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Sample records for abc efflux pumps

  1. 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

  2. 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.

  3. The Efflux Pump Inhibitor Reserpine Selects Multidrug-Resistant Streptococcus pneumoniae Strains That Overexpress the ABC Transporters PatA and PatB▿ †

    PubMed Central

    Garvey, Mark I.; Piddock, Laura J. V.

    2008-01-01

    One way to combat multidrug-resistant microorganisms is the use of efflux pump inhibitors (EPIs). Spontaneous mutants resistant to the EPI reserpine selected from Streptococcus pneumoniae NCTC 7465 and R6 at a frequency suggestive of a single mutational event were also multidrug resistant. No mutations in pmrA (which encodes the efflux protein PmrA) were detected, and the expression of pmrA was unaltered in all mutants. In the reserpine-resistant multidrug-resistant mutants, the overexpression of both patA and patB, which encode ABC transporters, was associated with accumulation of low concentrations of antibiotics and dyes. The addition of sodium orthovanadate, an inhibitor of ABC efflux pumps, or the insertional inactivation of either gene restored wild-type antibiotic susceptibility and wild-type levels of accumulation. Only when patA was insertionally inactivated were both multidrug resistance and reserpine resistance lost. Strains in which patA was insertionally inactivated grew significantly more slowly than the wild type. These data indicate that the overexpression of both patA and patB confers multidrug resistance in S. pneumoniae but that only patA is involved in reserpine resistance. The selection of reserpine-resistant multidrug-resistant pneumococci has implications for analogous systems in other bacteria or in cancer. PMID:18362193

  4. 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 Central

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

    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

  5. 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

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

    PubMed Central

    Krishnamoorthy, Suvarna; Shah, Bhavikkumar P.; Lee, Hiu Ham

    2015-01-01

    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

  7. The monoamine oxidase A inhibitor clorgyline is a broad-spectrum inhibitor of fungal ABC and MFS transporter efflux pump activities which reverses the azole resistance of Candida albicans and Candida glabrata clinical isolates.

    PubMed

    Holmes, Ann R; Keniya, Mikhail V; Ivnitski-Steele, Irena; Monk, Brian C; Lamping, Erwin; Sklar, Larry A; Cannon, Richard D

    2012-03-01

    Resistance to the commonly used azole antifungal fluconazole (FLC) can develop due to overexpression of ATP-binding cassette (ABC) and major facilitator superfamily (MFS) plasma membrane transporters. An approach to overcoming this resistance is to identify inhibitors of these efflux pumps. We have developed a pump assay suitable for high-throughput screening (HTS) that uses recombinant Saccharomyces cerevisiae strains hyperexpressing individual transporters from the opportunistic fungal pathogen Candida albicans. The recombinant strains possess greater resistance to azoles and other pump substrates than the parental host strain. A flow cytometry-based HTS, which measured increased intracellular retention of the fluorescent pump substrate rhodamine 6G (R6G) within yeast cells, was used to screen the Prestwick Chemical Library (PCL) of 1,200 marketed drugs. Nine compounds were identified as hits, and the monoamine oxidase A inhibitor (MAOI) clorgyline was identified as an inhibitor of two C. albicans ABC efflux pumps, CaCdr1p and CaCdr2p. Secondary in vitro assays confirmed inhibition of pump-mediated efflux by clorgyline. Clorgyline also reversed the FLC resistance of S. cerevisiae strains expressing other individual fungal ABC transporters (Candida glabrata Cdr1p or Candida krusei Abc1p) or the C. albicans MFS transporter Mdr1p. Recombinant strains were also chemosensitized by clorgyline to other azoles (itraconazole and miconazole). Importantly, clorgyline showed synergy with FLC against FLC-resistant C. albicans clinical isolates and a C. glabrata strain and inhibited R6G efflux from a FLC-resistant C. albicans clinical isolate. Clorgyline is a novel broad-spectrum inhibitor of two classes of fungal efflux pumps that acts synergistically with azoles against azole-resistant C. albicans and C. glabrata strains. PMID:22203607

  8. 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

  9. 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

  10. 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.

  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. Efflux pump-mediated benzalkonium chloride resistance in Listeria monocytogenes isolated from retail food.

    PubMed

    Jiang, Xiaobing; Yu, Tao; Liang, Yu; Ji, Shengdong; Guo, Xiaowei; Ma, Jianmin; Zhou, Lijun

    2016-01-18

    In this study, efflux pump-mediated benzalkonium chloride (BC) resistance, including plasmid-encoded (Qac protein family and BcrABC) and chromosome-borne efflux pumps, was investigated in Listeria monocytogenes from retail food in China. Among the 59 L. monocytogenes strains, 13 (22.0%) strains were resistant to BC. The PCR results showed that bcrABC was harbored by 2 of 13 BC resistant strains. However, none of the qac genes were detected among the 59 strains. The bcrABC was absent in both of the plasmid cured strains, indicating that this BC resistance determinant was plasmid-encoded in the two bcrABC-positive strains. In the presence of reserpine, most of the bcrABC-negative strains had decreases in the MICs of BC, suggesting the existence of other efflux pumps and their role in BC resistance. After exposed to reserpine, the reduction in BC MICs was observed in the two cured strains, indicating that efflux pumps located on chromosome was also involved in BC resistance. Our findings suggest that food products may act as reservoirs for BC resistant isolates of L. monocytogenes and plasmid- and chromosome-encoded efflux pumps could mediate the BC resistance of L. monocytogenes, which is especially relevant to the adaption of this organism in food-related environments with frequent BC use. PMID:26513255

  13. 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

  14. Emergence of a Potent Multidrug Efflux Pump Variant That Enhances Campylobacter Resistance to Multiple Antibiotics

    PubMed Central

    Yao, Hong; Shen, Zhangqi; Wang, Yang; Deng, Fengru; Liu, Dejun; Naren, Gaowa; Dai, Lei; Su, Chih-Chia; Wang, Bing; Wang, Shaolin; Wu, Congming; Yu, Edward W.

    2016-01-01

    ABSTRACT Bacterial antibiotic efflux pumps are key players in antibiotic resistance. Although their role in conferring multidrug resistance is well documented, the emergence of “super” efflux pump variants that enhance bacterial resistance to multiple drugs has not been reported. Here, we describe the emergence of a resistance-enhancing variant (named RE-CmeABC) of the predominant efflux pump CmeABC in Campylobacter, a major zoonotic pathogen whose resistance to antibiotics is considered a serious antibiotic resistance threat in the United States. Compared to the previously characterized CmeABC transporters, RE-CmeABC is much more potent in conferring Campylobacter resistance to antibiotics, which was shown by increased MICs and reduced intracellular accumulation of antibiotics. Structural modeling suggests that sequence variations in the drug-binding pocket of CmeB possibly contribute to the enhanced efflux function. Additionally, RE-CmeABC expands the mutant selection window of ciprofloxacin, enhances the emergence of antibiotic-resistant mutants, and confers exceedingly high-level resistance to fluoroquinolones, an important class of antibiotics for clinical therapy of campylobacteriosis. Furthermore, RE-CmeABC is horizontally transferable, shifts antibiotic MIC distribution among clinical isolates, and is increasingly prevalent in Campylobacter jejuni isolates, suggesting that it confers a fitness advantage under antimicrobial selection. These findings reveal a new mechanism for enhanced multidrug resistance and an effective strategy utilized by bacteria for adaptation to selection from multiple antibiotics. PMID:27651364

  15. 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

  16. Distribution of the multidrug efflux pump genes, adeABC, adeDE and adeIJK, and class 1 integron genes in multiple-antimicrobial-resistant clinical isolates of Acinetobacter baumannii-Acinetobacter calcoaceticus complex.

    PubMed

    Lin, Li; Ling, Bao-Dong; Li, Xian-Zhi

    2009-01-01

    Of 112 non-repetitive clinical isolates of Acinetobacter baumannii-Acinetobacter calcoaceticus complex, 80% were resistant to a variety of structurally unrelated antimicrobials although all isolates were susceptible to minocycline and polymyxin. Resistance to carbapenems occurred in 8% of the isolates. The presence of adeSR-adeABC, adeDE and adeIJK drug efflux system genes and class 1 integron genes (integrase gene int1) was assessed by polymerase chain reaction (PCR) in relation to the susceptibility of the isolates to 20 antimicrobials. The majority of isolates (75%) with high levels of multidrug resistance were positive for adeSR-adeABC and adeIJK as well as int1 and thus belong to A. baumannii (i.e. genomospecies 2). Positive adeE was only observed in adeSR-adeABC/adeIJK/int1-negative isolates (8%; likely belonging to Acinetobacter genomospecies 3) that were relatively susceptible to several agents, and adeE expression was undetectable. The results reveal a possible association between adeABC/adeIJK and int1 in multidrug-resistant isolates of A. baumannii. In addition, differential distribution of the resistance-nodulation-cell division (RND) genes can likely be used as indicators for differentiating Acinetobacter species.

  17. Modified host cells with efflux pumps

    DOEpatents

    Dunlop, Mary J.; Keasling, Jay D.; Mukhopadhyay, Aindrila

    2016-08-30

    The present invention provides for a modified host cell comprising a heterologous expression of an efflux pump capable of transporting an organic molecule out of the host cell wherein the organic molecule at a sufficiently high concentration reduces the growth rate of or is lethal to the host cell.

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

    PubMed

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

    2016-01-01

    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

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

    PubMed

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

    2016-01-01

    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.

  20. Microbial Efflux Pump Inhibition: Tactics and Strategies

    PubMed Central

    Tegos, George P.; Haynes, Mark; Strouse, J. Jacob; Khan, Mohiuddin Md. T.; Bologa, Cristian G.; Oprea, Tudor I.; Sklar, Larry A.

    2013-01-01

    Traditional antimicrobials are increasingly suffering from the emergence of multidrug resistance among pathogenic microorganisms. To overcome these deficiencies, a range of novel approaches to control microbial infections are under investigation as potential alternative treatments. Multidrug efflux is a key target of these efforts. Efflux mechanisms are broadly recognized as major components of resistance to many classes of chemotherapeutic agents as well as antimicrobials. Efflux occurs due to the activity of membrane transporter proteins widely known as Multidrug Efflux Systems (MES). They are implicated in a variety of physiological roles other than efflux and identifying natural substrates and inhibitors is an active and expanding research discipline. One plausible alternative is the combination of conventional antimicrobial agents/antibiotics with small molecules that block MES known as multidrug efflux pump inhibitors (EPIs). An array of approaches in academic and industrial research settings, varying from high-throughput screening (HTS) ventures to bioassay guided purification and determination, have yielded a number of promising EPIs in a series of pathogenic systems. This synergistic discovery platform has been exploited in translational directions beyond the potentiation of conventional antimicrobial treatments. This venture attempts to highlight different tactical elements of this platform, identifying the need for highly informative and comprehensive EPI-discovery strategies. Advances in assay development genomics, proteomics as well as the accumulation of bioactivity and structural information regarding MES facilitates the basis for a new discovery era. This platform is expanding drastically. A combination of chemogenomics and chemoinformatics approaches will integrate data mining with virtual and physical HTS ventures and populate the chemical-biological interface with a plethora of novel chemotypes. This comprehensive step will expedite the

  1. Targeting efflux pumps to overcome antifungal drug resistance.

    PubMed

    Holmes, Ann R; Cardno, Tony S; Strouse, J Jacob; Ivnitski-Steele, Irena; Keniya, Mikhail V; Lackovic, Kurt; Monk, Brian C; Sklar, Larry A; Cannon, Richard D

    2016-08-01

    Resistance to antifungal drugs is an increasingly significant clinical problem. The most common antifungal resistance encountered is efflux pump-mediated resistance of Candida species to azole drugs. One approach to overcome this resistance is to inhibit the pumps and chemosensitize resistant strains to azole drugs. Drug discovery targeting fungal efflux pumps could thus result in the development of azole-enhancing combination therapy. Heterologous expression of fungal efflux pumps in Saccharomyces cerevisiae provides a versatile system for screening for pump inhibitors. Fungal efflux pumps transport a range of xenobiotics including fluorescent compounds. This enables the use of fluorescence-based detection, as well as growth inhibition assays, in screens to discover compounds targeting efflux-mediated antifungal drug resistance. A variety of medium- and high-throughput screens have been used to identify a number of chemical entities that inhibit fungal efflux pumps. PMID:27463566

  2. 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

  3. Multidrug efflux pumps in Staphylococcus aureus and their clinical implications.

    PubMed

    Jang, Soojin

    2016-01-01

    Antibiotic resistance is rapidly spreading among bacteria such as Staphylococcus aureus, an opportunistic bacterial pathogen that causes a variety of diseases in humans. For the last two decades, bacterial multidrug efflux pumps have drawn attention due to their potential association with clinical multidrug resistance. Numerous researchers have demonstrated efflux-mediated resistance in vitro and in vivo and found novel multidrug transporters using advanced genomic information about bacteria. This article aims to provide a concise summary of multidrug efflux pumps and their important clinical implications, focusing on recent findings concerning S. aureus efflux pumps.

  4. 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

  5. Physiological characterisation of the efflux pump system of antibiotic-susceptible and multidrug-resistant Enterobacter aerogenes.

    PubMed

    Martins, A; Spengler, G; Martins, M; Rodrigues, L; Viveiros, M; Davin-Regli, A; Chevalier, J; Couto, I; Pagès, J M; Amaral, L

    2010-10-01

    Enterobacter aerogenes predominates amongst Enterobacteriaceae species that are increasingly reported as producers of extended-spectrum beta-lactamases. Although this mechanism of resistance to beta-lactams is important, other mechanisms bestowing a multidrug-resistant (MDR) phenotype in this species are now well documented. Amongst these mechanisms is the overexpression of efflux pumps that extrude structurally unrelated antibiotics prior to their reaching their targets. Interestingly, although knowledge of the genetic background behind efflux pumps is rapidly advancing, few studies assess the physiological nature of the overall efflux pump system of this, or for that matter any other, bacterium. The study reported here evaluates physiologically the efflux pump system of an E. aerogenes ATCC reference as well as two strains whose MDR phenotypes are mediated by overexpressed efflux pumps. The activities of the efflux pumps in these strains are modulated by pH and glucose, although the effects of the latter are essentially restricted to pH 8, suggesting the presence of two general efflux pump systems, i.e. proton-motive force-dependent and ABC transporter types, respectively.

  6. Bacterial Multidrug Efflux Pumps: Much More Than Antibiotic Resistance Determinants.

    PubMed

    Blanco, Paula; Hernando-Amado, Sara; Reales-Calderon, Jose Antonio; Corona, Fernando; Lira, Felipe; Alcalde-Rico, Manuel; Bernardini, Alejandra; Sanchez, Maria Blanca; Martinez, Jose Luis

    2016-01-01

    Bacterial multidrug efflux pumps are antibiotic resistance determinants present in all microorganisms. With few exceptions, they are chromosomally encoded and present a conserved organization both at the genetic and at the protein levels. In addition, most, if not all, strains of a given bacterial species present the same chromosomally-encoded efflux pumps. Altogether this indicates that multidrug efflux pumps are ancient elements encoded in bacterial genomes long before the recent use of antibiotics for human and animal therapy. In this regard, it is worth mentioning that efflux pumps can extrude a wide range of substrates that include, besides antibiotics, heavy metals, organic pollutants, plant-produced compounds, quorum sensing signals or bacterial metabolites, among others. In the current review, we present information on the different functions that multidrug efflux pumps may have for the bacterial behaviour in different habitats as well as on their regulation by specific signals. Since, in addition to their function in non-clinical ecosystems, multidrug efflux pumps contribute to intrinsic, acquired, and phenotypic resistance of bacterial pathogens, the review also presents information on the search for inhibitors of multidrug efflux pumps, which are currently under development, in the aim of increasing the susceptibility of bacterial pathogens to antibiotics. PMID:27681908

  7. Bacterial Multidrug Efflux Pumps: Much More Than Antibiotic Resistance Determinants

    PubMed Central

    Blanco, Paula; Hernando-Amado, Sara; Reales-Calderon, Jose Antonio; Corona, Fernando; Lira, Felipe; Alcalde-Rico, Manuel; Bernardini, Alejandra; Sanchez, Maria Blanca; Martinez, Jose Luis

    2016-01-01

    Bacterial multidrug efflux pumps are antibiotic resistance determinants present in all microorganisms. With few exceptions, they are chromosomally encoded and present a conserved organization both at the genetic and at the protein levels. In addition, most, if not all, strains of a given bacterial species present the same chromosomally-encoded efflux pumps. Altogether this indicates that multidrug efflux pumps are ancient elements encoded in bacterial genomes long before the recent use of antibiotics for human and animal therapy. In this regard, it is worth mentioning that efflux pumps can extrude a wide range of substrates that include, besides antibiotics, heavy metals, organic pollutants, plant-produced compounds, quorum sensing signals or bacterial metabolites, among others. In the current review, we present information on the different functions that multidrug efflux pumps may have for the bacterial behaviour in different habitats as well as on their regulation by specific signals. Since, in addition to their function in non-clinical ecosystems, multidrug efflux pumps contribute to intrinsic, acquired, and phenotypic resistance of bacterial pathogens, the review also presents information on the search for inhibitors of multidrug efflux pumps, which are currently under development, in the aim of increasing the susceptibility of bacterial pathogens to antibiotics.

  8. Bacterial Multidrug Efflux Pumps: Much More Than Antibiotic Resistance Determinants.

    PubMed

    Blanco, Paula; Hernando-Amado, Sara; Reales-Calderon, Jose Antonio; Corona, Fernando; Lira, Felipe; Alcalde-Rico, Manuel; Bernardini, Alejandra; Sanchez, Maria Blanca; Martinez, Jose Luis

    2016-01-01

    Bacterial multidrug efflux pumps are antibiotic resistance determinants present in all microorganisms. With few exceptions, they are chromosomally encoded and present a conserved organization both at the genetic and at the protein levels. In addition, most, if not all, strains of a given bacterial species present the same chromosomally-encoded efflux pumps. Altogether this indicates that multidrug efflux pumps are ancient elements encoded in bacterial genomes long before the recent use of antibiotics for human and animal therapy. In this regard, it is worth mentioning that efflux pumps can extrude a wide range of substrates that include, besides antibiotics, heavy metals, organic pollutants, plant-produced compounds, quorum sensing signals or bacterial metabolites, among others. In the current review, we present information on the different functions that multidrug efflux pumps may have for the bacterial behaviour in different habitats as well as on their regulation by specific signals. Since, in addition to their function in non-clinical ecosystems, multidrug efflux pumps contribute to intrinsic, acquired, and phenotypic resistance of bacterial pathogens, the review also presents information on the search for inhibitors of multidrug efflux pumps, which are currently under development, in the aim of increasing the susceptibility of bacterial pathogens to antibiotics.

  9. Bacterial Multidrug Efflux Pumps: Much More Than Antibiotic Resistance Determinants

    PubMed Central

    Blanco, Paula; Hernando-Amado, Sara; Reales-Calderon, Jose Antonio; Corona, Fernando; Lira, Felipe; Alcalde-Rico, Manuel; Bernardini, Alejandra; Sanchez, Maria Blanca; Martinez, Jose Luis

    2016-01-01

    Bacterial multidrug efflux pumps are antibiotic resistance determinants present in all microorganisms. With few exceptions, they are chromosomally encoded and present a conserved organization both at the genetic and at the protein levels. In addition, most, if not all, strains of a given bacterial species present the same chromosomally-encoded efflux pumps. Altogether this indicates that multidrug efflux pumps are ancient elements encoded in bacterial genomes long before the recent use of antibiotics for human and animal therapy. In this regard, it is worth mentioning that efflux pumps can extrude a wide range of substrates that include, besides antibiotics, heavy metals, organic pollutants, plant-produced compounds, quorum sensing signals or bacterial metabolites, among others. In the current review, we present information on the different functions that multidrug efflux pumps may have for the bacterial behaviour in different habitats as well as on their regulation by specific signals. Since, in addition to their function in non-clinical ecosystems, multidrug efflux pumps contribute to intrinsic, acquired, and phenotypic resistance of bacterial pathogens, the review also presents information on the search for inhibitors of multidrug efflux pumps, which are currently under development, in the aim of increasing the susceptibility of bacterial pathogens to antibiotics. PMID:27681908

  10. Multidrug efflux pumps and cancer stem cells: insights into multidrug resistance and therapeutic development.

    PubMed

    Moitra, K; Lou, H; Dean, M

    2011-04-01

    Stem cells possess the dual properties of self-renewal and pluripotency. Self-renewal affords these populations the luxury of self-propagation, whereas pluripotency allows them to produce the multitude of cell types found in the body. Protection of the stem cell population from damage or death is critical because these cells need to remain intact throughout the life of an organism. The principal mechanism of protection is through expression of multifunctional efflux transporters--the adenosine triphosphate-binding cassette (ABC) transporters that are the "guardians" of the stem cell population. Ironically, it has been shown that these ABC efflux pumps also afford protection to cancer stem cells (CSCs), shielding them from the adverse effects of chemotherapeutic insult. It is therefore imperative to gain a better understanding of the mechanisms involved in the resistance of stem cells to chemotherapy, which could lead to the discovery of new therapeutic targets and improvement of current anticancer strategies. PMID:21368752

  11. 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

  12. 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

  13. 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

  14. Regulation of ABC efflux transporters at blood-brain barrier in health and neurological disorders.

    PubMed

    Qosa, Hisham; Miller, David S; Pasinelli, Piera; Trotti, Davide

    2015-12-01

    The strength of the blood-brain barrier (BBB) in providing protection to the central nervous system from exposure to circulating chemicals is maintained by tight junctions between endothelial cells and by a broad range of transporter proteins that regulate exchange between CNS and blood. The most important transporters that restrict the permeability of large number of toxins as well as therapeutic agents are the ABC transporters. Among them, P-gp, BCRP, MRP1 and MRP2 are the utmost studied. These efflux transporters are neuroprotective, limiting the brain entry of neurotoxins; however, they could also restrict the entry of many therapeutics and contribute to CNS pharmacoresistance. Characterization of several regulatory pathways that govern expression and activity of ABC efflux transporters in the endothelium of brain capillaries have led to an emerging consensus that these processes are complex and contain several cellular and molecular elements. Alterations in ABC efflux transporters expression and/or activity occur in several neurological diseases. Here, we review the signaling pathways that regulate expression and transport activity of P-gp, BCRP, MRP1 and MRP2 as well as how their expression/activity changes in neurological diseases. This article is part of a Special Issue entitled SI: Neuroprotection.

  15. 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

  16. Assembly and operation of bacterial tripartite multidrug efflux pumps.

    PubMed

    Du, Dijun; van Veen, Hendrik W; Luisi, Ben F

    2015-05-01

    Microorganisms encode several classes of transmembrane pumps that can expel an enormous range of toxic substances, thereby improving their fitness in harsh environments and contributing to resistance against antimicrobial agents. In Gram-negative bacteria these pumps can take the form of tripartite assemblies that actively efflux drugs and other harmful compounds across the cell envelope. We describe recent structural and functional data that have provided insights into the transport mechanisms of these intricate molecular machines.

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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

  2. Engineering microbial biofuel tolerance and export using efflux pumps.

    PubMed

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

    2011-05-10

    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.

  3. 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

  4. 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.

  5. 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

  6. 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

  7. Channel-tunnels: outer membrane components of type I secretion systems and multidrug efflux pumps of Gram-negative bacteria.

    PubMed

    Andersen, C

    2003-01-01

    For translocation across the cell envelope of Gram-negative bacteria, substances have to overcome two permeability barriers, the inner and outer membrane. Channel-tunnels are outer membrane proteins, which are central to two distinct export systems: the type I secretion system exporting proteins such as toxins or proteases, and efflux pumps discharging antibiotics, dyes, or heavy metals and thus mediating drug resistance. Protein secretion is driven by an inner membrane ATP-binding cassette (ABC) transporter while drug efflux occurs via an inner membrane proton antiporter. Both inner membrane transporters are associated with a periplasmic accessory protein that recruits an outer membrane channel-tunnel to form a functional export complex. Prototypes of these export systems are the hemolysin secretion system and the AcrAB/TolC drug efflux pump of Escherichia coli, which both employ TolC as an outer membrane component. Its remarkable conduit-like structure, protruding 100 A into the periplasmic space, reveals how both systems are capable of transporting substrates across both membranes directly from the cytosol into the external environment. Proteins of the channel-tunnel family are widespread within Gram-negative bacteria. Their involvement in drug resistance and in secretion of pathogenic factors makes them an interesting system for further studies. Understanding the mechanism of the different export apparatus could help to develop new drugs, which block the efflux pumps or the secretion system.

  8. The ABC transporter ATR1 is necessary for efflux of the toxin cercosporin in the fungus Cercospora nicotianae.

    PubMed

    Amnuaykanjanasin, Alongkorn; Daub, Margaret E

    2009-02-01

    The Cercospora nicotianae mutant deficient for the CRG1 transcription factor has marked reductions in both resistance and biosynthesis of the toxin cercosporin. We cloned and sequenced full-length copies of two genes, ATR1 and CnCFP, previously identified from a subtractive library between the wild type (WT) and a crg1 mutant. ATR1 is an ABC transporter gene and has an open reading frame (ORF) of 4368bp with one intron. CnCFP encodes a MFS transporter with homology to Cercospora kikuchii CFP, previously implicated in cercosporin export, and has an ORF of 1975bp with three introns. Disruption of ATR1 indicated atr1-null mutants had dramatic reductions in cercosporin production (25% and 20% of WT levels) in solid and liquid cultures, respectively. The ATR1 disruptants also showed moderately higher sensitivity to cercosporin. Constitutive expression of ATR1 in the crg1 mutant restored cercosporin biosynthesis and moderately increased resistance. In contrast, CnCFP overexpression in the mutant did not restore toxin production, however, it moderately enhanced toxin resistance. The results together indicate ATR1 acts as a cercosporin efflux pump in this fungus and plays a partial role in resistance.

  9. 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

  10. Role of the Mmr Efflux Pump in Drug Resistance in Mycobacterium tuberculosis

    PubMed Central

    Rodrigues, Liliana; Villellas, Cristina; Bailo, Rebeca; Viveiros, Miguel

    2013-01-01

    Efflux pumps are membrane proteins capable of actively transporting a broad range of substrates from the cytoplasm to the exterior of the cell. Increased efflux activity in response to drug treatment may be the first step in the development of bacterial drug resistance. Previous studies showed that the efflux pump Mmr was significantly overexpressed in strains exposed to isoniazid. In the work to be described, we constructed mutants lacking or overexpressing Mmr in order to clarify the role of this efflux pump in the development of resistance to isoniazid and other drugs in M. tuberculosis. The mmr knockout mutant showed an increased susceptibility to ethidium bromide, tetraphenylphosphonium, and cetyltrimethylammonium bromide (CTAB). Overexpression of mmr caused a decreased susceptibility to ethidium bromide, acriflavine, and safranin O that was obliterated in the presence of the efflux inhibitors verapamil and carbonyl cyanide m-chlorophenylhydrazone. Isoniazid susceptibility was not affected by the absence or overexpression of mmr. The fluorometric method allowed the detection of a decreased efflux of ethidium bromide in the knockout mutant, whereas the overexpressed strain showed increased efflux of this dye. This increased efflux activity was inhibited in the presence of efflux inhibitors. Under our experimental conditions, we have found that efflux pump Mmr is mainly involved in the susceptibility to quaternary compounds such as ethidium bromide and disinfectants such as CTAB. The contribution of this efflux pump to isoniazid resistance in Mycobacterium tuberculosis still needs to be further elucidated. PMID:23165464

  11. 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

  12. 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.

  13. Multidrug Efflux Pumps at the Crossroad between Antibiotic Resistance and Bacterial Virulence

    PubMed Central

    Alcalde-Rico, Manuel; Hernando-Amado, Sara; Blanco, Paula; Martínez, José L.

    2016-01-01

    Multidrug efflux pumps can be involved in bacterial resistance to antibiotics at different levels. Some efflux pumps are constitutively expressed at low levels and contribute to intrinsic resistance. In addition, their overexpression may allow higher levels of resistance. This overexpression can be transient, in the presence of an effector (phenotypic resistance), or constitutive when mutants in the regulatory elements of the expression of efflux pumps are selected (acquired resistance). Efflux pumps are present in all cells, from human to bacteria and are highly conserved, which indicates that they are ancient elements in the evolution of different organisms. Consequently, it has been suggested that, besides antibiotic resistance, bacterial multidrug efflux pumps would likely contribute to other relevant processes of the microbial physiology. In the current article, we discuss some specific examples of the role that efflux pumps may have in the bacterial virulence of animals’ and plants’ pathogens, including the processes of intercellular communication. Based in these evidences, we propose that efflux pumps are at the crossroad between resistance and virulence of bacterial pathogens. Consequently, the comprehensive study of multidrug efflux pumps requires addressing these functions, which are of relevance for the bacterial–host interactions during infection. PMID:27708632

  14. Effects of efflux-pump inducers and genetic variation of the multidrug transporter cmeB in biocide resistance of Campylobacter jejuni and Campylobacter coli.

    PubMed

    Mavri, Ana; Smole Možina, Sonja

    2013-03-01

    Multidrug efflux pumps, such as CmeABC and CmeDEF, are involved in the resistance of Campylobacter to a broad spectrum of antimicrobials. The aim of this study was to analyse the effects of two putative efflux-pump inducers, bile salts and sodium deoxycholate, on the resistance of Campylobacter to biocides (triclosan, benzalkonium chloride, chlorhexidine diacetate, cetylpyridinium chloride and trisodium phosphate), SDS and erythromycin. The involvement of the CmeABC and CmeDEF efflux pumps in this resistance was studied on the basis of the effects of bile salts and sodium deoxycholate in Campylobacter cmeB, cmeF and cmeR mutants. The genetic variation in the cmeB gene was also examined, to see whether this polymorphism is related to the function of the efflux pump. In 15 Campylobacter jejuni and 23 Campylobacter coli strains, bile salts and sodium deoxycholate increased the MICs of benzalkonium chloride, chlorhexidine diacetate, cetylpyridinium chloride and SDS, and decreased the MICs of triclosan, trisodium phosphate and erythromycin. Bile salts and sodium deoxycholate further decreased or increased the MICs of biocides and erythromycin in the cmeF and cmeR mutants. For cmeB polymorphisms, 17 different cmeB-specific PCR-RFLP patterns were identified: six within C. jejuni only, nine within C. coli only and two in both species. In conclusion, bile salts and sodium deoxycholate can increase or decrease bacterial resistance to structurally unrelated antimicrobials. The MIC increases in the cmeF and cmeR mutants indicated that at least one non-CmeABC efflux system is involved in resistance to biocides. These results indicate that the cmeB gene polymorphism identified is not associated with biocide and erythromycin resistance in Campylobacter.

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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

  20. Downregulation of mdr1 and abcg2 genes is a mechanism of inhibition of efflux pumps mediated by polymeric amphiphiles.

    PubMed

    Cuestas, María L; Castillo, Amalia I; Sosnik, Alejandro; Mathet, Verónica L

    2012-11-01

    The ability of cells to acquire resistance to multiple pharmaceuticals, namely multidrug resistance (MDR), is often mediated by the over-expression of efflux transporters of the ATP-binding cassette (ABC) superfamily; for example P-glycoprotein (P-gp or MDR1), breast cancer resistance protein (BCRP or ABCG2), and multidrug resistance-associated protein MRP1. ABCs pump drug molecules out of cells against a concentration gradient, reducing their intracellular concentration. The ability of polymeric amphiphiles to inhibit ABCs as well as the cellular pathways involved in the inhibition has been extensively investigated. This work investigated for the first time the effect of branched poly(ethylene oxide)-poly(propylene oxide) block copolymers (poloxamines) on the levels of mRNA encoding for MDR1, BCRP and MRP1, in a human hepatoma cell line (Huh7). Copolymers with a broad range of molecular weights and hydrophilic-lipophilic balances were assayed. Results confirmed the down-regulation of mdr1 and abcg2 genes. Conversely, the mrp1 gene was not affected. These findings further support the versatility of these temperature- and pH-responsive copolymers to overcome drug resistance in cancer and infectious diseases.

  1. Downregulation of mdr1 and abcg2 genes is a mechanism of inhibition of efflux pumps mediated by polymeric amphiphiles.

    PubMed

    Cuestas, María L; Castillo, Amalia I; Sosnik, Alejandro; Mathet, Verónica L

    2012-11-01

    The ability of cells to acquire resistance to multiple pharmaceuticals, namely multidrug resistance (MDR), is often mediated by the over-expression of efflux transporters of the ATP-binding cassette (ABC) superfamily; for example P-glycoprotein (P-gp or MDR1), breast cancer resistance protein (BCRP or ABCG2), and multidrug resistance-associated protein MRP1. ABCs pump drug molecules out of cells against a concentration gradient, reducing their intracellular concentration. The ability of polymeric amphiphiles to inhibit ABCs as well as the cellular pathways involved in the inhibition has been extensively investigated. This work investigated for the first time the effect of branched poly(ethylene oxide)-poly(propylene oxide) block copolymers (poloxamines) on the levels of mRNA encoding for MDR1, BCRP and MRP1, in a human hepatoma cell line (Huh7). Copolymers with a broad range of molecular weights and hydrophilic-lipophilic balances were assayed. Results confirmed the down-regulation of mdr1 and abcg2 genes. Conversely, the mrp1 gene was not affected. These findings further support the versatility of these temperature- and pH-responsive copolymers to overcome drug resistance in cancer and infectious diseases. PMID:23031592

  2. 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

  3. Three's company: component structures bring a closer view of tripartite drug efflux pumps.

    PubMed

    Eswaran, Jeyanthy; Koronakis, Eva; Higgins, Matthew K; Hughes, Colin; Koronakis, Vassilis

    2004-12-01

    Bacterial multidrug resistance is a serious clinical problem and is commonly conferred by tripartite efflux 'pumps' in the prokaryotic cell envelope. Crystal structures of the three components of a drug efflux pump have now been solved: the outer membrane TolC exit duct in the year 2000, the inner membrane AcrB antiporter in 2002 and the periplasmic adaptor MexA in 2004. These structures have enhanced our understanding of the principles underlying pump assembly and operation, and present pumps as new drug targets.

  4. 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

  5. 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

  6. Phytochemicals increase the antibacterial activity of antibiotics by acting on a drug efflux pump

    PubMed Central

    Ohene-Agyei, Thelma; Mowla, Rumana; Rahman, Taufiq; Venter, Henrietta

    2014-01-01

    Drug efflux pumps confer resistance upon bacteria to a wide range of antibiotics from various classes. The expression of efflux pumps are also implicated in virulence and biofilm formation. Moreover, organisms can only acquire resistance in the presence of active drug efflux pumps. Therefore, efflux pump inhibitors (EPIs) are attractive compounds to reverse multidrug resistance and to prevent the development of resistance in clinically relevant bacterial pathogens. We investigated the potential of pure compounds isolated from plants to act as EPIs. In silico screening was used to predict the bioactivity of plant compounds and to compare that with the known EPI, phe-arg-β-naphthylamide (PAβN). Subsequently, promising products have been tested for their ability to inhibit efflux. Plumbagin nordihydroguaretic acid (NDGA) and to a lesser degree shikonin, acted as sensitizers of drug-resistant bacteria to currently used antibiotics and were able to inhibit the efflux pump-mediated removal of substrate from cells. We demonstrated the feasibility of in silico screening to identify compounds that potentiate the action of antibiotics against drug-resistant strains and which might be potentially useful lead compounds for an EPI discovery program. PMID:25224951

  7. 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

  8. Abc3-Mediated Efflux of an Endogenous Digoxin-like Steroidal Glycoside by Magnaporthe oryzae Is Necessary for Host Invasion during Blast Disease

    PubMed Central

    Patkar, Rajesh N.; Xue, Yang Kui; Shui, Guanghou; Wenk, Markus R.; Naqvi, Naweed I.

    2012-01-01

    Magnaporthe oryzae, which causes the devastating rice-blast disease, invades its host plants via a specialized infection structure called the appressorium. Previously, we showed that the ATP-Binding Cassette 3 transporter is necessary for appressorial function (host penetration) in M. oryzae. However, thus far, the molecular basis underlying impaired appressorial function in the abc3Δ remains elusive. We hypothesized that the abc3Δ appressoria accumulate excessive amounts of specific efflux substrate(s) of the Abc3 transporter in M. oryzae. We devised an innovative yeast-based strategy and identified Abc3 Transporter efflux Substrate (ATS) to be a digoxin-like endogenous steroidal glycoside that accumulates to inhibitory levels in M. oryzae abc3Δ appressoria. Exogenous ATS altered cell wall biogenesis and viability in wild-type Schizosaccharomyces pombe, but not in S. pombe expressing M. oryzae Abc3. We show that ATS associates with the Translation Elongation factor Tef2 in M. oryzae, and propose that ATS regulates ion homeostasis during pathogenesis. Excessive ATS accumulation, either intracellularly due to impaired efflux in the abc3Δ or when added exogenously to the wild type, renders M. oryzae nonpathogenic. Furthermore, we demonstrate that the host penetration defects in the abc3Δ are due to aberrant F-actin dynamics as a result of altered Tef2 function and/or ion homeostasis defects caused by excess accumulation of ATS therein. Rather surprisingly, excessive exogenous ATS or digoxin elicited the hypersensitive response in rice, even in the absence of the blast fungus. Lastly, reduced disease symptoms in the inoculated host plants in the presence of excessive digoxin suggest a potential use for such related steroidal glycosides in controlling rice-blast disease. PMID:22927822

  9. Phenylpropanoids of Alpinia galanga as efflux pump inhibitors in Mycobacterium smegmatis mc² 155.

    PubMed

    Roy, Somendu K; Pahwa, Sonika; Nandanwar, Hemraj; Jachak, Sanjay M

    2012-10-01

    The first and second line drugs used for the treatment of tuberculosis are now becoming ineffective due to emergence of resistant strains. Efflux pump provokes resistance in mycobacterium and hence could be explored as a new target for the discovery of anti-TB agents. In search of efflux pump inhibitors, MIC and modulation factor of phenylpropanoids isolated from A. galanga rhizome were determined prior to the accumulation and efflux assay. Phenylpropanoid compounds viz. 1'-S-1'-acetoxychavicol acetate, trans-p-coumaryl diacetate and 1'-S-1'-acetoxyeugenol acetate were found to be potent modulators and decreased the MIC of ethidium bromide by 64 fold at the concentration of 2.5, 6.25 and 5.0 mg/L respectively. 1'-S-1'-acetoxyeugenol acetate enhanced the accumulation and inhibited the efflux of EtBr in Mycobacterium smegmatis mc² 155 cells.

  10. 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

  11. Identification of Acinetobacter baumannii serum-associated antibiotic efflux pump inhibitors.

    PubMed

    Blanchard, Catlyn; Barnett, Pamela; Perlmutter, Jessamyn; Dunman, Paul M

    2014-11-01

    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 Ca(2+) 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.

  12. Recent advances regarding the role of ABC subfamily C member 10 (ABCC10) in the efflux of antitumor drugs

    PubMed Central

    Kathawala, Rishil J.; Wang, Yi-Jun; Ashby, Charles R.; Chen, Zhe-Sheng

    2014-01-01

    ABCC10, also known as multidrug-resistant protein 7 (MRP7), is the tenth member of the C subfamily of the ATP-binding cassette (ABC) superfamily. ABCC10 mediates multidrug resistance (MDR) in cancer cells by preventing the intracellular accumulation of certain antitumor drugs. The ABCC10 transporter is a 171-kDa protein that is localized on the basolateral cell membrane. ABCC10 is a broad-specificity transporter of xenobiotics, including antitumor drugs, such as taxanes, epothilone B, vinca alkaloids, and cytarabine, as well as modulators of the estrogen pathway, such as tamoxifen. In recent years, ABCC10 inhibitors, including cepharanthine, lapatinib, erlotinib, nilotinib, imatinib, sildenafil, and vardenafil, have been reported to overcome ABCC10-mediated MDR. This review discusses some recent and clinically relevant aspects of the ABCC10 drug efflux transporter from the perspective of current chemotherapy, particularly its inhibition by tyrosine kinase inhibitors and phosphodiesterase type 5 inhibitors. PMID:24103790

  13. Recent advances regarding the role of ABC subfamily C member 10 (ABCC10) in the efflux of antitumor drugs.

    PubMed

    Kathawala, Rishil J; Wang, Yi-Jun; Ashby, Charles R; Chen, Zhe-Sheng

    2014-05-01

    ABCC10, also known as multidrug-resistant protein 7 (MRP7), is the tenth member of the C subfamily of the ATP-binding cassette (ABC) superfamily. ABCC10 mediates multidrug resistance (MDR) in cancer cells by preventing the intracellular accumulation of certain antitumor drugs. The ABCC10 transporter is a 171-kDa protein that is localized on the basolateral cell membrane. ABCC10 is a broad-specificity transporter of xenobiotics, including antitumor drugs, such as taxanes, epothilone B, vinca alkaloids, and cytarabine, as well as modulators of the estrogen pathway, such as tamoxifen. In recent years, ABCC10 inhibitors, including cepharanthine, lapatinib, erlotinib, nilotinib, imatinib, sildenafil, and vardenafil, have been reported to overcome ABCC10-mediated MDR. This review discusses some recent and clinically relevant aspects of the ABCC10 drug efflux transporter from the perspective of current chemotherapy, particularly its inhibition by tyrosine kinase inhibitors and phosphodiesterase type 5 inhibitors. PMID:24103790

  14. The ABCs of pump selection for mine dewatering

    SciTech Connect

    Morgan, S.E.

    2008-10-15

    Choosing the right type of pump for removing water from mine operations can provide significant benefits in overall performance and cost of operation. The article describes the types of pump most commonly used: vertical turbine pumps, electric and hydraulic submersible pumps, horizontal multistage centrifugal pumps and horizontal single-stage centrifugal pumps. It gives points to consider when selecting a suitable pump, including solids handling capacity and acid content, portability, automatic operation, easy maintenance and parts availability. 1 photo.

  15. Trade-Offs in Improving Biofuel Tolerance Using Combinations of Efflux Pumps.

    PubMed

    Turner, William J; Dunlop, Mary J

    2015-10-16

    Microbes can be engineered to produce next-generation biofuels; however, the accumulation of toxic biofuels can limit yields. Previous studies have shown that efflux pumps can increase biofuel tolerance and improve production. Here, we asked whether expressing multiple pumps in combination could further increase biofuel tolerance. Pump overexpression inhibits cell growth, suggesting a trade-off between biofuel and pump toxicity. With multiple pumps, it is unclear how the fitness landscape is impacted. To address this, we measured tolerance of Escherichia coli to the biojet fuel precursor α-pinene in one-pump and two-pump strains. To support our experiments, we developed a mathematical model describing toxicity due to biofuel and overexpression of pumps. We found that data from one-pump strains can accurately predict the performance of two-pump strains. This result suggests that it may be possible to dramatically reduce the number of experiments required for characterizing the effects of combined biofuel tolerance mechanisms.

  16. [Investigation of mutations in transcription factors of efflux pump genes in fluconazole-resistant Candida albicans strains overexpressing the efflux pumps].

    PubMed

    Kalkandelen, Kemal Turan; Doluca Dereli, Mine

    2015-10-01

    In recent years, a significant rise in the number of immunocompromised patients have been observed due to cancer chemotherapy, organ transplantation and HIV infection. As a result of this, the frequency of Candida albicans infections in the clinics have been increased. Fluconazole, as being a well tolerated, easy to use drug with minor side effects, is often the first choice antifungal agent for this patient group, both for therapy and prophylaxis. Especially the long-term use of this drug, causes the selection of resistant strains and leads to the development of fluconazole resistance. The most frequently observed resistance mechanism against fluconazole in C.albicans strains is the transportation of the drug out of the cell via efflux pumps. The efflux pumps mainly involved are Cdr1, Cdr2 ve Mdr1 encoded by CDR1, CDR2 and MDR1 genes. It has been shown that, the overexpression of these efflux pump genes was caused by functional mutations in TAC1 and MRR1 genes which encode the transcription factors Tac1p and Mrr1p. This study was aimed to analyze TAC1 and MRR1 genes of 15 C.albicans strains which consist of six fluconazole-susceptible, four susceptible with trailing effect and five fluconazole-resistant isolates plus one resistant strain (DSY292), known to overexpress Mdr1 efflux pump due to P683H mutation in MRR1 gene and one fluconazole-sensitive ATCC 14053 C.albicans strain in terms of mutations with polymerase chain reaction and sequence analysis. Two of the fluconazole-resistant isolates which had overexpression of Cdr1 and Cdr2 pumps known to have overexpression of TAC1 gene, revealed R673Q and A736V mutations. A P683H point mutation, that overexpressed the Mdr1 pump was detected in a fluconazole-resistant strain, which was known to cause MRR1 overexpression. In conclusion, mutations in the transcription factors of the efflux pump genes may play an important role in the resistance against fluconazole among our selected C.albicans strains. PMID:26649419

  17. [Investigation of mutations in transcription factors of efflux pump genes in fluconazole-resistant Candida albicans strains overexpressing the efflux pumps].

    PubMed

    Kalkandelen, Kemal Turan; Doluca Dereli, Mine

    2015-10-01

    In recent years, a significant rise in the number of immunocompromised patients have been observed due to cancer chemotherapy, organ transplantation and HIV infection. As a result of this, the frequency of Candida albicans infections in the clinics have been increased. Fluconazole, as being a well tolerated, easy to use drug with minor side effects, is often the first choice antifungal agent for this patient group, both for therapy and prophylaxis. Especially the long-term use of this drug, causes the selection of resistant strains and leads to the development of fluconazole resistance. The most frequently observed resistance mechanism against fluconazole in C.albicans strains is the transportation of the drug out of the cell via efflux pumps. The efflux pumps mainly involved are Cdr1, Cdr2 ve Mdr1 encoded by CDR1, CDR2 and MDR1 genes. It has been shown that, the overexpression of these efflux pump genes was caused by functional mutations in TAC1 and MRR1 genes which encode the transcription factors Tac1p and Mrr1p. This study was aimed to analyze TAC1 and MRR1 genes of 15 C.albicans strains which consist of six fluconazole-susceptible, four susceptible with trailing effect and five fluconazole-resistant isolates plus one resistant strain (DSY292), known to overexpress Mdr1 efflux pump due to P683H mutation in MRR1 gene and one fluconazole-sensitive ATCC 14053 C.albicans strain in terms of mutations with polymerase chain reaction and sequence analysis. Two of the fluconazole-resistant isolates which had overexpression of Cdr1 and Cdr2 pumps known to have overexpression of TAC1 gene, revealed R673Q and A736V mutations. A P683H point mutation, that overexpressed the Mdr1 pump was detected in a fluconazole-resistant strain, which was known to cause MRR1 overexpression. In conclusion, mutations in the transcription factors of the efflux pump genes may play an important role in the resistance against fluconazole among our selected C.albicans strains.

  18. 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

  19. 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

  20. MAPK1 of Leishmania donovani modulates antimony susceptibility by downregulating P-glycoprotein efflux pumps.

    PubMed

    Garg, Mansi; Goyal, Neena

    2015-07-01

    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.

  1. RELATIVE EXPRESSION OF EFFLUX PUMPS IN MULTI DRUG RESISTANT PSEUDOMONAS AERUGINOSA.

    PubMed

    Azimi, Leila; Namvar, Amirmorteza Ebrahimzadeh; Jamali, Sadaf; Lari, Aida Rastegar; Bijari, Aslan; Lari, Abdolaziz Rastegar

    2015-01-01

    Pseudomonas aeruginosa is known as an important opportunistic pathogen, resistant to a high number of antibiotics. Efflux pumps are one of the main intrinsic antibiotics resistance mechanisms in P. aeruginosa. MexAB-OprM, MexCD-OprJ, and MexXY-OprM are the main efflux pumps involved in beta-lactam resistant strains which may cause cross resistance to different antimicrobial classes. The aim of this study was to detect relative gene expression in betalactam-resistant clinical P. aeruginosa strains. One hundred fourteen clinical strains of P. aeruginosa were identified by phenotypic and genotypic methods. Antibiotic susceptibility testing was conducted according to CLSI guideline. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as an efflux pump inhibitor for phenotypic detection of efflux pump mechanism and q-RT PCR was conducted for relative gene expression detection. The highest rate of resistance was observed against cefotaxime and various relative gene expressions levels were observed in all isolates with positive phenotypic test results. PMID:27328522

  2. 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

  3. 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

  4. 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

  5. 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

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

    DOE PAGES

    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

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. The mycobacterial P55 efflux pump is required for optimal growth on cholesterol.

    PubMed

    Ramón-García, Santiago; Stewart, Gordon R; Hui, Zhao Kun; Mohn, William W; Thompson, Charles J

    2015-01-01

    Cholesterol catabolism is thought to be a key factor contributing to the pathogenesis of Mycobacterium tuberculosis. Previous epistasis and mutant screening studies predicted that the P55 efflux pump (Rv1410c) positively interacts with the Mce4 transporter, a major cholesterol import system of M. tuberculosis and is needed for optimal growth in vitro, in macrophages, and in vivo. Using a combination of cell growth kinetic techniques, cholesterol consumption, and [4-(14)C]cholesterol uptake studies, we demonstrated that the Mycobacterium bovis BCG rv1410c gene indeed is needed for optimal in vitro growth on cholesterol and other carbon sources. Our data, together with previous predictions, support hypotheses that the P55 efflux pump functions in maintaining general metabolism or as a subunit of the Mce4 transport apparatus (catalyzing its assembly or providing cell wall integrity) to allow more efficient cholesterol uptake.

  12. 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

  13. CraA, a Major Facilitator Superfamily Efflux Pump Associated with Chloramphenicol Resistance in Acinetobacter baumannii▿

    PubMed Central

    Roca, I.; Marti, S.; Espinal, P.; Martínez, P.; Gibert, I.; Vila, J.

    2009-01-01

    Acinetobacter baumannii has been increasingly associated with hospital-acquired infections, and the presence of multidrug resistance strains is of great concern to clinicians. A. baumannii is thought to possess a great deal of intrinsic resistance to several antimicrobial agents, including chloramphenicol, although the mechanisms involved in such resistance are not well understood. In this work, we have identified a major facilitator superfamily efflux pump present in most A. baumannii strains, displaying strong substrate specificity toward chloramphenicol. PMID:19581458

  14. AcrAB efflux pump plays a role in decreased susceptibility to tigecycline in Morganella morganii.

    PubMed

    Ruzin, Alexey; Keeney, David; Bradford, Patricia A

    2005-02-01

    Transposon mutagenesis of a clinical isolate of Morganella morganii, G1492 (tigecycline MIC of 4 microg/ml), yielded two insertion knockout mutants for which tigecycline MICs were 0.03 microg/ml. Transposon insertions mapped to acrA, which is constitutively overexpressed in G1492, suggesting a role of the AcrAB efflux pump in decreased susceptibility to tigecycline in M. morganii.

  15. Multidrug efflux pumps from Enterobacteriaceae, Vibrio cholerae and Staphylococcus aureus bacterial food pathogens.

    PubMed

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

    2015-02-01

    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.

  16. 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

  17. Palytoxin induces K+ efflux from yeast cells expressing the mammalian sodium pump.

    PubMed

    Scheiner-Bobis, G; Meyer zu Heringdorf, D; Christ, M; Habermann, E

    1994-06-01

    Palytoxin causes potassium efflux and sodium influx in all investigated animals cells. Much evidence points to the sodium pump (Na+/K(+)-ATPase) as the target of the toxin. A heterologous expression system for mammalian Na+/K(+)-ATPase in the brewers yeast Saccharomyces cerevisiae has been used to test this hypothesis. Yeast cells do not contain endogenous sodium pumps but can be transformed with vectors coding for the alpha and beta subunits of the mammalian sodium pump. We now show that transformed yeast cells expressing both alpha and beta subunits of Na+/K(+)-ATPase are highly sensitive to the toxin, as measured by the loss of intracellular potassium. Palytoxin-induced potassium efflux is completely inhibited by 500 microM ouabain. In contrast, nontransformed yeast cells or cells expressing either the alpha or beta subunits are insensitive to palytoxin. Thus, the alpha/beta heterodimer of the sodium pump is required for the release of potassium induced by palytoxin. The results suggest that palytoxin converts the sodium pump into an open channel, allowing the passage of alkali ions.

  18. 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.

  19. 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

  20. 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

  1. Role of Novel Multidrug Efflux Pump Involved in Drug Resistance in Klebsiella pneumoniae

    PubMed Central

    Srinivasan, Vijaya Bharathi; Singh, Bharat Bhushan; Priyadarshi, Nitesh; Chauhan, Neeraj Kumar; Rajamohan, Govindan

    2014-01-01

    Background Multidrug resistant Klebsiella pneumoniae have caused major therapeutic problems worldwide due to the emergence of the extended-spectrum β-lactamase producing strains. Although there are >10 major facilitator super family (MFS) efflux pumps annotated in the genome sequence of the K. pneumoniae bacillus, apparently less is known about their physiological relevance. Principal Findings Insertional inactivation of kpnGH resulting in increased susceptibility to antibiotics such as azithromycin, ceftazidime, ciprofloxacin, ertapenem, erythromycin, gentamicin, imipenem, ticarcillin, norfloxacin, polymyxin-B, piperacillin, spectinomycin, tobramycin and streptomycin, including dyes and detergents such as ethidium bromide, acriflavine, deoxycholate, sodium dodecyl sulphate, and disinfectants benzalkonium chloride, chlorhexidine and triclosan signifies the wide substrate specificity of the transporter in K. pneumoniae. Growth inactivation and direct fluorimetric efflux assays provide evidence that kpnGH mediates antimicrobial resistance by active extrusion in K. pneumoniae. The kpnGH isogenic mutant displayed decreased tolerance to cell envelope stressors emphasizing its added role in K. pneumoniae physiology. Conclusions and Significance The MFS efflux pump KpnGH involves in crucial physiological functions besides being an intrinsic resistance determinant in K. pneumoniae. PMID:24823362

  2. 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

  3. 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

  4. 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

  5. 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

  6. Evaluation of efflux pump gene expression among drug susceptible and drug resistant strains of Mycobacterium tuberculosis from Iran.

    PubMed

    Kardan Yamchi, Jalil; Haeili, Mehri; Gizaw Feyisa, Seifu; Kazemian, Hossein; Hashemi Shahraki, Abdolrazagh; Zahednamazi, Fatemeh; Imani Fooladi, Abbas Ali; Feizabadi, Mohammad Mehdi

    2015-12-01

    Absence of mutations within the genes encoding drug targets in some phenotypically drug resistant strains of Mycobacterium tuberculosis suggests possible involvement of alternative mechanisms such as over-expression of efflux pumps. We investigated the expression level of Rv1410c, Rv2459, Rv1218c and Rv1273c efflux pumps gene by real-time quantitative reverse transcription PCR (qRT-PCR) in 31 clinical isolates of M. tuberculosis. Susceptibility to first-line drugs was performed using the proportion method. Twenty one isolates were characterized with drug resistance (DR), and among them 12 showed a significantly elevated level of expression (>4 fold) for at least one of the studied genes encoding for efflux pumps. Point mutations in the katG (codons 315 or 335) and rpoB (codons 456 and 441) genes were found in 42.85% and 66.6% of drug resistant isolates, respectively. Only one isolate showed mutation at position -15 of the inhA promoter region. Among the 7 isolates (33.33%) which had no mutation in the studied regions of drug target genes, 5 isolates showed over-expression for efflux pumps. Our results demonstrated that over-expression of efflux pumps can contribute to drug resistance in M. tuberculosis.

  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. 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

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

    PubMed

    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

  10. Ofloxacin resistance in Mycobacterium tuberculosis is associated with efflux pump activity independent of resistance pattern and genotype.

    PubMed

    Sun, Zhaogang; Xu, Yuhui; Sun, Yong; Liu, Yi; Zhang, Xuxia; Huang, Hairong; Li, Chuanyou

    2014-12-01

    Drug-resistance to ofloxacin (OFX) in Mycobacterium tuberculosis is due to missense mutations in gyrA and other factors, such as alterations in the activity of drug efflux pumps. In this study, we identified 8 extensively drug resistant tuberculosis (XDR-TB), 40 multidrug resistant TB (MDR-TB), 38 polydrug resistant TB (PDR-TB), and 16 single OFX-resistant TB from 102 clinical isolates. We tested the effect of three efflux inhibitors, reserpine, verapamil, and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), on changes in the OFX minimum inhibitory concentration (MIC) using Resazurin microtitre assay. These three inhibitors changed the MICs from 2- to 32-fold, with CCCP having the strongest effect. A total of 55%, 74%, and 83% of the tested isolates had changes in MIC of more than two-fold by reserpine, verapamil, and CCCP, respectively. The inhibitors led to similar fold-changes of OFX MICs in the XDR, MDR, PDR, and single OFX-resistant isolates. For each inhibitor, a higher resistance to OFX was associated with the greater efflux pump activity. There were no significant differences in the effect of efflux pump inhibitors upon Beijing and non-Beijing M. tuberculosis genotypes. Taken together, these results indicate that the efflux pump activity was greater in the isolates higher resistant to OFX and had similar effects on isolates with different drug resistant pattern, and had similar effects on Beijing and non-Beijing genotypes.

  11. 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

  12. 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

  13. Interaction of BDE-47 and its Hydroxylated Metabolite 6-OH-BDE-47 with the Human ABC Efflux Transporters P-gp and BCRP: Considerations for Human Exposure and Risk Assessment

    EPA Science Inventory

    ATP binding cassette (ABC) transporters, including P-glycoprotein (P-gp; also known as MDR1, ABCB1) and breast cancer resistance protein (BCRP; also known as ABCG2), are membrane-bound proteins that mediate the cellular efflux of xenobiotics as an important defense against chemic...

  14. 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.

  15. Human and Rat ABC Transporter Efflux of Bisphenol A and Bisphenol A Glucuronide: Interspecies Comparison and Implications for Pharmacokinetic Assessment

    EPA Science Inventory

    Significant interspecies differences exist between human and rodent with respect to absorption, distribution, and excretion of bisphenol A (BPA) and its primary metabolite, BPA-glucuronide (BPA-G). ATP-Binding Cassette (ABC) transporter enzymes play important roles in these physi...

  16. 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

  17. 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

  18. The multidrug efflux pump MdtEF protects against nitrosative damage during the anaerobic respiration in Escherichia coli.

    PubMed

    Zhang, Yiliang; Xiao, Minfeng; Horiyama, Tsukasa; Zhang, Yinfeng; Li, Xuechen; Nishino, Kunihiko; Yan, Aixin

    2011-07-29

    Drug efflux represents an important protection mechanism in bacteria to withstand antibiotics and environmental toxic substances. Efflux genes constitute 6-18% of all transporters in bacterial genomes, yet the expression and functions of only a handful of them have been studied. Among the 20 efflux genes encoded in the Escherichia coli K-12 genome, only the AcrAB-TolC system is constitutively expressed. The expression, activities, and physiological functions of the remaining efflux genes are poorly understood. In this study we identified a dramatic up-regulation of an additional efflux pump, MdtEF, under the anaerobic growth condition of E. coli, which is independent of antibiotic exposure. We found that expression of MdtEF is up-regulated more than 20-fold under anaerobic conditions by the global transcription factor ArcA, resulting in increased efflux activity and enhanced drug tolerance in anaerobically grown E. coli. Cells lacking mdtEF display a significantly decreased survival rate under the condition of anaerobic respiration of nitrate. Deletion of the genes responsible for the biosynthesis of indole, tnaAB, or replacing nitrate with fumarate as the terminal electron acceptor during the anaerobic respiration restores the decreased survival of ΔmdtEF cells. Moreover, ΔmdtEF cells are susceptible to indole nitrosative derivatives, a class of toxic byproducts formed and accumulated within E. coli when the bacterium respires nitrate under anaerobic conditions. Taken together, we conclude that the multidrug efflux pump MdtEF is up-regulated during the anaerobic physiology of E. coli to protect the bacterium from nitrosative damage through expelling the nitrosyl indole derivatives out of the cells.

  19. 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

  20. MexXY-OprM Efflux Pump Is Required for Antagonism of Aminoglycosides by Divalent Cations in Pseudomonas aeruginosa

    PubMed Central

    Mao, Weimin; Warren, Mark S.; Lee, Angela; Mistry, Anita; Lomovskaya, Olga

    2001-01-01

    Antagonism of aminoglycosides by divalent cations is well documented for Pseudomonas aeruginosa and is regarded as one of the problems in aminoglycoside therapy. It is generally considered that divalent cations interfere with uptake of aminoglycosides at both the outer and inner membranes. It has been demonstrated recently that aminoglycosides can be removed from cells of P. aeruginosa by the three-component multidrug resistance efflux pump MexXY-OprM. We sought to investigate the interplay between efflux and uptake in resistance to aminoglycosides in P. aeruginosa. To do so, we studied the effects of the divalent cations Mg2+ and Ca2+ on susceptibility to aminoglycosides in a wild-type strain of P. aeruginosa and in mutants either overexpressing or lacking the MexXY-OprM efflux pump. MICs of gentamicin, streptomycin, amikacin, apramycin, netilmicin, and arbekacin were determined in Mueller-Hinton broth in the presence of cations added at concentrations that varied from 0.125 to 8 mM. We found, unexpectedly, that while both Mg2+ and Ca2+ antagonized aminoglycosides (up to a 64-fold decrease in susceptibility at 8 mM), antagonism was seen only in the strains of P. aeruginosa that contained the functional MexXY-OprM efflux pump. Our results indicate that inhibition of the MexXY-OprM efflux pump should abolish the antagonism of aminoglycosides by divalent cations, regardless of its precise mechanism. This may significantly increase the therapeutic index of aminoglycosides and improve the clinical utility of this important class of antibiotics. PMID:11408215

  1. 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

  2. Crystallization and preliminary X-ray analysis of the TetR-like efflux pump regulator SimR

    PubMed Central

    Le, Tung B. K.; Stevenson, Clare E. M.; Buttner, Mark J.; Lawson, David M.

    2011-01-01

    Crystals of SimR were grown by vapour diffusion. The protein crystallized with trigonal symmetry and X-ray data were recorded to a resolution of 2.3 Å from a single crystal at the synchrotron. SimR belongs to the TetR family of bacterial transcriptional regulators. In the absence of the antibiotic simocyclinone, SimR represses the transcription of a divergently transcribed gene encoding the simocyclinone efflux pump SimX in Streptomyces antibioticus by binding to operators in the simR–simX intergenic region. Simocyclinone binding causes SimR to dissociate from its operators, leading to expression of the SimX efflux pump. Thus, SimR represents an intimate link between the biosynthesis of simocyclinone and its export, which may also provide the mechanism of self-resistance to the antibiotic in the producer strain. PMID:21393832

  3. 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.

  4. 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.

  5. Efflux Pump Inhibitor Potentiates Antimicrobial Photodynamic Inactivation of Enterococcus faecalis Biofilm

    PubMed Central

    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 (OGIRF 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 to 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 to anionic RB (P < 0.05). The ability to inactivate biofilm bacteria was further enhanced when the EPI was used with M B (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

  6. Homology modeling, molecular dynamics, and virtual screening of NorA efflux pump inhibitors of Staphylococcus aureus

    PubMed Central

    Bhaskar, Baki Vijaya; Babu, Tirumalasetty Muni Chandra; Reddy, Netala Vasudeva; Rajendra, Wudayagiri

    2016-01-01

    Emerging drug resistance in clinical isolates of Staphylococcus aureus might be implicated to the overexpression of NorA efflux pump which is capable of extruding numerous structurally diverse compounds. However, NorA efflux pump is considered as a potential drug target for the development of efflux pump inhibitors. In the present study, NorA model was constructed based on the crystal structure of glycerol-3-phosphate transporter (PDBID: 1PW4). Molecular dynamics (MD) simulation was performed using NAMD2.7 for NorA which is embedded in the hydrated lipid bilayer. Structural design of NorA unveils amino (N)- and carboxyl (C)-terminal domains which are connected by long cytoplasmic loop. N and C domains are composed of six transmembrane α-helices (TM) which exhibits pseudo-twofold symmetry and possess voluminous substrate binding cavity between TM helices. Molecular docking of reserpine, totarol, ferruginol, salvin, thioxanthene, phenothiazine, omeprazole, verapamil, nalidixic acid, ciprofloxacin, levofloxacin, and acridine to NorA found that all the molecules were bound at the large hydrophobic cleft and indicated significant interactions with the key residues. In addition, structure-based virtual screening was employed which indicates that 14 potent novel lead molecules such as CID58685302, CID58685367, CID5799283, CID5578487, CID60028372, ZINC12196383, ZINC72140751, ZINC72137843, ZINC39227983, ZINC43742707, ZINC12196375, ZINC66166948, ZINC39228014, and ZINC14616160 have highest binding affinity for NorA. These lead molecules displayed considerable pharmacological properties as evidenced by Lipinski rule of five and prophecy of toxicity risk assessment. Thus, the present study will be helpful in designing and synthesis of a novel class of NorA efflux pump inhibitors that restore the susceptibilities of drug compounds. PMID:27757014

  7. Staphylococcus aureus NorD, a Putative Efflux Pump Coregulated with the Opp1 Oligopeptide Permease, Contributes Selectively to Fitness In Vivo

    PubMed Central

    Ding, Yanpeng; Fu, Yingmei; Lee, Jean C.

    2012-01-01

    Staphylococcus aureus readily infects humans, causing infections from mild superficial skin infections to lethal bacteremia and endocarditis. Transporters produced by S. aureus allow the pathogen to adapt to a variety of settings, including survival at sites of infection and in the presence of antibiotics. The native functions of many transporters are unknown, but their potential dual contribution to fitness and antimicrobial resistance highlights their importance in staphylococcal infections. Here, we show that S. aureus NorD, a newly recognized efflux pump of the major facilitator superfamily, contributes to fitness in a murine subcutaneous abscess model. In community-associated methicillin-resistant S. aureus (CA-MRSA) strain MW2, norD was selectively upregulated 36-fold at the infection site relative to growth in vitro, and the norD mutant demonstrated significant fitness impairment in abscesses, with fitness 20- to 40-fold lower than that of the parent MW2 strain. Plasmid-encoded NorD could complement the fitness defect of the MW2 norD mutant. Chromosomal norD expression is polycistronic with the upstream oligopeptide permease genes (opp1ABCDF), which encode an ABC oligopeptide transporter. Both norD and opp1 were upregulated in abscesses and iron-restricted culture medium and negatively regulated by Fur, but only NorD contributed to fitness in the murine abscess model. PMID:23042988

  8. The accumulation and efflux of lead partly depend on ATP-dependent efflux pump-multidrug resistance protein 1 and glutathione in testis Sertoli cells.

    PubMed

    Huang, Shaoxin; Ye, Jingping; Yu, Jun; Chen, Li; Zhou, Langhuan; Wang, Hong; Li, Zhen; Wang, Chunhong

    2014-05-01

    Since lead accumulation is toxic to cells, its excretion is crucial for organisms to survive the toxicity. In this study, mouse testis sertoli (TM4) and Mrp1 lower-expression TM4-sh cells were used to explore the lead accumulation characteristics, and the role of ATP-dependent efflux pump-multidrug resistance protein 1 (Mrp1) in lead excretion. TM4 cells possess Mrp-like transport activity. The expression levels of mrp1 mRNA and Mrp1 increased after lead treatments at first and then decreased. The maximum difference of relative mRNA expression reached 10 times. In the presence of lead acetate, the amount of cumulative lead in TM4-sh was much higher than that in TM4. After the treatment with lead acetate at 10-40 μM for 12h or 24h, the differences were about 2-8 times. After with the switch to lead-free medium, the cellular lead content in TM4-sh remains higher than that in TM4 cells at 1,3, 6, and 9h time points (P<0.01). Energy inhibitor sodium azide, Mrp inhibitors MK571 and glutathione (GSH) biosynthesis inhibitor BSO could block lead efflux from TM4 cells significantly. These results indicate that lead excretion may be mediated by Mrp1 and GSH in TM4 cells. Mrp1 could be one of the important intervention points for lead detoxification.

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

    PubMed

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

    2015-04-22

    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.

  10. 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.

  11. Isolation and Characterization of the Colletotrichum acutatum ABC Transporter CaABC1

    PubMed Central

    Kim, Suyoung; Park, Sook-Young; Kim, Hyejeong; Kim, Dongyoung; Lee, Seon-Woo; Kim, Heung Tae; Lee, Jong-Hwan; Choi, Woobong

    2014-01-01

    Fungi tolerate exposure to various abiotic stresses, including cytotoxic compounds and fungicides, via their ATP-driven efflux pumps belonging to ATP-binding cassette (ABC) transporters. To clarify the molecular basis of interaction between the fungus and various abiotic stresses including fungicides, we constructed a cDNA library from germinated conidia of Colletotrichum acutatum, a major anthracnose pathogen of pepper (Capsicum annum L.). Over 1,000 cDNA clones were sequenced, of which single clone exhibited significant nucleotide sequence homology to ABC transporter genes. We isolated three fosmid clones containing the C. acutatum ABC1 (CaABC1) gene in full-length from genomic DNA library screening. The CaABC1 gene consists of 4,059 bp transcript, predicting a 1,353-aa protein. The gene contains the typical ABC signature and Walker A and B motifs. The 5′-flanking region contains a CAAT motif, a TATA box, and a Kozak region. Phylogenetic and structural analysis suggested that the CaABC1 is a typical ABC transporter gene highly conserved in various fungal species, as well as in Chromista, Metazoans, and Viridiplantae. We also found that CaABC1 was up-regulated during conidiation and a minimal medium condition. Moreover, CaABC1 was induced in iprobenfos, kresoxim-methyl, thiophanate-methyl, and hygromycin B. These results demonstrate that CaABC1 is necessary for conidiation, abiotic stress, and various fungicide resistances. These results will provide the basis for further study on the function of ABC transporter genes in C. acutatum. PMID:25506302

  12. 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

  13. Identification of Natural Compound Inhibitors for Multidrug Efflux Pumps of Escherichia coli and Pseudomonas aeruginosa Using In Silico High-Throughput Virtual Screening and In Vitro Validation

    PubMed Central

    Aparna, Vasudevan; Dineshkumar, Kesavan; Mohanalakshmi, Narasumani; Velmurugan, Devadasan; Hopper, Waheeta

    2014-01-01

    Pseudomonas aeruginosa and Escherichia coli are resistant to wide range of antibiotics rendering the treatment of infections very difficult. A main mechanism attributed to the resistance is the function of efflux pumps. MexAB-OprM and AcrAB-TolC are the tripartite efflux pump assemblies, responsible for multidrug resistance in P. aeruginosa and E. coli respectively. Substrates that are more susceptible for efflux are predicted to have a common pharmacophore feature map. In this study, a new criterion of excluding compounds with efflux substrate-like features was used, thereby refining the selection process and enriching the inhibitor identification process. An in-house database of phytochemicals was created and screened using high-throughput virtual screening against AcrB and MexB proteins and filtered by matching with the common pharmacophore models (AADHR, ADHNR, AAHNR, AADHN, AADNR, AAADN, AAADR, AAANR, AAAHN, AAADD and AAADH) generated using known efflux substrates. Phytochemical hits that matched with any one or more of the efflux substrate models were excluded from the study. Hits that do not have features similar to the efflux substrate models were docked using XP docking against the AcrB and MexB proteins. The best hits of the XP docking were validated by checkerboard synergy assay and ethidium bromide accumulation assay for their efflux inhibition potency. Lanatoside C and diadzein were filtered based on the synergistic potential and validated for their efflux inhibition potency using ethidium bromide accumulation study. These compounds exhibited the ability to increase the accumulation of ethidium bromide inside the bacterial cell as evidenced by these increase in fluorescence in the presence of the compounds. With this good correlation between in silico screening and positive efflux inhibitory activity in vitro, the two compounds, lanatoside C and diadzein could be promising efflux pump inhibitors and effective to use in combination therapy against drug

  14. Hoechst 33342 Is a Hidden "Janus" amongst Substrates for the Multidrug Efflux Pump LmrP.

    PubMed

    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

  15. Structure-activity relationships of a novel pyranopyridine series of Gram-negative bacterial efflux pump inhibitors.

    PubMed

    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

    2015-05-01

    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.

  16. 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

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

    PubMed Central

    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.

    2016-01-01

    The Escherichia coli AcrAB-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

  18. 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

  19. Role of EfrAB efflux pump in biocide tolerance and antibiotic resistance of Enterococcus faecalis and Enterococcus faecium isolated from traditional fermented foods and the effect of EDTA as EfrAB inhibitor.

    PubMed

    Lavilla Lerma, Leyre; Benomar, Nabil; Valenzuela, Antonio Sánchez; Casado Muñoz, María del Carmen; Gálvez, Antonio; Abriouel, Hikmate

    2014-12-01

    Enterococcus faecalis and Enterococcus faecium isolated from various traditional fermented foods of both animal and vegetable origins have shown multidrug resistance to several antibiotics and tolerance to biocides. Reduced susceptibility was intra and inter-species dependent and was due to specific and unspecific mechanisms such as efflux pumps. EfrAB, a heterodimeric ABC transporter efflux pump, was detected in 100% of multidrug resistant (MDR) E. faecalis strains and only in 12% of MDR E. faecium strains. EfrAB expression was induced by half of minimum inhibitory concentration (MIC) of gentamicin, streptomycin and chloramphenicol. However, expression of efrA and efrB genes was highly dependent on the strain tested and on the antimicrobial used. Our results indicated that 3 mM EDTA highly reduced the MICs of almost all drugs tested. Nevertheless, the higher reductions (>8 folds) were obtained with gentamicin, streptomycin, chlorhexidine and triclosan. Reductions of MICs were correlated with down-regulation of EfrAB expression (10-140 folds) in all three MDR enterococci strains. This is the first report describing the role of EfrAB in the efflux of antibiotics and biocides which reflect also the importance of EfrAB in multidrug resistance in enterococci. EDTA used at low concentration as food preservative could be one of the best choices to prevent spread of multidrug resistant enterococci throughout food chain by decreasing EfrAB expression. EfrAB could be an attractive target not only in enterococci present in food matrix but also those causing infections as well by using EDTA as therapeutic agent in combination with low doses of antibiotics.

  20. Hydroxypropyl-sulfobutyl-β-cyclodextrin improves the oral bioavailability of edaravone by modulating drug efflux pump of enterocytes.

    PubMed

    Rong, Wen-Ting; Lu, Ya-Peng; Tao, Qing; Guo, Miao; Lu, Yu; Ren, Yong; Yu, Shu-Qin

    2014-02-01

    The objective of the study was to evaluate the effect of hydroxypropyl-sulfobutyl-β-cyclodextrin (HP-SBE-βCD) on the bioavailability and intestinal absorption of edaravone, and identify its mechanism of action. We devised HP-SBE-βCD as a carrier and modulator of P-glycoprotein (Pgp) efflux pump, and edaravone as a model drug, and prepared edaravone/HP-SBE-βCD inclusion complex. HP-SBE-βCD improved the water solubility and enhanced the bioavailability of edaravone by 10.3-fold in rats. Then, in situ single-pass intestinal perfusion showed that HP-SBE-βCD had an effect of improving the permeability and inhibiting the efflux of edaravone. Furthermore, the effects of HP-SBE-βCD on Pgp were achieved through interfering with the lipid raft and depleting the cholesterol of enterocytes membrane. From the results, we presented the novel mechanisms. First, edaravone/HP-SBE-βCD had a lower release from the inclusion compound to protect edaravone from the low pH of the stomach. Then, HP-SBE-βCD modulated the membrane microenvironment of intestinal absorption epithelial cells. At last, the result was that HP-SBE-βCD enhanced the absorption of edaravone by interfering with Pgp. In conclusion, HP-SBE-βCD improves the bioavailability of drug not only because of its enhancing water solubility of the drug, but also because it modulates the Pgp-mediated efflux from enterocytes.

  1. Multidrug efflux pump MdtBC of Escherichia coli is active only as a B2C heterotrimer.

    PubMed

    Kim, Hong-Suk; Nagore, Daniel; Nikaido, Hiroshi

    2010-03-01

    RND (resistance-nodulation-division) family transporters in Gram-negative bacteria frequently pump out a wide range of inhibitors and often contribute to multidrug resistance to antibiotics and biocides. An archetypal RND pump of Escherichia coli, AcrB, is known to exist as a homotrimer, and this construction is essential for drug pumping through the functionally rotating mechanism. MdtBC, however, appears different because two pump genes coexist within a single operon, and genetic deletion data suggest that both pumps must be expressed in order for the drug efflux to occur. We have expressed the corresponding genes, with one of them in a His-tagged form. Copurification of MdtB and MdtC under these conditions showed that they form a complex, with an average stoichiometry of 2:1. Unequivocal evidence that only the trimer containing two B protomers and one C protomer is active was obtained by expressing all possible combinations of B and C in covalently linked forms. Finally, conversion into alanine of the residues, known to form a proton translocation pathway in AcrB, inactivated transport only when made in MdtB, not when made in MdtC, a result suggesting that MdtC plays a different role not directly involved in drug binding and extrusion.

  2. Reduction of cellular stress by TolC-dependent efflux pumps in Escherichia coli indicated by BaeSR and CpxARP activation of spy in efflux mutants.

    PubMed

    Rosner, Judah L; Martin, Robert G

    2013-03-01

    Escherichia coli has nine inner membrane efflux pumps which complex with the outer membrane protein TolC and cognate membrane fusion proteins to form tripartite transperiplasmic pumps with diverse functions, including the expulsion of antibiotics. We recently observed that tolC mutants have elevated activities for three stress response regulators, MarA, SoxS, and Rob, and we suggested that TolC-dependent efflux is required to prevent the accumulation of stressful cellular metabolites. Here, we used spy::lacZ fusions to show that two systems for sensing/repairing extracytoplasmic stress, BaeRS and CpxARP, are activated in the absence of TolC-dependent efflux. In either tolC mutants or bacteria with mutations in the genes for four TolC-dependent efflux pumps, spy expression was increased 6- to 8-fold. spy encodes a periplasmic chaperone regulated by the BaeRS and CpxARP stress response systems. The overexpression of spy in tolC or multiple efflux pump mutants also depended on these systems. spy overexpression was not due to acetate, ethanol, or indole accumulation, since external acetate had only a minor effect on wild-type cells, ethanol had a large effect that was not CpxA dependent, and a tolC tnaA mutant which cannot accumulate internal indole overexpressed spy. We propose that, unless TolC-dependent pumps excrete certain metabolites, the metabolites accumulate and activate at least five different stress response systems.

  3. 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.

  4. 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

  5. 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

  6. MdsABC-Mediated Pathway for Pathogenicity in Salmonella enterica Serovar Typhimurium.

    PubMed

    Song, Saemee; Lee, Boeun; Yeom, Ji-Hyun; Hwang, Soonhye; Kang, Ilnam; Cho, Jang-Cheon; Ha, Nam-Chul; Bae, Jeehyeon; Lee, Kangseok; Kim, Yong-Hak

    2015-11-01

    MdsABC is a Salmonella-specific tripartite efflux pump that has been implicated in the virulence of Salmonella enterica serovar Typhimurium; however, little is known about the virulence factors associated with this pump. We observed MdsABC expression-dependent alterations in the degree of resistance to extracellular oxidative stress and macrophage-mediated killing. Thin-layer chromatography and tandem mass spectrometry analyses revealed that overexpression of MdsABC led to increased secretion of 1-palmitoyl-2-stearoyl-phosphatidylserine (PSPS), affecting the ability of the bacteria to invade and survive in host cells. Overexpression of MdsABC and external addition of PSPS similarly rendered the mdsABC deletion strain resistant to diamide. Diagonal gel analysis showed that PSPS treatment reduced the diamide-mediated formation of disulfide bonds, particularly in the membrane fraction of the bacteria. Salmonella infection of macrophages induced the upregulation of MdsABC expression and led to an increase of intracellular bacterial number and host cell death, similar to the effects of MdsABC overexpression and PSPS pretreatment on the mdsABC deletion strain. Our study shows that MdsABC mediates a previously uncharacterized pathway that involves PSPS as a key factor for the survival and virulence of S. Typhimurium in phagocytic cells.

  7. 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.

  8. Mass spectrometry reveals synergistic effects of nucleotides, lipids, and drugs binding to a multidrug resistance efflux pump.

    PubMed

    Marcoux, Julien; Wang, Sheila C; Politis, Argyris; Reading, Eamonn; Ma, Jerome; Biggin, Philip C; Zhou, Min; Tao, Houchao; Zhang, Qinghai; Chang, Geoffrey; Morgner, Nina; Robinson, Carol V

    2013-06-11

    Multidrug resistance is a serious barrier to successful treatment of many human diseases, including cancer, wherein chemotherapeutics are exported from target cells by membrane-embedded pumps. The most prevalent of these pumps, the ATP-Binding Cassette transporter P-glycoprotein (P-gp), consists of two homologous halves each comprising one nucleotide-binding domain and six transmembrane helices. The transmembrane region encapsulates a hydrophobic cavity, accessed by portals in the membrane, that binds cytotoxic compounds as well as lipids and peptides. Here we use mass spectrometry (MS) to probe the intact P-gp small molecule-bound complex in a detergent micelle. Activation in the gas phase leads to formation of ions, largely devoid of detergent, yet retaining drug molecules as well as charged or zwitterionic lipids. Measuring the rates of lipid binding and calculating apparent KD values shows that up to six negatively charged diacylglycerides bind more favorably than zwitterionic lipids. Similar experiments confirm binding of cardiolipins and show that prior binding of the immunosuppressant and antifungal antibiotic cyclosporin A enhances subsequent binding of cardiolipin. Ion mobility MS reveals that P-gp exists in an equilibrium between different states, readily interconverted by ligand binding. Overall these MS results show how concerted small molecule binding leads to synergistic effects on binding affinities and conformations of a multidrug efflux pump.

  9. 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

  10. The csbX gene of Azotobacter vinelandii encodes an MFS efflux pump required for catecholate siderophore export.

    PubMed

    Page, William J; Kwon, Elena; Cornish, Anthony S; Tindale, Anne E

    2003-11-21

    The csbX gene of Azotobacter vinelandii was regulated in an iron-repressible manner from a divergent promoter upstream of the catecholate siderophore biosynthesis (csb) operon and was predicted to encode an efflux pump of the major facilitator superfamily. Other proteins that were most similar to CsbX were encoded by genes found in the catecholate siderophore biosynthesis operons of Aeromonas hydrophila and Stigmatella aurantiaca. Inactivation of csbX resulted in 57-100% decrease in the amount of catecholates released when compared to the wild-type in iron-limited medium. CsbX was most important for the export of the high affinity chelator protochelin with the majority of the catecholates released by csbX mutants being the protochelin intermediates azotochelin and aminochelin.

  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. MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 4: Addressing the problem of poor stability due to photoisomerization of an acrylic acid moiety.

    PubMed

    Nakayama, Kiyoshi; Kuru, Noriko; Ohtsuka, Masami; Yokomizo, Yoshihiro; Sakamoto, Atsunobu; Kawato, Haruko; Yoshida, Ken-ichi; Ohta, Toshiharu; Hoshino, Kazuki; Akimoto, Katsuya; Itoh, Junko; Ishida, Hiroko; Cho, Aesop; Palme, Monica H; Zhang, Jason Z; Lee, Ving J; Watkins, William J

    2004-05-17

    Exchange of the ethylene tether in a series of pyridopyrimidine-based MexAB-OprM specific efflux pump inhibitors to an amide bond stabilized the olefin of the acrylic acid moiety, preventing facile photoisomerization to the Z-isomer. Furthermore, the activity was drastically improved in the amide tether variants, providing extremely potent acrylic acid and vinyl tetrazole analogues.

  13. 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...

  14. 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 ...

  15. 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.

  16. 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

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

    PubMed

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

    2015-08-01

    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.

  18. 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

  19. The MerR-like regulator BrlR confers biofilm tolerance by activating multidrug efflux pumps in Pseudomonas aeruginosa biofilms.

    PubMed

    Liao, Julie; Schurr, Michael J; Sauer, Karin

    2013-08-01

    A defining characteristic of biofilms is antibiotic tolerance that can be up to 1,000-fold greater than that of planktonic cells. In Pseudomonas aeruginosa, biofilm tolerance to antimicrobial agents requires the biofilm-specific MerR-type transcriptional regulator BrlR. However, the mechanism by which BrlR mediates biofilm tolerance has not been elucidated. Genome-wide transcriptional profiling indicated that brlR was required for maximal expression of genes associated with antibiotic resistance, in particular those encoding the multidrug efflux pumps MexAB-OprM and MexEF-OprN. Chromatin immunoprecipitation (ChIP) analysis revealed a direct regulation of these genes by BrlR, with DNA binding assays confirming BrlR binding to the promoter regions of the mexAB-oprM and mexEF-oprN operons. Quantitative reverse transcriptase PCR (qRT-PCR) analysis further indicated BrlR to be an activator of mexAB-oprM and mexEF-oprN gene expression. Moreover, immunoblot analysis confirmed increased MexA abundance in cells overexpressing brlR. Inactivation of both efflux pumps rendered biofilms significantly more susceptible to five different classes of antibiotics by affecting MIC but not the recalcitrance of biofilms to killing by bactericidal agents. Overexpression of either efflux pump in a ΔbrlR strain partly restored tolerance of ΔbrlR biofilms to antibiotics. Expression of brlR in mutant biofilms lacking both efflux pumps partly restored antimicrobial tolerance of biofilms to wild-type levels. Our results indicate that BrlR acts as an activator of multidrug efflux pumps to confer tolerance to P. aeruginosa biofilms and to resist the action of antimicrobial agents.

  20. Random Mutagenesis of the Multidrug Transporter AcrB from Escherichia coli for Identification of Putative Target Residues of Efflux Pump Inhibitors

    PubMed Central

    Kohler, Samay; Buck, Annika; Dambacher, Christine; König, Armin; Bohnert, Jürgen A.; Kern, Winfried V.

    2014-01-01

    Efflux is an important mechanism of bacterial multidrug resistance (MDR), and the inhibition of MDR pumps by efflux pump inhibitors (EPIs) could be a promising strategy to overcome MDR. 1-(1-Naphthylmethyl)-piperazine (NMP) and phenylalanine-arginine-β-naphthylamide (PAβN) are model EPIs with activity in various Gram-negative bacteria expressing AcrB, the major efflux pump of Escherichia coli, or similar homologous pumps of the resistance-nodulation-cell division class. The aim of the present study was to generate E. coli AcrB mutants resistant to the inhibitory action of the two model EPIs and to identify putative EPI target residues in order to better understand mechanisms of pump inhibition. Using an in vitro random mutagenesis approach focusing on the periplasmic domain of AcrB, we identified the double mutation G141D N282Y, which substantially compromised the synergistic activity of NMP with linezolid, was associated with similar intracellular linezolid concentrations in the presence and absence of NMP, and did not impair the intrinsic MICs of various pump substrates and dye accumulation. We propose that these mutations near the outer face of the distal substrate binding pocket reduce NMP trapping. Other residues found to be relevant for efflux inhibition by NMP were G288 and A279, but mutations at these sites also changed the susceptibility to several pump substrates. Unlike with NMP, we were unable to generate AcrB periplasmic domain mutants with resistance or partial resistance to the EPI activity of PAβN, which is consistent with the modes of action of PAβN differing from those of NMP. PMID:25182653

  1. 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

  2. Conserved small protein associates with the multidrug efflux pump AcrB and differentially affects antibiotic resistance

    PubMed Central

    Hobbs, Errett C.; Yin, Xuefeng; Paul, Brian J.; Astarita, Jillian L.; Storz, Gisela

    2012-01-01

    The AcrAB–TolC multidrug efflux pump confers resistance to a wide variety of antibiotics and other compounds in Escherichia coli. Here we show that AcrZ (formerly named YbhT), a 49-amino-acid inner membrane protein, associates with the AcrAB-TolC complex. Co-purification of AcrZ with AcrB, in the absence of both AcrA and TolC, two-hybrid assays and suppressor mutations indicate that this interaction occurs through the inner membrane protein AcrB. The highly conserved acrZ gene is coregulated with acrAB through induction by the MarA, Rob, and SoxS transcription regulators. In addition, mutants lacking AcrZ are sensitive to many, but not all, of the antibiotics transported by AcrAB–TolC. This differential antibiotic sensitivity suggests that AcrZ may enhance the ability of the AcrAB–TolC pump to export certain classes of substrates. PMID:23010927

  3. A D-octapeptide drug efflux pump inhibitor acts synergistically with azoles in a murine oral candidiasis infection model.

    PubMed

    Hayama, Kazumi; Ishibashi, Hiroko; Ishijima, Sanae A; Niimi, Kyoko; Tansho, Shigeru; Ono, Yasuo; Monk, Brian C; Holmes, Ann R; Harding, David R K; Cannon, Richard D; Abe, Shigeru

    2012-03-01

    Clinical management of patients undergoing treatment of oropharyngeal candidiasis with azole antifungals can be impaired by azole resistance. High-level azole resistance is often caused by the overexpression of Candida albicans efflux pump Cdr1p. Inhibition of this pump therefore represents a target for combination therapies that reverse azole resistance. We assessed the therapeutic potential of the D-octapeptide derivative RC21v3, a Cdr1p inhibitor, in the treatment of murine oral candidiasis caused by either the azole-resistant C. albicans clinical isolate MML611 or its azole-susceptible parental strain MML610. RC21v3, fluconazole (FLC), or a combination of both drugs were administered orally to immunosuppressed ICR mice at 3, 24, and 27 h after oral inoculation with C. albicans. FLC protected the mice inoculated with MML610 from oral candidiasis, but was only partially effective in MML611-infected mice. The co-application of RC21v3 (0.02 μmol per dose) potentiated the therapeutic performance of FLC for mice infected with either strain. It caused a statistically significant decrease in C. albicans cfu isolated from the oral cavity of the infected mice and reduced oral lesions. RC21v3 also enhanced the therapeutic activity of itraconazole against MML611 infection. These results indicate that RC21v3 in combination with azoles has potential as a therapy against azole-resistant oral candidiasis.

  4. Structure of the Tripartite Multidrug Efflux Pump AcrAB-TolC Suggests an Alternative Assembly Mode

    PubMed Central

    Kim, Jin-Sik; Jeong, Hyeongseop; Song, Saemee; Kim, Hye-Yeon; Lee, Kangseok; Hyun, Jaekyung; Ha, Nam-Chul

    2015-01-01

    Escherichia coli AcrAB-TolC is a multidrug efflux pump that expels a wide range of toxic substrates. The dynamic nature of the binding or low affinity between the components has impeded elucidation of how the three components assemble in the functional state. Here, we created fusion proteins composed of AcrB, a transmembrane linker, and two copies of AcrA. The fusion protein exhibited acridine pumping activity, suggesting that the protein reflects the functional structure in vivo. To discern the assembling mode with TolC, the AcrBA fusion protein was incubated with TolC or a chimeric protein containing the TolC aperture tip region. Three-dimensional structures of the complex proteins were determined through transmission electron microscopy. The overall structure exemplifies the adaptor bridging model, wherein the funnel-like AcrA hexamer forms an intermeshing cogwheel interaction with the α-barrel tip region of TolC, and a direct interaction between AcrB and TolC is not allowed. These observations provide a structural blueprint for understanding multidrug resistance in pathogenic Gram-negative bacteria. PMID:26013259

  5. NorM, an Erwinia amylovora multidrug efflux pump involved in in vitro competition with other epiphytic bacteria.

    PubMed

    Burse, Antje; Weingart, Helge; Ullrich, Matthias S

    2004-02-01

    Blossoms are important sites of infection for Erwinia amylovora, the causal agent of fire blight of rosaceous plants. Before entering the tissue, the pathogen colonizes the stigmatic surface and has to compete for space and nutrient resources within the epiphytic community. Several epiphytes are capable of synthesizing antibiotics with which they antagonize phytopathogenic bacteria. Here, we report that a multidrug efflux transporter, designated NorM, of E. amylovora confers tolerance to the toxin(s) produced by epiphytic bacteria cocolonizing plant blossoms. According to sequence comparisons, the single-component efflux pump NorM is a member of the multidrug and toxic compound extrusion protein family. The corresponding gene is widely distributed among E. amylovora strains and related plant-associated bacteria. NorM mediated resistance to the hydrophobic cationic compounds norfloxacin, ethidium bromide, and berberine. A norM mutant was constructed and exhibited full virulence on apple rootstock MM 106. However, it was susceptible to antibiotics produced by epiphytes isolated from apple and quince blossoms. The epiphytes were identified as Pantoea agglomerans by 16S rRNA analysis and were isolated from one-third of all trees examined. The promoter activity of norM was twofold greater at 18 degrees C than at 28 degrees C. The lower temperature seems to be beneficial for host infection because of the availability of moisture necessary for movement of the pathogen to the infection sites. Thus, E. amylovora might employ NorM for successful competition with other epiphytic microbes to reach high population densities, particularly at a lower temperature.

  6. 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

  7. 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

  8. Dual Repression of the Multidrug Efflux Pump CmeABC by CosR and CmeR in Campylobacter jejuni

    PubMed Central

    Grinnage-Pulley, Tara; Mu, Yang; Dai, Lei; Zhang, Qijing

    2016-01-01

    During transmission and intestinal colonization, Campylobacter jejuni, a major foodborne human pathogen, experiences oxidative stress. CosR, a response regulator in C. jejuni, modulates the oxidative stress response and represses expression of the CmeABC multidrug efflux pump. CmeABC, a key component in resistance to toxic compounds including antimicrobials and bile salts, is also under negative regulation by CmeR, a TetR family transcriptional regulator. How CosR and CmeR interact in binding to the cmeABC promoter and how CosR senses oxidative stress are still unknown. To answer these questions, we conducted various experiments utilizing electrophoretic mobility shift assays and transcriptional fusion assays. CosR and CmeR bound independently to two separate sites of the cmeABC promoter, simultaneously repressing cmeABC expression. This dual binding of CosR and CmeR is optimal with a 17 base pair space between the two binding sites as mutations that shortened the distance between the binding sites decreased binding by CmeR and enhanced cmeABC expression. Additionally, the single cysteine residue (C218) of CosR was sensitive to oxidation, which altered the DNA-binding activity of CosR and dissociated CosR from the cmeABC promoter as determined by electrophoretic mobility shift assay. Replacement of C218 with serine rendered CosR insensitive to oxidation, suggesting a potential role of C218 in sensing oxidative stress and providing a possible mechanism for CosR-mediated response to oxidative stress. These findings reveal a dual regulatory role of CosR and CmeR in modulating cmeABC expression and suggest a potential mechanism that may explain overexpression of cmeABC in response to oxidative stress. Differential expression of cmeABC mediated by CmeR and CosR in response to different signals may facilitate adaptation of Campylobacter to various environmental conditions. PMID:27468281

  9. Dual Repression of the Multidrug Efflux Pump CmeABC by CosR and CmeR in Campylobacter jejuni.

    PubMed

    Grinnage-Pulley, Tara; Mu, Yang; Dai, Lei; Zhang, Qijing

    2016-01-01

    During transmission and intestinal colonization, Campylobacter jejuni, a major foodborne human pathogen, experiences oxidative stress. CosR, a response regulator in C. jejuni, modulates the oxidative stress response and represses expression of the CmeABC multidrug efflux pump. CmeABC, a key component in resistance to toxic compounds including antimicrobials and bile salts, is also under negative regulation by CmeR, a TetR family transcriptional regulator. How CosR and CmeR interact in binding to the cmeABC promoter and how CosR senses oxidative stress are still unknown. To answer these questions, we conducted various experiments utilizing electrophoretic mobility shift assays and transcriptional fusion assays. CosR and CmeR bound independently to two separate sites of the cmeABC promoter, simultaneously repressing cmeABC expression. This dual binding of CosR and CmeR is optimal with a 17 base pair space between the two binding sites as mutations that shortened the distance between the binding sites decreased binding by CmeR and enhanced cmeABC expression. Additionally, the single cysteine residue (C218) of CosR was sensitive to oxidation, which altered the DNA-binding activity of CosR and dissociated CosR from the cmeABC promoter as determined by electrophoretic mobility shift assay. Replacement of C218 with serine rendered CosR insensitive to oxidation, suggesting a potential role of C218 in sensing oxidative stress and providing a possible mechanism for CosR-mediated response to oxidative stress. These findings reveal a dual regulatory role of CosR and CmeR in modulating cmeABC expression and suggest a potential mechanism that may explain overexpression of cmeABC in response to oxidative stress. Differential expression of cmeABC mediated by CmeR and CosR in response to different signals may facilitate adaptation of Campylobacter to various environmental conditions.

  10. Dual Repression of the Multidrug Efflux Pump CmeABC by CosR and CmeR in Campylobacter jejuni.

    PubMed

    Grinnage-Pulley, Tara; Mu, Yang; Dai, Lei; Zhang, Qijing

    2016-01-01

    During transmission and intestinal colonization, Campylobacter jejuni, a major foodborne human pathogen, experiences oxidative stress. CosR, a response regulator in C. jejuni, modulates the oxidative stress response and represses expression of the CmeABC multidrug efflux pump. CmeABC, a key component in resistance to toxic compounds including antimicrobials and bile salts, is also under negative regulation by CmeR, a TetR family transcriptional regulator. How CosR and CmeR interact in binding to the cmeABC promoter and how CosR senses oxidative stress are still unknown. To answer these questions, we conducted various experiments utilizing electrophoretic mobility shift assays and transcriptional fusion assays. CosR and CmeR bound independently to two separate sites of the cmeABC promoter, simultaneously repressing cmeABC expression. This dual binding of CosR and CmeR is optimal with a 17 base pair space between the two binding sites as mutations that shortened the distance between the binding sites decreased binding by CmeR and enhanced cmeABC expression. Additionally, the single cysteine residue (C218) of CosR was sensitive to oxidation, which altered the DNA-binding activity of CosR and dissociated CosR from the cmeABC promoter as determined by electrophoretic mobility shift assay. Replacement of C218 with serine rendered CosR insensitive to oxidation, suggesting a potential role of C218 in sensing oxidative stress and providing a possible mechanism for CosR-mediated response to oxidative stress. These findings reveal a dual regulatory role of CosR and CmeR in modulating cmeABC expression and suggest a potential mechanism that may explain overexpression of cmeABC in response to oxidative stress. Differential expression of cmeABC mediated by CmeR and CosR in response to different signals may facilitate adaptation of Campylobacter to various environmental conditions. PMID:27468281

  11. 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

  12. Temporal dynamics of the ABC transporter response to insecticide treatment: insights from the malaria vector Anopheles stephensi

    NASA Astrophysics Data System (ADS)

    Epis, Sara; Porretta, Daniele; Mastrantonio, Valentina; Urbanelli, Sandra; Sassera, Davide; De Marco, Leone; Mereghetti, Valeria; Montagna, Matteo; Ricci, Irene; Favia, Guido; Bandi, Claudio

    2014-12-01

    In insects, ABC transporters have been shown to contribute to defence/resistance to insecticides by reducing toxic concentrations in cells/tissues. Despite the extensive studies about this detoxifying mechanism, the temporal patterns of ABC transporter activation have been poorly investigated. Using the malaria vector Anopheles stephensi as a study system, we investigated the expression profile of ABC genes belonging to different subfamilies in permethrin-treated larvae at different time points (30 min to 48 h). Our results showed that the expression of ABCB and ABCG subfamily genes was upregulated at 1 h after treatment, with the highest expression observed at 6 h. Therefore, future investigations on the temporal dynamics of ABC gene expression will allow a better implementation of insecticide treatment regimens, including the use of specific inhibitors of ABC efflux pumps.

  13. Temporal dynamics of the ABC transporter response to insecticide treatment: insights from the malaria vector Anopheles stephensi.

    PubMed

    Epis, Sara; Porretta, Daniele; Mastrantonio, Valentina; Urbanelli, Sandra; Sassera, Davide; De Marco, Leone; Mereghetti, Valeria; Montagna, Matteo; Ricci, Irene; Favia, Guido; Bandi, Claudio

    2014-01-01

    In insects, ABC transporters have been shown to contribute to defence/resistance to insecticides by reducing toxic concentrations in cells/tissues. Despite the extensive studies about this detoxifying mechanism, the temporal patterns of ABC transporter activation have been poorly investigated. Using the malaria vector Anopheles stephensi as a study system, we investigated the expression profile of ABC genes belonging to different subfamilies in permethrin-treated larvae at different time points (30 min to 48 h). Our results showed that the expression of ABCB and ABCG subfamily genes was upregulated at 1 h after treatment, with the highest expression observed at 6 h. Therefore, future investigations on the temporal dynamics of ABC gene expression will allow a better implementation of insecticide treatment regimens, including the use of specific inhibitors of ABC efflux pumps. PMID:25504146

  14. A putative cyclic peptide efflux pump encoded by the TOXA gene of the plant-pathogenic fungus Cochliobolus carbonum.

    PubMed

    Pitkin, J W; Panaccione, D G; Walton, J D

    1996-06-01

    Race 1 isolates of Cochliobolus carbonum are pathogenic on certain maize lines due to production of a host-selective cyclic tetrapeptide, HC-toxin. Flanking HTS1, which encodes the central enzyme in HC-toxin biosynthesis, a gene was identified and named TOXA. Like HTS1, TOXA occurred only in isolates of the fungus that make HC-toxin and was present as two linked copies in most toxin-producing isolates. HTS1 and TOXA were transcribed in the opposite orientation and their transcriptional start sites were 386 bp apart. The predicted product of TOXA was a 58 kDa hydrophobic protein with 10-13 membrane-spanning regions. The sequence was highly similar to several members of the major facilitator superfamily that confer resistance to tetracycline, methylenomycin, and other antibiotics. Although it was possible to mutate one copy or the other of TOXA by targeted gene disruption, numerous attempts to disrupt both copies in a single strain were unsuccessful, suggesting that TOXA is an essential gene in strains that synthesize HC-toxin. On the basis of its presence only in HC-toxin-producing strains, its proximity to HTS1 and its predicted amino acid sequence, we propose that TOXA encodes an HC-toxin efflux pump which contributes to self-protection against HC-toxin and/or the secretion of HC-toxin into the extracellular milieu.

  15. MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 3: Optimization of potency in the pyridopyrimidine series through the application of a pharmacophore model.

    PubMed

    Nakayama, Kiyoshi; Kawato, Haruko; Watanabe, Jun; Ohtsuka, Masami; Yoshida, Ken-ichi; Yokomizo, Yoshihiro; Sakamoto, Atsunobu; Kuru, Noriko; Ohta, Toshiharu; Hoshino, Kazuki; Yoshida, Kumi; Ishida, Hiroko; Cho, Aesop; Palme, Monica H; Zhang, Jason Z; Lee, Ving J; Watkins, William J

    2004-01-19

    The addition of substituents to the pyridopyrimidine scaffold of MexAB-OprM specific efflux pump inhibitors was explored. As predicted by a pharmacophore model, the incorporation substituents at the 2-position improved potency. Piperidines were found to be optimal, and further introduction of polar groups without compromising the activity was shown to be feasible. Careful positioning of the essential acidic moiety of the pharmacophore relative to the scaffold led to the discovery of vinyl tetrazoles with still greater potency.

  16. Characterization of a novel domain ‘GATE’ in the ABC protein DrrA and its role in drug efflux by the DrrAB complex

    SciTech Connect

    Zhang, Han; Rahman, Sadia; Li, Wen; Fu, Guoxing; Kaur, Parjit

    2015-03-27

    A novel domain, GATE (Glycine-loop And Transducer Element), is identified in the ABC protein DrrA. This domain shows sequence and structural conservation among close homologs of DrrA as well as distantly-related ABC proteins. Among the highly conserved residues in this domain are three glycines, G215, G221 and G231, of which G215 was found to be critical for stable expression of the DrrAB complex. Other conserved residues, including E201, G221, K227 and G231, were found to be critical for the catalytic and transport functions of the DrrAB transporter. Structural analysis of both the previously published crystal structure of the DrrA homolog MalK and the modeled structure of DrrA showed that G215 makes close contacts with residues in and around the Walker A motif, suggesting that these interactions may be critical for maintaining the integrity of the ATP binding pocket as well as the complex. It is also shown that G215A or K227R mutation diminishes some of the atomic interactions essential for ATP catalysis and overall transport function. Therefore, based on both the biochemical and structural analyses, it is proposed that the GATE domain, located outside of the previously identified ATP binding and hydrolysis motifs, is an additional element involved in ATP catalysis. - Highlights: • A novel domain ‘GATE’ is identified in the ABC protein DrrA. • GATE shows high sequence and structural conservation among diverse ABC proteins. • GATE is located outside of the previously studied ATP binding and hydrolysis motifs. • Conserved GATE residues are critical for stability of DrrAB and for ATP catalysis.

  17. 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

  18. Association of overexpression of efflux pump genes with antibiotic resistance in Pseudomonas aeruginosa strains clinically isolated from urinary tract infection patients.

    PubMed

    Shigemura, Katsumi; Osawa, Kayo; Kato, Ayaka; Tokimatsu, Issei; Arakawa, Soichi; Shirakawa, Toshiro; Fujisawa, Masato

    2015-09-01

    There are several mechanisms for antibiotic-resistant Pseudomonas aeruginosa. The purpose of this study is to investigate the association between the expression of efflux pump-coding genes and antibiotic resistance in P. aeruginosa causing urinary tract infections (UTIs). We extracted the RNA from 105 clinical strains of P. aeruginosa isolated from UTI patients with full data on antibiotic MICs and assayed real-time quantitative reverse-transcription PCR. We investigated the gene expressions of four resistance nodulation cell division-type multi-drug efflux pump systems (MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY(-OprA)) and the correlation of the MICs of nine antibiotics, risk factors and antibiotic resistance-related genes with expressions of mexB, mexC, mexE and mexY. Multivariate statistical data demonstrated a significant relationship between increased expression of mexB or mexC and complicated UTI (Odds ratio=8.03, P<0.001 and Odds ratio=8.86, P=0.032, respectively). We also found a significant association between the increased expression of mexC and resistance to levofloxacin (LVFX) (Odds ratio=4.48, P=0.035). In conclusion, increased expression of mexC leads to LVFX resistance in P. aeruginosa causing UTI. These results contribute to our knowledge of the efflux pump system and antibiotic resistance.

  19. Fluorescence-Based Flow Sorting in Parallel with Transposon Insertion Site Sequencing Identifies Multidrug Efflux Systems in Acinetobacter baumannii

    PubMed Central

    Cain, Amy K.; Huang, TaoTao; Liu, Qi; Elbourne, Liam D. H.; Boinett, Christine J.; Brzoska, Anthony J.; Li, Liping; Ostrowski, Martin; Nhu, Nguyen Thi Khanh; Nhu, Tran Do Hoang; Baker, Stephen; Paulsen, Ian T.

    2016-01-01

    ABSTRACT Multidrug efflux pumps provide clinically significant levels of drug resistance in a number of Gram-negative hospital-acquired pathogens. These pathogens frequently carry dozens of genes encoding putative multidrug efflux pumps. However, it can be difficult to determine how many of these pumps actually mediate antimicrobial efflux, and it can be even more challenging to identify the regulatory proteins that control expression of these pumps. In this study, we developed an innovative high-throughput screening method, combining transposon insertion sequencing and cell sorting methods (TraDISort), to identify the genes encoding major multidrug efflux pumps, regulators, and other factors that may affect the permeation of antimicrobials, using the nosocomial pathogen Acinetobacter baumannii. A dense library of more than 100,000 unique transposon insertion mutants was treated with ethidium bromide, a common substrate of multidrug efflux pumps that is differentially fluorescent inside and outside the bacterial cytoplasm. Populations of cells displaying aberrant accumulations of ethidium were physically enriched using fluorescence-activated cell sorting, and the genomic locations of transposon insertions within these strains were determined using transposon-directed insertion sequencing. The relative abundance of mutants in the input pool compared to the selected mutant pools indicated that the AdeABC, AdeIJK, and AmvA efflux pumps are the major ethidium efflux systems in A. baumannii. Furthermore, the method identified a new transcriptional regulator that controls expression of amvA. In addition to the identification of efflux pumps and their regulators, TraDISort identified genes that are likely to control cell division, cell morphology, or aggregation in A. baumannii. PMID:27601573

  20. Overexpression of the Novel MATE Fluoroquinolone Efflux Pump FepA in Listeria monocytogenes Is Driven by Inactivation of Its Local Repressor FepR

    PubMed Central

    Guérin, François; Galimand, Marc; Tuambilangana, Fabrice; Courvalin, Patrice; Cattoir, Vincent

    2014-01-01

    Whereas fluoroquinolone resistance mainly results from target modifications in gram-positive bacteria, it is primarily due to active efflux in Listeria monocytogenes. The aim of this study was to dissect a novel molecular mechanism of fluoroquinolone resistance in this important human pathogen. Isogenic L. monocytogenes clinical isolates BM4715 and BM4716, respectively susceptible and resistant to fluoroquinolones, were studied. MICs of norfloxacin and ciprofloxacin were determined in the presence or in the absence of reserpine (10 mg/L). Strain BM4715 was susceptible to norfloxacin (MIC, 4 mg/L) and ciprofloxacin (MIC, 0.5 mg/L) whereas BM4716 was highly resistant to both drugs (MICs 128 and 32 mg/L, respectively). Reserpine was responsible for a 16-fold decrease in both norfloxacin and ciprofloxacin MICs against BM4716 suggesting efflux associated resistance. Whole-genome sequencing of the strains followed by comparative genomic analysis revealed a single point mutation in the gene for a transcriptional regulator, designated fepR (for fluoroquinolone efflux protein regulator) belonging to the TetR family. The frame-shift mutation was responsible for the introduction of a premature stop codon resulting in an inactive truncated protein. Just downstream from fepR, the structural gene for an efflux pump of the MATE family (named FepA) was identified. Gene expression was quantified by qRT-PCR and demonstrated that fepA expression was more than 64-fold higher in BM4716 than in BM4715. The clean deletion of the fepR gene from BM4715 was responsible for an overexpression of fepA with resistance to norfloxacin and ciprofloxacin, confirming the role of FepR as a local repressor of fepA. In conclusion, we demonstrated that overexpression of the new MATE efflux pump FepA is responsible for fluoroquinolone resistance in L. monocytogenes and secondary to inactivation of the FepR repressor. PMID:25188450

  1. Tripartite efflux pumps: energy is required for dissociation, but not assembly or opening of the outer membrane channel of the pump.

    PubMed

    Janganan, Thamarai K; Bavro, Vassiliy N; Zhang, Li; Borges-Walmsley, Maria Inês; Walmsley, Adrian R

    2013-05-01

    The MtrCDE multidrug pump, from Neisseria gonorrhoeae, is assembled from the inner and outer membrane proteins MtrD and MtrE, which are connected by the periplasmic membrane fusion protein MtrC. Although it is clear that MtrD delivers drugs to the channel of MtrE, it remains unclear how drug delivery and channel opening are connected. We used a vancomycin sensitivity assay to test for opening of the MtrE channel. Cells expressing MtrE or MtrE-E434K were insensitive to vancomycin; but became moderately and highly sensitive to vancomycin respectively, when coexpressed with MtrC, suggesting that the MtrE channel opening requires MtrC binding and is energy-independent. Cells expressing wild-type MtrD, in an MtrCE background, were vancomycin-insensitive, but moderately sensitive in an MtrCE-E434K background. The mutation of residues involved in proton translocation inactivated MtrD and abolished drug efflux, rendered both MtrE and MtrE-E434K vancomycin-insensitive; imply that the pump-component interactions are preserved, and that the complex is stable in the absence of proton flux, thus sealing the open end of MtrE. Following the energy-dependent dissociation of the tripartite complex, the MtrE channel is able to reseal, while MtrE-E434K is unable to do so, resulting in the vancomycin-sensitive phenotype. Thus, our findings suggest that opening of the OMP via interaction with the MFP is energy-independent, while both drug export and complex dissociation require active proton flux.

  2. Molecular Evidence and Functional Expression of a Novel Drug Efflux pump (ABCC2) in Human Corneal Epithelium and Rabbit Cornea and its role in Ocular drug efflux

    PubMed Central

    Karla, Pradeep K.; Pal, Dhananjay; Quinn, Tim; Mitra, Ashim K.

    2007-01-01

    Cornea is considered as a major barrier for ocular drug delivery. Low ocular bioavailability of drugs has been attributed primarily to low permeability across corneal epithelium thus leading to sub-therapeutic concentrations of drug in the eye and treatment failure. The role of drug efflux proteins, particularly the Pglycoprotein in ocular drug bioavailability has been reported. The objective of this research was to determine whether human corneal epithelium expresses multi drug resistance associated proteins contributing to drug efflux by employing both cultured corneal cells and freshly excised rabbit cornea. SV40 HCEC and rPCEC were selected for in-vitro testing. SV40-HCEC and freshly excised rabbit corneas were utilized for transport studies. [3H]-cyclosporine-A and [14C]-erythromycin which are known substrates for ABCC2 and MK-571, a specific inhibitor for MRP were applied in this study. RT-PCR indicated a unique and distinct band at ∼272 bp corresponding to ABCC2 in HCEC, SV40-HCEC, rabbit cornea, rPCEC and MDCKII-MRP2 cells. Also RT-PCR indicated a unique band ∼181 bp for HCEC and SV40-HCEC. Immunoprecipitation followed by Western Blot analysis revealed a specific band at ∼190-kDa in membrane fraction of SV40-HCEC, MDCKII-MRP2 and no band with isotype control. Uptake of [3H]-cyclosporine-A and [14C]-erythromycin in the presence of MK-571 was significantly enhanced than control in both SV40 HCEC and rPCEC. Similarly a significant elevation in (A→B) permeability of [3H]-cyclosporine-A and [14C]-erythromycin was observed in the presence of MK-571 in SV40-HCEC. A→B transport of [3H]-cyclosporine-A was elevated in the presence of MK-571 in freshly excised rabbit cornea indicating potential role of this efflux transporter and high clinical significance of this finding. PMID:17156953

  3. 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

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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.

  9. Functional characterization of Brucella melitensis NorMI, an efflux pump belonging to the multidrug and toxic compound extrusion family.

    PubMed

    Braibant, Martine; Guilloteau, Laurence; Zygmunt, Michel S

    2002-09-01

    Two putative proteins (NorMI and NorMII) similar to the multidrug efflux protein NorM of Vibrio parahaemolyticus are encoded by the Brucella melitensis 16 M genome. We show that a drug-hypersusceptible Escherichia coli strain overexpressing NorMI displays increased resistance to norfloxacin, ciprofloxacin, gentamicin, tetraphenylphosphonium ion, acriflavine, and berberine. This elevated resistance was proven to be mediated by an energy-dependent efflux mechanism. NorMI belongs to the multidrug and toxic compound extrusion family and is the first multidrug efflux protein identified in Brucella spp.

  10. Enhancement of antibiotic activity against multidrug-resistant bacteria by the efflux pump inhibitor 3,4-dibromopyrrole-2,5-dione isolated from a Pseudoalteromonas sp.

    PubMed

    Whalen, Kristen E; Poulson-Ellestad, Kelsey L; Deering, Robert W; Rowley, David C; Mincer, Tracy J

    2015-03-27

    Members of the resistance nodulation cell division (RND) of efflux pumps play essential roles in multidrug resistance (MDR) in Gram-negative bacteria. Here, we describe the search for new small molecules from marine microbial extracts to block efflux and thus restore antibiotic susceptibility in MDR bacterial strains. We report the isolation of 3,4-dibromopyrrole-2,5-dione (1), an inhibitor of RND transporters, from Enterobacteriaceae and Pseudomonas aeruginosa, from the marine bacterium Pseudoalteromonas piscicida. 3,4-Dibromopyrrole-2,5-dione decreased the minimum inhibitory concentrations (MICs) of two fluoroquinolones, an aminoglycoside, a macrolide, a beta-lactam, tetracycline, and chloramphenicol between 2- and 16-fold in strains overexpressing three archetype RND transporters (AcrAB-TolC, MexAB-OprM, and MexXY-OprM). 3,4-Dibromopyrrole-2,5-dione also increased the intracellular accumulation of Hoechst 33342 in wild-type but not in transporter-deficient strains and prevented H33342 efflux (IC50 = 0.79 μg/mL or 3 μM), a hallmark of efflux pump inhibitor (EPI) functionality. A metabolomic survey of 36 Pseudoalteromonas isolates mapped the presence of primarily brominated metabolites only within the P. piscicida phylogenetic clade, where a majority of antibiotic activity was also observed, suggesting a link between halogenation and enhanced secondary metabolite biosynthetic potential. In sum, 3,4-dibromopyrrole-2,5-dione is a potent EPI and deserves further attention as an adjuvant to enhance the effectiveness of existing antibiotics. PMID:25646964

  11. The ABC of Ribosome-Related Antibiotic Resistance.

    PubMed

    Wilson, Daniel N

    2016-01-01

    The increase in multidrug-resistant pathogenic bacteria is limiting the utility of our current arsenal of antimicrobial agents. Mechanistically understanding how bacteria obtain antibiotic resistance is a critical first step to the development of improved inhibitors. One common mechanism for bacteria to obtain antibiotic resistance is by employing ATP-binding cassette (ABC) transporters to actively pump the drug from the cell. The ABC-F family includes proteins conferring resistance to a variety of clinically important ribosome-targeting antibiotics; however, controversy remains as to whether resistance is conferred via efflux like other ABC transporters or whether another mechanism, such as ribosome protection, is at play. A recent study by Sharkey and coworkers (L. K. Sharkey, T. A. Edwards, and A. J. O'Neill, mBio 7:e01975-15, 2016, http://dx.doi.org/10.1128/mBio.01975-15) provides strong evidence that ABC-F proteins conferring antibiotic resistance utilize ribosome protection mechanisms, namely, by interacting with the ribosome and displacing the drug from its binding site, thus revealing a novel role for ABC-F proteins in antibiotic resistance. PMID:27143393

  12. The ABC of Ribosome-Related Antibiotic Resistance.

    PubMed

    Wilson, Daniel N

    2016-05-03

    The increase in multidrug-resistant pathogenic bacteria is limiting the utility of our current arsenal of antimicrobial agents. Mechanistically understanding how bacteria obtain antibiotic resistance is a critical first step to the development of improved inhibitors. One common mechanism for bacteria to obtain antibiotic resistance is by employing ATP-binding cassette (ABC) transporters to actively pump the drug from the cell. The ABC-F family includes proteins conferring resistance to a variety of clinically important ribosome-targeting antibiotics; however, controversy remains as to whether resistance is conferred via efflux like other ABC transporters or whether another mechanism, such as ribosome protection, is at play. A recent study by Sharkey and coworkers (L. K. Sharkey, T. A. Edwards, and A. J. O'Neill, mBio 7:e01975-15, 2016, http://dx.doi.org/10.1128/mBio.01975-15) provides strong evidence that ABC-F proteins conferring antibiotic resistance utilize ribosome protection mechanisms, namely, by interacting with the ribosome and displacing the drug from its binding site, thus revealing a novel role for ABC-F proteins in antibiotic resistance.

  13. 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

  14. Research Progress on the Role of ABC Transporters in the Drug Resistance Mechanism of Intractable Epilepsy

    PubMed Central

    Xiong, Jie; Mao, Ding-an; Liu, Li-qun

    2015-01-01

    The pathogenesis of intractable epilepsy is not fully clear. In recent years, both animal and clinical trials have shown that the expression of ATP-binding cassette (ABC) transporters is increased in patients with intractable epilepsy; additionally, epileptic seizures can lead to an increase in the number of sites that express ABC transporters. These findings suggest that ABC transporters play an important role in the drug resistance mechanism of epilepsy. ABC transporters can perform the funcions of a drug efflux pump, which can reduce the effective drug concentration at epilepsy lesions by reducing the permeability of the blood brain barrier to antiepileptic drugs, thus causing resistance to antiepileptic drugs. Given the important role of ABC transporters in refractory epilepsy drug resistance, antiepileptic drugs that are not substrates of ABC transporters were used to obtain ABC transporter inhibitors with strong specificity, high safety, and few side effects, making them suitable for long-term use; therefore, these drugs can be used for future clinical treatment of intractable epilepsy. PMID:26491660

  15. Overexpression and functional characterization of an ABC (ATP-binding cassette) transporter encoded by the genes drrA and drrB of Mycobacterium tuberculosis.

    PubMed Central

    Choudhuri, Baisakhee Saha; Bhakta, Sanjib; Barik, Rajib; Basu, Joyoti; Kundu, Manikuntala; Chakrabarti, Parul

    2002-01-01

    The genes encoding ATP-binding cassette (ABC) transporters occupy 2.5% of the genome of Mycobacterium tuberculosis. However, none of these putative ABC transporters has been characterized so far. We describe the development of expression systems for simultaneous expression of the ATP-binding protein DrrA and the membrane integral protein DrrB which together behave as a functional doxorubicin efflux pump. Doxorubicin uptake in Escherichia coli or Mycobacterium smegmatis expressing DrrAB was inhibited by reserpine, an inhibitor of ABC transporters. The localization of DrrA to the membrane depended on the simultaneous expression of DrrB. ATP binding was positively regulated by doxorubicin and daunorubicin. At the same time, DrrB appeared to be sensitive to proteolysis when expressed alone in the absence of DrrA. Simultaneous expression of the two polypeptides was essential to obtain a functional doxorubicin efflux pump. Expression of DrrAB in E. coli conferred 8-fold increased resistance to ethidium bromide, a cationic compound. 2',7'-bis-(2-Carboxyethyl)-5(6)-carboxyfluorescein (BCECF), a neutral compound, also behaved as a substrate of the reconstituted efflux pump. When expressed in M. smegmatis, DrrAB conferred resistance to a number of clinically relevant, structurally unrelated antibiotics. The resistant phenotype could be reversed by verapamil and reserpine, two potent inhibitors of ABC transporters. PMID:12057006

  16. 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.

  17. 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

  18. A gonococcal efflux pump system enhances bacterial survival in a female mouse model of genital tract infection.

    PubMed

    Jerse, Ann E; Sharma, Nirmala D; Simms, Amy N; Crow, Emily T; Snyder, Lori A; Shafer, William M

    2003-10-01

    Active efflux of antimicrobial substances is likely to be an important bacterial defense against inhibitory host factors inherent to different body sites. Two well-characterized multidrug resistance efflux systems (MtrCDE and FarAB-MtrE) exist in Neisseria gonorrhoeae, a bacterial pathogen of the human genital mucosae. In vitro studies suggest that the MtrCDE and FarAB-MtrE efflux systems protect the gonococcus from hydrophobic antimicrobial substances that are likely to be present on mucosal surfaces. Here we report that a functional MtrCDE efflux system, but not a functional FarAB-MtrE system, enhances experimental gonococcal genital tract infection in female mice. Specifically, the recovery of mtrD and mtrE mutants, but not a farB mutant, from mice inoculated with mutant or wild-type gonococci was reduced compared with that of the wild-type strain. Competitive-infection experiments confirmed the survival disadvantage of MtrCDE-deficient gonococci. This report is the first direct evidence that a multidrug resistance efflux system enhances survival of a bacterial pathogen in the genital tract. Additionally, experiments using ovariectomized mice showed that MtrCDE-deficient gonococci were more rapidly cleared from mice that were capable of secreting gonadal hormones. MtrCDE-deficient gonococci were more sensitive to nonphysiological concentrations of progesterone in vitro than were wild-type or FarAB-MtrE-deficient gonococci. These results suggest that progesterone may play an inhibitory role in vivo. However, hormonally regulated factors rather than progesterone itself may be responsible for the more rapid clearance of mtr-deficient gonococci from intact mice.

  19. Effect of Transcriptional Activators SoxS, RobA, and RamA on Expression of Multidrug Efflux Pump AcrAB-TolC in Enterobacter cloacae

    PubMed Central

    Pérez, Astrid; Poza, Margarita; Aranda, Jesús; Latasa, Cristina; Medrano, Francisco Javier; Tomás, María; Romero, Antonio; Lasa, Iñigo

    2012-01-01

    Control of membrane permeability is a key step in regulating the intracellular concentration of antibiotics. Efflux pumps confer innate resistance to a wide range of toxic compounds such as antibiotics, dyes, detergents, and disinfectants in members of the Enterobacteriaceae. The AcrAB-TolC efflux pump is involved in multidrug resistance in Enterobacter cloacae. However, the underlying mechanism that regulates the system in this microorganism remains unknown. In Escherichia coli, the transcription of acrAB is upregulated under global stress conditions by proteins such as MarA, SoxS, and Rob. In the present study, two clinical isolates of E. cloacae, EcDC64 (a multidrug-resistant strain overexpressing the AcrAB-TolC efflux pump) and Jc194 (a strain with a basal AcrAB-TolC expression level), were used to determine whether similar global stress responses operate in E. cloacae and also to establish the molecular mechanisms underlying this response. A decrease in susceptibility to erythromycin, tetracycline, telithromycin, ciprofloxacin, and chloramphenicol was observed in clinical isolate Jc194 and, to a lesser extent in EcDC64, in the presence of salicylate, decanoate, tetracycline, and paraquat. Increased expression of the acrAB promoter in the presence of the above-described conditions was observed by flow cytometry and reverse transcription-PCR, by using a reporter fusion protein (green fluorescent protein). The expression level of the AcrAB promoter decreased in E. cloacae EcDC64 derivates deficient in SoxS, RobA, and RamA. Accordingly, the expression level of the AcrAB promoter was higher in E. cloacae Jc194 strains overproducing SoxS, RobA, and RamA. Overall, the data showed that SoxS, RobA, and RamA regulators were associated with the upregulation of acrAB, thus conferring antimicrobial resistance as well as a stress response in E. cloacae. In summary, the regulatory proteins SoxS, RobA, and RamA were cloned and sequenced for the first time in this species. The

  20. 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.

  1. 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

  2. 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

  3. Reduced aeration affects the expression of the NorB efflux pump of Staphylococcus aureus by posttranslational modification of MgrA.

    PubMed

    Truong-Bolduc, Que Chi; Hsing, Liao Chun; Villet, Regis; Bolduc, Gilles R; Estabrooks, Zoe; Taguezem, G Florent; Hooper, David C

    2012-04-01

    We previously showed that at acid pH, the transcription of norB, encoding the NorB efflux pump, increases due to a reduction in the phosphorylation level of MgrA, which in turn leads to a reduction in bacterial killing by moxifloxacin, a substrate of the NorB efflux pump. In this study, we demonstrated that reduced oxygen levels did not affect the transcript levels of mgrA but modified the dimerization of the MgrA protein, which remained mostly in its monomeric form. Under reduced aeration, we also observed a 21.7-fold increase in the norB transcript levels after 60 min of growth that contributed to a 4-fold increase in the MICs of moxifloxacin and sparfloxacin for Staphylococcus aureus RN6390. The relative proportions of MgrA in monomeric and dimeric forms were altered by treatment with H(2)O(2), but incubation of purified MgrA with extracts of cells grown under reduced but not normal aeration prevented MgrA from being converted to its dimeric DNA-binding form. This modification was associated with cleavage of a fragment of the dimerization domain of MgrA without change in MgrA phosphorylation and an increase in transcript levels of genes encoding serine proteases in cells incubated at reduced aeration. Taken together, these data suggest that modification of MgrA by proteases underlies the reversal of its repression of norB and increased resistance to NorB substrates in response to reduced-aeration conditions, illustrating a third mechanism of posttranslational modification, in addition to oxidation and phosphorylation, that modulates the regulatory activities of MgrA. PMID:22287526

  4. Differential requirement of the transcription factor Mcm1 for activation of the Candida albicans multidrug efflux pump MDR1 by its regulators Mrr1 and Cap1.

    PubMed

    Mogavero, Selene; Tavanti, Arianna; Senesi, Sonia; Rogers, P David; Morschhäuser, Joachim

    2011-05-01

    Overexpression of the multidrug efflux pump Mdr1 causes increased fluconazole resistance in the pathogenic yeast Candida albicans. The transcription factors Mrr1 and Cap1 mediate MDR1 upregulation in response to inducing stimuli, and gain-of-function mutations in Mrr1 or Cap1, which render the transcription factors hyperactive, result in constitutive MDR1 overexpression. The essential MADS box transcription factor Mcm1 also binds to the MDR1 promoter, but its role in inducible or constitutive MDR1 upregulation is unknown. Using a conditional mutant in which Mcm1 can be depleted from the cells, we investigated the importance of Mcm1 for MDR1 expression. We found that Mcm1 was dispensable for MDR1 upregulation by H2O2 but was required for full MDR1 induction by benomyl. A C-terminally truncated, hyperactive Cap1 could upregulate MDR1 expression both in the presence and in the absence of Mcm1. In contrast, a hyperactive Mrr1 containing a gain-of-function mutation depended on Mcm1 to cause MDR1 overexpression. These results demonstrate a differential requirement for the coregulator Mcm1 for Cap1- and Mrr1-mediated MDR1 upregulation. When activated by oxidative stress or a gain-of-function mutation, Cap1 can induce MDR1 expression independently of Mcm1, whereas Mrr1 requires either Mcm1 or an active Cap1 to cause overexpression of the MDR1 efflux pump. Our findings provide more detailed insight into the molecular mechanisms of drug resistance in this important human fungal pathogen. PMID:21343453

  5. 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

  6. RT-PCR and statistical analyses of adeABC expression in clinical isolates of Acinetobacter calcoaceticus-Acinetobacter baumannii complex.

    PubMed

    Ruzin, Alexey; Immermann, Frederick W; Bradford, Patricia A

    2010-06-01

    The relationship between expression of adeABC and minimal inhibitory concentration (MIC) of tigecycline was investigated by RT-PCR and statistical analyses in a population of 106 clinical isolates (MIC range, 0.0313-16 microg/ml) of Acinetobacter calcoaceticus-Acinetobacter baumannii complex. There was a statistically significant linear relationship (p < 0.0001) between log-transformed expression values and log-transformed MIC values, indicating that overexpression of AdeABC efflux pump is a prevalent mechanism for decreased susceptibility to tigecycline in A. calcoaceticus-A. baumannii complex.

  7. 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

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

    PubMed

    Wu, Chao-Jung; Huang, Yi-Wei; 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

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

    PubMed

    Wu, Chao-Jung; Huang, Yi-Wei; 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

  10. 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

  11. Contribution of Resistance-Nodulation-Cell Division Efflux Systems to Antibiotic Resistance and Biofilm Formation in Acinetobacter baumannii

    PubMed Central

    Yoon, Eun-Jeong; Nait Chabane, Yassine; Goussard, Sylvie; Snesrud, Erik; Courvalin, Patrice; Dé, Emmanuelle

    2015-01-01

    ABSTRACT Acinetobacter baumannii is a nosocomial pathogen of increasing importance due to its multiple resistance to antibiotics and ability to survive in the hospital environment linked to its capacity to form biofilms. To fully characterize the contribution of AdeABC, AdeFGH, and AdeIJK resistance-nodulation-cell division (RND)-type efflux systems to acquired and intrinsic resistance, we constructed, from an entirely sequenced susceptible A. baumannii strain, a set of isogenic mutants overexpressing each system following introduction of a point mutation in their cognate regulator or a deletion for the pump by allelic replacement. Pairwise comparison of every derivative with the parental strain indicated that AdeABC and AdeFGH are tightly regulated and contribute to acquisition of antibiotic resistance when overproduced. AdeABC had a broad substrate range, including β-lactams, fluoroquinolones, tetracyclines-tigecycline, macrolides-lincosamides, and chloramphenicol, and conferred clinical resistance to aminoglycosides. Importantly, when combined with enzymatic resistance to carbapenems and aminoglycosides, this pump contributed in a synergistic fashion to the level of resistance of the host. In contrast, AdeIJK was expressed constitutively and was responsible for intrinsic resistance to the same major drug classes as AdeABC as well as antifolates and fusidic acid. Surprisingly, overproduction of AdeABC and AdeIJK altered bacterial membrane composition, resulting in decreased biofilm formation but not motility. Natural transformation and plasmid transfer were diminished in recipients overproducing AdeABC. It thus appears that alteration in the expression of efflux systems leads to multiple changes in the relationship between the host and its environment, in addition to antibiotic resistance. PMID:25805730

  12. 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

  13. Mitochondria influence CDR1 efflux pump activity, Hog1-mediated oxidative stress pathway, iron homeostasis, and ergosterol levels in Candida albicans.

    PubMed

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

    2013-11-01

    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.

  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. Differential contributions of five ABC transporters to mutidrug resistance, antioxidion and virulence of Beauveria bassiana, an entomopathogenic fungus.

    PubMed

    Song, Ting-Ting; Zhao, Jing; Ying, Sheng-Hua; Feng, Ming-Guang

    2013-01-01

    Multidrug resistance (MDR) confers agrochemical compatibility to fungal cells-based mycoinsecticdes but mechanisms involved in MDR remain poorly understood for entomopathogenic fungi, which have been widely applied as biocontrol agents against arthropod pests. Here we characterized the functions of five ATP-binding cassette (ABC) transporters, which were classified to the subfamilies ABC-B (Mdr1), ABC-C (Mrp1) and ABC-G (Pdr1, Pdr2 and Pdr5) and selected from 54 full-size ABC proteins of Beauveria bassiana based on their main domain architecture, membrane topology and transcriptional responses to three antifungal inducers. Disruption of each transporter gene resulted in significant reduction in resistance to four to six of eight fungicides or antifungal drugs tested due to their differences in structure and function. Compared with wild-type and complemented (control) strains, disruption mutants of all the five transporter genes became significantly less tolerant to the oxidants menadione and H₂O₂ based on 22-41% and 10-31% reductions of their effective concentrations required for the suppression of 50% colony growth at 25°C. Under a standardized spray, the killing actions of ΔPdr5 and ΔMrp1 mutants against Spodoptera litura second-instar larvae were delayed by 59% and 33% respectively. However, no significant virulence change was observed in three other delta mutants. Taken together, the examined five ABC transporters contribute differentially to not only the fungal MDR but antioxidant capability, a phenotype rarely associated with ABC efflux pumps in previous reports; at least some of them are required for the full virulence of B. bassiana, thereby affecting the fungal biocontrol potential. Our results indicate that ABC pump-dependent MDR mechanisms exist in entomopathogenic fungi as do in yeasts and human and plant pathogenic fungi.

  16. 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.

  17. Functional induction of P-glycoprotein efflux pump by phenyl benzenesulfonamides: Synthesis and biological evaluation of T0901317 analogs.

    PubMed

    Padala, Anil K; Wani, Abubakar; Vishwakarma, Ram A; Kumar, Ajay; Bharate, Sandip B

    2016-10-21

    N-(2,2,2-Trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]-benzenesulfonamide (T0901317, 6) is a potent activator of pregnane-X-receptor (PXR), which is a nuclear receptor controlling P-gp expression. Herein, we aimed to investigate P-gp induction activity of T0901317 and establish its structure-activity relationship. T0901317 along with a series of N-triazolyl-methylene-linked benzenesulfonamides were synthesized and screened for P-gp induction activity using a rhodamine-123 based efflux assay in the P-gp overexpressing human adenocarcinoma LS-180 cells, wherein several compounds showed potent P-gp induction activity at 5 μM. Treatment with benzene sulphonamides led to the decrease in intracellular accumulation of a fluorescent P-gp substrate rhodamine-123 up to 48% (control 100%). In the western-blot studies, T0901317 (6) and its triazole linked analog 26e at 5 μM displayed induction of P-gp expression in LS180 cells. These compounds were non-toxic in LS-180 and human neuroblastoma SH-SY5Y cells (IC50 > 50 μM). The compound 26e showed significant P-gp induction even at 0.3 μM, indicating an excellent therapeutic window. These results clearly indicate promise of this class of compounds as potential agents to enhance amyloid-β clearance in Alzheimers patients.

  18. C2 Arylated Benzo[b]thiophene Derivatives as Staphylococcus aureus NorA Efflux Pump Inhibitors.

    PubMed

    Liger, François; Bouhours, Pascale; Ganem-Elbaz, Carine; Jolivalt, Claude; Pellet-Rostaing, Stéphane; Popowycz, Florence; Paris, Jean-Marc; Lemaire, Marc

    2016-02-01

    An innovative and straightforward synthesis of second-generation 2-arylbenzo[b]thiophenes as structural analogues of INF55 and the first generation of our laboratory-made molecules was developed. The synthesis of C2-arylated benzo[b]thiophene derivatives was achieved through a method involving direct arylation, followed by simple structural modifications. Among the 34 compounds tested, two of them were potent NorA pump inhibitors, which led to a 16-fold decrease in the ciprofloxacin minimum inhibitory concentration (MIC) against the SA-1199B strain at concentrations of 0.25 and 0.5 μg mL(-1) (1 and 1.5 μm, respectively). This is a promising result relative to that obtained for reserpine (MIC=20 μg mL(-1)), a reference compound amongst NorA pump inhibitors. These molecules thus represent promising candidates to be used in combination with ciprofloxacin against fluoroquinolone-resistant strains.

  19. CpxR Activates MexAB-OprM Efflux Pump Expression and Enhances Antibiotic Resistance in Both Laboratory and Clinical nalB-Type Isolates of Pseudomonas aeruginosa

    PubMed Central

    Yi, Xue-Xian; O’Gara, Fergal; Wang, Yi-Ping

    2016-01-01

    Resistance-Nodulation-Division (RND) efflux pumps are responsible for multidrug resistance in Pseudomonas aeruginosa. In this study, we demonstrate that CpxR, previously identified as a regulator of the cell envelope stress response in Escherichia coli, is directly involved in activation of expression of RND efflux pump MexAB-OprM in P. aeruginosa. A conserved CpxR binding site was identified upstream of the mexA promoter in all genome-sequenced P. aeruginosa strains. CpxR is required to enhance mexAB-oprM expression and drug resistance, in the absence of repressor MexR, in P. aeruginosa strains PA14. As defective mexR is a genetic trait associated with the clinical emergence of nalB-type multidrug resistance in P. aeruginosa during antibiotic treatment, we investigated the involvement of CpxR in regulating multidrug resistance among resistant isolates generated in the laboratory via antibiotic treatment and collected in clinical settings. CpxR is required to activate expression of mexAB-oprM and enhances drug resistance, in the absence or presence of MexR, in ofloxacin-cefsulodin-resistant isolates generated in the laboratory. Furthermore, CpxR was also important in the mexR-defective clinical isolates. The newly identified regulatory linkage between CpxR and the MexAB-OprM efflux pump highlights the presence of a complex regulatory network modulating multidrug resistance in P. aeruginosa. PMID:27736975

  20. Bioinformatic survey of ABC transporters in dermatophytes.

    PubMed

    Gadzalski, Marek; Ciesielska, Anita; Stączek, Paweł

    2016-01-15

    ATP binding cassette (ABC) transporters constitute a very large and ubiquitous superfamily of membrane proteins. They are responsible for ATP hydrolysis driven translocation of countless substrates. Being a very old and diverse group of proteins present in all organisms they share a common feature, which is the presence of an evolutionary conservative nucleotide binding domain (NBD)--the engine that drives the transport. Another common domain is a transmembrane domain (TMD) which consists of several membrane-spanning helices. This part of protein is substrate-specific, thus it is much more variable. ABC transporters are known for driving drug efflux in many pathogens and cancer cells, therefore they are the subject of extensive studies. There are many examples of conferring a drug resistance phenotype in fungal pathogens by ABC transporters, however, little is known about these proteins in dermatophytes--a group of fungi causing superficial mycoses. So far only a single ABC transporter has been extensively studied in this group of pathogens. We analyzed available genomic sequences of seven dermatophyte species in order to provide an insight into dermatophyte ABC protein inventory. Phylogenetic studies of ABC transporter genes and their products were conducted and included ABC transporters of other fungi. Our results show that each dermatophyte genome studied possesses a great variety of ABC transporter genes. Detailed analysis of selected genes and their products indicates that relatively recent duplication of ABC transporter genes could lead to novel substrate specificity. PMID:26524502

  1. 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

  2. Fluoroquinolone Efflux in Streptococcus suis Is Mediated by SatAB and Not by SmrA ▿

    PubMed Central

    Escudero, Jose Antonio; San Millan, Alvaro; Gutierrez, Belen; Hidalgo, Laura; La Ragione, Roberto M.; AbuOun, Manal; Galimand, Marc; Ferrándiz, María José; Domínguez, Lucas; de la Campa, Adela G.; Gonzalez-Zorn, Bruno

    2011-01-01

    Streptococcus suis is an emerging zoonotic pathogen. With the lack of an effective vaccine, antibiotics remain the main tool to fight infections caused by this pathogen. We have previously observed a reserpine-sensitive fluoroquinolone (FQ) efflux phenotype in this species. Here, SatAB and SmrA, two pumps belonging to the ATP binding cassette (ABC) and the major facilitator superfamily (MFS), respectively, have been analyzed in the fluoroquinolone-resistant clinical isolate BB1013. Genes encoding these pumps were overexpressed either constitutively or in the presence of ciprofloxacin in this strain. These genes could not be cloned in plasmids in Escherichia coli despite strong expression repression. Finally, site-directed insertion of smrA and satAB in the amy locus of the Bacillus subtilis chromosome using ligated PCR amplicons allowed for the functional expression and study of both pumps. Results showed that SatAB is a narrow-spectrum fluoroquinolone exporter (norfloxacin and ciprofloxacin), susceptible to reserpine, whereas SmrA was not involved in fluoroquinolone resistance. Chromosomal integration in Bacillus is a novel method for studying efflux pumps from Gram-positive bacteria, which enabled us to demonstrate the possible role of SatAB, and not SmrA, in fluoroquinolone efflux in S. suis. PMID:21930876

  3. The Binding of Triclosan to SmeT, the Repressor of the Multidrug Efflux Pump SmeDEF, Induces Antibiotic Resistance in Stenotrophomonas maltophilia

    PubMed Central

    Romero, Antonio; Martínez, José L.

    2011-01-01

    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

  4. Two Host-Induced Ralstonia solanacearum Genes, acrA and dinF, Encode Multidrug Efflux Pumps and Contribute to Bacterial Wilt Virulence▿ †

    PubMed Central

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

    2007-01-01

    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 (>107 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

  5. Pleiotropic effects of the vacuolar ABC transporter MLT1 of Candida albicans on cell function and virulence

    PubMed Central

    Khandelwal, Nitesh Kumar; Kaemmer, Philipp; Förster, Toni M.; Singh, Ashutosh; Coste, Alix T.; Andes, David R.; Hube, Bernhard; Sanglard, Dominique; Chauhan, Neeraj; Kaur, Rupinder; d'Enfert, Christophe; Mondal, Alok Kumar; Prasad, Rajendra

    2016-01-01

    Among the several mechanisms that contribute to MDR (multidrug resistance), the overexpression of drug-efflux pumps belonging to the ABC (ATP-binding cassette) superfamily is the most frequent cause of resistance to antifungal agents. The multidrug transporter proteins Cdr1p and Cdr2p of the ABCG subfamily are major players in the development of MDR in Candida albicans. Because several genes coding for ABC proteins exist in the genome of C. albicans, but only Cdr1p and Cdr2p have established roles in MDR, it is implicit that the other members of the ABC family also have alternative physiological roles. The present study focuses on an ABC transporter of C. albicans, Mlt1p, which is localized in the vacuolar membrane and specifically transports PC (phosphatidylcholine) into the vacuolar lumen. Transcriptional profiling of the mlt1∆/∆ mutant revealed a down-regulation of the genes involved in endocytosis, oxidoreductase activity, virulence and hyphal development. High-throughput MS-based lipidome analysis revealed that the Mlt1p levels affect lipid homoeostasis and thus lead to a plethora of physiological perturbations. These include a delay in endocytosis, inefficient sequestering of reactive oxygen species (ROS), defects in hyphal development and attenuated virulence. The present study is an emerging example where new and unconventional roles of an ABC transporter are being identified. PMID:27026051

  6. Pleiotropic effects of the vacuolar ABC transporter MLT1 of Candida albicans on cell function and virulence.

    PubMed

    Khandelwal, Nitesh Kumar; Kaemmer, Philipp; Förster, Toni M; Singh, Ashutosh; Coste, Alix T; Andes, David R; Hube, Bernhard; Sanglard, Dominique; Chauhan, Neeraj; Kaur, Rupinder; d'Enfert, Christophe; Mondal, Alok Kumar; Prasad, Rajendra

    2016-06-01

    Among the several mechanisms that contribute to MDR (multidrug resistance), the overexpression of drug-efflux pumps belonging to the ABC (ATP-binding cassette) superfamily is the most frequent cause of resistance to antifungal agents. The multidrug transporter proteins Cdr1p and Cdr2p of the ABCG subfamily are major players in the development of MDR in Candida albicans Because several genes coding for ABC proteins exist in the genome of C. albicans, but only Cdr1p and Cdr2p have established roles in MDR, it is implicit that the other members of the ABC family also have alternative physiological roles. The present study focuses on an ABC transporter of C. albicans, Mlt1p, which is localized in the vacuolar membrane and specifically transports PC (phosphatidylcholine) into the vacuolar lumen. Transcriptional profiling of the mlt1∆/∆ mutant revealed a down-regulation of the genes involved in endocytosis, oxidoreductase activity, virulence and hyphal development. High-throughput MS-based lipidome analysis revealed that the Mlt1p levels affect lipid homoeostasis and thus lead to a plethora of physiological perturbations. These include a delay in endocytosis, inefficient sequestering of reactive oxygen species (ROS), defects in hyphal development and attenuated virulence. The present study is an emerging example where new and unconventional roles of an ABC transporter are being identified. PMID:27026051

  7. 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

  8. An OXA-66/OXA-51-like carbapenemase and possibly an efflux pump are associated with resistance to imipenem in Acinetobacter baumannii.

    PubMed

    Hu, Wensi S; Yao, Shu-Man; Fung, Chang-Phone; Hsieh, Yi-Ping; Liu, Chang-Pan; Lin, Jing-Fang

    2007-11-01

    We investigated the mechanisms involved in imipenem resistance in 23 clinical strains of Acinetobacter baumannii. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the presence of a 30-kDa protein in imipenem-intermediate A. baumannii (IIAB) and imipenem-resistant A. baumannii (IRAB) strains; this protein was almost undetectable in imipenem-susceptible A. baumannii (ISAB) strains. The 30-kDa protein was identified as an OXA-51-like carbapenemase using two-dimensional gel electrophoresis and mass spectrometry. Similar to other recent findings, bla(OXA-51-like) genes were found to exist in all 23 clinical strains; however, the transcript levels of bla(OXA-51-like) in the IIAB and IRAB were higher than in the ISAB strains using reverse transcriptase PCR (RT-PCR) and real-time RT-PCR assays. This change was due to the presence of an insertion sequence, ISAba1, upstream of bla(OXA-51-like) in the IIAB and IRAB strains that was not present in the ISAB strains. The introduction of bla(OXA-66) (a bla(OXA-51)(-like) gene), identified in ISAB ab1254 and IRAB ab1266, into Escherichia coli TOP10 cells resulted in 3.95-fold and 7.90-fold elevations in resistance to imipenem, respectively. Furthermore, when ISAB ab8 and ISAB ab1254 and their in vitro-selected imipenem-resistant mutants ISAB ab8(r) and ISAB ab1254(r) were compared, the results showed no change in the bla(OXA-66)/bla(OXA-51-like) gene sequences, in expression of the gene, and in the outer membrane protein profiles. However, there was a four- to eightfold reduction in imipenem resistance upon adding carbonyl cyanide m-chlorophenylhydrazone. Taken together, these results suggest that the OXA-66/OXA-51-like carbapenemase contributes to intrinsic resistance to imipenem; however, drug export by an efflux pump may be more important and/or occur more frequently in imipenem-resistant A. baumannii. Furthermore, this is the first report of a Taiwanese strain of an OXA-66/OXA-51-like

  9. 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

    This study examines adenosine 5′-triphosphate-binding cassette (ABC) transporters as a potential therapeutic target in dendritic cell (DC) modulation under hypoxia and lipopolysaccharide (LPS). Functional capacity of dendritic cells (DCs) (mixed lymphocyte reaction: MLR) and maturation of iDCs were evaluated in the presence or absence of specific ABC-transporter inhibitors. Monocyte-derived DCs were cultured in the presence of interleukin (IL)-4/granulocyte–macrophage colony-stimulating factor (GM-CSF). Their maturation under hypoxia or LPS conditions was evaluated by assessing the expression of maturation phenotypes using flow cytometry. The effect of ABC transporters on DC maturation was determined using specific inhibitors for multi-drug resistance (MDR1) and multi-drug resistance proteins (MRPs). Depending on their maturation status to elicit T cell alloresponses, the functional capacity of DCs was studied by MLR. Mature DCs showed higher P-glycoprotein (Pgp) expression with confocal microscopy. Up-regulation of maturation markers was observed in hypoxia and LPS-DC, defining two different DC subpopulation profiles, plasmacytoid versus conventional-like, respectively, and different cytokine release T helper type 2 (Th2) versus Th1, depending on the stimuli. Furthermore, hypoxia-DCs induced more B lymphocyte proliferation than control-iDC (56% versus 9%), while LPS-DCs induced more CD8-lymphocyte proliferation (67% versus 16%). ABC transporter-inhibitors strongly abrogated DC maturation [half maximal inhibitory concentration (IC50): P-glycoprotein inhibition using valspodar (PSC833) 5 μM, CAS 115104-28-4 (MK571) 50 μM and probenecid 2·5 μM], induced significantly less lymphocyte proliferation and reduced cytokine release compared with stimulated-DCs without inhibitors. We conclude that diverse stimuli, hypoxia or LPS induce different profiles in the maturation and functionality of DC. Pgp appears to play a role in these DC events. Thus, ABC

  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. 3-(Benzo[d][1,3]dioxol-5-ylamino)-N-(4-fluorophenyl)thiophene-2-carboxamide overcomes cancer chemoresistance via inhibition of angiogenesis and P-glycoprotein efflux pump activity.

    PubMed

    Mudududdla, Ramesh; Guru, Santosh K; Wani, Abubakar; Sharma, Sadhana; Joshi, Prashant; Vishwakarma, Ram A; Kumar, Ajay; Bhushan, Shashi; Bharate, Sandip B

    2015-04-14

    3-((Quinolin-4-yl)methylamino)-N-(4-(trifluoromethoxy)phenyl)thiophene-2-carboxamide (OSI-930, 1) is a potent inhibitor of c-kit and VEGFR2, currently under phase I clinical trials in patients with advanced solid tumors. In order to understand the structure-activity relationship, a series of 3-arylamino N-aryl thiophene 2-carboxamides were synthesized by modifications at both quinoline and amide domains of the OSI-930 scaffold. All the synthesized compounds were screened for in vitro cytotoxicity in a panel of cancer cell lines and for VEGFR1 and VEGFR2 inhibition. Thiophene 2-carboxamides substituted with benzo[d][1,3]dioxol-5-yl and 2,3-dihydrobenzo[b][1,4]dioxin-6-yl groups 1l and 1m displayed inhibition of VEGFR1 with IC50 values of 2.5 and 1.9 μM, respectively. Compounds 1l and 1m also inhibited the VEGF-induced HUVEC cell migration, indicating its anti-angiogenic activity. OSI-930 along with compounds 1l and 1m showed inhibition of P-gp efflux pumps (MDR1, ABCB1) with EC50 values in the range of 35-74 μM. The combination of these compounds with doxorubicin led to significant enhancement of the anticancer activity of doxorubicin in human colorectal carcinoma LS180 cells, which was evident from the improved IC50 of doxorubicin, the increased activity of caspase-3 and the significant reduction in colony formation ability of LS180 cells after treatment with doxorubicin. Compound 1l showed a 13.8-fold improvement in the IC50 of doxorubicin in LS180 cells. The ability of these compounds to display dual inhibition of VEGFR and P-gp efflux pumps demonstrates the promise of this scaffold for its development as multi-drug resistance-reversal agents.

  12. The purine efflux pump PbuE in Bacillus subtilis modulates expression of the PurR and G-box (XptR) regulons by adjusting the purine base pool size.

    PubMed

    Nygaard, Per; Saxild, Hans H

    2005-01-01

    In Bacillus subtilis, the expression of genes encoding enzymes and other proteins involved in purine de novo synthesis and salvage is affected by purine bases and phosphoribosylpyrophosphate (PRPP). The transcription of the genes belonging to the PurR regulon is negatively regulated by the PurR protein and PRPP. The expression of the genes belonging to the G-box (XptR) regulon, including the pbuE gene, is negatively regulated by a riboswitch-controlled transcription termination mechanism. The G-box regulon effector molecules are hypoxanthine and guanine. pbuE encodes a purine base efflux pump and is now recognized as belonging to a third purine regulon. The expression of the pbuE gene is positively regulated by a riboswitch that recognizes adenine. Here we show that the expression of pbuE'-lacZ transcriptional fusions are induced by adenine to the highest extent in mutants which do not express a functional PbuE pump. In a mutant defective in the metabolism of adenine, the ade apt mutant, we found a high intracellular level of adenine and constitutive high levels of PbuE. A growth test using a purine auxotroph provided further evidence for the role of PbuE in lowering the intracellular concentration of purine bases, including adenine. Purine analogs also affect the expression of pbuE, which might be of importance for the protection against toxic analogs. In a mutant that overexpresses PbuE, the expression of genes belonging to the PurR regulon was increased. Our findings provide further evidence for important functions of the PbuE protein, such as acting as a pump that lowers the purine base pool and affects the expression of the G-box and PurR regulons, including pbuE itself, and as a pump involved in protection against toxic purine base analogs.

  13. The Acinetobacter baumannii Two-Component System AdeRS Regulates Genes Required for Multidrug Efflux, Biofilm Formation, and Virulence in a Strain-Specific Manner

    PubMed Central

    Richmond, Grace E.; Evans, Laura P.; Anderson, Michele J.; Wand, Matthew E.; Bonney, Laura C.; Ivens, Alasdair; Chua, Kim Lee; Webber, Mark A.; Sutton, J. Mark; Peterson, Marnie L.

    2016-01-01

    ABSTRACT The opportunistic pathogen Acinetobacter baumannii is able to persist in the environment and is often multidrug resistant (MDR), causing difficulties in the treatment of infections. Here, we show that the two-component system AdeRS, which regulates the production of the AdeABC multidrug resistance efflux pump, is required for the formation of a protective biofilm in an ex vivo porcine mucosal model, which mimics a natural infection of the human epithelium. Interestingly, deletion of adeB impacted only on the ability of strain AYE to form a biofilm on plastic and only on the virulence of strain Singapore 1 for Galleria mellonella. RNA-Seq revealed that loss of AdeRS or AdeB significantly altered the transcriptional landscape, resulting in the changed expression of many genes, notably those associated with antimicrobial resistance and virulence interactions. For example, A. baumannii lacking AdeRS displayed decreased expression of adeABC, pil genes, com genes, and a pgaC-like gene, whereas loss of AdeB resulted in increased expression of pil and com genes and decreased expression of ferric acinetobactin transport system genes. These data define the scope of AdeRS-mediated regulation, show that changes in the production of AdeABC mediate important phenotypes controlled by AdeRS, and suggest that AdeABC is a viable target for antimicrobial drug and antibiofilm discovery. PMID:27094331

  14. 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

  15. Learning the ABC of oral fungal drug resistance.

    PubMed

    Cannon, R D; Holmes, A R

    2015-12-01

    ATP-binding cassette (ABC) proteins are ubiquitous in prokaryotes and eukaryotes. They are involved in energy-dependent transport of molecules across membranes. ABC proteins are often promiscuous transporters that can translocate a variety of substrates. In oral fungi, especially in Candida species, they have been implicated as major contributors to the high-level azole resistance of clinical isolates from infections that do not respond to drug therapy. Although this is predominantly due to efflux of azoles from the cells, ABC proteins can contribute to fungal drug resistance in other ways as well. Cells in biofilms are notoriously resistant to antifungal agents. ABC proteins can contribute to this resistance through the efflux of drugs. Biofilms are complex communities of myriad microorganisms which, to survive in such a milieu, need to communicate with, and respond to, other microorganisms and their products. ABC proteins are involved in the secretion of fungal mating factors and quorum sensing molecules. These molecules affect biofilm structure and behavior that can result in increased drug resistance. Hence, ABC proteins make multiple contributions to oral fungal drug resistance through a variety of responses to environmental signals. PMID:26042641

  16. Learning the ABC of oral fungal drug resistance.

    PubMed

    Cannon, R D; Holmes, A R

    2015-12-01

    ATP-binding cassette (ABC) proteins are ubiquitous in prokaryotes and eukaryotes. They are involved in energy-dependent transport of molecules across membranes. ABC proteins are often promiscuous transporters that can translocate a variety of substrates. In oral fungi, especially in Candida species, they have been implicated as major contributors to the high-level azole resistance of clinical isolates from infections that do not respond to drug therapy. Although this is predominantly due to efflux of azoles from the cells, ABC proteins can contribute to fungal drug resistance in other ways as well. Cells in biofilms are notoriously resistant to antifungal agents. ABC proteins can contribute to this resistance through the efflux of drugs. Biofilms are complex communities of myriad microorganisms which, to survive in such a milieu, need to communicate with, and respond to, other microorganisms and their products. ABC proteins are involved in the secretion of fungal mating factors and quorum sensing molecules. These molecules affect biofilm structure and behavior that can result in increased drug resistance. Hence, ABC proteins make multiple contributions to oral fungal drug resistance through a variety of responses to environmental signals.

  17. Direct measurement of efflux in Pseudomonas aeruginosa using an environment-sensitive fluorescent dye.

    PubMed

    Iyer, Ramkumar; Erwin, Alice L

    2015-01-01

    Resistance-Nodulation-Division (RND) family pumps AcrB and MexB are the major efflux routes in Escherichia coli and Pseudomonas aeruginosa respectively. Fluorescent environment-sensitive dyes provide a means to study efflux pump function in live bacterial cells in real-time. Recently, we demonstrated the utility of this approach using the dye Nile Red to quantify AcrB-mediated efflux and measured the ability of antibiotics and other efflux pump substrates to compete with efflux of Nile Red, independent of antibacterial activity. Here, we extend this method to P. aeruginosa and describe a novel application that permits the comparison and rank-ordering of bacterial strains by their inherent efflux potential. We show that glucose and l-malate re-energize Nile Red efflux in P. aeruginosa, and we highlight differences in the glucose dependence and kinetics of efflux between P. aeruginosa and E. coli. We quantify the differences in efflux among a set of P. aeruginosa laboratory strains, which include PAO1, the hyper-sensitive strain ATCC 35151 and its parent, ATCC 12055. Efflux of Nile Red in P. aeruginosa is mediated by MexAB-OprM and is slower than in E. coli. In conclusion, we describe an efflux measurement tool for use in antibacterial drug discovery and basic research on P. aeruginosa efflux pumps.

  18. 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.

  19. Poloxamines display a multiple inhibitory activity of ATP-binding cassette (ABC) transporters in cancer cell lines.

    PubMed

    Cuestas, María L; Sosnik, Alejandro; Mathet, Verónica L

    2011-08-01

    Primary hepatocellular carcinoma is the third most common fatal cancer worldwide with more than 500,000 annual deaths. Approximately 40% of the patients with HCC showed tumoral overexpression of transmembrane proteins belonging to the ATP-binding cassette protein superfamily (ABC) which pump drugs out of cells. The overexpression of these efflux transporters confers on the cells a multiple drug resistance phenotype, which is considered a crucial cause of treatment refractoriness in patients with cancer. The aim of this study was to investigate the inhibitory effect of different concentrations of pH- and temperature-responsive X-shaped poly(ethylene oxide)-poly(propylene oxide) block copolymers (poloxamines, Tetronic, PEO-PPO) showing a wide range of molecular weights and EO/PO ratios on the functional activity of three different ABC proteins, namely P-glycoprotein (P-gp or MDR1), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein MRP1, in two human hepatocarcinoma cell lines, HepG2 and Huh7. First, the cytotoxicity of the different copolymers (at different concentrations) on both liver carcinoma cell lines was thoroughly evaluated by means of apoptosis analysis using annexin V and propidium iodide (PI). Thus, viable cells (AV-/PI-), early apoptotic cells (AV+/PI-) and late apoptotic cells (V-FITC+/PI+) were identified. Results pointed out copolymers of intermediate to high hydrophobicity and intermediate molecular weight (e.g., T904) as the most cytotoxic. Then, DiOC2, rhodamine 123 and vinblastine were used as differential substrates of these pumps. HeLa, an epithelial cell line of human cervical cancer that does not express P-gp, was used exclusively as a control and enabled the discerning between P-gp and MRP1 inhibition. Moderate to highly hydrophobic poloxamines T304, T904 and T1301 showed inhibitory activity against P-gp and BCRP but not against MRP1 in both hepatic cell lines. A remarkable dependence of this effect on the

  20. Poloxamines display a multiple inhibitory activity of ATP-binding cassette (ABC) transporters in cancer cell lines.

    PubMed

    Cuestas, María L; Sosnik, Alejandro; Mathet, Verónica L

    2011-08-01

    Primary hepatocellular carcinoma is the third most common fatal cancer worldwide with more than 500,000 annual deaths. Approximately 40% of the patients with HCC showed tumoral overexpression of transmembrane proteins belonging to the ATP-binding cassette protein superfamily (ABC) which pump drugs out of cells. The overexpression of these efflux transporters confers on the cells a multiple drug resistance phenotype, which is considered a crucial cause of treatment refractoriness in patients with cancer. The aim of this study was to investigate the inhibitory effect of different concentrations of pH- and temperature-responsive X-shaped poly(ethylene oxide)-poly(propylene oxide) block copolymers (poloxamines, Tetronic, PEO-PPO) showing a wide range of molecular weights and EO/PO ratios on the functional activity of three different ABC proteins, namely P-glycoprotein (P-gp or MDR1), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein MRP1, in two human hepatocarcinoma cell lines, HepG2 and Huh7. First, the cytotoxicity of the different copolymers (at different concentrations) on both liver carcinoma cell lines was thoroughly evaluated by means of apoptosis analysis using annexin V and propidium iodide (PI). Thus, viable cells (AV-/PI-), early apoptotic cells (AV+/PI-) and late apoptotic cells (V-FITC+/PI+) were identified. Results pointed out copolymers of intermediate to high hydrophobicity and intermediate molecular weight (e.g., T904) as the most cytotoxic. Then, DiOC2, rhodamine 123 and vinblastine were used as differential substrates of these pumps. HeLa, an epithelial cell line of human cervical cancer that does not express P-gp, was used exclusively as a control and enabled the discerning between P-gp and MRP1 inhibition. Moderate to highly hydrophobic poloxamines T304, T904 and T1301 showed inhibitory activity against P-gp and BCRP but not against MRP1 in both hepatic cell lines. A remarkable dependence of this effect on the

  1. 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

  2. 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

  3. Pathways of arsenic uptake and efflux.

    PubMed

    Yang, Hung-Chi; Fu, Hsueh-Liang; Lin, Yung-Feng; Rosen, Barry P

    2012-01-01

    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.

  4. RamA, which controls expression of the MDR efflux pump AcrAB-TolC, is regulated by the Lon protease

    PubMed Central

    Ricci, Vito; Blair, Jessica M. A.; Piddock, Laura J. V.

    2014-01-01

    Objectives RamA regulates the AcrAB-TolC multidrug efflux system. Using Salmonella Typhimurium, we investigated the stability of RamA and its impact on antibiotic resistance. Methods To detect RamA, we introduced ramA::3XFLAG::aph into plasmid pACYC184 and transformed this into Salmonella Typhimurium SL1344ramA::cat and lon::aph mutants. An N-terminus-deleted mutant [pACYC184ramA(Δ2-21)::3XFLAG::aph] in which the first 20 amino acids of RamA were deleted was also constructed. To determine the abundance and half-life of FLAG-tagged RamA, we induced RamA with chlorpromazine (50 mg/L) and carried out western blotting using anti-FLAG antibody. Susceptibility to antibiotics and phenotypic characterization of the lon mutant was also carried out. Results We show that on removal of chlorpromazine, a known inducer of ramA, the abundance of RamA decreased to pre-induced levels. However, in cells lacking functional Lon, we found that the RamA protein was not degraded. We also demonstrated that the 21 amino acid residues of the RamA N-terminus are required for recognition by the Lon protease. Antimicrobial susceptibility and phenotypic tests showed that the lon mutant was more susceptible to fluoroquinolone antibiotics, was filamentous when observed by microscopy and grew poorly, but showed no difference in motility or the ability to form a biofilm. There was also no difference in the ability of the lon mutant to invade human intestinal cells (INT-407). Conclusions In summary, we show that the ATP-dependent Lon protease plays an important role in regulating the expression of RamA and therefore multidrug resistance via AcrAB-TolC in Salmonella Typhimurium. PMID:24169580

  5. The Transcriptional Repressor, MtrR, of the mtrCDE Efflux Pump Operon of Neisseria gonorrhoeae Can Also Serve as an Activator of "off Target" Gene (glnE) Expression.

    PubMed

    Johnson, Paul J T; Shafer, William M

    2015-06-01

    MtrR is a well-characterized repressor of the Neisseria gonorrhoeae mtrCDE efflux pump operon. However, results from a previous transcriptional profiling study suggested that MtrR also represses or activates expression of at least sixty genes outside of the mtr locus. Evidence that MtrR can directly repress so-called "off target" genes has previously been reported; in particular, MtrR was shown to directly repress glnA, which encodes glutamine synthetase. In contrast, evidence for the ability of MtrR to directly activate expression of gonococcal genes has been lacking; herein, we provide such evidence. We now report that MtrR has the ability to directly activate expression of glnE, which encodes the dual functional adenyltransferase/deadenylase enzyme GlnE that modifies GlnA resulting in regulation of its role in glutamine biosynthesis. With its capacity to repress expression of glnA, the results presented herein emphasize the diverse and often opposing regulatory properties of MtrR that likely contributes to the overall physiology and metabolism of N. gonorrhoeae. PMID:26078871

  6. An attenuated mutant of the Rv1747 ATP-binding cassette transporter of Mycobacterium tuberculosis and a mutant of its cognate kinase, PknF, show increased expression of the efflux pump-related iniBAC operon

    PubMed Central

    Spivey, Vicky L; Whalan, Rachael H; Hirst, Elizabeth M A; Smerdon, Stephen J; Buxton, Roger S

    2013-01-01

    The ATP-binding cassette transporter Rv1747 is required for the growth of Mycobacterium tuberculosis in mice and in macrophages. Its structure suggests it is an exporter. Rv1747 forms a two-gene operon with pknF coding for the serine/threonine protein kinase PknF, which positively modulates the function of the transporter. We show that deletion of Rv1747 or pknF results in a number of transcriptional changes which could be complemented by the wild type allele, most significantly up-regulation of the iniBAC genes. This operon is inducible by isoniazid and ethambutol and by a broad range of inhibitors of cell wall biosynthesis and is required for efflux pump functioning. However, neither the Rv1747 or pknF mutant showed increased susceptibility to a range of drugs and cell wall stress reagents including isoniazid and ethambutol, cell wall structure and cell division appear normal by electron microscopy, and no differences in lipoarabinomannan were found. Transcription from the pknF promoter was not induced by a range of stress reagents. We conclude that the loss of Rv1747 affects cell wall biosynthesis leading to the production of intermediates that cause induction of iniBAC transcription and implicates it in exporting a component of the cell wall, which is necessary for virulence. PMID:23915284

  7. The role of ABC and SLC transporters in the pharmacokinetics of dietary and herbal phytochemicals and their interactions with xenobiotics.

    PubMed

    Li, Yan; Lu, Jun; Paxton, James W

    2012-06-01

    There is accumulating evidence that many compounds, known as phytochemicals (PCs), which are derived from dietary plants and herbs, may have a role in combating a number of chronic diseases. Despite many in vitro studies elucidating the mechanism(s) of action of various PCs, there are still reservations with regard to their health benefits in vivo, particularly as there is a paucity of research on their oral bioavailability, their pharmacokinetics, and the concentrations achieved at their site(s) of action. Recently various transporters, including the ATP-binding cassette (ABC) and the solute carrier (SLC) transporters, have been cloned and functional analyses have suggested that they play significant roles in the absorption and disposition of most drugs and PCs. While some SLC transporters facilitate absorption of PCs into the systemic circulation, various efflux pumps, including the ABC transporters, actively transport the PC back into the gastro-intestinal (GI) lumen, thus preventing further penetration into the body. Some ABC transporters also act in concert with Phase 1 and 2 metabolizing enzymes as a defensive barrier in the intestines and liver. If the PC overcomes the defence mechanisms of the gut and the liver, it will enter the systemic circulation and be distributed to the other organs of the body and possible site(s) of action. PCs can usually pass with ease through the pores of the capillaries of organs such as the heart and lungs, but with difficulty into pharmacological sanctuaries, such as the brain, testis, or foetus. Such sanctuaries contain a number of efflux transporters in their protective membrane, which restrict the penetration of xenobiotics, including PCs. The ABC and SLC transporters are also abundantly expressed in the liver and kidney and regulate the excretion of many compounds, including PCs and their metabolites. It is also becoming apparent that there is a complex interplay between various PCs and their ability to modulate the

  8. The Crystal Structure of the YknZ Extracellular Domain of ABC Transporter YknWXYZ from Bacillus amyloliquefaciens

    PubMed Central

    Wang, Lulu; Jiang, Rui; Jin, Xiaoling; Liu, Jing; Fan, Shengdi; Quan, Chun-Shan; Ha, Nam-Chul

    2016-01-01

    Bacillus possesses the peptide toxin Sporulation-Delaying Protein (SDP), which can kill cells within a biofilm to support continued growth, thereby delaying the onset of biofilm sporulation. The four-component transporter YknWXYZ acts as a major SDP efflux pump to protect cells against the endogenous SDP toxin, for which YknYZ is a non-canonical ATP-binding cassette (ABC)-type transporter. YknYZ consists of the following two components: (1) an individual protein (YknY) and (2) a respective permease (YknZ). To date, the crystal structure, molecular function, and mechanism of action of the integral membrane protein YknZ remain to be elucidated. In this study, to characterize the structural and biochemical roles of YknZ in the functional assembly of YknWXYZ, we predicted and overexpressed the YknZ extracellular domain. We determined the crystal structure of B. amyloliquefaciens YknZ at a resolution of 2.0 Å. The structure revealed that the YknZ extracellular region exhibits significant structural similarity with the MacB periplasmic domain, which is a non-canonical ABC-type transporter in the tripartite macrolide-specific efflux pump in Gram-negative bacteria. We also found that the YknZ extracellular domain can directly bind to an extracellular component of YknX. This structural and biochemical study provides insights into the assembly of YknWXYZ, which may be relevant to understanding cannibalistic peptide toxin resistance in Bacillus and controlling bacterial growth. PMID:27243566

  9. The Crystal Structure of the YknZ Extracellular Domain of ABC Transporter YknWXYZ from Bacillus amyloliquefaciens.

    PubMed

    Xu, Yongbin; Guo, Jianyun; Wang, Lulu; Jiang, Rui; Jin, Xiaoling; Liu, Jing; Fan, Shengdi; Quan, Chun-Shan; Ha, Nam-Chul

    2016-01-01

    Bacillus possesses the peptide toxin Sporulation-Delaying Protein (SDP), which can kill cells within a biofilm to support continued growth, thereby delaying the onset of biofilm sporulation. The four-component transporter YknWXYZ acts as a major SDP efflux pump to protect cells against the endogenous SDP toxin, for which YknYZ is a non-canonical ATP-binding cassette (ABC)-type transporter. YknYZ consists of the following two components: (1) an individual protein (YknY) and (2) a respective permease (YknZ). To date, the crystal structure, molecular function, and mechanism of action of the integral membrane protein YknZ remain to be elucidated. In this study, to characterize the structural and biochemical roles of YknZ in the functional assembly of YknWXYZ, we predicted and overexpressed the YknZ extracellular domain. We determined the crystal structure of B. amyloliquefaciens YknZ at a resolution of 2.0 Å. The structure revealed that the YknZ extracellular region exhibits significant structural similarity with the MacB periplasmic domain, which is a non-canonical ABC-type transporter in the tripartite macrolide-specific efflux pump in Gram-negative bacteria. We also found that the YknZ extracellular domain can directly bind to an extracellular component of YknX. This structural and biochemical study provides insights into the assembly of YknWXYZ, which may be relevant to understanding cannibalistic peptide toxin resistance in Bacillus and controlling bacterial growth. PMID:27243566

  10. 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.

  11. 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

  12. Fragment-Based Strategy for Investigating and Suppressing the Efflux of Bioactive Small Molecules.

    PubMed

    Compton, Corey L; Carney, Daniel W; Groomes, Patrice V; Sello, Jason K

    2015-01-01

    Membrane protein-mediated drug efflux is a phenomenon that compromises our ability to treat both infectious diseases and cancer. Accordingly, there is much interest in the development of strategies for suppression of the mechanisms by which therapeutic agents are effluxed. Here, using resistance to the cyclic acyldepsipeptide (ADEP) antibacterial agents as a model, we demonstrate a new counter-efflux strategy wherein a fragment of an actively exported bioactive compound competitively interferes with its efflux and potentiates its activity. A fragment comprising the N-heptenoyldifluorophenylalanine side chain of the pharmacologically optimized ADEPs potentiates the antibacterial activity of the ADEPs against actinobacteria to a greater extent than reserpine, a well-known efflux inhibitor. Beyond their validation of a new approach to studying molecular recognition by drug efflux pumps, our findings have important implications for killing Mycobacterium tuberculosis with ADEPs and reclaiming the efficacies of therapeutic agents whose activity has been compromised by efflux pumps. PMID:27620145

  13. 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

    Sato, Toyotaka; Yokota, Shin-ichi; Ichihashi, Risa; Miyauchi, Tomoka; Okubo, Torahiko; Usui, Masaru; Fujii, Nobuhiro; Tamura, Yutaka

    2014-07-01

    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.

  14. 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

  15. Identification of a new biocontrol gene in Trichoderma atroviride: the role of an ABC transporter membrane pump in the interaction with different plant-pathogenic fungi.

    PubMed

    Ruocco, Michelina; Lanzuise, Stefania; Vinale, Francesco; Marra, Roberta; Turrà, David; Woo, Sheridan Lois; Lorito, Matteo

    2009-03-01

    Successful biocontrol interactions often require that the beneficial microbes involved are resistant or tolerant to a variety of toxicants, including antibiotics produced by themselves or phytopathogens, plant antimicrobial compounds, and synthetic chemicals or contaminants. The ability of Trichoderma spp., the most widely applied biocontrol fungi, to withstand different chemical stresses, including those associated with mycoparasitism, is well known. In this work, we identified an ATP-binding cassette transporter cell membrane pump as an important component of the above indicated resistance mechanisms that appears to be supported by an extensive and powerful cell detoxification system. The encoding gene, named Taabc2, was cloned from a strain of Trichoderma atroviride and characterized. Its expression was found to be upregulated in the presence of pathogen-secreted metabolites, specific mycotoxins and some fungicides, and in conditions that stimulate the production in Trichoderma spp. of antagonism-related factors (toxins and enzymes). The key role of this gene in antagonism and biocontrol was demonstrated by the characterization of the obtained deletion mutants. They suffered an increased susceptibility to inhibitory compounds either secreted by pathogenic fungi or possibly produced by the biocontrol microbe itself and lost, partially or entirely, the ability to protect tomato plants from Pythium ultimum and Rhizoctonia solani attack.

  16. Efflux Systems in Bacteria and their Metabolic Engineering Applications

    PubMed Central

    Jones, Christopher M.; Hernández Lozada, Néstor J.; Pfleger, Brian F.

    2015-01-01

    The production of valuable chemicals from metabolically engineered microbes can be limited by excretion from the cell. Efflux is often overlooked as a bottleneck in metabolic pathways, despite its impact on alleviating feedback inhibition and product toxicity. In the past, it has been assumed that endogenous efflux pumps and membrane porins can accommodate product efflux rates, however, there are an increasing number of examples wherein overexpressing efflux systems is required to improve metabolite production. In this review, we highlight specific examples from the literature where metabolite export has been studied to identify unknown transporters, increase tolerance to metabolites, and improve the production capabilities of engineered bacteria. The review focuses on the export of a broad spectrum of valuable chemicals including amino acids, sugars, flavins, biofuels and solvents. The combined set of examples supports the hypothesis that efflux systems can be identified and engineered to confer export capabilities on industrially relevant microbes. PMID:26363557

  17. Control of Angiogenesis by AIBP-mediated Cholesterol Efflux

    PubMed Central

    Fang, Longhou; Choi, Soo-Ho; Baek, Ji Sun; Liu, Chao; Almazan, Felicidad; Ulrich, Florian; Wiesner, Philipp; Taleb, Adam; Deer, Elena; Pattison, Jennifer; Torres-Vázquez, Jesús; Li, Andrew C.; Miller, Yury I.

    2013-01-01

    Cholesterol is a structural component of the cell, indispensable for normal cellular function, but its excess often leads to abnormal proliferation, migration, inflammatory responses and/or cell death. To prevent cholesterol overload, ATP-binding cassette (ABC) transporters mediate cholesterol efflux from the cells to apolipoprotein A-I (ApoA-I) and to the ApoA-I-containing high-density lipoprotein (HDL)1-3. Maintaining efficient cholesterol efflux is essential for normal cellular function4-6. However, the role of cholesterol efflux in angiogenesis and the identity of its local regulators are poorly understood. Here we show that ApoA-I binding protein (AIBP) accelerates cholesterol efflux from endothelial cells (EC) to HDL and thereby regulates angiogenesis. AIBP/HDL-mediated cholesterol depletion reduces lipid rafts, interferes with VEGFR2 dimerization and signaling, and inhibits VEGF-induced angiogenesis in vitro and mouse aortic neovascularization ex vivo. Remarkably, Aibp regulates the membrane lipid order in embryonic zebrafish vasculature and functions as a non-cell autonomous regulator of zebrafish angiogenesis. Aibp knockdown results in dysregulated sprouting/branching angiogenesis, while forced Aibp expression inhibits angiogenesis. Dysregulated angiogenesis is phenocopied in Abca1/Abcg1-deficient embryos, and cholesterol levels are increased in Aibp-deficient and Abca1/Abcg1-deficient embryos. Our findings demonstrate that secreted AIBP positively regulates cholesterol efflux from EC and that effective cholesterol efflux is critical for proper angiogenesis. PMID:23719382

  18. ABC transporters: bacterial exporters.

    PubMed Central

    Fath, M J; Kolter, R

    1993-01-01

    The ABC transporters (also called traffic ATPases) make up a large superfamily of proteins which share a common function and a common ATP-binding domain. ABC transporters are classified into three major groups: bacterial importers (the periplasmic permeases), eukaryotic transporters, and bacterial exporters. We present a comprehensive review of the bacterial ABC exporter group, which currently includes over 40 systems. The bacterial ABC exporter systems are functionally subdivided on the basis of the type of substrate that each translocates. We describe three main groups: protein exporters, peptide exporters, and systems that transport nonprotein substrates. Prototype exporters from each group are described in detail to illustrate our current understanding of this protein family. The prototype systems include the alpha-hemolysin, colicin V, and capsular polysaccharide exporters from Escherichia coli, the protease exporter from Erwinia chrysanthemi, and the glucan exporters from Agrobacterium tumefaciens and Rhizobium meliloti. Phylogenetic analysis of the ATP-binding domains from 29 bacterial ABC exporters indicates that the bacterial ABC exporters can be divided into two primary branches. One branch contains the transport systems where the ATP-binding domain and the membrane-spanning domain are present on the same polypeptide, and the other branch contains the systems where these domains are found on separate polypeptides. Differences in substrate specificity do not correlate with evolutionary relatedness. A complete survey of the known and putative bacterial ABC exporters is included at the end of the review. PMID:8302219

  19. Placental ABC transporters, cellular toxicity and stress in pregnancy.

    PubMed

    Aye, Irving L M H; Keelan, Jeffrey A

    2013-04-25

    The human placenta, in addition to its roles as a nutrient transfer and endocrine organ, functions as a selective barrier to protect the fetus against the harmful effects of exogenous and endogenous toxins. Members of the ATP-binding cassette (ABC) family of transport proteins limit the entry of xenobiotics into the fetal circulation via vectorial efflux from the placenta to the maternal circulation. Several members of the ABC family, including proteins from the ABCA, ABCB, ABCC and ABCG subfamilies, have been shown to be functional in the placenta with clinically significant roles in xenobiotic efflux. However, recent findings suggest that these transporters also protect placental tissue by preventing the cellular accumulation of cytotoxic compounds such as lipids, sterols and their derivatives. Such protective functions are likely to be particularly important in pregnancies complicated by inflammatory or oxidative stress, where the generation of toxic metabolites is enhanced. For example, ABC transporters have been shown to protect against the harmful effects of hypoxia and oxidative stress through increased expression and efflux of oxysterols and glutathione conjugated xenobiotics. However, this protective capacity may be diminished in response to the same stressors. Several studies in primary human trophoblast cells and animal models have demonstrated decreased expression and activity of placental ABC transporters with inflammatory, oxidative or metabolic stress. Several clinical studies in pregnancies complicated by inflammatory conditions such as preeclampsia and gestational diabetes support these findings, although further studies are required to determine the clinical relevance of the relationships between placental ABC transporter expression and activity, and placental function in stressed pregnancies. Such studies are necessary to fully understand the consequences of pregnancy disorders on placental function and viability in order to optimise pregnancy

  20. Thermodynamics of ABC transporters.

    PubMed

    Zhang, Xuejun C; Han, Lei; Zhao, Yan

    2016-01-01

    ABC transporters form the largest of all transporter families, and their structural study has made tremendous progress over recent years. However, despite such advances, the precise mechanisms that determine the energy-coupling between ATP hydrolysis and the conformational changes following substrate binding remain to be elucidated. Here, we present our thermodynamic analysis for both ABC importers and exporters, and introduce the two new concepts of differential-binding energy and elastic conformational energy into the discussion. We hope that the structural analysis of ABC transporters will henceforth take thermodynamic aspects of transport mechanisms into account as well.

  1. Boosting Effect of 2-Phenylquinoline Efflux Inhibitors in Combination with Macrolides against Mycobacterium smegmatis and Mycobacterium avium.

    PubMed

    Machado, Diana; Cannalire, Rolando; Santos Costa, Sofia; Manfroni, Giuseppe; Tabarrini, Oriana; Cecchetti, Violetta; Couto, Isabel; Viveiros, Miguel; Sabatini, Stefano

    2015-12-11

    The identification of efflux inhibitors to be used as adjuvants alongside existing drug regimens could have a tremendous value in the treatment of any mycobacterial infection. Here, we investigated the ability of four 2-(4'-propoxyphenyl)quinoline Staphylococcus aureus NorA efflux inhibitors (1-4) to reduce the efflux activity in Mycobacterium smegmatis and Mycobacterium avium strains. All four compounds were able to inhibit efflux pumps in both mycobacterial species; in particular, O-ethylpiperazinyl derivative 2 showed an efflux inhibitory activity comparable to that of verapamil, the most potent mycobacterial efflux inhibitor reported to date, and was able to significantly reduce the MIC values of macrolides against different M. avium strains. The contribution of the M. avium efflux pumps MAV_1406 and MAV_1695 to clarithromycin resistance was proved because they were found to be overexpressed in two M. avium 104 isogenic strains showing high-level clarithromycin resistance. These results indicated a correlation between increased expression of efflux pumps, increased efflux, macrolide resistance, and reduction of resistance by efflux pump inhibitors such as compound 2. Additionally, compound 2 showed synergistic activity with clarithromycin, at a concentration below the cytotoxicity threshold, in an ex vivo experiment against M. avium 104-infected macrophages. In summary, the 2-(4'-propoxyphenyl)quinoline scaffold is suitable to obtain compounds endowed with good efflux pump inhibitory activity against both S. aureus and nontuberculous mycobacteria. PMID:27623057

  2. PET and SPECT Radiotracers to Assess Function and Expression of ABC Transporters in Vivo

    PubMed Central

    Mairinger, Severin; Erker, Thomas; Müller, Markus; Langer, Oliver

    2013-01-01

    Adenosine triphosphate-binding cassette (ABC) transporters, such as P-glycoprotein (Pgp, ABCB1), breast cancer resistance protein (BCRP, ABCG2) and multidrug resistance-associated proteins (MRPs) are expressed in high concentrations at various physiological barriers (e.g. blood-brain barrier, blood-testis barrier, blood-tumor barrier), where they impede the tissue accumulation of various drugs by active efflux transport. Changes in ABC transporter expression and function are thought to be implicated in various diseases, such as cancer, epilepsy, Alzheimer’s and Parkinson’s disease. The availability of a non-invasive imaging method which allows for measuring ABC transporter function or expression in vivo would be of great clinical use in that it could facilitate the identification of those patients that would benefit from treatment with ABC transporter modulating drugs. To date three different kinds of imaging probes have been described to measure ABC transporters in vivo: i) radiolabelled transporter substrates ii) radiolabelled transporter inhibitors and iii) radiolabelled prodrugs which are enzymatically converted into transporter substrates in the organ of interest (e.g. brain). The design of new imaging probes to visualize efflux transporters is inter alia complicated by the overlapping substrate recognition pattern of different ABC transporter types. The present article will describe currently available ABC transporter radiotracers for positron emission tomography (PET) and single-photon emission computed tomography (SPECT) and critically discuss strengths and limitations of individual probes and their potential clinical applications. PMID:21434859

  3. The function of the ATP-binding cassette (ABC) transporter ABCB1 is not susceptible to actin disruption.

    PubMed

    Meszaros, Peter; Hummel, Ina; Klappe, Karin; Draghiciu, Oana; Hoekstra, Dick; Kok, Jan W

    2013-02-01

    Previously we have shown that the activity of the multidrug transporter ABCC1 (multidrug resistance protein 1), and its localization in lipid rafts, depends on cortical actin (Hummel I, Klappe K, Ercan C, Kok JW. Mol. Pharm. 2011 79, 229-40). Here we show that the efflux activity of the ATP-binding cassette (ABC) family member ABCB1 (P-glycoprotein), did not depend on actin, neither in ABCB1 over expressing murine National Institutes of Health (NIH) 3T3 MDR1 G185 cells nor in human SK-N-FI cells, which endogenously express ABCB1. Disruption of the actin cytoskeleton, upon treatment of the cells with latrunculin B or cytochalasin D, caused severe changes in cell and membrane morphology, and concomitant changes in the subcellular distribution of ABCB1, as revealed by confocal laser scanning and electron microscopy. Nevertheless, irrespective of actin perturbation, the cell surface pool of ABCB1 remained unaltered. In NIH 3T3 MDR1 G185 cells, ABCB1 is partly localized in detergent-free lipid rafts, which partitioned in two different density gradient regions, both enriched in cholesterol and sphingolipids. Interestingly, disruption of the actin cytoskeleton did not change the density gradient distribution of ABCB1. Our data demonstrate that the functioning of ABCB1 as an efflux pump does not depend on actin, which is due to its distribution in both cell surface-localized non-raft membrane areas and lipid raft domains, which do not depend on actin stabilization.

  4. 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...

  5. Control of Plasma Membrane Permeability by ABC Transporters

    PubMed Central

    Khakhina, Svetlana; Johnson, Soraya S.; Manoharlal, Raman; Russo, Sarah B.; Blugeon, Corinne; Lemoine, Sophie; Sunshine, Anna B.; Dunham, Maitreya J.; Cowart, L. Ashley; Devaux, Frédéric

    2014-01-01

    ATP-binding cassette transporters Pdr5 and Yor1 from Saccharomyces cerevisiae control the asymmetric distribution of phospholipids across the plasma membrane as well as serving as ATP-dependent drug efflux pumps. Mutant strains lacking these transporter proteins were found to exhibit very different resistance phenotypes to two inhibitors of sphingolipid biosynthesis that act either late (aureobasidin A [AbA]) or early (myriocin [Myr]) in the pathway leading to production of these important plasma membrane lipids. These pdr5Δ yor1 strains were highly AbA resistant but extremely sensitive to Myr. We provide evidence that these phenotypic changes are likely due to modulation of the plasma membrane flippase complexes, Dnf1/Lem3 and Dnf2/Lem3. Flippases act to move phospholipids from the outer to the inner leaflet of the plasma membrane. Genetic analyses indicate that lem3Δ mutant strains are highly AbA sensitive and Myr resistant. These phenotypes are fully epistatic to those seen in pdr5Δ yor1 strains. Direct analysis of AbA-induced signaling demonstrated that loss of Pdr5 and Yor1 inhibited the AbA-triggered phosphorylation of the AGC kinase Ypk1 and its substrate Orm1. Microarray experiments found that a pdr5Δ yor1 strain induced a Pdr1-dependent induction of the entire Pdr regulon. Our data support the view that Pdr5/Yor1 negatively regulate flippase function and activity of the nuclear Pdr1 transcription factor. Together, these data argue that the interaction of the ABC transporters Pdr5 and Yor1 with the Lem3-dependent flippases regulates permeability of AbA via control of plasma membrane protein function as seen for the high-affinity tryptophan permease Tat2. PMID:25724885

  6. The Cus efflux system removes toxic ions via a methionine shuttle.

    PubMed

    Su, Chih-Chia; Long, Feng; Yu, Edward W

    2011-01-01

    Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes in the resistance-nodulation-cell division (RND) family to expel diverse toxic compounds from the cell. These efflux systems span the entire cell envelope to mediate the phenomenon of bacterial multidrug resistance. The three parts of the efflux complexes are: (1) a membrane fusion protein (MFP) connecting (2) a substrate-binding inner membrane transporter to (3) an outer membrane-anchored channel in the periplasmic space. One such efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions. We recently determined the crystal structures of both the inner membrane transporter CusA and MFP CusB of the CusCBA tripartite efflux system from E. coli. These are the first structures of the heavy-metal efflux (HME) subfamily of the RND efflux pumps. Here, we summarize the structural information of these two efflux proteins and present the accumulated evidence that this efflux system utilizes methionine residues to bind and export Cu(I)/Ag(I). Genetic and structural analyses suggest that the CusA pump is capable of picking up the metal ions from both the periplasm and cytoplasm. We propose a stepwise shuttle mechanism for this pump to extrude metal ions from the cell.

  7. Tunicamycin depresses P-glycoprotein glycosylation without an effect on its membrane localization and drug efflux activity in L1210 cells.

    PubMed

    Sereš, 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.

  8. 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

  9. ABC's of Being Smart

    ERIC Educational Resources Information Center

    Foster, Joanne

    2011-01-01

    Determining what giftedness is all about means focusing on many aspects of the individual. In this paper, the author focuses on letter D of the ABC's of being smart. She starts with specifics about giftedness (details), and then moves on to some ways of thinking (dispositions).

  10. Development of chitosan-SLN microparticles for chemotherapy: in vitro approach through efflux-transporter modulation.

    PubMed

    Dharmala, Kiran; Yoo, Jin Wook; Lee, Chi H

    2008-11-12

    Drug efflux-transporters serve as a major barrier to anticancer drugs at the target site. One strategy to enhance the therapeutic efficacy of drugs against cancer is to increase their available concentrations at the target site by suppressing or modulating efflux-transporters. This manuscript deals with the development and evaluation of the particle type drug delivery system made of stearic acid (Solid Lipid Nanoparticle - SLN) and chitosan for the delivery of Phenethyl Isothiocyanate (PEITC), a tumor-suppressive agent, through the pulmonary route. The rationale behind the particle type drug delivery system involves a prior release of the efflux-transporter inhibitors, such as tamoxifen, verapamil HCl or nifedipine, to suppress or modulate the efflux activity of ABC transporters followed by the release of the efflux-transporter substrate, PEITC. The efficacy of Chitosan-SLN Microparticles (CSM) as a carrier for PEITC was evaluated by investigating the release profiles of PEITC loaded in CSM and its cytotoxicity in the presence or absence of the efflux-transporter inhibitors. An initial burst release of the inhibitors, followed by gradual, sustained release of PEITC and subsequent increase in cytotoxicity was observed. This finding indicated that the efflux transporter inhibitors significantly affected the PEITC uptake rate by Calu-3 cells. Judging from these results, CSM can be an efficient drug delivery system for the substrates susceptible to the efflux-transporters. PMID:18723057

  11. Multidrug resistance-associated ABC transporters - too much of one thing, good for nothing.

    PubMed

    Prochazkova, Jirina; Lanova, Martina; Pachernik, Jiri

    2012-08-01

    Abstract Overexpression of ATP-binding cassette (ABC) transporters in cancer cells results in multidrug resistance (MDR) which leads to unsuccessful chemotherapy. The most important MDR-associated members of ABC superfamily are ABC B1/P-glycoprotein/MDR1, ABC C1/multidrug resistance associated protein 1 (MRP1), and ABC G2/BCRP. This study is not only focused on function, substrates, and localization of these popular proteins but also on other ABC C family members such as ABC C2-6/MRP2-6 and ABC C7/CFTR. Current research is mainly oriented on the cancer-promoting role of these proteins, but important lessons could also be learned from the physiological roles of these proteins or from polymorphisms affecting their function. Thorough knowledge of structure and detailed mechanism of efflux can aid in the discovery of new chemotherapy targets in the future. Although the best way on how to deal with MDR would be to prevent its development, we describe some new promising strategies on how to conquer both inherited and induced MDRs.

  12. Secondary Metabolites from Plants Inhibiting ABC Transporters and Reversing Resistance of Cancer Cells and Microbes to Cytotoxic and Antimicrobial Agents

    PubMed Central

    Wink, Michael; Ashour, Mohamed L.; El-Readi, Mahmoud Zaki

    2012-01-01

    Fungal, bacterial, and cancer cells can develop resistance against antifungal, antibacterial, or anticancer agents. Mechanisms of resistance are complex and often multifactorial. Mechanisms include: (1) Activation of ATP-binding cassette (ABC) transporters, such as P-gp, which pump out lipophilic compounds that have entered a cell, (2) Activation of cytochrome p450 oxidases which can oxidize lipophilic agents to make them more hydrophilic and accessible for conjugation reaction with glucuronic acid, sulfate, or amino acids, and (3) Activation of glutathione transferase, which can conjugate xenobiotics. This review summarizes the evidence that secondary metabolites (SM) of plants, such as alkaloids, phenolics, and terpenoids can interfere with ABC transporters in cancer cells, parasites, bacteria, and fungi. Among the active natural products several lipophilic terpenoids [monoterpenes, diterpenes, triterpenes (including saponins), steroids (including cardiac glycosides), and tetraterpenes] but also some alkaloids (isoquinoline, protoberberine, quinoline, indole, monoterpene indole, and steroidal alkaloids) function probably as competitive inhibitors of P-gp, multiple resistance-associated protein 1, and Breast cancer resistance protein in cancer cells, or efflux pumps in bacteria (NorA) and fungi. More polar phenolics (phenolic acids, flavonoids, catechins, chalcones, xanthones, stilbenes, anthocyanins, tannins, anthraquinones, and naphthoquinones) directly inhibit proteins forming several hydrogen and ionic bonds and thus disturbing the 3D structure of the transporters. The natural products may be interesting in medicine or agriculture as they can enhance the activity of active chemotherapeutics or pesticides or even reverse multidrug resistance, at least partially, of adapted and resistant cells. If these SM are applied in combination with a cytotoxic or antimicrobial agent, they may reverse resistance in a synergistic fashion. PMID:22536197

  13. Exploring the contribution of efflux on the resistance to fluoroquinolones in clinical isolates of Escherichia coli.

    PubMed

    Paltansing, Sunita; Tengeler, Anouk C; Kraakman, Margriet E M; Claas, Eric C J; Bernards, Alexandra T

    2013-12-01

    Resistance to ciprofloxacin in Escherichia coli is increasing parallel to increased use of fluoroquinolones both in The Netherlands and in other European countries. The objective was to investigate the contribution of active efflux and expression of outer membrane proteins (OMPs) in a collection of clinical E. coli isolates collected at a clinical microbiology department in a Dutch hospital. Forty-seven E. coli isolates a wide range of ciprofloxacin minimum inhibitory concentrations and known mutations in the quinolone resistance determining region were included. A fluorometric determination of bisbenzimide efflux was used two different efflux pump inhibitors and compared to quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for the expression levels of acrA, acrB, tolC, yhiV, and mdfA efflux pump genes and the OMPs ompF and ompX. Six isolates (12.7%) showed increased efflux. Although in 35 isolates (76%), overexpression of ≥1 efflux pump genes using qRT-PCR was present. Only the combined overexpression of acrAB-TolC and mdfA correlated with the phenotypic efflux assay using glucose/carbonyl cyanide m-chlorophenylhydrazone with glucose. Thus, efflux was involved in ciprofloxacin resistance in a limited number of E. coli isolates collected at a clinical microbiology department in a Dutch hospital complementing other resistance mechanisms.

  14. The challenge of efflux-mediated antibiotic resistance in Gram-negative bacteria.

    PubMed

    Li, Xian-Zhi; Plésiat, Patrick; Nikaido, Hiroshi

    2015-04-01

    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.

  15. 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

  16. 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

  17. Structural diversity of ABC transporters

    PubMed Central

    ter Beek, Josy; Guskov, Albert

    2014-01-01

    ATP-binding cassette (ABC) transporters form a large superfamily of ATP-dependent protein complexes that mediate transport of a vast array of substrates across membranes. The 14 currently available structures of ABC transporters have greatly advanced insight into the transport mechanism and revealed a tremendous structural diversity. Whereas the domains that hydrolyze ATP are structurally related in all ABC transporters, the membrane-embedded domains, where the substrates are translocated, adopt four different unrelated folds. Here, we review the structural characteristics of ABC transporters and discuss the implications of this structural diversity for mechanistic diversity. PMID:24638992

  18. Overcoming ABC transporter-mediated multidrug resistance: Molecular mechanisms and novel therapeutic drug strategies.

    PubMed

    Li, Wen; Zhang, Han; Assaraf, Yehuda G; Zhao, Kun; Xu, Xiaojun; Xie, Jinbing; Yang, Dong-Hua; Chen, Zhe-Sheng

    2016-07-01

    Multidrug resistance is a key determinant of cancer chemotherapy failure. One of the major causes of multidrug resistance is the enhanced efflux of drugs by membrane ABC transporters. Targeting ABC transporters projects a promising approach to eliminating or suppressing drug resistance in cancer treatment. To reveal the functional mechanisms of ABC transporters in drug resistance, extensive studies have been conducted from identifying drug binding sites to elucidating structural dynamics. In this review article, we examined the recent crystal structures of ABC proteins to depict the functionally important structural elements, such as domains, conserved motifs, and critical amino acids that are involved in ATP-binding and drug efflux. We inspected the drug-binding sites on ABC proteins and the molecular mechanisms of various substrate interactions with the drug binding pocket. While our continuous battle against drug resistance is far from over, new approaches and technologies have emerged to push forward our frontier. Most recent developments in anti-MDR strategies include P-gp inhibitors, RNA-interference, nano-medicines, and delivering combination strategies. With the advent of the 'Omics' era - genomics, epigenomics, transcriptomics, proteomics, and metabolomics - these disciplines play an important role in fighting the battle against chemoresistance by further unraveling the molecular mechanisms of drug resistance and shed light on medical therapies that specifically target MDR. PMID:27449595

  19. Overcoming ABC transporter-mediated multidrug resistance: Molecular mechanisms and novel therapeutic drug strategies.

    PubMed

    Li, Wen; Zhang, Han; Assaraf, Yehuda G; Zhao, Kun; Xu, Xiaojun; Xie, Jinbing; Yang, Dong-Hua; Chen, Zhe-Sheng

    2016-07-01

    Multidrug resistance is a key determinant of cancer chemotherapy failure. One of the major causes of multidrug resistance is the enhanced efflux of drugs by membrane ABC transporters. Targeting ABC transporters projects a promising approach to eliminating or suppressing drug resistance in cancer treatment. To reveal the functional mechanisms of ABC transporters in drug resistance, extensive studies have been conducted from identifying drug binding sites to elucidating structural dynamics. In this review article, we examined the recent crystal structures of ABC proteins to depict the functionally important structural elements, such as domains, conserved motifs, and critical amino acids that are involved in ATP-binding and drug efflux. We inspected the drug-binding sites on ABC proteins and the molecular mechanisms of various substrate interactions with the drug binding pocket. While our continuous battle against drug resistance is far from over, new approaches and technologies have emerged to push forward our frontier. Most recent developments in anti-MDR strategies include P-gp inhibitors, RNA-interference, nano-medicines, and delivering combination strategies. With the advent of the 'Omics' era - genomics, epigenomics, transcriptomics, proteomics, and metabolomics - these disciplines play an important role in fighting the battle against chemoresistance by further unraveling the molecular mechanisms of drug resistance and shed light on medical therapies that specifically target MDR.

  20. Exploring the contribution of efflux on the resistance to fluoroquinolones in clinical isolates of Staphylococcus aureus

    PubMed Central

    2011-01-01

    Background Antimicrobial resistance mediated by efflux systems is still poorly characterized in Staphylococcus aureus, despite the description of several efflux pumps (EPs) for this bacterium. In this work we used several methodologies to characterize the efflux activity of 52 S. aureus isolates resistant to ciprofloxacin collected in a hospital in Lisbon, Portugal, in order to understand the role played by these systems in the resistance to fluoroquinolones. Results Augmented efflux activity was detected in 12 out of 52 isolates and correlated with increased resistance to fluoroquinolones. Addition of efflux inhibitors did not result in the full reversion of the fluoroquinolone resistance phenotype, yet it implied a significant decrease in the resistance levels, regardless of the type(s) of mutation(s) found in the quinolone-resistance determining region of grlA and gyrA genes, which accounted for the remaining resistance that was not efflux-mediated. Expression analysis of the genes coding for the main efflux pumps revealed increased expression only in the presence of inducing agents. Moreover, it showed that not only different substrates can trigger expression of different EP genes, but also that the same substrate can promote a variable response, according to its concentration. We also found isolates belonging to the same clonal type that showed different responses towards drug exposure, thus evidencing that highly related clinical isolates may diverge in the efflux-mediated response to noxious agents. The data gathered by real-time fluorometric and RT-qPCR assays suggest that S. aureus clinical isolates may be primed to efflux antimicrobial compounds. Conclusions The results obtained in this work do not exclude the importance of mutations in resistance to fluoroquinolones in S. aureus, yet they underline the contribution of efflux systems for the emergence of high-level resistance. All together, the results presented in this study show the potential role

  1. Do You Know Your ABC?

    ERIC Educational Resources Information Center

    Neale, Claire

    2013-01-01

    Within primary schools, the core subjects of literacy and numeracy are highly regarded, and rightly so, as children need to learn to read, write and be numerically literate. This means that all children learn their ABCs at an early age, But, what about the "other ABC"--"Airway, Breathing and Circulation?" Accidents and medical…

  2. ABC Technology Development Program

    SciTech Connect

    1994-10-14

    The Accelerator-Based Conversion (ABC) facility will be designed to accomplish the following mission: `Provide a weapon`s grade plutonium disposition capability in a safe, economical, and environmentally sound manner on a prudent schedule for [50] tons of weapon`s grade plutonium to be disposed on in [20] years.` This mission is supported by four major objectives: provide a reliable plutonium disposition capability within the next [15] years; provide a level of safety and of safety assurance that meets or exceeds that afforded to the public by modern commercial nuclear power plants; meet or exceed all applicable federal, state, and local regulations or standards for environmental compliance; manage the program in a cost effective manner. The ABC Technology Development Program defines the technology development activities that are required to accomplish this mission. The technology development tasks are related to the following topics: blanket system; vessel systems; reactivity control systems; heat transport system components; energy conversion systems; shutdown heat transport systems components; auxiliary systems; technology demonstrations - large scale experiments.

  3. A Survey of the ATP-Binding Cassette (ABC) Gene Superfamily in the Salmon Louse (Lepeophtheirus salmonis)

    PubMed Central

    Heumann, Jan; Taggart, John B.; Gharbi, Karim; Bron, James E.; Bekaert, Michaël; Sturm, Armin

    2015-01-01

    Salmon lice, Lepeophtheirus salmonis (Krøyer, 1837), are fish ectoparasites causing significant economic damage in the mariculture of Atlantic salmon, Salmo salar Linnaeus, 1758. The control of L. salmonis at fish farms relies to a large extent on treatment with anti-parasitic drugs. A problem related to chemical control is the potential for development of resistance, which in L. salmonis is documented for a number of drug classes including organophosphates, pyrethroids and avermectins. The ATP-binding cassette (ABC) gene superfamily is found in all biota and includes a range of drug efflux transporters that can confer drug resistance to cancers and pathogens. Furthermore, some ABC transporters are recognised to be involved in conferral of insecticide resistance. While a number of studies have investigated ABC transporters in L. salmonis, no systematic analysis of the ABC gene family exists for this species. This study presents a genome-wide survey of ABC genes in L. salmonis for which, ABC superfamily members were identified through homology searching of the L. salmonis genome. In addition, ABC proteins were identified in a reference transcriptome of the parasite generated by high-throughput RNA sequencing (RNA-seq) of a multi-stage RNA library. Searches of both genome and transcriptome allowed the identification of a total of 33 genes / transcripts coding for ABC proteins, of which 3 were represented only in the genome and 4 only in the transcriptome. Eighteen sequences were assigned to ABC subfamilies known to contain drug transporters, i.e. subfamilies B (4 sequences), C (11) and G (2). The results suggest that the ABC gene family of L. salmonis possesses fewer members than recorded for other arthropods. The present survey of the L. salmonis ABC gene superfamily will provide the basis for further research into potential roles of ABC transporters in the toxicity of salmon delousing agents and as potential mechanisms of drug resistance. PMID:26418738

  4. Inflammatory Regulation of ATP Binding Cassette Efflux Transporter Expression and Function in Microglia

    PubMed Central

    Gibson, Christopher J.; Hossain, Muhammad M.; Richardson, Jason R.

    2012-01-01

    ATP-binding cassette (ABC) efflux transporters, including multidrug resistance protein 1 (Mdr1), breast cancer resistance protein (Bcrp), and multidrug resistance-associated proteins (Mrps) extrude chemicals from the brain. Although ABC transporters are critical for blood-brain barrier integrity, less attention has been placed on the regulation of these proteins in brain parenchymal cells such as microglia. Prior studies demonstrate that inflammation after lipopolysaccharide (LPS) treatment alters transporter expression in the livers of mice. Here, we sought to determine the effects of inflammation on the expression and function of transporters in microglia. To test this, the expression and function of ABC efflux transport proteins were quantified in mouse BV-2 microglial cells in response to activation with LPS. Intracellular retention of fluorescent rhodamine 123, Hoechst 33342, and calcein acetoxymethyl ester was increased in LPS-treated microglia, suggesting that the functions of Mdr1, Bcrp, and Mrps were decreased, respectively. LPS reduced Mdr1, Bcrp, and Mrp4 mRNA and protein expression between 40 and 70%. Conversely, LPS increased expression of Mrp1 and Mrp5 mRNA and protein. Immunofluorescent staining confirmed reduced Bcrp and Mrp4 and elevated Mrp1 and Mrp5 protein in activated microglia. Pharmacological inhibition of nuclear factor κB (NF-κB) transcriptional signaling attenuated down-regulation of Mdr1a mRNA and potentiated up-regulation of Mrp5 mRNA in LPS-treated cells. Together, these data suggest that LPS stimulates microglia and impairs efflux of prototypical ABC transporter substrates by altering mRNA and protein expression, in part through NF-κB signaling. Decreased transporter efflux function in microglia may lead to the retention of toxic chemicals and aberrant cell-cell communication during neuroinflammation. PMID:22942241

  5. Putative mycobacterial efflux inhibitors from the seeds of Aframomum melegueta.

    PubMed

    Gröblacher, Barbara; Maier, Veronika; Kunert, Olaf; Bucar, Franz

    2012-07-27

    In order to identify new putative efflux pump inhibitors that represent an appropriate target in antimycobacterial chemotherapy, nine paradol- and gingerol-related compounds (1-9) isolated from the seeds of Aframomum melegueta were assessed for their potential to inhibit ethidium bromide (EtBr) efflux in a Mycobacterium smegmatis model. Five of the compounds from A. melegueta and NMR spectroscopic data of the diketone 6-gingerdione (2) and its enolic tautomers, methyl-6-gingerol (5) and rac-6-dihydroparadol (7), are presented herein for the first time. After determination of their antimycobacterial activities and modulatory effects on the MIC of antibiotics as well as their synergistic effects in combination with antibiotics against M. smegmatis mc(2) 155, their impact on EtBr accumulation and efflux was evaluated using a microtiter plate-based fluorometric assay. The compounds exhibited moderate to weak antimycobacterial activities, and the best modulators induced a 4- to 16-fold decrease of the MICs of EtBr and rifampicin as well as a reduction of the MIC of isoniazid with fractional inhibitory concentration index values indicating synergistic activities in some cases. 6-Paradol (3), 8-gingerol (6), and rac-6-dihydroparadol (7) were the most potent EtBr efflux inhibitors in M. smegmatis mc(2) 155, displaying EtBr efflux inhibiting activities comparable to reference inhibitors.

  6. [Efflux systems in Serratia marcescens].

    PubMed

    Mardanova, A M; Bogomol'naia, L M; Romanova, Iu D; Sharipova, M R

    2014-01-01

    A widespread bacterium Serratia marcescens (family Enterobacteriaceae) is an opportunistic and exhibits multiple drug resistance. Active removal of antibiotics and other antimicrobials from pathogen and exhibits multiple drug resistance. Active removal of antibiotics and other antimicrobials from the cells by efflux systems is one of the mechanisms responsible for microbial resistance to these compounds. Among enterobacteria, efflux systems of Escherichia coli and Salmonella enterica var. Typhimurium have been studied most extensively. Few efflux systems that belong to different families have been reported for S. marcescens. In this review, we analyzed available literature about S. marcescens efflux systems and carried out the comparative analysis of the genes encoding the RND type systems in different Serratia species and in other enterobacteria. Bioinformatical analysis of the S. marcescens genome allowed us to identify the previously unknown efflux systems based on their homology with the relevant E. coli genes. Identification of additional efflux systems in S. marcescens genome will promote our understanding of physiology of these bacteria, will detect new molecular mechanisms of resistance and will reveal their resistance potential.

  7. Multidrug resistance in parasites: ABC transporters, P-glycoproteins and molecular modelling.

    PubMed

    Jones, P M; George, A M

    2005-04-30

    Parasitic diseases, caused by protozoa, helminths and arthropods, rank among the most important problems in human and veterinary medicine, and in agriculture, leading to debilitating sicknesses and loss of life. In the absence of vaccines and with the general failure of vector eradication programs, drugs are the main line of defence, but the newest drugs are being tracked by the emergence of resistance in parasites, sharing ominous parallels with multidrug resistance in bacterial pathogens. Any of a number of mechanisms will elicit a drug resistance phenotype in parasites, including: active efflux, reduced uptake, target modification, drug modification, drug sequestration, by-pass shunting, or substrate competition. The role of ABC transporters in parasitic multidrug resistance mechanisms is being subjected to more scrutiny, due in part to the established roles of certain ABC transporters in human diseases, and also to an increasing portfolio of ABC transporters from parasite genome sequencing projects. For example, over 100 ABC transporters have been identified in the Escherichia coli genome, but to date only about 65 in all parasitic genomes. Long established laboratory investigations are now being assisted by molecular biology, bioinformatics, and computational modelling, and it is in these areas that the role of ABC transporters in parasitic multidrug resistance mechanisms may be defined and put in perspective with that of other proteins. We discuss ABC transporters in parasites, and conclude with an example of molecular modelling that identifies a new interaction between the structural domains of a parasite P-glycoprotein. PMID:15826647

  8. ABC-F Proteins Mediate Antibiotic Resistance through Ribosomal Protection

    PubMed Central

    Sharkey, Liam K. R.; Edwards, Thomas A.

    2016-01-01

    ABSTRACT Members of the ABC-F subfamily of ATP-binding cassette proteins mediate resistance to a broad array of clinically important antibiotic classes that target the ribosome of Gram-positive pathogens. The mechanism by which these proteins act has been a subject of long-standing controversy, with two competing hypotheses each having gained considerable support: antibiotic efflux versus ribosomal protection. Here, we report on studies employing a combination of bacteriological and biochemical techniques to unravel the mechanism of resistance of these proteins, and provide several lines of evidence that together offer clear support to the ribosomal protection hypothesis. Of particular note, we show that addition of purified ABC-F proteins to an in vitro translation assay prompts dose-dependent rescue of translation, and demonstrate that such proteins are capable of displacing antibiotic from the ribosome in vitro. To our knowledge, these experiments constitute the first direct evidence that ABC-F proteins mediate antibiotic resistance through ribosomal protection. PMID:27006457

  9. Essential letters in the fungal alphabet: ABC and MFS transporters and their roles in survival and pathogenicity.

    PubMed

    Perlin, Michael H; Andrews, Jared; Toh, Su San

    2014-01-01

    Fungi depend heavily on their ability to exploit resources that may become available to them in their myriad of possible lifestyles. Whether this requires simple uptake of sugars as saprobes or competition for host-derived carbohydrates or peptides, fungi must rely on transporters that effectively allow the fungus to accumulate such nutrients from their environments. In other cases, fungi secrete compounds that facilitate their interactions with potential hosts and/or neutralize their competition. Finally, fungi that find themselves on the receiving end of insults, from hosts, competitors, or the overall environment are better served if they can get rid of such toxins or xenobiotics. In this chapter, we update studies on the most ubiquitous transporters, the ABC and MFS superfamilies. In addition, we discuss the importance of subsets of these proteins with particular relevance to plant pathogenic fungi. The availability of ever-increasing numbers of sequenced fungal genomes, combined with high-throughput methods for transcriptome analysis, provides insights previously inaccessible prior to the -omics era. As examples of such broader perspectives, we point to revelations about exploitive use of sugar transporters by plant pathogens, expansion of trichothecene efflux pumps in fungi that do not produce these mycotoxins, and the discovery of a fungal-specific oligopeptide transporter class that, so far, is overrepresented in the plant pathogenic fungi.

  10. Development, Maintenance, and Reversal of Multiple Drug Resistance: At the Crossroads of TFPI1, ABC Transporters, and HIF1α

    PubMed Central

    Arnason, Terra; Harkness, Troy

    2015-01-01

    Early detection and improved therapies for many cancers are enhancing survival rates. Although many cytotoxic therapies are approved for aggressive or metastatic cancer; response rates are low and acquisition of de novo resistance is virtually universal. For decades; chemotherapeutic treatments for cancer have included anthracyclines such as Doxorubicin (DOX); and its use in aggressive tumors appears to remain a viable option; but drug resistance arises against DOX; as for all other classes of compounds. Our recent work suggests the anticoagulant protein Tissue Factor Pathway Inhibitor 1α (TFPI1α) plays a role in driving the development of multiple drug resistance (MDR); but not maintenance; of the MDR state. Other factors; such as the ABC transporter drug efflux pumps MDR-1/P-gp (ABCB1) and BCRP (ABCG2); are required for MDR maintenance; as well as development. The patient population struggling with therapeutic resistance specifically requires novel treatment options to resensitize these tumor cells to therapy. In this review we discuss the development, maintenance, and reversal of MDR as three distinct phases of cancer biology. Possible means to exploit these stages to reverse MDR will be explored. Early molecular detection of MDR cancers before clinical failure has the potential to offer new approaches to fighting MDR cancer. PMID:26501324

  11. ABC transporters as multidrug resistance mechanisms and the development of chemosensitizers for their reversal

    PubMed Central

    Choi, Cheol-Hee

    2005-01-01

    One of the major problems related with anticancer chemotherapy is resistance against anticancer drugs. The ATP-binding cassette (ABC) transporters are a family of transporter proteins that are responsible for drug resistance and a low bioavailability of drugs by pumping a variety of drugs out cells at the expense of ATP hydrolysis. One strategy for reversal of the resistance of tumor cells expressing ABC transporters is combined use of anticancer drugs with chemosensitizers. In this review, the physiological functions and structures of ABC transporters, and the development of chemosensitizers are described focusing on well-known proteins including P-glycoprotein, multidrug resistance associated protein, and breast cancer resistance protein. PMID:16202168

  12. The ABCs of Sex Ed.

    ERIC Educational Resources Information Center

    Sroka, Stephen R.

    2002-01-01

    Cites statistics on extent of sexually transmitted diseases and pregnancies among adolescents; describes ideological dispute over how to teach sex education; advocates teaching the ABCs of sex education: Abstinence, Be Monogamous, and Condoms. (PKP)

  13. 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.

  14. Regulation of ATP-binding cassette transporters and cholesterol efflux by glucose in primary human monocytes and murine bone marrow-derived macrophages

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Individuals with type 2 diabetes mellitus are at increased risk of developing atherosclerosis. This may be partially attributable to suppression of macrophage ATP-binding cassette (ABC) transporter mediated cholesterol efflux by sustained elevated blood glucose concentrations. Two models were used...

  15. Tissue-Specific Transcript Profiling for ABC Transporters in the Sequestering Larvae of the Phytophagous Leaf Beetle Chrysomela populi

    PubMed Central

    Gretscher, René R.; Groth, Marco; Boland, Wilhelm; Burse, Antje

    2014-01-01

    Background Insects evolved ingenious adaptations to use extraordinary food sources. Particularly, the diet of herbivores enriched with noxious plant secondary metabolites requires detoxification mechanisms. Sequestration, which involves the uptake, transfer, and concentration of occasionally modified phytochemicals into specialized tissues or hemolymph, is one of the most successful detoxification strategies found in most insect orders. Due to the ability of ATP-binding cassette (ABC) carriers to transport a wide range of molecules including phytochemicals and xenobiotics, it is highly likely that they play a role in this sequestration process. To shed light on the role of ABC proteins in sequestration, we describe an inventory of putative ABC transporters in various tissues in the sequestering juvenile poplar leaf beetle, Chrysomela populi. Results In the transcriptome of C. populi, we predicted 65 ABC transporters. To link the proteins with a possible function, we performed comparative phylogenetic analyses with ABC transporters of other insects and of humans. While tissue-specific profiling of each ABC transporter subfamily suggests that ABCB, C and G influence the plant metabolite absorption in the gut, ABCC with 14 members is the preferred subfamily responsible for the excretion of these metabolites via Malpighian tubules. Moreover, salicin, which is sequestered from poplar plants, is translocated into the defensive glands for further deterrent production. In these glands and among all identified ABC transporters, an exceptionally high transcript level was observed only for Cpabc35 (Cpmrp). RNAi revealed the deficiency of other ABC pumps to compensate the function of CpABC35, demonstrating its key role during sequestration. Conclusion We provide the first comprehensive phylogenetic study of the ABC family in a phytophagous beetle species. RNA-seq data from different larval tissues propose the importance of ABC pumps to achieve a homeostasis of plant

  16. Ontogeny of ABC and SLC transporters in the microvessels of developing rat brain.

    PubMed

    Soares, Ricardo V; Do, Tuan M; Mabondzo, Aloïse; Pons, Gérard; Chhun, Stéphanie

    2016-04-01

    The blood-brain barrier (BBB) is responsible for the control of solutes' concentration in the brain. Tight junctions and multiple ATP-binding cassette (ABC) and SoLute Carrier (SLC) efflux transporters protect brain cells from xenobiotics, therefore reducing brain exposure to intentionally administered drugs. In epilepsy, polymorphisms and overexpression of efflux transporters genes could be associated with pharmacoresistance. The ontogeny of these efflux transporters should also be addressed because their expression during development may be related to different brain exposure to antiepileptic drugs in the immature brain. We detected statistically significant higher expression of Abcb1b and Slc16a1 genes, and lower expression of Abcb1a and Abcg2 genes between the post-natal day 14 (P14) and the adult rat microvessels. P-gP efflux activity was also shown to be lower in P14 rats when compared with the adults. The P-gP proteins coded by rodent genes Abcb1a and Abcb1b are known to have different substrate affinities. The role of the Abcg2 gene is less clear in pharmacoresistance in epilepsy, nonetheless the coded protein Bcrp is frequently associated with drug resistance. Finally, we observed a higher expression of the Mct1 transporter gene in the P14 rat brain microvessels. Accordingly to our results, we suppose that age may be another factor influencing brain exposure to antiepileptics as a consequence of different expression patterns of efflux transporters between the adult and immature BBB.

  17. The ABCs of Student Engagement

    ERIC Educational Resources Information Center

    Parsons, Seth A.; Nuland, Leila Richey; Parsons, Allison Ward

    2014-01-01

    Student engagement is an important consideration for teachers and administrators because it is explicitly associated with achievement. What the authors call the ABC's of engagement they outline as: Affective engagement, Behavioral engagement, and Cognitive engagement. They also present "Three Things Every Teacher Needs to Know about…

  18. 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.

  19. Effects of toxicologically relevant xenobiotics and the lipid-derived electrophile 4-hydroxynonenal on macrophage cholesterol efflux: silencing carboxylesterase 1 has paradoxical effects on cholesterol uptake and efflux.

    PubMed

    Ross, Matthew K; Borazjani, Abdolsamad; Mangum, Lee C; Wang, Ran; Crow, J Allen

    2014-10-20

    Cholesterol cycles between free cholesterol (unesterified) found predominantly in membranes and cholesteryl esters (CEs) stored in cytoplasmic lipid droplets. Only free cholesterol is effluxed from macrophages via ATP-binding cassette (ABC) transporters to extracellular acceptors. Carboxylesterase 1 (CES1), proposed to hydrolyze CEs, is inactivated by oxon metabolites of organophosphorus pesticides and by the lipid electrophile 4-hydroxynonenal (HNE). We assessed the ability of these compounds to reduce cholesterol efflux from foam cells. Human THP-1 macrophages were loaded with [(3)H]-cholesterol/acetylated LDL and then allowed to equilibrate to enable [(3)H]-cholesterol to distribute into its various cellular pools. The cholesterol-engorged cells were then treated with toxicants in the absence of cholesterol acceptors for 24 h, followed by a 24 h efflux period in the presence of toxicant. A concentration-dependent reduction in [(3)H]-cholesterol efflux via ABCA1 (up to 50%) was found for paraoxon (0.1-10 μM), whereas treatment with HNE had no effect. A modest reduction in [(3)H]-cholesterol efflux via ABCG1 (25%) was found after treatment with either paraoxon or chlorpyrifos oxon (10 μM each) but not HNE. No difference in efflux rates was found after treatments with either paraoxon or HNE when the universal cholesterol acceptor 10% (v/v) fetal bovine serum was used. When the re-esterification arm of the CE cycle was disabled in foam cells, paraoxon treatment increased CE levels, suggesting the neutral CE hydrolysis arm of the cycle had been inhibited by the toxicant. However, paraoxon also partially inhibited lysosomal acid lipase, which generates cholesterol for efflux, and reduced the expression of ABCA1 protein. Paradoxically, silencing CES1 expression in macrophages did not affect the percent of [(3)H]-cholesterol efflux. However, CES1 mRNA knockdown markedly reduced cholesterol uptake by macrophages, with SR-A and CD36 mRNA reduced 3- and 4-fold

  20. Rapid quantification of murine ABC mRNAs by real time reverse transcriptase-polymerase chain reaction.

    PubMed

    Su, Yan Ru; Linton, MacRae F; Fazio, Sergio

    2002-12-01

    Several ATP-binding cassette (ABC) transporters are critically involved in cholesterol and phospholipid efflux, reverse cholesterol transport, and play an important role in the development of atherosclerosis. Quantification of ABC mRNA is important in studying the regulation of cellular cholesterol homeostasis and mechanisms related to the pathogenesis of atherosclerosis. We have developed a one-step real time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method for measuring mRNA levels of ABCA1, ABCG1, and ABCA2 in murine tissues using the TaqMan(TM) technology. It has significant methodological benefits when compared to classic Northern blotting or semi-quantitative RT-PCR analysis. Using this method, we found high expression levels of ABCA1 in liver and macrophages, and of ABCG1 in the brain and macrophages. The expression of ABCA1 and ABCG1 were further induced in macrophages loaded with acLDL. In contrast, ABCA2 was expressed exclusively in the brain with low expression levels in the macrophages. This method provides a rapid, highly sensitive, specific, and reproducible quantification of ABC mRNA, and can be performed with nanograms of total RNA sample, thus making it a superior method for studying the regulation of ABC transporters in cholesterol efflux and its role in the pathogenesis of atherosclerosis in murine models.

  1. Accumulation and efflux of polychlorinated biphenyls in Escherichia coli.

    PubMed

    Geng, Shen; Fang, Jun; Turner, Kendrick B; Daunert, Sylvia; Wei, Yinan

    2012-06-01

    Polychlorinated biphenyls (PCBs) are environmental pollutants that have been associated with numerous adverse health effects in human and animals. Hydroxylated PCBs (HPCBs) are the product of the oxidative metabolism of PCBs. The presence of hydroxyl groups in HPCBs makes these compounds more hydrophilic than the parent PCBs. One of the best approaches to break down and remove these contaminants is bioremediation; an environmentally friendly process that uses microorganisms to degrade hazardous chemicals into non-toxic ones. In this study, we investigated the cellular accumulation and toxicity of selected PCBs and HPCBs in Gram-negative bacteria, using Escherichia coli as a model organism. We found that none of the five PCBs tested were toxic to E. coli, presumably due to their limited bioavailability. Nevertheless, different HPCBs tested showed different levels of toxicity. Furthermore, we demonstrated that the primary multidrug efflux system in E. coli, AcrAB-TolC, facilitated the efflux of HPCBs out of the cell. Since AcrAB-TolC is constitutively expressed in E. coli and is conserved in all sequenced Gram-negative bacterial genomes, our results suggest that the efflux activities of multidrug resistant pumps may affect the accumulation and degradation of PCBs in Gram-negative bacteria.

  2. Calcium Efflux from Barnacle Muscle Fibers

    PubMed Central

    Russell, J. M.; Blaustein, M. P.

    1974-01-01

    Calcium-45 was injected into single giant barnacle muscle fibers, and the rate of efflux was measured under a variety of conditions. The rate constant (k) for 45Ca efflux into standard seawater averaged 17 x 10–4 min–1 which corresponds to an efflux of about 1–2 pmol/cm2·s. Removal of external Ca (Cao) reduced the efflux by 50%. In most fibers about 40% of the 45Ca efflux into Ca-free seawater was dependent on external Na (Nao); treatment with 3.5 mM caffeine increased the magnitude of the Nao-dependent efflux. In a few fibers removal of Nao, in the absence of Cao, either had no effect or increased k; caffeine (2–3.5 mM) unmasked an Nao-dependent efflux in these fibers. The Nao-dependent Ca efflux had a Q10 of about 3.7. The data are consistent with the idea that a large fraction of the Ca efflux may be carrier-mediated, and may involve both Ca-Ca and Na-Ca counterflow. The relation between the Nao-dependent Ca efflux and the external Na concentration is sigmoid, and suggests that two, or more likely three, external Na+ ions may activate the efflux of one Ca+2. With a three-for-one Na-Ca exchange, the Na electrochemical gradient may be able to supply sufficient energy to maintain the Ca gradient in these fibers. Other, more complex models are not excluded, however, and may be required to explain some puzzling features of the Ca efflux such as the variable Nao-dependence. PMID:4812633

  3. Mapping the functional yeast ABC transporter interactome

    PubMed Central

    Snider, Jamie; Hanif, Asad; Lee, Mid Eum; Jin, Ke; Yu, Analyn R.; Graham, Chris; Chuk, Matthew; Damjanovic, Dunja; Wierzbicka, Marta; Tang, Priscilla; Balderes, Dina; Wong, Victoria; Jessulat, Matthew; Darowski, Katelyn D.; Luis, Bryan-Joseph San; Shevelev, Igor; Sturley, Stephen L; Boone, Charles; Greenblatt, Jack F.; Zhang, Zhaolei; Paumi, Christian M.; Babu, Mohan; Park, Hay-Oak; Michaelis, Susan; Stagljar, Igor

    2013-01-01

    ABC transporters are a ubiquitous class of integral membrane proteins of immense clinical interest because of their strong association with human disease and pharmacology. To improve our understanding of these proteins, we used Membrane Yeast Two-Hybrid (MYTH) technology to map the protein interactome of all non-mitochondrial ABC transporters in the model organism Saccharomy cescerevisiae, and combined this data with previously reported yeast ABC transporter interactions in the BioGRID database to generate a comprehensive, integrated interactome. We show that ABC transporters physically associate with proteins involved in a surprisingly diverse range of functions. We specifically examine the importance of the physical interactions of ABC transporters in both the regulation of one another and in the modulation of proteins involved in zinc homeostasis. The interaction network presented here will be a powerful resource for increasing our fundamental understanding of the cellular role and regulation of ABC transporters. PMID:23831759

  4. 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

  5. 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.

  6. Sodium efflux from perfused giant algal cells.

    PubMed

    Clint, G M; Macrobbie, E A

    1987-06-01

    Internodal cells of the giant alga Chara corallina were perfused internally to replace the native cytoplasm, tonoplast and vacuole with artificial cytoplasm. Sodium efflux from perfused cells, measured by including (22)Na in the perfusion media, was increased by increasing the internal sodium concentration and by decreasing the external pH, and was inhibited by external application of the renal diuretic amiloride. The sodium efflux was markedly ATP-dependent, with a 50-fold decrease in efflux observed after perfusion with media lacking ATP. Efflux in the presence of ATP was reduced by 33% by inclusion of 10 μM N,N'-dicyclohexylcarbodiimide in the perfusion medium. The membrane potential of the perfused cells approximated that of intact cells from the same culture. It is suggested that sodium efflux in perfused Chara cells proceeds via a secondary antiporter with protons, regulated by ATP in a catalytic role and with the proton motive force acting as the energy source.

  7. The arsenical ATPase efflux pump mediates tellurite resistance.

    PubMed

    Turner, R J; Hou, Y; Weiner, J H; Taylor, D E

    1992-05-01

    The ars operon of the resistance plasmid R773 was found to produce moderate levels of resistance to tellurite. A MIC of 64 micrograms of TeO3(2-) per ml was found for Escherichia coli cells harboring plasmids which contained all three of the structural genes (arsA, arsB, and arsC) of the anion-translocating ATPase. MICs specified by plasmids carrying only one or two structural elements or the cloning vector alone were 2 to 4 micrograms/ml. The rate of TeO3(2-) uptake was found to be on the order of 55% less for cultures containing the resistance plasmids.

  8. ABC-Transporter Expression Does Not Correlate with Response to Irinotecan in Patients with Metastatic Colorectal Cancer

    PubMed Central

    Trumpi, K.; Emmink, B.L.; Prins, A.M.; van Oijen, M.G.H.; van Diest, P.J.; Punt, C.J.A.; Koopman, M.; Kranenburg, O.; Rinkes, I.H.M. Borel

    2015-01-01

    Background: Active efflux of irinotecan by ATP-binding cassette (ABC)-transporters, in particular ABCB1 and ABCG2, is a well-established drug resistance mechanism in vitro and in pre-clinical mouse models, but its relevance in colorectal cancer (CRC) patients is unknown. Therefore, we assessed the association between ABC-transporter expression and tumour response to irinotecan in patients with metastatic CRC. Methods: Tissue microarrays of a large cohort of metastatic CRC patients treated with irinotecan in a prospective study (CAIRO study; n=566) were analysed for expression of ABCB1 and ABCG2 by immunohistochemistry. Kaplan-Meier and Cox proportional hazard regression analyses were performed to assess the association of ABC transporter expression with irinotecan response. Gene expression profiles of 17 paired tumours were used to assess the concordance of ABCB1/ABCG2 expression in primary CRC and corresponding metastases. Results: The response to irinotecan was not significantly different between primary tumours with positive versus negative expression of ABCB1 (5.8 vs 5.7 months, p=0.696) or ABCG2 (5.7 vs 6.1 months, p=0.811). Multivariate analysis showed neither ABCB1 nor ABCG2 were independent predictors for progression free survival. There was a mediocre to poor concordance between ABC-transporter expression in paired tumours. Conclusion: In metastatic CRC, ABC-transporter expression in the primary tumour does not predict irinotecan response. PMID:26516354

  9. The ABC transporters in Candidatus Liberibacter asiaticus

    PubMed Central

    Li, Wenlin; Cong, Qian; Pei, Jimin; Kinch, Lisa N; Grishin, Nick V

    2012-01-01

    Candidatus Liberibacter asiaticus (Ca. L. asiaticus) is a Gram-negative bacterium and the pathogen of Citrus Greening disease (Huanglongbing, HLB). As a parasitic bacterium, Ca. L. asiaticus harbors ABC transporters that play important roles in exchanging chemical compounds between Ca. L. asiaticus and its host. Here, we analyzed all the ABC transporter-related proteins in Ca. L. asiaticus. We identified 14 ABC transporter systems and predicted their structures and substrate specificities. In-depth sequence and structure analysis including multiple sequence alignment, phylogenetic tree reconstruction, and structure comparison further support their function predictions. Our study shows that this bacterium could use these ABC transporters to import metabolites (amino acids and phosphates) and enzyme cofactors (choline, thiamine, iron, manganese, and zinc), resist to organic solvent, heavy metal, and lipid-like drugs, maintain the composition of the outer membrane (OM), and secrete virulence factors. Although the features of most ABC systems could be deduced from the abundant experimental data on their orthologs, we reported several novel observations within ABC system proteins. Moreover, we identified seven nontransport ABC systems that are likely involved in virulence gene expression regulation, transposon excision regulation, and DNA repair. Our analysis reveals several candidates for further studies to understand and control the disease, including the type I virulence factor secretion system and its substrate that are likely related to Ca. L. asiaticus pathogenicity and the ABC transporter systems responsible for bacterial OM biosynthesis that are good drug targets. PMID:22807026

  10. On the physics of multidrug efflux through a biomolecular complex

    NASA Astrophysics Data System (ADS)

    Mishima, Hirokazu; Oshima, Hiraku; Yasuda, Satoshi; Amano, Ken-ichi; Kinoshita, Masahiro

    2013-11-01

    focused on the entropic component. We show that the entropically inserted solute can be released by a continuous variation of the vessel geometry which forms a time-dependent entropic force continuing to accelerate the solute motion to the exit. Solutes with a wide range of sizes are entropically released using the same vessel-geometry variation. The results obtained are fairly general and also applicable to the efflux pump protein AcrB and ATP-binding cassette transporter.

  11. 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

  12. Bioluminescent imaging of drug efflux at the blood-brain barrier mediated by the transporter ABCG2.

    PubMed

    Bakhsheshian, Joshua; Wei, Bih-Rong; Chang, Ki-Eun; Shukla, Suneet; Ambudkar, Suresh V; Simpson, R Mark; Gottesman, Michael M; Hall, Matthew D

    2013-12-17

    ATP-binding cassette (ABC) transporters are a group of transmembrane proteins that maintain chemical homeostasis through efflux of compounds out of organelles and cells. Among other functions, ABC transporters play a key role in protecting the brain parenchyma by efflux of xenobiotics from capillary endothelial cells at the blood-brain barrier (BBB). They also prevent the entry of therapeutic drugs at the BBB, thereby limiting their efficacy. One of the key transporters playing this role is ABCG2. Although other ABC transporters can be studied through various imaging modalities, no specific probe exists for imaging ABCG2 function in vivo. Here we show that D-luciferin, the endogenous substrate of firefly luciferase, is a specific substrate for ABCG2. We hypothesized that ABCG2 function at the BBB could be evaluated by using bioluminescence imaging in transgenic mice expressing firefly luciferase in the brain. Bioluminescence signal in the brain of mice increased with coadministration of the ABCG2 inhibitors Ko143, gefitinib, and nilotinib, but not an ABCB1 inhibitor. This method for imaging ABCG2 function at the BBB will facilitate understanding of the function and pharmacokinetic inhibition of this transporter.

  13. 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

  14. 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

  15. 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.

  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. Fibroblast growth factor 21 enhances cholesterol efflux in THP-1 macrophage-derived foam cells.

    PubMed

    Shang, Wei; Yu, Xuejing; Wang, Honglian; Chen, Tielin; Fang, Ying; Yang, Xianggui; Zhou, Puhui; Nie, Fang; Zhou, Qin; Zhou, Jianzhong

    2015-01-01

    Fibroblast growth factor 21 (FGF21) is a novel metabolic regulator. The present study aimed to investigate the effect of FGF21 on cholesterol efflux and the expression of ATP binding cassette (ABC) A1 and G1 in human THP-1 macrophage-derived foam cells. Furthermore, the present study aimed to investigate the role of the liver X receptor (LXR) α in this process. A model of oxidized low-density lipoprotein-induced foam cells from human THP-1 cells was established. The effect of FGF21 on cholesterol efflux was analyzed using a liquid scintillation counter. The expression of ABCA1 and ABCG1 was determined using quantitative polymerase chain reaction and western blot analyses. FGF21 was found to enhance apolipoprotein A1- and high-density lipoprotein-mediated cholesterol efflux. FGF21 was also observed to increase the mRNA and protein expression of ABCA1 and ABCG1. Furthermore, LXRα-short interfering RNA attenuated the stimulatory effects induced by FGF21. These findings suggest that FGF21 may have a protective effect against atherosclerosis by enhancing cholesterol efflux through the induction of LXRα-dependent ABCA1 and ABCG1 expression.

  18. Fibroblast growth factor 21 enhances cholesterol efflux in THP-1 macrophage-derived foam cells.

    PubMed

    Shang, Wei; Yu, Xuejing; Wang, Honglian; Chen, Tielin; Fang, Ying; Yang, Xianggui; Zhou, Puhui; Nie, Fang; Zhou, Qin; Zhou, Jianzhong

    2015-01-01

    Fibroblast growth factor 21 (FGF21) is a novel metabolic regulator. The present study aimed to investigate the effect of FGF21 on cholesterol efflux and the expression of ATP binding cassette (ABC) A1 and G1 in human THP-1 macrophage-derived foam cells. Furthermore, the present study aimed to investigate the role of the liver X receptor (LXR) α in this process. A model of oxidized low-density lipoprotein-induced foam cells from human THP-1 cells was established. The effect of FGF21 on cholesterol efflux was analyzed using a liquid scintillation counter. The expression of ABCA1 and ABCG1 was determined using quantitative polymerase chain reaction and western blot analyses. FGF21 was found to enhance apolipoprotein A1- and high-density lipoprotein-mediated cholesterol efflux. FGF21 was also observed to increase the mRNA and protein expression of ABCA1 and ABCG1. Furthermore, LXRα-short interfering RNA attenuated the stimulatory effects induced by FGF21. These findings suggest that FGF21 may have a protective effect against atherosclerosis by enhancing cholesterol efflux through the induction of LXRα-dependent ABCA1 and ABCG1 expression. PMID:25334019

  19. An ABC for decision making.

    PubMed

    Garcia, Luiz Henrique Costa; Ferreira, Bruna Cortez

    2015-01-01

    The present study was aimed at proposing a systematic evaluation of cranial computed tomography, identifying the main aspects to be analyzed in order to facilitate the decision making process regarding diagnosis and management in emergency settings. The present descriptive study comprised a literature review at the following databases: Access Medicine and Access Emergency Medicine (McGraw- Hill Education); British Medical Journal Evidence Center; UptoDate; Bireme; PubMed; Lilacs; SciELO; ProQuest; Micromedex (Thomson Reuters); Embase. Once the literature review was completed, the authors identified the main diseases with tomographic repercussions and proposed the present system to evaluate cranial computed tomography images. An easy-to-memorize ABC system will facilitate the decision making in emergency settings, as it covers the main diseases encountered by intensivists and emergency physicians, and provides a sequential guidance about anatomical structures to be investigated as well as their respective alterations.

  20. An ABC for decision making*

    PubMed Central

    Garcia, Luiz Henrique Costa; Ferreira, Bruna Cortez

    2015-01-01

    The present study was aimed at proposing a systematic evaluation of cranial computed tomography, identifying the main aspects to be analyzed in order to facilitate the decision making process regarding diagnosis and management in emergency settings. The present descriptive study comprised a literature review at the following databases: Access Medicine and Access Emergency Medicine (McGraw- Hill Education); British Medical Journal Evidence Center; UptoDate; Bireme; PubMed; Lilacs; SciELO; ProQuest; Micromedex (Thomson Reuters); Embase. Once the literature review was completed, the authors identified the main diseases with tomographic repercussions and proposed the present system to evaluate cranial computed tomography images. An easy-to-memorize ABC system will facilitate the decision making in emergency settings, as it covers the main diseases encountered by intensivists and emergency physicians, and provides a sequential guidance about anatomical structures to be investigated as well as their respective alterations. PMID:25987751

  1. Helicobacter pylori uptake and efflux: basis for intrinsic susceptibility to antibiotics in vitro.

    PubMed

    Bina, J E; Alm, R A; Uria-Nickelsen, M; Thomas, S R; Trust, T J; Hancock, R E

    2000-02-01

    We previously demonstrated (M. M. Exner, P. Doig, T. J. Trust, and R. E. W. Hancock, Infect. Immun. 63:1567-1572, 1995) that Helicobacter pylori has at least one nonspecific porin, HopE, which has a low abundance in the outer membrane but forms large channels. H. pylori is relatively susceptible to most antimicrobial agents but less susceptible to the polycationic antibiotic polymyxin B. We demonstrate here that H. pylori is able to take up higher basal levels of the hydrophobic fluorescent probe 1-N-phenylnaphthylamine (NPN) than Pseudomonas aeruginosa or Escherichia coli, consistent with its enhanced susceptibility to hydrophobic agents. Addition of polymyxin B led to a further increase in NPN uptake, indicative of a self-promoted uptake pathway, but it required a much higher amount of polymyxin B to yield a 50% increase in NPN uptake in H. pylori (6 to 8 microg/ml) than in P. aeruginosa or E. coli (0.3 to 0.5 microg/ml), suggesting that H. pylori has a less efficient self-promoted uptake pathway. Since intrinsic resistance involves the collaboration of restricted outer membrane permeability and secondary defense mechanisms, such as periplasmic beta-lactamase (which H. pylori lacks) or efflux, we examined the possible role of efflux in antibiotic susceptibility. We had previously identified in H. pylori 11637 the presence of portions of three genes with homology to potential restriction-nodulation-division (RND) efflux systems. It was confirmed that H. pylori contained only these three putative RND efflux systems, named here hefABC, hefDEF, and hefGHI, and that the hefGHI system was expressed only in vivo while the two other RND systems were expressed both in vivo and in vitro. In uptake studies, there was no observable energy-dependent tetracycline, chloramphenicol, or NPN efflux activity in H. pylori. Independent mutagenesis of the three putative RND efflux operons in the chromosome of H. pylori had no effect on the in vitro susceptibility of H. pylori to 19

  2. Identification of ABC transporters in Sarcoptes scabiei.

    PubMed

    Mounsey, K E; Holt, D C; McCarthy, J; Walton, S F

    2006-06-01

    We have identified and partially sequenced 8 ABC transporters from an EST dataset of Sarcoptes scabiei var. hominis, the causative agent of scabies. Analysis confirmed that most of the known ABC subfamilies are represented in the EST dataset including several members of the multidrug resistance protein subfamily (ABC-C). Although P-glycoprotein (ABC-B) sequences were not found in the EST dataset, a partial P-glycoprotein sequence was subsequently obtained using a degenerate PCR strategy and library screening. Thus a total of 9 potential S. scabiei ABC transporters representing the subfamilies A, B, C, E, F and H have been identified. Ivermectin is currently used in the treatment of hyper-infested (crusted) scabies, and has also been identified as a potentially effective acaricide for mass treatment programmes in scabies-endemic communities. The observation of clinical and in vitro ivermectin resistance in 2 crusted scabies patients who received multiple treatments has raised serious concerns regarding the sustainability of such programmes. One possible mechanism for ivermectin resistance is through ABC transporters such as P-glycoprotein. This work forms an important foundation for further studies to elucidate the potential role of ABC transporters in ivermectin resistance of S. scabiei.

  3. Spectinamides: A New Class of Semisynthetic Anti-Tuberculosis Agents that Overcome Native Drug Efflux

    PubMed Central

    Vaddady, Pavan K; Zheng, Zhong; Qi, Jianjun; Akbergenov, Rashid; Das, Sourav; Madhura, Dora B.; Rathi, Chetan; Trivedi, Ashit; Villellas, Cristina; Lee, Robin. B.; Rakesh; Waidyarachchi, Samanthi L.; Sun, Dianqing; McNeil, Michael R.; Ainsa, Jose A.; Boshoff, Helena I.; Gonzalez-Juarrero, Mercedes

    2014-01-01

    Although the classical antibiotic spectinomycin is a potent bacterial protein synthesis inhibitor, poor antimycobacterial activity limits its clinical application for treating tuberculosis. Using structure-based design, a novel semisynthetic series of spectinomycin analogs was generated with selective ribosomal inhibition and excellent narrow-spectrum antitubercular activity. In multiple murine infection models, these spectinamides were well tolerated, significantly reduced lung mycobacterial burden and increased survival. In vitro studies demonstrated a lack of cross-resistance with existing tuberculosis therapeutics, activity against MDR/XDR-tuberculosis, and an excellent pharmacological profile. Key to their potent antitubercular properties was their structural modification to evade the Rv1258c efflux pump, which is upregulated in MDR strains and is implicated in macrophage induced drug tolerance. The antitubercular efficacy of spectinamides demonstrates that synthetic modifications to classical antibiotics can overcome the challenge of intrinsic efflux pump-mediated resistance and expands opportunities for target based tuberculosis drug discovery. PMID:24464186

  4. Inactivation of MuxABC-OpmB transporter system in Pseudomonas aeruginosa leads to increased ampicillin and carbenicillin resistance and decreased virulence.

    PubMed

    Yang, Liang; Chen, Lin; Shen, Lixin; Surette, Michael; Duan, Kangmin

    2011-02-01

    Resistance-Nodulation-Cell Division (RND) pumps play important roles in bacterial resistance to antibiotics. Pseudomonas aeruginosa is an important human pathogen which exhibits high level resistance to antibiotics. There are total of 12 RND pumps present in the P. aeruginosa PAOl genome. The recently characterized MuxABC-OpmB system has been shown to play a role in resistance to novobiocin, aztreonam, macrolides, and tetracycline in a multiple knockout mutation. In this study, we examined the expression levels of all the 12 RND pump gene clusters and tested the involvement of MuxABC-OpmB in pathogenicity. The results indicated that in addition to the four known constitutively expressed RND pumps, mexAB-oprM, mexGHI-opmD, mexVW, and mexXY, relatively high levels of expression were observed with mexJK and muxABC-opmB in the conditions tested. Inactivation of muxA in the muxABC-opmB operon resulted in elevated resistance to ampicillin and carbenicillin. The mutant also showed attenuated virulence in both Brassica rapa pekinensis and Drosophila melanogaster infection models. The decreased virulence at least in part was due to decreased twitching motility in the mutant. These results indicate that the RND pump MuxABC-OpmB is associated with ampicillin and carbenicillin susceptibility and also involved in pathogenesis in P. aeruginosa.

  5. Applying the ABCs in provider organizations.

    PubMed

    Pandey, Seema

    2012-11-01

    Activity-based costing (ABC) is an accounting technique designed to guard against potentially serious financial problems that can arise when an organization's accounting costs deviate significantly from its actual costs. In general, an ABC analysis considers two factors: a cost element (a directly measurable unit of cost, such as the cost of an item) and a cost driver (a directly measurable feature of the service, such as how often the item is used). ABC is best applied to specific service areas, orservice packages, for which consumption of resources is largely predictable and atomic units of services can be accurately identified. PMID:23173369

  6. Applying the ABCs in provider organizations.

    PubMed

    Pandey, Seema

    2012-11-01

    Activity-based costing (ABC) is an accounting technique designed to guard against potentially serious financial problems that can arise when an organization's accounting costs deviate significantly from its actual costs. In general, an ABC analysis considers two factors: a cost element (a directly measurable unit of cost, such as the cost of an item) and a cost driver (a directly measurable feature of the service, such as how often the item is used). ABC is best applied to specific service areas, orservice packages, for which consumption of resources is largely predictable and atomic units of services can be accurately identified.

  7. A bacterial-type ABC transporter is involved in aluminum tolerance in rice.

    PubMed

    Huang, Chao Feng; Yamaji, Naoki; Mitani, Namiki; Yano, Masahiro; Nagamura, Yoshiaki; Ma, Jian Feng

    2009-02-01

    Aluminum (Al) toxicity is a major factor limiting crop production in acidic soil, but the molecular mechanisms of Al tolerance are poorly understood. Here, we report that two genes, STAR1 (for sensitive to Al rhizotoxicity1) and STAR2, are responsible for Al tolerance in rice. STAR1 encodes a nucleotide binding domain, while STAR2 encodes a transmembrane domain, of a bacterial-type ATP binding cassette (ABC) transporter. Disruption of either gene resulted in hypersensitivity to aluminum toxicity. Both STAR1 and STAR2 are expressed mainly in the roots and are specifically induced by Al exposure. Expression in onion epidermal cells, rice protoplasts, and yeast showed that STAR1 interacts with STAR2 to form a complex that localizes to the vesicle membranes of all root cells, except for those in the epidermal layer of the mature zone. When expressed together in Xenopus laevis oocytes, STAR1/2 shows efflux transport activity specific for UDP-glucose. Furthermore, addition of exogenous UDP-glucose rescued root growth in the star1 mutant exposed to Al. These results indicate that STAR1 and STAR2 form a complex that functions as an ABC transporter, which is required for detoxification of Al in rice. The ABC transporter transports UDP-glucose, which may be used to modify the cell wall. PMID:19244140

  8. 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

  9. Novel cGMP efflux inhibitors identified by virtual ligand screening (VLS) and confirmed by experimental studies.

    PubMed

    Sager, Georg; Ørvoll, Elin Ø; Lysaa, Roy A; Kufareva, Irina; Abagyan, Ruben; Ravna, Aina W

    2012-04-12

    Elevated intracellular levels of cyclic guanosine monophosphate (cGMP) may induce apoptosis, and at least some cancer cells seem to escape this effect by increased efflux of cGMP, as clinical studies have shown that extracellular cGMP levels are elevated in various types of cancer. The human ATP binding cassette (ABC) transporter ABCC5 transports cGMP out of cells, and inhibition of ABCC5 may have cytotoxic effects. Sildenafil inhibits cGMP efflux by binding to ABCC5, and in order to search for potential novel ABCC5 inhibitors, we have identified sildenafil derivates using structural and computational guidance and tested them for the cGMP efflux effect. Eleven compounds from virtual ligand screening (VLS) were tested in vitro, using inside-out vesicles (IOV), for inhibition of cGMP efflux. Seven of 11 compounds predicted by VLS to bind to ABCC5 were more potent than sildenafil, and the two most potent showed K(i) of 50-100 nM. PMID:22380603

  10. 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

  11. Acetobacter aceti possesses a proton motive force-dependent efflux system for acetic acid.

    PubMed

    Matsushita, Kazunobu; Inoue, Taketo; Adachi, Osao; Toyama, Hirohide

    2005-07-01

    Acetic acid bacteria are obligate aerobes able to oxidize ethanol, sugar alcohols, and sugars into their corresponding acids. Among them, Acetobacter and Gluconacetobacter species have very high ethanol oxidation capacity, leading to accumulation of vast amounts of acetic acid outside the cell. Since these bacteria are able to grow in media with high concentrations of acetic acid, they must possess a specific mechanism such as an efflux pump by which they can resist the toxic effects of acetic acid. In this study, the efflux pump of Acetobacter aceti IFO 3283 was examined using intact cells and membrane vesicles. The accumulation of acetic acid/acetate in intact cells was increased by the addition of a proton uncoupler and/or cyanide, suggesting the presence of an energy-dependent efflux system. To confirm this, right-side-out and inside-out membrane vesicles were prepared from A. aceti IFO 3283, and the accumulation of acetic acid/acetate in the vesicles was examined. Upon the addition of a respiratory substrate, the accumulation of acetic acid/acetate in the right-side-out vesicles was largely decreased, while its accumulation was very much increased in the inside-out vesicles. These respiration-dependent phenomena observed in both types of membrane vesicles were all sensitive to a proton uncoupler. Acetic acid/acetate uptake in the inside-out membrane vesicles was dependent not on ATP but on the proton motive force. Furthermore, uptake was shown to be rather specific for acetic acid and to be pH dependent, because higher uptake was observed at lower pH. Thus, A. aceti IFO 3283 possesses a proton motive force-dependent efflux pump for acetic acid.

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. Contribution of Target Gene Mutations and Efflux to Decreased Susceptibility of Salmonella enterica Serovar Typhimurium to Fluoroquinolones and Other Antimicrobials▿

    PubMed Central

    Chen, Sheng; Cui, Shenghui; McDermott, Patrick F.; Zhao, Shaohua; White, David G.; Paulsen, Ian; Meng, Jianghong

    2007-01-01

    The mechanisms involved in fluoroquinolone resistance in Salmonella enterica include target alterations and overexpression of efflux pumps. The present study evaluated the role of known and putative multidrug resistance efflux pumps and mutations in topoisomerase genes among laboratory-selected and naturally occurring fluoroquinolone-resistant Salmonella enterica serovar Typhimurium strains. Strains with ciprofloxacin MICs of 0.25, 4, 32, and 256 μg/ml were derived in vitro using serovar Typhimurium S21. These mutants also showed decreased susceptibility or resistance to many nonfluoroquinolone antimicrobials, including tetracycline, chloramphenicol, and several β-lactams. The expression of efflux pump genes acrA, acrB, acrE, acrF, emrB, emrD, and mdlB were substantially increased (≥2-fold) among the fluoroquinolone-resistant mutants. Increased expression was also observed, but to a lesser extent, with three other putative efflux pumps: mdtB (yegN), mdtC (yegO), and emrA among mutants with ciprofloxacin MICs of ≥32 μg/ml. Deletion of acrAB or tolC in S21 and its fluoroquinolone-resistant mutants resulted in increased susceptibility to fluoroquinolones and other tested antimicrobials. In naturally occurring fluoroquinolone-resistant serovar Typhimurium strains, deletion of acrAB or tolC increased fluoroquinolone susceptibility 4-fold, whereas replacement of gyrA double mutations (S83F D87N) with wild-type gyrA increased susceptibility >500-fold. These results indicate that a combination of topoisomerase gene mutations, as well as enhanced antimicrobial efflux, plays a critical role in the development of fluoroquinolone resistance in both laboratory-derived and naturally occurring quinolone-resistant serovar Typhimurium strains. PMID:17043131

  18. The cloning of a human ABC gene (ABC3) mapping to chromosome 16p13.3

    SciTech Connect

    Connors, T.D.; Van Raay, T.J.; Petry, L.R.

    1997-01-15

    The ATP binding cassette (ABC) transporters, or traffic ATPases, constitute a large family of proteins responsible for the transport of a wide variety of substrates across cell membranes in both prokaryotic and eukaryotic cells. We describe a human ABC protein with regions of strong homology to the recently described murine ABC1 and ABC2 transporters. The gene for this novel protein, human ABC3, maps near the polycystic kidney disease type 1 (PKD1) gene on chromosome 16p13.3. The ABC3 gene is expressed at highest levels in lung compared to other tissues. 19 refs., 3 figs.

  19. Structural insights into ABC transporter mechanism

    SciTech Connect

    Oldham, Michael L.; Davidson, Amy L.; Chen, Jue

    2010-07-27

    ATP-binding cassette (ABC) transporters utilize the energy from ATP hydrolysis to transport substances across the membrane. In recent years, crystal structures of several ABC transporters have become available. These structures show that both importers and exporters oscillate between two conformations: an inward-facing conformation with the substrate translocation pathway open to the cytoplasm and an outward-facing conformation with the translocation pathway facing the opposite side of the membrane. In this review, conformational differences found in the structures of homologous ABC transporters are analyzed to understand how alternating-access is achieved. It appears that rigid-body rotations of the transmembrane subunits, coinciding with the opening and closing of the nucleotide-binding subunits, couples ATP hydrolysis to substrate translocation.

  20. ABC transporters, atherosclerosis and inflammation.

    PubMed

    Fitzgerald, Michael L; Mujawar, Zahedi; Tamehiro, Norimasa

    2010-08-01

    Atherosclerosis, driven by inflamed lipid-laden lesions, can occlude the coronary arteries and lead to myocardial infarction. This chronic disease is a major and expensive health burden. However, the body is able to mobilize and excrete cholesterol and other lipids, thus preventing atherosclerosis by a process termed reverse cholesterol transport (RCT). Insight into the mechanism of RCT has been gained by the study of two rare syndromes caused by the mutation of ABC transporter loci. In Tangier disease, loss of ABCA1 prevents cells from exporting cholesterol and phospholipid, thus resulting in the build-up of cholesterol in the peripheral tissues and a loss of circulating HDL. Consistent with HDL being an athero-protective particle, Tangier patients are more prone to develop atherosclerosis. Likewise, sitosterolemia is another inherited syndrome associated with premature atherosclerosis. Here mutations in either the ABCG5 or G8 loci, prevents hepatocytes and enterocytes from excreting cholesterol and plant sterols, including sitosterol, into the bile and intestinal lumen. Thus, ABCG5 and G8, which from a heterodimer, constitute a transporter that excretes cholesterol and dietary sterols back into the gut, while ABCA1 functions to export excess cell cholesterol and phospholipid during the biogenesis of HDL. Interestingly, a third protein, ABCG1, that has been shown to have anti-atherosclerotic activity in mice, may also act to transfer cholesterol to mature HDL particles. Here we review the relationship between the lipid transport activities of these proteins and their anti-atherosclerotic effect, particularly how they may reduce inflammatory signaling pathways. Of particular interest are recent reports that indicate both ABCA1 and ABCG1 modulate cell surface cholesterol levels and inhibit its partitioning into lipid rafts. Given lipid rafts may provide platforms for innate immune receptors to respond to inflammatory signals, it follows that loss of ABCA1 and ABCG1

  1. MpeR regulates the mtr efflux locus in Neisseria gonorrhoeae and modulates antimicrobial resistance by an iron-responsive mechanism.

    PubMed

    Mercante, Alexandra Dubon; Jackson, Lydgia; Johnson, Paul J T; Stringer, Virginia A; Dyer, David W; Shafer, William M

    2012-03-01

    Previous studies have shown that the MpeR transcriptional regulator produced by Neisseria gonorrhoeae represses the expression of mtrF, which encodes a putative inner membrane protein (MtrF). MtrF works as an accessory protein with the Mtr efflux pump, helping gonococci to resist high levels of diverse hydrophobic antimicrobials. Regulation of mpeR has been reported to occur by an iron-dependent mechanism involving Fur (ferric uptake regulator). Collectively, these observations suggest the presence of an interconnected regulatory system in gonococci that modulates the expression of efflux pump protein-encoding genes in an iron-responsive manner. Herein, we describe this connection and report that levels of gonococcal resistance to a substrate of the mtrCDE-encoded efflux pump can be modulated by MpeR and the availability of free iron. Using microarray analysis, we found that the mtrR gene, which encodes a direct repressor (MtrR) of mtrCDE, is an MpeR-repressed determinant in the late logarithmic phase of growth when free iron levels would be reduced due to bacterial consumption. This repression was enhanced under conditions of iron limitation and resulted in increased expression of the mtrCDE efflux pump operon. Furthermore, as judged by DNA-binding analysis, MpeR-mediated repression of mtrR was direct. Collectively, our results indicate that both genetic and physiologic parameters (e.g., iron availability) can influence the expression of the mtr efflux system and modulate levels of gonococcal susceptibility to efflux pump substrates. PMID:22214775

  2. Overcoming drug efflux-based multidrug resistance in cancer with nanotechnology.

    PubMed

    Xue, Xue; Liang, Xing-Jie

    2012-02-01

    Multidrug resistance (MDR), which significantly decreases the efficacy of anticancer drugs and causes tumor recurrence, has been a major challenge in clinical cancer treatment with chemotherapeutic drugs for decades. Several mechanisms of overcoming drug resistance have been postulated. Well known P-glycoprotein (P-gp) and other drug efflux transporters are considered to be critical in pumping anticancer drugs out of cells and causing chemotherapy failure. Innovative theranostic (therapeutic and diagnostic) strategies with nanoparticles are rapidly evolving and are anticipated to offer opportunities to overcome these limits. In this review, we discuss the mechanisms of drug efflux-mediated resistance and the application of multiple nanoparticle-based platforms to overcome chemoresistance and improve therapeutic outcome.

  3. 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

  4. Efflux in Fungi: La Pièce de Résistance

    PubMed Central

    Coleman, Jeffrey J.; Mylonakis, Eleftherios

    2009-01-01

    Pathogens must be able to overcome both host defenses and antimicrobial treatment in order to successfully infect and maintain colonization of the host. One way fungi accomplish this feat and overcome intercellular toxin accumulation is efflux pumps, in particular ATP-binding cassette transporters and transporters of the major facilitator superfamily. Members of these two superfamilies remove many toxic compounds by coupling transport with ATP hydrolysis or a proton gradient, respectively. Fungal genomes encode a plethora of members of these families of transporters compared to other organisms. In this review we discuss the role these two fungal superfamilies of transporters play in virulence and resistance to antifungal agents. These efflux transporters are responsible not only for export of compounds involved in pathogenesis such as secondary metabolites, but also export of host-derived antimicrobial compounds. In addition, we examine the current knowledge of these transporters in resistance of pathogens to clinically relevant antifungal agents. PMID:19557154

  5. The plant alkaloid piperine as a potential inhibitor of ethidium bromide efflux in Mycobacterium smegmatis.

    PubMed

    Jin, Jing; Zhang, Jiyu; Guo, Na; Feng, Haihua; Li, Lei; Liang, Junchao; Sun, Kai; Wu, Xiuping; Wang, Xuelin; Liu, Mingyuan; Deng, Xuming; Yu, Lu

    2011-02-01

    Piperine, a major plant alkaloid found in black pepper (Piper nigrum) and long pepper (Piper longum), has shown potential for inhibiting the efflux pump (EP) of Staphylococcus aureus. In this study, a modulation assay showed that piperine could decrease the MIC of ethidium bromide (EtBr) twofold at 32 μg ml(-1) and fourfold at 64 μg ml(-1) against Mycobacterium smegmatis mc(2) 155 ATCC 700084. A real-time, 96-well plate fluorometric method was employed to evaluate the EP inhibition ability of piperine in M. smegmatis. Reserpine, chlorpromazine, verapamil and carbonyl cyanide m-chlorophenylhydrazone were used as positive controls. Piperine significantly enhanced accumulation and decreased the efflux of EtBr in M. smegmatis, which suggests that it has the ability to inhibit mycobacterial EPs.

  6. Communicating a New ABC: Advocacy, Partnerships, and Implementing Change.

    ERIC Educational Resources Information Center

    Ryan, Cathy

    2001-01-01

    Reiterates some key points and responds to challenges issued by the speakers to the Association for Business Communication (ABC) conference. Notes that Paula Pomerenke spoke about formalizing support of Plain Language across the continents and about ABC's continuing need to strengthen relationships with practitioners. Suggests the ABC build…

  7. Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter

    NASA Astrophysics Data System (ADS)

    Grossmann, Nina; Vakkasoglu, Ahmet S.; Hulpke, Sabine; Abele, Rupert; Gaudet, Rachelle; Tampé, Robert

    2014-11-01

    The ATP-binding cassette (ABC) transporter associated with antigen processing (TAP) participates in immune surveillance by moving proteasomal products into the endoplasmic reticulum (ER) lumen for major histocompatibility complex class I loading and cell surface presentation to cytotoxic T cells. Here we delineate the mechanistic basis for antigen translocation. Notably, TAP works as a molecular diode, translocating peptide substrates against the gradient in a strict unidirectional way. We reveal the importance of the D-loop at the dimer interface of the two nucleotide-binding domains (NBDs) in coupling substrate translocation with ATP hydrolysis and defining transport vectoriality. Substitution of the conserved aspartate, which coordinates the ATP-binding site, decreases NBD dimerization affinity and turns the unidirectional primary active pump into a passive bidirectional nucleotide-gated facilitator. Thus, ATP hydrolysis is not required for translocation per se, but is essential for both active and unidirectional transport. Our data provide detailed mechanistic insight into how heterodimeric ABC exporters operate.

  8. The phytoestrogen genistein enhances multidrug resistance in breast cancer cell lines by translational regulation of ABC transporters.

    PubMed

    Rigalli, Juan Pablo; Tocchetti, Guillermo Nicolás; Arana, Maite Rocío; Villanueva, Silvina Stella Maris; Catania, Viviana Alicia; Theile, Dirk; Ruiz, María Laura; Weiss, Johanna

    2016-06-28

    Breast cancer is the most frequent malignancy in women. Multidrug resistance due to overexpression of ABC drug transporters is a common cause of chemotherapy failure and disease recurrence. Genistein (GNT) is a phytoestrogen present in soybeans and hormone supplements. We investigated the effect of GNT on the expression and function of ABC transporters in MCF-7 and MDA-MB-231 breast cancer cell lines. Results demonstrated an induction at the protein level of ABCC1 and ABCG2 and of ABCC1 in MCF-7 and MDA-MB-231, respectively. MCF-7 cells showed a concomitant increase in doxorubicin and mitoxantrone efflux and resistance, dependent on ABCG2 activity. ABCC1 induction by GNT in MDA-MB-231 cells modified neither drug efflux nor chemoresistance due to simultaneous acute inhibition of the transporter activity by GNT. All inductions took place at the translational level, as no increment in mRNA was observed and protein increase was prevented by cycloheximide. miR-181a, already demonstrated to inhibit ABCG2 translation, was down-regulated by GNT, explaining translational induction. Effects were independent of classical estrogen receptors. Results suggest potential nutrient-drug interactions that could threaten chemotherapy efficacy, especially in ABCG2-expressing tumors treated with substrates of this transporter. PMID:27033456

  9. Importance of Non-Diffusive Transport for Soil CO2 Efflux in a Temperate Mountain Grassland

    NASA Astrophysics Data System (ADS)

    Roland, Marilyn; Vicca, Sara; Bahn, Michael; Ladreiter-Knauss, Thomas; Schmitt, Michael; Janssens, Ivan A.

    2015-04-01

    A key focus in climate change is on the dynamics and predictions of the soil CO2 efflux (SCE) from terrestrial ecosystems. Limited knowledge of CO2 transport through the soil restricts our understanding of the various biotic and abiotic processes underlying these emissions. Diffusion is often thought to be the main transport mechanism for trace gases in soils, an assumption that is reflected in the increasing popularity of the flux-gradient approach (FGA). Based on Fick's law, the FGA calculates soil CO2 efflux from CO2 concentration profiles, given good estimates of the diffusion coefficient. The latter can be calculated via different commonly used models, and solid-state sensors allow continuous high-frequency measurements of soil CO2 concentrations with minimal disturbance to the soil conditions in a cost-effective way. Fast growing evidence of pressure pumping and advection, makes it impossible to disregard non-diffusive gas transport when evaluating diel and day-to-day dynamics of soil CO2 emissions. We have analyzed combined measurements from solid-state sensors and soil chambers to gain insight in the CO2 transport mechanisms in a grassland site in the Austrian Alps. The FGA-derived efflux underestimated the chamber efflux by 10 to 87% at our site, depending on which model was used for calculation of the diffusion coefficient. We found that the actual transport rates correlated well with irradiation and wind speed, even more when the soil moisture content was below 33%. These findings suggest that bulk soil air transport was enhanced by pressure changes induced by wind shear at the surface and by local heating of the soil surface. Considering the importance of non-diffusive transport processes is a prerequisite when using solid-state CO2 concentration measurements to estimate soil CO2 efflux at any given site.

  10. 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

  11. Active efflux and diffusion are involved in transport of Pseudomonas aeruginosa cell-to-cell signals.

    PubMed

    Pearson, J P; Van Delden, C; Iglewski, B H

    1999-02-01

    Many gram-negative bacteria communicate by N-acyl homoserine lactone signals called autoinducers (AIs). In Pseudomonas aeruginosa, cell-to-cell signaling controls expression of extracellular virulence factors, the type II secretion apparatus, a stationary-phase sigma factor (sigmas), and biofilm differentiation. The fact that a similar signal, N-(3-oxohexanoyl) homoserine lactone, freely diffuses through Vibrio fischeri and Escherichia coli cells has led to the assumption that all AIs are freely diffusible. In this work, transport of the two P. aeruginosa AIs, N-(3-oxododecanoyl) homoserine lactone (3OC12-HSL) (formerly called PAI-1) and N-butyryl homoserine lactone (C4-HSL) (formerly called PAI-2), was studied by using tritium-labeled signals. When [3H]C4-HSL was added to cell suspensions of P. aeruginosa, the cellular concentration reached a steady state in less than 30 s and was nearly equal to the external concentration, as expected for a freely diffusible compound. In contrast, [3H]3OC12-HSL required about 5 min to reach a steady state, and the cellular concentration was 3 times higher than the external level. Addition of inhibitors of the cytoplasmic membrane proton gradient, such as azide, led to a strong increase in cellular accumulation of [3H]3OC12-HSL, suggesting the involvement of active efflux. A defined mutant lacking the mexA-mexB-oprM-encoded active-efflux pump accumulated [3H]3OC12-HSL to levels similar to those in the azide-treated wild-type cells. Efflux experiments confirmed these observations. Our results show that in contrast to the case for C4-HSL, P. aeruginosa cells are not freely permeable to 3OC12-HSL. Instead, the mexA-mexB-oprM-encoded efflux pump is involved in active efflux of 3OC12-HSL. Apparently the length and/or degree of substitution of the N-acyl side chain determines whether an AI is freely diffusible or is subject to active efflux by P. aeruginosa.

  12. The New ABC of the Arts

    ERIC Educational Resources Information Center

    Tusa, John

    2007-01-01

    This article presents a new alphabet of the arts that shows how the arts world has been transformed the past five years. Beginning with "A" for assessment and continuing through "Y" for year end, the ABC of the arts illustrates the ways in which the arts world is judged, managed, and evaluated, and shows that the skill of arts management is to…

  13. Calculus ABCs: A Gateway for Freshman Calculus

    ERIC Educational Resources Information Center

    Fulton, Scott R.

    2003-01-01

    This paper describes a gateway testing program designed to ensure that students acquire basic skills in freshman calculus. Students must demonstrate they have mastered standards for "Absolutely Basic Competency"--the Calculus ABCs--in order to pass the course with a grade of C or better. We describe the background, standards, and testing program.…

  14. The ABC's of Learning in Infancy.

    ERIC Educational Resources Information Center

    Saunders, Minta M.

    Learning in infancy is based on activity, beginnings, and curiosity, the so-called ABC's. Earliest behavior consists of mass activity, the period from birth to 24 months of sensory-motor development which provides the foundation for all future learning. Adults must provide space, toys, and affectionate care to help infants proceed through…

  15. The ABCs of Managing Teacher Stress.

    ERIC Educational Resources Information Center

    Nagel, Liza; Brown, Sheri

    2003-01-01

    Describes stress management for teachers and presents strategies that teachers can use to lessen the impact of stress. Outlines the ABCs of stress: Acknowledge, Behavior Modification, and Communication. Notes that stress can motivate teachers to explore new instructional strategies, adopt innovative approaches to increasing student motivation, and…

  16. Proteasome Regulator Marizomib (NPI-0052) Exhibits Prolonged Inhibition, Attenuated Efflux, and Greater Cytotoxicity than Its Reversible Analogs

    PubMed Central

    Obaidat, Amanda; Weiss, Jeffrey; Wahlgren, Brett; Manam, Rama R.; Macherla, Venkat R.; McArthur, Katherine; Chao, Ta-Hsiang; Palladino, Michael A.; Lloyd, G. Kenneth; Potts, Barbara C.; Enna, Salvatore J.; Neuteboom, Saskia T. C.

    2011-01-01

    The present study was undertaken to compare the cellular transport characteristics of [3H]NPI-0052 (1R,4R,5S)-4-(2-chloroethyl)-1-((S)-((S)-cyclohex-2-enyl)(hydroxy)methyl)-5-methyl-6-oxa-2-azabicyclo[3.2.0]heptane-3,7-dione (marizomib; salinosporamide A) and [3H]NPI-0047 (1R,4R, 5S)-1-((S)-((S)-cyclohex-2-enyl)(hydroxy)methyl)-4-ethyl-5-methyl-6-oxa-2-azabicyclo[3.2.0]heptane-3,7-dione in RPMI 8226 multiple myeloma and PC-3 prostate adenocarcinoma cells to determine whether these properties explain differences in the cytotoxic potencies of these chemical analogs. The results indicate that marizomib, which possesses a chemical-leaving group, is more cytotoxic to both cell lines and inhibits proteasome activity more completely at lower concentrations than NPI-0047, a nonleaving-group analog. Moreover, it was found that both compounds accumulate in these cells by simple diffusion and the same carrier-mediated transport system. Although the rate of uptake is similar, the cellular efflux, which does not seem to be mediated by a major ATP-binding cassette (ABC)-efflux transporter, is more rapid for NPI-0047 than for marizomib. Experiments revealed that the irreversible binding of marizomib to the proteasome is responsible for its slower efflux, longer duration of action, and greater cytotoxicity compared with NPI-0047. The discovery that major ABC transporters of the multidrug resistance-associated protein family do not seem to be involved in the accumulation or removal of these agents suggests they may not be affected by multidrug resistance mechanisms during prolonged administration. PMID:21303921

  17. K(+)- and HCO3(-)-dependent acid-base transport in squid giant axons. I. Base efflux

    PubMed Central

    1995-01-01

    We used microelectrodes to monitor the recovery (i.e., decrease) of intracellular pH (pHi) after using internal dialysis to load squid giant axons with alkali to pHi values of 7.7, 8.0, or 8.3. The dialysis fluid (DF) contained 400 mM K+ but was free of Na+ and Cl-. The artificial seawater (ASW) lacked Na+, K+, and Cl-, thereby eliminating effects of known acid-base transporters on pHi. Under these conditions, halting dialysis unmasked a slow pHi decrease caused at least in part by acid-base transport we refer to as "base efflux." Replacing K+ in the DF with either NMDG+ or TEA+ significantly reduced base efflux and made membrane voltage (Vm) more positive. Base efflux in K(+)-dialyzed axons was stimulated by decreasing the pH of the ASW (pHo) from 8 to 7, implicating transport of acid or base. Although postdialysis acidifications also occurred in axons in which we replaced the K+ in the DF with Li+, Na+, Rb+, or Cs+, only with Rb+ was base efflux stimulated by low pHo. Thus, the base effluxes supported by K+ and Rb+ appear to be unrelated mechanistically to those observed with Li+, Na+, or Cs+. The combination of 437 mM K+ and 12 mM HCO3- in the ASW, which eliminates the gradient favoring a hypothetical K+/HCO3- efflux, blocked pHi recovery in K(+)-dialyzed axons. However, the pHi recovery was not blocked by the combination of 437 mM Na+, veratridine, and CO2/HCO3- in the ASW, a treatment that inverts electrochemical gradients for H+ and HCO3- and would favor passive H+ and HCO3- fluxes that would have alkalinized the axon. Similarly, the recovery was not blocked by K+ alone or HCO3- alone in the ASW, nor was it inhibited by the K-H pump blocker Sch28080 nor by the Na-H exchange inhibitors amiloride and hexamethyleneamiloride. Our data suggest that a major component of base efflux in alkali-loaded axons cannot be explained by metabolism, a H+ or HCO3- conductance, or by a K-H exchanger. However, this component could be mediated by a novel K/HCO3- cotransporter

  18. ABC transporters in fish species: a review

    PubMed Central

    Ferreira, Marta; Costa, Joana; Reis-Henriques, Maria A.

    2014-01-01

    ATP-binding cassette (ABC) proteins were first recognized for their role in multidrug resistance (MDR) in chemotherapeutic treatments, which is a major impediment for the successful treatment of many forms of malignant tumors in humans. These proteins, highly conserved throughout vertebrate species, were later related to cellular detoxification and accounted as responsible for protecting aquatic organisms from xenobiotic insults in the so-called multixenobiotic resistance mechanism (MXR). In recent years, research on these proteins in aquatic species has highlighted their importance in the detoxification mechanisms in fish thus it is necessary to continue these studies. Several transporters have been pointed out as relevant in the ecotoxicological context associated to the transport of xenobiotics, such as P-glycoproteins (Pgps), multidrug-resistance-associated proteins (MRPs 1-5) and breast cancer resistance associated protein (BCRP). In mammals, several nuclear receptors have been identified as mediators of phase I and II metabolizing enzymes and ABC transporters. In aquatic species, knowledge on co-regulation of the detoxification mechanism is scarce and needs to be addressed. The interaction of emergent contaminants that can act as chemosensitizers, with ABC transporters in aquatic organisms can compromise detoxification processes and have population effects and should be studied in more detail. This review intends to summarize the recent advances in research on MXR mechanisms in fish species, focusing in (1) regulation and functioning of ABC proteins; (2) cooperation with phase I and II biotransformation enzymes; and (3) ecotoxicological relevance and information on emergent pollutants with ability to modulate ABC transporters expression and activity. Several lines of evidence are clearly suggesting the important role of these transporters in detoxification mechanisms and must be further investigated in fish to underlay the mechanism to consider their use as

  19. PUMP CONSTRUCTION

    DOEpatents

    Strickland, G.; Horn, F.L.; White, H.T.

    1960-09-27

    A pump which utilizes the fluid being pumped through it as its lubricating fluid is described. This is achieved by means of an improved bearing construction in a pump of the enclosed or canned rotor type. At the outlet end of the pump, adjacent to an impeller mechanism, there is a bypass which conveys some of the pumped fluid to a chamber at the inlet end of the pump. After this chamber becomes full, the pumped fluid passes through fixed orifices in the top of the chamber and exerts a thrust on the inlet end of the pump rotor. Lubrication of the rotor shaft is accomplished by passing the pumped fluid through a bypass at the outlet end of the rotor shaft. This bypass conveys Pumped fluid to a cooling means and then to grooves on the surface of the rotor shait, thus lubricating the shaft.

  20. Citrulline increases cholesterol efflux from macrophages in vitro and ex vivo via ATP-binding cassette transporters

    PubMed Central

    Uto-Kondo, Harumi; Ayaori, Makoto; Nakaya, Kazuhiro; Takiguchi, Shunichi; Yakushiji, Emi; Ogura, Masatsune; Terao, Yoshio; Ozasa, Hideki; Sasaki, Makoto; Komatsu, Tomohiro; Sotherden, Grace Megumi; Hosoai, Tamaki; Sakurada, Masami; Ikewaki, Katsunori

    2014-01-01

    Reverse cholesterol transport (RCT) is a mechanism critical to the anti-atherogenic property of HDL. Although citrulline contributes to the amelioration of atherosclerosis via endothelial nitric oxide production, it remains unclear whether it affects RCT. This study was undertaken to clarify the effects of citrulline on expressions of specific transporters such as ATP binding cassette transporters (ABC)A1 and ABCG1, and the cholesterol efflux from macrophages to apolipoprotein (apo) A-I or HDL in vitro and ex vivo. Citrulline increased ABCA1 and ABCG1 mRNA and protein levels in THP-1 macrophages, translating into enhanced apoA-I- and HDL-mediated cholesterol efflux. In the human crossover study, 8 healthy male volunteers (age 30–49 years) consumed either 3.2 g/day citrulline or placebo for 1 week. Citrulline consumption brought about significant increases in plasma levels of citrulline and arginine. Supporting the in vitro data, monocyte-derived macrophages (MDM) differentiated under autologous post-citrulline sera demonstrated enhancement of both apoA-I- and HDL-mediated cholesterol efflux through increased ABCA1 and ABCG1 expressions, compared to MDM differentiated under pre-citrulline sera. However, the placebo did not modulate these parameters. Therefore, in addition to improving endothelium function, citrulline might have an anti-atherogenic property by increasing RCT of HDL. PMID:25120277

  1. The yeast vacuolar ABC transporter Ybt1p regulates membrane fusion through Ca2+ transport modulation

    PubMed Central

    Sasser, Terry L.; Padolina, Mark; Fratti, Rutilio A.

    2013-01-01

    Ybt1p is a class C ABC transporter (ATP-binding cassette transporter) that is localized to the vacuole of Saccharomyces cerevisiae. Although Ybt1p was originally identified as a bile acid transporter, it has also been found to function in other capacities, including the translocation of phosphatidylcholine to the vacuole lumen, and the regulation of Ca2+ homoeostasis. In the present study we found that deletion of YBT1 enhanced in vitro homotypic vacuole fusion by up to 50 % relative to wild-type vacuoles. The increased vacuole fusion was not due to aberrant protein sorting of SNAREs (soluble N-ethylmaleimide-sensitive factor-attachment protein receptors) or recruitment of factors from the cytosol such as Ypt7p and the HOPS (homotypic fusion and vacuole protein sorting) tethering complex. In addition, ybt1Δ vacuoles displayed no observable differences in the formation of SNARE complexes, interactions between SNAREs and HOPS, or formation of vertex microdomains. However, the absence of Ybt1p caused significant changes in Ca2+ transport during fusion. One difference was the prolonged Ca2+ influx exhibited by ybt1Δ vacuoles at the start of the fusion reaction. We also observed a striking delay in SNARE-dependent Ca2+ efflux. As vacuole fusion can be inhibited by high Ca2+ concentrations, we suggest that the delayed efflux in ybt1Δ vacuoles leads to the enhanced SNARE function. PMID:22970809

  2. Efflux transporters of the human placenta.

    PubMed

    Young, Amber M; Allen, Courtni E; Audus, Kenneth L

    2003-01-21

    The use of pharmaceuticals during pregnancy is often a necessity for the health of the mother. Until recently, the placenta was viewed as a passive organ through which molecules are passed indiscriminately between mother and fetus. In reality, the placenta contains a plethora of transporters, some of which appear to be specifically dedicated to removal of xenobiotics and toxic endogenous compounds. Drug efflux transporters such as P-glycoprotein (P-gp), several multidrug resistant associated proteins (MRPs) and breast cancer resistant protein (BCRP) may provide mechanisms that protect the developing fetus. Bile acid transporters may also play a role in exporting compounds back into the maternal compartment. Steroid hormones directly influence the level of expression and function in some of these transporters. Investigating the link between the hormones of pregnancy and these drug efflux transporters is one possible key in developing strategies to deliver drugs to the mother with minimal fetal risk. PMID:12535577

  3. 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.

  4. Multidrug Efflux Systems in Helicobacter cinaedi

    PubMed Central

    Morita, Yuji; Tomida, Junko; Kawamura, Yoshiaki

    2012-01-01

    Helicobacter cinaedi causes infections, such as bacteremia, diarrhea and cellulitis in mainly immunocompromised patients. This pathogen is often problematic to analyze, and insufficient information is available, because it grows slowly and poorly in subculture under a microaerobic atmosphere. The first-choice therapy to eradicate H. cinaedi is antimicrobial chemotherapy; however, its use is linked to the development of resistance. Although we need to understand the antimicrobial resistance mechanisms of H. cinaedi, unfortunately, sufficient genetic tools for H. cinaedi have not yet been developed. In July 2012, the complete sequence of H. cinaedi strain PAGU 611, isolated from a case of human bacteremia, was announced. This strain possesses multidrug efflux systems, intrinsic antimicrobial resistance mechanisms and typical mutations in gyrA and the 23S rRNA gene, which are involved in acquired resistance to fluoroquinolones and macrolides, respectively. Here, we compare the organization and properties of the efflux systems of H. cinaedi with the multidrug efflux systems identified in other bacteria. PMID:27029418

  5. CO₂ efflux from shrimp ponds in Indonesia.

    PubMed

    Sidik, Frida; Lovelock, Catherine E

    2013-01-01

    The conversion of mangrove forest to aquaculture ponds has been increasing in recent decades. One of major concerns of this habitat loss is the release of stored 'blue' carbon from mangrove soils to the atmosphere. In this study, we assessed carbon dioxide (CO₂) efflux from soil in intensive shrimp ponds in Bali, Indonesia. We measured CO₂ efflux from the floors and walls of shrimp ponds. Rates of CO₂ efflux within shrimp ponds were 4.37 kg CO₂ m⁻² y⁻¹ from the walls and 1.60 kg CO₂ m⁻² y⁻¹ from the floors. Combining our findings with published data of aquaculture land use in Indonesia, we estimated that shrimp ponds in this region result in CO₂ emissions to the atmosphere between 5.76 and 13.95 Tg y⁻¹. The results indicate that conversion of mangrove forests to aquaculture ponds contributes to greenhouse gas emissions that are comparable to peat forest conversion to other land uses in Indonesia. Higher magnitudes of CO₂ emission may be released to atmosphere where ponds are constructed in newly cleared mangrove forests. This study indicates the need for incentives that can meet the target of aquaculture industry without expanding the converted mangrove areas, which will lead to increased CO₂ released to atmosphere. PMID:23755306

  6. PK11195, a peripheral benzodiazepine receptor (pBR) ligand, broadly blocks drug efflux to chemosensitize leukemia and myeloma cells by a pBR-independent, direct transporter-modulating mechanism.

    PubMed

    Walter, Roland B; Pirga, Jason L; Cronk, Michelle R; Mayer, Sasha; Appelbaum, Frederick R; Banker, Deborah E

    2005-11-15

    The peripheral benzodiazepine receptor (pBR) ligand, PK11195, promotes mitochondrial apoptosis and blocks P-glycoprotein (Pgp)-mediated drug efflux to chemosensitize cancer cells at least as well or better than the Pgp modulator, cyclosporine A (CSA). We now show that PK11195 broadly inhibits adenosine triphosphate (ATP)-binding cassette (ABC) transporters in hematologic cancer cell lines and primary leukemia-cell samples, including multidrug resistance protein (MRP), breast cancer resistance protein (BCRP), and/or Pgp. Ectopic expression models confirmed that pBR can directly mediate chemosensitizing by PK11195, presumably via mitochondrial activities, but showed that pBR expression is unnecessary to PK11195-mediated efflux inhibition. PK11195 binds plasma-membrane sites in Pgp-expressing cells, stimulates Pgp-associated adenosine triphosphatase (ATPase) activity, and causes conformational changes in Pgp, suggesting that PK11195 modulates Pgp-mediated efflux by direct transporter interaction(s). PK11195 and CSA bind noncompetitively in Pgp-expressing cells, indicating that PK11195 interacts with Pgp at sites that are distinct from CSA-binding sites. Importantly, PK11195 concentrations that were effective in these in vitro assays can be safely achieved in patients. Because PK11195 promotes chemotherapy-induced apoptosis by a pBR-dependent mitochondrial mechanism and broadly blocks drug efflux by an apparently pBR-independent, ABC transporter-dependent mechanism, PK11195 may be a useful clinical chemosensitizer in cancer patients.

  7. 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.

  8. Loss of plastoglobule kinases ABC1K1 and ABC1K3 causes conditional degreening, modified prenyl-lipids, and recruitment of the jasmonic acid pathway

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plastoglobules (PGs) are plastid lipid-protein particles. This study examines the function of PG-localized kinases ABC1K1 and ABC1K3 in Arabidopsis thaliana. Several lines of evidence suggested that ABC1K1 and ABC1K3 form a protein complex. Null mutants for both genes (abc1k1 and abc1k3) and the dou...

  9. Nanostructured assemblies from amphiphilic ABC multiblock polymers

    NASA Astrophysics Data System (ADS)

    Hillmyer, Marc A.

    2012-02-01

    Amphiphilic AB diblock copolymers containing a water compatible segment can self-assemble in aqueous media to give supramolecular structures that include simple spherical micelles and macromolecular vesicles termed polymersomes. Amphiphilic ABA triblocks with hydrophobic end blocks can adopt analogous structures but can also form gels at high polymer concentrations. The structural and chemical diversity demonstrated in block copolymer micelles and gels makes them attractive for applications ranging from drug delivery to personal care products to nanoreactors. The inclusion of a third block in amphiphilic ABC triblock systems can lead to a much wider array of self-assembled structures that depend not only on composition but also on block sequence, architecture and incompatibility considerations. I will present our recent efforts on tuning micelle and gel structure and behavior using controlled architecture ABC triblocks. The combination of diverse polymer segments into a single macromolecule is a powerful method for development of self-assembled structures with both new form and new function.

  10. Three ways to learn the ABCs.

    PubMed

    Ng, M; Yanofsky, M F

    2000-02-01

    The ABC model of flower development represents a milestone in explaining how the fate of emerging floral organ primordia is specified. This model states that organ identity is specified by different combinations of the activities of the A, B and C class homeotic genes. In spite of the remarkable simplicity of this model, the complex regulatory interactions that establish the initial pattern of A, B and C gene activity have yet to be fully explained. It has been shown that the LEAFY gene functions early to promote flower meristem identity, and that it is subsequently required for the normal expression of the ABC genes. Recently, LEAFY has been identified as an immediate upstream regulator of the floral homeotic genes, thus opening up an avenue to examine the transcriptional interactions that underlie floral patterning. PMID:10679448

  11. Pharmacological correction of misfolding of ABC proteins☆

    PubMed Central

    Rudashevskaya, Elena L.; Stockner, Thomas; Trauner, Michael; Freissmuth, Michael; Chiba, Peter

    2014-01-01

    The endoplasmic reticulum (ER) quality control system distinguishes between correctly and incorrectly folded proteins to prevent processing of aberrantly folded conformations along the secretory pathway. Non-synonymous mutations can lead to misfolding of ABC proteins and associated disease phenotypes. Specific phenotypes may at least partially be corrected by small molecules, so-called pharmacological chaperones. Screening for folding correctors is expected to open an avenue for treatment of diseases such as cystic fibrosis and intrahepatic cholestasis. PMID:25027379

  12. Adhesion to chondroitinase ABC treated dentin

    PubMed Central

    Mazzoni, Annalisa; Pashley, David H.; Ruggeri, Alessandra; Vita, Francesca; Falconi, Mirella; Di Lenarda, Roberto; Breschi, Lorenzo

    2013-01-01

    Dentin bonding relies on complete resin impregnation throughout the demineralised hydrophilic collagen mesh. Chondroitin sulphate-glycosaminoglycans are claimed to regulate the three-dimensional arrangement of the dentin organic matrix and its hydrophilicity. The aim of this study was to investigate bond strength of two etch-and-rinse adhesives to chondroitinase ABC treated dentin. Human extracted molars were treated with chondroitinase ABC and a double labelling immunohistochemical technique was applied to reveal type I collagen and chondroitin 4/6 sulphate distribution under field emission in-lens scanning electron microscope. The immunohistochemical technique confirmed the effective removal of chondroitin 4/6 sulphate after the enzymatic treatment. Dentin surfaces exposed to chondroitinase ABC and untreated specimens prepared on untreated acid-etched dentin were bonded with Adper Scotchbond Multi-Purpose or Prime & Bond NT. Bonded specimens were submitted to microtensile testing and nanoleakage interfacial analysis under transmission electron microscope. Increased mean values of microtensile bond strength and reduced nanoleakage expression were found for both adhesives after chondroitinase ABC treatment of the dentin surface. Adper Scotchbond Multi-Purpose increased its bond strength about 28%, while bonding made with Prime & Bond NT almost doubled (92% increase) compared to untreated specimens. This study supports the hypothesis that adhesion can be enhanced by removal of chondroitin 4/6 sulphate and dermatan sulphate, probably due to a reduced amount of water content and enlarged interfibrillar spaces. Further studies should validate this hypothesis investigating the stability of chondroitin 4/6 and dermatan sulphate-depleted dentin bonded interface over time. PMID:18161809

  13. Analyzing health care operations using ABC.

    PubMed

    Ross, Thomas K

    2004-01-01

    The evolution of health care created a climate in which cost was subordinate to medical treatment. Current reimbursement constraints have increased the need for providers to be cost conscious, but they have discovered that current accounting practices do not provide the appropriate information to determine the cost of service or make decisions. This article argues that activity-based costing (ABC) can bridge the gap between the medical and financial communities and provide a foundation for performance improvement. PMID:15151193

  14. Pharmacological correction of misfolding of ABC proteins.

    PubMed

    Rudashevskaya, Elena L; Stockner, Thomas; Trauner, Michael; Freissmuth, Michael; Chiba, Peter

    2014-06-01

    The endoplasmic reticulum (ER) quality control system distinguishes between correctly and incorrectly folded proteins to prevent processing of aberrantly folded conformations along the secretory pathway. Non-synonymous mutations can lead to misfolding of ABC proteins and associated disease phenotypes. Specific phenotypes may at least partially be corrected by small molecules, so-called pharmacological chaperones. Screening for folding correctors is expected to open an avenue for treatment of diseases such as cystic fibrosis and intrahepatic cholestasis. PMID:25027379

  15. Magnetocaloric pump

    NASA Technical Reports Server (NTRS)

    Brown, G. V.

    1973-01-01

    Very cold liquids and gases such as helium, neon, and nitrogen can be pumped by using magnetocaloric effect. Adiabatic magnetization and demagnetization are used to alternately heat and cool slug of pumped fluid contained in closed chamber.

  16. Casing pump

    SciTech Connect

    Bass, H.E.; Bass, R.E.

    1987-09-29

    A natural gas operated pump is described for use in the casing of an oil well, comprising: a tubular pump body having an open lower end for admitting well fluids to the interior of the pump body and an open upper end, wherein a downwardly facing seating surface is formed on the inner periphery of the pump body adjacent the upper end thereof; means for forming a seal between the pump body and the casing of the well; a rod extending longitudinally through the seating surface formed in the pump body and protruding from the upper end of the pump body; a valve member mounted on the rod below the seating surface and shaped to mate with the seating surface; and means for vertically positioning the rod in proportion to fluid pressure within the pump body.

  17. ELECTROMAGNETIC PUMP

    DOEpatents

    Pulley, O.O.

    1954-08-17

    This patent reiates to electromagnetic pumps for electricity-conducting fluids and, in particular, describes several modifications for a linear conduction type electromagnetic interaction pump. The invention resides in passing the return conductor for the current traversing the fiuid in the duct back through the gap in the iron circuit of the pump. Both the maximum allowable pressure and the efficiency of a linear conduction electromagnetic pump are increased by incorporation of the present invention.

  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. Conserved allosteric hot spots in the transmembrane domains of cystic fibrosis transmembrane conductance regulator (CFTR) channels and multidrug resistance protein (MRP) pumps.

    PubMed

    Wei, Shipeng; Roessler, Bryan C; Chauvet, Sylvain; Guo, Jingyu; Hartman, John L; Kirk, Kevin L

    2014-07-18

    ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5'-adenylyl-β,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs.

  20. Phase behavior of model ABC triblock copolymers

    NASA Astrophysics Data System (ADS)

    Chatterjee, Joon

    The phase behavior of poly(isoprene-b-styrene- b-ethylene oxide) (ISO), a model ABC triblock copolymer has been studied. This class of materials exhibit self-assembly, forming a large array of ordered morphologies at length scales of 5-100 nm. The formation of stable three-dimensionally continuous network morphologies is of special interest in this study. Since these nanostructures considerably impact the material properties, fundamental knowledge for designing ABC systems have high technological importance for realizing applications in the areas of nanofabrication, nanoporous media, separation membranes, drug delivery and high surface area catalysts. A comprehensive framework was developed to describe the phase behavior of the ISO triblock copolymers at weak to intermediate segregation strengths spanning a wide range of composition. Phases were characterized through a combination of characterization techniques, including small angle x-ray scattering, dynamic mechanical spectroscopy, transmission electron microscopy, and birefringence measurements. Combined with previous investigations on ISO, six different stable ordered state symmetries have been identified: lamellae (LAM), Fddd orthorhombic network (O70), double gyroid (Q230), alternating gyroid (Q214), hexagonal (HEX), and body-centered cubic (BCC). The phase map was found to be somewhat asymmetric around the fI = fO isopleth. This work provides a guide for theoretical studies and gives insight into the intricate effects of various parameters on the self-assembly of ABC triblock copolymers. Experimental SAXS data evaluated with a simple scattering intensity model show that local mixing varies continuously across the phase map between states of two- and three-domain segregation. Strategies of blending homopolymers with ISO triblock copolymer were employed for studying the swelling properties of a lamellar state. Results demonstrate that lamellar domains swell or shrink depending upon the type of homopolymer that

  1. Synthesis of poly[N-(2-hydroxypropyl)methacrylamide] conjugates of inhibitors of the ABC transporter that overcome multidrug resistance in doxorubicin-resistant P388 cells in vitro.

    PubMed

    Subr, V; Sivák, L; Koziolová, E; Braunová, A; Pechar, M; Strohalm, J; Kabešová, M; Ríhová, B; Ulbrich, K; Kovář, M

    2014-08-11

    The effects of novel polymeric therapeutics based on water-soluble N-(2-hydroxypropyl)methacrylamide copolymers (P(HPMA)) bearing the anticancer drug doxorubicin (Dox), an inhibitor of ABC transporters, or both, on the viability and the proliferation of the murine monocytic leukemia cell line P388 (parental cell line) and its doxorubicin-resistant subline P388/MDR were studied in vitro. The inhibitor derivatives 5-methyl-4-oxohexanoyl reversin 121 (MeOHe-R121) and 5-methyl-4-oxohexanoyl ritonavir ester (MeOHe-RIT), showing the highest inhibitory activities, were conjugated to the P(HPMA) via the biodegradable pH-sensitive hydrazone bond, and the ability of these conjugates to block the ATP driven P-glycoprotein (P-gp) efflux pump was tested. The P(HPMA) conjugate P-Ahx-NH-N═MeOHe-R121 showed a dose-dependent increase in the ability to sensitize the P388/MDR cells to Dox from 1.5 to 24 μM, and achieved an approximately 50-fold increase in sensitization at 24 μM. The P(HPMA) conjugate P-Ahx-NH-N═MeOHe-RIT showed moderate activity at 6 μM (∼10 times higher sensitization) and increased sensitization by 50-fold at 12 μM. The cytostatic activity of the P(HPMA) conjugate P-Ahx-NH-N═MeOHe-R121(Dox) containing Dox and the P-gp inhibitor MeOHe-R121, both bound via hydrazone bonds to the P(HPMA) carrier, was almost 30 times higher than that of the conjugate P-Ahx-NH-N═Dox toward the P388/MDR cells in vitro. A similar result was observed for P-Ahx-NH-N═MeOHe-RIT(Dox), which exhibited almost 10 times higher cytostatic activity than P-Ahx-NH-N═Dox.

  2. OSCILLATORY PUMP

    DOEpatents

    Underwood, N.

    1958-09-23

    This patent relates to a pump suitable fur pumping highly corrosive gases wherein no lubricant is needed in the pumping chamber thus eliminating possible contamination sources. The chamber contains a gas inlet and outlet in each side, with a paddle like piston suspended by a sylphon seal between these pcrts. An external arrangement causes the paddle to oscillate rapidly between the ports, alternately compressing and exhausting the gas trapped on each side of the paddle. Since the paddle does nnt touch the chamber sides at any point, no lubricant is required. This pump is useful for pumping large quantities of uranium hexafluorine.

  3. Polarized location of SLC and ABC drug transporters in monolayer-cultured human hepatocytes.

    PubMed

    Le Vee, Marc; Jouan, Elodie; Noel, Gregory; Stieger, Bruno; Fardel, Olivier

    2015-08-01

    Human hepatocytes cultured in a monolayer configuration represent a well-established in vitro model in liver toxicology, notably used in drug transporter studies. Polarized status of drug transporters, i.e., their coordinated location at sinusoidal or canalicular membranes, remains however incompletely documented in these cultured hepatocytes. The present study was therefore designed to analyze transporter expression and location in such cells. Most of drug transporters were first shown to be present at notable mRNA levels in monolayer-cultured human hepatocytes. Cultured human hepatocytes, which morphologically exhibited bile canaliculi-like structures, were next demonstrated, through immunofluorescence staining, to express the influx transporters organic anion transporting polypeptide (OATP) 1B1, OATP2B1 and organic cation transporter (OCT) 1 and the efflux transporter multidrug resistance-associated protein (MRP) 3 at their sinusoidal pole. In addition, the efflux transporters P-glycoprotein and MRP2 were detected at the canalicular pole of monolayer-cultured human hepatocytes. Moreover, canalicular secretion of reference substrates for the efflux transporters bile salt export pump, MRP2 and P-glycoprotein as well as sinusoidal drug transporter activities were observed. This polarized and functional expression of drug transporters in monolayer-cultured human hepatocytes highlights the interest of using this human in vitro cell model in xenobiotic transport studies.

  4. Multi-drug resistance in Salmonella enterica: efflux mechanisms and their relationships with the development of chromosomal resistance gene clusters.

    PubMed

    Quinn, Teresa; O'Mahony, Rebecca; Baird, Alan W; Drudy, Denise; Whyte, Paul; Fanning, Séamus

    2006-07-01

    Bacterial drug resistance represents one of the most crucial problems in present day antibacterial chemotherapy. Of particular concern to public health is the continuing worldwide epidemic spread of Salmonella enterica serovar Typhimurium phage type DT104 harbouring a genomic island called Salmonella genomic island I (SGI-1). This island contains an antibiotic gene cluster conferring resistance to ampicillin, chloramphenicol, florfenicol, streptomycin, sulfonamides and tetracyclines. These resistance genes are assembled in a mosaic pattern, indicative of several independent recombinational events. The mobility of SGI-1 coupled with the ability of various antibiotic resistance genes to be integrated and lost from the chromosomal resistance locus allows for the transfer of stable antibiotic resistance to most of the commonly used antibiotics and adaptation to new antibiotic challenges. This, coupled with the incidence of increasing fluoroquinolone resistance in these strains increases the risk of therapeutic failure in cases of life-threatening salmonellosis. Fluoroquinolone resistance has largely been attributed to mutations occurring in the genes coding for intracellular targets of these drugs. However, efflux by the AcrAB-TolC multi-drug efflux pump has recently been shown to directly contribute to fluoroquinolone resistance. Furthermore, the resistance to chloramphenicol-florfenicol and tetracyclines in DT104 isolates, is due to interaction between specific transporters for these antibiotics encoded by genes mapping to the SGI-1 and the AcrAB-TolC tripartite efflux pump. The potential for the use of efflux pump inhibitors to restore therapeutic efficacy to fluoroquinolones and other antibiotics offers an exciting developmental area for drug discovery. PMID:16842216

  5. Efflux by small multidrug resistance proteins is inhibited by membrane-interactive helix-stapled peptides.

    PubMed

    Bellmann-Sickert, Kathrin; Stone, Tracy A; Poulsen, Bradley E; Deber, Charles M

    2015-01-16

    Bacterial cell membranes contain several protein pumps that resist the toxic effects of drugs by efficiently extruding them. One family of these pumps, the small multidrug resistance proteins (SMRs), consists of proteins of about 110 residues that need to oligomerize to form a structural pathway for substrate extrusion. As such, SMR oligomerization sites should constitute viable targets for efflux inhibition, by disrupting protein-protein interactions between helical segments. To explore this proposition, we are using Hsmr, an SMR from Halobacter salinarum that dimerizes to extrude toxicants. Our previous work established that (i) Hsmr dimerization is mediated by a helix-helix interface in Hsmr transmembrane (TM) helix 4 (residues (90)GLALIVAGV(98)); and (ii) a peptide comprised of the full TM4(85-105) sequence inhibits Hsmr-mediated ethidium bromide efflux from bacterial cells. Here we define the minimal linear sequence for inhibitor activity (determined as TM4(88-100), and then "staple" this sequence via Grubbs metathesis to produce peptides typified by acetyl-A-(Sar)3-(88)VVGLXLIZXGVVV(100)-KKK-NH2 (X = 2-(4'-pentenyl)alanine at positions 92 and 96; Z = Val, Gly, or Asn at position 95)). The Asn(95) peptide displayed specific efflux inhibition and resensitization of Hsmr-expressing cells to ethidium bromide; and was non-hemolytic to human red blood cells. Stapling essentially prevented peptide degradation in blood plasma and liver homogenates versus an unstapled counterpart. The overall results confirm that the stapled analog of TM4(88-100) retains the structural complementarity required to disrupt the Hsmr TM4-TM4 locus in Hsmr, and portend the general validity of stapled peptides as therapeutics for the disruption of functional protein-protein interactions in membranes.

  6. Efflux by Small Multidrug Resistance Proteins Is Inhibited by Membrane-interactive Helix-stapled Peptides*

    PubMed Central

    Bellmann-Sickert, Kathrin; Stone, Tracy A.; Poulsen, Bradley E.; Deber, Charles M.

    2015-01-01

    Bacterial cell membranes contain several protein pumps that resist the toxic effects of drugs by efficiently extruding them. One family of these pumps, the small multidrug resistance proteins (SMRs), consists of proteins of about 110 residues that need to oligomerize to form a structural pathway for substrate extrusion. As such, SMR oligomerization sites should constitute viable targets for efflux inhibition, by disrupting protein-protein interactions between helical segments. To explore this proposition, we are using Hsmr, an SMR from Halobacter salinarum that dimerizes to extrude toxicants. Our previous work established that (i) Hsmr dimerization is mediated by a helix-helix interface in Hsmr transmembrane (TM) helix 4 (residues 90GLALIVAGV98); and (ii) a peptide comprised of the full TM4(85–105) sequence inhibits Hsmr-mediated ethidium bromide efflux from bacterial cells. Here we define the minimal linear sequence for inhibitor activity (determined as TM4(88–100), and then “staple” this sequence via Grubbs metathesis to produce peptides typified by acetyl-A-(Sar)3-88VVGLXLIZXGVVV100-KKK-NH2 (X = 2-(4′-pentenyl)alanine at positions 92 and 96; Z = Val, Gly, or Asn at position 95)). The Asn95 peptide displayed specific efflux inhibition and resensitization of Hsmr-expressing cells to ethidium bromide; and was non-hemolytic to human red blood cells. Stapling essentially prevented peptide degradation in blood plasma and liver homogenates versus an unstapled counterpart. The overall results confirm that the stapled analog of TM4(88–100) retains the structural complementarity required to disrupt the Hsmr TM4-TM4 locus in Hsmr, and portend the general validity of stapled peptides as therapeutics for the disruption of functional protein-protein interactions in membranes. PMID:25425644

  7. Characterization of the multidrug efflux regulator AcrR from Escherichia coli

    PubMed Central

    Su, Chih-Chia; Rutherford, Denae J.; Yu, Edward W.

    2007-01-01

    The Escherichia coli AcrR represses transcription of the acrB gene, which encodes the multidrug efflux pump AcrB that extrudes a wide variety of toxic compounds, by binding its target operator DNA. Fluorescence polarization was performed using purified, recombinant AcrR that contains a 6xHis tag at the C-terminus and a fluorescein-labeled 28-base pair oligonucleotide bearing a predicted palindrome (IR) operator sequence. Binding of AcrR to the predicted IR sequence occurred with a dissociation constant (KD) in the nanomolar range. Fluorescence polarization assays were also applied to characterize the affinity and specificity of AcrR interaction with three different fluorescent ligands, rhodamine 6G, ethidium and proflavin. The KD values for these ligands range from 4.2 to 10.1 μM, suggesting that AcrR is capable of recognizing a wide range of structurally dissimilar toxic compounds as it is in the case of the AcrB multidrug efflux pump. We found that the binding of rhodamine 6G to AcrR is inhibited by the presence of ethidium. In contrast, the dissociation constant of proflavin binding to AcrR was not affected by ethidium, a result suggesting that ethidium and proflavin are bound to distinct binding sites. PMID:17644067

  8. ArsP: a methylarsenite efflux permease

    PubMed Central

    Chen, Jian; Madegowda, Mahendra; Bhattacharjee, Hiranmoy; Rosen, Barry P.

    2015-01-01

    Trivalent organoarsenic compounds are far more toxic than either pentavalent organoarsenicals or inorganic arsenite. Many microbes methylate inorganic arsenite (As(III)) to more toxic and carcinogenic methylarsenite (MAs(III)). Additionally, monosodium methylarsenate (MSMA or MAs(V)) has been used widely as an herbicide and is reduced by microbial communities to MAs(III). Roxarsone (3-nitro-4-hydroxybenzenearsonic acid) is a pentavalent aromatic arsenical that is used as antimicrobial growth promoter for poultry and swine, and its active form is the trivalent species Rox(III). A bacterial permease, ArsP, from Campylobacter jejuni, was recently shown to confer resistance to roxarsone. In this study C. jejuni arsP was expressed in Escherichia coli and shown to confer resistance to MAs(III) and Rox(III) but not to inorganic As(III) or pentavalent organoarsenicals. Cells of E. coli expressing arsP did not accumulate trivalent organoarsenicals. Everted membrane vesicles from those cells accumulated MAs(III)>Rox(III) with energy supplied by NADH oxidation, reflecting efflux from cells. The vesicles did not transport As(III), MAs(V) or pentavalent roxarsone. Mutation or modification of the two conserved cysteine residues resulted in loss of transport activity, suggesting that they play a role in ArsP function. Thus ArsP is the first identified efflux system specific for trivalent organoarsenicals. PMID:26234817

  9. AcrAB-TolC Directs Efflux-Mediated Multidrug Resistance in Salmonella enterica Serovar Typhimurium DT104

    PubMed Central

    Baucheron, Sylvie; Tyler, Shaun; Boyd, David; Mulvey, Michael R.; Chaslus-Dancla, Elisabeth; Cloeckaert, Axel

    2004-01-01

    Multidrug-resistant Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) strains harbor a genomic island, called Salmonella genomic island 1 (SGI1), which contains an antibiotic resistance gene cluster conferring resistance to ampicillin, chloramphenicol, florfenicol, streptomycin, sulfonamides, and tetracyclines. They may be additionally resistant to quinolones. Among the antibiotic resistance genes there are two, i.e., floR and tet(G), which code for efflux pumps of the major facilitator superfamily with 12 transmembrane segments that confer resistance to chloramphenicol-florfenicol and the tetracyclines, respectively. In the present study we determined, by constructing acrB and tolC mutants, the role of the AcrAB-TolC multidrug efflux system in the multidrug resistance of several DT104 strains displaying additional quinolone resistance or not displaying quinolone resistance. This study shows that the quinolone resistance and the decreased fluoroquinolone susceptibilities of the strains are highly dependent on the AcrAB-TolC efflux system and that single mutations in the quinolone resistance-determining region of gyrA are of little relevance in mediating this resistance. Overproduction of the AcrAB efflux pump, as determined by Western blotting with an anti-AcrA polyclonal antibody, appeared to be the major mechanism of resistance to quinolones. Moreover, chloramphenicol-florfenicol and tetracycline resistance also appeared to be highly dependent on the presence of AcrAB-TolC, since the introduction of mutations in the respective acrB and tolC genes resulted in a susceptible or intermediate resistance phenotype, according to clinical MIC breakpoints, despite the presence of the FloR and Tet(G) efflux pumps. Resistance to other antibiotics, ampicillin, streptomycin, and sulfonamides, was not affected in the acrB and tolC mutants of DT104 strains harboring SGI1. Therefore, AcrAB-TolC appears to direct efflux-mediated resistance to quinolones

  10. The Role of the Atypical Kinases ABC1K7 and ABC1K8 in Abscisic Acid Responses

    PubMed Central

    Manara, Anna; DalCorso, Giovanni; Furini, Antonella

    2016-01-01

    The ABC1K family of atypical kinases (activity of bc1 complex kinase) is represented in bacteria, archaea, and eukaryotes. In plants they regulate diverse physiological processes in the chloroplasts and mitochondria, but their precise functions are poorly defined. ABC1K7 and ABC1K8 are probably involved in oxidative stress responses, isoprenyl lipid synthesis and distribution of iron within chloroplasts. Because reactive oxygen species take part in abscisic acid (ABA)-mediated processes, we investigated the functions of ABC1K7 and ABC1K8 during germination, stomatal movement, and leaf senescence. Both genes were upregulated by ABA treatment and some ABA-responsive physiological processes were affected in abc1k7 and abc1k8 mutants. Germination was more severely affected by ABA, osmotic stress and salt stress in the single and double mutants; the stomatal aperture was smaller in the mutants under standard growth conditions and was not further reduced by exogenous ABA application; ABA-induced senescence symptoms were more severe in the leaves of the single and double mutants compared to wild type leaves. Taken together, our results suggest that ABC1K7 and ABC1K8 might be involved in the cross-talk between ABA and ROS signaling. PMID:27047531

  11. The Role of the Atypical Kinases ABC1K7 and ABC1K8 in Abscisic Acid Responses.

    PubMed

    Manara, Anna; DalCorso, Giovanni; Furini, Antonella

    2016-01-01

    The ABC1K family of atypical kinases (activity of bc1 complex kinase) is represented in bacteria, archaea, and eukaryotes. In plants they regulate diverse physiological processes in the chloroplasts and mitochondria, but their precise functions are poorly defined. ABC1K7 and ABC1K8 are probably involved in oxidative stress responses, isoprenyl lipid synthesis and distribution of iron within chloroplasts. Because reactive oxygen species take part in abscisic acid (ABA)-mediated processes, we investigated the functions of ABC1K7 and ABC1K8 during germination, stomatal movement, and leaf senescence. Both genes were upregulated by ABA treatment and some ABA-responsive physiological processes were affected in abc1k7 and abc1k8 mutants. Germination was more severely affected by ABA, osmotic stress and salt stress in the single and double mutants; the stomatal aperture was smaller in the mutants under standard growth conditions and was not further reduced by exogenous ABA application; ABA-induced senescence symptoms were more severe in the leaves of the single and double mutants compared to wild type leaves. Taken together, our results suggest that ABC1K7 and ABC1K8 might be involved in the cross-talk between ABA and ROS signaling. PMID:27047531

  12. 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

  13. APOLLO 13: A News Bulletin from ABC

    NASA Technical Reports Server (NTRS)

    1974-01-01

    APOLLO 13: ABC breaks the news of a mishap aboard the spacecraft From the film documentary 'APOLLO 13: 'Houston, We've got a problem'', part of a documentary series on the APOLLO missions made in the early '70's and narrated by Burgess Meredith. APOLO 13 : Third manned lunar landing attempt with James A. Lovell, Jr., John L. Swigert, Jr., and Fred W. Haise, Jr. Pressure lost in SM oxygen system; mission aborted; LM used for life support. Mission Duration 142hrs 54mins 41sec

  14. ABC makes the switch to digital

    NASA Astrophysics Data System (ADS)

    Pearl, R. G.

    1984-05-01

    The digital satellite-distribution system being completed for the ABC radio network is briefly characterized. Audio programming on 19 channels at 15 kHz is digitized at the studio and transmitted by microwave to the uplink facility for distribution via Satcom I-R to 1800 affiliates with affiliate-owned receivers. A data channel comprising several 32-kbit/sec subchannels operates through network-owned data cards and printers installed in the affiliate ground stations to provide internal communication (to all affiliates, a selected group, or a single station) using soft addresses, data-distribution services for station customers, or the proposed 900-MHz nationwide paging service.

  15. Sodium efflux in plant roots: what do we really know?

    PubMed

    Britto, D T; Kronzucker, H J

    2015-08-15

    The efflux of sodium (Na(+)) ions across the plasma membrane of plant root cells into the external medium is surprisingly poorly understood. Nevertheless, Na(+) efflux is widely regarded as a major mechanism by which plants restrain the rise of Na(+) concentrations in the cytosolic compartments of root cells and, thus, achieve a degree of tolerance to saline environments. In this review, several key ideas and bodies of evidence concerning root Na(+) efflux are summarized with a critical eye. Findings from decades past are brought to bear on current thinking, and pivotal studies are discussed, both "purely physiological", and also with regard to the SOS1 protein, the only major Na(+) efflux transporter that has, to date, been genetically characterized. We find that the current model of rapid transmembrane sodium cycling (RTSC), across the plasma membrane of root cells, is not adequately supported by evidence from the majority of efflux studies. An alternative hypothesis cannot be ruled out, that most Na(+) tracer efflux from the root in the salinity range does not proceed across the plasma membrane, but through the apoplast. Support for this idea comes from studies showing that Na(+) efflux, when measured with tracers, is rarely affected by the presence of inhibitors or the ionic composition in saline rooting media. We conclude that the actual efflux of Na(+) across the plasma membrane of root cells may be much more modest than what is often reported in studies using tracers, and may predominantly occur in the root tips, where SOS1 expression has been localized.

  16. Insulin pumps.

    PubMed

    Pickup, J

    2010-02-01

    Insulin pump therapy is now more than 30 years old, and is an established part of the routine care of selected people with type 1 diabetes. Nevertheless, there are still significant areas of concern, particularly how pumps compare with modern injection therapy, whether the increasingly sophisticated pump technologies like onboard calculators and facility for computer download offer any real benefit, and whether we have a consensus on the clinical indications. The following papers offer some insight into these and other current questions.

  17. Danofloxacin-mesylate is a substrate for ATP-dependent efflux transporters

    PubMed Central

    Schrickx, J A; Fink-Gremmels, J

    2007-01-01

    Background and purpose: Next to its broad antimicrobial spectrum, the therapeutic advantages of the fluoroquinolone antimicrobial drug Danofloxacin-Mesylate (DM) are attributed to its rapid distribution to the major target tissues such as lungs, intestines and the mammary gland in animals. Previous analyses revealed that effective drug concentrations are achieved also in luminal compartments of these organs, suggesting that active transport proteins facilitate excretion into the luminal space. Members of the ATP-Binding Cassette (ABC) superfamily, including P-gp, BCRP and MRP2 are known to be expressed in many tissue barriers and in cell-membranes facing luminal compartments. Hence we hypothesized that DM is a substrate for one of these efflux-transporters. Experimental approach: Confluent monolayers of Caco-2 cells, grown on microporous membranes in two-chamber devices were used. DM concentrations were measured by fluorimetric assay after HPLC of the culture media. Key results: DM transport across Caco-2 cells was asymmetric, with a rate of secretion exceeding that of absorption. The P-gp inhibitors PSC833 and GF120918 and the MRP-inhibitor MK571 partially decreased the secretion of DM and increased its absorption rate. The BCRP inhibitor, Ko143, decreased secretion only at a concentration of 1 μM. When DM was applied together with ciprofloxacin, secretion as well as absorption of DM decreased. Conclusions and Implications: DM is a substrate for the efflux transporters P-gp and MRP2, whereas the specific role of BCRP in DM transport needs further evaluation. These findings provide a mechanistic basis for the understanding of the pharmacokinetics of DM in healthy and diseased individuals. PMID:17211460

  18. Telatinib reverses chemotherapeutic multidrug resistance mediated by ABCG2 efflux transporter in vitro and in vivo

    PubMed Central

    Sodani, Kamlesh; Patel, Atish; Anreddy, Nagaraju; Singh, Satyakam; Yang, Dong-Hua; Kathawala, Rishil J; Kumar, Priyank; Talele, Tanaji T; Chen, Zhe-Sheng

    2014-01-01

    Multidrug resistance (MDR) is a phenomenon where cancer cells become simultaneously resistant to anticancer drugs with different structures and mechanisms of action. MDR has been shown to be associated with overexpression of ATP-binding cassette (ABC) transporters. Here, we report that telatinib, a small molecule tyrosine kinase inhibitor, enhances the anticancer activity of ABCG2 substrate anticancer drugs by inhibiting ABCG2 efflux transporter activity. Co-incubation of ABCG2-overexpressing drug resistant cell lines with telatinib and ABCG2 substrate anticancer drugs significantly reduced cellular viability, whereas telatinib alone did not significantly affect drug sensitive and drug resistant cell lines. Telatinib at 1 μM did not significantly alter the expression of ABCG2 in ABCG2-overexpressing cell lines. Telatinib at 1 μM significantly enhanced the intracellular accumulation of [3H]-mitoxantrone (MX) in ABCG2-overexpressing cell lines. In addition, telatinib at 1 μM significantly reduced the rate of [3H]-MX efflux from ABCG2-overexpressing cells. Furthermore, telatinib significantly inhibited ABCG2-mediated transport of [3H]-E217βG in ABCG2 overexpressing membrane vesicles. Telatinib stimulated the ATPase activity of ABCG2 in a concentration-dependent manner, indicating that telatinib might be a substrate of ABCG2. Binding interactions of telatinib were found to be in transmembrane region of homology modeled human ABCG2. In addition, telatinib (15 mg/kg) with doxorubicin (1.8 mg/kg) significantly decreased the growth rate and tumor size of ABCG2 overexpressing tumors in a xenograft nude mouse model. These results, provided that they can be translated to humans, suggesting that telatinib, in combination with specific ABCG2 substrate drugs may be useful in treating tumors that overexpress ABCG2. PMID:24565910

  19. The Reign of Confusion: ABC and the "Crisis in Iran."

    ERIC Educational Resources Information Center

    Palmerton, Patricia R.

    A study examined reports broadcast by ABC News between November 8, 1979 and December 7, 1979 in its series entitled "Crisis in Iran: America Held Hostage." Transcripts of approximately 50% of actual broadcasts were subjected to rhetorical critical analysis, from which the finding emerged that confusion was the predominant characteristic in ABC's…

  20. The ABCs of School Choice, 2009-2010 Edition

    ERIC Educational Resources Information Center

    Friedman Foundation for Educational Choice, 2010

    2010-01-01

    This publication presents the 2009-2010 edition of the Friedman Foundation for Educational Choice's "ABCs of School Choice". The "ABCs of School Choice" provides the latest in up-to-date and accurate information about the many school choice success stories taking place throughout the country. Readers will find this guide an essential resource on…

  1. To What Extent Does Attention Affect K-ABC Scores?

    ERIC Educational Resources Information Center

    Gordon, Michael; And Others

    1990-01-01

    Analyzed the protocols of 52 clinic-referred children who were administered the Kaufman Assessment Battery for Children (K-ABC) as well as version of the Continuous Performance Test (CPT), a laboratory measure of attention. Results demonstrated significant interrelationships among K-ABC and CPT scores. (Author/ABL)

  2. ABCs of Being Smart: S Is for Supporting

    ERIC Educational Resources Information Center

    Foster, Joanne

    2014-01-01

    Joanne Foster's article "R We There Yet?" was first published in "Parenting for High Potential" ("PHP") in 2006, which became the springboard for the "ABCs of Being Smart" series of columns. At that time, Foster invited "PHP" readers to think about their own versions of the "ABCs of Being…

  3. Measuring Academic Behavioural Confidence: The ABC Scale Revisited

    ERIC Educational Resources Information Center

    Sander, Paul; Sanders, Lalage

    2009-01-01

    The Academic Behavioural Confidence (ABC) scale has been shown to be valid and can be useful to teachers in understanding their students, enabling the design of more effective teaching sessions with large cohorts. However, some of the between-group differences have been smaller than expected, leading to the hypothesis that the ABC scale many not…

  4. Cholesterol efflux capacity: An introduction for clinicians.

    PubMed

    Anastasius, Malcolm; Kockx, Maaike; Jessup, Wendy; Sullivan, David; Rye, Kerry-Anne; Kritharides, Leonard

    2016-10-01

    Epidemiologic studies have shown an inverse correlation between high-density lipoprotein (HDL) cholesterol (HDL-C) levels and cardiovascular disease outcomes. However, the hypothesis of a causal relationship between HDL-C and cardiovascular disease has been challenged by genetic and clinical studies. Serum cholesterol efflux capacity (CEC) is an important measure of HDL function in humans. Recent large clinical studies have shown a correlation between in vitro CEC and cardiovascular disease prevalence and incidence, which appears to be independent of HDL-C concentration. The present review summarizes recent large clinical studies and introduces important methodological considerations. Further studies are required to standardize and establish the reproducibility of this measure of HDL function and clarify whether modulating CEC will emerge as a useful therapeutic target. PMID:27659883

  5. [Role of the ABC transporters A1 and G1, key reverse cholesterol transport proteins, in atherosclerosis].

    PubMed

    Demina, E P; Miroshnikova, V V; Schwarzman, A L

    2016-01-01

    Atherosclerosis is one of the most common causes of death worldwide. Epidemiology studies firmly established an inverse relationship between atherogenesis and distorted lipid metabolism, in particular, higher levels of total cholesterol, an accumulation of CH-laden macrophages (foam cells), and lower plasma levels of antiatherogenic high density lipoprotein (HDL). It is believed that the reverse cholesterol transport, a process that removes excess cholesterol from peripheral tissues/cells including macrophages to circulating HDL, is one of the main mechanisms responsible for anti-atherogenic properties of HDL. The key proteins of reverse cholesterol transport-ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1)-mediate the cholesterol efflux from macrophages and prevent their transformation into foam cells. This review focuses on the role of ABC transporters A1 and G1 in the pathogenesis of atherosclerosis.

  6. Ferroelectric Pump

    NASA Technical Reports Server (NTRS)

    Jalink, Antony, Jr. (Inventor); Hellbaum, Richard F. (Inventor); Rohrbach, Wayne W. (Inventor)

    2000-01-01

    A ferroelectric pump has one or more variable volume pumping chambers internal to a housing. Each chamber has at least one wall comprising a dome shaped internally prestressed ferroelectric actuator having a curvature and a dome height that varies with an electric voltage applied between an inside and outside surface of the actuator. A pumped medium flows into and out of each pumping chamber in response to displacement of the ferroelectric actuator. The ferroelectric actuator is mounted within each wall and isolates each ferroelectric actuator from the pumped medium, supplies a path for voltage to be applied to each ferroelectric actuator, and provides for positive containment of each ferroelectric actuator while allowing displacement of the entirety of each ferroelectric actuator in response to the applied voltage.

  7. Axial Pump

    NASA Technical Reports Server (NTRS)

    Bozeman, Richard J., Jr. (Inventor); Akkerman, James W. (Inventor); Aber, Gregory S. (Inventor); VanDamm, George Arthur (Inventor); Bacak, James W. (Inventor); Svejkovsky, Paul A. (Inventor); Benkowski, Robert J. (Inventor)

    1997-01-01

    A rotary blood pump includes a pump housing for receiving a flow straightener, a rotor mounted on rotor bearings and having an inducer portion and an impeller portion, and a diffuser. The entrance angle, outlet angle, axial and radial clearances of blades associated with the flow straightener, inducer portion, impeller portion and diffuser are optimized to minimize hemolysis while maintaining pump efficiency. The rotor bearing includes a bearing chamber that is filled with cross-linked blood or other bio-compatible material. A back emf integrated circuit regulates rotor operation and a microcomputer may be used to control one or more back emf integrated circuits. A plurality of magnets are disposed in each of a plurality of impeller blades with a small air gap. A stator may be axially adjusted on the pump housing to absorb bearing load and maximize pump efficiency.

  8. Alkylrhodamines enhance the toxicity of clotrimazole and benzalkonium chloride by interfering with yeast pleiotropic ABC-transporters.

    PubMed

    Knorre, Dmitry A; Besedina, Elizaveta; Karavaeva, Iuliia E; Smirnova, Ekaterina A; Markova, Olga V; Severin, Fedor F

    2016-06-01

    ABC-transporters with broad substrate specificity are responsible for pathogenic yeast resistance to antifungal compounds. Here we asked whether highly hydrophobic chemicals with delocalized positive charge can be used to overcome the resistance. Such molecules efficiently penetrate the plasma membrane and accumulate inside the cells. We reasoned that these properties can convert an active efflux of the compounds into a futile cycle thus interfering with the extrusion of the antibiotics. To test this, we studied the effects of several alkylated rhodamines on the drug resistance of yeast Saccharomyces cerevisiae We found that octylrhodamine synergetically increases toxicity of Pdr5p substrate-clotrimazole, while the others were less effective. Next, we compared the contributions of three major pleiotropic ABC-transporters (Pdr5p, Yor1p, Snq2p) on the accumulation of the alkylated rhodamines. While all of the tested compounds were extruded by Pdr5p, Yor1p and Snq2p showed narrower substrate specificity. Interestingly, among the tested alkylated rhodamines, inactivation of Pdr5p had the strongest effect on the accumulation of octylrhodamine inside the cells, which is consistent with the fact that clotrimazole is a substrate of Pdr5p. As alkylated rhodamines were shown to be non-toxic on mice, our study makes them potential components of pharmacological antifungal compositions. PMID:27044313

  9. Submersible pump

    SciTech Connect

    Todd, D. B.

    1985-08-27

    A method and apparatus for using a submersible pump to lift reservoir fluids in a well while having the tubing/casing annulus isolated from the produced fluids. The apparatus allows the submersible pump to be positioned above the annular packoff device. The apparatus comprises an outer shield that encloses the pump and can be attached to the production tubing. The lower end of the shield attaches to a short tubing section that seals with the annular packoff device or a receptacle above the annular packoff device.

  10. Identification of a Cyanobacterial RND-Type Efflux System Involved in Export of Free Fatty Acids.

    PubMed

    Kato, Akihiro; Takatani, Nobuyuki; Use, Kazuhide; Uesaka, Kazuma; Ikeda, Kazutaka; Chang, Yajun; Kojima, Kouji; Aichi, Makiko; Ihara, Kunio; Nakahigashi, Kenji; Maeda, Shin-Ichi; Omata, Tatsuo

    2015-12-01

    An RND (resistance-nodulation-division)-type transporter having the capacity to export free fatty acids (FFAs) was identified in the cyanobacterium Synechococcus elongatus strain PCC 7942 during characterization of a mutant strain engineered to produce FFAs. The basic strategy for construction of the FFA-producing mutant was a commonly used one, involving inactivation of the endogenous acyl-acyl carrier protein synthetase gene (aas) and introduction of a foreign thioesterase gene ('tesA), but a nitrate transport mutant NA3 was used as the parental strain to achieve slow, nitrate-limited growth in batch cultures. Also, a nitrogen-regulated promoter PnirA was used to drive 'tesA to maximize thioesterase expression during the nitrate-limited growth. The resulting mutant (dAS2T) was, however, incapable of growth under the conditions of nitrate limitation, presumably due to toxicity associated with FFA overproduction. Incubation of the mutant culture under the non-permissive conditions allowed for isolation of a pseudorevertant (dAS2T-pr1) capable of growth on nitrate. Genome sequence and gene expression analyses of this strain suggested that expression of an RND-type efflux system had rescued growth on nitrate. Targeted inactivation of the RND-type transporter genes in the wild-type strain resulted in loss of tolerance to exogenously added FFAs including capric, lauric, myristic, oleic and linolenic acids. Overexpression of the genes in dAS2T, on the other hand, enhanced FFA excretion and cell growth in nitrate-containing medium, verifying that the genes encode an efflux pump for FFAs. These results demonstrate the importance of the efflux system in efficient FFA production using genetically engineered cyanobacteria. PMID:26468506

  11. Importance of nondiffusive transport for soil CO2 efflux in a temperate mountain grassland

    NASA Astrophysics Data System (ADS)

    Roland, Marilyn; Vicca, Sara; Bahn, Michael; Ladreiter-Knauss, Thomas; Schmitt, Michael; Janssens, Ivan A.

    2015-03-01

    Soil respiration and its biotic and abiotic drivers have been an important research topic in recent years. While the bulk of these efforts has focused on the emission of CO2 from soils, the production and subsequent transport of CO2 from soil to atmosphere received far less attention. However, to understand processes underlying emissions of CO2 from terrestrial ecosystems, both processes need to be fully evaluated. In this study, we tested to what extent the transport of CO2 in a grassland site in the Austrian Alps could be modeled based on the common assumption that diffusion is the main transport mechanism for trace gases in soils. Therefore, we compared the CO2 efflux calculated from the soil CO2 concentration gradient with the CO2 efflux from chamber measurements. We used four commonly used diffusion-driven models for the flux-gradient approach. Models generally underestimated the soil chamber effluxes and their amplitudes, indicating that processes other than diffusion were responsible for the transport of CO2. We further observed that transport rates correlated well with irradiation and, below a soil moisture content of 33%, with wind speed. This suggests that mechanisms such as bulk soil air transport, due to pressure pumping or thermal expansion of soil air due to local surface heating, considerably influence soil CO2 transport at this site. Our results suggest that nondiffusive transport may be an important mechanism influencing diel and day-to-day dynamics of soil CO2 emissions, leading to a significant mismatch (10-87% depending on the model used) between the two approaches at short time scales.

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

    PubMed

    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

    2016-04-21

    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.

  13. 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

  14. PI3K signaling supports amphetamine-induced dopamine efflux.

    PubMed

    Lute, Brandon J; Khoshbouei, Habibeh; Saunders, Christine; Sen, Namita; Lin, Richard Z; Javitch, Jonathan A; Galli, Aurelio

    2008-08-01

    The dopamine (DA) transporter (DAT) is a major molecular target of the psychostimulant amphetamine (AMPH). AMPH, as a result of its ability to reverse DAT-mediated inward transport of DA, induces DA efflux thereby increasing extracellular DA levels. This increase is thought to underlie the behavioral effects of AMPH. We have demonstrated previously that insulin, through phosphatidylinositol 3-kinase (PI3K) signaling, regulates DA clearance by fine-tuning DAT plasma membrane expression. PI3K signaling may represent a novel mechanism for regulating DA efflux evoked by AMPH, since only active DAT at the plasma membrane can efflux DA. Here, we show in both a heterologous expression system and DA neurons that inhibition of PI3K decreases DAT cell surface expression and, as a consequence, AMPH-induced DA efflux.

  15. ION PUMP

    DOEpatents

    Milleron, N.

    1961-01-01

    An ion pump and pumping method are given for low vacuum pressures in which gases introduced into a pumping cavity are ionized and thereafter directed and accelerated into a quantity of liquid gettering metal where they are absorbed. In the preferred embodiment the metal is disposed as a liquid pool upon one electrode of a Phillips ion gauge type pump. Means are provided for continuously and remotely withdrawing and degassing the gettering metal. The liquid gettering metal may be heated if desired, although various combinations of gallium, indium, tin, bismuth, and lead, the preferred metals, have very low melting points. A background pressure of evaporated gettering metal may be provided by means of a resistance heated refractory metal wick protruding from the surface of the pcol of gettering metal.

  16. Electrokinetic pump

    DOEpatents

    Patel, Kamlesh D.

    2007-11-20

    A method for altering the surface properties of a particle bed. In application, the method pertains particularly to an electrokinetic pump configuration where nanoparticles are bonded to the surface of the stationary phase to alter the surface properties of the stationary phase including the surface area and/or the zeta potential and thus improve the efficiency and operating range of these pumps. By functionalizing the nanoparticles to change the zeta potential the electrokinetic pump is rendered capable of operating with working fluids having pH values that can range from 2-10 generally and acidic working fluids in particular. For applications in which the pump is intended to handle highly acidic solutions latex nanoparticles that are quaternary amine functionalized can be used.

  17. Charged Amino Acids (R83, E567, D617, E625, R669, and K678) of CusA Are Required for Metal Ion Transport in the Cus Efflux System

    SciTech Connect

    Su, Chih-Chia; Long, Feng; Lei, Hsiang-Ting; Reddy Bolla, Jani; Do, Sylvia V.; Rajashankar, Kanagalaghatta R.; Yu, Edward W.

    2012-10-23

    Gram-negative bacteria expel various toxic chemicals via tripartite efflux pumps belonging to the resistance-nodulation-cell division superfamily. These pumps span both the inner and outer membranes of the cell. The three components of these tripartite systems are an inner-membrane, substrate-binding transporter (or pump); a periplasmic membrane fusion protein (or adaptor); and an outer-membrane-anchored channel. These three efflux proteins interact in the periplasmic space to form the three-part complexes. We previously presented the crystal structures of both the inner-membrane transporter CusA and membrane fusion protein CusB of the CusCBA tripartite efflux system from Escherichia coli. We also described the co-crystal structure of the CusBA adaptor-transporter, revealing that the trimeric CusA efflux pump assembles with six CusB protein molecules to form the complex CusB{sub 6}-CusA{sub 3}. We here report three different conformers of the crystal structures of CusBA-Cu(I), suggesting a mechanism on how Cu(I) binding initiates a sequence of conformational transitions in the transport cycle. Genetic analysis and transport assays indicate that charged residues, in addition to the methionine pairs and clusters, are essential for extruding metal ions out of the cell.

  18. Cadmium induced potassium efflux from Scenedesmus quadricauda

    SciTech Connect

    Reddy, G.N.; Prasad, M.N.V.

    1992-10-01

    Plants, algae and bacteria respond to heavy metal toxicity by inducing different enzymes, ion influx/efflux for ionic balance and synthesize small peptides such as poly({gamma}-glutamyl cysteinyl) glycines called phytochelatins (PCs) mainly consisting of glutamate, cysteine and glycine. These peptides bind metal ions and reduce toxicity. The uptake of metal ions comprises two phases. The first phase consists of a quick and nonspecific binding of the cations to negatively-charged membrane components located at the cell surface. The second phase consists of energy-dependent intracellular uptake of the metal ions. During uptake of Co{sup 2+} by yeast cells, an electroneutral 2:1 exchange with K{sup +} was found. Cd{sup 2+} uptake by yeast also caused loss of cell K{sup +}, however, there was no electroneutral exchange of K{sup +}. The molar ratio of K{sup +} released and Cd{sup 2+} accumulated by yeast in the initial stage of incubation is 22 and seems to be independent of the Cd concentration. Disruption of the cell membrane of part of the cells, according to an all-or-none process, by Cd{sup 2+} may explain the disproportional loss of cell K{sup +} during Cd{sup 2+} uptake. This paper examines the exchange of K{sup +} with Cd{sup 2+} uptake in Scenedesmus quadricauda, and whether it follows an electroneutral 2:1 exchange or an all-or-none process. 11 refs., 2 figs.

  19. Experimental investigation of charged liquid jet efflux from a capillary

    NASA Astrophysics Data System (ADS)

    Zhakin, A. I.; Belov, P. A.; Kuz'ko, A. E.

    2013-03-01

    The shapes and electrical characteristics of charged liquid (water, ethanol, glycerol, castor oil) jets emitted from a metal capillary have been experimentally studied depending on the applied high voltage. A map of efflux regimes in the flow velocity-applied voltage coordinates is constructed for water. The effects of medium viscosity, surface tension, and charge relaxation time on the laws of jet efflux are analyzed.

  20. Sodium efflux in plant roots: what do we really know?

    PubMed

    Britto, D T; Kronzucker, H J

    2015-08-15

    The efflux of sodium (Na(+)) ions across the plasma membrane of plant root cells into the external medium is surprisingly poorly understood. Nevertheless, Na(+) efflux is widely regarded as a major mechanism by which plants restrain the rise of Na(+) concentrations in the cytosolic compartments of root cells and, thus, achieve a degree of tolerance to saline environments. In this review, several key ideas and bodies of evidence concerning root Na(+) efflux are summarized with a critical eye. Findings from decades past are brought to bear on current thinking, and pivotal studies are discussed, both "purely physiological", and also with regard to the SOS1 protein, the only major Na(+) efflux transporter that has, to date, been genetically characterized. We find that the current model of rapid transmembrane sodium cycling (RTSC), across the plasma membrane of root cells, is not adequately supported by evidence from the majority of efflux studies. An alternative hypothesis cannot be ruled out, that most Na(+) tracer efflux from the root in the salinity range does not proceed across the plasma membrane, but through the apoplast. Support for this idea comes from studies showing that Na(+) efflux, when measured with tracers, is rarely affected by the presence of inhibitors or the ionic composition in saline rooting media. We conclude that the actual efflux of Na(+) across the plasma membrane of root cells may be much more modest than what is often reported in studies using tracers, and may predominantly occur in the root tips, where SOS1 expression has been localized. PMID:26318642

  1. Cyclic AMP efflux inhibitors as potential therapeutic agents for leukemia

    PubMed Central

    Perez, Dominique R.; Smagley, Yelena; Garcia, Matthew; Carter, Mark B.; Evangelisti, Annette; Matlawska-Wasowska, Ksenia; Winter, Stuart S.; Sklar, Larry A.; Chigaev, Alexandre

    2016-01-01

    Apoptotic evasion is a hallmark of cancer. We propose that some cancers may evade cell death by regulating 3′-5′-cyclic adenosine monophosphate (cAMP), which is associated with pro-apoptotic signaling. We hypothesize that leukemic cells possess mechanisms that efflux cAMP from the cytoplasm, thus protecting them from apoptosis. Accordingly, cAMP efflux inhibition should result in: cAMP accumulation, activation of cAMP-dependent downstream signaling, viability loss, and apoptosis. We developed a novel assay to assess cAMP efflux and performed screens to identify inhibitors. In an acute myeloid leukemia (AML) model, several identified compounds reduced cAMP efflux, appropriately modulated pathways that are responsive to cAMP elevation (cAMP-responsive element-binding protein phosphorylation, and deactivation of Very Late Antigen-4 integrin), and induced mitochondrial depolarization and caspase activation. Blocking adenylyl cyclase activity was sufficient to reduce effects of the most potent compounds. These compounds also decreased cAMP efflux and viability of B-lineage acute lymphoblastic leukemia (B-ALL) cell lines and primary patient samples, but not of normal primary peripheral blood mononuclear cells. Our data suggest that cAMP efflux is a functional feature that could be therapeutically targeted in leukemia. Furthermore, because some of the identified drugs are currently used for treating other illnesses, this work creates an opportunity for repurposing. PMID:27129155

  2. Genomic Analysis of ATP Efflux in Saccharomyces cerevisiae.

    PubMed

    Peters, Theodore W; Miller, Aaron W; Tourette, Cendrine; Agren, Hannah; Hubbard, Alan; Hughes, Robert E

    2015-11-19

    Adenosine triphosphate (ATP) plays an important role as a primary molecule for the transfer of chemical energy to drive biological processes. ATP also functions as an extracellular signaling molecule in a diverse array of eukaryotic taxa in a conserved process known as purinergic signaling. Given the important roles of extracellular ATP in cell signaling, we sought to comprehensively elucidate the pathways and mechanisms governing ATP efflux from eukaryotic cells. Here, we present results of a genomic analysis of ATP efflux from Saccharomyces cerevisiae by measuring extracellular ATP levels in cultures of 4609 deletion mutants. This screen revealed key cellular processes that regulate extracellular ATP levels, including mitochondrial translation and vesicle sorting in the late endosome, indicating that ATP production and transport through vesicles are required for efflux. We also observed evidence for altered ATP efflux in strains deleted for genes involved in amino acid signaling, and mitochondrial retrograde signaling. Based on these results, we propose a model in which the retrograde signaling pathway potentiates amino acid signaling to promote mitochondrial respiration. This study advances our understanding of the mechanism of ATP secretion in eukaryotes and implicates TOR complex 1 (TORC1) and nutrient signaling pathways in the regulation of ATP efflux. These results will facilitate analysis of ATP efflux mechanisms in higher eukaryotes.

  3. Substrate binding accelerates the conformational transitions and substrate dissociation in multidrug efflux transporter AcrB

    PubMed Central

    Wang, Beibei; Weng, Jingwei; Wang, Wenning

    2015-01-01

    The tripartite efflux pump assembly AcrAB-TolC is the major multidrug resistance transporter in E. coli. The inner membrane transporter AcrB is a homotrimer, energized by the proton movement down the transmembrane electrochemical gradient. The asymmetric crystal structures of AcrB with three monomers in distinct conformational states [access (A), binding (B) and extrusion (E)] support a functional rotating mechanism, in which each monomer of AcrB cycles among the three states in a concerted way. However, the relationship between the conformational changes during functional rotation and drug translocation has not been totally understood. Here, we explored the conformational changes of the AcrB homotrimer during the ABE to BEA transition in different substrate-binding states using targeted MD simulations. It was found that the dissociation of substrate from the distal binding pocket of B monomer is closely related to the concerted conformational changes in the translocation pathway, especially the side chain reorientation of Phe628 and Tyr327. A second substrate binding at the proximal binding pocket of A monomer evidently accelerates the conformational transitions as well as substrate dissociation in B monomer. The acceleration effect of the multi-substrate binding mode provides a molecular explanation for the positive cooperativity observed in the kinetic studies of substrate efflux and deepens our understanding of the functional rotating mechanism of AcrB. PMID:25918513

  4. Tissue distribution and phenobarbital induction of target SLC- and ABC- transporters in cattle.

    PubMed

    Zancanella, V; Giantin, M; Lopparelli, R M; Nebbia, C; Dacasto, M

    2013-08-01

    In veterinary pharmaco-toxicological sciences, few data about uptake and efflux drug transporters (DTs) expression and regulation phenomena have been published. In this study, the tissue distribution and transcriptional modulation of solute carrier (SLC) and ATP-binding cassette (ABC) DTs were investigated in cattle orally administered with phenobarbital (PB) by using a quantitative real-time RT-PCR approach. The criterion for target gene selection was the PB-responsiveness in human and rodent model species. All target DTs were expressed in the liver. Only two of the seven PB-responsive target DTs (SLCO1B3 and SLC10A1) were not constitutively expressed in cattle extra-hepatic tissues. The greatest number of DTs (SLCO2B1, ABCB1, ABCC2, ABCG2) were expressed in intestine and testis, followed by, adrenal gland (SLCO2B1, ABCB1, ABCG2), lung (ABCB1, ABCG2), kidney, and skeletal muscle (ABCG2). PB administration never altered DTs mRNA levels, except for an increase in hepatic ABCC2 mRNA and a down-regulation of renal ABCG2. Altogether, these results confirm only to some extent data obtained in humans and laboratory species; clearly, they should be considered a preliminary step for further molecular investigations about species-differences in DT gene expression and regulation as well as in DT expression and function.

  5. ABC's of Being Smart: I Can "C" Clearly Now

    ERIC Educational Resources Information Center

    Foster, Joanne

    2011-01-01

    In this paper, the author focuses on C of the ABC's of being smart. She continues to categorize the points for readers. These categories include the following: (1) being; (2) doing; and (3) stretching.

  6. ABC proteins protect the human body and maintain optimal health.

    PubMed

    Ueda, Kazumitsu

    2011-01-01

    Human MDR1, a multi-drug transporter gene, was isolated as the first of the eukaryote ATP Binding Cassette (ABC) proteins from a multidrug-resistant carcinoma cell line in 1986. To date, over 25 years, many ABC proteins have been found to play important physiological roles by transporting hydrophobic compounds. Defects in their functions cause various diseases, indicating that endogenous hydrophobic compounds, as well as water-soluble compounds, are properly transported by transmembrane proteins. MDR1 transports a large number of structurally unrelated drugs and is involved in their pharmacokinetics, and thus is a key factor in drug interaction. ABCA1, an ABC protein, eliminates excess cholesterol in peripheral cells by generating HDL. Because ABCA1 is a key molecule in cholesterol homeostasis, its function and expression are highly regulated. Eukaryote ABC proteins function on the body surface facing the outside and in organ pathways to adapt to the extracellular environment and protect the body to maintain optimal health.

  7. MDR-ABC transporters: biomarkers in rheumatoid arthritis.

    PubMed

    Márki-Zay, János; Tauberné Jakab, Katalin; Szerémy, Péter; Krajcsi, Peter

    2013-01-01

    MDR-ABC transporters are widely expressed in cell types relevant to pathogenesis of rheumatoid arthritis. Many reports demonstrate the interaction of small molecule drugs with MDR-ABC transporters. Cell-based assays for disease relevant cell types can be easily gated and could reveal specific drug targets and may increase significance and utilisation of data in clinical practice. Many commonly used DMARDs (e.g. methotrexate, sulfasalazine, leflunomide/teriflunomide, hydroxychloroquine) are ABCG2 substrates. Consequently, the activity of this transporter in patients should be determined to understand the disposition and pharmacokinetics of the therapy. In addition, MDR-ABC transporters transport a variety of endobiotics that play important roles in cell proliferation, cell migration, angiogenesis and inflammation. Therefore, MDR-ABC transporters are important biomarkers in rheumatoid arthritis. PMID:23711386

  8. Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter

    PubMed Central

    Grossmann, Nina; Vakkasoglu, Ahmet S.; Hulpke, Sabine; Abele, Rupert; Gaudet, Rachelle; Tampé, Robert

    2014-01-01

    The ATP-binding cassette (ABC) transporter associated with antigen processing (TAP) participates in immune surveillance by moving proteasomal products into the endoplasmic reticulum (ER) lumen for major histocompatibility complex class I loading and cell surface presentation to cytotoxic T cells. Here we delineate the mechanistic basis for antigen translocation. Notably, TAP works as a molecular diode, translocating peptide substrates against the gradient in a strict unidirectional way. We reveal the importance of the D-loop at the dimer interface of the two nucleotide-binding domains (NBDs) in coupling substrate translocation with ATP hydrolysis and defining transport vectoriality. Substitution of the conserved aspartate, which coordinates the ATP-binding site, decreases NBD dimerization affinity and turns the unidirectional primary active pump into a passive bidirectional nucleotide-gated facilitator. Thus, ATP hydrolysis is not required for translocation per se, but is essential for both active and unidirectional transport. Our data provide detailed mechanistic insight into how heterodimeric ABC exporters operate. PMID:25377891

  9. Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter

    SciTech Connect

    Grossmann, Nina; Vakkasoglu, Ahmet S.; Hulpke, Sabine; Abele, Rupert; Gaudet, Rachelle; Tampé, Robert

    2014-11-07

    The ATP-binding cassette (ABC) transporter associated with antigen processing (TAP) participates in immune surveillance by moving proteasomal products into the endoplasmic reticulum (ER) lumen for major histocompatibility complex class I loading and cell surface presentation to cytotoxic T cells. Here we delineate the mechanistic basis for antigen translocation. Notably, TAP works as a molecular diode, translocating peptide substrates against the gradient in a strict unidirectional way. We reveal the importance of the D-loop at the dimer interface of the two nucleotide-binding domains (NBDs) in coupling substrate translocation with ATP hydrolysis and defining transport vectoriality. Substitution of the converved aspartate, which coordinates the ATP-binding site, decreases NBD dimerization affinity and turns the unidirectional primary active pump into a passive bidirectional nucleotide-gated facilitator. Thus, ATP hydrolysis is not required for translocation per se, but is essential for both active and unidirectional transport. As a result, our data provide detailed mechanistic insight into how heterodimeric ABC exporters operate.

  10. Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter

    DOE PAGES

    Grossmann, Nina; Vakkasoglu, Ahmet S.; Hulpke, Sabine; Abele, Rupert; Gaudet, Rachelle; Tampé, Robert

    2014-11-07

    The ATP-binding cassette (ABC) transporter associated with antigen processing (TAP) participates in immune surveillance by moving proteasomal products into the endoplasmic reticulum (ER) lumen for major histocompatibility complex class I loading and cell surface presentation to cytotoxic T cells. Here we delineate the mechanistic basis for antigen translocation. Notably, TAP works as a molecular diode, translocating peptide substrates against the gradient in a strict unidirectional way. We reveal the importance of the D-loop at the dimer interface of the two nucleotide-binding domains (NBDs) in coupling substrate translocation with ATP hydrolysis and defining transport vectoriality. Substitution of the converved aspartate, whichmore » coordinates the ATP-binding site, decreases NBD dimerization affinity and turns the unidirectional primary active pump into a passive bidirectional nucleotide-gated facilitator. Thus, ATP hydrolysis is not required for translocation per se, but is essential for both active and unidirectional transport. As a result, our data provide detailed mechanistic insight into how heterodimeric ABC exporters operate.« less

  11. The ABC daycare disaster of Hermosillo, Mexico.

    PubMed

    Greenhalgh, David G; Chang, Philip; Maguina, Pirko; Combs, Elena; Sen, Soman; Palmieri, Tina L

    2012-01-01

    On June 5, 2009, the ABC Daycare facility in Hermosillo, Mexico, caught on fire with an estimated 142 children and 6 adult caregivers inside. The purpose of this article is to describe the factors contributing to the disaster including care of the survivors, tertiary burn center triage, patient transport, and treatment for this international mass casualty event. Finally, the results of an investigation performed by the Mexican Government are reviewed. A summary of the Mexican Government's investigation of the circumstances of fire and an examination of prevention lapses in other Mexican daycare centers was obtained from their public Web site. The demographic and clinical characteristics of the children transported to the burn center were obtained from the patients' medical records and transport data sheets. The ABC Daycare had many fire safety breaches that contributed to the severity of the tragedy. Twenty-nine children died at the scene and more than 35 children were hospitalized throughout Mexico. A total of 12 children were transported to two Shriners Hospitals, 9 to Sacramento, and 3 to Cincinnati. The mean age of patients sent to the Shriners Hospitals was 2.9 ± 0.16 years (2-4 years), with 5 being male and 7 female. The mean duration between injury and arrival was 9.2 ± 2.1 days, the burn size was 43.0 ± 6.8% TBSA (6.5-80%), and there were 3.75 operations per patient. Four had fourth-degree burns requiring finger amputations (2), flaps to cover bone (1), or a through-knee amputation (1). Ten patients were admitted to the intensive care unit, and nine patients (seven with inhalation injury) required mechanical ventilation for a mean of 23.6 ± 10.3 days. All the surviving children were discharged after a mean length of stay of 45.9 ± 8.7 days. In the first year postinjury, seven children were readmitted a total of 11 times for reconstructive surgery, wound care, or rehabilitation. Ultimately, a total of 49 children died. A review of other daycare centers

  12. ABC and IFC: Modules Detection Method for PPI Network

    PubMed Central

    Lei, Xiujuan; Tian, Jianfang

    2014-01-01

    Many clustering algorithms are unable to solve the clustering problem of protein-protein interaction (PPI) networks effectively. A novel clustering model which combines the optimization mechanism of artificial bee colony (ABC) with the fuzzy membership matrix is proposed in this paper. The proposed ABC-IFC clustering model contains two parts: searching for the optimum cluster centers using ABC mechanism and forming clusters using intuitionistic fuzzy clustering (IFC) method. Firstly, the cluster centers are set randomly and the initial clustering results are obtained by using fuzzy membership matrix. Then the cluster centers are updated through different functions of bees in ABC algorithm; then the clustering result is obtained through IFC method based on the new optimized cluster center. To illustrate its performance, the ABC-IFC method is compared with the traditional fuzzy C-means clustering and IFC method. The experimental results on MIPS dataset show that the proposed ABC-IFC method not only gets improved in terms of several commonly used evaluation criteria such as precision, recall, and P value, but also obtains a better clustering result. PMID:24991575

  13. ABC transporter research: going strong 40 years on

    PubMed Central

    Theodoulou, Frederica L.; Kerr, Ian D.

    2015-01-01

    In most organisms, ABC transporters constitute one of the largest families of membrane proteins. In humans, their functions are diverse and underpin numerous key physiological processes, as well as being causative factors in a number of clinically relevant pathologies. Advances in our understanding of these diseases have come about through combinations of genetic and protein biochemical investigations of these transporters and the power of in vitro and in vivo investigations is helping to develop genotype–phenotype understanding. However, the importance of ABC transporter research goes far beyond human biology; microbial ABC transporters are of great interest in terms of understanding virulence and drug resistance and industrial biotechnology researchers are exploring the potential of prokaryotic ABC exporters to increase the capacity of synthetic biology systems. Plant ABC transporters play important roles in transport of hormones, xenobiotics, metals and secondary metabolites, pathogen responses and numerous aspects of development, all of which are important in the global food security area. For 3 days in Chester, this Biochemical Society Focused Meeting brought together researchers with diverse experimental approaches and with different fundamental questions, all of which are linked by the commonality of ABC transporters. PMID:26517919

  14. Structural basis for the mechanism of ABC transporters.

    PubMed

    Beis, Konstantinos

    2015-10-01

    The ATP-binding cassette (ABC) transporters are primary transporters that couple the energy stored in adenosine triphosphate (ATP) to the movement of molecules across the membrane. ABC transporters can be divided into exporters and importers; importers mediate the uptake of essential nutrients into cells and are found predominantly in prokaryotes whereas exporters transport molecules out of cells or into organelles and are found in all organisms. ABC exporters have been linked with multi-drug resistance in both bacterial and eukaryotic cells. ABC transporters are powered by the hydrolysis of ATP and transport their substrate via the alternating access mechanism, whereby the protein alternates between a conformation in which the substrate-binding site is accessible from the outside of the membrane, outward-facing and one in which it is inward-facing. In this mini-review, the structures of different ABC transporter types in different conformations are presented within the context of the alternating access mechanism and how they have shaped our current understanding of the mechanism of ABC transporters.

  15. ABCE1 Is a Highly Conserved RNA Silencing Suppressor

    PubMed Central

    Kärblane, Kairi; Gerassimenko, Jelena; Nigul, Lenne; Piirsoo, Alla; Smialowska, Agata; Vinkel, Kadri; Kylsten, Per; Ekwall, Karl; Swoboda, Peter; Truve, Erkki; Sarmiento, Cecilia

    2015-01-01

    ATP-binding cassette sub-family E member 1 (ABCE1) is a highly conserved protein among eukaryotes and archaea. Recent studies have identified ABCE1 as a ribosome-recycling factor important for translation termination in mammalian cells, yeast and also archaea. Here we report another conserved function of ABCE1. We have previously described AtRLI2, the homolog of ABCE1 in the plant Arabidopsis thaliana, as an endogenous suppressor of RNA silencing. In this study we show that this function is conserved: human ABCE1 is able to suppress RNA silencing in Nicotiana benthamiana plants, in mammalian HEK293 cells and in the worm Caenorhabditis elegans. Using co-immunoprecipitation and mass spectrometry, we found a number of potential ABCE1-interacting proteins that might support its function as an endogenous suppressor of RNA interference. The interactor candidates are associated with epigenetic regulation, transcription, RNA processing and mRNA surveillance. In addition, one of the identified proteins is translin, which together with its binding partner TRAX supports RNA interference. PMID:25659154

  16. Active Efflux Influences the Potency of Quorum Sensing Inhibitors in Pseudomonas aeruginosa

    PubMed Central

    Moore, Joseph D.; Gerdt, Joseph P.; Eibergen, Nora R.; Blackwell, Helen E.

    2014-01-01

    Many bacteria regulate gene expression through a cell-cell signaling process called quorum sensing (QS). In proteobacteria, QS is largely mediated by signaling molecules known as N-acylated L-homoserine lactones (AHLs) and their associated intracellular LuxR-type receptors. The design of non-native small molecules capable of inhibiting LuxR-type receptors, and thereby QS, in proteobacteria is an active area of research, and numerous lead compounds are AHL derivatives that mimic native AHL signals. Much of this past work has focused on the pathogen Pseudomonas aeruginosa, which controls an arsenal of virulence factors and biofilm formation through QS. The MexAB-OprM drug efflux pump has been shown to play a role in the secretion of the major AHL signal in P. aeruginosa, N-(3-oxododecanoyl) L-homoserine lactone. In the current study, we show that a variety of non-native AHLs and related derivatives capable of inhibiting LuxR-type receptors in P. aeruginosa display significantly higher potency in a P. aeruginosa Δ(mexAB-oprM) mutant, suggesting that MexAB-OprM also recognizes these compounds as substrates. We also demonstrate that the potency of 5,6-dimethyl-2-aminobenzimidazole, recently shown to be a QS and biofilm inhibitor in P. aeruginosa, is not affected by the presence or absence of the MexAB-OprM pump. These results have implications for the use of non-native AHLs and related derivatives as QS modulators in P. aeruginosa and other bacteria, and provide a potential design strategy for the development of new QS modulators that are resistant to active efflux. PMID:24478193

  17. Acceleration of Tuberculosis Treatment by Adjunctive Therapy with Verapamil as an Efflux Inhibitor

    PubMed Central

    Gupta, Shashank; Tyagi, Sandeep; Almeida, Deepak V.; Maiga, Mariama C.; Ammerman, Nicole C.

    2013-01-01

    Rationale: A major priority in tuberculosis (TB) is to reduce effective treatment times and emergence of resistance. Recent studies in macrophages and zebrafish show that inhibition of mycobacterial efflux pumps with verapamil reduces the bacterial drug tolerance and may enhance drug efficacy. Objectives: Using mice, a mammalian model known to predict human treatment responses, and selecting conservative human bioequivalent doses, we tested verapamil as an adjunctive drug together with standard TB chemotherapy. As verapamil is a substrate for CYP3A4, which is induced by rifampin, we evaluated the pharmacokinetic/pharmacodynamic relationships of verapamil and rifampin coadministration in mice. Methods: Using doses that achieve human bioequivalent levels matched to those of standard verapamil, but lower than those of extended release verapamil, we evaluated the activity of verapamil added to standard chemotherapy in both C3HeB/FeJ (which produce necrotic granulomas) and the wild-type background C3H/HeJ mouse strains. Relapse rates were assessed after 16, 20, and 24 weeks of treatment in mice. Measurements and Main Results: We determined that a dose adjustment of verapamil by 1.5-fold is required to compensate for concurrent use of rifampin during TB treatment. We found that standard TB chemotherapy plus verapamil accelerates bacterial clearance in C3HeB/FeJ mice with near sterilization, and significantly lowers relapse rates in just 4 months of treatment when compared with mice receiving standard therapy alone. Conclusions: These data demonstrate treatment shortening by verapamil adjunctive therapy in mice, and strongly support further study of verapamil and other efflux pump inhibitors in human TB. PMID:23805786

  18. Interaction Studies of Resolvin E1 Analog (RX-10045) with Efflux Transporters

    PubMed Central

    Cholkar, Kishore; Trinh, Hoang M.; Vadlapudi, Aswani Dutt; Wang, Zhiying; Pal, Dhananjay

    2015-01-01

    Abstract Purpose: Screening interactions of a resolvin E1 analog (RX-10045) with efflux transporters (P-glycoprotein [P-gp], multidrug resistance-associated protein [MRP2], and breast cancer-resistant protein [BCRP]). Methods: Madin-Darby canine kidney cells transfected with P-gp, MRP2, and BCRP genes were selected for this study. [3H]-Digoxin, [3H]-vinblastine and [3H]-abacavir were selected as model substrates for P-gp, MRP2, and BCRP. Uptake and permeability studies across cell monolayer in both apical to basal (AP–BL) and BL–AP of these substrates were conducted in the presence of specific efflux pump inhibitors and RX-10045. Cell viability studies were conducted with increasing concentrations of RX-10045. Results: Uptake studies showed a higher accumulation in the presence of inhibitors (GF120918 and ketoconazole for P-gp; MK571 for MRP2; and β-estradiol for BCRP) as well as RX-10045. Similarly, dose-dependent inhibition studies demonstrated higher accumulation of various substrates ([3H]-digoxin, [3H]-vinblastine, and [3H]-abacavir) in the presence of RX-10045. IC50 values of dose-dependent inhibition of RX-10045 for P-gp, MRP2, and BCRP were 239±11.2, 291±79.2, and 300±42 μM, respectively. Cell viability assay indicated no apparent toxicity up to 350 μM concentration. Enhanced permeability for model substrates was observed in the presence of RX-10045. Uptake studies in human corneal epithelial cells suggest that RX-10045 is a strong inhibitor of organic cation transporter-1 (OCT-1). Conclusions: In summary, the resolvin analog (RX-10045) was identified as a substrate/inhibitor for efflux transporters (MRP2 and BCRP). Also, RX-10045 appears to be a strong inhibitor/substrate of OCT-1. Novel formulation strategies such as nanoparticles, nanomicelles, and liposomes for circumventing efflux barriers and delivering higher drug concentrations leading to a higher therapeutic efficacy may be employed. PMID:25844889

  19. PfeT, a P1B4 -type ATPase, effluxes ferrous iron and protects Bacillus subtilis against iron intoxication.

    PubMed

    Guan, Guohua; Pinochet-Barros, Azul; Gaballa, Ahmed; Patel, Sarju J; Argüello, José M; Helmann, John D

    2015-11-01

    Iron is an essential element for nearly all cells and limited iron availability often restricts growth. However, excess iron can also be deleterious, particularly when cells expressing high affinity iron uptake systems transition to iron rich environments. Bacillus subtilis expresses numerous iron importers, but iron efflux has not been reported. Here, we describe the B. subtilis PfeT protein (formerly YkvW/ZosA) as a P1B4 -type ATPase in the PerR regulon that serves as an Fe(II) efflux pump and protects cells against iron intoxication. Iron and manganese homeostasis in B. subtilis are closely intertwined: a pfeT mutant is iron sensitive, and this sensitivity can be suppressed by low levels of Mn(II). Conversely, a pfeT mutant is more resistant to Mn(II) overload. In vitro, the PfeT ATPase is activated by both Fe(II) and Co(II), although only Fe(II) efflux is physiologically relevant in wild-type cells, and null mutants accumulate elevated levels of intracellular iron. Genetic studies indicate that PfeT together with the ferric uptake repressor (Fur) cooperate to prevent iron intoxication, with iron sequestration by the MrgA mini-ferritin playing a secondary role. Protection against iron toxicity may also be a key role for related P1B4 -type ATPases previously implicated in bacterial pathogenesis. PMID:26261021

  20. Phase transition in the ABC model.

    PubMed

    Clincy, M; Derrida, B; Evans, M R

    2003-06-01

    Recent studies have shown that one-dimensional driven systems can exhibit phase separation even if the dynamics is governed by local rules. The ABC model, which comprises three particle species that diffuse asymmetrically around a ring, shows anomalous coarsening into a phase separated steady state. In the limiting case in which the dynamics is symmetric and the parameter q describing the asymmetry tends to one, no phase separation occurs and the steady state of the system is disordered. In the present work, we consider the weak asymmetry regime q=exp(-beta/N), where N is the system size, and study how the disordered state is approached. In the case of equal densities, we find that the system exhibits a second-order phase transition at some nonzero beta(c). The value of beta(c)=2pi square root 3 and the optimal profiles can be obtained by writing the exact large deviation functional. For nonequal densities, we write down mean-field equations and analyze some of their predictions. PMID:16241312

  1. Optimal ABC inventory classification using interval programming

    NASA Astrophysics Data System (ADS)

    Rezaei, Jafar; Salimi, Negin

    2015-08-01

    Inventory classification is one of the most important activities in inventory management, whereby inventories are classified into three or more classes. Several inventory classifications have been proposed in the literature, almost all of which have two main shortcomings in common. That is, the previous methods mainly rely on an expert opinion to derive the importance of the classification criteria which results in subjective classification, and they need precise item parameters before implementing the classification. While the problem has been predominantly considered as a multi-criteria, we examine the problem from a different perspective, proposing a novel optimisation model for ABC inventory classification in the form of an interval programming problem. The proposed interval programming model has two important features compared to the existing methods: it provides optimal results instead of an expert-based classification and it does not require precise values of item parameters, which are not almost always available before classification. Finally, by illustrating the proposed classification model in the form of numerical example, conclusion and suggestions for future works are presented.

  2. Modulation of Membrane Influx and Efflux in Escherichia coli Sequence Type 131 Has an Impact on Bacterial Motility, Biofilm Formation, and Virulence in a Caenorhabditis elegans Model.

    PubMed

    Pantel, Alix; Dunyach-Remy, Catherine; Ngba Essebe, Christelle; Mesureur, Jennifer; Sotto, Albert; Pagès, Jean-Marie; Nicolas-Chanoine, Marie-Hélène; Lavigne, Jean-Philippe

    2016-05-01

    Energy-dependent efflux overexpression and altered outer membrane permeability (influx) can promote multidrug resistance (MDR). The present study clarifies the regulatory pathways that control membrane permeability in the pandemic clone Escherichia coli sequence type 131 (ST131) and evaluates the impact of efflux and influx modulations on biofilm formation, motility, and virulence in the Caenorhabditis elegans model. Mutants of two uropathogenic E. coli (UPEC) strains, MECB5 (ST131; H30-Rx) and CFT073 (ST73), as well as a fecal strain, S250 (ST131; H22), were in vitro selected using continuous subculture in subinhibitory concentrations of ertapenem (ETP), chloramphenicol (CMP), and cefoxitin (FOX). Mutations in genes known to control permeability were shown for the two UPEC strains: MECB5-FOX (deletion of 127 bp in marR; deletion of 1 bp and insertion of an IS1 element in acrR) and CFT073-CMP (a 1-bp deletion causing a premature stop in marR). We also demonstrated that efflux phenotypes in the mutants selected with CMP and FOX were related to the AcrAB-TolC pump, but also to other efflux systems. Alteration of membrane permeability, caused by underexpression of the two major porins, OmpF and OmpC, was shown in MECB5-ETP and mutants selected with FOX. Lastly, our findings suggest that efflux pump-overproducing isolates (CMP mutants) pose a serious threat in terms of virulence (significant reduction in worm median survival) and host colonization. Lack of porins (ETP and FOX mutants) led to a high level of antibiotic resistance in an H30-Rx subclone. Nevertheless, this adaptation created a physiological disadvantage (decreased motility and ability to form biofilm) associated with a low potential for virulence. PMID:26926643

  3. Modulation of Membrane Influx and Efflux in Escherichia coli Sequence Type 131 Has an Impact on Bacterial Motility, Biofilm Formation, and Virulence in a Caenorhabditis elegans Model

    PubMed Central

    Pantel, Alix; Dunyach-Remy, Catherine; Ngba Essebe, Christelle; Mesureur, Jennifer; Sotto, Albert; Nicolas-Chanoine, Marie-Hélène

    2016-01-01

    Energy-dependent efflux overexpression and altered outer membrane permeability (influx) can promote multidrug resistance (MDR). The present study clarifies the regulatory pathways that control membrane permeability in the pandemic clone Escherichia coli sequence type 131 (ST131) and evaluates the impact of efflux and influx modulations on biofilm formation, motility, and virulence in the Caenorhabditis elegans model. Mutants of two uropathogenic E. coli (UPEC) strains, MECB5 (ST131; H30-Rx) and CFT073 (ST73), as well as a fecal strain, S250 (ST131; H22), were in vitro selected using continuous subculture in subinhibitory concentrations of ertapenem (ETP), chloramphenicol (CMP), and cefoxitin (FOX). Mutations in genes known to control permeability were shown for the two UPEC strains: MECB5-FOX (deletion of 127 bp in marR; deletion of 1 bp and insertion of an IS1 element in acrR) and CFT073-CMP (a 1-bp deletion causing a premature stop in marR). We also demonstrated that efflux phenotypes in the mutants selected with CMP and FOX were related to the AcrAB-TolC pump, but also to other efflux systems. Alteration of membrane permeability, caused by underexpression of the two major porins, OmpF and OmpC, was shown in MECB5-ETP and mutants selected with FOX. Lastly, our findings suggest that efflux pump-overproducing isolates (CMP mutants) pose a serious threat in terms of virulence (significant reduction in worm median survival) and host colonization. Lack of porins (ETP and FOX mutants) led to a high level of antibiotic resistance in an H30-Rx subclone. Nevertheless, this adaptation created a physiological disadvantage (decreased motility and ability to form biofilm) associated with a low potential for virulence. PMID:26926643

  4. Multixenobiotic resistance efflux activity in Daphnia magna and Lumbriculus variegatus.

    PubMed

    Vehniäinen, Eeva-Riikka; Kukkonen, Jussi V K

    2015-04-01

    Multixenobiotic resistance is a phenomenon in which ATP-binding cassette (ABC) family proteins transfer harmful compounds out of cells. Daphnia magna and Lumbriculus variegatus are model species in aquatic ecotoxicology, but the presence and activity of ABC proteins have not been well described in these species. The aim of this work was to study the presence, activity, and inhibition of ABC transport proteins in D. magna and L. variegatus. The presence of abcb1 and abcc transcripts in 8-9-day-old D. magna was investigated by qRT-PCR. The activity of MXR in D. magna and L. variegatus was explored by influx of the fluorescent ABC protein substrates rhodamine B and calcein-AM, with and without the model inhibitors verapamil (unspecific ABC inhibitor), reversin 205 (ABCB1 inhibitor) and MK571 (ABCC inhibitor). Juvenile D. magna possessed all examined abcb and abcc transcripts, but only reversin 205 inhibited MXR activity. The MXR activity in L. variegatus was inhibited by MK571, and to a lesser extent by verapamil, whereas reversin 205 seemed to stimulate the transport activity. Whereas calcein-AM worked better as an MXR substrate in D. magna, rhodamine B was a better substrate for L. variegatus MXR activity measurements. This is the first report on MXR activity in the order Lumbriculida, subclass Oligochaeta, and class Clitellata.

  5. DIFFUSION PUMP

    DOEpatents

    Levenson, L.

    1963-09-01

    A high-vacuum diffusion pump is described, featuring a novel housing geometry for enhancing pumping speed. An upright, cylindrical lower housing portion is surmounted by a concentric, upright, cylindrical upper housing portion of substantially larger diameter; an uppermost nozzle, disposed concentrically within the upper portion, is adapted to eject downwardly a conical sheet of liquid outwardly to impinge upon the uppermost extremity of the interior wall of the lower portion. Preferably this nozzle is mounted upon a pedestal rising coaxially from within the lower portion and projecting up into said upper portion. (AEC)

  6. Electrokinetic pump

    DOEpatents

    Hencken, Kenneth R.; Sartor, George B.

    2004-08-03

    An electrokinetic pump in which the porous dielectric medium of conventional electrokinetic pumps is replaced by a patterned microstructure. The patterned microstructure is fabricated by lithographic patterning and etching of a substrate and is formed by features arranged so as to create an array of microchannels. The microchannels have dimensions on the order of the pore spacing in a conventional porous dielectric medium. Embedded unitary electrodes are vapor deposited on either end of the channel structure to provide the electric field necessary for electroosmotic flow.

  7. Kinetics and stoichiometry of coupled Na efflux and Ca influx (Na/Ca exchange) in barnacle muscle cells

    PubMed Central

    1989-01-01

    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

  8. Prevention of egg contamination by Salmonella Enteritidis after oral vaccination of laying hens with Salmonella Enteritidis ΔtolC and ΔacrABacrEFmdtABC mutants.

    PubMed

    Kilroy, Sofie; Raspoet, Ruth; Haesebrouck, Freddy; Ducatelle, Richard; Van Immerseel, Filip

    2016-01-01

    Vaccination of laying hens has been successfully used to reduce egg contamination by Salmonella Enteritidis, decreasing human salmonellosis cases worldwide. Currently used vaccines for layers are either inactivated vaccines or live attenuated strains produced by mutagenesis. Targeted gene deletion mutants hold promise for future vaccines, because specific bacterial functions can be removed that may improve safety and allow differentiation from field strains. In this study, the efficacy of Salmonella Enteritidis ΔtolC and ΔacrABacrEFmdtABC strains in laying hens as live vaccines was evaluated. The mutants are deficient in either the membrane channel TolC (ΔtolC) or the multi-drug efflux systems acrAB, acrEF and mdtABC (ΔacrABacrEFmdtABC). These strains have a decreased ability for gut and tissue colonization and are unable to survive in egg white, the latter preventing transmission of the vaccine strains to humans. Two groups of 30 laying hens were orally inoculated at day 1, 6 weeks and 16 weeks of age with 10(8) cfu of either vaccine strain, while a third group was left unvaccinated. At 24 weeks of age, the birds were intravenously challenged with 5 × 10(7) cfu Salmonella Enteritidis PT4 S1400/94. The vaccine strains were not shed or detected in the gut, internal organs or eggs, 2 weeks after the third vaccination. The strains significantly protected against gut and internal organ colonization, and completely prevented egg contamination by Salmonella Enteritidis under the conditions of this study. This indicates that Salmonella Enteritidis ΔtolC and ΔacrABacrEFmdtABC strains might be valuable strains for vaccination of layers against Salmonella Enteritidis. PMID:27519174

  9. Fullerene inhibits benzo(a)pyrene Efflux from Cyprinus carpio hepatocytes by affecting cell membrane fluidity and P-glycoprotein expression.

    PubMed

    Chen, Qiqing; Hu, Xialin; Wang, Rui; Yuan, Jin; Yin, Daqiang

    2016-05-01

    P-Glycoprotein (P-gp) can protect cells by pumping out toxic compounds, and has been found widely expressed in fish tissues. Here, we illustrate the P-gp efflux ability for benzo(a)pyrene (BaP) in the hepatocytes of common carp (Cyprinus carpio) after exposing to fullerene aqueous suspension (nC60). The results revealed that nC60 increased the membrane fluidity by decreasing the ratio of saturated to unsaturated fatty acids, and increased the cholesterol contents. These findings, combined with 10-38% and 70-75% down-regulation of P-gp mRNA and protein respectively, suggested that nC60 caused inhibition on P-gp efflux transport system. Therefore, we further investigated the cellular efflux ability for BaP. Results showed unequivocally that nC60 is a potent P-gp inhibitor. The retaining BaP amounts after efflux were elevated by 1.7-2.8 fold during the 10 day exposure. Meanwhile, 5mg/L humic acid (one of the important fractions of natural organic matter, which is ubiquitous in aquatic environment) alleviated the nC60 damage to hepatocytes in terms of oxidative damage, cholesterol increment, and P-gp content reduction; and finally attenuated the suppressed P-gp efflux ability. Collectively, this study provides the first evidence of nC60 toxicity to P-gp functionality in fish and illustrates the possible mechanism of the suppressed P-gp efflux ability for BaP.

  10. Fullerene inhibits benzo(a)pyrene Efflux from Cyprinus carpio hepatocytes by affecting cell membrane fluidity and P-glycoprotein expression.

    PubMed

    Chen, Qiqing; Hu, Xialin; Wang, Rui; Yuan, Jin; Yin, Daqiang

    2016-05-01

    P-Glycoprotein (P-gp) can protect cells by pumping out toxic compounds, and has been found widely expressed in fish tissues. Here, we illustrate the P-gp efflux ability for benzo(a)pyrene (BaP) in the hepatocytes of common carp (Cyprinus carpio) after exposing to fullerene aqueous suspension (nC60). The results revealed that nC60 increased the membrane fluidity by decreasing the ratio of saturated to unsaturated fatty acids, and increased the cholesterol contents. These findings, combined with 10-38% and 70-75% down-regulation of P-gp mRNA and protein respectively, suggested that nC60 caused inhibition on P-gp efflux transport system. Therefore, we further investigated the cellular efflux ability for BaP. Results showed unequivocally that nC60 is a potent P-gp inhibitor. The retaining BaP amounts after efflux were elevated by 1.7-2.8 fold during the 10 day exposure. Meanwhile, 5mg/L humic acid (one of the important fractions of natural organic matter, which is ubiquitous in aquatic environment) alleviated the nC60 damage to hepatocytes in terms of oxidative damage, cholesterol increment, and P-gp content reduction; and finally attenuated the suppressed P-gp efflux ability. Collectively, this study provides the first evidence of nC60 toxicity to P-gp functionality in fish and illustrates the possible mechanism of the suppressed P-gp efflux ability for BaP. PMID:26918948

  11. Arsenic efflux from Microcystis aeruginosa under different phosphate regimes.

    PubMed

    Yan, Changzhou; Wang, Zhenhong; Luo, Zhuanxi

    2014-01-01

    Phytoplankton plays an important role in arsenic speciation, distribution, and cycling in freshwater environments. Little information, however, is available on arsenic efflux from the cyanobacteria Microcystis aeruginosa under different phosphate regimes. This study investigated M. aeruginosa arsenic efflux and speciation by pre-exposing it to 10 µM arsenate or arsenite for 24 h during limited (12 h) and extended (13 d) depuration periods under phosphate enriched (+P) and phosphate depleted (-P) treatments. Arsenate was the predominant species detected in algal cells throughout the depuration period while arsenite only accounted for no greater than 45% of intracellular arsenic. During the limited depuration period, arsenic efflux occurred rapidly and only arsenate was detected in solutions. During the extended depuration period, however, arsenate and dimethylarsinic acid (DMA) were found to be the two predominant arsenic species detected in solutions under -P treatments, but arsenate was the only species detected under +P treatments. Experimental results also suggest that phosphorus has a significant effect in accelerating arsenic efflux and promoting arsenite bio-oxidation in M. aeruginosa. Furthermore, phosphorus depletion can reduce arsenic efflux from algal cells as well as accelerate arsenic reduction and methylation. These findings can contribute to our understanding of arsenic biogeochemistry in aquatic environments and its potential environmental risks under different phosphorus levels. PMID:25549253

  12. Effects of neurotransmitters on calcium efflux from cultured glioma cells

    SciTech Connect

    Lazarewicz, J.W.; Kanje, M.

    1981-01-01

    The effects of various neurotransmitters and cyclic nucleotides on 45Ca2+ efflux in cultured human glioma cells were investigated. Glutamate and glycine, but not GABA, stimulated 45Ca2+ release from the cells. Stimulation of beta-adrenergic receptors but not alpha-adrenergic receptors also increased 45Ca2+ efflux. Cholinergic receptor stimulation by carbachol had the same effect. The stimulatory effect of carbachol was abolished in the presence of either atropine or hexamethonium. C-AMP and c-GMP increased the 45Ca2+ efflux, suggesting that these agents are involved in the transmitter-stimulated release of 45Ca2+ from the cell. Kinetic analysis of the efflux revealed four calcium compartments. The carbachol-stimulated efflux represented a net release of calcium and could be ascribed to the slowest compartment. The physiological role of the transmitter-stimulated calcium release is discussed in terms of calcium-regulated stimulus-response coupling in glial-neural interaction during excitation.

  13. Arsenic Efflux from Microcystis aeruginosa under Different Phosphate Regimes

    PubMed Central

    Yan, Changzhou; Wang, Zhenhong; Luo, Zhuanxi

    2014-01-01

    Phytoplankton plays an important role in arsenic speciation, distribution, and cycling in freshwater environments. Little information, however, is available on arsenic efflux from the cyanobacteria Microcystis aeruginosa under different phosphate regimes. This study investigated M. aeruginosa arsenic efflux and speciation by pre-exposing it to 10 µM arsenate or arsenite for 24 h during limited (12 h) and extended (13 d) depuration periods under phosphate enriched (+P) and phosphate depleted (−P) treatments. Arsenate was the predominant species detected in algal cells throughout the depuration period while arsenite only accounted for no greater than 45% of intracellular arsenic. During the limited depuration period, arsenic efflux occurred rapidly and only arsenate was detected in solutions. During the extended depuration period, however, arsenate and dimethylarsinic acid (DMA) were found to be the two predominant arsenic species detected in solutions under −P treatments, but arsenate was the only species detected under +P treatments. Experimental results also suggest that phosphorus has a significant effect in accelerating arsenic efflux and promoting arsenite bio-oxidation in M. aeruginosa. Furthermore, phosphorus depletion can reduce arsenic efflux from algal cells as well as accelerate arsenic reduction and methylation. These findings can contribute to our understanding of arsenic biogeochemistry in aquatic environments and its potential environmental risks under different phosphorus levels. PMID:25549253

  14. Berberine Is a Novel Type Efflux Inhibitor Which Attenuates the MexXY-Mediated Aminoglycoside Resistance in Pseudomonas aeruginosa

    PubMed Central

    Morita, Yuji; Nakashima, Ken-ichi; Nishino, Kunihiko; Kotani, Kenta; Tomida, Junko; Inoue, Makoto; Kawamura, Yoshiaki

    2016-01-01

    The emergence and spread of multidrug-resistant P. aeruginosa infections is of great concern, as very few agents are effective against strains of this species. Methanolic extracts from the Coptidis Rhizoma (the rhizomes of Coptis japonica var. major Satake) or Phellodendri Cortex (the bark of Phellodendron chinense Schneider) markedly reduced resistance to anti-pseudomonal aminoglycosides (e.g., amikacin) in multidrug-resistant P. aeruginosa strains. Berberine, the most abundant benzylisoquinoline alkaloid in the two extracts, reduced aminoglycoside resistance of P. aeruginosa via a mechanism that required the MexXY multidrug efflux system; berberine also reduced aminoglycoside MICs in Achromobacter xylosoxidans and Burkholderia cepacia, two species that harbor intrinsic multidrug efflux systems very similar to the MexXY. Furthermore this compound inhibited MexXY-dependent antibiotic resistance of other classes including cephalosporins (cefepime), macrolides (erythromycin), and lincosamides (lincomycin) demonstrated using a pseudomonad lacking the four other major Mex pumps. Although phenylalanine-arginine beta-naphthylamide (PAβN), a well-known efflux inhibitor, antagonized aminoglycoside in a MexXY-dependent manner, a lower concentration of berberine was sufficient to reduce amikacin resistance of P. aeruginosa in the presence of PAβN. Moreover, berberine enhanced the synergistic effects of amikacin and piperacillin (and vice versa) in multidrug-resistant P. aeruginosa strains. Thus, berberine appears to be a novel type inhibitor of the MexXY-dependent aminoglycoside efflux in P. aeruginosa. As aminoglycosides are molecules of choice to treat severe infections the clinical impact is potentially important. PMID:27547203

  15. Berberine Is a Novel Type Efflux Inhibitor Which Attenuates the MexXY-Mediated Aminoglycoside Resistance in Pseudomonas aeruginosa.

    PubMed

    Morita, Yuji; Nakashima, Ken-Ichi; Nishino, Kunihiko; Kotani, Kenta; Tomida, Junko; Inoue, Makoto; Kawamura, Yoshiaki

    2016-01-01

    The emergence and spread of multidrug-resistant P. aeruginosa infections is of great concern, as very few agents are effective against strains of this species. Methanolic extracts from the Coptidis Rhizoma (the rhizomes of Coptis japonica var. major Satake) or Phellodendri Cortex (the bark of Phellodendron chinense Schneider) markedly reduced resistance to anti-pseudomonal aminoglycosides (e.g., amikacin) in multidrug-resistant P. aeruginosa strains. Berberine, the most abundant benzylisoquinoline alkaloid in the two extracts, reduced aminoglycoside resistance of P. aeruginosa via a mechanism that required the MexXY multidrug efflux system; berberine also reduced aminoglycoside MICs in Achromobacter xylosoxidans and Burkholderia cepacia, two species that harbor intrinsic multidrug efflux systems very similar to the MexXY. Furthermore this compound inhibited MexXY-dependent antibiotic resistance of other classes including cephalosporins (cefepime), macrolides (erythromycin), and lincosamides (lincomycin) demonstrated using a pseudomonad lacking the four other major Mex pumps. Although phenylalanine-arginine beta-naphthylamide (PAβN), a well-known efflux inhibitor, antagonized aminoglycoside in a MexXY-dependent manner, a lower concentration of berberine was sufficient to reduce amikacin resistance of P. aeruginosa in the presence of PAβN. Moreover, berberine enhanced the synergistic effects of amikacin and piperacillin (and vice versa) in multidrug-resistant P. aeruginosa strains. Thus, berberine appears to be a novel type inhibitor of the MexXY-dependent aminoglycoside efflux in P. aeruginosa. As aminoglycosides are molecules of choice to treat severe infections the clinical impact is potentially important. PMID:27547203

  16. Pump jack

    SciTech Connect

    Stanton, G. E.

    1985-02-26

    A pump jack of the type comprising a rocker arm pivotably mounted intermediate its ends on a support member, said rocker arm being divided by said pivot mounting into a sucker-rod limb and a drive limb wherein the improvement comprises a pneumatic motor pivotably attached to the drive support member and further pivotably attached to the mounting base of the pump jack to provide the power to reciprocate the pump jack. The working fluid of said pneumatic motor being natural gas which is available from the well casing of the well without any interference with the flow of the oil in the oil tube of the well thereby making use of an energy source available at any oil well without having to provide gasoline to drive a rotating type gasoline engine or electricity to drive an electric motor usually of the rotating variety. Also the stroke of a pneumatic cylinder inherently smooths out and eliminates the shock loading at the extremes of motion at the piston mounted to the sucker rods of such pump jack at the bottom of the well.

  17. Function of prokaryotic and eukaryotic ABC proteins in lipid transport.

    PubMed

    Pohl, Antje; Devaux, Philippe F; Herrmann, Andreas

    2005-03-21

    ATP binding cassette (ABC) proteins of both eukaryotic and prokaryotic origins are implicated in the transport of lipids. In humans, members of the ABC protein families A, B, C, D and G are mutated in a number of lipid transport and metabolism disorders, such as Tangier disease, Stargardt syndrome, progressive familial intrahepatic cholestasis, pseudoxanthoma elasticum, adrenoleukodystrophy or sitosterolemia. Studies employing transfection, overexpression, reconstitution, deletion and inhibition indicate the transbilayer transport of endogenous lipids and their analogs by some of these proteins, modulating lipid transbilayer asymmetry. Other proteins appear to be involved in the exposure of specific lipids on the exoplasmic leaflet, allowing their uptake by acceptors and further transport to specific sites. Additionally, lipid transport by ABC proteins is currently being studied in non-human eukaryotes, e.g. in sea urchin, trypanosomatides, arabidopsis and yeast, as well as in prokaryotes such as Escherichia coli and Lactococcus lactis. Here, we review current information about the (putative) role of both pro- and eukaryotic ABC proteins in the various phenomena associated with lipid transport. Besides providing a better understanding of phenomena like lipid metabolism, circulation, multidrug resistance, hormonal processes, fertilization, vision and signalling, studies on pro- and eukaryotic ABC proteins might eventually enable us to put a name on some of the proteins mediating transbilayer lipid transport in various membranes of cells and organelles. It must be emphasized, however, that there are still many uncertainties concerning the functions and mechanisms of ABC proteins interacting with lipids. In particular, further purification and reconstitution experiments with an unambiguous role of ATP hydrolysis are needed to demonstrate a clear involvement of ABC proteins in lipid transbilayer asymmetry. PMID:15749056

  18. Rapid efflux of Ca2+ from heart mitochondria in the presence of inorganic pyrophosphate.

    PubMed

    Vercesi, A; Lehninger, A L

    1984-01-13

    Inorganic pyrophosphate (PPi) in the intracellular concentration range causes rapid efflux of Ca2+ from rat heart mitochondria oxidizing pyruvate + malate in a low Na+ medium. Half-maximal rates of Ca2+ efflux were given by 20 microM PPi. During and after PPi-stimulated Ca2+ efflux the mitochondria retain their structural integrity and complete respiratory control. Carboxyatractyloside inhibits PPi-stimulated Ca2+ efflux, indicating PPi must enter the matrix in order to promote Ca2+ efflux. Heart mitochondria have a much higher affinity for PPi uptake and PPi-induced Ca2+ efflux than liver mitochondria.

  19. 18. Electrically driven pumps in Armory Street Pump House. Pumps ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    18. Electrically driven pumps in Armory Street Pump House. Pumps in background formerly drew water from the clear well. They went out of service when use of the beds was discontinued. Pumps in the foreground provide high pressure water to Hamden. - Lake Whitney Water Filtration Plant, Armory Street Pumphouse, North side of Armory Street between Edgehill Road & Whitney Avenue, Hamden, New Haven County, CT

  20. Regulation of a nickel-cobalt efflux system and nickel homeostasis in a soil actinobacterium Streptomyces coelicolor.

    PubMed

    Kim, Hae Mi; Ahn, Bo-Eun; Lee, Ju-Hyung; Roe, Jung-Hye

    2015-04-01

    In Streptomyces coelicolor, a soil actinobacterium capable of morphological differentiation and complex secondary metabolism, nickel deficiency is sensed by Nur, a Ni-specific Fur family regulator that controls nickel uptake systems (NikABCDE and NikMNOQ) and both Fe-containing and Ni-containing superoxide dismutases (SodF and SodN). On the other hand, the nickel efflux system and its regulator have not been elucidated. In this study, we demonstrate that an ArsR/SmtB family metalloregulator NmtR, a close homologue of NmtR from Mycobacterium tuberculosis, controls a putative efflux pump of P1-type ATPase (NmtA) in S. coelicolor. NmtR binds to the nmtA promoter region to repress its transcription, and is dissociated in the presence of Ni(ii) and Co(ii). Disruption of the nmtA gene makes cells more sensitive to nickel and cobalt, consistent with its predicted role in encoding a Ni-Co-efflux pump. Growth of S. coelicolor in complex YEME medium is only marginally inhibited by up to 0.5 mM Ni(ii), with significant growth retardation at 1 mM. Nur-regulated sodF and nikA genes are repressed at less than 0.1 μM added NiSO4 whereas NmtR-regulated nmtA transcription is induced at 0.5 mM or more Ni(ii). This reveals the extreme sensitivity of S. coelicolor to nickel deficiency as well as tolerance to surplus nickel. How this organism and possibly other actinomycetes have evolved to develop such a highly Ni-tolerant physiology and how the highly sensitive regulator Nur and the obtuse regulator NmtR achieve their characteristic Ni-sensitivity are interesting questions to solve in the future.

  1. Cloning of two novel ABC transporters mapping on human chromosome 9

    SciTech Connect

    Luciani, M.F.; Savary, S.; Chimini, G. ); Denizot, F. ); Mattei, M.G. )

    1994-05-01

    The family of ATP binding cassette (ABC) transporters or traffic ATPases is composed of several membrane-associated proteins that transport a great variety of solutes across cellular membranes. Two novel mammalian members of the family, ABC1 and ABC2, have been identified by a PCR-based approach. They belong to a group of traffic ATPases encoded as a single multifunctional protein, such as CFTR, STE 6, and P-glycoproteins. Their peculiar structural features and close relationship to ABC transporters involved in nodulation suggest that ABC1 and ABC2 define a novel subgroup of mammalian traffic ATPases. 51 refs., 7 figs.

  2. ABC transporters involved in export of cell surface glycoconjugates.

    PubMed

    Cuthbertson, Leslie; Kos, Veronica; Whitfield, Chris

    2010-09-01

    Complex glycoconjugates play critical roles in the biology of microorganisms. Despite the remarkable diversity in glycan structures and the bacteria that produce them, conserved themes are evident in the biosynthesis-export pathways. One of the primary pathways involves representatives of the ATP-binding cassette (ABC) transporter superfamily. These proteins are responsible for the export of a wide variety of cell surface oligo- and polysaccharides in both Gram-positive and Gram-negative bacteria. Recent investigations of the structure and function of ABC transporters involved in the export of lipopolysaccharide O antigens have revealed two fundamentally different strategies for coupling glycan polymerization to export. These mechanisms are distinguished by the presence (or absence) of characteristic nonreducing terminal modifications on the export substrates, which serve as chain termination and/or export signals, and by the presence (or absence) of a discrete substrate-binding domain in the nucleotide-binding domain polypeptide of the ABC transporter. A bioinformatic survey examining ABC exporters from known oligo- and polysaccharide biosynthesis loci identifies conserved nucleotide-binding domain protein families that correlate well with themes in the structures and assembly of glycans. The familial relationships among the ABC exporters generate hypotheses concerning the biosynthesis of structurally diverse oligo- and polysaccharides, which play important roles in the biology of bacteria with different lifestyles.

  3. ABC Transporters Involved in Export of Cell Surface Glycoconjugates

    PubMed Central

    Cuthbertson, Leslie; Kos, Veronica; Whitfield, Chris

    2010-01-01

    Summary: Complex glycoconjugates play critical roles in the biology of microorganisms. Despite the remarkable diversity in glycan structures and the bacteria that produce them, conserved themes are evident in the biosynthesis-export pathways. One of the primary pathways involves representatives of the ATP-binding cassette (ABC) transporter superfamily. These proteins are responsible for the export of a wide variety of cell surface oligo- and polysaccharides in both Gram-positive and Gram-negative bacteria. Recent investigations of the structure and function of ABC transporters involved in the export of lipopolysaccharide O antigens have revealed two fundamentally different strategies for coupling glycan polymerization to export. These mechanisms are distinguished by the presence (or absence) of characteristic nonreducing terminal modifications on the export substrates, which serve as chain termination and/or export signals, and by the presence (or absence) of a discrete substrate-binding domain in the nucleotide-binding domain polypeptide of the ABC transporter. A bioinformatic survey examining ABC exporters from known oligo- and polysaccharide biosynthesis loci identifies conserved nucleotide-binding domain protein families that correlate well with themes in the structures and assembly of glycans. The familial relationships among the ABC exporters generate hypotheses concerning the biosynthesis of structurally diverse oligo- and polysaccharides, which play important roles in the biology of bacteria with different lifestyles. PMID:20805402

  4. Reversal of multidrug resistance by the inhibition of ATP-binding cassette pumps employing "Generally Recognized As Safe" (GRAS) nanopharmaceuticals: A review.

    PubMed

    Sosnik, Alejandro

    2013-11-01

    Pumps of the ATP-binding cassette superfamily (ABCs) regulate the access of drugs to the intracellular space. In this context, the overexpression of ABCs is a well-known mechanism of multidrug resistance (MDR) in cancer and infectious diseases (e.g., viral hepatitis and the human immunodeficiency virus) and is associated with therapeutic failure. Since their discovery, ABCs have emerged as attractive therapeutic targets and the search of compounds that inhibit their genetic expression and/or their functional activity has gained growing interest. Different generations of pharmacological ABC inhibitors have been explored over the last four decades to address resistance in cancer, though clinical results have been somehow disappointing. "Generally Recognized As Safe" (GRAS) is a U.S. Food and Drug Administration designation for substances that are accepted as safe for addition in food. Far from being "inert", some amphiphilic excipients used in the production of pharmaceutical products have been shown to inhibit the activity of ABCs in MDR tumors, emerging as a clinically translatable approach to overcome resistance. The present article initially overviews the classification, structure and function of the different ABCs, with emphasis on those pumps related to drug resistance. Then, the different attempts to capitalize on the activity of GRAS nanopharmaceuticals as ABC inhibitors are discussed. PMID:24055628

  5. Effect of nonylphenol ethoxylates (NPEs) on barrier functions of epithelial cell membranes: opening of tight junctions and competitive inhibition of P-gp-mediated efflux.

    PubMed

    Doo, Min-Ho; Li, Hong; Jang, Hye-In; Song, Im-Sook; Chung, Suk-Jae; Shim, Chang-Koo

    2005-09-30

    The effect of nonylphenol ethoxylates (NPEs) on selected barrier functions of biological membranes, such as tight junction and P-gp efflux pump of epithelial membranes, against the transport of xenobiotics was examined. The Caco-2 cell line was used to evaluate the transport of mannitol and daunomycin across the cell monolayer as well as the cellular uptake of daunomycin. In the presence of NPEs, the transport of mannitol was increased, with NP-9 showing a maximal effect, and the transepithelial electrical resistance (TEER) was reduced. The onset of this effect of NP-9 was fairly rapid and reversible for a short term (e.g., 2 h) treatment, while irreversible for a long term (e.g., 72 h) treatment. In the presence of NP-9, the apical uptake of daunomycin was increased, suggesting competitive inhibition between NP-9 and daunomycin in the efflux via the P-gp system. However, a 72 h pretreatment of the cells with NP-9 (up to 1000 nM) did not affect the apparent cellular uptake of daunomycin, suggesting no significant effect of NPEs on the expression of P-gp. In conclusion, NPEs appear to rapidly open the tight junction of epithelial cell membranes and to competitively inhibit the efflux of P-gp substrates, thereby reducing the self-protection ability of the organism against xenobiotics or hazardous environmental compounds that are transported via the paracellular pathway (i.e., uptake) or the P-gp system (i.e., efflux).

  6. Calcium Efflux from Internally Dialyzed Squid Giant Axons

    PubMed Central

    Dipolo, Reinaldo

    1973-01-01

    Calcium efflux has been studied in squid giant axons under conditions in which the internal composition was controlled by means of a dialysis perfusion technique. The mean calcium efflux from axons dialyzed with 0.3 µM calcium and 5 mM ATP was 0.26 pmol/cm2·s at 22°C. The curve relating the Ca efflux with the internal Ca concentration had a slope of about one for [Ca]i lower than 0.3µM and a slope smaller than one for higher concentrations. Under the above conditions replacement of [Na]o and [Ca]o by Tris and Mg causes an 80% fall in the calcium efflux. When the axons were dialyzed with a medium free of ATP and containing 2 mM cyanide plus 5µg/ml oligomycin, analysis of the perfusion effluent gave values of 1–4 µM ATP. Under this low ATP condition, replacement of external sodium and calcium causes the same drop in the calcium efflux. The same effect was observed at higher [Ca]i, (80 µM). These results suggest that the Na-Ca exchange component of the calcium efflux is apparently not dependent on the amounts of ATP in the axoplasm. Axons previously depleted of ATP show a significant transient drop in the calcium efflux when ATP is added to the dialysis medium. This effect probably represents the sequestering of calcium by the mitochondrial system. The consumption of calcium by the mitochondria of the axoplasm in dialyzed axons was determined to be of the order of 6.0 x 10-7 mol Ca++/mg of protein with an initial rate of 2.6 x 10-8 mol Ca++/min·mg of protein. Axons dialyzed with 2 mM cyanide after 8–10-min delays show a rise in the calcium efflux in the presence of "normal" amounts of exogenous ATP. This effect seems to indicate that cyanide, per se, can release calcium ions from internal sources. PMID:4751386

  7. Vertical variations in wood CO2 efflux for live emergent trees in a Bornean tropical rainforest.

    PubMed

    Katayama, Ayumi; Kume, Tomonori; Komatsu, Hikaru; Ohashi, Mizue; Matsumoto, Kazuho; Ichihashi, Ryuji; Kumagai, Tomo'omi; Otsuki, Kyoichi

    2014-05-01

    Difficult access to 40-m-tall emergent trees in tropical rainforests has resulted in a lack of data related to vertical variations in wood CO2 efflux, even though significant variations in wood CO2 efflux are an important source of errors when estimating whole-tree total wood CO2 efflux. This study aimed to clarify vertical variations in wood CO2 efflux for emergent trees and to document the impact of the variations on the whole-tree estimates of stem and branch CO2 efflux. First, we measured wood CO2 efflux and factors related to tree morphology and environment for seven live emergent trees of two dipterocarp species at four to seven heights of up to ∼ 40 m for each tree using ladders and a crane. No systematic tendencies in vertical variations were observed for all the trees. Wood CO2 efflux was not affected by stem and air temperature, stem diameter, stem height or stem growth. The ratios of wood CO2 efflux at the treetop to that at breast height were larger in emergent trees with relatively smaller diameters at breast height. Second, we compared whole-tree stem CO2 efflux estimates using vertical measurements with those based on solely breast height measurements. We found similar whole-tree stem CO2 efflux estimates regardless of the patterns of vertical variations in CO2 efflux because the surface area in the canopy, where wood CO2 efflux often differed from that at breast height, was very small compared with that at low stem heights, resulting in little effect of the vertical variations on the estimate. Additionally, whole-tree branch CO2 efflux estimates using measured wood CO2 efflux in the canopy were considerably different from those measured using only breast height measurements. Uncertainties in wood CO2 efflux in the canopy did not cause any bias in stem CO2 efflux scaling, but affected branch CO2 efflux.

  8. Role of an ABC importer in mycobacterial drug resistance.

    PubMed

    Chakraborti, P K; Bhatt, K; Banerjee, S K; Misra, P

    1999-08-01

    Phosphate specific transporter (Pst) in bacteria is involved in phosphate transport. Pst is a multisubunit system which belongs to the ABC family of transporters. The import function of this transporter is known to be operative at media phosphate concentrations below the millimolar range. However, we found amplification of this transporter in a laboratory generated ciprofloxacin resistant Mycobacterium smegmatis colony (CIPr) which was grown in a condition when phosphate scavenging function of this operon was inoperative. Our results therefore argue the role of this ABC importer in conferring high level of fluoroquinolone resistance in CIPr.

  9. ABC estimation of unit costs for emergency department services.

    PubMed

    Holmes, R L; Schroeder, R E

    1996-04-01

    Rapid evolution of the health care industry forces managers to make cost-effective decisions. Typical hospital cost accounting systems do not provide emergency department managers with the information needed, but emergency department settings are so complex and dynamic as to make the more accurate activity-based costing (ABC) system prohibitively expensive. Through judicious use of the available traditional cost accounting information and simple computer spreadsheets. managers may approximate the decision-guiding information that would result from the much more costly and time-consuming implementation of ABC. PMID:10156656

  10. Plant cuticular lipid export requires an ABC transporter.

    PubMed

    Pighin, Jamie A; Zheng, Huanquan; Balakshin, Laura J; Goodman, Ian P; Western, Tamara L; Jetter, Reinhard; Kunst, Ljerka; Samuels, A Lacey

    2004-10-22

    A waxy protective cuticle coats all primary aerial plant tissues. Its synthesis requires extensive export of lipids from epidermal cells to the plant surface. Arabidopsis cer5 mutants had reduced stem cuticular wax loads and accumulated sheetlike inclusions in the cytoplasm of wax-secreting cells. These inclusions represented abnormal deposits of cuticular wax and resembled inclusions found in a human disorder caused by a defective peroxisomal adenosine triphosphate binding cassette (ABC) transporter. We found that the CER5 gene encodes an ABC transporter localized in the plasma membrane of epidermal cells and conclude that it is required for wax export to the cuticle.

  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. Modeling radionuclide effluxes from agricultural and natural ecosystems in Belarus.

    PubMed

    Zhuchenko, Yu M; Firsakova, S K; Voigt, G

    2002-06-01

    A mathematical model is described which is appropriately constructed to calculate effluxes of radionuclides from agricultural and natural ecosystems. The application of this model is demonstrated by estimating effluxes in the Bragin region and in the Narovlya region in the Republic of Belarus both highly affected by the Chernobyl accident fallout. Depending on the nature of the area and the deposition, the total efflux and the exported radioactivity are calculated. It is shown that the exported radioactivity for natural foodstuffs represents more than 64% (Bragin region) and 86% (Narovlya region) of the total 137Cs efflux, and for agricultural products 2.7% and 2.3%, respectively. The contribution of the different foodstuffs deriving from natural and agricultural used land to the individual and collective dose for 137Cs and 90Sr are estimated and presented. In the Bragin region for the collective annual dose the highest contribution is due to milk and meat consumption (137Cs) and flour and milk (90Sr), for individual annual dose milk and mushrooms (137Cs), and milk and flour (90Sr) contribute most. In the Narovlya region this contribution for the collective and individual annual dose is due to milk and mushroom consumption (137Cs) and flour and milk (90Sr).

  13. HDL-Mediated Cellular Cholesterol Efflux Assay Method.

    PubMed

    Hafiane, Anouar; Genest, Jacques

    2015-01-01

    Biomarkers of high-density lipoprotein (HDL) function may provide mechanistic insights and better cardiovascular risk discrimination than HDL-cholesterol mass. The purpose of this work is to describe a simplified experimental protocol that can be used in the determination of cholesterol efflux from macrophages cultured cells and be brought to a medium throughput volume. The cellular cholesterol efflux assay is designed to quantify the rate of cholesterol efflux from cultured cells to an acceptor particle or to plasma. This assay is multi step, cell based assay. Various factors, if not carefully controlled may influence the accuracy and reproducibility of the assay. Attempts were made to address factors influencing this assay and to provide a standardized method that is relatively rapid and scalable. We demonstrate that further centrifugation of the HDL fraction is necessary to avoid apolipoprotein B contamination when using polyethylene glycol (PEG) method. We demonstrate also no effect on cholesterol efflux efficiency when using PEG with plasma or serum. This method has been previously applied in our laboratory in context of cardiovascular research, cardiovascular disease and pharmacologic therapies. PMID:26663796

  14. Migration of Adipose-derived Mesenchymal Stem Cells Stably Expressing Chondroitinase ABC In vitro

    PubMed Central

    Wu, Jian-Huang; Li, Miao; Liang, Yan; Lu, Tao; Duan, Chun-Yue

    2016-01-01

    Background: Several studies have revealed that adipose-derived mesenchymal stem cells (ADSCs) can be used as seed cells for the treatment of spinal cord injury (SCI). Chondroitinase ABC (ChABC) decomposes chondroitin sulfate proteoglycans in the glial scar that forms following SCI, allowing stem cells to penetrate through the scar and promote recovery of nerve function. This study aimed to establish ADSCs that stably express ChABC (ChABC-ADSCs) and evaluate the migratory capability of ChABC-ADSCs in vitro. Methods: ADSCs were obtained from Sprague-Dawley rats using secondary collagenase digestion. Their phenotypes were characterized using flow cytometry detection of cell surface antigens and their stem cell properties were confirmed by induction of differentiation. After successful culture, ADSCs were transfected with lentiviral vectors and ChABC-ADSCs were obtained. Proliferation curves of ChABC-ADSCs were determined using the Cell Counting Kit-8 method, ChABC expression was verified using Western blotting, and the migration of ChABC-ADSCs was analyzed using the transwell assay. Results: Secondary collagenase digestion increased the isolation efficiency of primary ADSCs. Following transfection using lentiviral vectors, the proliferation of ChABC-ADSCs was reduced in comparison with control ADSCs at 48 h (P < 0.05). And the level of ChABC expression in the ChABC-ADSC group was significantly higher than that of the ADSC group (P < 0.05). Moreover, ChABC-ADSC migration in matrigel was significantly enhanced in comparison with the control (P < 0.05). Conclusions: Secondary collagenase digestion can be used to effectively isolate ADSCs. ChABC-ADSCs constructed using lentiviral vector transfection stably express ChABC, and ChABC expression significantly enhances the migratory capacity of ADSCs. PMID:27364797

  15. CO2 efflux from subterranean nests of ant communities in a seasonal tropical forest, Thailand.

    PubMed

    Hasin, Sasitorn; Ohashi, Mizue; Yamada, Akinori; Hashimoto, Yoshiaki; Tasen, Wattanachai; Kume, Tomonori; Yamane, Seiki

    2014-10-01

    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.

  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 t