoenzyme intermediate of H(+)-ATPases is labile in the basal state, which may provide an explanation for the low H(+)/ATP coupling ratio of these pumps in the basal state. PMID:26134563

  7. Proton-dependent multidrug efflux systems.

    PubMed Central

    Paulsen, I T; Brown, M H; Skurray, R A

    1996-01-01

    Multidrug efflux systems display the ability to transport a variety of structurally unrelated drugs from a cell and consequently are capable of conferring resistance to a diverse range of chemotherapeutic agents. This review examines multidrug efflux systems which use the proton motive force to drive drug transport. These proteins are likely to operate as multidrug/proton antiporters and have been identified in both prokaryotes and eukaryotes. Such proton-dependent multidrug efflux proteins belong to three distinct families or superfamilies of transport proteins: the major facilitator superfamily (MFS), the small multidrug resistance (SMR) family, and the resistance/ nodulation/cell division (RND) family. The MFS consists of symporters, antiporters, and uniporters with either 12 or 14 transmembrane-spanning segments (TMS), and we show that within the MFS, three separate families include various multidrug/proton antiport proteins. The SMR family consists of proteins with four TMS, and the multidrug efflux proteins within this family are the smallest known secondary transporters. The RND family consists of 12-TMS transport proteins and includes a number of multidrug efflux proteins with particularly broad substrate specificity. In gram-negative bacteria, some multidrug efflux systems require two auxiliary constituents, which might enable drug transport to occur across both membranes of the cell envelope. These auxiliary constituents belong to the membrane fusion protein and the outer membrane factor families, respectively. This review examines in detail each of the characterized proton-linked multidrug efflux systems. The molecular basis of the broad substrate specificity of these transporters is discussed. The surprisingly wide distribution of multidrug efflux systems and their multiplicity in single organisms, with Escherichia coli, for instance, possessing at least nine proton-dependent multidrug efflux systems with overlapping specificities, is examined. We also

  8. Cation activation of the pig kidney sodium pump: transmembrane allosteric effects of sodium.

    PubMed Central

    Karlish, S J; Stein, W D

    1985-01-01

    We have studied activation by Na or Rb ions of different transport modes of the Na-K pump, using phospholipid vesicles reconstituted with pig kidney Na-K-ATPase. The shape of the activation curves, sigmoid or quasi-hyperbolic, depends on the nature of the cation at the opposite surface and not on the specific mode of transport. ATP-dependent Na uptake into K-containing vesicles (Na-K exchange) is activated by cytoplasmic Na along a highly sigmoid curve in the absence of extracellular Na (Hill number, nH = 1.9). Activation displays progressively less-sigmoid curves as extracellular Na is raised to 150 mM (nH = 1.2). The maximal rate of the Na-K exchange is not affected. Na is not transported from the extracellular face by the pump in the presence of excess extracellular K, and the transmembrane effects of the extracellular Na are therefore 'allosteric' in nature. ATP-dependent Na-Na exchange (Lee & Blostein, 1980) and classical ATP-plus-ADP-dependent Na-Na exchange are activated by cytoplasmic Na along hyperbolic curves. ATP-dependent Na uptake into Tris-containing vesicles is activated by cytoplasmic Na along a somewhat sigmoidal curve. (ATP + Pi)-dependent Rb-Rb exchange is activated by cytoplasmic and extracellular Rb along strictly hyperbolic curves. The same applies for Rb-Rb exchange in the presence or absence of ATP or Pi alone. The presence of a high concentration of extracellular Na together with extracellular Rb induces a sigmoidal activation by cytoplasmic Rb of (ATP + Pi)-dependent Rb-Rb exchange (nH = 1.45) but does not affect the maximal rate of exchange. Slow passive Rb fluxes through the pump observed in the absence of other pump ligands (see Karlish & Stein, 1982 alpha) are activated by cytoplasmic Rb along a strictly hyperbolic curve with extracellular Rb, nH = 1.0 (Rb-Rb exchange), along a strongly sigmoid curve with extracellular Na, nH = 1.5 (Rb-Na exchange), and along less-sigmoid curves with extracellular Tris, nH = 1.24 (net Rb flux) or

  9. Efflux Of Nitrate From Hydroponically Grown Wheat

    NASA Technical Reports Server (NTRS)

    Huffaker, R. C.; Aslam, M.; Ward, M. R.

    1992-01-01

    Report describes experiments to measure influx, and efflux of nitrate from hydroponically grown wheat seedlings. Ratio between efflux and influx greater in darkness than in light; increased with concentration of nitrate in nutrient solution. On basis of experiments, authors suggest nutrient solution optimized at lowest possible concentration of nitrate.

  10. ABCG1 is involved in vitamin E efflux.

    PubMed

    Olivier, Maryline; BottG, Remain; Frisdal, Eric; Nowick, Marion; Plengpanich, Wanee; Desmarchelier, Charles; Roi, Stéphanie; Quinn, Carmel M; Gelissen, Ingrid; Jessup, Wendy; Van Eck, Miranda; Guérin, Maryse; Le Goff, Wilfried; Reboul, Emmanuelle

    2014-12-01

    Vitamin E membrane transport has been shown to involve the cholesterol transporters SR-BI, ABCA1 and NPC1L1. Our aim was to investigate the possible participation of another cholesterol transporter in cellular vitamin E efflux: ABCG1. In Abcgl-deficient mice, vitamin E concentration was reduced in plasma lipoproteins whereas most tissues displayed a higher vitamin E content compared to wild-type mice. α- and γ-tocopherol efflux was increased in CHO cells overexpressing human ABCG1 compared to control cells. Conversely, α- and γ- tocopherol efflux was decreased in ABCG1-knockdown human cells (Hep3B hepatocytes and THP-1 macro- phages). Interestingly, α- and γ-tocopherol significantly downregulated ABCG1 and ABCA1 expression levels in Hep3B and THP-1, an effect confirmed in vivo in rats given vitamin E for 5 days. This was likely due to reduced LXR activation by oxysterols, as Hep3B cells and rat liver treated with vitamin E displayed a significantly reduced content in oxysterols compared to their respective controls. Overall, the present study reveals for the first time that ABCG1 is involved in cellular vitamin E efflux. PMID:25462452

  11. Molecular Components of Nitrate and Nitrite Efflux in Yeast

    PubMed Central

    Cabrera, Elisa; González-Montelongo, Rafaela; Giraldez, Teresa; de la Rosa, Diego Alvarez

    2014-01-01

    Some eukaryotes, such as plant and fungi, are capable of utilizing nitrate as the sole nitrogen source. Once transported into the cell, nitrate is reduced to ammonium by the consecutive action of nitrate and nitrite reductase. How nitrate assimilation is balanced with nitrate and nitrite efflux is unknown, as are the proteins involved. The nitrate assimilatory yeast Hansenula polymorpha was used as a model to dissect these efflux systems. We identified the sulfite transporters Ssu1 and Ssu2 as effective nitrate exporters, Ssu2 being quantitatively more important, and we characterize the Nar1 protein as a nitrate/nitrite exporter. The use of strains lacking either SSU2 or NAR1 along with the nitrate reductase gene YNR1 showed that nitrate reductase activity is not required for net nitrate uptake. Growth test experiments indicated that Ssu2 and Nar1 exporters allow yeast to cope with nitrite toxicity. We also have shown that the well-known Saccharomyces cerevisiae sulfite efflux permease Ssu1 is also able to excrete nitrite and nitrate. These results characterize for the first time essential components of the nitrate/nitrite efflux system and their impact on net nitrate uptake and its regulation. PMID:24363367

  12. Harmine augments electrically evoked dopamine efflux in the nucleus accumbens shell.

    PubMed

    Brierley, Daniel I; Davidson, Colin

    2013-01-01

    Harmine is a β-carboline alkaloid and major component of ayahuasca, a traditional South American psychoactive tea with anecdotal efficacy for treatment of cocaine dependence. Harmine is an inhibitor of monoamine oxidase A (MAO-A) and interacts in vitro with several pharmacological targets which modulate dopamine (DA) neurotransmission. In vivo studies have demonstrated dopaminergic effects of harmine, attributed to monoamine oxidase inhibitor (MAOI) activity, however none have directly demonstrated a pharmacological mechanism. This study investigated the acute effects, and pharmacological mechanism(s), of harmine on electrically evoked DA efflux parameters in the nucleus accumbens both in the absence and presence of cocaine. Fast cyclic voltammetry in rat brain slices was used to measure electrically evoked DA efflux in accumbens core and shell. Harmine (300 nM) significantly augmented DA efflux (148±8% of baseline) in the accumbens shell. Cocaine augmented efflux in shell additive to harmine (260±35%). Harmine had no effect on efflux in the accumbens core or on reuptake in either sub-region. The effect of harmine in the shell was attenuated by the 5-HT(2A/2C) antagonist ketanserin. The MAOI moclobemide (10 µM) had no effect on DA efflux. These data suggest that harmine augments DA efflux via a novel, shell-specific, presynaptic 5-HT(2A) receptor-dependent mechanism, independent of MAOI activity. A DA-releasing 'agonist therapy' mechanism may thus contribute to the putative therapeutic efficacy of ayahuasca for cocaine dependence. PMID:23076833

  13. Structure and operation of bacterial tripartite pumps.

    PubMed

    Hinchliffe, Philip; Symmons, Martyn F; Hughes, Colin; Koronakis, Vassilis

    2013-01-01

    In bacteria such as Pseudomonas aeruginosa and Escherichia coli, tripartite membrane machineries, or pumps, determine the efflux of small noxious molecules, such as detergents, heavy metals, and antibiotics, and the export of large proteins including toxins. They are therefore influential in bacterial survival, particularly during infections caused by multidrug-resistant pathogens. In these tripartite pumps an inner membrane transporter, typically an ATPase or proton antiporter, binds and translocates export or efflux substrates. In cooperation with a periplasmic adaptor protein it recruits and opens a TolC family cell exit duct, which is anchored in the outer membrane and projects across the periplasmic space between inner and outer membranes. Assembled tripartite pumps thus span the entire bacterial cell envelope. We review the atomic structures of each of the three pump components and discuss how these have allowed high-resolution views of tripartite pump assembly, operation, and possible inhibition. PMID:23808339

  14. Distribution of AdeABC efflux system genes in genotypically diverse strains of clinical Acinetobacter baumannii.

    PubMed

    Wieczorek, Piotr; Sacha, Paweł; Czaban, Sławomir; Hauschild, Tomasz; Ojdana, Dominika; Kowalczuk, Oksana; Milewski, Robert; Poniatowski, Bogusław; Nikliński, Jacek; Tryniszewska, Elżbieta

    2013-10-01

    Acinetobacter baumannii has emerged as a highly problematic hospital-associated pathogen. Different mechanisms contribute to the formation of multidrug resistance in A. baumannii, including the AdeABC efflux system. Distribution of the structural and regulatory genes encoding the AdeABC efflux system among genetically diverse clinical A. baumannii strains was achieved by using PCR and pulsed-field gel electrophoresis techniques. The distribution of adeABRS genes is extremely high among our A. baumannii strains, except the adeC gene. We have observed a large proportion of strains presenting multidrug-resistance phenotype for several years. The efflux pump could be an important mechanism in these strains in resistance to antibiotics. PMID:23886790

  15. Redox-induced activation of the proton pump in the respiratory complex I

    PubMed Central

    Sharma, Vivek; Belevich, Galina; Gamiz-Hernandez, Ana P.; Róg, Tomasz; Vattulainen, Ilpo; Verkhovskaya, Marina L.; Wikström, Mårten; Hummer, Gerhard; Kaila, Ville R. I.

    2015-01-01

    Complex I functions as a redox-linked proton pump in the respiratory chains of mitochondria and bacteria, driven by the reduction of quinone (Q) by NADH. Remarkably, the distance between the Q reduction site and the most distant proton channels extends nearly 200 Å. To elucidate the molecular origin of this long-range coupling, we apply a combination of large-scale molecular simulations and a site-directed mutagenesis experiment of a key residue. In hybrid quantum mechanics/molecular mechanics simulations, we observe that reduction of Q is coupled to its local protonation by the His-38/Asp-139 ion pair and Tyr-87 of subunit Nqo4. Atomistic classical molecular dynamics simulations further suggest that formation of quinol (QH2) triggers rapid dissociation of the anionic Asp-139 toward the membrane domain that couples to conformational changes in a network of conserved charged residues. Site-directed mutagenesis data confirm the importance of Asp-139; upon mutation to asparagine the Q reductase activity is inhibited by 75%. The current results, together with earlier biochemical data, suggest that the proton pumping in complex I is activated by a unique combination of electrostatic and conformational transitions. PMID:26330610

  16. Time lag between photosynthesis and CO2 efflux from soil

    NASA Astrophysics Data System (ADS)

    Kuzyakov, Y.; Gavrichkova, O.

    2009-04-01

    Important part of CO2 efflux from planted soils is root-derived CO2, meaning that it originates directly and indirectly from roots: directly from root respiration, and indirectly from respiration of rhizosphere microorganisms decomposing organic substances released by roots into the soil (rhizodeposits). Recent studies have shown that apart of well studied effect of soil temperature and soil water content, the C supply of assimilates from photosynthetically active plant organs have a significant effect on the root-derived CO2. In fact, the effect of photosynthesis on root-derived CO2 is often masked by temperature because root biomass typically peaks in summer. However, roots can only respire the C that was allocated belowground, and so the effect of temperature on root respiration is likely to be constrained by photosynthesis. If models of soil respiration are to incorporate photosynthetic C inputs it is necessary to understand how these two fluxes are coupled and what are the factors affecting the time lag between C uptake and its following respiration by roots and associated microorganisms. We reviewed literature and own studies relevant for estimation of the delay of C assimilation by photosynthesis and CO2 efflux from soil. The most of the studies were based on pulse labeling of annual plants in the atmosphere with 14CO2 or 13CO2 and subsequent chase of 14C or 13C in the CO2 efflux from soil. We analyzed the dynamics of the CO2 efflux curves and evaluated 3 parameters: 1) the first appearance of labeled CO2 from soil, 2) maximum of labeled CO2, and 3) disappearance of the labeled CO2 from the total CO2 efflux from soil. Numerous studies showed that newly assimilated C cycles quickly within the ecosystem, being found in root respiration already some minutes after its assimilation. Reported time lags in situ and laboratory experiments varied from minutes to days. For annual and perennial grasses the first appearance of labeled CO2 from soil was measured within

  17. Inducer expulsion in Streptococcus pyogenes: properties and mechanism of the efflux reaction

    SciTech Connect

    Sutrina, S.L.; Reizer, J.; Saier, M.H Jr.

    1988-04-01

    Expulsion of preaccumulated methyl-..beta..-D-thiogalactoside-phosphate (TMG-P) from Streptococcus pyogenes is a two-step process comprising intracellular dephosphorylation of TMG-P followed by rapid efflux of the intracellularly formed free galactoside. The present study identifies the mechanism and the order and characterizes the temperature dependency of the efflux step. Unidirectional efflux of the intracellularly formed (/sup 14/C)TMG was only slightly affected when measured in the presence of unlabeled TMG (25 to 400 mM) in the extracellular medium. In contrast, pronounced inhibition of net efflux was observed in the presence of relatively low concentrations (1 to 16 mM) of extracellular (/sup 14/C)TMG. Since net efflux was nearly arrested when the external concentration of (/sup 14/C)TMG approached the intracellular concentration of this sugar, we propose that a facilitated diffusion mechanism is responsible for efflux and equilibration of TMG between the intracellular and extracellular milieus. The exit reaction was markedly dependent upon temperature, exhibited a high energy of activation (23 kcal (ca. 96 kJ) per mol), and followed first-order kinetics, indicating that the permease mediating this efflux was not saturated under the conditions of expulsion employed.

  18. Verbascoside isolated from Tectona grandis mediates gastric protection in rats via inhibiting proton pump activity.

    PubMed

    Singh, Neetu; Shukla, Nivedita; Singh, Pratibha; Sharma, Rolee; Rajendran, S M; Maurya, Rakesh; Palit, Gautam

    2010-10-01

    Evidences have suggested that Tectona grandis (TG) attenuates gastric mucosal injury; however its mechanism has not yet been established. The aim of present study was to evaluate the gastroprotective mechanism of ethanolic extract of TG (E-EtOH), butanolic fraction (Fr-Bu) and to identify its active constituents. Anti-ulcer activities were evaluated against cold restraint (CRU) and pyloric ligation (PL) induced gastric ulcer models and further confirmed through H(+) K(+)-ATPase inhibitory activity. Cytoprotective activity was evaluated in alcohol (AL) induced gastric ulcer model and further through PGE(2) level. E-EtOH and Fr-Bu attenuated ulcer formation in CRU. Moreover E-EtOH and Fr-Bu displayed potent anti-secretory activity as evident through reduced free acidity and pepsin activity in PL, confirmed further by in vitro inhibition of H(+) K(+)-ATPase activity. In addition cytoprotective potential of E-EtOH and Fr-Bu were apparent with protection in AL model, increased PGE(2) content and enhanced mucin level in PL. Phytochemical investigations of Fr-Bu yielded terpenoides and a phenolic glycoside, verbascoside. The anti-secretory mechanism of verbascoside mediated apparently through inhibition of H(+) K(+)-ATPase with corresponding decrease in plasma gastrin level, is novel to our finding. Gastroprotection elicited by TG might be through proton pump inhibition and consequent augmentation of the defensive mechanism. PMID:20388534

  19. Inhibition of calmodulin - regulated calcium pump activity in rat brain by toxaphene

    SciTech Connect

    Trottman, C.H.; Moorthy, K.S.

    1986-03-05

    In vivo effects of toxaphene on calcium pump activity in rat brain synaptosomes was studied. Male Sprague-Dawley rats were dosed with toxaphene at 0,25,50, and 100 mg/kg/day for 3 days and sacrificed 24 h after last dose. Ca/sup 2 +/-ATPase activity and /sup 45/Ca uptake were determined in brain P/sub 2/ fraction. Toxaphene inhibited both Ca/sup 2 +/-ATPase activity and /sup 45/Ca/sup 2 +/ uptake and the inhibition was dose dependent. Both substrate and Ca/sup 2 +/ activation kinetics of Ca/sup 2 +/-ATPase indicated non-competitive type of inhibition as evidenced by decreased catalytic velocity but not enzyme-substrate affinity. The inhibited Ca/sup 2 +/-ATPase activity and Ca/sup 2 +/ uptake were restored to normal level by exogenously added calmodulin which increased both velocity and affinity. The inhibition of Ca/sup 2 +/-ATPase activity and Ca/sup 2 +/ uptake and restoration by calmodulin suggests that toxaphene may impair active calcium transport mechanisms by decreasing regulator protein calmodulin levels.

  20. Calmodulin activation of an endoplasmic reticulum-located calcium pump involves an interaction with the N-terminal autoinhibitory domain

    NASA Technical Reports Server (NTRS)

    Hwang, I.; Harper, J. F.; Liang, F.; Sze, H.

    2000-01-01

    To investigate how calmodulin regulates a unique subfamily of Ca(2+) pumps found in plants, we examined the kinetic properties of isoform ACA2 identified in Arabidopsis. A recombinant ACA2 was expressed in a yeast K616 mutant deficient in two endogenous Ca(2+) pumps. Orthovanadate-sensitive (45)Ca(2+) transport into vesicles isolated from transformants demonstrated that ACA2 is a Ca(2+) pump. Ca(2+) pumping by the full-length protein (ACA2-1) was 4- to 10-fold lower than that of the N-terminal truncated ACA2-2 (Delta2-80), indicating that the N-terminal domain normally acts to inhibit the pump. An inhibitory sequence (IC(50) = 4 microM) was localized to a region within valine-20 to leucine-44, because a peptide corresponding to this sequence lowered the V(max) and increased the K(m) for Ca(2+) of the constitutively active ACA2-2 to values comparable to the full-length pump. The peptide also blocked the activity (IC(50) = 7 microM) of a Ca(2+) pump (AtECA1) belonging to a second family of Ca(2+) pumps. This inhibitory sequence appears to overlap with a calmodulin-binding site in ACA2, previously mapped between aspartate-19 and arginine-36 (J.F. Harper, B. Hong, I. Hwang, H.Q. Guo, R. Stoddard, J.F. Huang, M.G. Palmgren, H. Sze inverted question mark1998 J Biol Chem 273: 1099-1106). These results support a model in which the pump is kept "unactivated" by an intramolecular interaction between an autoinhibitory sequence located between residues 20 and 44 and a site in the Ca(2+) pump core that is highly conserved between different Ca(2+) pump families. Results further support a model in which activation occurs as a result of Ca(2+)-induced binding of calmodulin to a site overlapping or immediately adjacent to the autoinhibitory sequence.

  1. Interaction of gatifloxacin with efflux transporters: a possible mechanism for drug resistance

    PubMed Central

    Kwatra, Deep; Vadlapatla, Ramya Krishna; Vadlapudi, Aswani Dutt; Pal, Dhananjay; Mitra, Ashim K.

    2010-01-01

    The purpose of the study is to screen the interactions of fourth generation fluoroquinolone-gatifloxacin with efflux pumps i.e. P-gp, MRP2 and BCRP. Mechanism of gatifloxacin interaction with efflux transporters may explain its acquired resistance. Such clarification may lead to the development of strategies to overcome efflux and enhance its bioavailability at target site. This process will aid in the reduction of dose volume, further eliminating the chances of systemic toxicity from topical gatifloxacin eye drops. MDCK cell lines transfected with the targeted efflux transporters were used for this study. [14C] Erythromycin was selected as a model substrate for P-gp and MRP2 whereas Hoechst 33342 was employed as a substrate for BCRP. Uptake and transport studies of these substrates were performed in the presence of gatifloxacin to delineate its interaction with efflux transporters. Further the efflux ratio in the presence of gatifloxacin was calculated from bidirectional transport studies. The concentration of [14C] erythromycin and Hoechst 33342 were measured using scintillation counter and fluorescence plate reader respectively. A concentration dependent inhibition effect in the presence of gatifloxacin was revealed on [14C] erythromycin uptake. The efflux ratio (BL-AP/AP-BL) of substrates was found to approach unity at higher gatifloxacin concentrations. Increased concentration of gatifloxacin did not elevate uptake of Hoechst 33342. All these studies were validated with known inhibitors as positive control. Uptake and transport studies support the hypothesis that gatifloxacin is a substrate for P-gp, MRP2 but not for BCRP. Possible interactions of gatifloxacin with P-gp and MRP2 may be a possible mechanism for acquired resistance of gatifloxacin. This information can be further extended to design prodrugs or formulations in order to prevent development of acquired resistance and improve therapeutic efficacy with its reduction in side effects. PMID:20573570

  2. Characterization of all RND-type multidrug efflux transporters in Vibrio parahaemolyticus

    PubMed Central

    Matsuo, Taira; Nakamura, Koji; Kodama, Toshio; Mikami, Taro; Hiyoshi, Hirotaka; Tsuchiya, Tomofusa; Ogawa, Wakano; Kuroda, Teruo

    2013-01-01

    Resistance nodulation cell division (RND)-type efflux transporters play the main role in intrinsic resistance to various antimicrobial agents in many gram-negative bacteria. Here, we estimated 12 RND-type efflux transporter genes in Vibrio parahaemolyticus. Because VmeAB has already been characterized, we cloned the other 11 RND-type efflux transporter genes and characterized them in Escherichia coli KAM33 cells, a drug hypersusceptible strain. KAM33 expressing either VmeCD, VmeEF, or VmeYZ showed increased minimum inhibitory concentrations (MICs) for several antimicrobial agents. Additional four RND-type transporters were functional as efflux pumps only when co-expressed with VpoC, an outer membrane component in V. parahaemolyticus. Furthermore, VmeCD, VmeEF, and VmeYZ co-expressed with VpoC exhibited a broader substrate specificity and conferred higher resistance than that with TolC of E. coli. Deletion mutants of these transporter genes were constructed in V. parahaemolyticus. TM32 (ΔvmeAB and ΔvmeCD) had significantly decreased MICs for many antimicrobial agents and the number of viable cells after exposure to deoxycholate were markedly reduced. Strains in which 12 operons were all disrupted had very low MICs and much lower fluid accumulation in rabbit ileal loops. These results indicate that resistance nodulation cell division-type efflux transporters contribute not only to intrinsic resistance but also to exerting the virulence of V. parahaemolyticus. PMID:23894076

  3. Recent advances in the brain-to-blood efflux transport across the blood-brain barrier.

    PubMed

    Hosoya, Ken-ichi; Ohtsuki, Sumio; Terasaki, Tetsuya

    2002-11-01

    Elucidating the details of the blood-brain barrier (BBB) transport mechanism is a very important step towards successful drug targeting to the brain and understanding what happens in the brain. Although several brain uptake methods have been developed to characterize transport at the BBB, these are mainly useful for investigating influx transport across the BBB. In 1992, P-glycoprotein was found to act as an efflux pump for anti-cancer drugs at the BBB using primary cultured bovine brain endothelial cells. In order to determine the direct efflux transport from the brain to the circulating blood of exogenous compounds in vivo, the Brain Efflux Index method was developed to characterize several BBB efflux transport systems. Recently, we have established conditionally immortalized rat (TR-BBB) and mouse (TM-BBB) brain capillary endothelial cell lines from transgenic rats and mice harboring temperature-sensitive simian virus 40 large T-antigen gene to characterize the transport mechanisms at the BBB in vitro. TR-BBB and TM-BBB cells possess certain in vivo transport functions and express mRNAs for the BBB. Using a combination of newly developed in vivo and in vitro methods, we have elucidated the efflux transport mechanism at the BBB for neurosteroids, excitatory neurotransmitters, suppressive neurotransmitters, amino acids, and other organic anions to understand the physiological role played by the BBB as a detoxifying organ for the brain. PMID:12429456

  4. Highly active and stable oxaloacetate decarboxylase Na⁺ pump complex for structural analysis.

    PubMed

    Inoue, Michio; Li, Xiaodan

    2015-11-01

    The oxaloacetate decarboxylase primary Na(+) pump (Oad) produces energy for the surviving of some pathogenic bacteria under anaerobic conditions. Oad composes of three subunits: Oad-α, a biotinylated soluble subunit and catalyzes the decarboxylation of oxaloacetate; Oad-β, a transmembrane subunit and functions as a Na(+) pump; and Oad-γ, a single transmembrane α-helical anchor subunit and assembles Oad-α/β/γ complex. The molecular mechanism of Oad complex coupling the exothermic decarboxylation to generate the Na(+) electrochemical gradient remains unsolved. Our biophysical and biochemical studies suggested that the stoichiometry of Oad complex from Vibrio cholerae composed of α, β, γ in 4:2:2 stoichiometry not that of 4:4:4. The high-resolution structure determination of the Oad complex would reveal the energetic transformation mechanism from the catalytical soluble α subunit to membrane β subunit. Sufficient amount stable, conformational homogenous and active Oad complex with the right stoichiometry is the prerequisite for structural analysis. Here we report an easy and reproducible protocol to obtain high quantity and quality Oad complex protein for structural analysis. PMID:25986323

  5. Mechanisms underlying the activity-dependent regulation of locomotor network performance by the Na+ pump

    PubMed Central

    Zhang, Hong-Yan; Picton, Laurence; Li, Wen-Chang; Sillar, Keith T.

    2015-01-01

    Activity-dependent modification of neural network output usually results from changes in neurotransmitter release and/or membrane conductance. In Xenopus frog tadpoles, spinal locomotor network output is adapted by an ultraslow afterhyperpolarization (usAHP) mediated by an increase in Na+ pump current. Here we systematically explore how the interval between two swimming episodes affects the second episode, which is shorter and slower than the first episode. We find the firing reliability of spinal rhythmic neurons to be lower in the second episode, except for excitatory descending interneurons (dINs). The sodium/proton antiporter, monensin, which potentiates Na+ pump function, induced similar effects to short inter-swim intervals. A usAHP induced by supra-threshold pulses reduced neuronal firing reliability during swimming. It also increased the threshold current for spiking and introduced a delay to the first spike in a train, without reducing subsequent firing frequency. This delay was abolished by ouabain or zero K+ saline, which eliminate the usAHP. We present evidence for an A-type K+ current in spinal CPG neurons which is inactivated by depolarization and de-inactivated by hyperpolarization, and accounts for the prolonged delay. We conclude that the usAHP attenuates neuronal responses to excitatory network inputs by both membrane hyperpolarization and enhanced de-inactivation of an A-current. PMID:26541477

  6. A status of the activities of the NASA/MSFC pump stage technology team

    NASA Technical Reports Server (NTRS)

    Garcia, R.; Williams, R.; Dakhoul, Y.

    1992-01-01

    The Consortium for Computational Fluid Dynamics (CFD) Application in Propulsion Technology was established to aid the transfer of CFD related advancements among academia, government agencies, and industry. The specific goals of the Consortium are to develop CFD methodologies necessary to solve propulsion problems, to validate these methodologies, and to apply these methodologies in the design process. To accomplish these goals, a team of experts in various related fields was formed, a schedule of activities necessary to meet the goals was generated, and funding for the activities was obtained from NASA. During the past year (Mar. 1991 - Mar. 1992) the team's activities have focused on preliminary code validation and on the design of an advanced impeller. Six codes were used to calculate the flow in a Rocketdyne 0.3 flow coefficient inducer, and the results were compared to L2F data available for the inducer. This activity identified shortcomings in the experimental data sets and in the analytical solutions which must be surmounted in any future team activity. The design of the advanced impeller relied heavily on CFD results to obtain an optimized geometry. The optimized geometry was analyzed using four different codes, at design and off-design conditions. Activities for the next year include the optimization of a tandem blade impeller design, benchmark of CFD codes for diffuser and volute flows, the collection of L2F data for 'state-of-the-art' impeller and inducer, and the verification of the advanced pump team impeller design in a water rig.

  7. Bovicin HC5, a lantibiotic produced by Streptococcus bovis HC5, catalyzed the efflux of intracellular potassium but not ATP

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bovicin HC5, a broad spectrum lantibiotic produced by Streptococcus bovis HC5, catalyzed the efflux of intracellular potassium from Streptococcus bovis JB1, a sensitive strain. ATP also decreased, but this decline appeared to be caused by the activity of the F1FO ATPase rather than efflux per se....

  8. Periodic paralysis and the sodium-potassium pump.

    PubMed

    Layzer, R B

    1982-06-01

    Analysis of the pathophysiology of hypokalemic paralysis, as it occurs in barium poisoning, chronic potassium deficiency, and thyrotoxicosis, suggests that these disorders may have a similar mechanism. An increased ratio of muscle sodium permeability to potassium permeability reduces the ionic diffusion potential, while the resting membrane potential is sustained by an increase of Na-K pump electrogenesis. The result is that potassium entry (the sum of active and passive influx) exceeds potassium efflux; this causes a large shift of extracellular potassium into muscle until the Na-K pump turns off, leading to depolarization and paralysis. The primary defect in familial hypokalemic periodic paralysis, as in the example of barium poisoning, may be a marked reduction of muscle permeability to potassium. PMID:6287910

  9. Oxysterols trigger ABCA1-mediated basolateral surfactant efflux.

    PubMed

    Agassandian, Marianna; Mathur, Satya N; Zhou, Jiming; Field, F Jeffrey; Mallampalli, Rama K

    2004-08-01

    Surfactant is an apically-secreted surface-active material containing primarily disaturated phosphatidylcholine (DSPtdCho) that is released from alveolar epithelia into the alveolus. Surfactant deficiency is an important aspect of inflammatory lung disease and may result from extravasation of serum lipoproteins into the alveolus. We investigated whether one bioactive component of modified lipoproteins, oxysterols, might reduce surfactant PtdCho availability by altering its trafficking. The oxysterol, 22-hydroxycholesterol (22HC), in combination with its obligate partner, 9 cis-retinoic acid (RA), decreased surfactant PtdCho levels, in part, by stimulating basolateral phospholipid export in murine lung epithelia. 22HC/RA stimulated basolateral PtdCho efflux in cells via transcriptional activation of the ATP-binding cassette transporter 1 (ABCA1) gene. This effect was mediated by a DR-4 locus within the ABCA1 promoter. ABCA1 knockdown studies using ABCA1 siRNA or the ABCA1 inhibitor, glyburide, selectively attenuated 22HC/RA-driven basolateral PtdCho efflux. 22HC/RA significantly increased export of PtdCho molecular species containing saturated (16:0) fatty-acyl species typical of DSPtdCho. Overexpression of ABCA1 mimicked 22HC/RA effects by increasing cellular PtdCho efflux, whereas mutagenesis of ABCA1 at Trp590 attenuated PtdCho release. The results indicate the existence of an oxysterol-activated basolateral exit pathway for surfactant that might impact the availability of phospholipid destined for apical secretion. PMID:15039140

  10. 5-Aminolevulinic Acid Thins Pear Fruits by Inhibiting Pollen Tube Growth via Ca2+-ATPase-Mediated Ca2+ Efflux

    PubMed Central

    An, Yuyan; Li, Jie; Duan, Chunhui; Liu, Longbo; Sun, Yongping; Cao, Rongxiang; Wang, Liangju

    2016-01-01

    Chemical fruit thinning has become a popular practice in modern fruit orchards for achieving high quality fruits, reducing costs of hand thinning and promoting return bloom. However, most of the suggested chemical thinners are often concerned for their detrimental effects and environmental problems. 5-Aminolevulic acid (ALA) is a natural, nontoxic, biodegradable, and environment-friendly plant growth regulator. One of its outstanding roles is improving plant photosynthesis and fruit quality. Here, results showed that applying 100–200 mg/L ALA at full bloom stage significantly reduced pear fruit set. Both in vivo and in vitro studies showed that ALA significantly inhibited pollen germination and tube growth. ALA decreased not only cytosolic Ca2+ concentration ([Ca2+]cyt) but also “tip-focused” [Ca2+]cyt gradient, indicating that ALA inhibited pollen tube growth by down-regulating calcium signaling. ALA drastically enhanced pollen Ca2+-ATPase activity, suggesting that ALA-induced decrease of calcium signaling probably resulted from activating calcium pump. The significant negative correlations between Ca2+-ATPase activity and pollen germination or pollen tube length further demonstrated the critical role of calcium pump in ALA's negative effect on pollen germination. Taken together, our results suggest that ALA at low concentrations is a potential biochemical thinner, and it inhibits pollen germination and tube growth via Ca2+ efflux by activating Ca2+-ATPase, thereby thinning fruits by preventing fertilization. PMID:26904082

  11. Na(+)/K(+) pump interacts with the h-current to control bursting activity in central pattern generator neurons of leeches.

    PubMed

    Kueh, Daniel; Barnett, William H; Cymbalyuk, Gennady S; Calabrese, Ronald L

    2016-01-01

    The dynamics of different ionic currents shape the bursting activity of neurons and networks that control motor output. Despite being ubiquitous in all animal cells, the contribution of the Na(+)/K(+) pump current to such bursting activity has not been well studied. We used monensin, a Na(+)/H(+) antiporter, to examine the role of the pump on the bursting activity of oscillator heart interneurons in leeches. When we stimulated the pump with monensin, the period of these neurons decreased significantly, an effect that was prevented or reversed when the h-current was blocked by Cs(+). The decreased period could also occur if the pump was inhibited with strophanthidin or K(+)-free saline. Our monensin results were reproduced in model, which explains the pump's contributions to bursting activity based on Na(+) dynamics. Our results indicate that a dynamically oscillating pump current that interacts with the h-current can regulate the bursting activity of neurons and networks. PMID:27588351

  12. Pumping speed offered by activated carbon at liquid helium temperatures by sorbents adhered to indigenously developed hydroformed cryopanel

    NASA Astrophysics Data System (ADS)

    Gangradey, Ranjana; Shanti Mukherjee, Samiran; Panchal, Paresh; Nayak, Pratik; Agarwal, Jyoti; Rana, Chirag; Kasthurirengan, S.; Shankar Mishra, Jyoti; Patel, Haresh; Bairagi, Pawan; Lambade, Vrushabh; Sayani, Reena

    2015-12-01

    Towards the aim of developing a pump with large pumping speed of the order of 1 L/(s-cm2) or above for gases like hydrogen and helium through physical adsorption, development of activated carbon based sorbents like granules, spheres, flocked fibres, knitted and non -knitted cloth was carried out. To investigate the pumping speed offered, a test facility SSCF (Small Scale Cryopump Facility) which can take samples of hydroformed cryopanel (a technology developed in India) of size ∼500 mm × 100 mm was set up as per international standards comprising a dome mounted with gauges, calibrated leak valve, gas analyser, sorbent adhered to cryopanel etc. The cryopanel was shielded by chevron baffles. Pumping speed measurements were carried out for gases like hydrogen, helium and argon at a constant panel temperature in the pressure range of 1×10-7 to 1×10-4 mbar, and pumping speed was found to be in the range of 2000 L/s for a pressure range 1×10-6 to 1×10-4 mbar, and 4000 L/s for pressure range 1×10-7mbar and below for a pumping surface area of ∼1000 cm2 thus giving an average pumping speed of about 2 L/(s-cm2). Using the Monte Carlo codes SSCF was modelled and simulation studies performed. Parameters like sticking coefficient, capture coefficients affecting the pumping speed were studied. This paper describes the experimental setup of SSCF, experimental results and its correlation with Monte-Carlo simulation.

  13. Non-equivalent roles of two periplasmic subunits in the function and assembly of triclosan pump TriABC from Pseudomonas aeruginosa.

    PubMed

    Weeks, Jon W; Nickels, Logan M; Ntreh, Abigail T; Zgurskaya, Helen I

    2015-10-01

    In Gram-negative bacteria, multidrug efflux transporters function in complexes with periplasmic membrane fusion proteins (MFPs) that enable antibiotic efflux across the outer membrane. In this study, we analyzed the function, composition and assembly of the triclosan efflux transporter TriABC-OpmH from Pseudomonas aeruginosa. We report that this transporter possesses a surprising substrate specificity that encompasses not only triclosan but the detergent SDS, which are often used together in antibacterial soaps. These two compounds interact antagonistically in a TriABC-dependent manner and negate antibacterial properties of each other. Unlike other efflux pumps that rely on a single MFP for their activities, two different MFPs, TriA and TriB, are required for triclosan/SDS resistance mediated by TriABC-OpmH. We found that analogous mutations in the α-helical hairpin and membrane proximal domains of TriA and TriB differentially affect triclosan efflux and assembly of the complex. Furthermore, our results show that TriA and TriB function as a dimer, in which TriA is primarily responsible for stabilizing interactions with the outer membrane channel, whereas TriB is important for the stimulation of the transporter. We conclude that MFPs are engaged into complexes as asymmetric dimers, in which each protomer plays a specific role. PMID:26193906

  14. RAGE Suppresses ABCG1-Mediated Macrophage Cholesterol Efflux in Diabetes.

    PubMed

    Daffu, Gurdip; Shen, Xiaoping; Senatus, Laura; Thiagarajan, Devi; Abedini, Andisheh; Hurtado Del Pozo, Carmen; Rosario, Rosa; Song, Fei; Friedman, Richard A; Ramasamy, Ravichandran; Schmidt, Ann Marie

    2015-12-01

    Diabetes exacerbates cardiovascular disease, at least in part through suppression of macrophage cholesterol efflux and levels of the cholesterol transporters ATP binding cassette transporter A1 (ABCA1) and ABCG1. The receptor for advanced glycation end products (RAGE) is highly expressed in human and murine diabetic atherosclerotic plaques, particularly in macrophages. We tested the hypothesis that RAGE suppresses macrophage cholesterol efflux and probed the mechanisms by which RAGE downregulates ABCA1 and ABCG1. Macrophage cholesterol efflux to apolipoprotein A1 and HDL and reverse cholesterol transport to plasma, liver, and feces were reduced in diabetic macrophages through RAGE. In vitro, RAGE ligands suppressed ABCG1 and ABCA1 promoter luciferase activity and transcription of ABCG1 and ABCA1 through peroxisome proliferator-activated receptor-γ (PPARG)-responsive promoter elements but not through liver X receptor elements. Plasma levels of HDL were reduced in diabetic mice in a RAGE-dependent manner. Laser capture microdissected CD68(+) macrophages from atherosclerotic plaques of Ldlr(-/-) mice devoid of Ager (RAGE) displayed higher levels of Abca1, Abcg1, and Pparg mRNA transcripts versus Ager-expressing Ldlr(-/-) mice independently of glycemia or plasma levels of total cholesterol and triglycerides. Antagonism of RAGE may fill an important therapeutic gap in the treatment of diabetic macrovascular complications. PMID:26253613

  15. A Comparative Analysis of Synthetic Quorum Sensing Modulators in Pseudomonas aeruginosa: New Insights into Mechanism, Active Efflux Susceptibility, Phenotypic Response, and Next-Generation Ligand Design

    PubMed Central

    2015-01-01

    Quorum sensing (QS) is a chemical signaling mechanism that allows bacterial populations to coordinate gene expression in response to social and environmental cues. Many bacterial pathogens use QS to initiate infection at high cell densities. Over the past two decades, chemical antagonists of QS in pathogenic bacteria have attracted substantial interest for use both as tools to further elucidate QS mechanisms and, with further development, potential anti-infective agents. Considerable recent research has been devoted to the design of small molecules capable of modulating the LasR QS receptor in the opportunistic pathogen Pseudomonas aeruginosa. These molecules hold significant promise in a range of contexts; however, as most compounds have been developed independently, comparative activity data for these compounds are scarce. Moreover, the mechanisms by which the bulk of these compounds act are largely unknown. This paucity of data has stalled the choice of an optimal chemical scaffold for further advancement. Herein, we submit the best-characterized LasR modulators to standardized cell-based reporter and QS phenotypic assays in P. aeruginosa, and we report the first comprehensive set of comparative LasR activity data for these compounds. Our experiments uncovered multiple interesting mechanistic phenomena (including a potential alternative QS-modulatory ligand binding site/partner) that provide new, and unexpected, insights into the modes by which many of these LasR ligands act. The lead compounds, data trends, and mechanistic insights reported here will significantly aid the design of new small molecule QS inhibitors and activators in P. aeruginosa, and in other bacteria, with enhanced potencies and defined modes of action. PMID:26491787

  16. Modifications on the hydrogen bond network by mutations of Escherichia coli copper efflux oxidase affect the process of proton transfer to dioxygen leading to alterations of enzymatic activities

    SciTech Connect

    Kajikawa, Takao; Kataoka, Kunishige; Sakurai, Takeshi

    2012-05-25

    Highlights: Black-Right-Pointing-Pointer Proton transfer pathway to dioxygen in CueO was identified. Black-Right-Pointing-Pointer Glu506 is the key amino acid to transport proton. Black-Right-Pointing-Pointer The Ala mutation at Glu506 formed a compensatory proton transfer pathway. Black-Right-Pointing-Pointer The Ile mutation at Glu506 shut down the hydrogen bond network. -- Abstract: CueO has a branched hydrogen bond network leading from the exterior of the protein molecule to the trinuclear copper center. This network transports protons in the four-electron reduction of dioxygen. We replaced the acidic Glu506 and Asp507 residues with the charged and uncharged amino acid residues. Peculiar changes in the enzyme activity of the mutants relative to the native enzyme indicate that an acidic amino acid residue at position 506 is essential for effective proton transport. The Ala mutation resulted in the formation of a compensatory hydrogen bond network with one or two extra water molecules. On the other hand, the Ile mutation resulted in the complete shutdown of the hydrogen bond network leading to loss of enzymatic activities of CueO. In contrast, the hydrogen bond network without the proton transport function was constructed by the Gln mutation. These results exerted on the hydrogen bond network in CueO are discussed in comparison with proton transfers in cytochrome oxidase.

  17. Effect of alcohol and kolanut interaction on brain sodium pump activity in Wistar albino rats.

    PubMed

    Obochi, G O; Abara, A E; Malu, S P; Obi-Abang, M; Edu, F E; Eteng, M U; Umoh, I B

    2007-01-01

    Effect of alcohol-kolanut interaction on sodium pump activity in wistar albino rats was studied. Thirty wistar albino rats were divided into six groups of five (5) rats per group and used for the study. The control group (1) received via oral route a placebo (4 ml of distilled water). Groups 2 to 6 were treated for a period of 21 days, with (10% v/v) of alcohol (group 2), 50mg/kg body weight of kolanut (group 3), 50 mg/kg body weight of caffeine (group 4), 4 ml of 10% v/v of alcohol and 50 mg/kg body weight kolanut (group 5), 4 ml of 10% v/v of alcohol and 50 mg/kg body weight of caffeine in 4.0 ml of the vehicle via gastric intubation respectively. A day after the final exposure, the brain of each rat was harvested and processed to examine several biochemical parameters, i.e., total ATpase, ouabain-insensitive ATpase, ouabain sensitive ATpase (Na(+)-K(+)ATPase), non-enzymatic breakdown of ATP and inorganic phosphate (Pi) released. The results showed that the essential enzyme of the brain responsible for neuronal function, Na(+)-K(+)ATPase, was inhibited by alcohol-kolanut co-administration relative to control, resulting in a decrease in Na(+)-K(+)ATPase activity, ATP production, ion transport and action potential, leading to loss of neuronal activities. PMID:18379627

  18. Modifications of laccase activities of copper efflux oxidase, CueO by synergistic mutations in the first and second coordination spheres of the type I copper center.

    PubMed

    Kataoka, Kunishige; Kogi, Hiroki; Tsujimura, Seiya; Sakurai, Takeshi

    2013-02-15

    The redox potential of type I copper in the Escherichia coli multicopper oxidase CueO was shifted in the positive or negative direction as a result of the single, double, and triple mutations in the first and second coordination spheres: the formation of the NH···S(-)(Cys500 ligand) hydrogen bond, the breakdown of the NH(His443 ligand)···O(-)(Asp439) hydrogen bond, and the substitution of the Met510 ligand for the non-coordinating Leu or coordinating Gln. Laccase activities of CueO were maximally enhanced 140-fold by virtue of the synergistic effect of mild mutations at and at around the ligand groups to type I copper. PMID:23337502

  19. Radiocarbon in Tree STEM CO2 Efflux

    NASA Astrophysics Data System (ADS)

    Muhr, J.; Czimczik, C. I.; Angert, A.; Trumbore, S.

    2011-12-01

    Carbon dioxide efflux from tree stems can be a significant component of the stand-level carbon balance. Recent studies have demonstrated that tree stem CO2 efflux may reflect more than just in-situ respiration but also transport from other locations and it has been suggested that it may also include C originally respired in roots or even uptake of soil CO2. We report measurements of the radiocarbon signature of carbon emitted from a range of mature tree stems in tropical and temperate forest ecosystems. Comparison of the radiocarbon signature of respired CO2 with the observed rate of decline in atmsopheric 14C-CO2 provides a measure of the time elapsed between C fixation by the plant and its return to the atmosphere as stem CO2 efflux. In all investigated trees, we observed that stem CO2 efflux had higher radiocarbon signatures than the contemporary atmospheric 14C-CO2, and therefore was derived from C fixed one to several years earlier. In tropical forest trees, we found that the 14C signature of CO2 within the stem (~4-5 cm depth) had even higher radiocarbon signatures than the stem CO2 efflux. In one of the investigated tree species, the in-stem CO2 was derived from C sources fixed on average ~20 years previously. These results confirm observations of root-respired CO2 that also have shown contributions of C substrates older than recent photosynthetic products, and the presence of extracable C reserves in wood that reflect the presence of older C sources. Our results imply that stem CO2 efflux is not only derived from respiration of recent photosynthetic products but includes contributions from older, stored C pools. Ongoing investigations will enable us to compare CO2 efflux for trees subjected to experimental drought, and using different life strategies (deciduous versus evergreen oaks) to determine if the use of these older C stores varies with stress.

  20. Low temperature diode pumped active mirror Yb3+:YAG disk laser amplifier studies.

    PubMed

    Marrazzo, Samuel; Gonçalvès-Novo, Thierry; Millet, François; Chanteloup, Jean-Christophe

    2016-06-13

    An experimental study of a static helium gas gap heat switch concept for laser amplification is presented. High single pass gains with large co-sintered ceramic Yb:YAG disks are recorded in the 80-200K temperature range on a diode pumped active mirror amplifier. PMID:27410286

  1. Basal efflux of bile acids contributes to drug-induced bile acid-dependent hepatocyte toxicity in rat sandwich-cultured hepatocytes.

    PubMed

    Susukida, Takeshi; Sekine, Shuichi; Ogimura, Eiichiro; Aoki, Shigeki; Oizumi, Kumiko; Horie, Toshiharu; Ito, Kousei

    2015-10-01

    The bile salt export pump (BSEP or Bsep) functions as an apical transporter to eliminate bile acids (BAs) from hepatocytes into the bile. BSEP or Bsep inhibitors engender BA retention, suggested as an underlying mechanism of cholestatic drug-induced liver injury. We previously reported a method to evaluate BSEP-mediated BA-dependent hepatocyte toxicity by using sandwich-cultured hepatocytes (SCHs). However, basal efflux transporters, including multidrug resistance-associated proteins (MRP or Mrp) 3 and 4, also participate in BA efflux. This study examined the contribution of basal efflux transporters to BA-dependent hepatocyte toxicity in rat SCHs. The apical efflux of [(3)H]taurocholic acid (TC) was potently inhibited by 10 μM cyclosporine A (CsA), with later inhibition of basal [(3)H]TC efflux, while MK571 simultaneously inhibited both apical and basal [(3)H]TC efflux. CsA-induced BA-dependent hepatocyte toxicity was 30% at most at 10 μM CsA and ∼60% at 50 μM, while MK571 exacerbated hepatocyte toxicity at concentrations of ≥50 μM. Quinidine inhibited only basal [(3)H]TC efflux and showed BA-dependent hepatocyte toxicity in rat SCHs. Hence, inhibition of basal efflux transporters as well as Bsep may precipitate BA-dependent hepatocyte toxicity in rat SCHs. PMID:26055650

  2. High-efficient diode-pumped actively Q-switched Nd:YAG/KTP Raman laser at 1096 nm wavelength

    NASA Astrophysics Data System (ADS)

    Su, Fufang; Zhang, Xingyu; Wang, Weitao; Cong, Zhenhua; Shi, Men; Yang, Xiuqin; Kong, Weijin; Ma, Lili; Wu, Wendi

    2013-09-01

    With Nd:YAG as the gain medium and KTP crystal as the Raman medium, the characteristics of an LD pumped intracavity actively Q-switched Nd:YAG/KTP Raman laser at 1096 nm wavelength were studied. The output characteristics of 1096 nm were investigated. At a pulse repetition rate of 30 kHz an average power up to 1.97 W was obtained with the incident pump power of 11.75 W, corresponding to a diode-to-Stokes conversion efficiency of 16.8%.

  3. Increased efflux of glutathione conjugate in acutely diabetic cardiomyocytes.

    PubMed

    Ghosh, Sanjoy; Ting, Simon; Lau, Howard; Pulinilkunnil, Thomas; An, Ding; Qi, Dake; Abrahani, Mohammed A; Rodrigues, Brian

    2004-10-01

    In diabetes, cell death and resultant cardiomyopathy have been linked to oxidative stress and depletion of antioxidants like glutathione (GSH). Although the de novo synthesis and recycling of GSH have been extensively studied in the chronically diabetic heart, their contribution in modulating cardiac oxidative stress in acute diabetes has been largely ignored. Additionally, the possible contribution of cellular efflux in regulating GSH levels during diabetes is unknown. We used streptozotocin to make Wistar rats acutely diabetic and after 4 days examined the different processes that regulate cardiac GSH. Reduction in myocyte GSH in diabetic rats was accompanied by increased oxidative stress, excessive reactive oxygen species, and an elevated apoptotic cell death. The effect on GSH was not associated with any change in either synthesis or recycling, as both gamma-glutamylcysteine synthetase gene expression (responsible for bio syn thesis) and glutathione reductase activity (involved with GSH recycling) remained unchanged. However, gene expression of multidrug resistance protein 1, a transporter implicated in effluxing GSH during oxidative stress, was elevated. GSH conjugate efflux mediated by multidrug resistance protein 1 also increased in diabetic cardiomyocytes, an effect that was blocked using MK-571, a specific inhibitor of this transporter. As MK-571 also decreased oxidative stress in diabetic cardiomyocytes, an important role can be proposed for this transporter in GSH and reactive oxygen species homeostasis in the acutely diabetic heart. PMID:15573148

  4. Temporal evolution of pump beam self-focusing at the High-Frequency Active Auroral Research Program

    NASA Astrophysics Data System (ADS)

    Kosch, M. J.; Pedersen, T.; Mishin, E.; Starks, M.; Gerken-Kendall, E.; Sentman, D.; Oyama, S.; Watkins, B.

    2007-08-01

    On 4 February 2005 the High-Frequency Active Auroral Research Program (HAARP) facility was operated at 2.85 MHz to produce artificial optical emissions in the ionosphere while passing through the second electron gyroharmonic. All-sky optical recordings were performed with 15 s integration, alternating between 557.7 and 630 nm. We report the first optical observations showing the temporal evolution of large-scale pump wave self-focusing in the magnetic zenith, observed in the 557.7 nm images. These clearly show that the maximum intensity was not reached after 15 s of pumping, which is unexpected since the emission delay time is <1 s, and that the optical signature had intensified in a much smaller region within the beam after 45 s of pumping. In addition, adjacent regions within the beam lost intensity. Radar measurements indicate a plasma depletion of ~1% near the HF reflection altitude. Ray tracing of the pump wave through the plasma depletion region, which forms a concave reflecting radio wave mirror, reproduces the optical spatial morphology. A radio wave flux density gain of up to ~30 dB may occur. In addition, the ray trace is consistent with the observed artificial optical emissions for critical plasma frequencies down to ~0.5 MHz below the pump frequency.

  5. Activities of the NASA/Marshall Space Flight Center pump stage technology team

    NASA Technical Reports Server (NTRS)

    Garcia, R.; Mcconnaughey, P.; Eastland, A.

    1992-01-01

    In order to advance rocket propulsion technology, the Consortium for Computational Fluid Dynamics (CFD) Application in Propulsion Technology has been formed at Marshall Space Flight Center (MSFC). The Consortium consists of three Teams: the turbine stage team, the pump stage team (PST), and the combustion devices team. The PST has formulated and is implementing a plan for pump technology development whose end product will be validated CFD codes suitable for application to pump components, test data suitable for validating CFD codes, and advanced pump components optimized using CFD codes. The PST's work during the fall of 1991 and the winter and spring of 1992 is discussed in this paper. This work is highlighted by CFD analyses of an advanced impeller design and collection of laser two-focus velocimeter data for the Space Shuttle Main Engine High Pressure Fuel Pump impeller.

  6. Mind the gap: non-biological processes contributing to soil CO2 efflux.

    PubMed

    Rey, Ana

    2015-05-01

    Widespread recognition of the importance of soil CO2 efflux as a major source of CO2 to the atmosphere has led to active research. A large soil respiration database and recent reviews have compiled data, methods, and current challenges. This study highlights some deficiencies for a proper understanding of soil CO2 efflux focusing on processes of soil CO2 production and transport that have not received enough attention in the current soil respiration literature. It has mostly been assumed that soil CO2 efflux is the result of biological processes (i.e. soil respiration), but recent studies demonstrate that pedochemical and geological processes, such as geothermal and volcanic CO2 degassing, are potentially important in some areas. Besides the microbial decomposition of litter, solar radiation is responsible for photodegradation or photochemical degradation of litter. Diffusion is considered to be the main mechanism of CO2 transport in the soil, but changes in atmospheric pressure and thermal convection may also be important mechanisms driving soil CO2 efflux greater than diffusion under certain conditions. Lateral fluxes of carbon as dissolved organic and inorganic carbon occur and may cause an underestimation of soil CO2 efflux. Traditionally soil CO2 efflux has been measured with accumulation chambers assuming that the main transport mechanism is diffusion. New techniques are available such as improved automated chambers, CO2 concentration profiles and isotopic techniques that may help to elucidate the sources of carbon from soils. We need to develop specific and standardized methods for different CO2 sources to quantify this flux on a global scale. Biogeochemical models should include biological and non-biological CO2 production processes before we can predict the response of soil CO2 efflux to climate change. Improving our understanding of the processes involved in soil CO2 efflux should be a research priority given the importance of this flux in the global

  7. Applicability of soil column incubation experiments to measure CO2 efflux

    NASA Astrophysics Data System (ADS)

    Guo, Linlin; Nishimura, Taku; Imoto, Hiromi; Sun, Zhigang

    2015-10-01

    Accurate measurements of CO2 efflux from soils are essential to understand dynamic changes in soil carbon storage. Column incubation experiments are commonly used to study soil water and solute transport; however, the use of column incubation experiments to study soil CO2 efflux has seldom been reported. In this study, a 150-day greenhouse experiment with two treatments (no-tillage and tillage soils) was conducted to evaluate the applicability of soil column incubation experiments to study CO2 efflux. Both the chamber measurement and the gradient method were used, and results from the two methods were consistent: tillage increased soil cumulative CO2 efflux during the incubation period. Compared with fieldwork, incubation experiments can create or precisely control experimental conditions and thus have advantages for investigating the influence of climate factors or human activities on CO2 efflux. They are superior to bottle incubation because soil column experiments maintain a soil structure that is almost the same as that in the field, and thus can facilitate analyses on CO2 behaviour in the soil profile and more accurate evaluations of CO2 efflux. Although some improvements are still required for column incubation experiments, wider application of this method to study soil CO2 behaviour is expected.

  8. Stimulation of H+ Efflux and Inhibition of Photosynthesis by Esters of Carboxylic Acids 1

    PubMed Central

    Duhaime, Donna E.; Bown, Alan W.

    1983-01-01

    Suspensions of mechanically isolated Asparagus sprengeri Regel mesophyll cells were used to investigate the influence of various carboxyester compounds on rates of net H+ efflux in the dark or light and photosynthetic O2 production. Addition of 0.15 to 1.5 millimolar malathion, α-naphthyl acetate, phenyl acetate, or p-nitrophenyl acetate stimulated H+ efflux and inhibited photosynthesis within 1 minute. In contrast, the more polar esters methyl acetoacetate or ethyl p-aminobenzoate had little or no effect on either of these two processes. A 0.15 millimolar concentration of α-naphthylacetate stimulated the normal rate of H+ efflux, 0.77 nanomoles H+ per 106 cells per minute by 750% and inhibited photosynthesis by 100%. The four active carboxyester compounds also stimulated H+ efflux after the normal rate of H+ efflux was eliminated with 0.01 milligrams per milliliter oligomycin or 100% N2. Oligomycin reduced the ATP level by 70%. Incubation of cells with malathion, α-naphthyl acetate, or p-nitrophenyl acetate resulted in the generation of the respective hydrolysis products ethanol, α-naphthol, and p-nitrophenol. It is proposed that inhibition of photosynthesis and stimulation of H+ efflux result when nonpolar carboxyester compounds enter the cell and generate acidic carboxyl groups when hydrolyzed by esterase enzymes. PMID:16663308

  9. Transferable Resistance to Aminoglycosides by Methylation of G1405 in 16S rRNA and to Hydrophilic Fluoroquinolones by QepA-Mediated Efflux in Escherichia coli▿

    PubMed Central

    Périchon, Bruno; Courvalin, Patrice; Galimand, Marc

    2007-01-01

    Plasmid pIP1206 was detected in Escherichia coli strain 1540 during the screening of clinical isolates of Enterobacteriaceae for high-level resistance to aminoglycosides. The sequence of this IncFI conjugative plasmid of ca. 100 kb was partially determined. pIP1206 carried the rmtB gene for a ribosome methyltransferase that was shown to modify the N7 position of nucleotide G1405, located in the A site of 16S rRNA. It also contained the qepA (quinolone efflux pump) gene that encodes a 14-transmembrane-segment putative efflux pump belonging to the major facilitator superfamily of proton-dependent transporters. Disruption of membrane proton potential by the efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone in a transconjugant harboring the qepA gene resulted in elevation of norfloxacin accumulation. The transporter conferred resistance to the hydrophilic quinolones norfloxacin and ciprofloxacin. PMID:17470656

  10. Influence of the proton pump inhibitor lansoprazole on distribution and activity of doxorubicin in solid tumors

    PubMed Central

    Yu, Man; Lee, Carol; Wang, Marina; Tannock, Ian F

    2015-01-01

    Cellular causes of resistance and limited drug distribution within solid tumors limit therapeutic efficacy of anticancer drugs. Acidic endosomes in cancer cells mediate autophagy, which facilitates survival of stressed cells, and may contribute to drug resistance. Basic drugs (e.g. doxorubicin) are sequestered in acidic endosomes, thereby diverting drugs from their target DNA and decreasing penetration to distal cells. Proton pump inhibitors (PPIs) may raise endosomal pH, with potential to improve drug efficacy and distribution in solid tumors. We determined the effects of the PPI lansoprazole to modify the activity of doxorubicin. To gain insight into its mechanisms, we studied the effects of lansoprazole on endosomal pH, and on the spatial distribution of doxorubicin, and of biomarkers reflecting its activity, using in vitro and murine models. Lansoprazole showed concentration-dependent effects to raise endosomal pH and to inhibit endosomal sequestration of doxorubicin in cultured tumor cells. Lansoprazole was not toxic to cancer cells but potentiated the cytotoxicity of doxorubicin and enhanced its penetration through multilayered cell cultures. In solid tumors, lansoprazole improved the distribution of doxorubicin but also increased expression of biomarkers of drug activity throughout the tumor. Combined treatment with lansoprazole and doxorubicin was more effective in delaying tumor growth as compared to either agent alone. Together, lansoprazole enhances the therapeutic effects of doxorubicin both by improving its distribution and increasing its activity in solid tumors. Use of PPIs to improve drug distribution and to inhibit autophagy represents a promising strategy to enhance the effectiveness of anticancer drugs in solid tumors. PMID:26212113

  11. Top consumer abundance influences lake methane efflux

    PubMed Central

    Devlin, Shawn P.; Saarenheimo, Jatta; Syväranta, Jari; Jones, Roger I.

    2015-01-01

    Lakes are important habitats for biogeochemical cycling of carbon. The organization and structure of aquatic communities influences the biogeochemical interactions between lakes and the atmosphere. Understanding how trophic structure regulates ecosystem functions and influences greenhouse gas efflux from lakes is critical to understanding global carbon cycling and climate change. With a whole-lake experiment in which a previously fishless lake was divided into two treatment basins where fish abundance was manipulated, we show how a trophic cascade from fish to microbes affects methane efflux to the atmosphere. Here, fish exert high grazing pressure and remove nearly all zooplankton. This reduction in zooplankton density increases the abundance of methanotrophic bacteria, which in turn reduce CH4 efflux rates by roughly 10 times. Given that globally there are millions of lakes emitting methane, an important greenhouse gas, our findings that aquatic trophic interactions significantly influence the biogeochemical cycle of methane has important implications. PMID:26531291

  12. Top consumer abundance influences lake methane efflux.

    PubMed

    Devlin, Shawn P; Saarenheimo, Jatta; Syväranta, Jari; Jones, Roger I

    2015-01-01

    Lakes are important habitats for biogeochemical cycling of carbon. The organization and structure of aquatic communities influences the biogeochemical interactions between lakes and the atmosphere. Understanding how trophic structure regulates ecosystem functions and influences greenhouse gas efflux from lakes is critical to understanding global carbon cycling and climate change. With a whole-lake experiment in which a previously fishless lake was divided into two treatment basins where fish abundance was manipulated, we show how a trophic cascade from fish to microbes affects methane efflux to the atmosphere. Here, fish exert high grazing pressure and remove nearly all zooplankton. This reduction in zooplankton density increases the abundance of methanotrophic bacteria, which in turn reduce CH4 efflux rates by roughly 10 times. Given that globally there are millions of lakes emitting methane, an important greenhouse gas, our findings that aquatic trophic interactions significantly influence the biogeochemical cycle of methane has important implications. PMID:26531291

  13. Fiber-laser pumped actively Q-switched Er:LuYAG laser at 1648 nm

    NASA Astrophysics Data System (ADS)

    Yang, X. F.; Wang, Y.; Zhao, T.; Zhu, H. Y.; Shen, D. Y.

    2016-02-01

    We demonstrated an acousto-optic Q-switched 1648 nm Er:LuYAG laser resonantly pumped by a cladding-pumped Er,Yb fiber laser at 1532 nm. Stable Q-switching operation was obtained with the pulse repetition rate (PRR) varying from 200 Hz to 10 kHz. At PRR of 200 Hz, the laser yielded Q-switched pulses with 3.3 mJ pulse energy and 65 ns pulse duration, corresponding to a peak power of 50.7 kW for 10.4 W of incident pump power.

  14. Influence of multidrug efflux systems on methylene blue-mediated photodynami