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

Novel structural analogues of piperine as inhibitors of the NorA efflux pump of Staphylococcus aureus.

PubMed

Kumar, Ashwani; Khan, Inshad Ali; Koul, Surrinder; Koul, Jawahir Lal; Taneja, Subhash Chandra; Ali, Intzar; Ali, Furqan; Sharma, Sandeep; Mirza, Zahid Mehmood; Kumar, Manoj; Sangwan, Pyare Lal; Gupta, Pankaj; Thota, Niranjan; Qazi, Ghulam Nabi

2008-06-01

Evaluation of novel synthetic analogues of piperine as inhibitors of multidrug efflux pump NorA of Staphylococcus aureus. A library of piperine-derived compounds was evaluated for their potential to inhibit ethidium bromide efflux in NorA-overexpressing S. aureus SA 1199B. The active compounds were then individually combined with ciprofloxacin to study the potentiation of ciprofloxacin's activity. Based on the efflux inhibition assay, a library of 200 compounds was screened. Three piperine analogues, namely SK-20, SK-56 and SK-29, were found to be the most potent inhibitors of the NorA efflux pump. These inhibitors acted in a synergistic manner with ciprofloxacin, by substantially increasing its activity against both NorA-overexpressing and wild-type S. aureus isolates. These analogues were 2- to 4-fold more potent than piperine at a significantly lower minimal effective concentration. Furthermore, these inhibitors also significantly suppressed the in vitro emergence of ciprofloxacin-resistant S. aureus. A newly identified class of compounds derived from a natural amide, piperine, is more potent than the parent molecule in potentiating the activity of ciprofloxacin through the inhibition of the NorA efflux pump. These molecules may prove useful in augmenting the antibacterial activities of fluoroquinolones in a clinical setting.

First identification of boronic species as novel potential inhibitors of the Staphylococcus aureus NorA efflux pump.

PubMed

Fontaine, Fanny; Hequet, Arnaud; Voisin-Chiret, Anne-Sophie; Bouillon, Alexandre; Lesnard, Aurélien; Cresteil, Thierry; Jolivalt, Claude; Rault, Sylvain

2014-03-27

Overexpression of efflux pumps is an important mechanism of bacterial resistance that results in the extrusion of antimicrobial agents outside the bacterial cell. Inhibition of such pumps appears to be a promising strategy that could restore the potency of existing antibiotics. The NorA efflux pump of Staphylococcus aureus confers resistance to a wide range of unrelated substrates, such as hydrophilic fluoroquinolones, leading to a multidrug-resistance phenotype. In this work, approximately 150 heterocyclic boronic species were evaluated for their activity against susceptible and resistant strains of S. aureus. Twenty-four hit compounds, although inactive when tested alone, were found to potentiate ciprofloxacin activity by a 4-fold increase at concentrations ranging from 0.5 to 8 μg/mL against S. aureus 1199B, which overexpresses NorA. Boron-free analogues showed no biological activity, thus revealing that the boron atom is crucial for biological activity. This work describes the first reported efflux pump inhibitory activity of boronic acid derivatives.

Identification and evolution of drug efflux pump in clinical Enterobacter aerogenes strains isolated in 1995 and 2003.

PubMed

Chevalier, Jacqueline; Mulfinger, Céline; Garnotel, Eric; Nicolas, Pierre; Davin-Régli, Anne; Pagès, Jean-Marie

2008-09-12

The high mortality impact of infectious diseases will increase due to accelerated evolution of antibiotic resistance in important human pathogens. Development of antibiotic resistance is a evolutionary process inducing the erosion of the effectiveness of our arsenal of antibiotics. Resistance is not necessarily limited to a single class of antibacterial agents but may affect many unrelated compounds; this is termed 'multidrug resistance' (MDR). The major mechanism of MDR is the active expulsion of drugs by bacterial pumps; the treatment of gram negative bacterial infections is compromised due to resistance mechanisms including the expression of efflux pumps that actively expel various usual antibiotics (beta-lactams, quinolones, ...). Enterobacter aerogenes has emerged among Enterobacteriaceae associated hospital infections during the last twenty years due to its faculty of adaptation to antibiotic stresses. Clinical isolates of E. aerogenes belonging to two strain collections isolated in 1995 and 2003 respectively, were screened to assess the involvement of efflux pumps in antibiotic resistance. Drug susceptibility assays were performed on all bacterial isolates and an efflux pump inhibitor (PAbetaN) previously characterized allowed to decipher the role of efflux in the resistance. Accumulation of labelled chloramphenicol was monitored in the presence of an energy poison to determine the involvement of active efflux on the antibiotic intracellular concentrations. The presence of the PAbetaN-susceptible efflux system was also identified in resistant E. aerogenes strains. For the first time a noticeable increase in clinical isolates containing an efflux mechanism susceptible to pump inhibitor is report within an 8 year period. After the emergence of extended spectrum beta-lactamases in E. aerogenes and the recent characterisation of porin mutations in clinical isolates, this study describing an increase in inhibitor-susceptible efflux throws light on a new step in

Identification and Evolution of Drug Efflux Pump in Clinical Enterobacter aerogenes Strains Isolated in 1995 and 2003

PubMed Central

Garnotel, Eric; Nicolas, Pierre; Davin-Régli, Anne; Pagès, Jean-Marie

2008-01-01

Background The high mortality impact of infectious diseases will increase due to accelerated evolution of antibiotic resistance in important human pathogens. Development of antibiotic resistance is a evolutionary process inducing the erosion of the effectiveness of our arsenal of antibiotics. Resistance is not necessarily limited to a single class of antibacterial agents but may affect many unrelated compounds; this is termed ‘multidrug resistance’ (MDR). The major mechanism of MDR is the active expulsion of drugs by bacterial pumps; the treatment of Gram negative bacterial infections is compromised due to resistance mechanisms including the expression of efflux pumps that actively expel various usual antibiotics (ß-lactams, quinolones, …). Methodology/Principal Findings Enterobacter aerogenes has emerged among Enterobacteriaceae associated hospital infections during the last twenty years due to its faculty of adaptation to antibiotic stresses. Clinical isolates of E. aerogenes belonging to two strain collections isolated in 1995 and 2003 respectively, were screened to assess the involvement of efflux pumps in antibiotic resistance. Drug susceptibility assays were performed on all bacterial isolates and an efflux pump inhibitor (PAßN) previously characterized allowed to decipher the role of efflux in the resistance. Accumulation of labelled chloramphenicol was monitored in the presence of an energy poison to determine the involvement of active efflux on the antibiotic intracellular concentrations. The presence of the PAßN-susceptible efflux system was also identified in resistant E. aerogenes strains. Conclusions/Significance For the first time a noticeable increase in clinical isolates containing an efflux mechanism susceptible to pump inhibitor is report within an 8 year period. After the emergence of extended spectrum ß-lactamases in E. aerogenes and the recent characterisation of porin mutations in clinical isolates, this study describing an increase

Current Advances in Developing Inhibitors of Bacterial Multidrug 
Efflux Pumps

PubMed Central

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

Dynamics of Intact MexAB-OprM Efflux Pump: Focusing on the MexA-OprM Interface

DOE PAGES

Lopez, Cesar A.; Travers, Timothy; Pos, Klaas M.; ...

2017-11-28

Antibiotic efflux is one of the most critical mechanisms leading to bacterial multidrug resistance. Antibiotics are effluxed out of the bacterial cell by a tripartite efflux pump, a complex machinery comprised of outer membrane, periplasmic adaptor, and inner membrane protein components. Understanding the mechanism of efflux pump assembly and its dynamics could facilitate discovery of novel approaches to counteract antibiotic resistance in bacteria. We built here an intact atomistic model of the Pseudomonas aeruginosa MexAB-OprM pump in a Gram-negative membrane model that contained both inner and outer membranes separated by a periplasmic space. All-atom molecular dynamics (MD) simulations confirm thatmore » the fully assembled pump is stable in the microsecond timescale. Using a combination of all-atom and coarse-grained MD simulations and sequence covariation analysis, we characterized the interface between MexA and OprM in the context of the entire efflux pump. These analyses suggest a plausible mechanism by which OprM is activated via opening of its periplasmic aperture through a concerted interaction with MexA.« less

Dynamics of Intact MexAB-OprM Efflux Pump: Focusing on the MexA-OprM Interface

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lopez, Cesar A.; Travers, Timothy; Pos, Klaas M.

Antibiotic efflux is one of the most critical mechanisms leading to bacterial multidrug resistance. Antibiotics are effluxed out of the bacterial cell by a tripartite efflux pump, a complex machinery comprised of outer membrane, periplasmic adaptor, and inner membrane protein components. Understanding the mechanism of efflux pump assembly and its dynamics could facilitate discovery of novel approaches to counteract antibiotic resistance in bacteria. We built here an intact atomistic model of the Pseudomonas aeruginosa MexAB-OprM pump in a Gram-negative membrane model that contained both inner and outer membranes separated by a periplasmic space. All-atom molecular dynamics (MD) simulations confirm thatmore » the fully assembled pump is stable in the microsecond timescale. Using a combination of all-atom and coarse-grained MD simulations and sequence covariation analysis, we characterized the interface between MexA and OprM in the context of the entire efflux pump. These analyses suggest a plausible mechanism by which OprM is activated via opening of its periplasmic aperture through a concerted interaction with MexA.« less

Structures and transport dynamics of a Campylobacter jejuni multidrug efflux pump

DOE Office of Scientific and Technical Information (OSTI.GOV)

Su, Chih-Chia; Yin, Linxiang; Kumar, Nitin

2017-08-01

Resistance-nodulation-cell division efflux pumps are integral membrane proteins that catalyze the export of substrates across cell membranes. Within the hydrophobe-amphiphile efflux subfamily, these resistance-nodulation-cell division proteins largely form trimeric efflux pumps. The drug efflux process has been proposed to entail a synchronized motion between subunits of the trimer to advance the transport cycle, leading to the extrusion of drug molecules. Here we use X-ray crystallography and single-molecule fluorescence resonance energy transfer imaging to elucidate the structures and functional dynamics of the Campylobacter jejuni CmeB multidrug efflux pump. We find that the CmeB trimer displays a very unique conformation. A directmore » observation of transport dynamics in individual CmeB trimers embedded in membrane vesicles indicates that each CmeB subunit undergoes conformational transitions uncoordinated and independent of each other. On the basis of our findings and analyses, we propose a model for transport mechanism where CmeB protomers function independently within the trimer.« less

Efflux Pumps Are Involved in the Defense of Gram-Negative Bacteria against the Natural Products Isobavachalcone and Diospyrone ▿

PubMed Central

Kuete, Victor; Ngameni, Bathélémy; Tangmouo, Jean G.; Bolla, Jean-Michel; Alibert-Franco, Sandrine; Ngadjui, Bonaventure T.; Pagès, Jean-Marie

2010-01-01

The activities of two naturally occurring compounds, isobavachalcone and diospyrone, against documented strains and multidrug-resistant (MDR) Gram-negative bacterial isolates were evaluated. The results indicated that the two compounds exhibited intrinsic antibacterial activity against several Gram-negative bacteria, and their activities were significantly improved in the presence of an efflux pump inhibitor (MIC values decreased to below 10 μg/ml). In addition, the activities of isobavachalcone and diospyrone against various strains exhibiting deletions of the major efflux pump components (AcrAB, TolC) were significantly increased. The overall results indicate that isobavachalcone and diospyrone could be candidates for the development of new drugs against MDR strains and that their use in combination with efflux pump inhibitors reinforces their activity. PMID:20160051

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

PubMed

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

2008-07-24

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

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

Efflux Pump Gene Expression in Multidrug-Resistant Mycobacterium tuberculosis Clinical Isolates

PubMed Central

Jiang, Yi; Wei, Jianhao; Zhao, Li-li; Zhao, Xiuqin; Lu, Jianxin; Wan, Kanglin

2015-01-01

Isoniazid (INH) and rifampicin (RIF) are the two most effective drugs in tuberculosis therapy. Understanding the molecular mechanisms of resistance to these two drugs is essential to quickly diagnose multidrug-resistant (MDR) tuberculosis and extensive drug-resistant tuberculosis. Nine clinical Mycobacterium tuberculosis isolates resistant to only INH and RIF and 10 clinical pan-sensitive isolates were included to evaluate the expression of 20 putative drug efflux pump genes and sequence mutations in rpoB (RIF), katG (INH), the inhA promoter (INH), and oxyR-ahpC (INH). Nine and three MDR isolates were induced to overexpress efflux pump genes by INH and RIF, respectively. Eight and two efflux pump genes were induced to overexpress by INH and RIF in MDR isolates, respectively. drrA, drrB, efpA, jefA (Rv2459), mmr, Rv0849, Rv1634, and Rv1250 were overexpressed under INH or RIF stress. Most efflux pump genes were overexpressed under INH stress in a MDR isolates that carried the wild-type katG, inhA, and oxyR-ahpC associated with INH resistance than in those that carried mutations. The expression levels of 11 genes (efpA, Rv0849, Rv1250, P55 (Rv1410c), Rv1634, Rv2994, stp, Rv2459, pstB, drrA, and drrB) without drug inducement were significantly higher (P < 0.05) in nine MDR isolates than in 10 pan-sensitive isolates. In conclusion, efflux pumps may play an important role in INH acquired resistance in MDR M. tuberculosis, especially in those strains having no mutations in genes associated with INH resistance; basal expression levels of some efflux pump genes are higher in MDR isolates than in pan-sensitive isolates and the basal expressional differences may be helpful to diagnose and treat resistant tuberculosis. PMID:25695504

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

PubMed

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

2012-07-15

A library of 117 chalcones was screened for efflux pump inhibitory (EPI) activity against NorA mediated ethidium bromide efflux. Five of the chalcones (5-7, 9, and 10) were active and two chalcones (9 and 10) were equipotent to reserpine with IC(50)-values of 9.0 and 7.7 μM, respectively. Twenty chalcones were subsequently proved to be inhibitors of the NorA efflux pump in everted membrane vesicles. Compounds 5, 7, and 9 synergistically increased the effect of ciprofloxacin on Staphylococcus aureus. Our results suggest that chalcones might be developed into drugs for overcoming multidrug resistance based on efflux transporters of microorganisms. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

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.

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

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

2015-01-28

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.

An alternative physiological role for the EmhABC efflux pump in Pseudomonas fluorescens cLP6a

PubMed Central

2011-01-01

Background Efflux pumps belonging to the resistance-nodulation-division (RND) superfamily in bacteria are involved in antibiotic resistance and solvent tolerance but have an unknown physiological role. EmhABC, a RND-type efflux pump in Pseudomonas fluorescens strain cLP6a, extrudes hydrophobic antibiotics, dyes and polycyclic aromatic hydrocarbons including phenanthrene. The effects of physico-chemical factors such as temperature or antibiotics on the activity and expression of EmhABC were determined in order to deduce its physiological role(s) in strain cLP6a in comparison to the emhB disruptant strain, cLP6a-1. Results Efflux assays conducted with 14C-phenanthrene showed that EmhABC activity is affected by incubation temperature. Increased phenanthrene efflux was measured in cLP6a cells grown at 10°C and decreased efflux was observed at 35°C compared with cells grown at the optimum temperature of 28°C. Membrane fatty acids in cLP6a cells were substantially altered by changes in growth temperature and in the presence of tetracycline. Changed membrane fatty acids and increased membrane permeability were associated with ~30-fold increased expression of emhABC in cLP6a cells grown at 35°C, and with increased extracellular free fatty acids. Growth of P. fluorescens cLP6a at supra-optimal temperature was enhanced by the presence of EmhABC compared to strain cLP6a-1. Conclusions Combined, these observations suggest that the EmhABC efflux pump may be involved in the management of membrane stress effects such as those due to unfavourable incubation temperatures. Efflux of fatty acids replaced as a result of membrane damage or phospholipid turnover may be the primary physiological role of the EmhABC efflux pump in P. fluorescens cLP6a. PMID:22085438

Characterization of the Serratia marcescens SdeCDE multidrug efflux pump studied via gene knockout mutagenesis.

PubMed

Begic, Sanela; Worobec, Elizabeth A

2008-05-01

Serratia marcescens is an important nosocomial agent having high antibiotic resistance. A major mechanism for S. marcescens antibiotic resistance is active efflux. To ascertain the substrate specificity of the S. marcescens SdeCDE efflux pump, we constructed pump gene deletion mutants. sdeCDE knockout strains showed no change in antibiotic susceptibility in comparison with the parental strains for any of the substrates, with the exception of novobiocin. In addition, novobiocin was the only antibiotic to be accumulated by sdeCDE-deficient strains. Based on the substrates used in our study, we conclude that SdeCDE is a Resistance-Nodulation-Cell Division family pump with limited substrate specificity.

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.

Pharmacophore-Based Repositioning of Approved Drugs as Novel Staphylococcus aureus NorA Efflux Pump Inhibitors.

PubMed

Astolfi, Andrea; Felicetti, Tommaso; Iraci, Nunzio; Manfroni, Giuseppe; Massari, Serena; Pietrella, Donatella; Tabarrini, Oriana; Kaatz, Glenn W; Barreca, Maria L; Sabatini, Stefano; Cecchetti, Violetta

2017-02-23

An intriguing opportunity to address antimicrobial resistance is represented by the inhibition of efflux pumps. Focusing on NorA, the most important efflux pump of Staphylococcus aureus, an efflux pump inhibitors (EPIs) library was used for ligand-based pharmacophore modeling studies. By exploitation of the obtained models, an in silico drug repositioning approach allowed for the identification of novel and potent NorA EPIs.

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. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed

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.

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

PubMed

Chacón, Kelly N; Mealman, Tiffany D; McEvoy, Megan M; Blackburn, Ninian J

2014-10-28

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

Determination of the concentration of potential efflux pump inhibitors, pheophorbide a and pyropheophorbide a, in the feces of animals by fluorescence spectroscopy.

PubMed

Barnes, Charles A; Rasmussen, Sharon L; Petrich, Jacob W; Rasmussen, Mark A

2012-10-24

Efflux pumps are vital bacterial components, and research has demonstrated that some plant compounds such as pheophorbide a (php) possess efflux pump inhibitor (EPI) activity. This study determined the quantity of php present in feces as an indicator of EPI activity. Feces were collected from different species of animals fed a variety of feeds. The chlorophyll metabolites php and pyropheophorbide a (pyp) were determined using fluorescense spectroscopy. The average concentrations [μg/g dry matter (DM) feces] of pyp/php in feces were as follows: guinea pig, 180; goat, 150; rabbit, 150; dairy cow, 120; feedlot cattle, 60; rat, <1; pig, <1; chicken, <1. These data indicate that animals consuming "green" diets will excrete feces with concentrations of php/pyp that exceed levels demonstrated to be inhibitory to bacterial efflux pumps (0.5 μg/mL). The natural presence EPIs in the gastrointestinal tract may modulate the activity of microbial efflux pumps and exert selection pressure upon resident microbial populations.

Inactivation of a putative efflux pump (LmrB) in Streptococcus mutans results in altered biofilm structure and increased exopolysaccharide synthesis: implications for biofilm resistance.

PubMed

Liu, Jia; Zhang, Jianying; Guo, Lihong; Zhao, Wei; Hu, Xiaoli; Wei, Xi

2017-07-01

Efflux pumps are a mechanism associated with biofilm formation and resistance. There is limited information regarding efflux pumps in Streptococcus mutans, a major pathogen in dental caries. The aim of this study was to investigate potential roles of a putative efflux pump (LmrB) in S. mutans biofilm formation and susceptibility. Upon lmrB inactivation and antimicrobial exposure, the biofilm structure and expression of other efflux pumps were examined using confocal laser scanning microscopy (CLSM) and qRT-PCR. lmrB inactivation resulted in biofilm structural changes, increased EPS formation and EPS-related gene transcription (p < 0.05), but no improvement in susceptibility was observed. The expression of most efflux pump genes increased upon lmrB inactivation when exposed to antimicrobials (p < 0.05), suggesting a feedback mechanism that activated the transcription of other efflux pumps to compensate for the loss of lmrB. These observations imply that sole inactivation of lmrB is not an effective solution to control biofilms.

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. Copyright © 2015 Elsevier B.V. All rights reserved.

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

DOE PAGES

Chacon, Kelly N.; Mealman, Tiffany D.; McEvoy, Megan M.; ...

2014-10-13

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

Cloning, Sequencing, and Characterization of the SdeAB Multidrug Efflux Pump of Serratia marcescens

PubMed Central

Kumar, Ayush; Worobec, Elizabeth A.

2005-01-01

Serratia marcescens is an important nosocomial agent known for causing various infections in immunocompromised individuals. Resistance of this organism to a broad spectrum of antibiotics makes the treatment of infections very difficult. This study was undertaken to identify multidrug resistance efflux pumps in S. marcescens. Three mutant strains of S. marcescens were isolated in vitro by the serial passaging of a wild-type strain in culture medium supplemented with ciprofloxacin, norfloxacin, or ofloxacin. Fluoroquinolone accumulation assays were performed to detect the presence of a proton gradient-dependent efflux mechanism. Two of the mutant strains were found to be effluxing norfloxacin, ciprofloxacin, and ofloxacin, while the third was found to efflux only ofloxacin. A genomic library of S. marcescens wild-type strain UOC-67 was constructed and screened for RND pump-encoding genes by using DNA probes for two putative RND pump-encoding genes. Two different loci were identified: sdeAB, encoding an MFP and an RND pump, and sdeCDE, encoding an MFP and two different RND pumps. Northern blot analysis revealed overexpression of sdeB in two mutant strains effluxing fluoroquinolones. Analysis of the sdeAB and sdeCDE loci in Escherichia coli strain AG102MB, deficient in the RND pump (AcrB), revealed that gene products of sdeAB are responsible for the efflux of a diverse range of substrates that includes ciprofloxacin, norfloxacin, ofloxacin, chloramphenicol, sodium dodecyl sulfate, ethidium bromide, and n-hexane, while those of sdeCDE did not result in any change in susceptibilities to any of these agents. PMID:15793131

Cloning, sequencing, and characterization of the SdeAB multidrug efflux pump of Serratia marcescens.

PubMed

Kumar, Ayush; Worobec, Elizabeth A

2005-04-01

Serratia marcescens is an important nosocomial agent known for causing various infections in immunocompromised individuals. Resistance of this organism to a broad spectrum of antibiotics makes the treatment of infections very difficult. This study was undertaken to identify multidrug resistance efflux pumps in S. marcescens. Three mutant strains of S. marcescens were isolated in vitro by the serial passaging of a wild-type strain in culture medium supplemented with ciprofloxacin, norfloxacin, or ofloxacin. Fluoroquinolone accumulation assays were performed to detect the presence of a proton gradient-dependent efflux mechanism. Two of the mutant strains were found to be effluxing norfloxacin, ciprofloxacin, and ofloxacin, while the third was found to efflux only ofloxacin. A genomic library of S. marcescens wild-type strain UOC-67 was constructed and screened for RND pump-encoding genes by using DNA probes for two putative RND pump-encoding genes. Two different loci were identified: sdeAB, encoding an MFP and an RND pump, and sdeCDE, encoding an MFP and two different RND pumps. Northern blot analysis revealed overexpression of sdeB in two mutant strains effluxing fluoroquinolones. Analysis of the sdeAB and sdeCDE loci in Escherichia coli strain AG102MB, deficient in the RND pump (AcrB), revealed that gene products of sdeAB are responsible for the efflux of a diverse range of substrates that includes ciprofloxacin, norfloxacin, ofloxacin, chloramphenicol, sodium dodecyl sulfate, ethidium bromide, and n-hexane, while those of sdeCDE did not result in any change in susceptibilities to any of these agents.

Role of efflux pumps in the antibiotic resistance of bacteria embedded in a biofilm.

PubMed

Soto, Sara M

2013-04-01

Biofilms are complex microbial associations anchored to abiotic or biotic surfaces, embedded in extracellular matrix produced by the biofilms themselves where they interact with each other and the environment. One of the main properties of biofilms is their capacity to be more resistant to antimicrobial agents than planktonic cells. Efflux pumps have been reported as one of the mechanisms responsible for the antimicrobial resistance in biofilm structures. Evidence of the role of efflux pump in biofilm resistance has been found in several microorganisms such as Pseudomonas aeruginosa, Escherichia coli and Candida albicans. However, in spite of the studies on the importance of efflux pumps in biofilm growth and about their relevance in antimicrobial resistance forming biofilm, the exact role of these efflux systems has not been determined as yet.

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

Influence of intestinal efflux pumps on the absorption and transport of furosemide

PubMed Central

Al-Mohizea, Abdullah M.

2010-01-01

Purpose Furosemide is a commonly used diuretic which is used in the treatment of edema, congestive heart failure, hypertension and renal failure. Its absorption exhibits inter- and intra-subject variability that can be attributed to many factors including the intestinal efflux pumps such as the P-glycoprotein (P-gp). This study was done due to the great disagreement between what is published in the literature regarding the influence of P-gp on furosemide and at the same time due to the importance of this drug in the treatment of different conditions as described above. In addition, an investigation of the effect of two of the commonly used pharmaceutical excipients (hydroxypropyl β-cyclodextrin [HPβCD] and Tween 80) and also a P-gp inhibitor (verapamil hydrochloride) on the intestinal absorption of this drug were also done. Methods The study utilized the everted intestinal sacs technique to investigate both the effect of the efflux transporter (P-gp) on furosemide absorption and also the effect of the chosen excipients. Results The absorption of furosemide was significantly influenced by the P-gp as confirmed by the everted vis the non-everted sacs together with the verapamil study in which the transport of furosemide was inhibited by verapamil. In addition, Tween 80 was also shown to inhibit the P-gp pump whereas the HPβCD did not significantly influence the efflux of furosemide in this study. Conclusions P-glycoprotein and some of the used excipients in the formulation play a very important role in the transport of furosemide and other drugs. Thus excipients that affect the activity of P-gp should be avoided when formulating drugs that are substrate for the P-gp or other efflux pumps. PMID:23960725

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

PubMed

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

2011-08-25

Overexpression of efflux pumps is an important mechanism by which bacteria evade the effects of substrate antimicrobial agents. Inhibition of such pumps is a promising strategy to circumvent this resistance mechanism. NorA is a Staphylococcus aureus efflux pump that confers reduced susceptibility to many structurally unrelated agents, including fluoroquinolones, resulting in a multidrug resistant phenotype. In this work, a series of 2-phenyl-4(1H)-quinolone and 2-phenyl-4-hydroxyquinoline derivatives, obtained by modifying the flavone nucleus of known efflux pump inhibitors (EPIs), were synthesized in an effort to identify more potent S. aureus NorA EPIs. The 2-phenyl-4-hydroxyquinoline derivatives 28f and 29f display potent EPI activity against SA-1199B, a strain that overexpresses norA, in an ethidium bromide efflux inhibition assay. The same compounds, in combination with ciprofloxacin, were able to completely restore its antibacterial activity against both S. aureus SA-K2378 and SA-1199B, norA-overexpressing strains. © 2011 American Chemical Society

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

Antibacterial and efflux pump inhibitors of thymol and carvacrol against food-borne pathogens.

PubMed

Miladi, Hanene; Zmantar, Tarek; Chaabouni, Yassine; Fedhila, Kais; Bakhrouf, Amina; Mahdouani, Kacem; Chaieb, Kamel

2016-10-01

In this study thymol (THY) and carvacrol (CAR), two monoterpenic phenol produced by various aromatic plants, was tested for their antibacterial and efflux pump inhibitors potencies against a panel of clinical and foodborne pathogenes. Our results demonstrated a substantial susceptibility of the tested bacteria toward THY and CAR. Especially, THY displayed a strong inhibitory activity (MIC's values ranged from 32 to 64 μg/mL) against the majority of the tested strains compared to CAR. Moreover, a significant reduction in MIC's of TET and benzalkonium chloride (QAC) were noticed when tested in combinations with THY and CAR. Their synergic effect was more significant in the case of THY which resulted a reduction of MIC's values of TET (2-8 fold) and QAC (2-8 fold). We noted also that THY and CAR inhibited the ethidium bromide (EtBr) cell efflux in a concentration-dependent manner. The rate of EtBr accumulation in food-borne pathogen was enhanced with THY and CAR (0, 250 and 500 μg/mL). The lowest concentration causing 50% of EtBr efflux inhibition (IC 50) was noticed in Salmonella enteritidis (1129) at 150 μg/mL of THY and 190 μg/mL of CAR respectively. These findings indicate that THY and CAR may serve as potential sources of efflux pump inhibitor in food-borne pathogens. Copyright © 2016 Elsevier Ltd. All rights reserved.

Novel inhibitory activity of the Staphylococcus aureus NorA efflux pump by a kaempferol rhamnoside isolated from Persea lingue Nees.

PubMed

Holler, Jes Gitz; Christensen, S Brøgger; Slotved, Hans-Christian; Rasmussen, Hasse B; Gúzman, Alfonso; Olsen, Carl-Erik; Petersen, Bent; Mølgaard, Per

2012-05-01

To isolate a plant-derived compound with efflux inhibitory activity towards the NorA transporter of Staphylococcus aureus. Bioassay-guided isolation was used, with inhibition of ethidium bromide efflux via NorA as a guide. Characterization of activity was carried out using MIC determination and potentiation studies of a fluoroquinolone antibiotic in combination with the isolated compound. Everted membrane vesicles of Escherichia coli cells enriched with NorA were prepared to study efflux inhibitory activity in an isolated manner. The ethanolic extract of Persea lingue was subjected to bioassay-guided fractionation and led to the isolation of the known compound kaempferol-3-O-α-L-(2,4-bis-E-p-coumaroyl)rhamnoside (compound 1). Evaluation of the dose-response relationship of compound 1 showed that ethidium bromide efflux was inhibited, with an IC(50) value of 2 μM. The positive control, reserpine, was found to have an IC(50) value of 9 μM. Compound 1 also inhibited NorA in enriched everted membrane vesicles of E. coli. Potentiation studies revealed that compound 1 at 1.56 mg/L synergistically increased the antimicrobial activity of ciprofloxacin 8-fold against a NorA overexpresser, and the synergistic activity was exerted at a fourth of the concentration necessary for reserpine. Compound 1 was not found to exert a synergistic effect on ciprofloxacin against a norA deletion mutant. The 2,3-coumaroyl isomer of compound 1 has been shown previously not to cause acute toxicity in mice at 20 mg/kg/day. Our results show that compound 1 acts through inhibition of the NorA efflux pump. Combination of compound 1 with subinhibitory concentrations of ciprofloxacin renders a wild-type more susceptible and a NorA overexpresser S. aureus susceptible.

Xenon for tunnelling analysis of the efflux pump component OprN.

PubMed

Ntsogo Enguéné, Yvette Véronique; Phan, Gilles; Garnier, Cyril; Ducruix, Arnaud; Prangé, Thierry; Broutin, Isabelle

2017-01-01

Tripartite efflux pumps are among the main actors responsible for antibiotics resistance in Gram-negative bacteria. In the last two decades, structural studies gave crucial information about the assembly interfaces and the mechanistic motions. Thus rigidifying the assembly seems to be an interesting way to hamper the drug efflux. In this context, xenon is a suitable probe for checking whether small ligands could act as conformational lockers by targeting hydrophobic cavities. Here we focus on OprN, the outer membrane channel of the MexEF efflux pump from Pseudomonas aeruginosa. After exposing OprN crystals to xenon gas pressure, 14 binding sites were observed using X-ray crystallography. These binding sites were unambiguously characterized in hydrophobic cavities of OprN. The major site is observed in the sensitive iris-like region gating the channel at the periplasmic side, built by the three key-residues Leu 405, Asp 109, and Arg 412. This arrangement defines along the tunnel axis a strong hydrophobic/polar gradient able to enhance the passive efflux mechanism of OprN. The other xenon atoms reveal strategic hydrophobic regions of the channel scaffold to target, with the aim to freeze the dynamic movements responsible of the open/close conformational equilibrium in OprN.

Xenon for tunnelling analysis of the efflux pump component OprN

PubMed Central

Garnier, Cyril; Ducruix, Arnaud; Broutin, Isabelle

2017-01-01

Tripartite efflux pumps are among the main actors responsible for antibiotics resistance in Gram-negative bacteria. In the last two decades, structural studies gave crucial information about the assembly interfaces and the mechanistic motions. Thus rigidifying the assembly seems to be an interesting way to hamper the drug efflux. In this context, xenon is a suitable probe for checking whether small ligands could act as conformational lockers by targeting hydrophobic cavities. Here we focus on OprN, the outer membrane channel of the MexEF efflux pump from Pseudomonas aeruginosa. After exposing OprN crystals to xenon gas pressure, 14 binding sites were observed using X-ray crystallography. These binding sites were unambiguously characterized in hydrophobic cavities of OprN. The major site is observed in the sensitive iris-like region gating the channel at the periplasmic side, built by the three key-residues Leu 405, Asp 109, and Arg 412. This arrangement defines along the tunnel axis a strong hydrophobic/polar gradient able to enhance the passive efflux mechanism of OprN. The other xenon atoms reveal strategic hydrophobic regions of the channel scaffold to target, with the aim to freeze the dynamic movements responsible of the open/close conformational equilibrium in OprN. PMID:28886086

Aminoguanidine hydrazones (AGH's) as modulators of norfloxacin resistance in Staphylococcus aureus that overexpress NorA efflux pump.

PubMed

Dantas, Natalina; de Aquino, Thiago Mendonça; de Araújo-Júnior, João Xavier; da Silva-Júnior, Edeildo; Gomes, Ednaldo Almeida; Gomes, Antoniel Augusto Severo; Siqueira-Júnior, José Pinto; Mendonça Junior, Francisco Jaime Bezerra

2018-01-25

One of the promising fields for improving the effectiveness of antimicrobial agents is their combination with efflux pump inhibitors (EPIs), which besides expanding the use of existing antibiotics. The goal of this research was to evaluate a series of aminoguanidine hydrazones (AGH's, 1-19) as antibacterial agents and NorA efflux pump inhibitors in Staphylococcus aureus strain SA-1199B. Molecular modeling and docking studies were also performed in order to explain at the molecular level the interactions of the compounds with the generated NorA efflux pump model. The MICs of the antibiotic and ethidium bromide were determined by microdilution assay in absence or presence of a subinhibitory concentration of aminoguanidine hydrazones and macrophages viability was determined through MTT assay. Bioinformatic software Swiss-Model and AutoDock 4.2 were used to perform modeling and docking studies, respectively. As results, all AGH's were able to potentiate the action for the antibiotic norfloxacin, causing MIC's reduction of 16-fold and 32-fold to ethidium bromide. In the cell viability test, the concentration of 10 μg/mL showed better results than 90% and the concentration of 1000 μg/mL showed the lowest viability, reaching a maximum of 50% for the analyzed aminoguanidine hydrazones. Molecular docking studies showed that both norfloxacin and derivative 13 were recognized by the same binding site of NorA pump, suggesting a competitive mechanism. The present work demonstrated for the first time that AGH derivatives have potential to be putative inhibitors of NorA efflux pump, showing a promising activity as an antibacterial drug development. Copyright © 2017 Elsevier B.V. All rights reserved.

Use and engineering of efflux pumps for the export of olefins in microbes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mukhopadhyay, Aindrila

2016-07-14

The scope of the project is to investigate efflux pump systems in engineered host microorganisms, such as E. coli, and develop a pump engineered to export a target compound. To initiate the project in coordination with other TOTAL driven projects, the first target compound to be studied was 1-hexene. However, we were investigating other chemicals as Styrene. The main goal of the project was to generate a set of optimized efflux pump systems for microorganisms (E. coli and Streptomyces or other host) engineered to contain biosynthetic pathways to export large titers of target compounds that are toxic (or accumulate andmore » push back biosynthesis) to the host cell. An optimized microbial host will utilize specific and efficient cell wall located pumps to extrude harmful target compounds and enable greater production of these compounds.« less

Stress Introduction Rate Alters the Benefit of AcrAB-TolC Efflux Pumps.

PubMed

Langevin, Ariel M; Dunlop, Mary J

2018-01-01

Stress tolerance studies are typically conducted in an all-or-none fashion. However, in realistic settings-such as in clinical or metabolic engineering applications-cells may encounter stresses at different rates. Therefore, how cells tolerate stress may depend on its rate of appearance. To address this, we studied how the rate of stress introduction affects bacterial stress tolerance by focusing on a key stress response mechanism. Efflux pumps, such as AcrAB-TolC of Escherichia coli , are membrane transporters well known for the ability to export a wide variety of substrates, including antibiotics, signaling molecules, and biofuels. Although efflux pumps improve stress tolerance, pump overexpression can result in a substantial fitness cost to the cells. We hypothesized that the ideal pump expression level would involve a rate-dependent trade-off between the benefit of pumps and the cost of their expression. To test this, we evaluated the benefit of the AcrAB-TolC pump under different rates of stress introduction, including a step, a fast ramp, and a gradual ramp. Using two chemically diverse stresses, the antibiotic chloramphenicol and the jet biofuel precursor pinene, we assessed the benefit provided by the pumps. A mathematical model describing these effects predicted the benefit as a function of the rate of stress introduction. Our findings demonstrate that as the rate of introduction is lowered, stress response mechanisms provide a disproportionate benefit to pump-containing strains, allowing cells to survive beyond the original inhibitory concentrations. IMPORTANCE Efflux pumps are ubiquitous in nature and provide stress tolerance in the cells of species ranging from bacteria to mammals. Understanding how pumps provide tolerance has far-reaching implications for diverse fields, from medicine to biotechnology. Here, we investigated how the rate of stressor appearance impacts tolerance. We focused on two distinct substrates of AcrAB-TolC efflux pumps, the

Involvement of the Efflux Pumps in Chloramphenicol Selected Strains of Burkholderia thailandensis: Proteomic and Mechanistic Evidence

PubMed Central

Biot, Fabrice V.; Valade, Eric; Garnotel, Eric; Chevalier, Jacqueline; Villard, Claude; Thibault, François M.; Vidal, Dominique R.; Pagès, Jean-Marie

2011-01-01

Burkholderia is a bacterial genus comprising several pathogenic species, including two species highly pathogenic for humans, B. pseudomallei and B. mallei. B. thailandensis is a weakly pathogenic species closely related to both B. pseudomallei and B. mallei. It is used as a study model. These bacteria are able to exhibit multiple resistance mechanisms towards various families of antibiotics. By sequentially plating B. thailandensis wild type strains on chloramphenicol we obtained several resistant variants. This chloramphenicol-induced resistance was associated with resistance against structurally unrelated antibiotics including quinolones and tetracyclines. We functionally and proteomically demonstrate that this multidrug resistance phenotype, identified in chloramphenicol-resistant variants, is associated with the overexpression of two different efflux pumps. These efflux pumps are able to expel antibiotics from several families, including chloramphenicol, quinolones, tetracyclines, trimethoprim and some β-lactams, and present a partial susceptibility to efflux pump inhibitors. It is thus possible that Burkholderia species can develop such adaptive resistance mechanisms in response to antibiotic pressure resulting in emergence of multidrug resistant strains. Antibiotics known to easily induce overexpression of these efflux pumps should be used with discernment in the treatment of Burkholderia infections. PMID:21347382

Inhibition of the NorA efflux pump of Staphylococcus aureus by synthetic riparins.

PubMed

Costa, L M; de Macedo, E V; Oliveira, F A A; Ferreira, J H L; Gutierrez, S J C; Peláez, W J; Lima, F C A; de Siqueira Júnior, J P; Coutinho, H D M; Kaatz, G W; de Freitas, R M; Barreto, H M

2016-11-01

The goal of this study was to increase knowledge about the antimicrobial activity of some synthetic Riparin-derived compounds, alone or in combination with fluoroquinolone antibiotics, against a strain of Staphylococcus aureus resistant to fluoroquinolone by way of overexpression of the NorA efflux pump. Microdilution tests showed that Riparins A and B did not show any significant antibacterial activity against Staph. aureus strains. On the other hand, the intrinsic antibacterial activity increased with increasing lipophilicity of the compounds, in the following order: Riparin-D (MIC 256 μg ml -1 ; Log P 2·95) < Riparin-C (MIC 102 μg ml -1 ; Log P 3·22) < Riparin-E (MIC 16 μg ml -1 ; Log P 3·57). The addition of all riparins to growth media at subinhibitory concentrations caused an increase in the antibacterial activity of antibiotics against the NorA-overexpressing test strain. Riparin-B, which has two methoxyl groups at the phenethyl moiety, showed the best modulatory effect. Riparin-E is a good anti-staphylococci agent, while Riparin-B functions as a NorA efflux pump inhibitor. Our data suggest the possibility of using Riparin-B in combination with norfloxacin or ciprofloxacin for therapy of infections caused by multi-drug resistant Staph. aureus. © 2016 The Society for Applied Microbiology.

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

PubMed

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

2013-06-27

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

Evaluation of a series of 2-napthamide derivatives as inhibitors of the drug efflux pump AcrB for the reversal of antimicrobial resistance.

PubMed

Wang, Yinhu; Mowla, Rumana; Guo, Liwei; Ogunniyi, Abiodun D; Rahman, Taufiq; De Barros Lopes, Miguel A; Ma, Shutao; Venter, Henrietta

2017-02-15

Drug efflux pumps confer multidrug resistance to dangerous pathogens which makes these pumps important drug targets. We have synthesised a novel series of compounds based on a 2-naphthamide pharmacore aimed at inhibiting the efflux pumps from Gram-negative bacteria. The archeatypical transporter AcrB from Escherichia coli was used as model efflux pump as AcrB is widely conserved throughout Gram-negative organisms. The compounds were tested for their antibacterial action, ability to potentiate the action of antibiotics and for their ability to inhibit Nile Red efflux by AcrB. None of the compounds were antimicrobial against E. coli wild type cells. Most of the compounds were able to inhibit Nile Red efflux indicating that they are substrates of the AcrB efflux pump. Three compounds were able to synergise with antibiotics and reverse resistance in the resistant phenotype. Compound A3, 4-(isopentyloxy)-2-naphthamide, reduced the MICs of erythromycin and chloramphenicol to the MIC levels of the drug sensitive strain that lacks an efflux pump. A3 had no effect on the MIC of the non-substrate rifampicin indicating that this compound acts specifically through the AcrB efflux pump. A3 also does not act through non-specific mechanisms such as outer membrane or inner membrane permeabilisation and is not cytotoxic against mammalian cell lines. Therefore, we have designed and synthesised a novel chemical compound with great potential to further optimisation as inhibitor of drug efflux pumps. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

PubMed

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

2016-09-01

A total of eighteen piperic acid (PA) and 4-ethylpiperic acid (EPA) amides (C1-C18) with α-, β- and γ-amino acids were synthesized, characterized and evaluated for their efflux pump inhibitory activity against ciprofloxacin resistant Staphylococcus aureus. The amides were screened against NorA overexpressing S. aureus SA-1199B and wild type S. aureus SA-1199 using ethidium bromide as NorA efflux pump substrate. EPI C6 was found to be most potent and reduced the MIC of ciprofloxacin by 16 fold followed by C18 which showed 4 fold reduction of MIC. Ethidium bromide efflux inhibition and accumulation assay proved these compounds as NorA inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

Efflux pump induction by quaternary ammonium compounds and fluoroquinolone resistance in bacteria.

PubMed

Buffet-Bataillon, Sylvie; Tattevin, Pierre; Maillard, Jean-Yves; Bonnaure-Mallet, Martine; Jolivet-Gougeon, Anne

2016-01-01

Biocides, primarily those containing quaternary ammonium compounds (QAC), are heavily used in hospital environments and various industries (e.g., food, water, cosmetic). To date, little attention has been paid to potential implications of QAC use in the emergence of antibiotic resistance, especially fluoroquinolone-resistant bacteria in patients and in the environment. QAC-induced overexpression of efflux pumps can lead to: cross resistance with fluoroquinolones mediated by multidrug efflux pumps; stress response facilitating mutation in the Quinolone Resistance Determining Region; and biofilm formation increasing the risk of transfer of mobile genetic elements carrying fluoroquinolone or QAC resistance determinants. By following the European Biocidal Product Regulation, manufacturers of QAC are required to ensure that their QAC-based biocidal products are safe and will not contribute to emerging bacterial resistance.

Contribution of putative efflux pump genes to isoniazid resistance in clinical isolates of Mycobacterium tuberculosis.

PubMed

Narang, Anshika; Giri, Astha; Gupta, Shraddha; Garima, Kushal; Bose, Mridula; Varma-Basil, Mandira

2017-01-01

Isoniazid (INH) resistance in Mycobacterium tuberculosis has been mainly attributed to mutations in katG (64%) and inhA (19%). However, 20%-30% resistance to INH cannot be explained by mutations alone. Hence, other mechanisms besides mutations may play a significant role in providing drug resistance. Here, we explored the role of 24 putative efflux pump genes conferring INH-resistance in M. tuberculosis. Real-time expression profiling of the efflux pump genes was performed in five INH-susceptible and six high-level INH-resistant clinical isolates of M. tuberculosis exposed to the drug. Isolates were also analyzed for mutations in katG and inhA. Four high-level INH-resistant isolates (minimum inhibitory concentration [MIC] ≥2.5 mg/L) with mutations at codon 315 (AGC-ACC) of katG showed upregulation of one of the efflux genes Rv1634, Rv0849, efpA, or p55. Another high-level INH-resistant isolate (MIC 1.5 mg/L), with no mutations at katG or inhA overexpressed 8/24 efflux genes, namely, Rv1273c, Rv0194, Rv1634, Rv1250, Rv3823c, Rv0507, jefA, and p55. Five of these, namely, Rv0194, Rv1634, Rv1250, Rv0507, and p55 were induced only in resistant isolates. The high number of efflux genes overexpressed in an INH-resistant isolate with no known INH resistance associated mutations, suggests a role for efflux pumps in resistance to this antituberculous agent, with the role of Rv0194 and Rv0507 in INH resistance being reported for the first time.

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.

Macrolide resistance in Legionella pneumophila: the role of LpeAB efflux pump.

PubMed

Massip, Clémence; Descours, Ghislaine; Ginevra, Christophe; Doublet, Patricia; Jarraud, Sophie; Gilbert, Christophe

2017-05-01

A previous study on 12 in vitro -selected azithromycin-resistant Legionella pneumophila lineages showed that ribosomal mutations were major macrolide resistance determinants. In addition to these mechanisms that have been well described in many species, mutations upstream of lpeAB operon, homologous to acrAB in Escherichia coli , were identified in two lineages. In this study, we investigated the role of LpeAB and of these mutations in macrolide resistance of L. pneumophila . The role of LpeAB was studied by testing the antibiotic susceptibility of WT, deleted and complemented L. pneumophila Paris strains. Translational fusion experiments using GFP as a reporter were conducted to investigate the consequences of the mutations observed in the upstream sequence of lpeAB operon. We demonstrated the involvement of LpeAB in an efflux pump responsible for a macrolide-specific reduced susceptibility of L. pneumophila Paris strain. Mutations in the upstream sequence of lpeAB operon were associated with an increased protein expression. Increased expression was also observed under sub-inhibitory macrolide concentrations in strains with both mutated and WT promoting regions. LpeAB are components of an efflux pump, which is a macrolide resistance determinant in L. pneumophila Paris strain. Mutations observed in the upstream sequence of lpeAB operon in resistant lineages led to an overexpression of this efflux pump. Sub-inhibitory concentrations of macrolides themselves participated in upregulating this efflux and could constitute a first step in the acquisition of a high macrolide resistance level. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Inhibitors of bacterial multidrug efflux pumps potentiate antimicrobial photoinactivation.

PubMed

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

2008-09-01

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

Fluoroquinolone resistance of Serratia marcescens: involvement of a proton gradient-dependent efflux pump.

PubMed

Kumar, Ayush; Worobec, Elizabeth A

2002-10-01

To determine the presence of a proton gradient-dependent efflux of fluoroquinolone drugs in Serratia marcescens. Thirteen clinical isolates of S. marcescens were screened for resistance to four fluoroquinolones: ofloxacin, ciprofloxacin, norfloxacin and nalidixic acid by determining MICs. The presence of a proton gradient-dependent efflux mechanism was assessed using ethidium bromide accumulation assays. Drug accumulation studies for norfloxacin, ciprofloxacin and ofloxacin were performed to determine the drug specificity of efflux. Western transfer of cellular proteins, followed by immunodetection using anti-AcrA (Escherichia coli) antibodies were used to demonstrate the presence of a resistance-nodulation-cell division (RND) pump protein. PCR was used to identify a RND pump-encoding gene using primers for two conserved motifs within inner membrane components of RND proteins. A mutant strain of S. marcescens, UOC-67WL, was isolated by culturing the wild-type strain in the presence of ciprofloxacin in T-soy media and was subjected to the same studies as described above for the clinical isolates. Ethidium bromide accumulation assays confirmed the presence of a proton gradient-dependent efflux mechanism in S. marcescens. One clinical isolate, T-861, and the mutant strain, UOC-67WL, were found to efflux ciprofloxacin and ofloxacin. Western immunoblot results confirmed overexpression of an AcrA-like protein in T-861 and UOC-67WL. Sequencing of the PCR product showed the presence of a mexF-like gene, which is overexpressed in nfxC mutants of Pseudomonas aeruginosa. This study reports the presence of a proton gradient-dependent efflux mechanism in S. marcescens.

Capsaicin, a novel inhibitor of the NorA efflux pump, reduces the intracellular invasion of Staphylococcus aureus.

PubMed

Kalia, Nitin Pal; Mahajan, Priya; Mehra, Rukmankesh; Nargotra, Amit; Sharma, Jai Parkash; Koul, Surrinder; Khan, Inshad Ali

2012-10-01

To delineate the role of capsaicin (8-methyl-N-vanillyl-6-nonenamide) as an inhibitor of the NorA efflux pump and its impact on invasion of macrophages by Staphylococcus aureus. Capsaicin in combination with ciprofloxacin was tested for activity against S. aureus SA-1199B (NorA overproducing), SA-1199 (wild-type) and SA-K1758 (norA knockout). The role of NorA in the intracellular invasion of S. aureus and the ability of capsaicin to inhibit this invasion was established in J774 macrophage cell lines. The three-dimensional structure of NorA was predicted using an in silico approach and docking studies of capsaicin were performed. Capsaicin significantly reduced the MIC of ciprofloxacin for S. aureus SA-1199 and SA-1199B. Furthermore, capsaicin also extended the post-antibiotic effect of ciprofloxacin by 1.1 h at MIC concentration. There was a decrease in mutation prevention concentration of ciprofloxacin when combined with capsaicin. Inhibition of ethidium bromide efflux by NorA-overproducing S. aureus SA-1199B confirmed the role of capsaicin as a NorA efflux pump inhibitor (EPI). The most significant finding of this study was the ability of capsaicin to reduce the intracellular invasion of S. aureus SA-1199B (NorA overproducing) in J774 macrophage cell lines by 2 log(10). This study, for the first time, has shown that capsaicin, a novel EPI, not only inhibits the NorA efflux pump of S. aureus but also reduces the invasiveness of S. aureus, thereby reducing its virulence.

Binding site feature description of 2-substituted benzothiazoles as potential AcrAB-TolC efflux pump inhibitors in E. coli.

PubMed

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

2015-01-01

The resistance-nodulation-division (RND) family efflux pumps are important in the antibiotic resistance of Gram-negative bacteria. However, although a number of bacterial RND efflux pump inhibitors have been developed, there has been no clinically available RND efflux pump inhibitor to date. A set of BSN-coded 2-substituted benzothiazoles were tested alone and in combinations with ciprofloxacin (CIP) against the AcrAB-TolC overexpressor Escherichia coli AG102 clinical strain. The results indicated that the BSN compounds did not show intrinsic antimicrobial activity when tested alone. However, when used in combinations with CIP, a reversal in the antibacterial activity of CIP with up to 10-fold better MIC values was observed. In order to describe the binding site features of these BSN compounds with AcrB, docking studies were performed using the CDocker method. The performed docking poses and the calculated binding energy scores revealed that the tested compounds BSN-006, BSN-023, and BSN-004 showed significant binding interactions with the phenylalanine-rich region in the distal binding site of the AcrB binding monomer. Moreover, the tested compounds BSN-006 and BSN-023 possessed stronger binding energies than CIP, verifying that BSN compounds are acting as the putative substrates of AcrB.

Ligand Promiscuity between the Efflux Pumps Human P-Glycoprotein and S. aureus NorA.

PubMed

Brincat, Jean Pierre; Broccatelli, Fabio; Sabatini, Stefano; Frosini, Maria; Neri, Annalisa; Kaatz, Glenn W; Cruciani, Gabriele; Carosati, Emanuele

2012-03-08

Thirty-two diverse compounds were evaluated for their ability to inhibit both Pgp-mediated efflux in mouse T-lymphoma L5178 MDR1 and NorA-mediated efflux in S. aureus SA-1199B. Only four compounds were strong inhibitors of both efflux pumps. Three compounds were found to inhibit Pgp exclusively and strongly, while seven compounds inhibited only NorA. These results demonstrate that Pgp and NorA inhibitors do not necessarily overlap, opening the way to safer therapeutic use of effective NorA inhibitors.

Vitamin K3 Induces the Expression of the Stenotrophomonas maltophilia SmeVWX Multidrug Efflux Pump.

PubMed

Blanco, P; Corona, F; Sánchez, M B; Martínez, J L

2017-05-01

Stenotrophomonas maltophilia is an opportunistic pathogen with increasing prevalence, which is able to cause infections in immunocompromised patients or in those with a previous pathology. The treatment of the infections caused by this bacterium is often complicated due to the several intrinsic antibiotic resistance mechanisms that it presents. Multidrug efflux pumps are among the best-studied mechanisms of S. maltophilia antibiotic resistance. Some of these efflux pumps have a basal expression level but, in general, their expression is often low and only reaches high levels when the local regulator is mutated or bacteria are in the presence of an effector. In the current work, we have developed a yellow fluorescent protein (YFP)-based sensor with the aim to identify effectors able to trigger the expression of SmeVWX, an efflux pump that confers resistance to quinolones, chloramphenicol, and tetracycline when it is expressed at high levels. With this purpose in mind, we tested a variety of different compounds and analyzed the fluorescence signal given by the expression of YFP under the control of the smeVWX promoter. Among the tested compounds, vitamin K 3 , which is a compound belonging to the 2-methyl-1,4-naphthoquinone family, is produced by plants in defense against infection, and has increasing importance in human therapy, was able to induce the expression of the SmeVWX efflux pump. In addition, a decrease in the susceptibility of S. maltophilia to ofloxacin and chloramphenicol was observed in the presence of vitamin K 3 , in both wild-type and smeW -deficient strains. Copyright © 2017 American Society for Microbiology.

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

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

Efflux pumps as antimicrobial resistance mechanisms.

PubMed

Poole, Keith

2007-01-01

Antibiotic resistance continues to hamper antimicrobial chemotherapy of infectious disease, and while biocide resistance outside of the laboratory is as yet unrealized, in vitro and in vivo episodes of reduced biocide susceptibility are not uncommon. Efflux mechanisms, both drug-specific and multidrug, are important determinants of intrinsic and/or acquired resistance to these antimicrobials in important human pathogens. Multidrug efflux mechanisms are generally chromosome-encoded, with their expression typically resultant from mutations in regulatory genes, while drug-specific efflux mechanisms are encoded by mobile genetic elements whose acquisition is sufficient for resistance. While it has been suggested that drug-specific efflux systems originated from efflux determinants of self-protection in antibiotic-producing Actinomycetes, chromosomal multidrug efflux determinants, at least in Gram-negative bacteria, are appreciated as having an intended housekeeping function unrelated to drug export and resistance. Thus, it will be important to elucidate the intended natural function of these efflux mechanisms in order, for example, to anticipate environmental conditions or circumstances that might promote their expression and, so, compromise antimicrobial chemotherapy. Given the clinical significance of antimicrobial exporters, it is clear that efflux must be considered in formulating strategies for treatment of drug-resistant infections, both in the development of new agents, for example, less impacted by efflux or in targeting efflux directly with efflux inhibitors.

Plasmid-Encoded Tetracycline Efflux Pump Protein Alters Bacterial Stress Responses and Ecological Fitness of Acinetobacter oleivorans

PubMed Central

Hong, Hyerim; Jung, Jaejoon; Park, Woojun

2014-01-01

Acquisition of the extracellular tetracycline (TC) resistance plasmid pAST2 affected host gene expression and phenotype in the oil-degrading soil bacterium, Acinetobacter oleivorans DR1. Whole-transcriptome profiling of DR1 cells harboring pAST2 revealed that all the plasmid genes were highly expressed under TC conditions, and the expression levels of many host chromosomal genes were modulated by the presence of pAST2. The host energy burden imposed by replication of pAST2 led to (i) lowered ATP concentrations, (ii) downregulated expression of many genes involved in cellular growth, and (iii) reduced growth rate. Interestingly, some phenotypes were restored by deleting the plasmid-encoded efflux pump gene tetH, suggesting that the membrane integrity changes resulting from the incorporation of efflux pump proteins also resulted in altered host response under the tested conditions. Alteration of membrane integrity by tetH deletion was shown by measuring permeability of fluorescent probe and membrane hydrophobicity. The presence of the plasmid conferred peroxide and superoxide resistance to cells, but only peroxide resistance was diminished by tetH gene deletion, suggesting that the plasmid-encoded membrane-bound efflux pump protein provided peroxide resistance. The downregulation of fimbriae-related genes presumably led to reduced swimming motility, but this phenotype was recovered by tetH gene deletion. Our data suggest that not only the plasmid replication burden, but also its encoded efflux pump protein altered host chromosomal gene expression and phenotype, which also alters the ecological fitness of the host in the environment. PMID:25229538

Plasmid-encoded tetracycline efflux pump protein alters bacterial stress responses and ecological fitness of Acinetobacter oleivorans.

PubMed

Hong, Hyerim; Jung, Jaejoon; Park, Woojun

2014-01-01

Acquisition of the extracellular tetracycline (TC) resistance plasmid pAST2 affected host gene expression and phenotype in the oil-degrading soil bacterium, Acinetobacter oleivorans DR1. Whole-transcriptome profiling of DR1 cells harboring pAST2 revealed that all the plasmid genes were highly expressed under TC conditions, and the expression levels of many host chromosomal genes were modulated by the presence of pAST2. The host energy burden imposed by replication of pAST2 led to (i) lowered ATP concentrations, (ii) downregulated expression of many genes involved in cellular growth, and (iii) reduced growth rate. Interestingly, some phenotypes were restored by deleting the plasmid-encoded efflux pump gene tetH, suggesting that the membrane integrity changes resulting from the incorporation of efflux pump proteins also resulted in altered host response under the tested conditions. Alteration of membrane integrity by tetH deletion was shown by measuring permeability of fluorescent probe and membrane hydrophobicity. The presence of the plasmid conferred peroxide and superoxide resistance to cells, but only peroxide resistance was diminished by tetH gene deletion, suggesting that the plasmid-encoded membrane-bound efflux pump protein provided peroxide resistance. The downregulation of fimbriae-related genes presumably led to reduced swimming motility, but this phenotype was recovered by tetH gene deletion. Our data suggest that not only the plasmid replication burden, but also its encoded efflux pump protein altered host chromosomal gene expression and phenotype, which also alters the ecological fitness of the host in the environment.

Evaluation of expression of NorA efflux pump in ciprofloxacin resistant Staphylococcus aureus against hexahydroquinoline derivative by real-time PCR.

PubMed

Pourmand, Mohammad Reza; Yousefi, Masoud; Salami, Seyed Alireza; Amini, Mohsen

2014-01-01

Staphylococcus aureus causes a wide variety of infections worldwide. Methicillin-resistant S. aureus is one of most common causes of nosocomial and community acquired infections. The fluoroquinolones are an important class of antibiotics that used to treat infections caused by S. aureus. Today, a significant increase in the rate of ciprofloxacin resistance in methicillin-resistant S. aureus strains is concerning. The norA efflux pump is considered as contributors to antibiotic resistance. Here, we aimed to evaluate the expression of norA efflux pump in the presence of hexahydroquinoline derivative in methicillin and ciprofloxacin resistant S. aureus. We were determined minimum inhibitory concentration of ciprofloxacin and hexahydroquinoline derivative and their combination by broth microdilution method against ciprofloxacin resistant S. aureus. The expression of the norA efflux pump gene was evaluated by quantitative Real-time PCR. This study showed that minimum inhibitory concentrations of ciprofloxacin in the presence of hexahydroquinoline derivative in comparison to ciprofloxacin were decreased. Quantitative Real-time PCR identified the increased expression of norA efflux pump gene in methicillin and ciprofloxacin resistant S. aureus strain. The increased expression of norA efflux pump gene may have resulted in the effort of S. aureus to survive. The results showed that the hexahydroquinoline derivative enhanced the antibacterial effect of ciprofloxacin against methicillin and ciprofloxacin resistant S. aureus. Therefore, the derivatives may be used as inhibitors of antibiotic resistance for combination therapy.

1-(1H-indol-3-yl)ethanamine derivatives as potent Staphylococcus aureus NorA efflux pump inhibitors.

PubMed

Hequet, Arnaud; Burchak, Olga N; Jeanty, Matthieu; Guinchard, Xavier; Le Pihive, Emmanuelle; Maigre, Laure; Bouhours, Pascale; Schneider, Dominique; Maurin, Max; Paris, Jean-Marc; Denis, Jean-Noël; Jolivalt, Claude

2014-07-01

The synthesis of 37 1-(1H-indol-3-yl)ethanamine derivatives, including 12 new compounds, was achieved through a series of simple and efficient chemical modifications. These indole derivatives displayed modest or no intrinsic anti-staphylococcal activity. By contrast, several of the compounds restored, in a concentration-dependent manner, the antibacterial activity of ciprofloxacin against Staphylococcus aureus strains that were resistant to fluoroquinolones due to overexpression of the NorA efflux pump. Structure-activity relationships studies revealed that the indolic aldonitrones halogenated at position 5 of the indole core were the most efficient inhibitors of the S. aureus NorA efflux pump. Among the compounds, (Z)-N-benzylidene-2-(tert-butoxycarbonylamino)-1-(5-iodo-1H-indol-3-yl)ethanamine oxide led to a fourfold decrease of the ciprofloxacin minimum inhibitory concentration against the SA-1199B strain when used at a concentration of 0.5 mg L(-1) . To the best of our knowledge, this activity is the highest reported to date for an indolic NorA inhibitor. In addition, a new antibacterial compound, tert-butyl (2-(3-hydroxyureido)-2-(1H-indol-3-yl)ethyl)carbamate, which is not toxic for human cells, was also found. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dithiazole thione derivative as competitive NorA efflux pump inhibitor to curtail multi drug resistant clinical isolate of MRSA in a zebrafish infection model.

PubMed

Lowrence, Rene Christena; Raman, Thiagarajan; Makala, Himesh V; Ulaganathan, Venkatasubramanian; Subramaniapillai, Selva Ganesan; Kuppuswamy, Ashok Ayyappa; Mani, Anisha; Chittoor Neelakantan, Sundaresan; Nagarajan, Saisubramanian

2016-11-01

Multi drug resistant (MDR) pathogens pose a serious threat to public health since they can easily render most potent drugs ineffective. Efflux pump inhibitors (EPI) can be used to counter the MDR phenotypes arising due to increased efflux. In the present study, a series of dithiazole thione derivatives were synthesized and checked for its antibacterial and efflux pump inhibitory (EPI) activity. Among 10 dithiazole thione derivatives, real-time efflux studies revealed that seven compounds were potent EPIs relative to CCCP. Zebrafish toxicity studies identified four non-toxic putative EPIs. Both DTT3 and DTT9 perturbed membrane potential and DTT6 was haemolytic. Among DTT6 and DTT10, the latter was less toxic as evidenced by histopathology studies. Since DTT10 was non-haemolytic, did not affect the membrane potential, and was least toxic, it was chosen further for in vivo study, wherein DTT10 potentiated effect of ciprofloxacin against clinical strain of MRSA and reduced bacterial burden in muscle and skin tissue of infected zebrafish by ~ 1.7 and 2.5 log fold respectively. Gene expression profiling of major efflux transport proteins by qPCR revealed that clinical isolate of MRSA, in the absence of antibiotic, upregulated NorA, NorB and MepA pump, whereas it downregulates NorC and MgrA relative to wild-type strain of Staphylococcus aureus. In vitro studies with NorA mutant strains and substrate profiling revealed that at higher concentrations DTT10 is likely to function as a competitive inhibitor of NorA efflux protein in S. aureus, whereas at lower concentrations it might inhibit ciprofloxacin efflux through NorB and MepA as implied by docking studies. A novel non-toxic, non-haemolytic dithiazole thione derivative (DTT10) was identified as a potent competitive inhibitor of NorA efflux pump in S. aureus using in silico, in vitro and in vivo studies. This study also underscores the importance of using zebrafish infection model to screen and evaluate putative EPI for

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. Copyright © 2012 Elsevier B.V. All rights reserved.

Relevant role of efflux pumps in high levels of rifaximin resistance in Escherichia coli clinical isolates.

PubMed

Gomes, C; Ruiz, L; Pons, M J; Ochoa, T J; Ruiz, J

2013-09-01

Enteropathogens have shown a high level of resistance against commonly used antibacterial drugs in Peru and it is necessary to explore alternative treatments. The aim of this study was to analyse the in vitro activity of rifaximin against diarrhoeagenic and commensal Escherichia coli in children less than 2 years of age. The minimal inhibitory concentration (MIC) to rifampicin and rifaximin was determined for 210 strains in the presence and absence of phenyl-arginine-β-naphthylamide (PAβN) and the mechanisms of resistance were investigated. The MIC levels ranged between 8 and >256 mg/litre and the predominant mechanism of resistance to rifaximin was the efflux pumps inhibited by PAβN in 95.2% of the isolates. The present MIC values are higher than those observed in other studies. Efflux pumps inhibited by PAβN were the cause of the rifaximin resistance in the majority of cases and suggest the presence of an environmental selective pressure. Consequently, rifaximin should be used with caution in the treatment of diarrhoea in Peru.

Mechanism of action of tetrandrine, a natural inhibitor of Candida albicans drug efflux pumps.

PubMed

Zhang, Hong; Gao, Aili; Li, Fengxia; Zhang, Gehua; Ho, Hon In; Liao, Wanqing

2009-05-01

Synergistic effects have previously been observed for a natural compound, tetrandrine (TET), with fluconazole (FLC) in vitro and in the treatment of Candida albicans-infected mice. To investigate the mechanisms of these synergistic effects, 16 strains of C. albicans from the same parent but with different FLC sensitivities were examined using flow cytometry and fluorescent spectrophotometry. Rhodamine 123 (Rh123)-positive cells and intracellular Rh123 fluorescence intensity were determined in accumulation/efflux experiments involving no or a noncytotoxic dose of TET. Total RNA extracted from each strain was used to compare the expressions of drug efflux pump genes in FLC-susceptible, -susceptible dose-dependent, and -resistant strains before and 24 h after TET administration. Accumulation experiments determined that mean percentages of Rh123-positive cells were 26.65% (TET-free) and 70.99% (TET 30 microg/ml), and mean respective intracellular Rh123 fluorescence intensities were 11.34 and 18.00. Efflux experiments showed that percentages of Rh123-positive cells were 1.79% (TET free) and 42.57% (TET 30 microg/ml), respectively, and respective mean intracellular Rh123 fluorescence intensities were 0.74 and 2.19. Differences in MDR1, FLU1, CDR1, and CDR2 expression levels in the absence of TET were statistically significant (p<0.05) between FLC-susceptible, -susceptible dose-dependent, and -resistant strains. Compared with TET-free conditions, 24 h TET-treated strains showed statistically different (p<0.05) expression of MDR1 (FLC-resistant strain), FLU1 (FLC-susceptible dose-dependent and -resistant strains), and CDR1 and CDR2 (FLC-susceptible, -susceptible dose-dependent, and -resistant strains). Thus TET can inhibit the C. albicans drug efflux system and reduce drug efflux. Its mechanism of action is related to the inhibition of expression of the drug efflux pump genes MDR1, FLU1, CDR1, and CDR2.

Boronic species as promising inhibitors of the Staphylococcus aureus NorA efflux pump: study of 6-substituted pyridine-3-boronic acid derivatives.

PubMed

Fontaine, Fanny; Héquet, Arnaud; Voisin-Chiret, Anne-Sophie; Bouillon, Alexandre; Lesnard, Aurélien; Cresteil, Thierry; Jolivalt, Claude; Rault, Sylvain

2015-05-05

In response to the extensive use of antibiotics, bacteria have evolved numerous mechanisms of defense against antimicrobial agents. Among them, extrusion of the antimicrobial agents outside the bacterial cell through efflux pumps is a major cause of concern. At first limited to one or few structurally-related antibiotics, bacterial resistance have then progressed towards cross-resistance between different classes of antibiotics, leading to multidrug-resistant microorganisms. Emergence of these pathogens requires development of novel therapeutic strategies and inhibition of efflux pumps appears to be a promising strategy that could restore the potency of existing antibiotics. NorA is the most studied chromosomal efflux pump of Staphylococcus aureus; it is known to be implied in resistance of Methicillin-resistant S. aureus (MRSA) strains against a wide range of unrelated substrates, including hydrophilic fluoroquinolones. Starting from 6-benzyloxypyridine-3-boronic acid I that we previously identified as a potential inhibitor of the NorA efflux pump against the NorA-overexpressing S. aureus 1199B strain (SA1199B), we describe here the synthesis and biological evaluation of a series of 6-(aryl)alkoxypyridine-3-boronic acids. 6-(3-Phenylpropoxy)pyridine-3-boronic acid 3i and 6-(4-phenylbutoxy)pyridine-3-boronic acid 3j were found to potentiate ciprofloxacin activity by a 4-fold increase compared to the parent compound I. In addition, it has been shown that both compounds promote Ethidium Bromide (EtBr) accumulation in SA1199B, thus corroborating their potential mode of action as NorA inhibitors. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

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

PubMed

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

2015-01-01

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

The AcrAB-TolC pump is involved in macrolide resistance but not in telithromycin efflux in Enterobacter aerogenes and Escherichia coli.

PubMed

Chollet, Renaud; Chevalier, Jacqueline; Bryskier, André; Pagès, Jean-Marie

2004-09-01

The role of the AcrAB-TolC pump in macrolide and ketolide susceptibility in Escherichia coli and Enterobacter aerogenes was studied. Efflux pump inhibitor restored erythromycin, clarithromycin, and telithromycin susceptibilities to multidrug-resistant isolates. No modification of telithromycin accumulation was detected in E. aerogenes acrAB or tolC derivatives compared to that in the parental strain. Two independent efflux pumps, inhibited by phenylalanine arginine beta-naphthylamide, expel macrolides and telithromycin in E. aerogenes.

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

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

PubMed

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.

Plagiochin E, a botanic-derived phenolic compound, reverses fungal resistance to fluconazole relating to the efflux pump.

PubMed

Guo, X-L; Leng, P; Yang, Y; Yu, L-G; Lou, H-X

2008-03-01

In this study, we investigated the effect of plagiochin E (PLE), a botanic-derived phenolic natural product, on reversal of fungal resistance to fluconazole (FLC) in vitro and the related mechanism. A synergistic action of PLE and FLC was observed in the FLC-resistant Candida albicans strains and was evaluated using the fractional inhibited concentration index. The effect of PLE on FLC intracellular uptake was investigated in FLC-resistant C. albicans cells by liquid chromatography-tandem mass spectrometry, and the effect on efflux drug pump was assessed by measuring the efflux of Rhodamine 123 (Rh123). PLE significantly inhibited the efflux, but not the absorption, of Rh123 in FLC-resistant strains in phosphate-buffered saline with 5% glucose. Overexpression of the multidrug-resistance gene CDR1 in FLC-resistant C. albicans isolates was detected, and the introduction of PLE to the cells showed a significant reduction of the CDR1 expression in those FLC-resistant isolates. These findings indicate that PLE could reverse the fungal resistant to FLC by inhibiting the efflux of FLC from C. albicans, and this effect may be related to the efflux pump. These results indicate that the combination of PLE and FLC may provide an approach for the clinical therapy of fungus infection induced by FLC-resistant strains.

The role of drug efflux pumps in Malassezia pachydermatis and Malassezia furfur defence against azoles.

PubMed

Iatta, Roberta; Puttilli, Maria Rita; Immediato, Davide; Otranto, Domenico; Cafarchia, Claudia

2017-03-01

This study aims to evaluate the effect of efflux pump modulators (EPMs) on the minimal inhibitory concentration (MIC) of fluconazole (FLZ) and voriconazole (VOR) in Malassezia furfur and Malassezia pachydermatis. The in vitro efficacy of azoles, in combination with EPMs (ie haloperidol-HAL, promethazine-PTZ and cyclosporine A-CYS), against 21 M. furfur from bloodstream infection patients and 14 M. pachydermatis from the skin of dogs with dermatitis, was assessed using a broth microdilution chequerboard analysis. Data were analysed using the model-fractional inhibitory concentration index (FICI) method. The MIC of FLZ and VOR of Malassezia spp. decreased in the presence of sub-inhibitory concentrations of HAL and/or PTZ. The synergic effect was observed only in strains with FLZ MIC≥128 μg/mL for M. furfur, FLZ MIC≥64 μg/mL for M. pachydermatis and VOR MIC≥4 μg/mL in both Malassezia spp. These results suggest that the drug efflux pumps are involved as defence mechanisms to azole drugs in Malassezia yeast. The synergism might be related to an increased expression of efflux pump genes, eventually resulting in azole resistance phenomena. Finally, the above FLZ and VOR MIC values might be considered the cut-off to discriminate susceptible and resistant strains. © 2016 Blackwell Verlag GmbH.

Response to Bile Salts in Clinical Strains of Acinetobacter baumannii Lacking the AdeABC Efflux Pump: Virulence Associated with Quorum Sensing

PubMed Central

López, Maria; Blasco, Lucia; Gato, Eva; Perez, Astrid; Fernández-Garcia, Laura; Martínez-Martinez, Luis; Fernández-Cuenca, Felipe; Rodríguez-Baño, Jesús; Pascual, Alvaro; Bou, German; Tomás, Maria

2017-01-01

Introduction: Acinetobacter baumannii is an opportunistic nosocomial pathogen associated with multiple infections. This pathogen usually colonizes (first stage of microbial infection) host tissues that are in contact with the external environment. As one of the sites of entry in human hosts is the gastrointestinal tract, the pathogen must be capable of tolerating bile salts. However, studies analyzing the molecular characteristics involved in the response to bile salts in clinical strains of A. baumannii are scarce. Material and Methods: Microbiological and transcriptional studies (arrays and RT-PCR) in the response to bile salts were carried out in isogenic (A. baumanni ΔadeB ATCC 17978 and A. baumannii ΔadeL ATCC 17978) and clinical strains from clone ST79/PFGE-HUI-1 which is characterized by lacking the AdeABC efflux pump and by overexpression the AdeFGH efflux pump. Results and Discussion: In presence of bile salts, in addition to the glutamate/aspartate transporter were found overexpressed in A. baumannii ΔadeB ATCC 17978, the virulence factors (surface motility, biofilm, and Type VI Secretion System) which are associated with activation of the Quorum Sensing system. Overexpression of these factors was confirmed in clinical strains of clone ST79/PFGE-HUI-1. Conclusions: This the first study about the adaptive response to bile salts investigating the molecular and microbiological characteristics in response to bile salts of an isogenic model of A. baumannii ATCC 17978 and clinical isolates of A. baumannii (clinical strains of ST79/PFGE-HUI-1) lacking the main RND efflux pump (AdeABC). Clinical isolates of A. baumannii lacking the AdeABC efflux pump (clone ST79/PFGE-HUI-1) displayed a new clinical profile (increased invasiveness) possibly associated with the response to stress conditions (such as the presence of bile salts). PMID:28536672

Inhibition of the essential oil from Chenopodium ambrosioides L. and α-terpinene on the NorA efflux-pump of Staphylococcus aureus.

PubMed

de Morais Oliveira-Tintino, Cícera Datiane; Tintino, Saulo Relison; Limaverde, Paulo W; Figueredo, Fernando G; Campina, Fábia F; da Cunha, Francisco A B; da Costa, Roger H S; Pereira, Pedro Silvino; Lima, Luciene F; de Matos, Yedda M L S; Coutinho, Henrique Douglas Melo; Siqueira-Júnior, José P; Balbino, Valdir Q; da Silva, Teresinha Gonçalves

2018-10-01

This study was carried out to test the essential oil from C. ambrosioides leaves and its main constituent, α-Terpinene, in an antibacterial activity assay. As well, it was evaluated ability reduce resistance to norfloxacin and ethidium bromide was compared the Staphylococcus aureus 1199B whith 1199 wild type strain. The MIC of the C. ambrosioides essential oil and α-Terpinene were determined by microdilution method. The MIC of the essential oil and α-Terpinene presented a value ≥ 1024 μg/mL. However, when associated with antibacterials, the essential oil from C. ambrosioides leaves significantly reduced the MIC of antibiotics and ethidium bromide, characterizing an efflux pump inhibition. The C. ambrosioides essential oil, despite having no direct antibacterial activity against the S. aureus 1199B strain, showed a potentiating action when associated with antibacterial agents, this being attributed to an inhibition of efflux pumps. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

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

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

Contribution of AcrAB efflux pump to ciprofloxacin resistance in Klebsiella pneumoniae isolated from burn patients.

PubMed

Pakzad, Iraj; Zayyen Karin, Maasoume; Taherikalani, Morovat; Boustanshenas, Mina; Lari, Abdolaziz Rastegar

2013-01-01

Resistance to fluoroquinolones has been recently increased among bacterial strains isolated from outpatients. Multidrug-resistant K. pneumoniae is one of the major organisms isolated from burn patients and the AcrAB efflux pump is the principal pump contributing to the intrinsic resistance in K. pneumoniae against multiple antimicrobial agents including ciprofloxacin and other fluoroquinolones. Fifty-two K. pneumoniae isolated from burn patients in Shahid Motahari hospital and confirmed by conventional biochemical tests. Antimicrobial susceptibility testing was done according to CLSI 2011 guidelines, to determine the antimicrobial resistance pattern of isolates. AcrA gene was detected among ciprofloxacin-resistant isolates by PCR assay. MICs to ciprofloxacin were measured with and without carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Forty out of the 52 K. pneumoniae isolated from burn patients in Shahid Motahari hospital were resistant to ciprofloxacin according to breakpoint of CLSI guideline. PCR assay for acrA gene demonstrated that all ciprofloxacin-resistant isolates harbored acrA gene coding the membrane fusion protein AcrA and is a part of AcrAB efflux system. Among these isolates, 19 strains (47.5%) showed 2 to 32 fold reduction in MICs after using CCCP as an efflux pump inhibitor. The other 21 strains (52.5%) showed no disparity in MICs before and after using CCCP. In conclusion, the AcrAB efflux system is one of the principal mechanisms contribute in ciprofloxacin resistance among K. pneumoniae isolates but there are some other mechanisms interfere with ciprofloxacin resistance such as mutation in target proteins of DNA gyrase of topoisomerase IV enzymes.

Attaching NorA efflux pump inhibitors to methylene blue enhances antimicrobial photodynamic inactivation of Escherichia coli and Acinetobacter baumannii in vitro and in vivo.

PubMed

Rineh, Ardeshir; Bremner, John B; Hamblin, Michael R; Ball, Anthony R; Tegos, George P; Kelso, Michael J

2018-02-24

Resistance of bacteria to antibiotics is a public health concern worldwide due to the increasing failure of standard antibiotic therapies. Antimicrobial photodynamic inactivation (aPDI) is a promising non-antibiotic alternative for treating localized bacterial infections that uses non-toxic photosensitizers and harmless visible light to produce reactive oxygen species and kill microbes. Phenothiazinium photosensitizers like methylene blue (MB) and toluidine blue O are hydrophobic cations that are naturally expelled from bacterial cells by multidrug efflux pumps, which reduces their effectiveness. We recently reported the discovery of a NorA efflux pump inhibitor-methylene blue (EPI-MB) hybrid compound INF55-(Ac)en-MB that shows enhanced photodynamic inactivation of the Gram-positive bacterium methicillin-resistant Staphylococcus aureus (MRSA) relative to MB, both in vitro and in vivo. Here, we report the surprising observation that INF55-(Ac)en-MB and two related hybrids bearing the NorA efflux pump inhibitors INF55 and INF271 also show enhanced aPDI activity in vitro (relative to MB) against the Gram-negative bacteria Escherichia coli and Acinetobacter baumannii, despite neither species expressing the NorA pump. Two of the hybrids showed superior effects to MB in murine aPDI infection models. The findings motivate wider exploration of aPDI with EPI-MB hybrids against Gram-negative pathogens and more detailed studies into the molecular mechanisms underpinning their activity. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Faria, Melissa; CESAM & Departamento de Biologia, Universidade de Aveiro, 3810-193 Aveiro; Pavlichenko, Vasiliy

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)more » 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. - Highlights: • Sequences and function of ABC efflux transporters in bivalve gills were explored. • Full length Dreissena polymorpha abcb1 and abcc1 cDNA sequences were identified. • A

Indole-Induced Activities of β-Lactamase and Efflux Pump Confer Ampicillin Resistance in Pseudomonas putida KT2440

PubMed Central

Kim, Jisun; Shin, Bora; Park, Chulwoo; Park, Woojun

2017-01-01

Indole, which is widespread in microbial communities, has received attention because of its effects on bacterial physiology. Pseudomonas putida and Pseudomonas aeruginosa can acquire ampicillin (Amp) resistance during growth on indole-Amp agar. Transcriptome, mutant, and inhibitor studies have suggested that Amp resistance induced by indole can be attributed to increased gene expression of ttgAB encoding two genes of RND-type multidrug efflux operons and an ampC encoding β-lactamase. Expression, enzyme activities, and mutational analyses indicated that AmpC β-lactamase is important for acquiring Amp resistance of P. putida in the presence of indole. Here, we show, for the first time, that volatile indole increased Amp-resistant cells. Consistent with results of the volatile indole assay, a low concentration of indole in liquid culture promoted growth initially, but led to mutagenesis after indole was depleted, which could not be observed at high indole concentrations. Interestingly, ttgAB and ampC gene expression levels correlate with the concentration of indole, which might explain the low number of Amp-mutated cells in high indole concentrations. The expression levels of genes involved in mutagenesis, namely rpoS, recA, and mutS, were also modulated by indole. Our data indicates that indole reduces Amp-induced heterogeneity by promoting expression of TtgABC or MexAB-OprM efflux pumps and the indole-induced β-lactamase in P. putida and P. aeruginosa. PMID:28352264

Quantifying the Evolutionary Conservation of Genes Encoding Multidrug Efflux Pumps in the ESKAPE Pathogens To Identify Antimicrobial Drug Targets.

PubMed

Brooks, Lauren E; Ul-Hasan, Sabah; Chan, Benjamin K; Sistrom, Mark J

2018-01-01

Increasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) have been identified as the leading global cause of multidrug-resistant bacterial infections, and overexpression of multidrug efflux (MEX) transport systems has been identified as one of the most critical mechanisms facilitating the evolution of multidrug resistance in ESKAPE pathogens. Despite efforts to develop efflux pump inhibitors to combat antibiotic resistance, the need persists to identify additional targets for future investigations. We evaluated evolutionary pressures on 110 MEX-encoding genes from all annotated ESKAPE organism genomes. We identify several MEX genes under stabilizing selection-representing targets which can facilitate broad-spectrum treatments with evolutionary constraints limiting the potential emergence of escape mutants. We also examine MEX systems being evaluated as drug targets, demonstrating that divergent selection may underlie some of the problems encountered in the development of effective treatments-specifically in relation to the NorA system in S. aureus. This study provides a comprehensive evolutionary context to efflux in the ESKAPE pathogens, which will provide critical context to the evaluation of efflux systems as antibiotic targets. IMPORTANCE Increasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogen group represents the leading cause of these infections, and upregulation of efflux pump expression is a significant mechanism of resistance in these pathogens. This has resulted in substantial interest in the development of efflux pump inhibitors to combat antibiotic-resistant infections; however, no widespread treatments have been developed to date

Quantifying the Evolutionary Conservation of Genes Encoding Multidrug Efflux Pumps in the ESKAPE Pathogens To Identify Antimicrobial Drug Targets

PubMed Central

Ul-Hasan, Sabah; Chan, Benjamin K.; Sistrom, Mark J.

2018-01-01

ABSTRACT Increasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) have been identified as the leading global cause of multidrug-resistant bacterial infections, and overexpression of multidrug efflux (MEX) transport systems has been identified as one of the most critical mechanisms facilitating the evolution of multidrug resistance in ESKAPE pathogens. Despite efforts to develop efflux pump inhibitors to combat antibiotic resistance, the need persists to identify additional targets for future investigations. We evaluated evolutionary pressures on 110 MEX-encoding genes from all annotated ESKAPE organism genomes. We identify several MEX genes under stabilizing selection—representing targets which can facilitate broad-spectrum treatments with evolutionary constraints limiting the potential emergence of escape mutants. We also examine MEX systems being evaluated as drug targets, demonstrating that divergent selection may underlie some of the problems encountered in the development of effective treatments—specifically in relation to the NorA system in S. aureus. This study provides a comprehensive evolutionary context to efflux in the ESKAPE pathogens, which will provide critical context to the evaluation of efflux systems as antibiotic targets. IMPORTANCE Increasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogen group represents the leading cause of these infections, and upregulation of efflux pump expression is a significant mechanism of resistance in these pathogens. This has resulted in substantial interest in the development of efflux pump inhibitors to combat antibiotic-resistant infections; however, no widespread treatments have been

Quantitative structure activity relationship (QSAR) of piperine analogs for bacterial NorA efflux pump inhibitors.

PubMed

Nargotra, Amit; Sharma, Sujata; Koul, Jawahir Lal; Sangwan, Pyare Lal; Khan, Inshad Ali; Kumar, Ashwani; Taneja, Subhash Chander; Koul, Surrinder

2009-10-01

Quantitative structure activity relationship (QSAR) analysis of piperine analogs as inhibitors of efflux pump NorA from Staphylococcus aureus has been performed in order to obtain a highly accurate model enabling prediction of inhibition of S. aureus NorA of new chemical entities from natural sources as well as synthetic ones. Algorithm based on genetic function approximation method of variable selection in Cerius2 was used to generate the model. Among several types of descriptors viz., topological, spatial, thermodynamic, information content and E-state indices that were considered in generating the QSAR model, three descriptors such as partial negative surface area of the compounds, area of the molecular shadow in the XZ plane and heat of formation of the molecules resulted in a statistically significant model with r(2)=0.962 and cross-validation parameter q(2)=0.917. The validation of the QSAR models was done by cross-validation, leave-25%-out and external test set prediction. The theoretical approach indicates that the increase in the exposed partial negative surface area increases the inhibitory activity of the compound against NorA whereas the area of the molecular shadow in the XZ plane is inversely proportional to the inhibitory activity. This model also explains the relationship of the heat of formation of the compound with the inhibitory activity. The model is not only able to predict the activity of new compounds but also explains the important regions in the molecules in quantitative manner.

Frequency of efflux pump genes mediating ciprofloxacin and antiseptic resistance in methicillin-resistant Staphylococcus aureus isolates.

PubMed

Hassanzadeh, Sepideh; Mashhadi, Rahil; Yousefi, Masoud; Askari, Emran; Saniei, Maryam; Pourmand, Mohammad Reza

2017-10-01

Efflux pumps are well known as a key role to fluoroquinolone resistance in methicillin-resistant Staphylococcus aureus (MRSA). In this study, among 60 clinical MRSA isolates, 42 isolates (70%) were resistant to ciprofloxacin. MRSA were isolated to detect efflux genes including norA, norB, norC, mepA, sepA, mdeA, qacA/B and smr. Isolates subjected to PCR detection and DNA sequence analysis for these genes. PCR detection showed that 42 isolates (70%) contained at least one efflux pump gene. Among ciprofloxacin-resistant isolates, mdeA and qacA/B genes were found with the highest (61.7%) and lowest (3.3%) frequency, respectively. We also observed that the highest minimum inhibitory concentrations of ciprofloxacin in the presence of mdeA+mepA+norA-C+sepA+smr combination. This type of combination may have the greatest impact on resistance to ciprofloxacin. Finally, compared to previous studies, our study demonstrates that prevalence of ciprofloxacin resistance has been increasing among MRSA clinical isolates. Copyright © 2017 Elsevier Ltd. All rights reserved.

Development of an accumulation assay and evaluation of the effects of efflux pump inhibitors on the retention of chlorhexidine digluconate in Pseudomonas aeruginosa and Staphylococcus aureus.

PubMed

Mombeshora, Molly; Mukanganyama, Stanley

2017-07-26

Chlorhexidine digluconate (CHG) is used as a disinfectant. The emergence of pathogens resistant to the biocide raises health concern. Information on specific efflux mechanisms utilised by bacteria to confer reduced susceptibility to the biocide, may be used to develop ways of preventing the efflux of the biocide from nosocomial pathogens resulting in higher disinfection activity. The aim of the study was to evaluate the role of ATP-binding cassette transporters on the transport of CHG in bacteria. Clinical strains of Pseudomonas aeruginosa, Staphylococcus aureus and their respective laboratory strains ATCC 27853 and ATCC 9144 were used for susceptibility tests. The minimum inhibitory concentration (MIC) of CHG with or without an efflux pump inhibitor [reserpine or carbonyl cyanide m-chlorophenylhydrazone (CCCP)] was determined using the broth microdilution method. A spectrophotometric method to quantify the amount of chlorhexidine in a sample was developed, validated and used to quantify CHG within P. aeruginosa and S. aureus cells. In the presence of reserpine, the MIC of CHG against the clinical strains of P. aeruginosa and S. aureus decreased from 6.3 to 3.2 µg/ml but showed no change against both ATCC isolates. The MIC of CHG in the presence of CCCP for both strains of P. aeruginosa remained unchanged but showed a reduction for both isolates of S. aureus. The suitability of the spectrophotometric method developed for quantifying the amount of CHG accumulated in microbial cells was validated and used successfully to quantify CHG accumulated within bacterial cells. The spectrophotometric determination of CHG within microbial cells may be used to quantify CHG in microbial cells. Only the clinical strain of P. aeruginosa showed significant efflux of CHG suggesting the participation of efflux transporters in the pumping out of CHG from this isolate. The use of efflux pump inhibitors together with the biocide may be explored to preventing the efflux of the biocide

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

Regulation of the aceI multidrug efflux pump gene in Acinetobacter baumannii.

PubMed

Liu, Qi; Hassan, Karl A; Ashwood, Heather E; Gamage, Hasinika K A H; Li, Liping; Mabbutt, Bridget C; Paulsen, Ian T

2018-06-01

To investigate the function of AceR, a putative transcriptional regulator of the chlorhexidine efflux pump gene aceI in Acinetobacter baumannii. Chlorhexidine susceptibility and chlorhexidine induction of aceI gene expression were determined by MIC and quantitative real-time PCR, respectively, in A. baumannii WT and ΔaceR mutant strains. Recombinant AceR was prepared as both a full-length protein and as a truncated protein, AceR (86-299), i.e. AceRt, which has the DNA-binding domain deleted. The binding interaction of the purified AceR protein and its putative operator region was investigated by electrophoretic mobility shift assays and DNase I footprinting assays. The binding of AceRt with its putative ligand chlorhexidine was examined using surface plasmon resonance and tryptophan fluorescence quenching assays. MIC determination assays indicated that the ΔaceI and ΔaceR mutant strains both showed lower resistance to chlorhexidine than the parental strain. Chlorhexidine-induced expression of aceI was abolished in a ΔaceR background. Electrophoretic mobility shift assays and DNase I footprinting assays demonstrated chlorhexidine-stimulated binding of AceR with two sites upstream of the putative aceI promoter. Surface plasmon resonance and tryptophan fluorescence quenching assays suggested that the purified ligand-binding domain of the AceR protein was able to bind with chlorhexidine with high affinity. This study provides strong evidence that AceR is an activator of aceI gene expression when challenged with chlorhexidine. This study is the first characterization, to our knowledge, of a regulator controlling expression of a PACE family multidrug efflux pump.

N,N'-disubstituted cinnamamide derivatives potentiate ciprofloxacin activity against overexpressing NorA efflux pump Staphylococcus aureus 1199B strains.

PubMed

Radix, Sylvie; Jordheim, Anne Doléans; Rocheblave, Luc; N'Digo, Serge; Prignon, Anne-Laure; Commun, Carine; Michalet, Serge; Dijoux-Franca, Marie-Geneviève; Mularoni, Angélique; Walchshofer, Nadia

2018-04-25

A multi-step procedure has been described which afforded satisfactory yields of N,N'-disubstituted cinnamamides derived from N-Boc-protected amino acids (Boc-Gly, Boc-Val, Boc-Phe). The key step of this synthesis was a regioselective RedAl reduction of an amide function in presence of a carbamate group. Next, these cinnamamides were evaluated in co-admnistration with ciprofloxacin as efflux pump inhibitors against two S. aureus strains, NorA overexpressing SA1199B and wild type SA1199. In parallel, their intrinsic toxicity was appreciated on human lung fibroblast MRC5 cells. Therefore, the cinnamamide combining both carbamate and indol-3-yl groups, was found to be the most active and one of the less toxic EPI and constituted a promising hit. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Role of the cell envelope stress regulators BaeR and CpxR in control of RND-type multidrug efflux pumps and transcriptional cross talk with exopolysaccharide synthesis in Erwinia amylovora.

PubMed

Pletzer, Daniel; Stahl, Antje; Oja, Anna Elisabeth; Weingart, Helge

2015-08-01

The purpose of this study was to identify the role of the cell envelope stress-sensing systems BaeSR and CpxARP in regulation of multidrug efflux and exopolysaccharide synthesis in Erwinia amylovora. We have previously reported that BaeR activates transcription of the RND-type efflux pumps AcrD and MdtABC. In this study, we found that a cpxR-deficient mutant was highly susceptible to β-lactams, aminoglycosides and lincomycin, whereas a baeR mutant showed no change in antimicrobial sensitivity. However, overexpression of BaeR in a mutant lacking the major RND pump AcrB increased resistance of E. amylovora to several compounds that are not substrates of AcrD or MdtABC. Furthermore, we observed that overexpression of BaeR significantly increased amylovoran production. Moreover, the expression of RND-type efflux pumps was changed in regulatory mutants of exopolysaccharide production. Our data suggest that BaeSR and CpxARP regulate additional mechanisms, beside efflux, which are responsible for antimicrobial resistance of E. amylovora.

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

PubMed

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

2015-08-01

Recently, the cationic polymer thiolated chitosan has been reported to modulate drug absorption by inhibition of intestinal efflux pumps. The objective of this study was to evaluate in vitro and in vivo whether thiolated anionic biopolymers also show an efflux pump inhibitory effect in order to improve intestinal transcellular drug uptake. Therefore, three thiomers have been synthesized due covalent attachment of cysteine to various polymer backbones: pectin-cysteine (pect-cys), carboxymethylcellulose-cysteine (CMC-cys) and alginate-cysteine (alg-cys). In vitro, the permeation enhancing properties of these thiomers and their corresponding unmodified polymers have been evaluated on rat small intestine in Ussing-type chambers, using sulforhodamine 101 (SR-101) as MRP2 model substrate. In comparison to buffer only, SR-101 transport in presence of pect-cys, CMC-cys and alg-cys was improved 1.5-fold, 1.8-fold and 3.0-fold, respectively. Due to the comparatively best in vitro performance of thiolated alginate, it has been chosen for in vivo studies: a SR-101 solution containing 4% (w/v) alg-cys led to an AUC0 ≥ 12 of SR-101 of 109 ng ml(-1)h in rats representing a 3.8-fold improvement in comparison to a SR-101 buffer solution. Unmodified alginate improved the AUC0 ≥ 12 of SR-101 by a factor of 1.9. These findings suggest thiolated alginate as promising auxiliary agent for drugs being anionic efflux pump substrates, since the oral bioavailability of a MRP2 substrate could be significantly improved. Copyright © 2015 Elsevier B.V. All rights reserved.

Efflux as a mechanism of antimicrobial drug resistance in clinical relevant microorganisms: the role of efflux inhibitors.

PubMed

Willers, Clarissa; Wentzel, Johannes Frederik; du Plessis, Lissinda Hester; Gouws, Chrisna; Hamman, Josias Hendrik

2017-01-01

Microbial resistance against antibiotics is a serious threat to the effective treatment of infectious diseases. Several mechanisms exist through which microorganisms can develop resistance against antimicrobial drugs, of which the overexpression of genes to produce efflux pumps is a major concern. Several efflux transporters have been identified in microorganisms, which infer resistance against specific antibiotics and even multidrug resistance. Areas covered: This paper focuses on microbial resistance against antibiotics by means of the mechanism of efflux and gives a critical overview of studies conducted to overcome this problem by combining efflux pump inhibitors with antibiotics. Information was obtained from a literature search done with MEDLINE, Pubmed, Scopus, ScienceDirect, OneSearch and EBSCO host. Expert opinion: Efflux as a mechanism of multidrug resistance has presented a platform for improved efficacy against resistant microorganisms by co-administration of efflux pump inhibitors with antimicrobial agents. Although proof of concept has been shown for this approach with in vitro experiments, further research is needed to develop more potent inhibitors with low toxicity which is clinically effective.

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

USDA-ARS?s Scientific Manuscript database

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...

Studies on 2-phenylquinoline Staphylococcus aureus NorA efflux pump inhibitors: New insights on the C-6 position.

PubMed

Felicetti, Tommaso; Cannalire, Rolando; Nizi, Maria Giulia; Tabarrini, Oriana; Massari, Serena; Barreca, Maria Letizia; Manfroni, Giuseppe; Schindler, Bryan D; Cecchetti, Violetta; Kaatz, Glenn W; Sabatini, Stefano

2018-06-06

The alarming and rapid spread of antimicrobial resistance among bacteria represents a high risk for global health. Targeting factors involved in resistance to restore the activity of failing antibiotics is a promising strategy to overcome this urgent medical need. Efflux pump inhibitors are able to increase antibiotic concentrations in bacteria, thus they can be considered true antimicrobial resistance breakers. In this work, continuing our studies on inhibitors of the Staphylococcus aureus NorA pump, we designed, synthesized and biologically evaluated novel 2-phenylquinoline derivatives starting from our hits 1 and 2. Two of the synthesized compounds (6 and 7) bearing a C-6 benzyloxy group showed the best NorA inhibition activity, thereby providing an excellent starting point to direct future chemical optimizations. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Overproduction of active efflux pump and variations of OprD dominate in imipenem-resistant Pseudomonas aeruginosa isolated from patients with bloodstream infections in Taiwan.

PubMed

Kao, Cheng-Yen; Chen, Shu-Sheng; Hung, Kuei-Hsiang; Wu, Hsiu-Mei; Hsueh, Po-Ren; Yan, Jing-Jou; Wu, Jiunn-Jong

2016-06-13

The emergence of imipenem-resistant Pseudomonas aeruginosa (IRPA) has become a great concern worldwide. The aim of this study was to investigate resistance mechanisms associated with bloodstream isolated IRPA strains in Taiwan. A total of 78 non-duplicated IRPA isolates were isolated from patients with bloodstream infection. The average prevalence of imipenem-resistance in those isolates was 5.9 % during a 10-year longitudinal surveillance in Taiwan. PFGE results showed high clonal diversity among the 78 isolates. VIM-2, VIM-3, OXA-10, and OXA-17 β-lactamases were identified in 2 (2.6 %), 3 (3.8 %), 2 (2.6 %), and 1 (1.3 %) isolates, respectively. Active efflux pumps, AmpC β-lactamase overproduction, and extended-spectrum AmpC cephalosporinases (ESACs) were found in 58 (74.4 %), 25 (32.1 %) and 15 (19.2 %) of IRPA isolates, respectively. oprD mutations with amino acid substitution, shortened putative loop L7, premature stop codon caused by point mutation, frameshift by nucleotide insertion or deletion, and interruption by insertion sequence were found in 19 (24.4 %), 18 (23.1 %), 15 (19.2 %), 14 (17.9 %), and 10 (12.8 %) of isolates, respectively. This study suggests that alterations in the OprD protein and having an active efflux pump are the main mechanisms associated with bloodstream isolated IRPA. Overproduction of AmpC, ESACs, and the presence of VIM- and OXA-type β-lactamases play additional roles in reduced susceptibility to imipenem in P. aeruginosa isolates in Taiwan.

Modulation of the multidrug efflux pump EmrD-3 from Vibrio cholerae by Allium sativum extract and the bioactive agent allyl sulfide plus synergistic enhancement of antimicrobial susceptibility by A. sativum extract.

PubMed

Bruns, Merissa M; Kakarla, Prathusha; Floyd, Jared T; Mukherjee, Mun Mun; Ponce, Robert C; Garcia, John A; Ranaweera, Indrika; Sanford, Leslie M; Hernandez, Alberto J; Willmon, T Mark; Tolson, Grace L; Varela, Manuel F

2017-10-01

The causative agent of cholera, Vibrio cholerae, is a public health concern. Multidrug-resistant V. cholerae variants may reduce chemotherapeutic efficacies of severe cholera. We previously reported that the multidrug efflux pump EmrD-3 from V. cholerae confers resistance to multiple structurally distinct antimicrobials. Medicinal plant compounds are potential candidates for EmrD-3 efflux pump modulation. The antibacterial activities of garlic Allium sativum, although poorly understood, predicts that a main bioactive component, allyl sulfide, modulates EmrD-3 efflux. Thus, we tested whether A. sativum extract acts in synergy with antimicrobials and that a main bioactive component allyl sulfide inhibits EmrD-3 efflux. We found that A. sativum extract and allyl sulfide inhibited ethidium bromide efflux in cells harboring EmrD-3 and that A. sativum lowered the MICs of multiple antibacterials. We conclude that A. sativum and allyl sulfide inhibit EmrD-3 and that A. sativum extract synergistically enhances antibacterial agents.

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

PubMed

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

2014-08-25

Periplasmic adaptor proteins are key components of bacterial tripartite efflux pumps. The 2.85 Å resolution structure of an MFS (major facilitator superfamily) pump adaptor, Aquifex aeolicus EmrA, shows linearly arranged α-helical coiled-coil, lipoyl, and β-barrel domains, but lacks the fourth membrane-proximal domain shown in other pumps to interact with the inner membrane transporter. The adaptor α-hairpin, which binds outer membrane TolC, is exceptionally long at 127 Å, and the β-barrel contains a conserved disordered loop. The structure extends the view of adaptors as flexible, modular components that mediate diverse pump assembly, and suggests that in MFS tripartite pumps a hexamer of adaptors could provide a periplasmic seal. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Study on the association between drug‑resistance and gene mutations of the active efflux pump acrAB‑tolC gene and its regulatory genes.

PubMed

Ma, Quan-Ping; Su, Liang; Liu, Jing-Wen; Yao, Ming-Xiao; Yuan, Guang-Ying

2018-06-01

The aim of the present study was to investigate the correlation between the multi‑drug resistance of Shigella flexneri and the drug‑resistant gene cassette carried by integrons; in the meanwhile, to detect the associations between drug‑resistance and gene mutations of the active efflux pump acrAB‑tolC gene and its regulatory genes, including marOR, acrR and soxS. A total of 158 isolates were isolated from the stool samples of 1,026 children with diarrhoea aged 14 years old between May 2012 and October 2015 in Henan. The K‑B method was applied for the determination of drug resistance of Shigella flexneri, and polymerase chain reaction amplification was used for class 1, 2 and 3 integrase genes. Enzyme digestion and sequence analysis were performed for the variable regions of positive strains. Based on the drug sensitivity assessment, multi‑drug resistant strains that were resistant to five or more antibiotics, and sensitive strains were selected for amplification. Their active efflux pump genes, acrA and acrB, and regulatory genes, marOR, acrR and soxS, were selected for sequencing. The results revealed that 91.1% of the 158 strains were multi‑resistant to ampicillin, chloramphenicol, tetracycline and streptomycin, and 69.6% of the strains were multi‑resistant to sulfamethoxazole/trimethoprim. The resistance to ceftazidime, ciprofloxacin and levofloxacin was <32.9%. All strains (100%) were sensitive to cefoxitin, cefoperazone/sulbactam and imipenem. The rate of the class 1 integron positivity was 91.9% (144/158). Among these class 1 integron‑positive strains, 18 strains exhibited the resistance gene cassette dfrV in the variable region of the strain, four strains exhibited dfrA17‑aadA5 in the variable region and 140 strains exhibited blaOXA‑30‑aadA1 in the variable region. Four strains showed no resistance gene in the variable regions. The rate of class 2 integron positivity was 86.1% (136/158), and all positive strains harboured the

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

PubMed

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

2014-06-01

Polyamines regulate a variety of cation and K(+) channels, but their potential effects on cation-transporting ATPases are underexplored. In this work, noninvasive microelectrode ion flux estimation and conventional microelectrode techniques were applied to study the effects of polyamines on Ca(2+) and H(+) transport and membrane potential in pea roots. Externally applied spermine or putrescine (1mM) equally activated eosin yellow (EY)-sensitive Ca(2+) pumping across the root epidermis and caused net H(+) influx or efflux. Proton influx induced by spermine was suppressed by EY, supporting the mechanism in which Ca(2+) pump imports 2 H(+) per each exported Ca(2+). Suppression of the Ca(2+) pump by EY diminished putrescine-induced net H(+) efflux instead of increasing it. Thus, activities of Ca(2+) and H(+) pumps were coupled, likely due to the H(+)-pump inhibition by intracellular Ca(2+). Additionally, spermine but not putrescine caused a direct inhibition of H(+) pumping in isolated plasma membrane vesicles. Spermine, spermidine, and putrescine (1mM) induced membrane depolarization by 70, 50, and 35 mV, respectively. Spermine-induced depolarization was abolished by cation transport blocker Gd(3+), was insensitive to anion channels' blocker niflumate, and was dependent on external Ca(2+). Further analysis showed that uptake of polyamines but not polyamine-induced cationic (K(+)+Ca(2+)+H(+)) fluxes were a main cause of membrane depolarization. Polyamine increase is a common component of plant stress responses. Activation of Ca(2+) efflux by polyamines and contrasting effects of polyamines on net H(+) fluxes and membrane potential can contribute to Ca(2+) signalling and modulate a variety of transport processes across the plasma membrane under stress. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Antibacterial Efficacy of Polysaccharide Capped Silver Nanoparticles Is Not Compromised by AcrAB-TolC Efflux Pump

PubMed Central

Mishra, Mitali; Kumar, Satish; Majhi, Rakesh K.; Goswami, Luna; Goswami, Chandan; Mohapatra, Harapriya

2018-01-01

Antibacterial therapy is of paramount importance in treatment of several acute and chronic infectious diseases caused by pathogens. Over the years extensive use and misuse of antimicrobial agents has led to emergence of multidrug resistant (MDR) and extensive drug resistant (XDR) pathogens. This drastic escalation in resistant phenotype has limited the efficacy of available therapeutic options. Thus, the need of the hour is to look for alternative therapeutic approaches to mitigate healthcare concerns caused due to MDR bacterial infections. Nanoparticles have gathered much attention as potential candidates for antibacterial therapy. Equipped with advantages of, wide spectrum bactericidal activity at very low dosage, inhibitor of biofilm formation and ease of permeability, nanoparticles have been considered as leading therapeutic candidates to curtail infections resulting from MDR bacteria. However, substrate non-specificity of efflux pumps, particularly those belonging to resistance nodulation division super family, have been reported to reduce efficacy of many potent antibacterial therapeutic drugs. Previously, we had reported antibacterial activity of polysaccharide-capped silver nanoparticles (AgNPs) toward MDR bacteria. We showed that AgNPs inhibits biofilm formation and alters expression of cytoskeletal proteins FtsZ and FtsA, with minimal cytotoxicity toward mammalian cells. In the present study, we report no reduction in antibacterial efficacy of silver nanoparticles in presence of AcrAB-TolC efflux pump proteins. Antibacterial tests were performed according to CLSI macrobroth dilution method, which revealed that both silver nanoparticles exhibited bactericidal activity at very low concentrations. Further, immunoblotting results indicated that both the nanoparticles modulate the transporter AcrB protein expression. However, expression of the membrane fusion protein AcrA did show a significant increase after exposure to AgNPs. Our results indicate that both

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. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Efflux drug transporters at the forefront of antimicrobial resistance.

PubMed

Rahman, Tahmina; Yarnall, Benjamin; Doyle, Declan A

2017-10-01

Bacterial antibiotic resistance is rapidly becoming a major world health consideration. To combat antibiotics, microorganisms employ their pre-existing defence mechanisms that existed long before man's discovery of antibiotics. Bacteria utilise levels of protection that range from gene upregulation, mutations, adaptive resistance, and production of resistant phenotypes (persisters) to communal behaviour, as in swarming and the ultimate defence of a biofilm. A major part of all of these responses involves the use of antibiotic efflux transporters. At the single cell level, it is becoming apparent that the use of efflux pumps is the first line of defence against an antibiotic, as these pumps decrease the intracellular level of antibiotic while the cell activates the various other levels of protection. This frontline of defence involves a coordinated network of efflux transporters. In the future, inhibition of this efflux transporter network, as a target for novel antibiotic therapy, will require the isolation and then biochemical/biophysical characterisation of each pump against all known and new antibiotics. This depth of knowledge is required so that we can fully understand and tackle the mechanisms of developing antimicrobial resistance.

The putative drug efflux systems of the Bacillus cereus group

PubMed Central

Elbourne, Liam D. H.; Vörös, Aniko; Kroeger, Jasmin K.; Simm, Roger; Tourasse, Nicolas J.; Finke, Sarah; Henderson, Peter J. F.; Økstad, Ole Andreas; Paulsen, Ian T.; Kolstø, Anne-Brit

2017-01-01

The Bacillus cereus group of bacteria includes seven closely related species, three of which, B. anthracis, B. cereus and B. thuringiensis, are pathogens of humans, animals and/or insects. Preliminary investigations into the transport capabilities of different bacterial lineages suggested that genes encoding putative efflux systems were unusually abundant in the B. cereus group compared to other bacteria. To explore the drug efflux potential of the B. cereus group all putative efflux systems were identified in the genomes of prototypical strains of B. cereus, B. anthracis and B. thuringiensis using our Transporter Automated Annotation Pipeline. More than 90 putative drug efflux systems were found within each of these strains, accounting for up to 2.7% of their protein coding potential. Comparative analyses demonstrated that the efflux systems are highly conserved between these species; 70–80% of the putative efflux pumps were shared between all three strains studied. Furthermore, 82% of the putative efflux system proteins encoded by the prototypical B. cereus strain ATCC 14579 (type strain) were found to be conserved in at least 80% of 169 B. cereus group strains that have high quality genome sequences available. However, only a handful of these efflux pumps have been functionally characterized. Deletion of individual efflux pump genes from B. cereus typically had little impact to drug resistance phenotypes or the general fitness of the strains, possibly because of the large numbers of alternative efflux systems that may have overlapping substrate specificities. Therefore, to gain insight into the possible transport functions of efflux systems in B. cereus, we undertook large-scale qRT-PCR analyses of efflux pump gene expression following drug shocks and other stress treatments. Clustering of gene expression changes identified several groups of similarly regulated systems that may have overlapping drug resistance functions. In this article we review current

Contribution of efflux to colistin heteroresistance in a multidrug resistant Acinetobacter baumannii clinical isolate.

PubMed

Machado, Diana; Antunes, Jéssica; Simões, Ana; Perdigão, João; Couto, Isabel; McCusker, Matthew; Martins, Marta; Portugal, Isabel; Pacheco, Teresa; Batista, Judite; Toscano, Cristina; Viveiros, Miguel

2018-06-01

The mechanisms underlying colistin heteroresistance in Acinetobacter baumannii are not fully understood. Here, we investigated the role of efflux in colistin-heteroresistant populations of a multidrug-resistant (MDR) A. baumannii clinical isolate. Three colistin-resistant A. baumannii strain variants isolated from the same clinical sample were studied for the presence of heteroresistance to colistin by drug susceptibility testing, genotyping and drug resistance target mutation analysis. The existence of active efflux was studied by synergism assays with efflux inhibitors, real-time efflux activity measurements and analysis of the mRNA transcriptional levels of selected efflux pump genes in response to colistin. All of the strain variants belong to the ST218, clonal complex 92, international clonal lineage II. Different colistin susceptibility levels were observed among the three strain variants, indicating that colistin-heteroresistant subpopulations were being selected upon exposure to colistin. No mutations were found in the genes lpxACD and pmrAB, which are associated with colistin resistance. The results showed the existence of synergistic interactions between efflux inhibitors and colistin and ethidium bromide. Real-time efflux assays demonstrated that the three strain variants had increased efflux activity that could be inhibited in the presence of the inhibitors. The efflux pump genes adeB, adeJ, adeG, craA, amvA, abeS and abeM were found to be overexpressed in the strain variants in response to colistin exposure. This study shows that efflux activity contributes to colistin heteroresistance in an MDR A. baumannii clinical isolate. The use of efflux inhibitors as adjuvants of the therapy can resensitize A. baumannii to colistin and prevent the emergence of drug resistance.

Anti-Campylobacter activity of resveratrol and an extract from waste Pinot noir grape skins and seeds, and resistance of Camp. jejuni planktonic and biofilm cells, mediated via the CmeABC efflux pump.

PubMed

Klančnik, A; Šikić Pogačar, M; Trošt, K; Tušek Žnidarič, M; Mozetič Vodopivec, B; Smole Možina, S

2017-01-01

To define anti-Campylobacter jejuni activity of an extract from waste skins and seeds of Pinot noir grapes (GSS), resveratrol and possible resistance mechanisms, and the influence of these on Camp. jejuni morphology. Using gene-specific knock-out Camp. jejuni mutants and an efflux pump inhibitor, we showed CmeABC as the most active efflux pump for extrusion across the outer membrane of GSS extract and resveratrol. Using polystyrene surface and pig small intestine epithelial (PSI) and human foetal small intestine (H4) cell lines, GSS extract shows an efficient inhibition of adhesion of Camp. jejuni to these abiotic and biotic surfaces. Low doses of GSS extract can inhibit Camp. jejuni adhesion to polystyrene surfaces and to PSI and H4 cells, and can thus modulate Camp. jejuni invasion and intracellular survival. An understanding of the activities of GSS extract and resveratrol as bacterial growth inhibitors and the specific mechanisms of cell accumulation is crucial for our understanding of Camp. jejuni resistance. GSS extract inhibition of Camp. jejuni adhesion to abiotic and biotic surfaces provides a further step towards the application of new innovative strategies to control Campylobacter contamination and infection via the food chain. © 2016 The Society for Applied Microbiology.

Expression of Sme efflux pumps and multilocus sequence typing in clinical isolates of Stenotrophomonas maltophilia.

PubMed

Cho, Hye Hyun; Sung, Ji Youn; Kwon, Kye Chul; Koo, Sun Hoe

2012-01-01

Stenotrophomonas maltophilia has emerged as an important opportunistic pathogen, which causes infections that are often difficult to manage because of the inherent resistance of the pathogen to a variety of antimicrobial agents. In this study, we analyzed the expressions of smeABC and smeDEF and their correlation with antimicrobial susceptibility. We also evaluated the genetic relatedness and epidemiological links among 33 isolates of S. maltophilia. In total, 33 S. maltophilia strains were isolated from patients in a tertiary hospital in Daejeon. Minimum inhibitory concentrations (MICs) of 11 antimicrobial agents were determined by using agar dilution method and E-test (BioMérieux, France). Real-time PCR analysis was performed to evaluate the expression of the Sme efflux systems in the S. maltophilia isolates. Additionally, an epidemiological investigation was performed using multilocus sequence typing (MLST) assays. The findings of susceptibility testing showed that the majority of the S. maltophilia isolates were resistant to β-lactams and aminoglycosides. Twenty-one clinical isolates overexpressed smeABC and showed high resistance to ciprofloxacin. Moreover, a high degree of genetic diversity was observed among the S. maltophilia isolates; 3 sequence types (STs) and 23 allelic profiles were observed. The smeABC efflux pump was associated with multidrug resistance in clinical isolates of S. maltophilia. In particular, smeABC efflux pumps appear to perform an important role in ciprofloxacin resistance of S. maltophilia. The MLST scheme for S. maltophilia represents a discriminatory typing method with stable markers and is appropriate for studying population structures.

Mode of action of the 2-phenylquinoline efflux inhibitor PQQ4R against Escherichia coli

PubMed Central

Machado, Diana; Fernandes, Laura; Costa, Sofia S.; Cannalire, Rolando; Manfroni, Giuseppe; Tabarrini, Oriana; Couto, Isabel; Sabatini, Stefano

2017-01-01

Efflux pump inhibitors are of great interest since their use as adjuvants of bacterial chemotherapy can increase the intracellular concentrations of the antibiotics and assist in the battle against the rising of antibiotic-resistant bacteria. In this work, we have described the mode of action of the 2-phenylquinoline efflux inhibitor (4-(2-(piperazin-1-yl)ethoxy)-2-(4-propoxyphenyl) quinolone – PQQ4R), against Escherichia coli, by studding its efflux inhibitory ability, its synergistic activity in combination with antibiotics, and compared its effects with the inhibitors phenyl-arginine-β-naphthylamide (PAβN) and chlorpromazine (CPZ). The results showed that PQQ4R acts synergistically, in a concentration dependent manner, with antibiotics known to be subject to efflux in E. coli reducing their MIC in correlation with the inhibition of their efflux. Real-time fluorometry assays demonstrated that PQQ4R at sub-inhibitory concentrations promote the intracellular accumulation of ethidium bromide inhibiting its efflux similarly to PAβN or CPZ, well-known and described efflux pump inhibitors for Gram-negative bacteria and whose clinical usage is limited by their levels of toxicity at clinical and bacteriological effective concentrations. The time-kill studies showed that PQQ4R, at bactericidal concentrations, has a rapid antimicrobial activity associated with a fast decrease of the intracellular ATP levels. The results also indicated that the mode of action of PQQ4R involves the destabilization of the E. coli inner membrane potential and ATP production impairment, ultimately leading to efflux pump inhibition by interference with the energy required by the efflux systems. At bactericidal concentrations, membrane permeabilization increases and finally ATP is totally depleted leading to cell death. Since drug resistance mediated by the activity of efflux pumps depends largely on the proton motive force (PMF), dissipaters of PMF such as PQQ4R, can be regarded as future

Characterization and regulation of the resistance-nodulation-cell division-type multidrug efflux pumps MdtABC and MdtUVW from the fire blight pathogen Erwinia amylovora.

PubMed

Pletzer, Daniel; Weingart, Helge

2014-07-11

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. 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. 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 efflux pumps are involved in

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

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

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

Water-mediated interactions enable smooth substrate transport in a bacterial efflux pump.

PubMed

Vargiu, Attilio Vittorio; Ramaswamy, Venkata Krishnan; Malvacio, Ivana; Malloci, Giuliano; Kleinekathöfer, Ulrich; Ruggerone, Paolo

2018-04-01

Efflux pumps of the Resistance-Nodulation-cell Division superfamily confer multi-drug resistance to Gram-negative bacteria. The most-studied polyspecific transporter belonging to this class is the inner-membrane trimeric antiporter AcrB of Escherichia coli. In previous studies, a functional rotation mechanism was proposed for its functioning, according to which the three monomers undergo concerted conformational changes facilitating the extrusion of substrates. However, the molecular determinants and the energetics of this mechanism still remain unknown, so its feasibility must be proven mechanistically. A computational protocol able to mimic the functional rotation mechanism in AcrB was developed. By using multi-bias molecular dynamics simulations we characterized the translocation of the substrate doxorubicin driven by conformational changes of the protein. In addition, we estimated for the first time the free energy profile associated to this process. We provided a molecular view of the process in agreement with experimental data. Moreover, we showed that the conformational changes occurring in AcrB enable the formation of a layer of structured waters on the internal surface of the transport channel. This water layer, in turn, allows for a fairly constant hydration of the substrate, facilitating its diffusion over a smooth free energy profile. Our findings reveal a new molecular mechanism of polyspecific transport whereby water contributes by screening potentially strong substrate-protein interactions. We provided a mechanistic understanding of a fundamental process related to multi-drug transport. Our results can help rationalizing the behavior of other polyspecific transporters and designing compounds avoiding extrusion or inhibitors of efflux pumps. Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.

Synthesis of omeprazole analogues and evaluation of these as potential inhibitors of the multidrug efflux pump NorA of Staphylococcus aureus.

PubMed

Vidaillac, Céline; Guillon, Jean; Arpin, Corinne; Forfar-Bares, Isabelle; Ba, Boubakar B; Grellet, Jean; Moreau, Stéphane; Caignard, Daniel-Henri; Jarry, Christian; Quentin, Claudine

2007-03-01

A series of 11 pyrrolo[1,2-a]quinoxaline derivatives, 1a to 1k, sharing structural analogies with omeprazole, a eukaryotic efflux pump inhibitor (EPI) used as an antiulcer agent, was synthesized. Their inhibitory effect was evaluated using Staphylococcus aureus strain SA-1199B overexpressing NorA. By determinations of the MIC of norfloxacin in the presence of these EPIs devoid of intrinsic antibacterial activity and used at 128 microg/ml, and by the checkerboard method, compound 1e (MIC decrease, 16-fold; fractional inhibitory concentration index [SigmaFIC], 0.18) appeared to be more active than compounds 1b to 1d, reserpine, and omeprazole (MIC decrease, eightfold; SigmaFIC, 0.31), followed by compounds 1a and 1f (MIC decrease, fourfold; SigmaFIC, 0.37) and 1g to 1k (MIC decrease, twofold; SigmaFIC, 0.50 to 0.56). By time-kill curves combining norfloxacin (1/4 MIC) and the most efficient EPIs (128 microg/ml), compound 1e persistently restored the bactericidal activity of norfloxacin (inoculum reduction, 3 log(10) CFU/ml at 8 and 24 h), compound 1f led to a delayed but progressive decrease in the number of viable cells, and compounds 1b to 1d and omeprazole acted synergistically (inoculum reduction, 3 log(10) CFU/ml at 8 h but further regrowth), while compound 1a and reserpine slightly enhanced norfloxacin activity. The bacterial uptake of norfloxacin monitored by high-performance liquid chromatography confirmed that compounds 1a to 1f increased antibiotic accumulation, as did reserpine and omeprazole. Since these EPIs did not disturb the Deltapsi and DeltapH, they might directly interact with the pump. A structure-activity relationships study identified the benzimidazole nucleus of omeprazole as the main structural element involved in efflux pump inhibition and highlighted the critical role of the chlorine substituents in the stability and efficiency of compounds 1e to 1f. However, further pharmacomodulation is required to obtain therapeutically applicable

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

Interplay between Mutations and Efflux in Drug Resistant Clinical Isolates of Mycobacterium tuberculosis.

PubMed

Machado, Diana; Coelho, Tatiane S; Perdigão, João; Pereira, Catarina; Couto, Isabel; Portugal, Isabel; Maschmann, Raquel De Abreu; Ramos, Daniela F; von Groll, Andrea; Rossetti, Maria L R; Silva, Pedro A; Viveiros, Miguel

2017-01-01

Numerous studies show efflux as a universal bacterial mechanism contributing to antibiotic resistance and also that the activity of the antibiotics subject to efflux can be enhanced by the combined use of efflux inhibitors. Nevertheless, the contribution of efflux to the overall drug resistance levels of clinical isolates of Mycobacterium tuberculosis is poorly understood and still is ignored by many. Here, we evaluated the contribution of drug efflux plus target-gene mutations to the drug resistance levels in clinical isolates of M. tuberculosis . A panel of 17 M. tuberculosis clinical strains were characterized for drug resistance associated mutations and antibiotic profiles in the presence and absence of efflux inhibitors. The correlation between the effect of the efflux inhibitors and the resistance levels was assessed by quantitative drug susceptibility testing. The bacterial growth/survival vs. growth inhibition was analyzed through the comparison between the time of growth in the presence and absence of an inhibitor. For the same mutation conferring antibiotic resistance, different MICs were observed and the different resistance levels found could be reduced by efflux inhibitors. Although susceptibility was not restored, the results demonstrate the existence of a broad-spectrum synergistic interaction between antibiotics and efflux inhibitors. The existence of efflux activity was confirmed by real-time fluorometry. Moreover, the efflux pump genes mmr, mmpL7, Rv1258c, p55 , and efpA were shown to be overexpressed in the presence of antibiotics, demonstrating the contribution of these efflux pumps to the overall resistance phenotype of the M. tuberculosis clinical isolates studied, independently of the genotype of the strains. These results showed that the drug resistance levels of multi- and extensively-drug resistant M. tuberculosis clinical strains are a combination between drug efflux and the presence of target-gene mutations, a reality that is often

Homologs of the Acinetobacter baumannii AceI transporter represent a new family of bacterial multidrug efflux systems.

PubMed

Hassan, Karl A; Liu, Qi; Henderson, Peter J F; Paulsen, Ian T

2015-02-10

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. Bacterial multidrug efflux pumps are an important class of resistance determinants that can be found in every bacterial genome sequenced to date. These transport proteins have important protective functions for the bacterial cell but are a significant problem in the clinical setting, since a single efflux system can mediate resistance to many structurally and mechanistically diverse antibiotics and biocides. In this study, we demonstrate that proteins related to the Acinetobacter baumannii AceI transporter are a new class of multidrug

Cytotoxicity and Efflux Pump Inhibition Induced by Molybdenum Disulfide and Boron Nitride Nanomaterials with Sheetlike Structure.

PubMed

Liu, Su; Shen, Zhuoyan; Wu, Bing; Yu, Yue; Hou, Hui; Zhang, Xu-Xiang; Ren, Hong-Qiang

2017-09-19

Sheetlike molybdenum disulfide (MoS 2 ) and boron nitride (BN) nanomaterials have attracted attention in the past few years due to their unique material properties. However, information on adverse effects and their underlying mechanisms for sheetlike MoS 2 and BN nanomaterials is rare. In this study, cytotoxicities of sheetlike MoS 2 and BN nanomaterials on human hepatoma HepG2 cells were systematically investigated at different toxic end points. Results showed that MoS 2 and BN nanomaterials decreased cell viability at 30 μg/mL and induced adverse effects on intracellular ROS generation (≥2 μg/mL), mitochondrial depolarization (≥4 μg/mL), and membrane integrity (≥8 μg/mL for MoS 2 and ≥2 μg/mL for BN). Furthermore, this study first found that low exposure concentrations (0.2-2 μg/mL) of MoS 2 and BN nanomaterials could increase plasma membrane fluidity and inhibit transmembrane ATP binding cassette (ABC) efflux transporter activity, which make both nanomaterials act as a chemosensitizer (increasing arsenic toxicity). Damage to plasma membrane and release of soluble Mo or B species might be two reasons that both nanomaterials inhibit efflux pump activities. This study provides a systematic understanding of the cytotoxicity of sheetlike MoS 2 and BN nanomaterials at different exposure levels, which is important for their safe use.

Metabolic Compensation of Fitness Costs Is a General Outcome for Antibiotic-Resistant Pseudomonas aeruginosa Mutants Overexpressing Efflux Pumps.

PubMed

Olivares Pacheco, Jorge; Alvarez-Ortega, Carolina; Alcalde Rico, Manuel; Martínez, José Luis

2017-07-25

It is generally assumed that the acquisition of antibiotic resistance is associated with a fitness cost. We have shown that overexpression of the MexEF-OprN efflux pump does not decrease the fitness of a resistant Pseudomonas aeruginosa strain compared to its wild-type counterpart. This lack of fitness cost was associated with a metabolic rewiring that includes increased expression of the anaerobic nitrate respiratory chain when cells are growing under fully aerobic conditions. It was not clear whether this metabolic compensation was exclusive to strains overexpressing MexEF-OprN or if it extended to other resistant strains that overexpress similar systems. To answer this question, we studied a set of P. aeruginosa mutants that independently overexpress the MexAB-OprM, MexCD-OprJ, or MexXY efflux pumps. We observed increased expression of the anaerobic nitrate respiratory chain in all cases, with a concomitant increase in NO 3 consumption and NO production. These efflux pumps are proton/substrate antiporters, and their overexpression may lead to intracellular H + accumulation, which may in turn offset the pH homeostasis. Indeed, all studied mutants showed a decrease in intracellular pH under anaerobic conditions. The fastest way to eliminate the excess of protons is by increasing oxygen consumption, a feature also displayed by all analyzed mutants. Taken together, our results support metabolic rewiring as a general mechanism to avoid the fitness costs derived from overexpression of P. aeruginosa multidrug efflux pumps. The development of drugs that block this metabolic "reaccommodation" might help in reducing the persistence and spread of antibiotic resistance elements among bacterial populations. IMPORTANCE It is widely accepted that the acquisition of resistance confers a fitness cost in such a way that in the absence of antibiotics, resistant populations will be outcompeted by susceptible ones. Based on this assumption, antibiotic cycling regimes have been

Reversal of the Drug Binding Pocket Defects of the AcrB Multidrug Efflux Pump Protein of Escherichia coli

PubMed Central

Soparkar, Ketaki; Kinana, Alfred D.; Weeks, Jon W.; Morrison, Keith D.; Nikaido, Hiroshi

2015-01-01

ABSTRACT The AcrB protein of Escherichia coli, together with TolC and AcrA, forms a contiguous envelope conduit for the capture and extrusion of diverse antibiotics and cellular metabolites. In this study, we sought to expand our knowledge of AcrB by conducting genetic and functional analyses. We began with an AcrB mutant bearing an F610A substitution in the drug binding pocket and obtained second-site substitutions that overcame the antibiotic hypersusceptibility phenotype conferred by the F610A mutation. Five of the seven unique single amino acid substitutions—Y49S, V127A, V127G, D153E, and G288C—mapped in the periplasmic porter domain of AcrB, with the D153E and G288C mutations mapping near and at the distal drug binding pocket, respectively. The other two substitutions—F453C and L486W—were mapped to transmembrane (TM) helices 5 and 6, respectively. The nitrocefin efflux kinetics data suggested that all periplasmic suppressors significantly restored nitrocefin binding affinity impaired by the F610A mutation. Surprisingly, despite increasing MICs of tested antibiotics and the efflux of N-phenyl-1-naphthylamine, the TM suppressors did not improve the nitrocefin efflux kinetics. These data suggest that the periplasmic substitutions act by influencing drug binding affinities for the distal binding pocket, whereas the TM substitutions may indirectly affect the conformational dynamics of the drug binding domain. IMPORTANCE The AcrB protein and its homologues confer multidrug resistance in many important human bacterial pathogens. A greater understanding of how these efflux pump proteins function will lead to the development of effective inhibitors against them. The research presented in this paper investigates drug binding pocket mutants of AcrB through the isolation and characterization of intragenic suppressor mutations that overcome the drug susceptibility phenotype of mutations affecting the drug binding pocket. The data reveal a remarkable structure

Effects of L-arabinose efflux on λ Red recombination-mediated gene knockout in multiple-antimicrobial-resistant Salmonella enterica serovar Choleraesuis.

PubMed

Liao, Shi-Wei; Lee, Jen-Jie; Ptak, Christopher P; Wu, Ying-Chen; Hsuan, Shih-Ling; Kuo, Chih-Jung; Chen, Ter-Hsin

2018-03-01

In this study, six swine-derived multiple-antimicrobial-resistant (MAR) strains of Salmonella Choleraesuis (S. Choleraesuis) were demonstrated to possess higher efflux pump activity than the wild-type (WT). L-Arabinose, a common inducer for gene expression, modulated S. Choleraesuis efflux pump activity in a dose-dependent manner. At low L-arabinose concentrations, increasing L-arabinose led to a corresponding increase in fluorophore efflux, while at higher L-arabinose concentrations, increasing L-arabinose decreased fluorophore efflux activity. The WT S. Choleraesuis that lacks TolC (ΔtolC), an efflux protein associated with bacterial antibiotic resistance and virulence, was demonstrated to possess a significantly reduced ability to extrude L-arabinose. Further, due to the rapid export of L-arabinose, an efficient method for recombination-mediated gene knockout, the L-arabinose-inducible bacteriophage λ Red recombinase system, has a reduced recombination frequency (~ 12.5%) in clinically isolated MAR Salmonella strains. An increased recombination frequency (up to 60%) can be achieved using a higher concentration of L-arabinose (fivefold) for genetic manipulation and functional analysis for MAR Salmonella using the λ Red system. The study suggests that L-arabinose serves not only as an inducer of the TolC-dependent efflux system but also acts as a competitive substrate of the efflux system. In addition, understanding the TolC-dependent efflux of L-arabinose should facilitate the optimization of L-arabinose induction in strains with high efflux activity.

Kinetic Parameters of Efflux of Penicillins by the Multidrug Efflux Transporter AcrAB-TolC of Escherichia coli▿

PubMed Central

Lim, Siew Ping; Nikaido, Hiroshi

2010-01-01

The multidrug efflux transporter AcrAB-TolC is known to pump out a diverse range of antibiotics, including β-lactams. However, the kinetic constants of the efflux process, needed for the quantitative understanding of resistance, were not available until those accompanying the efflux of some cephalosporins were recently determined by combining efflux with the hydrolysis of drugs by the periplasmic β-lactamase. In the present study we extended this approach to the study of a wide range of penicillins, from ampicillin and penicillin V to ureidopenicillins and isoxazolylpenicillins, by combining efflux with hydrolysis with the OXA-7 penicillinase. We found that the penicillins had a much stronger apparent affinity to AcrB and higher maximum rates of efflux than the cephalosporins. All penicillins showed strong positive cooperativity kinetics for export. The kinetic constants obtained were validated, as the MICs theoretically predicted on the basis of efflux and hydrolysis kinetics were remarkably similar to the observed MICs (except for the isoxazolylpenicillins). Surprisingly, however, the efflux kinetics of cloxacillin, for example, whose MIC decreased 512-fold in Escherichia coli upon the genetic deletion of the acrB gene, were quite similar to those of ampicillin, whose MIC decreased only 2-fold with the same treatment. Analysis of this phenomenon showed that the extensive decrease in the MIC for the acrB mutant is primarily due to the low permeation of the drug and that comparison of the MICs between the parent and the acrB strains is a very poor measure of the ability of AcrB to pump a drug out. PMID:20160052

Calcium Efflux Systems in Stress Signaling and Adaptation in Plants

PubMed Central

Bose, Jayakumar; Pottosin, Igor I.; Shabala, Stanislav S.; Palmgren, Michael G.; Shabala, Sergey

2011-01-01

Transient cytosolic calcium ([Ca2+]cyt) elevation is an ubiquitous denominator of the signaling network when plants are exposed to literally every known abiotic and biotic stress. These stress-induced [Ca2+]cyt elevations vary in magnitude, frequency, and shape, depending on the severity of the stress as well the type of stress experienced. This creates a unique stress-specific calcium “signature” that is then decoded by signal transduction networks. While most published papers have been focused predominantly on the role of Ca2+ influx mechanisms to shaping [Ca2+]cyt signatures, restoration of the basal [Ca2+]cyt levels is impossible without both cytosolic Ca2+ buffering and efficient Ca2+ efflux mechanisms removing excess Ca2+ from cytosol, to reload Ca2+ stores and to terminate Ca2+ signaling. This is the topic of the current review. The molecular identity of two major types of Ca2+ efflux systems, Ca2+-ATPase pumps and Ca2+/H+ exchangers, is described, and their regulatory modes are analyzed in detail. The spatial and temporal organization of calcium signaling networks is described, and the importance of existence of intracellular calcium microdomains is discussed. Experimental evidence for the role of Ca2+ efflux systems in plant responses to a range of abiotic and biotic factors is summarized. Contribution of Ca2+-ATPase pumps and Ca2+/H+ exchangers in shaping [Ca2+]cyt signatures is then modeled by using a four-component model (plasma- and endo-membrane-based Ca2+-permeable channels and efflux systems) taking into account the cytosolic Ca2+ buffering. It is concluded that physiologically relevant variations in the activity of Ca2+-ATPase pumps and Ca2+/H+ exchangers are sufficient to fully describe all the reported experimental evidence and determine the shape of [Ca2+]cyt signatures in response to environmental stimuli, emphasizing the crucial role these active efflux systems play in plant adaptive responses to environment. PMID:22639615

The role of the Serratia marcescens SdeAB multidrug efflux pump and TolC homologue in fluoroquinolone resistance studied via gene-knockout mutagenesis.

PubMed

Begic, Sanela; Worobec, Elizabeth A

2008-02-01

Serratia marcescens is a prominent opportunistic nosocomial pathogen resistant to several classes of antibiotics. The major mechanism for fluoroquinolone resistance in various Gram-negative pathogens is active efflux. Our group previously identified SdeAB, a resistance-nodulation-cell division (RND) efflux pump complex, and a TolC-like outer-membrane protein (HasF), which together mediate energy-dependent fluoroquinolone efflux. In addition, a regulatory protein-encoding gene in the upstream region of sdeAB was identified (sdeR) and found to be 40 % homologous to MarA, an Escherichia coli transcriptional regulator. To provide conclusive evidence as to the role of these components in S. marcescens, sdeB, hasF and sdeR deletion mutants were constructed. Suicide vectors were created and introduced via triparental mating into S. marcescens UOC-67 (wild-type) and, for sdeB and hasF, T-861 (clinical isolate). We have analysed these genetically altered strains using minimal inhibitory concentration (MIC) assays for a wide range of compounds (fluoroquinolones, SDS, novobiocin, ethidium bromide and chloramphenicol). Intracellular accumulation of a variety of fluoroquinolones was measured fluorospectroscopically. The sdeB, hasF and sdeR knockout strains were consistently more susceptible to antibiotics than the parent strains, with the sdeB/hasF double knockout strain showing the highest susceptibility. A marked increase in fluoroquinolone (ciprofloxacin) accumulation was observed for strains deficient in either the sdeB or hasF genes when compared to the parental strains, with the highest ciprofloxacin accumulation observed for the sdeB/hasF double knockout. Antibiotic accumulation assays for the sdeB knockout mutant strains performed in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), a proton-motive-force inhibitor, demonstrated that SdeAB-mediated efflux is proton-motive-force dependent. Due to the comparable susceptibility of the sdeB and the has

Gene expression analysis of the SdeAB multidrug efflux pump in antibiotic-resistant clinical isolates of Serratia marcescens.

PubMed

Dalvi, S D; Worobec, E A

2012-01-01

Many isolates of Serratia marcescens, a well-known opportunistic pathogen, can be multidrug resistant. Fluoroquinolones are among the most important groups of antibiotics used for treatment of these organisms. However, fluoroquinolone resistance among S. marcescens isolates is fast increasing. Drug extrusion through efflux pumps like SdeAB/ HasF is one of the major mechanisms of resistance to fluoroquinolones. This study was carried out to analyze, through gene expression analysis of sdeB, the relative contribution of this mechanism toward fluoroquinolone resistance in clinical isolates of Serratia. Total RNA from 45 clinical isolates of S. marcescens was isolated. Quantitative real-time RT PCR was performed on the extracted RNA to study the gene expression of sdeB and was normalized to the sdeB expression in the standard strain of S. marcescens. Of the 45 isolates analyzed, sdeB expression was found to be elevated in 20 isolates (44%). Of these 20 isolates, eight (40%) were fully resistant to at least one of the fluoroquinolones studied. Conversely, of the 20 isolates that over-expressed sdeB, 12 (60%) were fully sensitive to all fluoroquinolones tested. Drug efflux pumps are an important means of fluoroquinolone resistance among clinically important species of Serratia. The expression of these pumps can be up-regulated in the presence of antibiotics and have the potential for changing the phenotype from sensitive to resistant, thus contributing to therapeutic failures.

Volume-activated trimethylamine oxide efflux in red blood cells of spiny dogfish (Squalus acanthias).

PubMed

Koomoa, D L; Musch, M W; MacLean, A V; Goldstein, L

2001-09-01

The aims of this study were to determine the pathway of swelling-activated trimethylamine oxide (TMAO) efflux and its regulation in spiny dogfish (Squalus acanthias) red blood cells and compare the characteristics of this efflux pathway with the volume-activated osmolyte (taurine) channel present in erythrocytes of fishes. The characteristics of the TMAO efflux pathway were similar to those of the taurine efflux pathway. The swelling-activated effluxes of both TMAO and taurine were significantly inhibited by known anion transport inhibitors (DIDS and niflumic acid) and by the general channel inhibitor quinine. Volume expansion by hypotonicity, ethylene glycol, and diethyl urea activated both TMAO and taurine effluxes similarly. Volume expansion by hypotonicity, ethylene glycol, and diethyl urea also stimulated the activity of tyrosine kinases p72syk and p56lyn, although the stimulations by the latter two treatments were less than by hypotonicity. The volume activations of both TMAO and taurine effluxes were inhibited by tyrosine kinase inhibitors, suggesting that activation of tyrosine kinases may play a role in activating the osmolyte effluxes. These results indicate that the volume-activated TMAO efflux occurs via the organic osmolyte (taurine) channel and may be regulated by the volume activation of tyrosine kinases.

Efflux inhibitor suppresses Streptococcus mutans virulence properties.

PubMed

Zeng, Huihui; Liu, Jia; Ling, Junqi

2017-04-01

It is well established that efflux pumps play important roles in bacterial pathogenicity and efflux inhibitors (EIs) have been proved to be effective in suppressing bacterial virulence properties. However, little is known regarding the EI of Streptococcus mutans, a well-known caries-inducing bacterium. In this study, we identified the EI of S. mutans through ethidium bromide efflux assay and investigated how EI affected S. mutans virulence regarding the cariogenicity and stress response. Results indicated that reserpine, the identified EI, suppressed acid tolerance, mutacin production and transformation efficiency of S. mutans, and modified biofilm architecture and extracellular polysaccharide distribution. Suppressed glycosyltransferase activity was also noted after reserpine exposure. The data from quantitative real-time-PCR demonstrated that reserpine significantly altered the expression profile of quorum-sensing and virulence-associated genes. These findings suggest that reserpine represents a promising adjunct anticariogenic agent in that it suppresses virulence properties of S. mutans. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Improved Potency of Indole-Based NorA Efflux Pump Inhibitors: From Serendipity toward Rational Design and Development.

PubMed

Buonerba, Federica; Lepri, Susan; Goracci, Laura; Schindler, Bryan D; Seo, Susan M; Kaatz, Glenn W; Cruciani, Gabriele

2017-01-12

The NorA efflux pump is a potential drug target for reversal of resistance to selected antibacterial agents, and recently we described indole-based inhibitor candidates. Herein we report a second class of inhibitors derived from them but with significant differences in shape and size. In particular, compounds 13 and 14 are very potent inhibitors in that they demonstrated the lowest IC 50 values (2 μM) ever observed among all indole-based compounds we have evaluated.

Clonality, outer-membrane proteins profile and efflux pump in KPC- producing Enterobacter sp. in Brazil.

PubMed

Rosa, Juliana Ferraz; Rizek, Camila; Marchi, Ana Paula; Guimaraes, Thais; Miranda, Lourdes; Carrilho, Claudia; Levin, Anna S; Costa, Silvia F

2017-03-17

Carbapenems resistance in Enterobacter spp. has increased in the last decade, few studies, however, described the mechanisms of resistance in this bacterium. This study evaluated clonality and mechanisms of carbapenems resistance in clinical isolates of Enterobacter spp. identified in three hospitals in Brazil (Hospital A, B and C) over 7-year. Antibiotics sensitivity, pulsed-field gel electrophoresis (PFGE), PCR for carbapenemase and efflux pump genes were performed for all carbapenems-resistant isolates. Outer-membrane protein (OMP) was evaluated based on PFGE profile. A total of 130 isolates of Enterobacter spp were analyzed, 44/105 (41, 9%) E. aerogenes and 8/25 (32,0%) E. cloacae were resistant to carbapenems. All isolates were susceptible to fosfomycin, polymyxin B and tigecycline. KPC was present in 88.6% of E. aerogenes and in all E. cloacae resistant to carbapenems. The carbapenems-resistant E. aerogenes identified in hospital A belonged to six clones, however, a predominant clone was identified in this hospital over the study period. There is a predominant clone in Hospital B and Hospital C as well. The mechanisms of resistance to carbapenems differ among subtypes. Most of the isolates co-harbored blaKPC, blaTEM and /or blaCTX associated with decreased or lost of 35-36KDa and or 39 KDa OMP. The efflux pump AcrAB-TolC gene was only identified in carbapenems-resistant E. cloacae. There was a predominant clone in each hospital suggesting that cross-transmission of carbapenems-resistant Enterobacter spp. was frequent. The isolates presented multiple mechanisms of resistance to carbapenems including OMP alteration.

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

The PerR-Regulated P1B-4-Type ATPase (PmtA) Acts as a Ferrous Iron Efflux Pump in Streptococcus pyogenes.

PubMed

Turner, Andrew G; Ong, Cheryl-Lynn Y; Djoko, Karrera Y; West, Nicholas P; Davies, Mark R; McEwan, Alastair G; Walker, Mark J

2017-06-01

Streptococcus pyogenes (group A Streptococcus [GAS]) is an obligate human pathogen responsible for a broad spectrum of human disease. GAS has a requirement for metal homeostasis within the human host and, as such, tightly modulates metal uptake and efflux during infection. Metal acquisition systems are required to combat metal sequestration by the host, while metal efflux systems are essential to protect against metal overload poisoning. Here, we investigated the function of PmtA ( P erR-regulated m etal t ransporter A ), a P 1B-4 -type ATPase efflux pump, in invasive GAS M1T1 strain 5448. We reveal that PmtA functions as a ferrous iron [Fe(II)] efflux system. In the presence of high Fe(II) concentrations, the 5448Δ pmtA deletion mutant exhibited diminished growth and accumulated 5-fold-higher levels of intracellular Fe(II) than did the wild type and the complemented mutant. The 5448Δ pmtA deletion mutant also showed enhanced susceptibility to killing by the Fe-dependent antibiotic streptonigrin as well as increased sensitivity to hydrogen peroxide and superoxide. We suggest that the PerR-mediated control of Fe(II) efflux by PmtA is important for bacterial defense against oxidative stress. PmtA represents an exemplar for an Fe(II) efflux system in a host-adapted Gram-positive bacterial pathogen. Copyright © 2017 American Society for Microbiology.

Attaching the NorA Efflux Pump Inhibitor INF55 to Methylene Blue Enhances Antimicrobial Photodynamic Inactivation of Methicillin-Resistant Staphylococcus aureus in Vitro and in Vivo.

PubMed

Rineh, Ardeshir; Dolla, Naveen K; Ball, Anthony R; Magana, Maria; Bremner, John B; Hamblin, Michael R; Tegos, George P; Kelso, Michael J

2017-10-13

Antimicrobial photodynamic inactivation (aPDI) uses photosensitizers (PSs) and harmless visible light to generate reactive oxygen species (ROS) and kill microbes. Multidrug efflux systems can moderate the phototoxic effects of PSs by expelling the compounds from cells. We hypothesized that increasing intracellular concentrations of PSs by inhibiting efflux with a covalently attached efflux pump inhibitor (EPI) would enhance bacterial cell phototoxicity and reduce exposure of neighboring host cells to damaging ROS. In this study, we tested the hypothesis by linking NorA EPIs to methylene blue (MB) and examining the photoantimicrobial activity of the EPI-MB hybrids against the human pathogen methicillin-resistant Staphylococcus aureus (MRSA). Photochemical/photophysical and in vitro microbiological evaluation of 16 hybrids carrying four different NorA EPIs attached to MB via four linker types identified INF55-(Ac)en-MB 12 as a lead. Compound 12 showed increased uptake into S. aureus cells and enhanced aPDI activity and wound healing effects (relative to MB) in a murine model of an abrasion wound infected by MRSA. The study supports a new approach for treating localized multidrug-resistant MRSA infections and paves the way for wider exploration of the EPI-PS hybrid strategy in aPDI.

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

PubMed Central

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

2013-01-01

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

Efflux-Mediated Resistance to Tigecycline (GAR-936) in Pseudomonas aeruginosa PAO1

PubMed Central

Dean, Charles R.; Visalli, Melissa A.; Projan, Steven J.; Sum, Phaik-Eng; Bradford, Patricia A.

2003-01-01

Pseudomonas aeruginosa strains are less susceptible to tigecycline (previously GAR-936; MIC, 8 μg/ml) than many other bacteria (P. J. Petersen, N. V. Jacobus, W. J. Weiss, P. E. Sum, and R. T. Testa, Antimicrob. Agents Chemother. 43:738-744, 1999). To elucidate the mechanism of resistance to tigecycline, P. aeruginosa PAO1 strains defective in the MexAB-OprM and/or MexXY (OprM) efflux pumps were tested for susceptibility to tigecycline. Increased susceptibility to tigecycline (MIC, 0.5 to 1 μg/ml) was specifically associated with loss of MexXY. Transcription of mexX and mexY was also responsive to exposure of cells to tigecycline. To test for the emergence of compensatory efflux pumps in the absence of MexXY-OprM, mutants lacking MexXY-OprM were plated on medium containing tigecycline at 4 or 6 μg/ml. Resistant mutants were readily recovered, and these also had decreased susceptibility to several other antibiotics, suggesting efflux pump recruitment. One representative carbenicillin-resistant strain overexpressed OprM, the outer membrane channel component of the MexAB-OprM efflux pump. The mexAB-oprM repressor gene, mexR, from this strain contained a 15-bp in-frame deletion. Two representative chloramphenicol-resistant strains showed expression of an outer membrane protein slightly larger than OprM. The mexCD-OprJ repressor gene, nfxB, from these mutants contained a 327-bp in-frame deletion and an IS element insertion, respectively. Together, these data indicated drug efflux mediated by MexCD-OprJ. The MICs of the narrower-spectrum semisynthetic tetracyclines doxycycline and minocycline increased more substantially than did those of tigecycline and other glycylcyclines against the MexAB-OprM- and MexCD-OprJ-overexpressing mutant strains. This suggests that glycylcyclines, although they are subject to efflux from P. aeruginosa, are generally inferior substrates for P. aeruginosa efflux pumps than are narrower-spectrum tetracyclines. PMID:12604529

PigZ, a TetR/AcrR family repressor, modulates secondary metabolism via the expression of a putative four-component resistance-nodulation-cell-division efflux pump, ZrpADBC, in Serratia sp. ATCC 39006.

PubMed

Gristwood, Tamzin; Fineran, Peter C; Everson, Lee; Salmond, George P C

2008-07-01

The Gram-negative enterobacterium, Serratia sp. ATCC 39006 synthesizes several secondary metabolites, including prodigiosin (Pig) and a carbapenem antibiotic (Car). A complex hierarchical network of regulatory proteins control Pig and Car production. In this study we characterize a TetR family regulator, PigZ, which represses transcription of a divergently transcribed putative resistance-nodulation-cell-division (RND) efflux pump, encoded by zrp (PigZ repressed pump) ADBC, via direct binding to the zrpA-pigZ intergenic region. Unusually, this putative RND pump contains two predicted membrane fusion proteins (MFPs), ZrpA and ZrpD. A mutation in pigZ resulted in multiple phenotypic changes, including exoenzyme production, motility and differential regulation of Pig and Car production. A polar suppressor mutation, within zrpA, restored all tested phenotypes to parental strain levels, indicating that the changes observed are due to the increase in expression of ZrpADBC in the absence of the repressor, PigZ. Genomic deletions of zrpA and zrpD indicate that the MFP ZrpD, but not ZrpA, is essential for activity of the putative pump. Bioinformatic analysis revealed that putative RND efflux pumps encoding two MFP components are not uncommon, particularly among plant-associated, Gram-negative bacteria. In addition, based on phylogenetic analysis, we propose that these pairs of MFPs consist of two distinct subtypes.

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

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

PubMed

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

2015-04-28

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.

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.

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.

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

USDA-ARS?s Scientific Manuscript database

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 ...

Temporal Interplay between Efflux Pumps and Target Mutations in Development of Antibiotic Resistance in Escherichia coli

PubMed Central

Singh, Renu; Swick, Michelle C.; Ledesma, Kimberly R.; Yang, Zhen; Hu, Ming; Zechiedrich, Lynn

2012-01-01

The emergence of resistance presents a debilitating change in the management of infectious diseases. Currently, the temporal relationship and interplay between various mechanisms of drug resistance are not well understood. A thorough understanding of the resistance development process is needed to facilitate rational design of countermeasure strategies. Using an in vitro hollow-fiber infection model that simulates human drug treatment, we examined the appearance of efflux pump (acrAB) overexpression and target topoisomerase gene (gyrA and parC) mutations over time in the emergence of quinolone resistance in Escherichia coli. Drug-resistant isolates recovered early (24 h) had 2- to 8-fold elevation in the MIC due to acrAB overexpression, but no point mutations were noted. In contrast, high-level (≥64× MIC) resistant isolates with target site mutations (gyrA S83L with or without parC E84K) were selected more readily after 120 h, and regression of acrAB overexpression was observed at 240 h. Using a similar dosing selection pressure, the emergence of levofloxacin resistance was delayed in a strain with acrAB deleted compared to the isogenic parent. The role of efflux pumps in bacterial resistance development may have been underappreciated. Our data revealed the interplay between two mechanisms of quinolone resistance and provided a new mechanistic framework in the development of high-level resistance. Early low-level levofloxacin resistance conferred by acrAB overexpression preceded and facilitated high-level resistance development mediated by target site mutation(s). If this interpretation is correct, then these findings represent a paradigm shift in the way quinolone resistance is thought to develop. PMID:22232279

V-ATPase proton pumping activity is required for adult zebrafish appendage regeneration.

PubMed

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

2014-01-01

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

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

PubMed Central

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

2014-01-01

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

In vivo evaluation of thiolated poly(acrylic acid) as a drug absorption modulator for MRP2 efflux pump substrates.

PubMed

Greindl, Melanie; Föger, Florian; Hombach, Juliane; Bernkop-Schnürch, Andreas

2009-08-01

Recently, several polymers have been reported to modulate drug absorption by inhibition of intestinal efflux pumps such as multidrug resistance proteins (MRPs) and P-glycoprotein (P-gp). The aim of the present study was to evaluate the efficiency of thiolated poly(acrylic acid) (PAA-Cys) to act as a drug absorption modulator for MRP2 efflux pump substrates in vivo, using sulforhodamine 101 as representative MRP2 substrate. In vitro, the permeation-enhancing effect of unmodified PAA and PAA(250)-Cys(,) displaying 580 micromol free thiol groups per gram polymer, was evaluated by using freshly excised rat intestinal mucosa mounted in Ussing-type chambers. In comparison to that of the buffer control, the sulforhodamine 101 transport in the presence of 0.5% unmodified PAA(250) and 0.5% (w/v) PAA(250)-Cys was 1.3- and 4.0-fold improved, respectively. In vivo, sulforhodamine 101 solutions containing 4% (w/v) unmodified PAA(250) or 4% (w/v) thiolated PAA(250) were orally given to rats. The PAA(250)-Cys solution increased the area under the plasma concentration-time curve (AUC(0-12)) of sulforhodamine 101 3.8-fold in comparison to control and 2.2-fold in comparison to unmodified PAA(250). This in vivo study revealed that PAA(250)-Cys significantly increased the oral bioavailability of MRP2 substrate sulforhodamine 101.

Drug Tolerance in Replicating Mycobacteria Mediated by a Macrophage-Induced Efflux Mechanism

PubMed Central

Adams, Kristin N.; Takaki, Kevin; Connolly, Lynn E.; Wiedenhoft, Heather; Winglee, Kathryn; Humbert, Olivier; Edelstein, Paul H.; Cosma, Christine L.; Ramakrishnan, Lalita

2011-01-01

SUMMARY Treatment of tuberculosis, a complex granulomatous disease, requires long-term multidrug therapy to overcome tolerance, an epigenetic drug resistance that is widely attributed to nonreplicating bacterial subpopulations. Here, we deploy Mycobacterium marinum-infected zebrafish larvae for in vivo characterization of antitubercular drug activity and tolerance. We describe the existence of multi-drug tolerant organisms that arise within days of infection, are enriched in the replicating intracellular population, and are amplified and disseminated by the tuberculous granuloma. Bacterial efflux pumps that are required for intracellular growth mediate this macrophage-induced tolerance. This newly discovered tolerant population also develops when Mycobacterium tuberculosis infects cultured macrophages, suggesting that it contributes to the burden of drug tolerance in human tuberculosis. Efflux pump inhibitors like verapamil reduce this tolerance. Thus, the addition of this currently approved drug, or more specific inhibitors, to standard antitubercular therapy may shorten the duration of curative treatment. PMID:21376383

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. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association.

Comparative study of the active cadmium efflux systems operating at the plasma membrane and tonoplast of cucumber root cells.

PubMed

Migocka, Magdalena; Papierniak, Anna; Kosatka, Ewelina; Klobus, Grazyna

2011-10-01

The strategies developed by plants to avoid the toxicity of cadmium (Cd) and other heavy metals involve active sequestration of metals into the apoplast and vacuoles. The protein systems excluding heavy metals from the cell cytosol localize to the plasma membrane and tonoplast and are energized either by ATP or by the electrochemical gradient generated by H(+)-ATPase or by V-ATPase and pyrophosphatase (PPase), respectively. In this work, a comparative study on the contribution of both the plasma membrane and tonoplast in the active detoxification of plant cells after treatment with Cd was performed. The studies using plants treated and untreated with Cd reveal that both, H(+)-coupled and MgATP-driven efflux of Cd across plasma membranes and tonoplast is markedly stimulated in the presence of Cd in the environment. Previous studies on plasma-membrane localized H(+)-coupled Cd efflux together with the present data demonstrating tonoplast H(+)/Cd(2+) antiport activity suggest that H(+)-coupled secondary transport of Cd displays a lower affinity for Cd when compared with Cd primary pumps driven by MgATP. In addition, it is shown that MgATP-energized Cd efflux across both membranes is significantly enhanced by cysteine, dithiothreitol, and glutathione. These results suggest that Cd is excluded from the cytosol through an energy-dependent system as a free ion as well as a complexed form. Although both membranes contribute in the active exclusion of ionized and complexed Cd from the cytosol, the overall calculation of Cd accumulation in the everted plasma membranes and vacuolar vesicles suggests that the tonoplast and vacuole have a major function in Cd efflux from the cytosol in the roots of cucumber subjected to Cd stress.

NorM, an Erwinia amylovora Multidrug Efflux Pump Involved in In Vitro Competition with Other Epiphytic Bacteria

PubMed Central

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

2004-01-01

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°C than at 28°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. PMID:14766544

Development of classification models for identifying "true" P-glycoprotein (P-gp) inhibitors through inhibition, ATPase activation and monolayer efflux assays.

PubMed

Rapposelli, Simona; Coi, Alessio; Imbriani, Marcello; Bianucci, Anna Maria

2012-01-01

P-glycoprotein (P-gp) is an efflux pump involved in the protection of tissues of several organs by influencing xenobiotic disposition. P-gp plays a key role in multidrug resistance and in the progression of many neurodegenerative diseases. The development of new and more effective therapeutics targeting P-gp thus represents an intriguing challenge in drug discovery. P-gp inhibition may be considered as a valid approach to improve drug bioavailability as well as to overcome drug resistance to many kinds of tumours characterized by the over-expression of this protein. This study aims to develop classification models from a unique dataset of 59 compounds for which there were homogeneous experimental data on P-gp inhibition, ATPase activation and monolayer efflux. For each experiment, the dataset was split into a training and a test set comprising 39 and 20 molecules, respectively. Rational splitting was accomplished using a sphere-exclusion type algorithm. After a two-step (internal/external) validation, the best-performing classification models were used in a consensus predicting task for the identification of compounds named as "true" P-gp inhibitors, i.e., molecules able to inhibit P-gp without being effluxed by P-gp itself and simultaneously unable to activate the ATPase function.

Effect of biofilm formation by clinical isolates of Helicobacter pylori on the efflux-mediated resistance to commonly used antibiotics.

PubMed

Attaran, Bahareh; Falsafi, Tahereh; Ghorbanmehr, Nassim

2017-02-21

To evaluate the role of biofilm formation on the resistance of Helicobacter pylori ( H. pylori ) to commonly prescribed antibiotics, the expression rates of resistance genes in biofilm-forming and planktonic cells were compared. A collection of 33 H. pylori isolates from children and adult patients with chronic infection were taken for the present study. The isolates were screened for biofilm formation ability, as well as for polymerase chain reaction (PCR) reaction with HP1165 and hp1165 efflux pump genes. Susceptibilities of the selected strains to antibiotic and differences between susceptibilities of planktonic and biofilm-forming cell populations were determined. Quantitative real-time PCR (qPCR) analysis was performed using 16S rRNA gene as a H. pylori -specific primer, and two efflux pumps-specific primers, hp1165 and hefA . The strains were resistant to amoxicillin, metronidazole, and erythromycin, except for one strain, but they were all susceptible to tetracycline. Minimum bactericidal concentrations of antibiotics in the biofilm-forming cells were significantly higher than those of planktonic cells. qPCR demonstrated that the expression of efflux pump genes was significantly higher in the biofilm-forming cells as compared to the planktonic ones. The present work demonstrated an association between H. pylori biofilm formation and decreased susceptibility to all the antibiotics tested. This decreased susceptibility to antibiotics was associated with enhanced functional activity of two efflux pumps: hp1165 and hefA .

Contribution of Resistance-Nodulation-Division Efflux Pump Operon smeU1-V-W-U2-X to Multidrug Resistance of Stenotrophomonas maltophilia ▿

PubMed Central

Chen, Chao-Hsien; Huang, Chiang-Ching; Chung, Tsao-Chuen; Hu, Rouh-Mei; Huang, Yi-Wei; Yang, Tsuey-Ching

2011-01-01

KJ09C, a multidrug-resistant mutant of Stenotrophomonas maltophilia KJ, was generated by in vitro selection with chloramphenicol. The multidrug-resistant phenotype of KJ09C was attributed to overexpression of a resistance nodulation division (RND)-type efflux system encoded by an operon consisting of five genes: smeU1, smeV, smeW, smeU2, and smeX. Proteins encoded by smeV, smeW, and smeX were similar to the membrane fusion protein, RND transporter, and outer membrane protein, respectively, of known RND-type systems. The proteins encoded by smeU1 and smeU2 were found to belong to the family of short-chain dehydrogenases/reductases. Mutant KJ09C exhibited increased resistance to chloramphenicol, quinolones, and tetracyclines and susceptibility to aminoglycosides; susceptibility to β-lactams and erythromycin was not affected. The expression of the smeU1-V-W-U2-X operon was regulated by the divergently transcribed LysR-type regulator gene smeRv. Overexpression of the SmeVWX pump contributed to the acquired resistance to chloramphenicol, quinolones, and tetracyclines. Inactivation of smeV and smeW completely abolished the activity of the SmeVWX pump, whereas inactivation of smeX alone decreased the activity of the SmeVWX pump. The enhanced aminoglycoside susceptibility observed in KJ09C resulted from SmeX overexpression. PMID:21930878

Lack of AcrB Efflux Function Confers Loss of Virulence on Salmonella enterica Serovar Typhimurium

PubMed Central

Wang-Kan, Xuan; Chirullo, Barbara; Betts, Jonathan; La Ragione, Roberto M.; Ivens, Alasdair; Ricci, Vito; Opperman, Timothy J.

2017-01-01

ABSTRACT AcrAB-TolC is the paradigm resistance-nodulation-division (RND) multidrug resistance efflux system in Gram-negative bacteria, with AcrB being the pump protein in this complex. We constructed a nonfunctional AcrB mutant by replacing D408, a highly conserved residue essential for proton translocation. Western blotting confirmed that the AcrB D408A mutant had the same native level of expression of AcrB as the parental strain. The mutant had no growth deficiencies in rich or minimal medium. However, compared with wild-type SL1344, the mutant had increased accumulation of Hoechst 33342 dye and decreased efflux of ethidium bromide and was multidrug hypersusceptible. The D408A mutant was attenuated in vivo in mouse and Galleria mellonella models and showed significantly reduced invasion into intestinal epithelial cells and macrophages in vitro. A dose-dependent inhibition of invasion was also observed when two different efflux pump inhibitors were added to the wild-type strain during infection of epithelial cells. RNA sequencing (RNA-seq) revealed downregulation of bacterial factors necessary for infection, including those in the Salmonella pathogenicity islands 1, 2, and 4; quorum sensing genes; and phoPQ. Several general stress response genes were upregulated, probably due to retention of noxious molecules inside the bacterium. Unlike loss of AcrB protein, loss of efflux function did not induce overexpression of other RND efflux pumps. Our data suggest that gene deletion mutants are unsuitable for studying membrane transporters and, importantly, that inhibitors of AcrB efflux function will not induce expression of other RND pumps. PMID:28720734

Functional Relationships between the AcrA Hairpin Tip Region and the TolC Aperture Tip Region for the Formation of the Bacterial Tripartite Efflux Pump AcrAB-TolC ▿ † ‡

PubMed Central

Kim, Hong-Man; Xu, Yongbin; Lee, Minho; Piao, Shunfu; Sim, Se-Hoon; Ha, Nam-Chul; Lee, Kangseok

2010-01-01

Tripartite efflux pumps found in Gram-negative bacteria are involved in antibiotic resistance and toxic-protein secretion. In this study, we show, using site-directed mutational analyses, that the conserved residues located in the tip region of the α-hairpin of the membrane fusion protein (MFP) AcrA play an essential role in the action of the tripartite efflux pump AcrAB-TolC. In addition, we provide in vivo functional data showing that both the length and the amino acid sequence of the α-hairpin of AcrA can be flexible for the formation of a functional AcrAB-TolC pump. Genetic-complementation experiments further indicated functional interrelationships between the AcrA hairpin tip region and the TolC aperture tip region. Our findings may offer a molecular basis for understanding the multidrug resistance of pathogenic bacteria. PMID:20581201

IP/sub 3/ stimulates CA/sup + +/ efflux from fusogenic carrot protoplasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

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/supmore » + +/ 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.« less

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

NASA Astrophysics Data System (ADS)

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

2008-03-01

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

Sequence- and structure-based computational analyses of Gram-negative tripartite efflux pumps in the context of bacterial membranes

DOE PAGES

Travers, Timothy; Wang, Katherine J.; Lopez, Cesar A.; ...

2018-02-09

Gram-negative multidrug resistance currently presents a serious threat to public health with infections effectively rendered untreatable. Multiple molecular mechanisms exist that cause antibiotic resistance and in addition, the last three decades have seen slowing rates of new drug development. In this paper, we summarize the use of various computational techniques for investigating the mechanisms of multidrug resistance mediated by Gram-negative tripartite efflux pumps and membranes. Recent work in our lab combines data-driven sequence and structure analyses to study the interactions and dynamics of these bacterial components. Computational studies can complement experimental methodologies for gaining crucial insights into combatting multidrug resistance.

Sequence- and structure-based computational analyses of Gram-negative tripartite efflux pumps in the context of bacterial membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Travers, Timothy; Wang, Katherine J.; Lopez, Cesar A.

Gram-negative multidrug resistance currently presents a serious threat to public health with infections effectively rendered untreatable. Multiple molecular mechanisms exist that cause antibiotic resistance and in addition, the last three decades have seen slowing rates of new drug development. In this paper, we summarize the use of various computational techniques for investigating the mechanisms of multidrug resistance mediated by Gram-negative tripartite efflux pumps and membranes. Recent work in our lab combines data-driven sequence and structure analyses to study the interactions and dynamics of these bacterial components. Computational studies can complement experimental methodologies for gaining crucial insights into combatting multidrug resistance.

Identification and Structure–Activity Relationships of Novel Compounds that Potentiate the Activities of Antibiotics in Escherichia coli

DOE PAGES

Haynes, Keith M.; Abdali, Narges; Jhawar, Varsha; ...

2017-06-26

In Gram-negative bacteria, efflux pumps are able to prevent effective cellular concentrations from being achieved for a number of antibiotics. Small molecule adjuvants that act as efflux pump inhibitors (EPIs) have the potential to reinvigorate existing antibiotics that are currently ineffective due to efflux mechanisms. Through a combination of rigorous experimental screening and in silico virtual screening, we recently identified novel classes of EPIs that interact with the membrane fusion protein AcrA, a critical component of the AcrAB-TolC efflux pump in Escherichia coli. In this paper, we present initial optimization efforts and structure–activity relationships around one of those previously describedmore » hits, NSC 60339 (1). Finally, from these efforts we identified two compounds, SLUPP-225 (17h) and SLUPP-417 (17o), which demonstrate favorable properties as potential EPIs in E. coli cells including the ability to penetrate the outer membrane, improved inhibition of efflux relative to 1, and potentiation of the activity of novobiocin and erythromycin.« less

Identification and Structure–Activity Relationships of Novel Compounds that Potentiate the Activities of Antibiotics in Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haynes, Keith M.; Abdali, Narges; Jhawar, Varsha

In Gram-negative bacteria, efflux pumps are able to prevent effective cellular concentrations from being achieved for a number of antibiotics. Small molecule adjuvants that act as efflux pump inhibitors (EPIs) have the potential to reinvigorate existing antibiotics that are currently ineffective due to efflux mechanisms. Through a combination of rigorous experimental screening and in silico virtual screening, we recently identified novel classes of EPIs that interact with the membrane fusion protein AcrA, a critical component of the AcrAB-TolC efflux pump in Escherichia coli. In this paper, we present initial optimization efforts and structure–activity relationships around one of those previously describedmore » hits, NSC 60339 (1). Finally, from these efforts we identified two compounds, SLUPP-225 (17h) and SLUPP-417 (17o), which demonstrate favorable properties as potential EPIs in E. coli cells including the ability to penetrate the outer membrane, improved inhibition of efflux relative to 1, and potentiation of the activity of novobiocin and erythromycin.« less

Efflux systems in bacteria and their metabolic engineering applications.

PubMed

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

2015-11-01

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.

Antibacterial activity of berberine-NorA pump inhibitor hybrids with a methylene ether linking group.

PubMed

Samosorn, Siritron; Tanwirat, Bongkot; Muhamad, Nussara; Casadei, Gabriele; Tomkiewicz, Danuta; Lewis, Kim; Suksamrarn, Apichart; Prammananan, Therdsak; Gornall, Karina C; Beck, Jennifer L; Bremner, John B

2009-06-01

Conjugation of the NorA substrate berberine and the NorA inhibitor 5-nitro-2-phenyl-1H-indole via a methylene ether linking group gave the 13-substituted berberine-NorA inhibitor hybrid, 3. A series of simpler arylmethyl ether hybrid structures were also synthesized. The hybrid 3 showed excellent antibacterial activity (MIC Staphylococcus aureus, 1.7 microM), which was over 382-fold more active than the parent antibacterial berberine, against this bacterium. This compound was also shown to block the NorA efflux pump in S. aureus.

Inhibitors of bacterial multidrug efflux pumps from the resin glycosides of Ipomoea murucoides.

PubMed

Chérigo, Lilia; Pereda-Miranda, Rogelio; Fragoso-Serrano, Mabel; Jacobo-Herrera, Nadia; Kaatz, Glenn W; Gibbons, Simon

2008-06-01

A reinvestigation of the CHCl 3-soluble extract from flowers of the Mexican medicinal arborescent morning glory, Ipomoea murucoides, through preparative-scale recycling HPLC, yielded six new pentasaccharides, murucoidins VI-XI (1- 6), as well as the known pescaprein III (7), stoloniferin I (8), and murucoidins I-V (9- 13). Their structures were characterized through the interpretation of their NMR spectroscopic and FABMS data. Compounds 1-6 were found to be macrolactones of three known glycosidic acids identified as simonic acids A and B, and operculinic acid A, with different fatty acids esterifying the same positions, C-2 on the second rhamnose unit and C-4 on the third rhamnose moiety. The lactonization site of the aglycone was placed at C-2 or C-3 of the second saccharide unit. The esterifying residues were composed of two short-chain fatty acids, 2-methylpropanoic and (2S)-methylbutyric acids, and two long-chain fatty acids, n-dodecanoic (lauric) acid and the new (8R)-(-)-8-hydroxydodecanoic acid. For the latter residue, its absolute configuration was determined by analysis of its Mosher ester derivatives. All members of the murucoidin series exerted a potentiation effect of norfloxacin against the NorA overexpressing Staphylococcus aureus strain SA-1199B by increasing the activity 4-fold (8 microg/mL from 32 microg/mL) at concentrations of 5-25 microg/mL. Stoloniferin I (8) enhanced norfloxacin activity 8-fold when incorporated at a concentration of 5 microg/mL. Therefore, this type of amphipathic oligosaccharide could be developed further to provide more potent inhibitors of this multidrug efflux pump.

Antimicrobial and Efflux Inhibitor Activity of Usnic Acid Against Mycobacterium abscessus.

PubMed

Ramis, Ivy B; Vianna, Júlia S; Reis, Ana Júlia; von Groll, Andrea; Ramos, Daniela F; Viveiros, Miguel; da Silva, Pedro E Almeida

2018-06-18

New drugs are needed to treat infections with antimicrobial-resistant Mycobacterium abscessus ; therefore, we evaluated usnic acid as an antimicrobial agent and efflux inhibitor (EI) against M. abscessus . Usnic acid showed antimicrobial activity, and synergistically, the EI verapamil increased this activity. In addition, when we evaluated the interaction of antimicrobials with usnic acid, the increase of their activity was observed. Finally, usnic acid showed an efflux inhibitory effect between the classical EIs verapamil and carbonyl cyanide m-chlorophenylhydrazine. In conclusion, usnic acid showed both antimicrobial and EI activity, indicating that this natural compound may be a promising scaffold for new drugs against this difficult-to-treat microorganism. Georg Thieme Verlag KG Stuttgart · New York.

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.

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

PubMed

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

2003-05-01

Although alkaloids from the family Aizoaceae have anticancer activity, species of this family have received little attention. Because these alkaloids also exhibit properties normally associated with compounds that have activity at the level of the plasma membrane, a methanol extract of Carpobrotus edulis, a common plant found along the Portuguese coast, was studied for properties normally associated with plasma membrane active compounds. The results of this study show that the extract is non-toxic at concentrations that inhibit a verapamil sensitive efflux pump of L5178 mouse T cell lymphoma cell line thereby rendering these multi-drug resistant cells susceptible to anticancer drugs. These non-toxic concentrations also prime THP-1 human monocyte-derived macrophages to kill ingested Staphylococcus aureus and to promote the release of lymphokines associated with cellular immune functions. The extract also induces the proliferation of THP-1 cells within 1 day of exposure to quantities normally associated with phytohaemagglutinin. The potential role of the compound(s) isolated from this plant in cancer biology is intriguing and is currently under investigation. It is supposed that the resistance modifier and immunomodulatory effect of this plant extract can be exploited in the experimental chemotherapy of cancer and bacterial or viral infections. Copyright 2003 John Wiley & Sons, Ltd.

The BpeEF-OprC Efflux Pump Is Responsible for Widespread Trimethoprim Resistance in Clinical and Environmental Burkholderia pseudomallei Isolates

PubMed Central

Podnecky, Nicole L.; Wuthiekanun, Vanaporn; Peacock, Sharon J.

2013-01-01

Trimethoprim-sulfamethoxazole (co-trimoxazole) is the primary drug used for oral eradication therapy of Burkholderia pseudomallei infections (melioidosis). Here, we demonstrate that trimethoprim resistance is widespread in clinical and environmental isolates from northeast Thailand and northern Australia. This resistance was shown to be due to BpeEF-OprC efflux pump expression. No dihydrofolate reductase target mutations were involved, although frequent insertion of ISBma2 was noted within the putative folA transcriptional terminator. All isolates tested remained susceptible to trimethoprim-sulfamethoxazole, suggesting that resistance to trimethoprim alone in these strains probably does not affect the efficacy of co-trimoxazole therapy. PMID:23817379

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

CLICs-dependent chloride efflux is an essential and proximal upstream event for NLRP3 inflammasome activation.

PubMed

Tang, Tiantian; Lang, Xueting; Xu, Congfei; Wang, Xiaqiong; Gong, Tao; Yang, Yanqing; Cui, Jun; Bai, Li; Wang, Jun; Jiang, Wei; Zhou, Rongbin

2017-08-04

The NLRP3 inflammasome can sense different pathogens or danger signals, and has been reported to be involved in the development of many human diseases. Potassium efflux and mitochondrial damage are both reported to mediate NLRP3 inflammasome activation, but the underlying, orchestrating signaling events are still unclear. Here we show that chloride intracellular channels (CLIC) act downstream of the potassium efflux-mitochondrial reactive oxygen species (ROS) axis to promote NLRP3 inflammasome activation. NLRP3 agonists induce potassium efflux, which causes mitochondrial damage and ROS production. Mitochondrial ROS then induces the translocation of CLICs to the plasma membrane for the induction of chloride efflux to promote NEK7-NLRP3 interaction, inflammasome assembly, caspase-1 activation, and IL-1β secretion. Thus, our results identify CLICs-dependent chloride efflux as an essential and proximal upstream event for NLRP3 activation.The NLRP3 inflammasome is key to the regulation of innate immunity against pathogens or stress, but the underlying signaling regulation is still unclear. Here the authors show that chloride intracellular channels (CLIC) interface between mitochondria stress and inflammasome activation to modulate inflammatory responses.

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. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Boeravinone B, A Novel Dual Inhibitor of NorA Bacterial Efflux Pump of Staphylococcus aureus and Human P-Glycoprotein, Reduces the Biofilm Formation and Intracellular Invasion of Bacteria.

PubMed

Singh, Samsher; Kalia, Nitin P; Joshi, Prashant; Kumar, Ajay; Sharma, Parduman R; Kumar, Ashok; Bharate, Sandip B; Khan, Inshad A

2017-01-01

This study elucidated the role of boeravinone B, a NorA multidrug efflux pump inhibitor, in biofilm inhibition. The effects of boeravinone B plus ciprofloxacin, a NorA substrate, were evaluated in NorA-overexpressing, wild-type, and knocked-out Staphylococcus aureus (SA-1199B, SA-1199, and SA-K1758, respectively). The mechanism of action was confirmed using the ethidium bromide accumulation and efflux assay. The role of boeravinone B as a human P -glycoprotein ( P -gp) inhibitor was examined in the LS-180 (colon cancer) cell line. Moreover, its role in the inhibition of biofilm formation and intracellular invasion of S. aureus in macrophages was studied. Boeravinone B reduced the minimum inhibitory concentration (MIC) of ciprofloxacin against S. aureus and its methicillin-resistant strains; the effect was stronger in SA-1199B. Furthermore, time-kill kinetics revealed that boeravinone B plus ciprofloxacin, at subinhibitory concentration (0.25 × MIC), is as equipotent as that at the MIC level. This combination also had a reduced mutation prevention concentration. Boeravinone B reduced the efflux of ethidium bromide and increased the accumulation, thus strengthening the role as a NorA inhibitor. Biofilm formation was reduced by four-eightfold of the minimal biofilm inhibitory concentration of ciprofloxacin, effectively preventing bacterial entry into macrophages. Boeravinone B effectively inhibited P -gp with half maximal inhibitory concentration (IC 50 ) of 64.85 μM. The study concluded that boeravinone B not only inhibits the NorA-mediated efflux of fluoroquinolones but also considerably inhibits the biofilm formation of S. aureus. Its P -gp inhibition activity demonstrates its potential as a bioavailability and bioefficacy enhancer.

Boeravinone B, A Novel Dual Inhibitor of NorA Bacterial Efflux Pump of Staphylococcus aureus and Human P-Glycoprotein, Reduces the Biofilm Formation and Intracellular Invasion of Bacteria

PubMed Central

Singh, Samsher; Kalia, Nitin P.; Joshi, Prashant; Kumar, Ajay; Sharma, Parduman R.; Kumar, Ashok; Bharate, Sandip B.; Khan, Inshad A.

2017-01-01

This study elucidated the role of boeravinone B, a NorA multidrug efflux pump inhibitor, in biofilm inhibition. The effects of boeravinone B plus ciprofloxacin, a NorA substrate, were evaluated in NorA-overexpressing, wild-type, and knocked-out Staphylococcus aureus (SA-1199B, SA-1199, and SA-K1758, respectively). The mechanism of action was confirmed using the ethidium bromide accumulation and efflux assay. The role of boeravinone B as a human P-glycoprotein (P-gp) inhibitor was examined in the LS-180 (colon cancer) cell line. Moreover, its role in the inhibition of biofilm formation and intracellular invasion of S. aureus in macrophages was studied. Boeravinone B reduced the minimum inhibitory concentration (MIC) of ciprofloxacin against S. aureus and its methicillin-resistant strains; the effect was stronger in SA-1199B. Furthermore, time–kill kinetics revealed that boeravinone B plus ciprofloxacin, at subinhibitory concentration (0.25 × MIC), is as equipotent as that at the MIC level. This combination also had a reduced mutation prevention concentration. Boeravinone B reduced the efflux of ethidium bromide and increased the accumulation, thus strengthening the role as a NorA inhibitor. Biofilm formation was reduced by four–eightfold of the minimal biofilm inhibitory concentration of ciprofloxacin, effectively preventing bacterial entry into macrophages. Boeravinone B effectively inhibited P-gp with half maximal inhibitory concentration (IC50) of 64.85 μM. The study concluded that boeravinone B not only inhibits the NorA-mediated efflux of fluoroquinolones but also considerably inhibits the biofilm formation of S. aureus. Its P-gp inhibition activity demonstrates its potential as a bioavailability and bioefficacy enhancer. PMID:29046665

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

DOE PAGES

Su, Chih -Chia; Bolla, Jani  Reddy; Kumar, Nitin; ...

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 tomore » 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.« less

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

PubMed Central

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.

2015-01-01

SUMMARY 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, which belongs to the AbgT family of transporters for which no structural information is available. Here we describe the crystal structure of MtrF, revealing a dimeric molecule with architecture distinct from all other families of transporters. MtrF is a bowl-shaped dimer with a solvent-filled basin extending from the cytoplasm to halfway across the membrane bilayer. Each subunit of the transporter contains nine transmembrane helices and two hairpins, posing a plausible pathway for substrate transport. A combination of the crystal structure and biochemical functional assays suggests that MtrF is an antibiotic efflux pump, mediating bacterial resistance to sulfonamide antimetabolite drugs. PMID:25818299

[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.

HNP-3 enhanced the antimicrobial activity of CIP by promoting ATP efflux from P. aeruginosa cells.

PubMed

Wang, Hao; Dong, Birong; Lou, Li

2011-04-01

To establish a novel strategy of P. aeruginosa control, we acquired recombination HNP-3 by gene recombination. Then we examined HNP-3 bio-activities and the influences of antimicrobial peptide on the efflux of ATP. Consequently, we obtained target protein with a molecular mass of 3,000 D consistent with the Anticipation. FIC index of Ciprofloxacin added HNP-3 was less than 0.5 and HNP3 synergistically cooperated with CIP to suppress P. aeruginosa colony formation revealed that there was significant synergy. ATP efflux was however up-regulated by low concentrations of HNP-3, although CIP did not exert any influence on ATP efflux. Conclusively, recombination protein HNP-3 displayed antimicrobial and synergic effects. HNP-3 enhanced the antimicrobial activity of CIP by promoting ATP efflux from P. aeruginosa cells and decreasing efflux of the drugs, which could have useful clinical applications.

Gβγ subunit activation promotes dopamine efflux through the dopamine transporter

PubMed Central

Garcia-Olivares, J; Baust, T; Harris, S; Hamilton, P; Galli, A; Amara, SG; Torres, GE

2018-01-01

The dopamine transporter (DAT) is an important regulator of brain dopamine (DA) homeostasis, controlling the intensity and duration of DA signaling. DAT is the target for psychostimulants—like cocaine and amphetamine—and plays an important role in neuropsychiatric disorders, including attention-deficit hyperactivity disorder and drug addiction. Thus, a thorough understanding of the mechanisms that regulate DAT function is necessary for the development of clinical interventions to treat DA-related brain disorders. Previous studies have revealed a plethora of protein–protein interactions influencing DAT cellular localization and activity, suggesting that the fine-tuning of DA homeostasis involves multiple mechanisms. We recently reported that G-protein beta-gamma (Gβγ) subunits bind directly to DAT and decrease DA clearance. Here we show that Gβγ induces the release of DA through DAT. Specifically, a Gβγ-binding/activating peptide, mSIRK, increases DA efflux through DAT in heterologous cells and primary dopaminergic neurons in culture. Addition of the Gβγ inhibitor gallein or DAT inhibitors prevents this effect. Residues 582 to 596 in the DAT carboxy terminus were identified as the primary binding site of Gβγ. A TAT peptide containing the Gβγ-interacting domain of DAT blocked the ability of mSIRK to induce DA efflux, consistent with a direct interaction of Gβγ with the transporter. Finally, activation of a G-protein-coupled receptor, the muscarinic M5R, results in DAT-mediated DA efflux through a Gβγ-dependent mechanism. Collectively, our data show that Gβγ interacts with DAT to promote DA efflux. This novel mechanism may have important implications in the regulation of brain DA homeostasis. PMID:28894302

Lack of efflux mediated quinolone resistance in Salmonella enterica serovars Typhi and Paratyphi A

PubMed Central

Baucheron, Sylvie; Monchaux, Isabelle; Le Hello, Simon; Weill, François-Xavier; Cloeckaert, Axel

2014-01-01

Salmonella enterica serovars Typhi and Paratyphi A isolates from human patients in France displaying different levels of resistance to quinolones or fluoroquinolones were studied for resistance mechanisms to these antimicrobial agents. All resistant isolates carried either single or multiple target gene mutations (i.e., in gyrA, gyrB, or parC) correlating with the resistance levels observed. Active efflux, through upregulation of multipartite efflux systems, has also been previously reported as contributing mechanism for other serovars. Therefore, we investigated also the occurrence of non-target gene mutations in regulatory regions affecting efflux pump expression. However, no mutation was detected in these regions in both Typhi and Paratyphi isolates of this study. Besides, no overexpression of the major efflux systems was observed for these isolates. Nevertheless, a large deletion of 2334 bp was identified in the acrS-acrE region of all S. Typhi strains but which did not affect the resistance phenotype. As being specific to S. Typhi, this deletion could be used for specific molecular detection purposes. In conclusion, the different levels of quinolone or FQ resistance in both S. Typhi and S. Paratyphi A seem to rely only on target modifications. PMID:24478769

Expression of multidrug resistance efflux pump gene norA is iron responsive in Staphylococcus aureus.

PubMed

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

2012-04-01

Staphylococcus aureus utilizes efflux transporter NorA to pump out a wide range of structurally dissimilar drugs, conferring low-level multidrug resistance. The regulation of norA expression has yet to be fully understood although past studies have revealed that this gene is under the control of the global transcriptional regulator MgrA and the two-component system ArlRS. To identify additional regulators of norA, we screened a transposon library in strain Newman expressing the transcriptional fusion norA-lacZ for altered β-galactosidase activity. We identify a transposon insertion in fhuB, a gene that encodes a ferric hydroxamate uptake system permease, and propose that the norA transcription is iron responsive. In agreement with this observation, addition of FeCl(3) repressed the induction of norA-lacZ, suggesting that bacterial iron uptake plays an important role in regulating norA transcription. In addition, a fur (ferric uptake regulator) deletion exhibited compromised norA transcription and reduced resistance to quinolone compared to the wild-type strain, indicating that fur functions as a positive regulator of norA. A putative Fur box identified in the promoter region of norA was confirmed by electrophoretic mobility shift and DNase I footprint assays. Finally, by employing a siderophore secretion assay, we reveal that NorA may contribute to the export of siderophores. Collectively, our experiments uncover some novel interactions between cellular iron level and norA regulation in S. aureus.

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

PubMed Central

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

2012-01-01

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

Searching for Novel Inhibitors of the S. aureus NorA Efflux Pump: Synthesis and Biological Evaluation of the 3-Phenyl-1,4-benzothiazine Analogues.

PubMed

Felicetti, Tommaso; Cannalire, Rolando; Burali, Maria Sole; Massari, Serena; Manfroni, Giuseppe; Barreca, Maria Letizia; Tabarrini, Oriana; Schindler, Bryan D; Sabatini, Stefano; Kaatz, Glenn W; Cecchetti, Violetta

2017-08-22

Bacterial resistance to antimicrobial agents has become an increasingly serious health problem in recent years. Among the strategies by which resistance can be achieved, overexpression of efflux pumps such as NorA of Staphylococcus aureus leads to a sub-lethal concentration of the antibacterial agent at the active site that in turn may predispose the organism to the development of high-level target-based resistance. With an aim to improve both the chemical stability and potency of our previously reported 3-phenyl-1,4-benzothiazine NorA inhibitors, we replaced the benzothiazine core with different nuclei. None of the new synthesized compounds showed any appreciable intrinsic antibacterial activity, and, in particular, 2-(3,4-dimethoxyphenyl)quinoline (6 c) was able to decrease, in a concentration-dependent manner, the ciprofloxacin MIC against the norA-overexpressing strains S. aureus SA-K2378 (norA++) and SA-1199B (norA+/A116E GrlA). © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of AcrD, a resistance-nodulation-cell division-type multidrug efflux pump from the fire blight pathogen Erwinia amylovora.

PubMed

Pletzer, Daniel; Weingart, Helge

2014-01-21

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

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

PubMed Central

2014-01-01

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

An ATP-driven efflux pump is a novel pathogenicity factor in rice blast disease.

PubMed Central

Urban, M; Bhargava, T; Hamer, J E

1999-01-01

Cells tolerate exposure to cytotoxic compounds through the action of ATP-driven efflux pumps belonging to the ATP-binding cassette (ABC) superfamily of membrane transporters. Phytopathogenic fungi encounter toxic environments during plant invasion as a result of the plant defense response. Here we demonstrate the requirement for an ABC transporter during host infection by the fungal plant pathogen Magnaporthe grisea. The ABC1 gene was identified in an insertional mutagenesis screen for pathogenicity mutants. The ABC1 insertional mutant and a gene-replacement mutant arrest growth and die shortly after penetrating either rice or barley epidermal cells. The ABC1-encoded protein is similar to yeast ABC transporters implicated in multidrug resistance, and ABC1 gene transcripts are inducible by toxic drugs and a rice phytoalexin. However, abc1 mutants are not hypersensitive to antifungal compounds. The non-pathogenic, insertional mutation in ABC1 occurs in the promoter region and dramatically reduces transcript induction by metabolic poisons. These data strongly suggest that M.grisea requires the up-regulation of specific ABC transporters for pathogenesis; most likely to protect itself against plant defense mechanisms. PMID:9927411

Use of juglone as antibacterial and potential efflux pump inhibitors in Staphylococcus aureus isolated from the oral cavity.

PubMed

Zmantar, Tarek; Miladi, Hanene; Kouidhi, Bochra; Chaabouni, Yassine; Ben Slama, Rihab; Bakhrouf, Amina; Mahdouani, Kacem; Chaieb, Kamel

2016-12-01

In this study the minimal inhibitory concentration (MICs) of tetracycline (Tet), erythromycin (Ery) and benzalkonium chloride (BC) in absence and in presence of a sub-MIC of juglone (Jug) were determined. In addition, the Ethidium bromide (EtBr) efflux assay was performed to assess the effect of Jug on EtBr cells accumulation. Our results showed a selective antimicrobial activity of Jug against the tested strains. A synergistic effect of Jug, drugs (Tet and Ery) and disinfectant (BC) was noticed with a reduction rate varied from 2 to 16-fold. In addition, the efflux of EtBr was inhibited depending on the Jug concentration. In the presence of Jug, a decrease in loss of EtBr from bacteria was observed. The concentration inducing 50 % of EtBr efflux inhibition after 15 min was about 182 μg ml -1 for S. aureus ATCC 25923, 236 μg ml -1 for S. aureus B193 and 195 μg ml -1  for S. aureus B456. It appears from this study that Jug may be used as a natural source for resistance-modifying activity in same bacteria. Copyright © 2016 Elsevier Ltd. All rights reserved.

K+ Efflux-Independent NLRP3 Inflammasome Activation by Small Molecules Targeting Mitochondria.

PubMed

Groß, Christina J; Mishra, Ritu; Schneider, Katharina S; Médard, Guillaume; Wettmarshausen, Jennifer; Dittlein, Daniela C; Shi, Hexin; Gorka, Oliver; Koenig, Paul-Albert; Fromm, Stephan; Magnani, Giovanni; Ćiković, Tamara; Hartjes, Lara; Smollich, Joachim; Robertson, Avril A B; Cooper, Matthew A; Schmidt-Supprian, Marc; Schuster, Michael; Schroder, Kate; Broz, Petr; Traidl-Hoffmann, Claudia; Beutler, Bruce; Kuster, Bernhard; Ruland, Jürgen; Schneider, Sabine; Perocchi, Fabiana; Groß, Olaf

2016-10-18

Imiquimod is a small-molecule ligand of Toll-like receptor-7 (TLR7) that is licensed for the treatment of viral infections and cancers of the skin. Imiquimod has TLR7-independent activities that are mechanistically unexplained, including NLRP3 inflammasome activation in myeloid cells and apoptosis induction in cancer cells. We investigated the mechanism of inflammasome activation by imiquimod and the related molecule CL097 and determined that K + efflux was dispensable for NLRP3 activation by these compounds. Imiquimod and CL097 inhibited the quinone oxidoreductases NQO2 and mitochondrial Complex I. This induced a burst of reactive oxygen species (ROS) and thiol oxidation, and led to NLRP3 activation via NEK7, a recently identified component of this inflammasome. Metabolic consequences of Complex I inhibition and endolysosomal effects of imiquimod might also contribute to NLRP3 activation. Our results reveal a K + efflux-independent mechanism for NLRP3 activation and identify targets of imiquimod that might be clinically relevant. Copyright © 2016 Elsevier Inc. All rights reserved.

Further evidence for a potassium-like action of lithium ions on sodium efflux in frog skeletal muscle

PubMed Central

Beaugé, L. A.; Ortiz, Olga

1972-01-01

time potassium had no effect on the ouabain-insensitive lithium uptake. 8. Muscles incubated in potassium-free-magnesium Ringer had a residual sodium efflux which could not be accounted for by passive movement. About 40% of it was abolished by 10-4 M ouabain. This ouabain-sensitive part could be a consequence of some stimulation of the sodium pump by potassium leaking out of the cells. If this is correct it should be inhibited by external sodium and should not contribute to the total sodium efflux in potassium-free sodium media. 9. Magnesium was used as the reference cation to study the sodium-stimulated sodium efflux under potassium-free conditions. The total sodium efflux amounted to 0·668 hr-1 (rate constant) and was 71% ouabain sensitive. 10. The present experiments demonstrated that lithium ions have a direct stimulating effect on sodium efflux in high sodium skeletal muscle, and strongly support the notion that this effect is produced by an activation of the sodium pump through a potassium-like action. PMID:4637626

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

PubMed Central

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

2016-01-01

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

Efflux mediated adaptive and cross resistance to ciprofloxacin and benzalkonium chloride in Pseudomonas aeruginosa of dairy origin.

PubMed

Pagedar, Ankita; Singh, Jitender; Batish, Virender K

2011-06-01

The present study was undertaken to investigate the role of efflux pump activity (EPA) in conferring adaptive and cross resistances against ciprofloxacin (CF) and benzalkonium chloride (BC) in dairy isolates of Pseudomonas aeruginosa. Biofilm formation potential was correlated with development of adaptive resistance in originally resistant strains. Irrespective of parent strains's susceptibility, isolates developed substantial adaptive resistance against CF and BC. Significant difference was observed in ability of non resistant isolates to develop adaptive resistance against CF and BC (P < 0.02) and subsequent cross resistance. EPA was quantified using EtBr (Ethidium Bromide) model and its role was more prominent [confirmed by its inhibition using efflux pump inhibitor (EPI) 2,4-dinitrophenol (DNP)], in conferring adaptive resistance (P = 0.147) than cross resistance (P = 0.343). Reduction in adaptive resistances due to EPI was more evident in originally non resistant strains, which reaffirms EPA as probable mechanism of adaptive resistance. The present study perhaps first of its kind, suggests an active role of EPA in conferring adaptive and cross resistances in food related P. aeruginosa isolates and supports reverse hypothesis that antibiotic-resistant organisms eventually become tolerant to other antibacterial agents as well. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structure-function relationships in reconstituted HDL: Focus on antioxidative activity and cholesterol efflux capacity.

PubMed

Cukier, Alexandre M O; Therond, Patrice; Didichenko, Svetlana A; Guillas, Isabelle; Chapman, M John; Wright, Samuel D; Kontush, Anatol

2017-09-01

High-density lipoprotein (HDL) contains multiple components that endow it with biological activities. Apolipoprotein A-I (apoA-I) and surface phospholipids contribute to these activities; however, structure-function relationships in HDL particles remain incompletely characterised. Reconstituted HDLs (rHDLs) were prepared from apoA-I and soy phosphatidylcholine (PC) at molar ratios of 1:50, 1:100 and 1:150. Oxidative status of apoA-I was varied using controlled oxidation of Met112 residue. HDL-mediated inactivation of PC hydroperoxides (PCOOH) derived from mildly pre-oxidized low-density lipoprotein (LDL) was evaluated by HPLC with chemiluminescent detection in HDL+LDL mixtures and re-isolated LDL. Cellular cholesterol efflux was characterised in RAW264.7 macrophages. rHDL inactivated LDL-derived PCOOH in a dose- and time-dependent manner. The capacity of rHDL to both inactivate PCOOH and efflux cholesterol via ATP-binding cassette transporter A1 (ABCA1) increased with increasing apoA-I/PC ratio proportionally to the apoA-I content in rHDL. Controlled oxidation of apoA-I Met112 gradually decreased PCOOH-inactivating capacity of rHDL but increased ABCA1-mediated cellular cholesterol efflux. Increasing apoA-I content in rHDL enhanced its antioxidative activity towards oxidized LDL and cholesterol efflux capacity via ABCA1, whereas oxidation of apoA-I Met112 decreased the antioxidative activity but increased the cholesterol efflux. These findings provide important considerations in the design of future HDL therapeutics. Non-standard abbreviations and acronyms: AAPH, 2,2'-azobis(-amidinopropane) dihydrochloride; ABCA1, ATP-binding cassette transporter A1; apoA-I, apolipoprotein A-I; BHT, butylated hydroxytoluene; CV, cardiovascular; EDTA, ethylenediaminetetraacetic acid; HDL-C, high-density lipoprotein cholesterol; LOOH, lipid hydroperoxides; Met(O), methionine sulfoxide; Met112, methionine 112 residue; Met86, methionine 86 residue; oxLDL, oxidized low

Mutational analysis of P-glycoprotein: suppression of caspase activation in the absence of ATP-dependent drug efflux.

PubMed

Tainton, K M; Smyth, M J; Jackson, J T; Tanner, J E; Cerruti, L; Jane, S M; Darcy, P K; Johnstone, R W

2004-09-01

P-glycoprotein (P-gp) can induce multidrug resistance (MDR) through the ATP-dependent efflux of chemotherapeutic agents. We have previously shown that P-gp can inhibit nondrug apoptotic stimuli by suppressing the activation of caspases. To determine if this additional activity is functionally linked to ATP hydrolysis, we expressed wild-type and ATPase-mutant P-gp and showed that cells expressing mutant P-gp could not efflux chemotherapeutic drugs but remained relatively resistant to apoptosis. CEM lymphoma cells expressing mutant P-gp treated with vincristine showed a decrease in the fraction of cells with apoptotic morphology, cytochrome c release from the mitochondria and suppression of caspase activation, yet still accumulated in mitosis and showed a loss of clonogenic potential. The loss of clonogenicity in vincristine-treated cells expressing mutant P-gp was associated with accumulation of cells in mitosis and the presence of multinucleated cells consistent with mitotic catastrophe. The antiapoptotic effect of mutant P-gp was not affected by antibodies that inhibit the efflux function of the protein. These data are consistent with a dual activity model for P-gp-induced MDR involving both ATPase-dependent drug efflux and ATPase-independent inhibition of apoptosis. The structure-function analyses described herein provide novel insight into the mechanisms of action of P-gp in mediating MDR.

High-level tetracycline resistance mediated by efflux pumps Tet(A) and Tet(A)-1 with two start codons.

PubMed

Wang, Weixia; Guo, Qinglan; Xu, Xiaogang; Sheng, Zi-ke; Ye, Xinyu; Wang, Minggui

2014-11-01

Efflux is the most common mechanism of tetracycline resistance. Class A tetracycline efflux pumps, which often have high prevalence in Enterobacteriaceae, are encoded by tet(A) and tet(A)-1 genes. These genes have two potential start codons, GTG and ATG, located upstream of the genes. The purpose of this study was to determine the start codon(s) of the class A tetracycline resistance (tet) determinants tet(A) and tet(A)-1, and the tetracycline resistance level they mediated. Conjugation, transformation and cloning experiments were performed and the genetic environment of tet(A)-1 was analysed. The start codons in class A tet determinants were investigated by site-directed mutagenesis of ATG and GTG, the putative translation initiation codons. High-level tetracycline resistance was transferred from the clinical strain of Klebsiella pneumoniae 10-148 containing tet(A)-1 plasmid pHS27 to Escherichia coli J53 by conjugation. The transformants harbouring recombinant plasmids that carried tet(A) or tet(A)-1 exhibited tetracycline MICs of 256-512 µg ml(-1), with or without tetR(A). Once the ATG was mutated to a non-start codon, the tetracycline MICs were not changed, while the tetracycline MICs decreased from 512 to 64 µg ml(-1) following GTG mutation, and to ≤4 µg ml(-1) following mutation of both GTG and ATG. It was presumed that class A tet determinants had two start codons, which are the primary start codon GTG and secondary start codon ATG. Accordingly, two putative promoters were predicted. In conclusion, class A tet determinants can confer high-level tetracycline resistance and have two start codons. © 2014 The Authors.

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

Genome analysis of urease positive Serratia marcescens, co-producing SRT-2 and AAC(6')-Ic with multidrug efflux pumps for antimicrobial resistance.

PubMed

Srinivasan, Vijaya Bharathi; Rajamohan, Govindan

2018-04-05

In this study, we present the genome sequence of Serratia marcescens SM03, recovered from a human gut in India. The final assembly consists of 26 scaffolds (4620 coding DNA sequences, 5.08 Mb, 59.6% G + C ratio) and 79 tRNA genes. Analysis identified novel genes associated with lactose utilization, virulence, P-loop GTPases involved in urease production, CFA/I fimbriae apparatus and Yersinia - type CRISPR proteins. Antibiotic susceptibility testing indicated drug tolerant phenotype and inhibition assays demonstrated involvement of extrusion in resistance. Presence of enzymes SRT-2, AAC(6')-Ic, with additional Ybh transporter and EamA-like efflux pumps signifies the genetic plasticity observed in S. marcescens SM03. Copyright © 2018 Elsevier Inc. All rights reserved.

Design and Study of Efflux Function of EGFP Fused MexAB-OprM Membrane Transporter in Pseudomonas aeruginosa Using Fluorescence Spectroscopy

PubMed Central

Ding, Feng; Lee, Kerry J.; Vahedi-Faridi, Ardeschir; Yoneyama, Hiroshi; Osgood, Christopher J.; Xu, Xiao-Hong Nancy

2014-01-01

the translocation pore or restrict the movement of the individual pump domains, which leads to partially restrict efflux activity. PMID:24781334

Glutathione Efflux and Cell Death

PubMed Central

2012-01-01

Abstract Significance: Glutathione (GSH) depletion is a central signaling event that regulates the activation of cell death pathways. GSH depletion is often taken as a marker of oxidative stress and thus, as a consequence of its antioxidant properties scavenging reactive species of both oxygen and nitrogen (ROS/RNS). Recent Advances: There is increasing evidence demonstrating that GSH loss is an active phenomenon regulating the redox signaling events modulating cell death activation and progression. Critical Issues: In this work, we review the role of GSH depletion by its efflux, as an important event regulating alterations in the cellular redox balance during cell death independent from oxidative stress and ROS/RNS formation. We discuss the mechanisms involved in GSH efflux during cell death progression and the redox signaling events by which GSH depletion regulates the activation of the cell death machinery. Future Directions: The evidence summarized here clearly places GSH transport as a central mechanism mediating redox signaling during cell death progression. Future studies should be directed toward identifying the molecular identity of GSH transporters mediating GSH extrusion during cell death, and addressing the lack of sensitive approaches to quantify GSH efflux. Antioxid. Redox Signal. 17, 1694–1713. PMID:22656858

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. Copyright © 2014 Elsevier Ltd. All rights reserved.

The drug efflux pump Pgp1 in pro-inflammatory lymphocytes is a target for novel treatment strategies in COPD.

PubMed

Hodge, Greg; Holmes, Mark; Jersmann, Hubertus; Reynolds, Paul N; Hodge, Sandra

2013-06-03

Pro-inflammatory/cytotoxic T cells (IFNγ, TNFα, granzyme B+) are increased in the peripheral circulation in COPD. NKT-like and NK cells are effector lymphocytes that we have also shown to be major sources of pro-inflammatory cytokines and granzymes. P-glycoprotein 1 (Pgp1) is a transmembrane efflux pump well characterised in drug resistant cancer cells. We hypothesized that Pgp1 would be increased in peripheral blood T, NKT-like and NK cells in patients with COPD, and that this would be accompanied by increased expression of IFNγ, TNFα and granzyme B. We further hypothesized that treatment with cyclosporine A, a Pgp1 inhibitor, would render cells more sensitive to treatment with corticosteroids. Pgp1, granzyme B, IFNγ and TNFα expression were measured in peripheral blood T, NK and NKT-like cells from COPD patients and control subjects (± cyclosporine A and prednisolone) following in vitro stimulation and results correlated with uptake of efflux dye Calcein-AM using flow cytometry. There was increased Pgp1 expression by peripheral blood T, NKT-like and NK cells co-expressing IFNγ, TNFα and granzyme B in COPD patients compared with controls (e.g. %IFNγ/Pgp1 T, NKT-like, NK for COPD (Control): 25(6), 54(27), 39(23)). There was an inverse correlation between Pgp1 expression and Calcein-AM uptake. Treatment with 2.5 ng/ml cylosporin A and10-6 M prednisolone resulted in synergistic inhibition of pro-inflammatory cytokines in Pgp1 + cells (p < 0.05 for all). Treatment strategies that target Pgp1 in T, NKT-like and NK cells may reduce systemic inflammatory mediators in COPD and improve patient morbidity.

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

PubMed

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

2015-07-01

Pseudomonas putida DOT-T1E-18 is a strain deficient in the major antibiotic efflux pump (TtgABC) that exhibits an overall increased susceptibility to a wide range of drugs when compared with the wild-type strain. We used this strain as a platform to search for microbes able to produce antibiotics that inhibit growth. A collection of 2400 isolates from soil, sediments and water was generated and a drop assay developed to identify, via growth inhibition halos, strains that prevent the growth of DOT-T1E-18 on solid Luria-Bertani plates. In this study, 35 different isolates that produced known and unknown antibiotics were identified. The most potent inhibitor of DOT-T1E-18 growth was an isolate named 250J that, through multi-locus sequence analysis, was identified as a Pseudomonas sp. strain. Culture supernatants of 250J contain four different xantholysins that prevent growth of Gram-positive bacteria, Gram-negative and fungi. Two of the xantholysins were produced in higher concentrations and purified. Xantholysin A was effective against Bacillus, Lysinibacillus and Rhodococcus strains, and the effect against these microbes was enhanced when used in combination with other antibiotics such as ampicillin, gentamicin and kanamycin. Xantholysin C was also efficient against Gram-positive bacteria and showed an interesting antimicrobial effect against Pseudomonas strains, and a synergistic inhibitory effect with ampicillin, chloramphenicol and gentamicin. © 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

The human red cell voltage-regulated cation channel. The interplay with the chloride conductance, the Ca(2+)-activated K(+) channel and the Ca(2+) pump.

PubMed

Bennekou, P; Kristensen, B I; Christophersen, P

2003-09-01

The activation/deactivation kinetics of the human erythrocyte voltage-dependent cation channel was characterized at the single-channel level using inside-out patches. It was found that the time dependence for voltage activation after steps to positive membrane potentials was slow ( t(1/2) about 30 s), whereas the deactivation was fast ( t(1/2) about 15 ms). Both activation and deactivation of this channel were also demonstrated in intact red cells in suspension. At very positive membrane potentials generated by suspension in extracellular low Cl(-) concentrations, the cation conductance switched on with a time constant of about 2 min. Deactivation of the cation channel was clearly demonstrated during transient activation of the Gárdos channel elicited by Ca(2+) influx via the cation channel and ensuing efflux via the Ca(2+) pump. Thus, the voltage-dependent cation channel, the Gárdos channel and the Ca(2+) pump constitute a coupled feedback-regulated system that may become operative under physiological conditions.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gaur, Naseem Akhtar; Manoharlal, Raman; Saini, Preeti

2005-06-24

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

Efflux-mediated antimicrobial resistance.

PubMed

Poole, Keith

2005-07-01

Antibiotic resistance continues to plague antimicrobial chemotherapy of infectious disease. And while true biocide resistance is as yet unrealized, in vitro and in vivo episodes of reduced biocide susceptibility are common and the history of antibiotic resistance should not be ignored in the development and use of biocidal agents. Efflux mechanisms of resistance, both drug specific and multidrug, are important determinants of intrinsic and/or acquired resistance to these antimicrobials, with some accommodating both antibiotics and biocides. This latter raises the spectre (as yet generally unrealized) of biocide selection of multiple antibiotic-resistant organisms. Multidrug efflux mechanisms are broadly conserved in bacteria, are almost invariably chromosome-encoded and their expression in many instances results from mutations in regulatory genes. In contrast, drug-specific efflux mechanisms are generally encoded by plasmids and/or other mobile genetic elements (transposons, integrons) that carry additional resistance genes, and so their ready acquisition is compounded by their association with multidrug resistance. While there is some support for the latter efflux systems arising from efflux determinants of self-protection in antibiotic-producing Streptomyces spp. and, thus, intended as drug exporters, increasingly, chromosomal multidrug efflux determinants, at least in Gram-negative bacteria, appear not to be intended as drug exporters but as exporters with, perhaps, a variety of other roles in bacterial cells. Still, given the clinical significance of multidrug (and drug-specific) exporters, efflux must be considered in formulating strategies/approaches to treating drug-resistant infections, both in the development of new agents, for example, less impacted by efflux and in targeting efflux directly with efflux inhibitors.

Computational analysis of structure-based interactions and ligand properties can predict efflux effects on antibiotics.

PubMed

Sarkar, Aurijit; Anderson, Kelcey C; Kellogg, Glen E

2012-06-01

AcrA-AcrB-TolC efflux pumps extrude drugs of multiple classes from bacterial cells and are a leading cause for antimicrobial resistance. Thus, they are of paramount interest to those engaged in antibiotic discovery. Accurate prediction of antibiotic efflux has been elusive, despite several studies aimed at this purpose. Minimum inhibitory concentration (MIC) ratios of 32 β-lactam antibiotics were collected from literature. 3-Dimensional Quantitative Structure-Activity Relationship on the β-lactam antibiotic structures revealed seemingly predictive models (q(2)=0.53), but the lack of a general superposition rule does not allow its use on antibiotics that lack the β-lactam moiety. Since MIC ratios must depend on interactions of antibiotics with lipid membranes and transport proteins during influx, capture and extrusion of antibiotics from the bacterial cell, descriptors representing these factors were calculated and used in building mathematical models that quantitatively classify antibiotics as having high/low efflux (>93% accuracy). Our models provide preliminary evidence that it is possible to predict the effects of antibiotic efflux if the passage of antibiotics into, and out of, bacterial cells is taken into account--something descriptor and field-based QSAR models cannot do. While the paucity of data in the public domain remains the limiting factor in such studies, these models show significant improvements in predictions over simple LogP-based regression models and should pave the path toward further work in this field. This method should also be extensible to other pharmacologically and biologically relevant transport proteins. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

[Molecular evolution of the sulphite efflux gene SSU1 in Saccharomyces cerevisiae].

PubMed

Peng, Li-Xin; Sun, Fei-Fei; Huang, Yan-Yan; Li, Zhen-Chong

2013-11-01

The SSU1 gene encoding a membrane sulfite pump is a main facilitator invovled in sulfite efflux. In Saccharomyce cerevisiae, various range of resistance to sulfite was observed among strains. To explore the evolution traits of SSU1 gene, the population data of S. cerevisiae were collected and analyzed. The phylogenetic analysis indicated that S. cerevisiae population can be classified into three sub-populations, and the positive selection was detected in population by McDonald-Kreitman test. The anaylsis of Ka/Ks ratios further showed that S. cerevisiae sub-population was undergoing positive selection. This finding was also supported by PAML branch model. Nine potential positive selection sites were predicted by branch-site model, and four sites exclusively belong to the sub-population under positive seletion. The data from ssulp protein structure demonstrated that three sites are substitutions between polar and hydrophobic amino acids, and only one site of substitutaion from basic amino acid to basic amino acid (345R/K). Because amino acid pKa values are crucial for sulfite pump to maintain their routine function, positive selection of these amino acid substitutions might affect sulfite efflux efficient.

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

PubMed Central

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

2014-01-01

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

In Vitro Selection of ramR and soxR Mutants Overexpressing Efflux Systems by Fluoroquinolones as Well as Cefoxitin in Klebsiella pneumoniae▿

PubMed Central

Bialek-Davenet, Suzanne; Marcon, Estelle; Leflon-Guibout, Véronique; Lavigne, Jean-Philippe; Bert, Frédéric; Moreau, Richard; Nicolas-Chanoine, Marie-Hélène

2011-01-01

The relationship between efflux system overexpression and cross-resistance to cefoxitin, quinolones, and chloramphenicol has recently been reported in Klebsiella pneumoniae. In 3 previously published clinical isolates and 17 in vitro mutants selected with cefoxitin or fluoroquinolones, mutations in the potential regulator genes of the AcrAB efflux pump (acrR, ramR, ramA, marR, marA, soxR, soxS, and rob) were searched, and their impacts on efflux-related antibiotic cross-resistance were assessed. All mutants but 1, and 2 clinical isolates, overexpressed acrB. No mutation was detected in the regulator genes studied among the clinical isolates and 8 of the mutants. For the 9 remaining mutants, a mutation was found in the ramR gene in 8 of them and in the soxR gene in the last one, resulting in overexpression of ramA and soxS, respectively. Transformation of the ramR mutants and the soxR mutant with the wild-type ramR and soxR genes, respectively, abolished overexpression of acrB and ramA in the ramR mutants and of soxS in the soxR mutant, as well as antibiotic cross-resistance. Resistance due to efflux system overexpression was demonstrated for 4 new antibiotics: cefuroxime, cefotaxime, ceftazidime, and ertapenem. This study shows that the ramR and soxR genes control the expression of efflux systems in K. pneumoniae and suggests the existence of efflux pumps other than AcrAB and of other loci involved in the regulation of AcrAB expression. PMID:21464248

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

PubMed

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

2016-07-01

The activity of levofloxacin against planktonic and biofilm Stenotrophomonas maltophilia cells and the role played by the multidrug efflux pump SmeDEF were evaluated under conditions relevant to the cystic fibrosis (CF) lung. MIC, MBC and MBEC of levofloxacin were assessed, against five CF strains, under 'standard' (CLSI-recommended) and 'CF-like' (pH 6.8, 5% CO2, in a synthetic CF sputum) conditions. Levofloxacin was tested against biofilms at concentrations (10, 50 and 100 μg mL(-1)) corresponding to achievable serum levels and sputum levels by aerosolisation. smeD expression was evaluated, under both conditions, in planktonic and biofilm cells by RT-PCR. The bactericidal effect of levofloxacin was decreased, in three out of five strains tested, under 'CF-like' conditions (MBC: 2-4 vs 8-16 μg mL(-1), under 'standard' and 'CF-like' conditions, respectively). Biofilm was intrinsically resistant to levofloxacin, regardless of conditions tested (MBECs ≥ 100 μg mL(-1) for all strains). Only under 'CF-like' conditions, smeD expression increased during planktonic-to-biofilm transition, and in biofilm cells compared to stationary planktonic cells. Our findings confirmed that S. maltophilia biofilm is intrinsically resistant to therapeutic concentrations of levofloxacin. Under conditions relevant to CF, smeD overexpression could contribute to levofloxacin resistance. Further studies are warranted to define the clinical relevance of our findings. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Cyclic AMP efflux inhibitors as potential therapeutic agents for leukemia.

PubMed

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

2016-06-07

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.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

1988-08-01

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

Influence of high-frequency ambient pressure pumping on carbon dioxide efflux from soil

Treesearch

Eugene S. Takle; William J. Massman; James R. Brandle; R. A. Schmidt; Xinhua Zhou; Irina V. Litvina; Rick Garcia; Geoffrey Doyle; Charles W. Rice

2004-01-01

We report measurements at 2Hz of pressure fluctuations at and beneath the soil in an agricultural field with dry soil and no vegetation. The objective of our study was to examine the possible role of pressure fluctuations produced by fluctuations in ambient wind on the efflux of CO2 at the soil surface.We observed that pressure fluctuations penetrate to 50 cm in the...

An ex vivo study of nitric oxide efflux from human erythrocytes in both genders.

PubMed

Duarte, Catarina; Napoleão, Patrícia; Freitas, Teresa; Saldanha, Carlota

2016-01-01

Acetylcholinesterase (AChE) is located on outer surface of erythrocyte membrane. Gender-related differences in erythrocyte AChE enzyme activity had been verified in young adults. It is also known that binding of acetylcholine (ACh) with AChE on erythrocyte membrane initiates a signal transduction mechanism that stimulates nitric oxide (NO) efflux. This ex vivo study was done to compare the amount of NO efflux obtained from erythrocytes of healthy donors in males and females. We included 66 gender age-matched healthy donors (40-60 years old). We performed quantification of erythrocyte NO efflux from erythrocytes and of the membrane AChE enzyme activity. There are no significant differences in NO efflux from erythrocytes between men and women. Regarding AChE enzyme activity values, in this range of age, no differences between genders were obtained. However, the values of AChE enzyme activity in the third quartile of NO efflux values were significantly higher (p < 0.05) in women than in men. The efflux of NO from erythrocyte of healthy humans did not change with gender. For the same range of values of NO efflux from erythrocytes, in both gender, it was verified higher values of AChE enzyme activity in women.

Fluorescent Trimethoprim Conjugate Probes To Assess Drug Accumulation in Wild Type and Mutant Escherichia coli

PubMed Central

2016-01-01

Reduced susceptibility to antimicrobials in Gram-negative bacteria may result from multiple resistance mechanisms, including increased efflux pump activity or reduced porin protein expression. Up-regulation of the efflux pump system is closely associated with multidrug resistance (MDR). To help investigate the role of efflux pumps on compound accumulation, a fluorescence-based assay was developed using fluorescent derivatives of trimethoprim (TMP), a broad-spectrum synthetic antibiotic that inhibits an intracellular target, dihydrofolate reductase (DHFR). Novel fluorescent TMP probes inhibited eDHFR activity with comparable potency to TMP, but did not kill or inhibit growth of wild type Escherichia coli. However, bactericidal activity was observed against an efflux pump deficient E. coli mutant strain (ΔtolC). A simple and quick fluorescence assay was developed to measure cellular accumulation of the TMP probe using either fluorescence spectroscopy or flow cytometry, with validation by LC-MS/MS. This fluorescence assay may provide a simple method to assess efflux pump activity with standard laboratory equipment. PMID:27737551

Sodium Pumps Mediate Activity-Dependent Changes in Mammalian Motor Networks

PubMed Central

Picton, Laurence D.; Nascimento, Filipe; Broadhead, Matthew J.; Sillar, Keith T.

2017-01-01

Ubiquitously expressed sodium pumps are best known for maintaining the ionic gradients and resting membrane potential required for generating action potentials. However, activity- and state-dependent changes in pump activity can also influence neuronal firing and regulate rhythmic network output. Here we demonstrate that changes in sodium pump activity regulate locomotor networks in the spinal cord of neonatal mice. The sodium pump inhibitor, ouabain, increased the frequency and decreased the amplitude of drug-induced locomotor bursting, effects that were dependent on the presence of the neuromodulator dopamine. Conversely, activating the pump with the sodium ionophore monensin decreased burst frequency. When more “natural” locomotor output was evoked using dorsal-root stimulation, ouabain increased burst frequency and extended locomotor episode duration, whereas monensin slowed and shortened episodes. Decreasing the time between dorsal-root stimulation, and therefore interepisode interval, also shortened and slowed activity, suggesting that pump activity encodes information about past network output and contributes to feedforward control of subsequent locomotor bouts. Using whole-cell patch-clamp recordings from spinal motoneurons and interneurons, we describe a long-duration (∼60 s), activity-dependent, TTX- and ouabain-sensitive, hyperpolarization (∼5 mV), which is mediated by spike-dependent increases in pump activity. The duration of this dynamic pump potential is enhanced by dopamine. Our results therefore reveal sodium pumps as dynamic regulators of mammalian spinal motor networks that can also be affected by neuromodulatory systems. Given the involvement of sodium pumps in movement disorders, such as amyotrophic lateral sclerosis and rapid-onset dystonia parkinsonism, knowledge of their contribution to motor network regulation also has considerable clinical importance. SIGNIFICANCE STATEMENT The sodium pump is ubiquitously expressed and responsible

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.

Structural basis for the blockade of MATE multidrug efflux pumps

DOE PAGES

Radchenko, Martha; Symersky, Jindrich; Nie, Rongxin; ...

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 themore » 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.« less

Strong synergism of dexamethasone in combination with fluconazole against resistant Candida albicans mediated by inhibiting drug efflux and reducing virulence.

PubMed

Sun, Wenwen; Wang, Decai; Yu, Cuixiang; Huang, Xin; Li, Xiuyun; Sun, Shujuan

2017-09-01

Candida albicans is the most commonly isolated Candida spp. in the clinic and its resistance to fluconazole (FLC) has been emerging rapidly. Combination therapy may be a potentially effective approach to combat drug resistance. In this study, the combination antifungal effects of dexamethasone (DXM) and FLC against resistant C. albicans in vitro were assayed using minimum inhibitory concentrations (MICs), sessile MICs and time-kill curves. The in vivo efficacy of this drug combination was evaluated using a Galleria mellonella model by determining survival rate, fungal burden and histological damage. In addition, the impact of DXM on efflux pump activity was investigated using a rhodamine 6G assay. Expression of CDR1, CDR2 and MDR1 was determined by real-time quantitative PCR, and extracellular phospholipase activity was detected by the egg yolk agar method to reveal the potential synergistic mechanism. The results showed that DXM potentiates the antifungal effect of FLC against resistant C. albicans strains both in vitro and in vivo, and the synergistic mechanism is related to inhibiting the efflux of drugs and reducing the virulence of C. albicans. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lodge, N.J.; Cohen, R.B.; Havens, C.N.

1991-02-01

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

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. © 2015 John Wiley & Sons Ltd.

Activation of muscarinic cholinergic receptors on human SH-SY5Y neuroblastoma cells enhances both the influx and efflux of K+ under conditions of hypo-osmolarity.

PubMed

Foster, Daniel J; Heacock, Anne M; Keep, Richard F; Fisher, Stephen K

2008-05-01

The ability of receptor activation to regulate osmosensitive K+ fluxes (monitored as 86Rb+) in SH-SY5Y neuroblastoma has been examined. Incubation of SH-SY5Y cells in buffers rendered increasingly hypotonic by a reduction in NaCl concentration resulted in an enhanced basal efflux of Rb+ (threshold of release, 200 mOsM) but had no effect on Rb(+) influx. Addition of the muscarinic cholinergic agonist, oxotremorine-M (Oxo-M), potently enhanced Rb+ efflux (EC50 = 0.45 microM) and increased the threshold of release to 280 mOsM. Oxo-M elicited a similarly potent, but osmolarity-independent, enhancement of Rb+ influx (EC50 = 1.35 microM). However, when incubated under hypotonic conditions in which osmolarity was varied by the addition of sucrose to a fixed concentration of NaCl, basal- and Oxo-M-stimulated Rb+ influx and efflux were demonstrated to be dependent upon osmolarity. Basal- and Oxo-M-stimulated Rb+ influx (but not Rb+ efflux) were inhibited by inclusion of ouabain or furosemide. Both Rb+ influx and efflux were inhibited by removal of intracellular Ca2+ and inhibition of protein kinase C activity. In addition to Oxo-M, agonists acting at other cell surface receptors previously implicated in organic osmolyte release enhanced both Rb+ efflux and influx under hypotonic conditions. Oxo-M had no effect on cellular K+ concentration in SH-SY5Y cells under physiologically relevant reductions in osmolarity (0-15%) unless K+ influx was blocked. Thus, although receptor activation enhances the osmosensitive efflux of K+, it also stimulates K+ influx, and the latter permits retention of K+ by the cells.

Peroxisome proliferator-activated receptor delta activation leads to increased transintestinal cholesterol efflux

PubMed Central

Vrins, Carlos L. J.; van der Velde, Astrid E.; van den Oever, Karin; Levels, Johannes H. M.; Huet, Stephane; Oude Elferink, Ronald P. J.; Kuipers, Folkert; Groen, Albert K.

2009-01-01

Peroxisome proliferator-activated receptor delta (PPARδ) is involved in regulation of energy homeostasis. Activation of PPARδ markedly increases fecal neutral sterol secretion, the last step in reverse cholesterol transport. This phenomenon can neither be explained by increased hepatobiliary cholesterol secretion, nor by reduced cholesterol absorption. To test the hypothesis that PPARδ activation leads to stimulation of transintestinal cholesterol efflux (TICE), we quantified it by intestine perfusions in FVB mice treated with PPARδ agonist GW610742. To exclude the effects on cholesterol absorption, mice were also treated with cholesterol absorption inhibitor ezetimibe or ezetimibe/GW610742. GW601742 treatment had little effect on plasma lipid levels but stimulated both fecal neutral sterol excretion (∼200%) and TICE (∼100%). GW610742 decreased intestinal Npc1l1 expression but had no effect on Abcg5/Abcg8. Interestingly, expression of Rab9 and LIMPII, encoding proteins involved in intracellular cholesterol trafficking, was increased upon PPARδ activation. Although treatment with ezetimibe alone had no effect on TICE, it reduced the effect of GW610742 on TICE. These data show that activation of PPARδ stimulates fecal cholesterol excretion in mice, primarily by the two-fold increase in TICE, indicating that this pathway provides an interesting target for the development of drugs aiming at the prevention of atherosclerosis. PMID:19439761

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. Copyright © 2016 Elsevier B.V. All rights reserved.

Kinetic control of TolC recruitment by multidrug efflux complexes.

PubMed

Tikhonova, Elena B; Dastidar, Vishakha; Rybenkov, Valentin V; Zgurskaya, Helen I

2009-09-22

In Gram-negative pathogens, multidrug efflux pumps that provide clinically significant levels of antibiotic resistance function as three-component complexes. They are composed of the inner membrane transporters belonging to one of three superfamilies of proteins, RND, ABC, or MF; periplasmic proteins belonging to the membrane fusion protein (MFP) family; and outer membrane channels exemplified by the Escherichia coli TolC. The three-component complexes span the entire two-membrane envelope of Gram-negative bacteria and expel toxic molecules from the cytoplasmic membrane to the medium. The architecture of these complexes is expected to vary significantly because of the structural diversity of the inner membrane transporters. How the three-component pumps are assembled, their architecture, and their dynamics remain unclear. In this study, we reconstituted interactions and compared binding kinetics of the E. coli TolC with AcrA, MacA, and EmrA, the periplasmic MFPs that function in multidrug efflux with transporters from the RND, ABC, and MF superfamilies, respectively. By using surface plasmon resonance, we demonstrate that TolC interactions with MFPs are highly dynamic and sensitive to pH. The affinity of TolC to MFPs decreases in the order MacA > EmrA > AcrA. We further show that MFPs are prone to oligomerization, but differ dramatically from each other in oligomerization kinetics and stability of oligomers. The propensity of MFPs to oligomerize correlates with the stability of MFP-TolC complexes and structural features of inner membrane transporters. We propose that recruitment of TolC by various MFPs is determined not only by kinetics of MFP-TolC interactions but also by oligomerization kinetics of MFPs and pH.

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

PubMed

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

2005-12-01

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

Inhibition mechanism of P-glycoprotein mediated efflux by mPEG-PLA and influence of PLA chain length on P-glycoprotein inhibition activity.

PubMed

Li, Wenjing; Li, Xinru; Gao, Yajie; Zhou, Yanxia; Ma, Shujin; Zhao, Yong; Li, Jinwen; Liu, Yan; Wang, Xinglin; Yin, Dongdong

2014-01-06

The present study aimed to investigate the effect of monomethoxy poly(ethylene glycol)-block-poly(D,L-lactic acid) (mPEG-PLA) on the activity of P-glycoprotein (P-gp) in Caco-2 cells and further unravel the relationship between PLA chain length in mPEG-PLA and influence on P-gp efflux and the action mechanism. The transport results of rhodamine 123 (R123) across Caco-2 cell monolayers suggested that mPEG-PLA unimers were responsible for its P-gp inhibitory effect. Furthermore, transport studies of R123 revealed that the inhibitory potential of P-gp efflux by mPEG-PLA analogues was strongly correlated with their structural features and showed that the hydrophilic mPEG-PLA copolymers with an intermediate PLA chain length and 10.20 of hydrophilic-lipophilic balance were more effective at inhibiting P-gp efflux in Caco-2 cells. The fluorescence polarization measurement results ruled out the plasma membrane fluidization as a contributor for inhibition of P-gp by mPEG-PLA. Concurrently, mPEG-PLA inhibited neither basal P-gp ATPase (ATP is adenosine triphosphate) activity nor substrate stimulated P-gp ATPase activity, suggesting that mPEG-PLA seemed not to be a substrate of P-gp and a competitive inhibitor. No evident alteration in P-gp surface level was detected by flow cytometry upon exposure of the cells to mPEG-PLA. The depletion of intracellular ATP, which was likely to be a result of partial inhibition of cellular metabolism, was directly correlated with inhibitory potential for P-gp mediated efflux by mPEG-PLA analogues. Hence, intracellular ATP-depletion appeared to be possible explanation to the inhibition mechanism of P-gp by mPEG-PLA. Taken together, the establishment of a relationship between PLA chain length and impact on P-gp efflux activity and interpretation of action mechanism of mPEG-PLA on P-gp are of fundamental importance and will facilitate future development of mPEG-PLA in the drug delivery area.

Molecular Expression and Functional Activity of Efflux and Influx Transporters in Hypoxia Induced Retinal Pigment Epithelial Cells

PubMed Central

Vadlapatla, Ramya; Vadlapudi, Aswani Dutt; Ponnaluri, VK Chaithanya; Pal, Dhananjay; Mukherji, Mridul; Mitra, Ashim K.

2013-01-01

A decrease in tissue oxygen levels (aka hypoxia) mediates a number of vascular retinal diseases. Despite introduction of novel therapeutics, treatment of retinal disorders remains challenging, possibly due to complex nature of hypoxia signaling. To date, the differential effect of hypoxia on expression of efflux and influx transporters in retinal cells has not been studied. Therefore, the objective of this study was to delineate molecular and functional expression of membrane transporters in human retinal pigment epithelial (RPE) cells cultured under normoxic and hypoxic conditions. Quantitative real time polymerase chain reaction (qPCR), ELISA and immunoblot analysis were performed to examine the RNA and protein expression levels of transporters. Further, functional activity was evaluated by performing the uptake of various substrates in both normoxic and hypoxic conditions. qPCR analysis showed elevated expression of efflux transporters (P-glycoprotein, multidrug resistant protein 2, breast cancer resistant protein) and influx transporters (folate receptor-α, cationic and neutral amino acid transporter, sodium dependent multivitamin transporter) in a time dependent manner. Immunoblot analysis further confirmed elevated expression of breast cancer resistant protein and sodium dependent multivitamin transporter. A decrease in the uptake of efflux transporter substrates (digoxin, lopinavir and abacavir) and enhanced uptake of influx transporter substrates (arginine, folic acid and biotin) in hypoxia relative to normoxia further confirmed elevated expression of transporters, respectively. This study demonstrates for the first time that hypoxic conditions may alter expression of efflux and influx transporters in RPE cells. These findings suggest that hypoxia may further alter disposition of ophthalmic drugs. PMID:23827654

The role of multidrug resistance protein (MRP-1) as an active efflux transporter on blood-brain barrier (BBB) permeability.

PubMed

Lingineni, Karthik; Belekar, Vilas; Tangadpalliwar, Sujit R; Garg, Prabha

2017-05-01

Drugs acting on central nervous system (CNS) may take longer duration to reach the market as these compounds have a higher attrition rate in clinical trials due to the complexity of the brain, side effects, and poor blood-brain barrier (BBB) permeability compared to non-CNS-acting compounds. The roles of active efflux transporters with BBB are still unclear. The aim of the present work was to develop a predictive model for BBB permeability that includes the MRP-1 transporter, which is considered as an active efflux transporter. A support vector machine model was developed for the classification of MRP-1 substrates and non-substrates, which was validated with an external data set and Y-randomization method. An artificial neural network model has been developed to evaluate the role of MRP-1 on BBB permeation. A total of nine descriptors were selected, which included molecular weight, topological polar surface area, ClogP, number of hydrogen bond donors, number of hydrogen bond acceptors, number of rotatable bonds, P-gp, BCRP, and MRP-1 substrate probabilities for model development. We identified 5 molecules that fulfilled all criteria required for passive permeation of BBB, but they all have a low logBB value, which suggested that the molecules were effluxed by the MRP-1 transporter.

CO2 efflux from soils with seasonal water repellency

NASA Astrophysics Data System (ADS)

Urbanek, Emilia; Doerr, Stefan H.

2017-10-01

Soil carbon dioxide (CO2) emissions are strongly dependent on pore water distribution, which in turn can be modified by reduced wettability. Many soils around the world are affected by soil water repellency (SWR), which reduces infiltration and results in diverse moisture distribution. SWR is temporally variable and soils can change from wettable to water-repellent and vice versa throughout the year. Effects of SWR on soil carbon (C) dynamics, and specifically on CO2 efflux, have only been studied in a few laboratory experiments and hence remain poorly understood. Existing studies suggest soil respiration is reduced with increasing severity of SWR, but the responses of soil CO2 efflux to varying water distribution created by SWR are not yet known.Here we report on the first field-based study that tests whether SWR indeed reduces soil CO2 efflux, based on in situ measurements carried out over three consecutive years at a grassland and pine forest sites under the humid temperate climate of the UK.Soil CO2 efflux was indeed very low on occasions when soil exhibited consistently high SWR and low soil moisture following long dry spells. Low CO2 efflux was also observed when SWR was absent, in spring and late autumn when soil temperatures were low, but also in summer when SWR was reduced by frequent rainfall events. The highest CO2 efflux occurred not when soil was wettable, but when SWR, and thus soil moisture, was spatially patchy, a pattern observed for the majority of the measurement period. Patchiness of SWR is likely to have created zones with two different characteristics related to CO2 production and transport. Zones with wettable soil or low persistence of SWR with higher proportion of water-filled pores are expected to provide water with high nutrient concentration resulting in higher microbial activity and CO2 production. Soil zones with high SWR persistence, on the other hand, are dominated by air-filled pores with low microbial activity, but facilitating O2

Efflux proteins at the blood-brain barrier: review and bioinformatics analysis.

PubMed

Saidijam, Massoud; Karimi Dermani, Fatemeh; Sohrabi, Sareh; Patching, Simon G

2018-05-01

1. Efflux proteins at the blood-brain barrier provide a mechanism for export of waste products of normal metabolism from the brain and help to maintain brain homeostasis. They also prevent entry into the brain of a wide range of potentially harmful compounds such as drugs and xenobiotics. 2. Conversely, efflux proteins also hinder delivery of therapeutic drugs to the brain and central nervous system used to treat brain tumours and neurological disorders. For bypassing efflux proteins, a comprehensive understanding of their structures, functions and molecular mechanisms is necessary, along with new strategies and technologies for delivery of drugs across the blood-brain barrier. 3. We review efflux proteins at the blood-brain barrier, classified as either ATP-binding cassette (ABC) transporters (P-gp, BCRP, MRPs) or solute carrier (SLC) transporters (OATP1A2, OATP1A4, OATP1C1, OATP2B1, OAT3, EAATs, PMAT/hENT4 and MATE1). 4. This includes information about substrate and inhibitor specificity, structural organisation and mechanism, membrane localisation, regulation of expression and activity, effects of diseases and conditions and the principal technique used for in vivo analysis of efflux protein activity: positron emission tomography (PET). 5. We also performed analyses of evolutionary relationships, membrane topologies and amino acid compositions of the proteins, and linked these to structure and function.

Polyacylated oligosaccharides from medicinal Mexican morning glory species as antibacterials and inhibitors of multidrug resistance in Staphylococcus aureus.

PubMed

Pereda-Miranda, Rogelio; Kaatz, Glenn W; Gibbons, Simon

2006-03-01

Twenty-two convolvulaceous oligosaccharides selected from the tricolorin (1-7), scammonin (8, 9), and orizabin (10-22) series were evaluated for activity against a panel of Staphylococcus aureus strains possessing or lacking specific efflux pumps. The minimum inhibitory concentrations (MIC values) for most of the amphipatic compounds ranged from 4 to 32 microg/mL against XU-212 (possessing the TetK multidrug efflux pump) and SA-1199B (overexpressing the NorA multidrug efflux pump), compared with 64 and 0.25 microg/mL, respectively, for tetracycline. This activity was shown to be bactericidal. Two microbiologically inactive members of the orizabin series (10, 20) increased norfloxacin susceptibility of strain SA-1199B. At low concentrations, compound 10 was a more potent inhibitor of multidrug pump-mediated EtBr efflux than reserpine. The wide range of antimicrobial activity displayed by these compounds is an example of synergy between related components occurring in the same medicinal crude drug extract, i.e., microbiologically inactive components disabling a resistance mechanism, potentiating the antibiotic properties of the active substances. These results provide an insight into the antimicrobial potential of these complex macrocyclic lactones and open the possibility of using these compounds as starting points for the development of potent inhibitors of S. aureus multidrug efflux pumps.

β3-Adrenoceptor activation upregulates apolipoprotein A-I expression in HepG2 cells, which might further promote cholesterol efflux from macrophage foam cells.

PubMed

Gao, Xia-Qing; Li, Yan-Fang; Jiang, Zhi-Li

2017-01-01

The aim of this study was to explore the effects of β 3 -adrenoceptor (β 3 -AR) activation on HepG2 cells and its influence on cholesterol efflux from macrophage foam cells. HepG2 cells were cultured and treated with the β 3 -AR agonist, BRL37344, and antagonist, SR52390A, and the expression of apolipoprotein (Apo) A-I, ApoA-II, ApoB, and β 3 -AR in the supernatants and cells was determined. The expression of peroxisome proliferator-activated receptor (PPAR) γ and PPARα in the HepG2 cells was also assessed. Next, using the RAW264.7 macrophage foam cell model, we also assessed the influence of the HepG2 cell supernatants on lipid efflux. The cholesterol content of the foam cells was also measured, and the cholesterol efflux from the macrophages was examined by determining 3 H-labeled cholesterol levels. Expression of ATP-binding cassette transporter (ABC) A1 and ABCG1 of the macrophage foam cells was also assessed. β 3 -AR activation increased ApoA-I expression in both the HepG2 cells and the supernatants; PPARγ expression was upregulated, but PPARα expression was not. Treatment with GW9662 abolished the increased expression of ApoA-I induced by the β 3 -AR agonist. The HepG2 cell supernatants decreased the lipid accumulation and increased the cholesterol efflux from the macrophage foam cells. ABCA1 expression, but not ABCG1 expression, increased in the macrophage foam cells treated with BRL37344-treated HepG2 cell supernatants. Activation of β 3 -AR in HepG2 cells upregulates ApoA-I expression, which might further promote cholesterol efflux from macrophage foam cells. PPARγ might be required for the induction of ApoA-I expression.

Inhibition of Efflux Transporter-Mediated Fungicide Resistance in Pyrenophora tritici-repentis by a Derivative of 4′-Hydroxyflavone and Enhancement of Fungicide Activity

PubMed Central

Reimann, Sven; Deising, Holger B.

2005-01-01

Populations of the causal agent of wheat tan spot, Pyrenophora tritici-repentis, that are collected from fields frequently treated with reduced fungicide concentrations have reduced sensitivity to strobilurin fungicides and azole fungicides (C14-demethylase inhibitors). Energy-dependent efflux transporter activity can be induced under field conditions and after in vitro application of sublethal amounts of fungicides. Efflux transporters can mediate cross-resistance to a number of fungicides that belong to different chemical classes and have different modes of action. Resistant isolates can grow on substrata amended with fungicides and can infect plants treated with fungicides at levels above recommended field concentrations. We identified the hydroxyflavone derivative 2-(4-ethoxy-phenyl)-chromen-4-one as a potent inhibitor of energy-dependent fungicide efflux transporters in P. tritici-repentis. Application of this compound in combination with fungicides shifted fungicide-resistant P. tritici-repentis isolates back to normal sensitivity levels and prevented infection of wheat leaves. These results highlight the role of energy-dependent efflux transporters in fungicide resistance and could enable a novel disease management strategy based on the inhibition of fungicide efflux to be developed. PMID:15933029

Transcriptome analysis reveals that multidrug efflux genes are upregulated to protect Pseudomonas aeruginosa from pentachlorophenol stress.

PubMed

Muller, Jocelyn Fraga; Stevens, Ann M; Craig, Johanna; Love, Nancy G

2007-07-01

Through chemical contamination of natural environments, microbial communities are exposed to many different types of chemical stressors; however, research on whole-genome responses to this contaminant stress is limited. This study examined the transcriptome response of a common soil bacterium, Pseudomonas aeruginosa, to the common environmental contaminant pentachlorophenol (PCP). Cells were grown in chemostats at a low growth rate to obtain substrate-limited, steady-state, balanced-growth conditions. The PCP stress was administered as a continuous increase in concentration, and samples taken over time were examined for physiological function changes with whole-cell acetate uptake rates (WAURs) and cell viability and for gene expression changes by Affymetrix GeneChip technology and real-time reverse transcriptase PCR. Cell viability, measured by heterotrophic plate counts, showed a moderately steady decrease after exposure to the stressor, but WAURs did not change in response to PCP. In contrast to the physiological data, the microarray data showed significant changes in the expression of several genes. In particular, genes coding for multidrug efflux pumps, including MexAB-OprM, were strongly upregulated. The upregulation of these efflux pumps protected the cells from the potentially toxic effects of PCP, allowing the physiological whole-cell function to remain constant.

Switch loop flexibility affects substrate transport of the AcrB efflux pump

DOE PAGES

Muller, Reinke T.; Travers, Timothy; Cha, Hi-jea; ...

2017-10-05

The functionally important switch-loop of the trimeric multidrug transporter AcrB separates the access and deep drug binding pockets in every protomer. This loop, comprising 11 amino acid residues, has been shown to be crucial for substrate transport, as drugs have to travel past the loop to reach the deep binding pocket and from there are transported outside the cell via the connected AcrA and TolC channels. It contains four symmetrically arranged glycine residues suggesting that flexibility is a key feature for pump activity. Upon combinatorial substitution of these glycine residues to proline, functional and structural asymmetry was observed. Proline substitutionsmore » on the PC1 proximal side completely abolished transport and reduced backbone flexibility of the switch loop, which adopted a conformation restricting the pathway towards the deep binding pocket. Here, two phenylalanine residues located adjacent to the substitution sensitive glycine residues play a role in blocking the pathway upon rigidification of the loop, since the removal of the phenyl rings from the rigid loop restores drug transport activity.« less

Switch loop flexibility affects substrate transport of the AcrB efflux pump

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muller, Reinke T.; Travers, Timothy; Cha, Hi-jea

The functionally important switch-loop of the trimeric multidrug transporter AcrB separates the access and deep drug binding pockets in every protomer. This loop, comprising 11 amino acid residues, has been shown to be crucial for substrate transport, as drugs have to travel past the loop to reach the deep binding pocket and from there are transported outside the cell via the connected AcrA and TolC channels. It contains four symmetrically arranged glycine residues suggesting that flexibility is a key feature for pump activity. Upon combinatorial substitution of these glycine residues to proline, functional and structural asymmetry was observed. Proline substitutionsmore » on the PC1 proximal side completely abolished transport and reduced backbone flexibility of the switch loop, which adopted a conformation restricting the pathway towards the deep binding pocket. Here, two phenylalanine residues located adjacent to the substitution sensitive glycine residues play a role in blocking the pathway upon rigidification of the loop, since the removal of the phenyl rings from the rigid loop restores drug transport activity.« less

Mutations in MexB that affect the efflux of antibiotics with cytoplasmic targets.

PubMed

Ohene-Agyei, Thelma; Lea, Jon D; Venter, Henrietta

2012-08-01

Drug efflux pumps such as MexAB-OprM from Pseudomonas aeruginosa confer resistance to a wide range of chemically different compounds. Within the tripartite assembly, the inner membrane protein MexB is mainly responsible for substrate recognition. Recently, considerable advances have been made in elucidating the drug efflux pathway through the large periplasmic domains of resistance-nodulation-division (RND) transporters. However, little is known about the role of amino acids in other parts of the protein. We have investigated the role of two conserved phenylalanine residues that are aligned around the cytoplasmic side of the central cavity of MexB. The two conserved phenylalanine residues have been mutated to alanine residues (FAFA MexB). The interaction of the wild-type and mutant proteins with a variety of drugs from different classes was investigated by assays of cytotoxicity and drug transport. The FAFA mutation affected the efflux of compounds that have targets inside the cell, but antibiotics that act on cell wall synthesis and membrane probes were unaffected. Combined, our results indicate the presence of a hitherto unidentified cytoplasmic-binding site in RND drug transporters and enhance our understanding of the molecular mechanisms that govern drug resistance in Gram-negative pathogens. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Population pharmacokinetic modelling of non-linear brain distribution of morphine: influence of active saturable influx and P-glycoprotein mediated efflux

PubMed Central

Groenendaal, D; Freijer, J; de Mik, D; Bouw, M R; Danhof, M; de Lange, E C M

2007-01-01

Background and purpose: Biophase equilibration must be considered to gain insight into the mechanisms underlying the pharmacokinetic-pharmacodynamic (PK-PD) correlations of opioids. The objective was to characterise in a quantitative manner the non-linear distribution kinetics of morphine in brain. Experimental approach: Male rats received a 10-min infusion of 4 mg kg−1 of morphine, combined with a continuous infusion of the P-glycoprotein (Pgp) inhibitor GF120918 or vehicle, or 40 mg kg−1 morphine alone. Unbound extracellular fluid (ECF) concentrations obtained by intracerebral microdialysis and total blood concentrations were analysed using a population modelling approach. Key results: Blood pharmacokinetics of morphine was best described with a three-compartment model and was not influenced by GF120918. Non-linear distribution kinetics in brain ECF was observed with increasing dose. A one compartment distribution model was developed, with separate expressions for passive diffusion, active saturable influx and active efflux by Pgp. The passive diffusion rate constant was 0.0014 min−1. The active efflux rate constant decreased from 0.0195 min−1 to 0.0113 min−1 in the presence of GF120918. The active influx was insensitive to GF120918 and had a maximum transport (Nmax/Vecf) of 0.66 ng min−1 ml−1 and was saturated at low concentrations of morphine (C50=9.9 ng ml−1). Conclusions and implications: Brain distribution of morphine is determined by three factors: limited passive diffusion; active efflux, reduced by 42% by Pgp inhibition; low capacity active uptake. This implies blood concentration-dependency and sensitivity to drug-drug interactions. These factors should be taken into account in further investigations on PK-PD correlations of morphine. PMID:17471182

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

PubMed

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

2011-04-22

Simocyclinone D8 (SD8), a potent DNA gyrase inhibitor made by Streptomyces antibioticus, is exported from the producing organism by the SimX efflux pump. The expression of simX is under the control of SimR, a member of the TetR family of transcriptional regulators. SimR represses simX transcription by binding to operators in the intergenic region between simR and simX. Previously, we have shown that the mature antibiotic SD8 or its biosynthetic intermediate, simocyclinone C4, can dissociate SimR from its operators, leading to derepression of simX and export of SD8 from the cell. This provides a mechanism that couples the biosynthesis of the antibiotic to its export. Here, we report the crystal structures of SimR alone and in complex with either SD8 or simocyclinone C4. The ligand-binding pocket is unusual compared to those of other characterized TetR-family transcriptional regulators: the structures show an extensive ligand-binding pocket spanning both monomers in the functional dimeric unit, with the aminocoumarin moiety of SD8 buried in the protein core, while the angucyclic polyketide moiety is partially exposed to bulk solvent. Through comparisons of the structures, we postulate a derepression mechanism for SimR that invokes rigid-body motions of the subunits relative to one another, coupled with a putative locking mechanism to restrict further conformational change. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

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.

Large-Scale Femtoliter Droplet Array for Single Cell Efflux Assay of Bacteria.

PubMed

Iino, Ryota; Sakakihara, Shouichi; Matsumoto, Yoshimi; Nishino, Kunihiko

2018-01-01

Large-scale femtoliter droplet array as a platform for single cell efflux assay of bacteria is described. Device microfabrication, femtoliter droplet array formation and concomitant enclosure of single bacterial cells, fluorescence-based detection of efflux activity at the single cell level, and collection of single cells from droplet and subsequent gene analysis are described in detail.

Is the Ca2+-ATPase from sarcoplasmic reticulum also a heat pump?

PubMed

Kjelstrup, Signe; de Meis, Leopoldo; Bedeaux, Dick; Simon, Jean-Marc

2008-11-01

We calculate, using the first law of thermodynamics, the membrane heat fluxes during active transport of Ca(2+) in the Ca(2+)-ATPase in leaky and intact vesicles, during ATP hydrolysis or synthesis conditions. The results show that the vesicle interior may cool down during hydrolysis and Ca(2+)-uptake, and heat up during ATP synthesis and Ca(2+)-efflux. The heat flux varies with the SERCA isoform. Electroneutral processes and rapid equilibration of water were assumed. The results are consistent with the second law of thermodynamics for the overall processes. The expression for the heat flux and experimental data, show that important contributions come from the enthalpy of hydrolysis for the medium in question, and from proton transport between the vesicle interior and exterior. The analysis give quantitative support to earlier proposals that certain, but not all, Ca(2+)-ATPases, not only act as Ca(2+)-pumps, but also as heat pumps. It can thus help explain why SERCA 1 type enzymes dominate in tissues where thermal regulation is important, while SERCA 2 type enzymes, with their lower activity and better ability to use the energy from the reaction to pump ions, dominate in tissues where this is not an issue.

Uncoupling of attenuated myo-(3H)inositol uptake and dysfunction in Na(+)-K(+)-ATPase pumping activity in hypergalactosemic cultured bovine lens epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cammarata, P.R.; Tse, D.; Yorio, T.

1991-06-01

Attenuation of both the active transport of myo-inositol and Na(+)-K(+)-ATPase pumping activity has been implicated in the onset of sugar cataract and other diabetic complications in cell culture and animal models of the disease. Cultured bovine lens epithelial cells (BLECs) maintained in galactose-free Eagle's minimal essential medium (MEM) or 40 mM galactose with and without sorbinil for up to 5 days were examined to determine the temporal effects of hypergalactosemia on Na(+)-K(+)-ATPase and myo-inositol uptake. The Na(+)-K(+)-ATPase pumping activity after 5 days of continuous exposure to galactose did not change, as demonstrated by 86Rb uptake. The uptake of myo-(3H)inositol wasmore » lowered after 20 h of incubation in galactose and remained below that of the control throughout the 5-day exposure period. The coadministration of sorbinil to the galactose medium normalized the myo-(3H)inositol uptake. No significant difference in the rates of passive efflux of myo-(3H)inositol or 86Rb from preloaded galactose-treated and control cultures was observed. Culture-media reversal studies were also carried out to determine whether the galactose-induced dysfunction in myo-inositol uptake could be corrected. BLECs were incubated in galactose for 5 days, then changed to galactose-free physiological medium with and without sorbinil for a 1-day recovery period. myo-Inositol uptake was reduced to 34% of control after 6 days of continuous exposure to galactose. Within 24 h of media reversal, myo-inositol uptake returned to or exceeded control values in BLECs switched to either MEM or MEM with sorbinil.2+ reversible and occurred independently of changes in Na(+)-K(+)-ATPase pumping activity in cultured lens epithelium, indicating that the two parameters are not strictly associated and that the deficit in myo-inositol uptake occurs rapidly during hypergalactosemia.« less

Levofloxacin Efflux and smeD in Clinical Isolates of Stenotrophomonas maltophilia.

PubMed

Chong, So Young; Lee, Kyungwon; Chung, Hae-Sun; Hong, Seong Geun; Suh, Younghee; Chong, Yunsop

2017-03-01

Trimethoprim-sulfamethoxazole is the first-line antimicrobial combination for Stenotrophomonas maltophilia infections. However, allergy or intolerance and increasing resistance limit the use of trimethoprim-sulfamethoxazole. Quinolones can be used as an alternative therapeutic option, but resistance can emerge rapidly during therapy. We analyzed the contribution of SmeABC and SmeDEF efflux pumps to levofloxacin resistance in clinical isolates of S. maltophilia. Nonduplicate clinical isolates of S. maltophilia were collected in 2010 from 11 university hospitals (n = 102). Fifty-five levofloxacin nonsusceptible (minimum inhibitory concentration [MIC] ≥4 μg/ml) and 47 susceptible (MIC ≤2 μg/ml) isolates were tested for efflux pump overexpression. Real-time reverse transcription-PCR was performed for amplification and quantification of smeB, smeC, smeD, and smeF mRNA. To determine which antimicrobials were affected by smeD overexpression, the growth rates of a levofloxacin-susceptible S. maltophilia isolate were compared by measuring absorbance of antimicrobial-supplemented Luria-Bertani broth (LB) cultures with or without triclosan. Significant relationships between sme gene overexpression and resistance were observed for smeD against levofloxacin, smeC and smeF against ceftazidime, and smeC against ticarcillin-clavulanate. The mean MICs of moxifloxacin and tigecycline did not significantly differ for isolates with or without overexpression of smeB, smeC, and smeF, but were significantly higher for isolates with smeD overexpression. The mean MICs of amikacin were significantly higher for smeC or smeF overexpressing isolates. Increased growth of a levofloxacin-susceptible isolate was observed in LB with 1/2 MIC levofloxacin in the presence of triclosan. These data suggest that the expression of smeD plays a role in levofloxacin resistance in S. maltophilia.

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

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.

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

Oral and inhaled corticosteroids: Differences in P-glycoprotein (ABCB1) mediated efflux

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crowe, Andrew, E-mail: a.p.crowe@curtin.edu.au; Tan, Ai May

There is concern that P-glycoprotein mediated efflux contributes to steroid resistance. Therefore, this study examined bidirectional corticosteroid transport and induction capabilities for P-glycoprotein (P-gp) to understand which of the systemic and inhaled corticosteroids interacted with P-gp to the greatest extent. Hydrocortisone, prednisolone, prednisone, methylprednisolone, and dexamethasone represented systemically active drugs, while fluticasone propionate, beclomethasone dipropionate, ciclesonide and budesonide represented inhaled corticosteroids. Aldosterone and fludrocortisone represented mineralocorticoids. All drugs were detected using individually optimised HPLC protocols. Transport studies were conducted through Caco-2 monolayers. Hydrocortisone and aldosterone had efflux ratios below 1.5, while prednisone showed a P-gp mediated efflux ratio of onlymore » 1.8 compared to its active drug, prednisolone, with an efflux ratio of 4.5. Dexamethasone and beclomethasone had efflux ratios of 2.1 and 3.3 respectively, while this increased to 5.1 for methylprednisolone. Fluticasone showed an efflux ratio of 2.3. Protein expression studies suggested that all of the inhaled corticosteroids were able to induce P-gp expression, from 1.6 to 2 times control levels. Most of the systemic corticosteroids had higher passive permeability (> 20 × 10{sup −6} cm/s) compared to the inhaled corticosteroids (> 5 × 10{sup −6} cm/s), except for budesonide, with permeability similar to the systemic corticosteroids. Inhaled corticosteroids are not transported by P-gp to the same extent as systemic corticosteroids. However, they are able to induce P-gp production. Thus, inhaled corticosteroids may have greater interactions with other P-gp substrates, but P-gp itself is less likely to influence resistance to the drugs. -- Highlights: ► Inhaled corticosteroids are only weak substrates for P-gp, including budesonide. ► Inhaled corticosteroid potent P-gp inducers especially

Erythrocyte sodium pump activity in bipolar affective disorder and other psychiatric disorders.

PubMed

Hokin-Neaverson, M; Jefferson, J W

1989-01-01

Erythrocyte ouabain-inhibitable sodium pump activity, a measure of NaK-ATPase activity, was studied in 6 diagnostic groups of psychiatric subjects: bipolar affective disorder, unipolar depressive disorder, neurotic depression, chronic alcohol abuse, schizoaffective disorder, and schizophrenia, and in sex- and age-matched normal controls. In the bipolar manic-depressive group, which was restricted to lithium-free subjects, values for sodium pump activity were significantly lower than in the controls (-11.4%, n = 53, p less than 0.001); subgrouping of the bipolar group by sex or age showed a significantly lower sodium pump activity in each of the groups. In the unipolar depressive group, values for sodium pump activity were significantly higher than in the controls (+13.7%, n = 12, p less than 0.01). The difference in direction of changed sodium pump activity between the bipolar and the unipolar groups was also observed in the values for subgroups of subjects in the two categories who were in a depressed state at the time the blood sample was taken. In the chronic alcohol abuse group, values for sodium pump activity were significantly higher than those for the control group (+13.5%, n = 20, p less than 0.05). In the neurotic depression (n = 24), schizoaffective (n = 12), and schizophrenia (n = 35) groups, there were no significant differences in sodium pump activity between the group of psychiatric subjects and their matched controls. These observations indicate that there is a trait-dependent deficiency of NaK-ATPase activity in bipolar affective disorder.

Fractionation of Sodium Efflux in Frog Sartorius Muscles by Strophanthidin and Removal of External Sodium

PubMed Central

Horowicz, P.; Taylor, J. W.; Waggoner, D. M.

1970-01-01

The influence of strophanthidin, ouabain, and the removal of external sodium on the sodium efflux from frog sartorius muscle was measured. In freshly dissected muscles strophanthidin and ouabain in maximally effective concentrations reduced the efflux of sodium by about 50%. Of the sodium efflux which is strophanthidin-insensitive about 75% is inhibited after complete replacement of external sodium by lithium. In the absence of strophanthidin replacement of external sodium by lithium, calcium, or magnesium produces an initial rise in the sodium efflux, followed by a fall in the efflux as the exposure of the muscles to sodium-free media is continued. When the muscles are exposed for prolonged periods in sodium-free media, the fraction of internal sodium lost per minute is higher when returned to normal Ringer fluid than it was initially. The activation of sodium efflux by external sodium after long periods in sodium-free solutions is partly strophanthidin-sensitive and partly strophanthidin-insensitive. The internal sodium concentration is an important factor in these effects. The effects of temperature on the sodium efflux were also measured. Above 7°C the Q 10 of both the strophanthidin-sensitive and strophanthidin-insensitive sodium efflux is about 2.0. Below 7°C the strophanthidin-insensitive sodium efflux has a Q 10 of about 7.4. PMID:5315424

Multidrug efflux transporter, AcrB--the pumping mechanism.

PubMed

Murakami, Satoshi

2008-08-01

Resistance nodulation cell division (RND) transporters are one of the main causes of the bacterial multidrug resistance. They pump a wide range of antibiotics out of the cell by proton motive force. AcrB is the major RND transporter in Escherichia coli. Recently, the crystal structures of AcrB have been determined by different space groups. All these structures are consistent with asymmetric trimer. Each monomer has different conformation corresponding to one of the three functional states of the transport cycle. Transporting hydrophobic drug was bound in the periplasmic domain on one of the three monomers. The transport pathway with alternating access mechanism is located at the hydrophilic domain protruded into the periplasmic space while this mechanism of other transporter families like ATP binding cassette (ABC) and major facilitator superfamily (MFS) transporter is located in the membrane-embedded region. For the RND, protonation might also take place asymmetrically at the functionally important charged residues in the transmembrane (TM) region. The structures indicate that drugs are transported by a three-step functional rotation in which substrates undergo ordered binding change.

Nrf2 activation ameliorates cytotoxic effects of arsenic trioxide in acute promyelocytic leukemia cells through increased glutathione levels and arsenic efflux from cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nishimoto, Shoichi; Suzuki, Toshihiro; Koike, Shin

Carnosic acid (CA), a phenolic diterpene isolated from Rosmarinus officinalis, has been shown to activate nuclear transcription factor E2-related factor 2 (Nrf2), which plays a central role in cytoprotective responses to oxidative and electrophilic stress. Recently, the Nrf2-Kelch ECH associating protein 1 (Keap1) pathway has been associated with cancer drug resistance attributable to modulation of the expression and activation of antioxidant and detoxification enzymes. However, the exact mechanisms by which Nrf2 activation results in chemoresistance are insufficiently understood to date. This study investigated the mechanisms by which the cytotoxic effects of arsenic trioxide (ATO), an anticancer drug, were decreased inmore » acute promyelocytic leukemia cells treated with CA, a typical activator of Nrf2 used to stimulate the Nrf2/Keap1 system. Our findings suggest that arsenic is non-enzymatically incorporated into NB4 cells and forms complexes that are dependent on intracellular glutathione (GSH) concentrations. In addition, the arsenic complexes are recognized as substrates by multidrug resistance proteins and subsequently excreted from the cells. Therefore, Nrf2-associated activation of the GSH biosynthetic pathway, followed by increased levels of intracellular GSH, are key mechanisms underlying accelerated arsenic efflux and attenuation of the cytotoxic effects of ATO. - Highlights: • Nrf2 activation attenuates the effect of arsenic trioxide to acute promyelocytic leukemia cells. • The sensitivity of arsenic trioxide to NB4 cells was dependent on efflux rate of arsenic. • Activation of the GSH biosynthesis is essential in Nrf2-regulated responses for arsenic efflux.« less

Enhancing activity of antibiotics against Staphylococcus aureus: Zanthoxylum capense constituents and derivatives.

PubMed

Cabral, Vanessa; Luo, Xuan; Junqueira, Elisabete; Costa, Sofia S; Mulhovo, Silva; Duarte, Aida; Couto, Isabel; Viveiros, Miguel; Ferreira, Maria-José U

2015-04-15

Six compounds (1-6), isolated from the methanol extract of the roots of the African medicinal plant Zanthoxylum capense Thunb. (Rutaceae), and seven ester derivatives (7-13) were evaluated for their antibacterial activities and modulatory effects on the MIC of antibiotics (erythromycin, oxacillin, and tetracycline) and ethidium bromide (EtBr) against a Staphylococcus aureus reference strain (ATCC 6538). Using the same model, compounds 1-13 were also assessed for their potential as efflux pump inhibitors by a fluorometric assay that measures the accumulation of the broad range efflux pump substrate EtBr. Compounds 8 and 11 were further evaluated for their antibacterial, modulatory and EtBr accumulation effects against four additional S. aureus strains, which included two clinical methicillin-resistant S. aureus (MRSA) strains. Compounds (1-13) have not shown antibacterial activity at the concentration ranges tested. When evaluated against S. aureus ATCC 6538, oxychelerythrine (1) a benzophenanthridine alkaloid, showed the highest modulatory activity enhancing the susceptibility of this strain to all the tested antibiotics from two to four-fold. Ailanthoidiol diacetate (8) and ailanthoidiol di-2-ethylbutanoate (11) were also good modulators when combined with EtBr, increasing the bacteria susceptibility by four and two-fold, respectively. In the EtBr accumulation assay, using ATCC 6538 strain, the phenylpropanoid (+)-ailanthoidiol (6) and most of its ester derivatives (8-11) exhibited higher activity than the positive control verapamil. The highest effects were found for compounds 8 and 11 that also increased the accumulation of EtBr, using S. aureus ATCC 25923 as model. Furthermore, both compounds (8, 11) were able to enhance the ciprofloxacin activity against the MRSA clinical strains tested, causing a reduction of the antibiotic MIC values from two to four-fold. The EtBr accumulation assay revealed that this modulation activity was not due to an inhibition of

Substrate Specificities of MexAB-OprM, MexCD-OprJ, and MexXY-OprM Efflux Pumps in Pseudomonas aeruginosa

PubMed Central

Masuda, Nobuhisa; Sakagawa, Eiko; Ohya, Satoshi; Gotoh, Naomasa; Tsujimoto, Hideto; Nishino, Takeshi

2000-01-01

To find the exact substrate specificities of three species of tripartite efflux systems of Pseudomonas aeruginosa, MexAB-OprM, MexCD-OprJ, and MexXY-OprM, we constructed a series of isogenic mutants, each of which constitutively overproduced one of the three efflux systems and lacked the other two, and their isogenic mutants, which lacked all these systems. Comparison of the susceptibilities of the constructed mutants to 52 antimicrobial agents belonging to various groups suggested the following substrate specificities. All of the efflux systems extrude a wide variety of antimicrobial agent groups, i.e., quinolones, macrolides, tetracyclines, lincomycin, chloramphenicol, most penicillins (all but carbenicillin and sulbenicillin), most cephems (all but cefsulodin and ceftazidime), meropenem, and S-4661, but none of them extrude polymyxin B or imipenem. Extrusion of aminoglycosides is specific to MexXY-OprM, and extrusion of a group of the β-lactams, i.e., carbenicillin, sulbenicillin, ceftazidime, moxalactam, and aztreonam, is specific to MexAB-OprM. Moreover, MexAB-OprM and MexCD-OprJ extrude novobiocin, cefsulodin, and flomoxef, while MexXY-OprM does not. These substrate specificities are distinct from those reported previously. PMID:11083635

Listeria monocytogenes CadC Regulates Cadmium Efflux and Fine-tunes Lipoprotein Localization to Escape the Host Immune Response and Promote Infection.

PubMed

Pombinho, Rita; Camejo, Ana; Vieira, Ana; Reis, Olga; Carvalho, Filipe; Almeida, Maria Teresa; Pinheiro, Jorge Campos; Sousa, Sandra; Cabanes, Didier

2017-05-01

Listeria monocytogenes is a major intracellular human foodborne bacterial pathogen. We previously revealed L. monocytogenes cadC as highly expressed during mouse infection. Here we show that L. monocytogenes CadC is a sequence-specific, DNA-binding and cadmium-dependent regulator of CadA, an efflux pump conferring cadmium resistance. CadC but not CadA is required for L. monocytogenes infection in vivo. Interestingly, CadC also directly represses lspB, a gene encoding a lipoprotein signal peptidase whose expression appears detrimental for infection. lspB overexpression promotes the release of the LpeA lipoprotein to the extracellular medium, inducing tumor necrosis factor α and interleukin 6 expression, thus impairing L. monocytogenes survival in macrophages. We propose that L. monocytogenes uses CadC to repress lspB expression during infection to avoid LpeA exposure to the host immune system, diminishing inflammatory cytokine expression and promoting intramacrophagic survival and virulence. CadC appears as the first metal efflux pump regulator repurposed during infection to fine-tune lipoprotein processing and host responses. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Epigallocatechin gallate as a modulator of Campylobacter resistance to macrolide antibiotics.

PubMed

Kurinčič, Marija; Klančnik, Anja; Smole Možina, Sonja

2012-11-01

Comprehensive therapeutic use of macrolides in humans and animals is important in the selection of macrolide-resistant Campylobacter isolates. This study shows high co-resistance to erythromycin, azithromycin, clarithromycin, dirithromycin and tylosin, with contributions from the 23S rRNA gene and drug efflux systems. The CmeABC efflux pump plays an important role in reduced macrolide susceptibility, accompanied by contributions from the CmeDEF efflux pump and potentially a third efflux pump. To improve clinical performance of licensed antibiotics and chemotherapeutic agents, it is important to understand the factors in Campylobacter that affect susceptibility to macrolide antibiotics. Using mutants that lack the functional genes coding for the CmeB and CmeF efflux pump proteins and the CmeR transcriptional repressor, we show that these efflux pumps are potential targets for the development of therapeutic strategies that use a combination of a macrolide with an efflux pump inhibitor (EPI) to restore macrolide efficacy. The natural phenolic compound epigallocatechin gallate (EGCG) has good modulatory activity over the extrusion across the outer membrane of the macrolides tested, both in sensitive and resistant Campylobacter isolates. Comparing EGCG with known chemical EPIs, correlations in the effects on the particular macrolide antibiotics were seen. EGCG modifies Campylobacter multidrug efflux systems and thus could have an impact on restoring macrolide efficacy in resistant strains. Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Efflux of hydraulically lifted water from mycorrhizal fungal hyphae during imposed drought

PubMed Central

Querejeta, José Ignacio; Allen, Michael F

2008-01-01

Apart from improving plant and soil water status during drought, it has been suggested that hydraulic lift (HL) could enhance plant nutrient capture through the flow of mineral nutrients directly from the soil to plant roots, or by maintaining the functioning of mycorrhizal fungi. We evaluated the extent to which the diel cycle of water availability created by HL covaries with the efflux of HL water from the tips of extramatrical (external) mycorrhizal hyphae, and the possible effects on biogeochemical processes. Phenotypic mycorrhizal fungal variables, such as total and live hyphal lengths, were positively correlated with HL efflux from hyphae, soil water potential (dawn), and plant response variables (foliar 15N). The efflux of HL water from hyphae was also correlated with bacterial abundance and soil enzyme activity (P), and the moistening of soil organic matter. Such findings indicate that the efflux of HL water from the external mycorrhizal mycelia may be a complementary explanation for plant nutrient acquisition and survival during drought. PMID:19704776

Sodium efflux from voltage clamped squid giant axons.

PubMed Central

Landowne, D

1977-01-01

1. The efflux of radioactive sodium was measured from squid axons during simultaneous voltage clamp experiments such that it was possible to determine the efflux of sodium associated with a measured voltage clamp current. 2. The extra efflux of sodium associated with voltage clamp pulses increased linearly with the magnitude of the depolarization above 40 mV. A 100 mV pulse of sufficient duration to produce all of the sodium current increased the rate constant of efflux by about 10(-6). 3. Application of 100 nM tetrodotoxin eliminated the sodium current and the extra efflux of radioactive sodium. 4. Cooling the axon increased the extra efflux/voltage clamp pulse slightly with a Q10 of 1/1-1. On the same axons cooling increased the integral of the sodium current with a Q10 of 1/1-4. 5. Replacing external sodium with Tris, dextrose or Mg-mannitol reduced the extra efflux of sodium by about 50%. The inward sodium current was replaced with an outward current as expected. 6. Replacing external sodium with lithium also reduced the extra efflux by about 50% but the currents seen in lithium were slightly larger than those in sodium. 7. The effect of replacing external sodium was not voltage dependent. Cooling reduced the effect so that there was less reduction of efflux on switching to Tris ASW in the cold than in the warm. 8. The extra efflux of sodium into sodium-free ASW is approximately the same as the integral of the sodium current. Adding external sodium produces a deviation from the independence principle such that there is more exchange of sodium than predicted. Such a deviation from prediction was noted by Hodgkin & Huxley (1952c). 9. Using the equations of Hodgkin & Huxley (1952c) modified to include the deviation from independence reported in this paper and its temperature dependence, one can predict the temperature dependence of the sodium efflux associated with action potentials and obtain much better agreement than is possibly without these phenomena. 10

Potassium efflux fires the canon: Potassium efflux as a common trigger for canonical and noncanonical NLRP3 pathways.

PubMed

Rivers-Auty, Jack; Brough, David

2015-10-01

Murine caspase-11 and its human orthologues, caspase-4 and caspase-5, activate an inflammatory response following cytoplasmic recognition of cell wall constituents from Gram-negative bacteria, such as LPS. This inflammatory response involves pyroptotic cell death and the concomitant release of IL-1α, as well as the production of IL-1β and IL-18 through the noncanonical NLR family, pyrin domain containing 3 (NLRP3) pathway. This commentary discusses three papers in this issue of the European Journal of Immunology that advance our understanding of the roles of caspase-11, -4, and -5 in the noncanonical pathway. By utilizing the new gene editing technique, clustered regularly interspaced short palindromic repeats (CRISPR), as well as sensitive cell imaging techniques, these papers establish that cytoplasmic LPS-dependent IL-1β production requires the NLRP3 inflammasome and that its activation is dependent on K(+) efflux, whereas IL-1α release and pyroptotic cell death pathways are NLRP3-independent. These findings expand on previous research implicating K(+) efflux as the principal trigger for NLRP3 activation and suggest that canonical and noncanonical NLRP3 pathways are not as dissimilar as first thought. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biases of chamber methods for measuring soil CO2 efflux demonstrated with a laboratory apparatus.

Treesearch

S. Mark Nay; Kim G. Mattson; Bernard T. Bormann

1994-01-01

Investigators have historically measured soil CO2 efflux as an indicator of soil microbial and root activity and more recently in calculations of carbon budgets. The most common methods estimate CO2 efflux by placing a chamber over the soil surface and quantifying the amount of CO2 entering the...

PV water pumping: NEOS Corporation recent PV water pumping activities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lane, C.

1995-11-01

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

Off-pump versus on-pump coronary artery bypass surgery in patients with actively treated diabetes and multivessel coronary disease.

PubMed

Benedetto, Umberto; Caputo, Massimo; Vohra, Hunaid; Davies, Alan; Hillier, James; Bryan, Alan; Angelini, Gianni D

2016-11-01

We conducted a single-center analysis on short-term outcomes and long-term survival in actively treated diabetic patients undergoing off-pump coronary artery bypass versus on-pump coronary artery bypass surgery. The final population consisted of 2450 patients with actively treated diabetes (mean age, 66 ± 9 years; female/male 545/1905, 22%). Of those, 1493 subjects were orally treated and 1011 subjects were taking insulin. Off-pump coronary artery bypass and on-pump coronary artery bypass were performed in 1253 and 1197 patients, respectively. Propensity score matching was used to compare the 2 matched groups. When compared with on-pump coronary artery bypass, off-pump coronary artery bypass was associated with a significant risk reduction for postoperative cerebrovascular accident (odds ratio, 0.49; 95% confidence interval [CI], 0.25-0.99; P = .04), need for postoperative intra-aortic balloon pump (odds ratio, 0.48; 95% CI, 0.30-0.77; P = .002), and reexploration for bleeding (odds ratio, 0.55; 95% CI, 0.33-0.94; P = .02). Off-pump coronary artery bypass did not significantly affect early (hazard ratio [HR], 1.32; 95% CI, 0.73-2.40; P = .36) and late (HR, 1.08; 95% CI, 0.92-1.28; P = .32) mortality. However, off-pump coronary artery bypass with incomplete revascularization was associated with reduced survival when compared with off-pump coronary artery bypass with complete revascularization (HR, 1.82; 95% CI, 1.34-2.46; P = .0002) and on-pump coronary artery bypass with complete revascularization (HR, 1.83; 95% CI, 1.36-2.47; P < .0001). Off-pump coronary artery bypass is a safe and feasible option for diabetic patients with multivessel disease, reduces the incidence of early complications including postoperative cerebrovascular events, and provides excellent long-term survival similar to on-pump coronary artery bypass surgery in case of complete revascularization. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.

Mind the gap: non-biological processes contributing to soil CO2 efflux.

PubMed

Rey, Ana

2015-05-01

Widespread recognition of the importance of soil CO2 efflux as a major source of CO2 to the atmosphere has led to active research. A large soil respiration database and recent reviews have compiled data, methods, and current challenges. This study highlights some deficiencies for a proper understanding of soil CO2 efflux focusing on processes of soil CO2 production and transport that have not received enough attention in the current soil respiration literature. It has mostly been assumed that soil CO2 efflux is the result of biological processes (i.e. soil respiration), but recent studies demonstrate that pedochemical and geological processes, such as geothermal and volcanic CO2 degassing, are potentially important in some areas. Besides the microbial decomposition of litter, solar radiation is responsible for photodegradation or photochemical degradation of litter. Diffusion is considered to be the main mechanism of CO2 transport in the soil, but changes in atmospheric pressure and thermal convection may also be important mechanisms driving soil CO2 efflux greater than diffusion under certain conditions. Lateral fluxes of carbon as dissolved organic and inorganic carbon occur and may cause an underestimation of soil CO2 efflux. Traditionally soil CO2 efflux has been measured with accumulation chambers assuming that the main transport mechanism is diffusion. New techniques are available such as improved automated chambers, CO2 concentration profiles and isotopic techniques that may help to elucidate the sources of carbon from soils. We need to develop specific and standardized methods for different CO2 sources to quantify this flux on a global scale. Biogeochemical models should include biological and non-biological CO2 production processes before we can predict the response of soil CO2 efflux to climate change. Improving our understanding of the processes involved in soil CO2 efflux should be a research priority given the importance of this flux in the global

A novel coronary active perfusion system using a conventional intra-aortic balloon pump for off-pump coronary artery bypass grafting.

PubMed

Kiuchi, Ryuta; Tomita, Shigeyuki; Yamaguchi, Shojiro; Nishida, Yuji; Ohtake, Hiroshi; Nakamura, Hiroyuki; Watanabe, Go

2014-07-01

It is important for coronary active perfusion systems to avoid myocardial ischemia during off-pump coronary artery bypass grafting. We have developed a new concept for a perfusion system to pump blood based on changes in helium gas volume. This system uses a conventional intra-aortic balloon pump to activate the perfusion pump. Our study used basic and animal experiments to investigate the most suitable system for coronary perfusion using this new concept. A conventional intra-aortic balloon pump was used to supply power. A device for perfusion was developed with a balloon placed inside a stiff syringe barrel. The device was connected to the helium gas line of the intra-aortic balloon pump. Changes in flow with changes in augmentation level were noted when volumes outside and within the balloon were changed. Six pigs with occlusion of the left anterior descending artery were used for system validation, with monitoring to identify changes in hemodynamics and cardiac enzyme levels. In the basic experiment, an 80-mL outside volume and 3.0-mL inner volume resulted in the greatest percentage change in flow rate with respect to changes in augmentation. In the animal experiment, the new coronary active perfusion system prevented myocardial ischemia during coronary occlusion. We clarified the most suitable method for our new coronary active perfusion system. Using this system, safe anastomosis was consistently performed in animal experiments. Clinically, off-pump coronary artery bypass may potentially be performed more safely and easily using this new system. Copyright © 2014 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.

Enhanced bacterial efflux system is the first step to the development of metronidazole resistance in Helicobacter pylori.

PubMed

Tsugawa, Hitoshi; Suzuki, Hidekazu; Muraoka, Hiroe; Ikeda, Fumiaki; Hirata, Kenro; Matsuzaki, Juntaro; Saito, Yoshimasa; Hibi, Toshifumi

2011-01-14

Although metronidazole (Mtz) is an important component of Helicobacter pylori eradication regimens, it has been pointed out that the increasing use of Mtz may result in increase in the incidence of Mtz-resistant strains. The present study was designed to examine the initial mechanism of resistance acquisition of H. pylori to Mtz. After 10 Mtz-susceptible strains were cultured on plates containing sub-inhibitory concentrations of Mtz, the MIC of Mtz for 9 of the 10 strains increased to levels of the Mtz-resistant strains. In the Mtz-resistance-induced strains, the expression of the TolC efflux pump (hefA) was significantly increased under Mtz exposure, without the reduction of the Mtz-reductive activity. Our finding suggests that overexpression of hefA may be the initial step in the acquisition of Mtz resistance in H. pylori. Copyright © 2010 Elsevier Inc. All rights reserved.

Genomic Analysis of ATP Efflux in Saccharomyces cerevisiae

PubMed Central

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

2015-01-01

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. PMID:26585826

The Effects of Altered Membrane Cholesterol Levels on Sodium Pump Activity in Subclinical Hypothyroidism.

PubMed

Roy, Suparna; Dasgupta, Anindya

2017-03-01

Metabolic dysfunctions characteristic of overt hypothyroidism (OH) start at the early stage of subclinical hypothyroidism (SCH). Na⁺/K⁺-ATPase (the sodium pump) is a transmembrane enzyme that plays a vital role in cellular activities in combination with membrane lipids. We evaluated the effects of early changes in thyroid hormone and membrane cholesterol on sodium pump activity in SCH and OH patients. In 32 SCH patients, 35 OH patients, and 34 euthyroid patients, sodium pump activity and cholesterol levels in red blood cell membranes were measured. Serum thyroxine (T₄) and thyroid stimulating hormone (TSH) levels were measured using enzyme-linked immunosorbent assays. Differences in their mean values were analysed using post hoc analysis of variance. We assessed the dependence of the sodium pump on other metabolites by multiple regression analysis. Sodium pump activity and membrane cholesterol were lower in both hypothyroid groups than in control group, OH group exhibiting lower values than SCH group. In SCH group, sodium pump activity showed a significant direct dependence on membrane cholesterol with an inverse relationship with serum TSH levels. In OH group, sodium pump activity depended directly on membrane cholesterol and serum T₄ levels. No dependence on serum cholesterol was observed in either case. Despite the presence of elevated serum cholesterol in hypothyroidism, membrane cholesterol contributed significantly to maintain sodium pump activity in the cells. A critical reduction in membrane cholesterol levels heralds compromised enzyme activity, even in the early stage of hypothyroidism, and this can be predicted by elevated TSH levels alone, without any evident clinical manifestations. Copyright © 2017 Korean Endocrine Society

Functional implications of an intermeshing cogwheel-like interaction between TolC and MacA in the action of macrolide-specific efflux pump MacAB-TolC.

PubMed

Xu, Yongbin; Song, Saemee; Moeller, Arne; Kim, Nahee; Piao, Shunfu; Sim, Se-Hoon; Kang, Mooseok; Yu, Wookyung; Cho, Hyun-Soo; Chang, Iksoo; Lee, Kangseok; Ha, Nam-Chul

2011-04-15

Macrolide-specific efflux pump MacAB-TolC has been identified in diverse gram-negative bacteria including Escherichia coli. The inner membrane transporter MacB requires the outer membrane factor TolC and the periplasmic adaptor protein MacA to form a functional tripartite complex. In this study, we used a chimeric protein containing the tip region of the TolC α-barrel to investigate the role of the TolC α-barrel tip region with regard to its interaction with MacA. The chimeric protein formed a stable complex with MacA, and the complex formation was abolished by substitution at the functionally essential residues located at the MacA α-helical tip region. Electron microscopic study delineated that this complex was made by tip-to-tip interaction between the tip regions of the α-barrels of TolC and MacA, which correlated well with the TolC and MacA complex calculated by molecular dynamics. Taken together, our results demonstrate that the MacA hexamer interacts with TolC in a tip-to-tip manner, and implies the manner by which MacA induces opening of the TolC channel.

Functional Implications of an Intermeshing Cogwheel-like Interaction between TolC and MacA in the Action of Macrolide-specific Efflux Pump MacAB-TolC*

PubMed Central

Xu, Yongbin; Song, Saemee; Moeller, Arne; Kim, Nahee; Piao, Shunfu; Sim, Se-Hoon; Kang, Mooseok; Yu, Wookyung; Cho, Hyun-Soo; Chang, Iksoo; Lee, Kangseok; Ha, Nam-Chul

2011-01-01

Macrolide-specific efflux pump MacAB-TolC has been identified in diverse Gram-negative bacteria including Escherichia coli. The inner membrane transporter MacB requires the outer membrane factor TolC and the periplasmic adaptor protein MacA to form a functional tripartite complex. In this study, we used a chimeric protein containing the tip region of the TolC α-barrel to investigate the role of the TolC α-barrel tip region with regard to its interaction with MacA. The chimeric protein formed a stable complex with MacA, and the complex formation was abolished by substitution at the functionally essential residues located at the MacA α-helical tip region. Electron microscopic study delineated that this complex was made by tip-to-tip interaction between the tip regions of the α-barrels of TolC and MacA, which correlated well with the TolC and MacA complex calculated by molecular dynamics. Taken together, our results demonstrate that the MacA hexamer interacts with TolC in a tip-to-tip manner, and implies the manner by which MacA induces opening of the TolC channel. PMID:21325274

SatR Is a Repressor of Fluoroquinolone Efflux Pump SatAB

PubMed Central

Escudero, Jose Antonio; San Millan, Alvaro; Montero, Natalia; Gutierrez, Belen; Ovejero, Cristina Martinez; Carrilero, Laura

2013-01-01

Streptococcus suis is an emerging zoonotic agent responsible for high-mortality outbreaks among the human population in China. In this species, the ABC transporter SatAB mediates fluoroquinolone resistance when overexpressed. Here, we describe and characterize satR, an open reading frame (ORF) encoding a MarR superfamily regulator that acts as a repressor of satAB. satR is cotranscribed with satAB, and its interruption entails the overexpression of the pump, leading to a clinically relevant increase in resistance to fluoroquinolones. PMID:23650171

Efflux of creatine kinase from isolated soleus muscle depends on age, sex and type of exercise in mice.

PubMed

Baltusnikas, Juozas; Venckunas, Tomas; Kilikevicius, Audrius; Fokin, Andrej; Ratkevicius, Aivaras

2015-06-01

Elevated plasma creatine kinase (CK) activity is often used as an indicator of exercise-induced muscle damage. Our aim was to study effects of contraction type, sex and age on CK efflux from isolated skeletal muscles of mice. The soleus muscle (SOL) of adult (7.5-month old) female C57BL/6J mice was subjected to either 100 passive stretches, isometric contractions or eccentric contractions, and muscle CK efflux was assessed after two-hour incubation in vitro. SOL of young (3-month old) male and female mice was studied after 100 eccentric contractions. For adult females, muscle CK efflux was larger (p < 0.05) after eccentric contractions than after incubation without exercise (698 ± 344 vs. 268 ± 184 mU·h(-1), respectively), but smaller (p < 0.05) than for young females after the same type of exercise (1069 ± 341 mU·h(-1)). Eccentric exercise-induced CK efflux was larger in muscles of young males compared to young females (2046 ± 317 vs 1069 ± 341 mU · h(-1), respectively, p < 0.001). Our results show that eccentric contractions induce a significant increase in muscle CK efflux immediately after exercise. Isolated muscle resistance to exercise-induced CK efflux depends on age and sex of mice. Key pointsMuscle lengthening contractions induce the highest CK efflux in vitro compared with similar protocol of isometric contractions or passive stretches.Muscle CK efflux in vitro is applicable in studying changes of sarcolemma permeability/integrity, a proxy of muscle damage, in response to muscle contractile activity.Isolated muscle resistance to exercise-induced CK efflux is greater in female compared to male mice of young age and is further increased in adult female mice.

Efflux of Creatine Kinase from Isolated Soleus Muscle Depends on Age, Sex and Type of Exercise in Mice

PubMed Central

Baltusnikas, Juozas; Venckunas, Tomas; Kilikevicius, Audrius; Fokin, Andrej; Ratkevicius, Aivaras

2015-01-01

Elevated plasma creatine kinase (CK) activity is often used as an indicator of exercise-induced muscle damage. Our aim was to study effects of contraction type, sex and age on CK efflux from isolated skeletal muscles of mice. The soleus muscle (SOL) of adult (7.5-month old) female C57BL/6J mice was subjected to either 100 passive stretches, isometric contractions or eccentric contractions, and muscle CK efflux was assessed after two-hour incubation in vitro. SOL of young (3-month old) male and female mice was studied after 100 eccentric contractions. For adult females, muscle CK efflux was larger (p < 0.05) after eccentric contractions than after incubation without exercise (698 ± 344 vs. 268 ± 184 mU·h−1, respectively), but smaller (p < 0.05) than for young females after the same type of exercise (1069 ± 341 mU·h−1). Eccentric exercise-induced CK efflux was larger in muscles of young males compared to young females (2046 ± 317 vs 1069 ± 341 mU · h−1, respectively, p < 0.001). Our results show that eccentric contractions induce a significant increase in muscle CK efflux immediately after exercise. Isolated muscle resistance to exercise-induced CK efflux depends on age and sex of mice. Key points Muscle lengthening contractions induce the highest CK efflux in vitro compared with similar protocol of isometric contractions or passive stretches. Muscle CK efflux in vitro is applicable in studying changes of sarcolemma permeability/integrity, a proxy of muscle damage, in response to muscle contractile activity. Isolated muscle resistance to exercise-induced CK efflux is greater in female compared to male mice of young age and is further increased in adult female mice. PMID:25983588

Regulated efflux of photoreceptor outer segment-derived cholesterol by human RPE cells.

PubMed

Storti, Federica; Raphael, Gabriele; Griesser, Vera; Klee, Katrin; Drawnel, Faye; Willburger, Carolin; Scholz, Rebecca; Langmann, Thomas; von Eckardstein, Arnold; Fingerle, Jürgen; Grimm, Christian; Maugeais, Cyrille

2017-12-01

Genetic studies have linked age-related macular degeneration (AMD) to genes involved in high-density lipoprotein (HDL) metabolism, including ATP-binding cassette transporter A1 (ABCA1). The retinal pigment epithelium (RPE) handles large amounts of lipids, among others cholesterol, partially derived from internalized photoreceptor outer segments (OS) and lipids physiologically accumulate in the aging eye. To analyze the potential function of ABCA1 in the eye, we measured cholesterol efflux, the first step of HDL generation, in RPE cells. We show the expression of selected genes related to HDL metabolism in mouse and human eyecups as well as in ARPE-19 and human primary RPE cells. Immunofluorescence staining revealed localization of ABCA1 on both sides of polarized RPE cells. This was functionally confirmed by directional efflux to apolipoprotein AI (ApoA-I) of 3 H-labeled cholesterol given to the cells via serum or via OS. ABCA1 expression and activity was modulated using a liver-X-receptor (LXR) agonist and an ABCA1 neutralizing antibody, demonstrating that the efflux was ABCA1-dependent. We concluded that the ABCA1-mediated lipid efflux pathway, and hence HDL biosynthesis, is functional in RPE cells towards both the basal (choroidal) and apical (subretinal) space. Impaired activity of the pathway might cause age-related perturbations of lipid homeostasis in the outer retina and thus may contribute to disease development and/or progression. Copyright © 2017 Elsevier Ltd. All rights reserved.

RBE4 cells are highly resistant to paraquat-induced cytotoxicity: studies on uptake and efflux mechanisms.

PubMed

Vilas-Boas, V; Silva, R; Guedes-de-Pinho, P; Carvalho, F; Bastos, M L; Remião, F

2014-09-01

Paraquat (PQ) is a widely used, highly toxic and non-selective contact herbicide, which has been associated with central neurotoxic effects, namely the development of Parkinson's disease, but whose effects to the blood-brain barrier (BBB) itself have rarely been studied. This work studied the mechanisms of PQ uptake and efflux in a rat's BBB cell model, the RBE4 cells. PQ is believed to enter cells using the basic or neutral amino acid or polyamine transport systems or through the choline-uptake system. In contrast, PQ efflux from cells is reported to be mediated by P-glycoprotein. Therefore, we evaluated PQ-induced cytotoxicity and the effect of some substrates/blockers of these transporters (such as arginine, L-valine, putrescine, hemicholinium-3 and GF120918) on such cytotoxicity. RBE4 cells were shown to be extremely resistant to PQ after 24 h of exposure; even at concentrations as high as 50 mM approximately 45% of the cells remained viable. Prolonging exposure until 48 h elicited significant cytotoxicity only for PQ concentrations above 5 mM. Although hemicholinium-3, a choline-uptake system inhibitor, significantly protected cells against PQ-induced toxicity, none of the effects were observed for arginine, L-valine or putrescine. Meanwhile, inhibiting the efflux pump P-glycoprotein using GF120918 significantly enhanced PQ-induced cytotoxicity. In conclusion, PQ used the choline-uptake system, instead of the transporters for the basic or neutral amino acids or for the polyamines, to enter RBE4 cells. P-glycoprotein extrudes PQ back to the extracellular medium. However, this efflux mechanism only partially explains the observed RBE4 resistance to PQ. Copyright © 2013 John Wiley & Sons, Ltd.

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.

A Perspective on Efflux Transport Proteins in the Liver

PubMed Central

Kock, K; Brouwer, K.L.R

2013-01-01

Detailed knowledge regarding the influence of hepatic transport proteins on drug disposition has advanced at a rapid pace over the past decade. Efflux transport proteins located in the basolateral and apical (canalicular) membranes of hepatocytes play an important role in the hepatic elimination of many endogenous and exogenous compounds, including drugs and metabolites. This review focuses on the role of these efflux transporters in hepatic drug excretion. The impact of these proteins as underlying factors for disease is highlighted, and the importance of hepatic efflux proteins in the efficacy and toxicity of drugs is discussed. In addition, a brief overview of methodology to evaluate the function of hepatic efflux transport proteins is provided. Current challenges in predicting the impact of altered efflux protein function on systemic, intestinal and hepatocyte exposure to drugs and metabolites are highlighted. PMID:22948894

Cholesterol efflux is differentially regulated in neurons and astrocytes: implications for brain cholesterol homeostasis

PubMed Central

Chen, Jing; Zhang, Xiaolu; Kusumo, Handojo; Costa, Lucio G.; Guizzetti, Marina

2012-01-01

Disruption of cholesterol homeostasis in the central nervous system (CNS) has been associated with neurological, neurodegenerative, and neurodevelopmental disorders. The CNS is a closed system with regard to cholesterol homeostasis, as cholesterol-delivering lipoproteins from the periphery cannot pass the blood-brain-barrier and enter the brain. Different cell types in the brain have different functions in the regulation of cholesterol homeostasis, with astrocytes producing and releasing apolipoprotein E and lipoproteins, and neurons metabolizing cholesterol to 24(S)-hydroxycholesterol. We present evidence that astrocytes and neurons adopt different mechanisms also in regulating cholesterol efflux. We found that in astrocytes cholesterol efflux is induced by both lipid-free apolipoproteins and lipoproteins, while cholesterol removal from neurons is triggered only by lipoproteins. The main pathway by which apolipoproteins induce cholesterol efflux is through ABCA1. By upregulating ABCA1 levels and by inhibiting its activity and silencing its expression, we show that ABCA1 is involved in cholesterol efflux from astrocytes but not from neurons. Furthermore, our results suggest that ABCG1 is involved in cholesterol efflux to apolipoproteins and lipoproteins from astrocytes but not from neurons, while ABCG4, whose expression is much higher in neurons than astrocytes, is involved in cholesterol efflux from neurons but not astrocytes. These results indicate that different mechanisms regulate cholesterol efflux from neurons and astrocytes, reflecting the different roles that these cell types play in brain cholesterol homeostasis. These results are important in understanding cellular targets of therapeutic drugs under development for the treatments of conditions associated with altered cholesterol homeostasis in the CNS. PMID:23010475

[The Prevalence of Metallo-β-Lactamases and Efflux-Mediated Mechanisms in Carbapenem Nonsusceptible Nosocomial Pseudomonas aeruginosa Isolated in Moscow in 2012-2015].

PubMed

2015-01-01

Pseudomonas aeruginosa, the major nosocomial opportunistic pathogen, is an important cause of infectious morbidity and mortality among immunocompromised patients. To establish the role of metallo-β-lactamases (MBL) and efflux-mediated mechanisms in confer- ring carbapenem resistance in nosocomial isolates of P. aeruginosa. We analyzed carbapenem nonsusceptible nosocomial P. aeruginosa isolates obtained from pediatric and adult patients at three hospitals in Moscow in 2012-2015. Carbapenem susceptibility was assessed using the E-test. In addition, minimal inhibitory concentrations (MICs) of meropenem were tested by the broth microdilution method. The presence of MBL was determined using the ED TA-mediated suppression test. Efflux-dependent resistance was measured using an assay based on MIC modification by an ionophore carbonyl cyanide 3-chlorophenyl hydrazine (CCCP). A total of 54 carbapenem nonsusceptible P. aeruginosa isolates was examined. The presence of an MBL was detected in 37 (69%) isolates, 29 (54%) isolates had efflux-mediated resistance. In 10 (19%) isolates neither MBL nor efflux activity was found. Five out of 6 isolates (83%) with highly active efflux were MBL-positive. Among isolates with low efflux activity, 74% (17/23) possessed MBL, whereas in isolates with no efflux the rate of MBL-positivity was 60% (15/25). The prevalence of MBL- and efflux-mediated carbapenem resistance in nosocomial P. aeruginosa is high. Moreover, our results reveal that several resistance mechanisms may combine at the isolate level. These data may contribute to the development of novel strategies in combating carbapenem resistance.

Striatal norepinephrine efflux in l-DOPA-induced dyskinesia.

PubMed

Ostock, Corinne Y; Bhide, Nirmal; Goldenberg, Adam A; George, Jessica A; Bishop, Christopher

2018-03-01

l-DOPA remains the primary treatment for Parkinson's disease (PD). Unfortunately, its therapeutic benefits are compromised by the development of abnormal involuntary movements (AIMs) known as l-DOPA-induced dyskinesia (LID). The norepinephrine (NE) system originating in the locus coeruleus is profoundly affected in PD and known to influence dopamine (DA) signaling. However, the effect of noradrenergic loss on l-DOPA-induced striatal monoamine efflux and Parkinsonian motor behavior remains controversial and is frequently overlooked in traditional animal models of LID. Thus, the current study sought to determine whether degeneration of the DA and/or NE system(s) altered l-DOPA-induced striatal monoamine efflux in hemiparkinsonian rats with additional NE loss induced by the potent NE-toxin α DA beta hydroxylase (DBH)-saporin. Sham-, DA-, NE-, and dual DA + NE-lesioned rats were treated with l-DOPA (6 mg/kg, s.c.) for 2 weeks. Thereafter, l-DOPA-mediated striatal monoamine efflux was measured with in vivo microdialysis, and concurrent AIMs testing occurred to determine responsiveness to l-DOPA. Noradrenergic lesions exacerbated parkinsonian motor deficits but did not significantly alter LID expression or corresponding l-DOPA-induced striatal monoamine efflux. Interestingly, l-DOPA-induced striatal NE efflux rather than DA efflux, corresponded more closely with dyskinesia severity. Moreover, marked reductions in striatal NE tissue concentration did not appear to impact l-DOPA-induced striatal NE efflux. The current study implicates l-DOPA-induced striatal NE as an important factor in LID expression and demonstrates the importance of developing treatment strategies that co-modulate the NE and DA systems. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

PubMed

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

2007-05-01

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

Inhibition of the Human ABC Efflux Transporters P-gp and ...

EPA Pesticide Factsheets

High body burdens of polybrominated diphenyl ethers (PBDEs) in infants and young children have led to increased concern over their potential impact on human development. PBDE exposure can alter the expression of genes involved in thyroid homeostasis, including those of ATP-binding cassette (ABC) transporters, which mediate cellular xenobiotic efflux. However, little information exists on how PBDEs interact with ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). The purpose of this study was to evaluate the interactions of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) and its hydroxylated metabolite 6-OH-BDE-47 with P-gp and BCRP, using human MDR1- and BCRP-expressing membrane vesicles and stably transfected NIH-3T3-MDR1 and MDCK-BCRP cells. In P-gp membranes, BDE-47 did not affect P-gp activity; however, 6-OH-BDE-47 inhibited P-gp activity at low µM concentrations (IC50 = 11.7 µM). In BCRP membranes, BDE-47 inhibited BCRP activity; however, 6-OH-BDE-47 was a stronger inhibitor [IC50 = 45.9 µM (BDE-47) vs. IC50 = 9.4 µM (6-OH-BDE-47)]. Intracellular concentrations of known P-gp and BCRP substrates [(3H)-paclitaxel and (3H)-prazosin, respectively] were significantly higher (indicating less efflux) in NIH-3T3-MDR1 and MDCK-BCRP cells in the presence of 6-OH-BDE-47, but not BDE-47. Collectively, our results indicate that the BDE-47 metabolite 6-OH-BDE-47 is an inhibitor of both P-gp and BCRP efflux activity.

Evaluation of the first cytostatically active 1-aza-9-oxafluorenes as novel selective CDK1 inhibitors with P-glycoprotein modulating properties.

PubMed

Brachwitz, Kristin; Voigt, Burkhardt; Meijer, Laurent; Lozach, Olivier; Schächtele, Christoph; Molnár, Josef; Hilgeroth, Andreas

2003-02-27

The first series of synthetic 1-aza-9-oxafluorenes with cytostatic activities in the micromolar range was evaluated as cyclin-dependent kinase (CDK1) inhibitors. Activity was found to be selective in comparison to the inhibition of other kinases within the CDK family. Compounds were shown to inhibit the membrane-efflux pump P-glycoprotein responsible for multidrug resistance in cancer cells. First structure-activity relationships are discussed.

Overexpression and deletion of phospholipid transfer protein reduce HDL mass and cholesterol efflux capacity but not macrophage reverse cholesterol transport[S

PubMed Central

Kuwano, Takashi; Bi, Xin; Cipollari, Eleonora; Yasuda, Tomoyuki; Lagor, William R.; Szapary, Hannah J.; Tohyama, Junichiro; Millar, John S.; Billheimer, Jeffrey T.; Lyssenko, Nicholas N.; Rader, Daniel J.

2017-01-01

Phospholipid transfer protein (PLTP) may affect macrophage reverse cholesterol transport (mRCT) through its role in the metabolism of HDL. Ex vivo cholesterol efflux capacity and in vivo mRCT were assessed in PLTP deletion and PLTP overexpression mice. PLTP deletion mice had reduced HDL mass and cholesterol efflux capacity, but unchanged in vivo mRCT. To directly compare the effects of PLTP overexpression and deletion on mRCT, human PLTP was overexpressed in the liver of wild-type animals using an adeno-associated viral (AAV) vector, and control and PLTP deletion animals were injected with AAV-null. PLTP overexpression and deletion reduced plasma HDL mass and cholesterol efflux capacity. Both substantially decreased ABCA1-independent cholesterol efflux, whereas ABCA1-dependent cholesterol efflux remained the same or increased, even though preβ HDL levels were lower. Neither PLTP overexpression nor deletion affected excretion of macrophage-derived radiocholesterol in the in vivo mRCT assay. The ex vivo and in vivo assays were modified to gauge the rate of cholesterol efflux from macrophages to plasma. PLTP activity did not affect this metric. Thus, deviations in PLTP activity from the wild-type level reduce HDL mass and ex vivo cholesterol efflux capacity, but not the rate of macrophage cholesterol efflux to plasma or in vivo mRCT. PMID:28137768

Montelukast is a potent and durable inhibitor of multidrug resistance protein 2 (MRP2)-mediated efflux of taxol and saquinavir

PubMed Central

Roy, Upal; Chakravarty, Geetika; Honer Zu Bentrup, Kerstin; Mondal, Debasis

2009-01-01

The ATP binding cassette (ABC)-transporters are energy dependent efflux pumps which regulate the pharmacokinetics of both anti-cancer chemotherapeutic agents, e.g. taxol, and of HIV-1 protease inhibitors (HPIs), e.g. saquinavir. Increased expression of several ABC-transporters, especially P-gp and MRP2, are observed in multidrug resistant (MDR) tumor cells and on HIV-1 infected lymphocytes. In addition, due to their apical expression on vascular endothelial barriers, both P-gp and MRP2 are of crucial importance towards dictating drug access into sequestered tissues. However, although a number of P-gp inhibitors are currently in clinical trials, possible inhibitors of MRP2 are not being thoroughly investigated. The experimental leukotriene receptor antagonist (LTRA), MK-571 is known to be a potent inhibitor of MRP transporters. Using the MRP2 over-expressing cell line, MDCKII-MRP2, we evaluated whether the clinically approved LTRAs, e.g. montelukast (Singulair™) and zafirlukast (Accolate™), can similarly suppress MRP2-mediated efflux. We compared the efficacy of increasing concentrations (20-100 μM) of MK-571, montelukast, and zafirlukast, in suppressing the efflux of calcein-AM, a fluorescent MRP substrate, and the radiolabeled [3H-] drugs, taxol and saquinavir. Montelukast was the most potent inhibitor (p<0.01) of MRP2-mediated efflux of all three substrates. Montelukast also increased (p<0.01) the duration of intracellular retention of both taxol and saquinavir. More than 50% of the drugs were retained in cells even after 90 mins post removal of montelukast from the medium. Our findings implicate that montelukast, a relatively safe anti-asthmatic agent, may be used as an adjunct therapy to suppress the efflux of taxol and saquinavir from MRP2 overexpressing cells. PMID:19952419

ZmPUMP encodes a fully functional monocot plant uncoupling mitochondrial protein whose affinity to fatty acid is increased with the introduction of a His pair at the second matrix loop

DOE Office of Scientific and Technical Information (OSTI.GOV)

Favaro, Regiane Degan; Borecky, Jiri; Colombi, Debora

Uncoupling proteins (UCPs) are specialized mitochondrial transporter proteins that uncouple respiration from ATP synthesis. In this study, cDNA encoding maize uncoupling protein (ZmPUMP) was expressed in Escherichia coli and recombinant ZmPUMP reconstituted in liposomes. ZmPUMP activity was associated with a linoleic acid (LA)-mediated H{sup +} efflux with K {sub m} of 56.36 {+-} 0.27 {mu}M and V {sub max} of 66.9 {mu}mol H{sup +} min{sup -1} (mg prot){sup -1}. LA-mediated H{sup +} fluxes were sensitive to ATP inhibition with K {sub i} of 2.61 {+-} 0.36 mM (at pH 7.2), a value similar to those for dicot UCPs. ZmPUMP wasmore » also used to investigate the importance of a histidine pair present in the second matrix loop of mammalian UCP1 and absent in plant UCPs. ZmPUMP with introduced His pair (Lys155His and Ala157His) displayed a 1.55-fold increase in LA-affinity while its activity remained unchanged. Our data indicate conserved properties of plant UCPs and suggest an enhancing but not essential role of the histidine pair in proton transport mechanism.« less

Optimization of end-pumped, actively Q-switched quasi-III-level lasers.

PubMed

Jabczynski, Jan K; Gorajek, Lukasz; Kwiatkowski, Jacek; Kaskow, Mateusz; Zendzian, Waldemar

2011-08-15

The new model of end-pumped quasi-III-level laser considering transient pumping processes, ground-state-depletion and up-conversion effects was developed. The model consists of two parts: pumping stage and Q-switched part, which can be separated in a case of active Q-switching regime. For pumping stage the semi-analytical model was developed, enabling the calculations for final occupation of upper laser level for given pump power and duration, spatial profile of pump beam, length and dopant level of gain medium. For quasi-stationary inversion, the optimization procedure of Q-switching regime based on Lagrange multiplier technique was developed. The new approach for optimization of CW regime of quasi-three-level lasers was developed to optimize the Q-switched lasers operating with high repetition rates. Both methods of optimizations enable calculation of optimal absorbance of gain medium and output losses for given pump rate. © 2011 Optical Society of America

CO2 efflux from cleared mangrove peat.

PubMed

Lovelock, Catherine E; Ruess, Roger W; Feller, Ilka C

2011-01-01

CO(2) emissions from cleared mangrove areas may be substantial, increasing the costs of continued losses of these ecosystems, particularly in mangroves that have highly organic soils. We measured CO(2) efflux from mangrove soils that had been cleared for up to 20 years on the islands of Twin Cays, Belize. We also disturbed these cleared peat soils to assess what disturbance of soils after clearing may have on CO(2) efflux. CO(2) efflux from soils declines from time of clearing from ∼10,600 tonnes km(-2) year(-1) in the first year to 3000 tonnes km(2) year(-1) after 20 years since clearing. Disturbing peat leads to short term increases in CO(2) efflux (27 umol m(-2) s(-1)), but this had returned to baseline levels within 2 days. Deforesting mangroves that grow on peat soils results in CO(2) emissions that are comparable to rates estimated for peat collapse in other tropical ecosystems. Preventing deforestation presents an opportunity for countries to benefit from carbon payments for preservation of threatened carbon stocks.

Hypotonic stimulation of the Na+ active transport in frog skeletal muscle: role of the cytoskeleton

PubMed Central

Venosa, R A

2003-01-01

Hypotonicity produces a marked activation of the Na+ pump in frog sartorius muscle. The increase in net Na+ efflux under hypotonic conditions occurs despite the reductions in [Na+]i that are due to fibre swelling and Na+ loss. The pump density (ouabain binding) increases not only upon reduction of the medium osmotic pressure (π) from its normal value (π= 1) to one-half (π= 0.5), but also in muscles that are returned to π= 1 after equilibration in π= 2 medium. The equilibration in π= 2 medium does not affect pump density. Ouabain-binding increments cannot be ascribed to a rise in the Na+–K+ exchange rate of a fixed number of pumps: they also occurred in the continued presence of a saturating concentration of ouabain (50 μm). Under those conditions, the π= 1 →π= 0.5 transfer produced a 43 % increase in pump sites, while the π= 2 →π= 1 transfer induced a rise of 46 %. Actinomycin D did not alter the stimulation of Na+ extrusion elicited by hypotonicity, suggesting that de novo synthesis of pumps was not involved in the increase of the apparent number of pump sites. Disruption of microtubules by colchicine (100 μm) and intermediate filaments by acrylamide (4 mm) did not alter the hypotonic effect. Likewise, genistein (100 μm), a specific inhibitor of tyrosine kinase, did not affect significantly the hypotonic response. Microfilament-disrupting agents like cytochalasin B (5 μm) and latrunculin B (10 μm) reduced the increase in Na+ efflux induced by π= 1 →π= 0.5 transfer by about 35 % and 72 %, respectively. Latrunculin B reduced the increases in pump density generated by π= 1 →π= 0.5 and π= 2 →π= 1 transfers by about 79 % and 91 %, respectively. The results suggest that the membrane stretch due to hypotonic fibre volume increase would promote a microfilament-mediated insertion of submembranous spare Na+ pumps in the sarcolemma and, consequently, the rise in active Na+ transport. PMID:12598593

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.

Characterization of Zebrafish Abcc4 as an Efflux Transporter of Organochlorine Pesticides

PubMed Central

Lu, Xing; Long, Yong; Lin, Li; Sun, Rongze; Zhong, Shan; Cui, Zongbin

2014-01-01

DDT and lindane are highly toxic organochlorine pesticides and posing adverse effects on the environment and public health due to their frequent usage in developing countries. ABCC4/MRP4 is an organic anion transporter that mediates cellular efflux of a wide range of exogenous and endogenous compounds such as cyclic nucleotides and anti-cancer drugs; however, it remains unclear whether ABCC4 and its orthologs function in the detoxification of organochlorine pesticides. Here, we demonstrated the roles of zebrafish Abcc4 in cellular efflux of DDT and lindane. Zebrafish abcc4 was maternally expressed in the oocytes and its transcripts were detected in the lens, pancreas, gills, liver, intestine and bladder of developing embryos and in adult tissues examined. DDT and lindane were able to induce the expression of abcc4 gene and overexpression of Abcc4 significantly decreased the cytotoxicity and accumulation of DDT and lindane in LLC-PK1 cells and developing embryos. In contrast, overexpression of an Abcc4-G1188D mutant abolished its transporter function without effects on its substrate binding activity, and sensitized LLC-PK1 cells and developing embryos to toxic pesticides. Moreover, glutathione (GSH) was involved in the efflux of cellular pesticides and ATPase activity in developing embryos can be induced by DDT or lindane. Thus, zebrafish Abcc4 plays crucial roles in cellular efflux of organochlorine pesticides and can be used a potential molecular marker for the monitor of DDT and lindane contamination in the aquatic environment. PMID:25478949

Amphipathic Polyproline Peptides Stimulate Cholesterol Efflux by the ABCA1 Transporter

PubMed Central

Sviridov, D.O.; Drake, S.K.; Freeman, L.A.; Remaley, A.T.

2016-01-01

ApoA-I mimetics are short synthetic peptides that contain an amphipathic αα-helix and stimulate cholesterol efflux by the ABCA1 transporter in a detergent-like extraction mechanism. We investigated the use of amphipathic peptides with a polypro helix for stimulating cholesterol efflux by ABCA1. Polypro peptides were synthesized with modified prolines, containing either a hydrophobic phenol group (Prop) or a polar N-acetylgalactosamine (Prog) attached to the pyrrolidine ring and were designated as either PP-2, 3, 4, or 5, depending on the number of 3 amino acid repeat units (Prop - Prog - Prop). Based on molecular modeling, these peptides were predicted to be relatively rigid and to bind to a phospholipid bilayer. By CD spectroscopy, PP peptides formed a Type-II polypro helix in an aqueous solution. PP-2 was inactive in promoting cholesterol efflux, but peptides with more than 2 repeat units were active. PP-4 showed a similar Vmax as a much longer amphipathic α-αhelical peptide, containing 37 amino acids, but had a Km that was approximately 20-fold lower. PP peptides were specific in that they did not stimulate cholesterol efflux from cells not expressing ABCA1 and were also non-cytotoxic. Addition of PP-3, 4 and 5 to serum promoted the formation of smaller size HDL species (7 nM) and increased its capacity for ABCA1-dependent cholesterol efflux by approximately 20-35% (p<0.05). Because of their relatively small size and increased potency, amphipathic peptides with a polypro helix may represent an alternative structural motif for the development of apoA-I mimetic peptides. PMID:26879139

Milbemycins: More than Efflux Inhibitors for Fungal Pathogens

PubMed Central

Silva, Luis Vale; Sanguinetti, Maurizio; Vandeputte, Patrick; Torelli, Riccardo; Rochat, Bertrand

2013-01-01

Existing antifungal agents are still confronted to activities limited to specific fungal species and to the development of resistance. Several improvements are possible either by tackling and overcoming resistance or exacerbating the activity of existing antifungal agents. In Candida glabrata, azole resistance is almost exclusively mediated by ABC transporters (including C. glabrata CDR1 [CgCDR1] and CgCDR2) via gain-of-function mutations in the transcriptional activator CgPDR1 or by mitochondrial dysfunctions. We also observed that azole resistance was correlating with increasing virulence and fitness of C. glabrata in animal models of infection. This observation motivated the re-exploitation of ABC transporter inhibitors as a possible therapeutic intervention to decrease not only the development of azole resistance but also to interfere with the virulence of C. glabrata. Milbemycins are known ABC transporter inhibitors, and here we used commercially available milbemycin A3/A4 oxim derivatives to verify this effect. As expected, the derivatives were inhibiting C. glabrata efflux with the highest activity for A3 oxim below 1 μg/ml. More surprising was that oxim derivatives had intrinsic fungicidal activity above 3.2 μg/ml, thus highlighting effects additional to the efflux inhibition. Similar values were obtained with C. albicans. Our data show that the fungicidal activity could be related to reactive oxygen species formation in these species. Transcriptional analysis performed both in C. glabrata and C. albicans exposed to A3 oxim highlighted a core of commonly regulated genes involved in stress responses, including genes involved in oxidoreductive processes, protein ubiquitination, and vesicle trafficking, as well as mitogen-activated protein kinases. However, the transcript profiles contained also species-specific signatures. Following these observations, experimental treatments of invasive infections were performed in mice treated with the commercial A3/A4

Synthesis and P-glycoprotein induction activity of colupulone analogs.

PubMed

Bharate, Jaideep B; Batarseh, Yazan S; Wani, Abubakar; Sharma, Sadhana; Vishwakarma, Ram A; Kaddoumi, Amal; Kumar, Ajay; Bharate, Sandip B

2015-05-21

Brain amyloid-beta (Aβ) plaques are one of the primary hallmarks associated with Alzheimer's disease (AD) pathology. Efflux pump proteins located at the blood-brain barrier (BBB) have been reported to play an important role in the clearance of brain Aβ, among which the P-glycoprotein (P-gp) efflux transporter pump has been shown to play a crucial role. Thus, P-gp has been considered as a potential therapeutic target for treatment of AD. Colupulone, a prenylated phloroglucinol isolated from Humulus lupulus, is known to activate pregnane-X-receptor (PXR), which is a nuclear receptor controlling P-gp expression. In the present work, we aimed to synthesize and identify analogs of colupulone that are potent P-gp inducer(s) with an ability to enhance Aβ transport across the BBB. A series of colupulone analogs were synthesized by modifications at both prenyl as well as acyl domains. All compounds were screened for P-gp induction activity using a rhodamine 123 based efflux assay in the P-gp overexpressing human adenocarcinoma LS-180 cells, wherein all compounds showed significant P-gp induction activity at 5 μM. In the western blot studies in LS-180 cells, compounds 3k and 5f were able to induce P-gp as well as LRP1 at 1 μM. The effect of compounds on the Aβ uptake and transport was then evaluated. Among all tested compounds, diprenylated acyl phloroglucinol displayed a significant increase (29%) in Aβ transport across bEnd3 cells grown on inserts as a BBB model. The results presented here suggest the potential of this scaffold to enhance clearance of brain Aβ across the BBB and thus its promise for development as a potential anti-Alzheimer agent.

Differences in trans-stimulated chloroquine efflux kinetics are linked to PfCRT in Plasmodium falciparum

PubMed Central

Sanchez, Cecilia P.; Rohrbach, Petra; McLean, Jeremy E.; Fidock, David A.; Stein, Wilfred D.; Lanzer, Michael

2010-01-01

Summary The mechanism underpinning chloroquine drug resistance in the human malarial parasite Plasmodium falciparum has remained controversial. Currently discussed models include a carrier or a channel for chloroquine, the former actively expelling the drug, the latter facilitating its passive diffusion, out of the parasite’s food vacuole, where chloroquine accumulates and inhibits haem detoxification. Here we have challenged both models using an established trans-stimulation efflux protocol. While carriers may demonstrate trans-stimulation, channels do not. Our data reveal that extracellular chloroquine stimulates chloroquine efflux in the presence and absence of metabolic energy in both chloroquine-sensitive and -resistant parasites, resulting in a hyperbolic increase in the apparent initial efflux rates as the concentration of external chloroquine increases. In the absence of metabolic energy, the apparent initial efflux rates were comparable in both parasites. Significant differences were only observed in the presence of metabolic energy, where consistently higher apparent initial efflux rates were found in chloroquine-resistant parasites. As trans-stimulation is characteristic of a carrier, and not a channel, we interpret our data in favour of a carrier for chloroquine being present in both chloroquine-sensitive and -resistant parasites, however, with different transport modalities. PMID:17493125

Broadband two-dimensional electronic spectroscopy in an actively phase stabilized pump-probe configuration.

PubMed

Zhu, Weida; Wang, Rui; Zhang, Chunfeng; Wang, Guodong; Liu, Yunlong; Zhao, Wei; Dai, Xingcan; Wang, Xiaoyong; Cerullo, Giulio; Cundiff, Steven; Xiao, Min

2017-09-04

We introduce a novel configuration for two-dimensional electronic spectroscopy (2DES) that combines the partially collinear pump-probe geometry with active phase locking. We demonstrate the method on a solution sample of CdSe/ZnS nanocrystals by employing two non-collinear optical parametric amplifiers as the pump and probe sources. The two collinear pump pulse replicas are created using a Mach-Zehnder interferometer phase stabilized by active feedback electronics. Taking the advantage of separated paths of the two pump pulses in the interferometer, we improve the signal-to-noise ratio with double modulation of the individual pump beams. In addition, a quartz wedge pair manipulates the phase difference between the two pump pulses, enabling the recovery of the rephasing and non-rephasing signals. Our setup integrates many advantages of available 2DES techniques with robust phase stabilization, ultrafast time resolution, two-color operation, long delay scan, individual polarization manipulation and the ease of implementation.

Timescale dependence of environmental controls on methane efflux from Poyang Hu, China

NASA Astrophysics Data System (ADS)

Liu, Lixiang; Xu, Ming; Li, Renqiang; Shao, Rui

2017-04-01

Lakes are an important natural source of CH4 to the atmosphere. However, the multi-seasonal CH4 efflux from lakes has been rarely studied. In this study, the CH4 efflux from Poyang Hu, the largest freshwater lake in China, was measured monthly over a 4-year period by using the floating-chamber technique. The mean annual CH4 efflux throughout the 4 years was 0.54 mmol m-2 day-1, ranging from 0.47 to 0.60 mmol m-2 day-1. The CH4 efflux had a high seasonal variation with an average summer (June to August) efflux of 1.34 mmol m-2 day-1 and winter (December to February) efflux of merely 0.18 mmol m-2 day-1. The efflux showed no apparent diel pattern, although most of the peak effluxes appeared in the late morning, from 10:00 to 12:00 CST (GMT + 8). Multivariate stepwise regression on a seasonal scale showed that environmental factors, such as sediment temperature, sediment total nitrogen content, dissolved oxygen, and total phosphorus content in the water, mainly regulated the CH4 efflux. However, the CH4 efflux only showed a strong positive linear correlation with wind speed within 1 day on a bihourly scale in the multivariate regression analyses but almost no correlation with wind speed on diurnal and seasonal scales.

Isolation and Applications of Prostate Side Population Cells Based on Dye Cycle Violet Efflux

PubMed Central

Gangavarapu, Kalyan J.; Huss, Wendy J.

2011-01-01

This unit describes methods for the digestion of human prostate clinical specimens, dye cycle violet (DCV) staining procedure for the identification, isolation, and quantitation of radiolabeled dihydrotestosterone (DHT) retention of side population cells. The principle of the side population assay is based on differential efflux of DCV, a cell membrane permeable fluorescent dye, by cells with high ATP binding cassette (ABC) transporter activity. Cells with high ABC transporter activity efflux DCV and fall in the lower left quadrant of a flow cytograph are designated as “side population” cells. This unit emphasizes tissue digestion, DCV staining, flow settings for sorting side population cells and quantitation of radiolabeled DHT retention. PMID:21400686

Local administration of a cannabinoid agonist alters norepinephrine efflux in the rat frontal cortex.

PubMed

Page, M E; Oropeza, V C; Van Bockstaele, E J

2008-01-24

Delta(9)-tetrahydrocannabinol, the main psychoactive ingredient in marijuana, activates specific cannabinoid (CB) receptors to exert complex actions on modulatory neurotransmitters involved in attention and cognition. Previous research has demonstrated that systemic administration of the synthetic cannabinoid agonist, WIN 55,212-2, increases norepinephrine efflux in the frontal cortex. The distribution of CB1 receptors on noradrenergic fibers in the frontal cortex suggests this may be one potential site for the regulation of norepinephrine release. In the present study, we first examined the ability of a CB1 antagonist, applied locally in the frontal cortex of adult male Sprague-Dawley rats, to block the actions of systemic WIN 55,212-2. Pretreatment with SR 141716A (300 microM) significantly attenuated the excitatory effects of WIN 55,212-2 (15 mg/kg, i.p.). Next, the impact of direct perfusion of WIN 55,212-2 into the frontal cortex on extracellular norepinephrine efflux was measured. Direct application of WIN 55,212-2 (100 microM) into the frontal cortex elicited a significant increase in extracellular norepinephrine efflux suggesting that activation of cortical cannabinoid receptors contributes to alterations in norepinephrine levels in this brain region. Finally, local administration of SR 141716A followed by local administration of WIN 55,212-2 revealed a paradoxical inhibition of norepinephrine efflux.

The Impact of Diesel Oil Pollution on the Hydrophobicity and CO2 Efflux of Forest Soils.

PubMed

Hewelke, Edyta; Szatyłowicz, Jan; Hewelke, Piotr; Gnatowski, Tomasz; Aghalarov, Rufat

2018-01-01

The contamination of soil with petroleum products is a major environmental problem. Petroleum products are common soil contaminants as a result of human activities, and they are causing substantial changes in the biological (particularly microbiological) processes, chemical composition, structure and physical properties of soil. The main objective of this study was to assess the impact of soil moisture on CO 2 efflux from diesel-contaminated albic podzol soils. Two contamination treatments (3000 and 9000 mg of diesel oil per kg of soil) were prepared for four horizons from two forest study sites with different initial levels of soil water repellency. CO 2 emissions were measured using a portable infrared gas analyser (LCpro+, ADC BioScientific, UK) while the soil samples were drying under laboratory conditions (from saturation to air-dry). The assessment of soil water repellency was performed using the water drop penetration time test. An analysis of variance (ANVOA) was conducted for the CO 2 efflux data. The obtained results show that CO 2 efflux from diesel-contaminated soils is higher than efflux from uncontaminated soils. The initially water-repellent soils were found to have a bigger CO 2 efflux. The non-linear relationship between soil moisture content and CO 2 efflux only existed for the upper soil horizons, while for deeper soil horizons, the efflux is practically independent of soil moisture content. The contamination of soil by diesel leads to increased soil water repellency.

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

Identification and prevalence of RND family multidrug efflux pump oqxAB genes in Enterococci isolates from swine manure in China.

PubMed

Yuan, Li; Zhai, Ya-Jun; Wu, Hua; Sun, Hua-Run; He, Zhi-Pei; Wang, Ya-Bin; Pan, Yu-Shan; Kuang, Nan-Nan; Hu, Gong-Zheng

2018-06-01

The resistance/nodulation/cell division (RND) family multidrug efflux pump, OqxAB, has been identified as one of the leading mechanisms of plasmid-mediated quinolone resistance and has become increasingly prevalent among Enterobacteriaceae in recent years. However, oqxAB genes have not yet been reported in Enterococcus isolates. The aim of the present study was to identify the oqxAB genes and investigate their prevalence among Enterococcus from swine manure in China. The oqxAB genes were screened in 87 Enterococcus isolates by PCR. The transferability of the oqxAB genes in Enterococcus was determined by conjugation experiments. The genetic environment of oqxAB genes was investigated by cloning experiments, PCR mapping and sequencing. A high prevalence (86.2 %) of olaquindox resistance was observed in Enterococcus and 98.9 % isolates exhibited multidrug-resistance phenotypes. The occurrence of oqxA and oqxB in Enterococcus was also high (79.3 and 65.5 %, respectively). Sequence analysis of the cloned fragment indicated that the oqxAB cassette was linked to an incomplete Tn5 transposon containing aph(3')-IIa and flanked by IS26 [IS26-oqxAB-IS26-aph(3')-IIa]. The oqxAB-aph(3')-IIa-positive transconjugant or transformant showed resistance or reduced susceptibility to enrofloxacin, ciprofloxacin, olaquindox, mequindox, florfenicol, neomycin and kanamycin. This is the first time that the oqxAB genes have been identified in Enterococcus faecalis from swine manure. The genetic linkage of oqxAB-aph(3')-IIa in Enterococcus has not been described before. The high prevalence of oqxAB genes in Enterococcus suggests that it may constitute a reservoir for oqxAB genes and pose a potential threat to public health.

CO2 Efflux from Cleared Mangrove Peat

PubMed Central

Lovelock, Catherine E.; Ruess, Roger W.; Feller, Ilka C.

2011-01-01

Background CO2 emissions from cleared mangrove areas may be substantial, increasing the costs of continued losses of these ecosystems, particularly in mangroves that have highly organic soils. Methodology/Principal Findings We measured CO2 efflux from mangrove soils that had been cleared for up to 20 years on the islands of Twin Cays, Belize. We also disturbed these cleared peat soils to assess what disturbance of soils after clearing may have on CO2 efflux. CO2 efflux from soils declines from time of clearing from ∼10 600 tonnes km−2 year−1 in the first year to 3000 tonnes km2 year−1 after 20 years since clearing. Disturbing peat leads to short term increases in CO2 efflux (27 umol m−2 s−1), but this had returned to baseline levels within 2 days. Conclusions/Significance Deforesting mangroves that grow on peat soils results in CO2 emissions that are comparable to rates estimated for peat collapse in other tropical ecosystems. Preventing deforestation presents an opportunity for countries to benefit from carbon payments for preservation of threatened carbon stocks. PMID:21738628

ATP binding cassette G1-dependent cholesterol efflux during inflammation.

PubMed

de Beer, Maria C; Ji, Ailing; Jahangiri, Anisa; Vaughan, Ashley M; de Beer, Frederick C; van der Westhuyzen, Deneys R; Webb, Nancy R

2011-02-01

ATP binding cassette transporter G1 (ABCG1) mediates the transport of cellular cholesterol to HDL, and it plays a key role in maintaining macrophage cholesterol homeostasis. During inflammation, HDL undergoes substantial remodeling, acquiring lipid changes and serum amyloid A (SAA) as a major apolipoprotein. In the current study, we investigated whether remodeling of HDL that occurs during acute inflammation impacts ABCG1-dependent efflux. Our data indicate that lipid free SAA acts similarly to apolipoprotein A-I (apoA-I) in mediating sequential efflux from ABCA1 and ABCG1. Compared with normal mouse HDL, acute phase (AP) mouse HDL containing SAA exhibited a modest but significant 17% increase in ABCG1-dependent efflux. Interestingly, AP HDL isolated from mice lacking SAA (SAAKO mice) was even more effective in promoting ABCG1 efflux. Hydrolysis with Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) significantly reduced the ability of AP HDL from SAAKO mice to serve as a substrate for ABCG1-mediated cholesterol transfer, indicating that phospholipid (PL) enrichment, and not the presence of SAA, is responsible for alterations in efflux. AP human HDL, which is not PL-enriched, was somewhat less effective in mediating ABCG1-dependent efflux compared with normal human HDL. Our data indicate that inflammatory remodeling of HDL impacts ABCG1-dependent efflux independent of SAA.

Contribution of the biological crust to the soil CO2 efflux in a Mediterranean ecosystem

NASA Astrophysics Data System (ADS)

Morillas, Lourdes; Bellucco, Veronica; Lo Cascio, Mauro; Marras, Serena; Spano, Donatella; Mereu, Simone

2016-04-01

Lately, the important role of the soil biological crust (hereafter biocrust) in Mediterranean ecosystems is emerging from a multitude of articles. It is becoming apparent that the biocrust has an important role in regulating ecosystem functions and that it interacts with the woody and herbaceous vegetation to a degree depending on the availability of water among other factors. Here we present the first results of a wider project and focus on the contribution of the biocrust to soil CO2 efflux, and on how the respiration of the biocrust responds to soil water content and temperature. A manipulative experiment was performed in a Mediterranean shrubland ecosystem in Sardinia (Italy) to assess the contribution of the bicocrust to soil CO2 efflux and to identify the main environmental drivers of the CO2 efflux in this ecosystem. For 19 months,in situ soil CO2 efflux was measured over three different surfaces: soil deprived of biocrust (hereafter Soil), biocrust (hereafter BC) and intact soil (hereafter Soil+BC). For these surfaces, three different approaches were used to investigate the dependency of CO2 efflux on soil temperature and soil water content, e.g. a simple linear regression, a multi-linear equation, and a modified version of the most common used Lloyd and Taylor model (Lloyd and Taylor, 1994). Results showed that CO2 effluxes emitted by Soil, BC and Soil+BC were differently driven by soil moisture and temperature: BC respiration was mainly controlled by soil moisture at 5 cm depth, whereas both soil temperature and water content at 20 cm depth determined Soil CO2 efflux. Soil temperature and water content at 5 cm depth drove Soil+BC respiration. We also found that biocrust can contribute substantially (up to 60%) to the total soil respiration depending on its moisture content. This contribution persists even in periods in which deeper soil layers are inactive, as small water pulses can activate lichens, mosses and cyanobacteria associated to the biocrust as

TOR complex 1 regulates the yeast plasma membrane proton pump and pH and potassium homeostasis.

PubMed

Mahmoud, Shima; Planes, María Dolores; Cabedo, Marc; Trujillo, Cristina; Rienzo, Alessandro; Caballero-Molada, Marcos; Sharma, Sukesh C; Montesinos, Consuelo; Mulet, José Miguel; Serrano, Ramón

2017-07-01

We have identified in yeast a connection between two master regulators of cell growth: a biochemical connection involving the TORC1 protein kinase (which activates protein synthesis, nutrient uptake, and anabolism) and a biophysical connection involving the plasma membrane proton-pumping H + -ATPase Pma1 (which drives nutrient and K + uptake and regulates pH homeostasis). Raising the temperature to nonpermissive values in a TOR thermosensitive mutant decreases Pma1 activity. Rapamycin, a TORC1 inhibitor, inhibits Pma1 dependent on its receptor Fpr1 and on the protein phosphatase Sit4, a TORC1 effector. Mutation of either Sit4 or Tco89, a nonessential subunit of TORC1, decreases proton efflux, K + uptake, intracellular pH, cell growth, and tolerance to weak organic acids. Tco89 does not affect Pma1 activity but activates K + transport. © 2017 Federation of European Biochemical Societies.

Polyamines Interact with Hydroxyl Radicals in Activating Ca2+ and K+ Transport across the Root Epidermal Plasma Membranes1[W

PubMed Central

Zepeda-Jazo, Isaac; Velarde-Buendía, Ana María; Enríquez-Figueroa, René; Bose, Jayakumar; Shabala, Sergey; Muñiz-Murguía, Jesús; Pottosin, Igor I.

2011-01-01

Reactive oxygen species (ROS) are integral components of the plant adaptive responses to environment. Importantly, ROS affect the intracellular Ca2+ dynamics by activating a range of nonselective Ca2+-permeable channels in plasma membrane (PM). Using patch-clamp and noninvasive microelectrode ion flux measuring techniques, we have characterized ionic currents and net K+ and Ca2+ fluxes induced by hydroxyl radicals (OH•) in pea (Pisum sativum) roots. OH•, but not hydrogen peroxide, activated a rapid Ca2+ efflux and a more slowly developing net Ca2+ influx concurrent with a net K+ efflux. In isolated protoplasts, OH• evoked a nonselective current, with a time course and a steady-state magnitude similar to those for a K+ efflux in intact roots. This current displayed a low ionic selectivity and was permeable to Ca2+. Active OH•-induced Ca2+ efflux in roots was suppressed by the PM Ca2+ pump inhibitors eosine yellow and erythrosine B. The cation channel blockers gadolinium, nifedipine, and verapamil and the anionic channel blockers 5-nitro-2(3-phenylpropylamino)-benzoate and niflumate inhibited OH•-induced ionic currents in root protoplasts and K+ efflux and Ca2+ influx in roots. Contrary to expectations, polyamines (PAs) did not inhibit the OH•-induced cation fluxes. The net OH•-induced Ca2+ efflux was largely prolonged in the presence of spermine, and all PAs tested (spermine, spermidine, and putrescine) accelerated and augmented the OH•-induced net K+ efflux from roots. The latter effect was also observed in patch-clamp experiments on root protoplasts. We conclude that PAs interact with ROS to alter intracellular Ca2+ homeostasis by modulating both Ca2+ influx and efflux transport systems at the root cell PM. PMID:21980172

B16-BL6 melanoma cells release inhibitory factor(s) of active pump activity in isolated lymph vessels.

PubMed

Nakaya, K; Mizuno, R; Ohhashi, T

2001-12-01

We investigated whether supernatant cultured with melanoma cell lines B16-BL6 and K1735 or the Lewis lung carcinoma cell line (LLC) can regulate lymphatic pump activity with bioassay preparations isolated from murine iliac lymph vessels. B16-BL6 and LLC supernatants caused significant dilation of lymph microvessels with cessation of pump activity. B16-BL6 supernatant produced dose-related cessation of lymphatic pump activity. There was no significant tachyphylaxis in the supernatant-mediated inhibitory response of lymphatic pump activity. Pretreatment with 3 x 10(-5) M N(omega)-nitro-L-arginine methyl ester (L-NAME) or 10(-7) M or 10(-6) M glibenclamide and 5 x 10(-4) M 5-hydroxydecanoic acid caused significant reduction of supernatant-mediated inhibitory responses. Simultaneous treatment with 10(-3) M L-arginine and 3 x 10(-5) M L-NAME significantly lessened L-NAME-induced inhibition of the supernatant-mediated response, suggesting that endogenous nitric oxide (NO) plays important roles in supernatant-mediated inhibitory responses. Chemical treatment dialyzed substances of <1,000 molecular weight (MW), producing complete reduction of the supernatant-mediated response. In contrast, pretreatment with heating or digestion with protease had no significant effect on supernatant-mediated response. These findings suggest that B16-BL6 cells may release nonpeptide substance(s) of <1,000 MW, resulting in significant cessation of lymphatic pump activity via production and release of endogenous NO and activation of mitochondrial ATP-sensitive K(+) channels.

Leiurus quinquestriatus venom inhibits BRL 34915-induced /sup 86/Rb/sup +/ efflux from the rat portal vein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Quast, U.; Cook, N.S.

1988-01-01

The effect of the crude venom of the Israeli scorpion Leiurus quinquestriatus hebraeus on the /sup 86/Rb/sup +/ efflux stimulated by the K/sup +/ channel opener BRL 34915 in the rat portal vein was examined. Applied alone, the venom greatly increased the spontaneous mechanical activity of and the concomitant /sup 86/Rb/sup +/ efflux from the vessel. When the excitability of the vein was suppressed by the dihydropyridine calcium antagonist, PN 200-110, the /sup 86/Rb/sup +/ efflux stimulated by BRL 34915 could be shown to be inhibited by the venom. From the concentration dependence of this inhibition an IC/sub 50/ valuemore » of 0.17 +/- 0.01 mg/ml was estimated. This venom is thus the most potent blocker of BRL 34915-evoked /sup 86/Rb/sup +/ efflux reported so far. 17 references, 2 figures.« less

Vertical profile of branch CO2 efflux in a Norway spruce tree: a case study

NASA Astrophysics Data System (ADS)

Acosta, M.; Pavelka, M.

2012-04-01

Despite woody-tissue CO2 effluxes having been recognized as an important component of forest carbon budget due to the fraction of assimilates used and the dramatic increase in woody with stand development, there is limited research to determine the CO2 efflux vertical variability of woody-tissue components. For a better understanding and quantification of branch woody-tissue CO2 efflux in forest ecosystems, it is necessary to identify the environmental factors influencing it and the role of the branch distribution within the canopy. The proper assessment of this forest component will improve the knowledge of the ratio between ecosystem respiration and gross primary production at forest ecosystem. In order to achieve this goal, branch CO2 efflux of Norway spruce tree was measured in ten branches at five different whorls during the growing season 2004 (from June till October) in campaigns of 3-4 times per month at the Beskydy Mts., the Czech Republic, using a portable infrared gas analyzer operating as a closed system. Branch woody tissue temperature was measured continuously in ten minutes intervals for each sample position during the whole experiment period. On the basis of relation between CO2 efflux rate and woody tissue temperature a value of Q10 and normalized CO2 efflux rate (E10 - CO2 efflux rate at 10° C) were calculated for each sampled position. Estimated Q10 values ranged from 2.12 to 2.89 and E10 ranged from 0.41 to 1.19 ?molCO2m-2 s-1. Differences in branch CO2 efflux were found between orientations; East side branches presented higher efflux rate than west side branches. The highest branch CO2 efflux rate values were measured in August and the lowest in October, which were connected with woody tissue temperature and ontogenetic processes during these periods. Branch CO2 efflux was significantly and positively correlated with branch position within canopy and woody tissue temperature. Branches from the upper whorls showed higher respiration activity

Arctigenin promotes cholesterol efflux from THP-1 macrophages through PPAR-γ/LXR-α signaling pathway.

PubMed

Xu, Xiaolin; Li, Qian; Pang, Liewen; Huang, Guoqian; Huang, Jiechun; Shi, Meng; Sun, Xiaotian; Wang, Yiqing

2013-11-15

Cholesterol efflux from macrophages is a critical mechanism to prevent the development of atherosclerosis. Here, we sought to investigate the effects of arctigenin, a bioactive component of Arctium lappa, on the cholesterol efflux in oxidized low-density lipoprotein (oxLDL)-loaded THP-1 macrophages. Our data showed that arctigenin significantly accelerated apolipoprotein A-I- and high-density lipoprotein-induced cholesterol efflux in both dose- and time-dependent manners. Moreover, arctigenin treatment enhanced the expression of ATP binding cassette transporter A1 (ABCA1), ABCG1, and apoE, all of which are key molecules in the initial step of cholesterol efflux, at both mRNA and protein levels. Arctigenin also caused a concentration-dependent elevation in the expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) and liver X receptor-alpha (LXR-α). The arctigenin-mediated induction of ABCA1, ABCG1, and apoE was abolished by specific inhibition of PPAR-γ or LXR-α using small interfering RNA technology. Our results collectively indicate that arctigenin promotes cholesterol efflux in oxLDL-loaded THP-1 macrophages through upregulation of ABCA1, ABCG1 and apoE, which is dependent on the enhanced expression of PPAR-γ and LXR-α. Copyright © 2013 Elsevier Inc. All rights reserved.

Embryogenic competence acquisition in sugarcane callus is associated with differential H+ pump abundance and activity.

PubMed

Passamani, Lucas Z; Bertolazi, Amanda A; Ramos, Alessandro C; Santa-Catarina, Claudete; Thelen, Jay J; Silveira, Vanildo

2018-06-22

Somatic embryogenesis is an important biological process in several plant species, including sugarcane. Proteomics approaches have shown that H + pumps are differentially regulated during somatic embryogenesis; however, the relationship between H + flux and embryogenic competence is still unclear. This work aimed to elucidate the association between extracellular H + flux and somatic embryo maturation in sugarcane. We performed a microsomal proteomics analysis and analyzed changes in extracellular H + flux and H + pump (P-H + -ATPase, V-H + -ATPase and H + -PPase) activity in embryogenic and non-embryogenic callus. A total of 657 proteins were identified, 16 of which were H + pumps. We observed that P-H + -ATPase and H + -PPase were more abundant in embryogenic callus. Compared with non-embryogenic callus, embryogenic callus showed higher H + influx, especially on maturation day 14, as well as higher H+ pump activity (mainly P-H+-ATPase and H+-PPase activity). H+-PPase appears to be the major H + pump in embryogenic callus during somatic embryo formation, functioning in both vacuole acidification and PPi homeostasis. These results provide evidence for an association between higher H + pump protein abundance and, consequently, higher H + flux and embryogenic competence acquisition in the callus of sugarcane, allowing for optimization of the somatic embryo conversion process by modulating the activities of these H + pumps.

Ventral tegmental ionotropic glutamate receptor stimulation of nucleus accumbens tonic dopamine efflux blunts hindbrain-evoked phasic neurotransmission: implications for dopamine dysregulation disorders.

PubMed

Tye, S J; Miller, A D; Blaha, C D

2013-11-12

Activation of glutamate receptors within the ventral tegmental area (VTA) stimulates extrasynaptic (basal) dopamine release in terminal regions, including the nucleus accumbens (NAc). Hindbrain inputs from the laterodorsal tegmental nucleus (LDT) are critical for elicitation of phasic VTA dopamine cell activity and consequent transient dopamine release. This study investigated the role of VTA ionotropic glutamate receptor (iGluR) stimulation on both basal and LDT electrical stimulation-evoked dopamine efflux in the NAc using in vivo chronoamperometry and fixed potential amperometry in combination with stearate-graphite paste and carbon fiber electrodes, respectively. Intra-VTA infusion of the iGluR agonists (±)-α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA; 1 μg/μl) or N-methyl-d-aspartic acid (NMDA; 2 μg/μl) enhanced basal NAc dopamine efflux. This iGluR-mediated potentiation of basal dopamine efflux was paralleled by an attenuation of LDT-evoked transient NAc dopamine efflux, suggesting that excitation of basal activity effectively inhibited the capacity of hindbrain afferents to elicit transient dopamine efflux. In line with this, post-NMDA infusion of the dopamine D2 autoreceptor (D2R) agonist quinpirole (1 μg/μl; intra-VTA) partially recovered NMDA-mediated attenuation of LDT-evoked NAc dopamine, while concurrently attenuating NMDA-mediated potentiation of basal dopamine efflux. Post-NMDA infusion of quinpirole (1 μg/μl) alone attenuated basal and LDT-evoked dopamine efflux. Taken together, these data reveal that hyperstimulation of basal dopamine transmission can stunt hindbrain burst-like stimulation-evoked dopamine efflux. Inhibitory autoreceptor mechanisms within the VTA help to partially recover the magnitude of phasic dopamine efflux, highlighting the importance of both iGluRs and D2 autoreceptors in maintaining the functional balance of tonic and phasic dopamine neurotransmission. Dysregulation of this balance may have important

Alternative to antibiotics against Pseudomonas aeruginosa: Effects of Glycyrrhiza glabra on membrane permeability and inhibition of efflux activity and biofilm formation in Pseudomonas aeruginosa and its in vitro time-kill activity.

PubMed

Chakotiya, Ankita Singh; Tanwar, Ankit; Narula, Alka; Sharma, Rakesh Kumar

2016-09-01

The multi-drug resistance offered by Pseudomonas aeruginosa to antibiotics can be attributed towards its propensity to develop biofilm, modification in cell membrane and to efflux antibacterial drugs. The present study explored the activity of Glycyrrhiza glabra and one of its pure compounds, glycyrrhizic acid against P. aeruginosa and their mechanism of action in terms of the effect on membrane permeability, efflux activity, and biofilm formation were determined. Minimum inhibitory concentrations were determined by using broth dilution technique. The minimum bactericidal concentrations were assessed on agar plate. The MIC of the extract and glycyrrhizic acid was found to be 200 and 100 μg ml(-1), respectively. The MBC was found to be 800 and 400 μg ml(-1) in the case of extract and glycyrrhizic acid, respectively. Time -dependent killing efficacy was also estimated. Flowcytometric analysis with staining methods was used to determine the effect of extract and glycyrrhizic acid at 2 × MIC on different physiological parameters and compared it with the standard (antibiotic). The growth of P. aeruginosa was significantly inhibited by extract and the pure compound. The herbal extract and the glycyrrhic acid were also found to effective in targeting the physiological parameters of the bacteria that involve cell membrane permeabilization, efflux activity, and biofilm formation. This study reports the antipseudomonal action of Glycyrrhiza glabra and one of its compound and provides insight into their mode of action. Copyright © 2016 Elsevier Ltd. All rights reserved.

CC-Chemokine Ligand 2 (CCL2) Suppresses High Density Lipoprotein (HDL) Internalization and Cholesterol Efflux via CC-Chemokine Receptor 2 (CCR2) Induction and p42/44 Mitogen-activated Protein Kinase (MAPK) Activation in Human Endothelial Cells *

PubMed Central

Sun, Run-Lu; Huang, Can-Xia; Bao, Jin-Lan; Jiang, Jie-Yu; Zhang, Bo; Zhou, Shu-Xian; Cai, Wei-Bin; Wang, Hong; Wang, Jing-Feng; Zhang, Yu-Ling

2016-01-01

High density lipoprotein (HDL) has been proposed to be internalized and to promote reverse cholesterol transport in endothelial cells (ECs). However, the mechanism underlying these processes has not been studied. In this study, we aim to characterize HDL internalization and cholesterol efflux in ECs and regulatory mechanisms. We found mature HDL particles were reduced in patients with coronary artery disease (CAD), which was associated with an increase in CC-chemokine ligand 2 (CCL2). In cultured primary human coronary artery endothelial cells and human umbilical vein endothelial cells, we determined that CCL2 suppressed the binding (4 °C) and association (37 °C) of HDL to/with ECs and HDL cellular internalization. Furthermore, CCL2 inhibited [3H]cholesterol efflux to HDL/apoA1 in ECs. We further found that CCL2 induced CC-chemokine receptor 2 (CCR2) expression and siRNA-CCR2 reversed CCL2 suppression on HDL binding, association, internalization, and on cholesterol efflux in ECs. Moreover, CCL2 induced p42/44 mitogen-activated protein kinase (MAPK) phosphorylation via CCR2, and p42/44 MAPK inhibition reversed the suppression of CCL2 on HDL metabolism in ECs. Our study suggests that CCL2 was elevated in CAD patients. CCL2 suppressed HDL internalization and cholesterol efflux via CCR2 induction and p42/44 MAPK activation in ECs. CCL2 induction may contribute to impair HDL function and form atherosclerosis in CAD. PMID:27458015

Ascorbic Acid Efflux from Human Brain Microvascular Pericytes: Role of Re-uptake

PubMed Central

May, James M.; Qu, Zhi-chao

2015-01-01

Microvascular pericytes take up ascorbic acid on the ascorbate transporter SVCT2. Intracellular ascorbate then protects the cells against apoptosis induced by culture at diabetic glucose concentrations. To investigate whether pericytes might also provide ascorbate to the underlying endothelial cells, we studied ascorbate efflux from human pericytes. When loaded with ascorbate to intracellular concentrations of 0.8–1.0 mM, almost two-thirds of intracellular ascorbate effluxed from the cells over 2 h. This efflux was opposed by ascorbate re-uptake from the medium, since preventing re-uptake by destroying extracellular ascorbate with ascorbate oxidase increased ascorbate loss even further. Ascorbate re-uptake occurred on the SVCT2, since its blockade by replacing medium sodium with choline, by the SVCT2 inhibitor sulfinpyrazone, or by extracellular ascorbate accelerated ascorbate loss from the cells. This was supported by finding that net efflux of radiolabeled ascorbate was increased by unlabeled extracellular ascorbate with a half-maximal effect in the range of the high affinity Km of the SVCT2. Intracellular ascorbate did not inhibit its efflux. To assess the mechanism of ascorbate efflux, known inhibitors of volume-regulated anion channels (VRACs) were tested. These potently inhibited ascorbate transport into cells on the SVCT2, but not its efflux. An exception was the anion transport inhibitor DIDS, which, despite inhibition of ascorbate uptake, also inhibited net efflux at 25–50 µM. These results suggest that ascorbate efflux from vascular pericytes occurs on a DIDS-inhibitable transporter or channel different from VRACs. Further, ascorbate efflux is opposed by re-uptake of ascorbate on the SVCT2, providing a potential regulatory mechanism. PMID:26340060

Comparison of surficial CO2 efflux to other measures of subsurface crude oil degradation

USGS Publications Warehouse

Warren, Ean; Sihota, Natasha J.; Hostettler, Frances D.; Bekins, Barbara A.

2014-01-01

At a spill site near Bemidji, Minnesota, crude oil at the water table has been undergoing anaerobic biodegradation for over 30 years. Previous work at this site has shown that methane produced from biodegradation of the oil migrates upward and is oxidized in a methanotrophic zone midway between the water table and the surface. To compare microbial activity measurement methods from multiple locations in the oil body, surficial carbon dioxide efflux, methanogen and methanotroph concentrations, and oil degradation state were collected. Carbon dioxide effluxes over the oil body averaged more than four times those at the background site. Methanotrophic bacteria concentrations measured using pmoA were over 105 times higher above the oil-contaminated sediments compared with the background site. Methanogenic archaea measured using mcrA ranged from 105 to over 107 in the oil and were below detection in the background. Methanogens correlated very well with methanotroph concentrations (r = 0.99), n-alkylcyclohexane losses as a proxy for degradation state (r = − 0.96), and somewhat less well with carbon dioxide efflux (r = 0.92). Carbon dioxide efflux similarly correlated to methanotroph concentrations (r = 0.90) and n-alkylcyclohexane losses (r = − 0.91).

Metabolic targeting of lactate efflux by malignant glioma inhibits invasiveness and induces necrosis: an in vivo study.

PubMed

Colen, Chaim B; Shen, Yimin; Ghoddoussi, Farhad; Yu, Pingyang; Francis, Todd B; Koch, Brandon J; Monterey, Michael D; Galloway, Matthew P; Sloan, Andrew E; Mathupala, Saroj P

2011-07-01

Glioblastoma multiforme (GBM) are the most malignant among brain tumors. They are frequently refractory to chemotherapy and radiotherapy with mean patient survival of approximately 6 months, despite surgical intervention. The highly glycolytic nature of glioblastomas describes their propensity to metabolize glucose to lactic acid at an elevated rate. To survive, GBMs efflux lactic acid to the tumor microenvironment through transmembrane transporters denoted monocarboxylate transporters (MCTs). We hypothesized that inhibition of MCT function would impair the glycolytic metabolism and affect both glioma invasiveness and survival. We examined the effect on invasiveness with α-cyano-4-hydroxy-cinnamic acid (ACCA, 4CIN, CHCA), a small-molecule inhibitor of lactate transport, through Matrigel-based and organotypic (brain) slice culture invasive assays using U87-MG and U251-MG glioma cells. We then conducted studies in immunodeficient rats by stereotaxic intracranial implantation of the glioma cells followed by programmed orthotopic application of ACCA through osmotic pumps. Effect on the implanted tumor was monitored by small-animal magnetic resonance imaging. Our assays indicated that glioma invasion was markedly impaired when lactate efflux was inhibited. Convection-enhanced delivery of inhibitor to the tumor bed caused tumor necrosis, with 50% of the animals surviving beyond the experimental end points (3 months after inhibitor exhaustion). Most importantly, control animals did not display any adverse neurologic effects during orthotopic administration of ACCA to brain through programmed delivery. These results indicate the clinical potential of targeting lactate efflux in glioma through delivery of small-molecule inhibitors of MCTs either to the tumor bed or to the postsurgical resection cavity.

Inhibition of the active lymph pump by flow in rat mesenteric lymphatics and thoracic duct

NASA Technical Reports Server (NTRS)

Gashev, Anatoliy A.; Davis, Michael J.; Zawieja, David C.; Delp, M. D. (Principal Investigator)

2002-01-01

There are only a few reports of the influence of imposed flow on an active lymph pump under conditions of controlled intraluminal pressure. Thus, the mechanisms are not clearly defined. Rat mesenteric lymphatics and thoracic ducts were isolated, cannulated and pressurized. Input and output pressures were adjusted to impose various flows. Lymphatic systolic and diastolic diameters were measured and used to determine contraction frequency and pump flow indices. Imposed flow inhibited the active lymph pump in both mesenteric lymphatics and in the thoracic duct. The active pump of the thoracic duct appeared more sensitive to flow than did the active pump of the mesenteric lymphatics. Imposed flow reduced the frequency and amplitude of the contractions and accordingly the active pump flow. Flow-induced inhibition of the active lymph pump followed two temporal patterns. The first pattern was a rapidly developing inhibition of contraction frequency. Upon imposition of flow, the contraction frequency immediately fell and then partially recovered over time during continued flow. This effect was dependent on the magnitude of imposed flow, but did not depend on the direction of flow. The effect also depended upon the rate of change in the direction of flow. The second pattern was a slowly developing reduction of the amplitude of the lymphatic contractions, which increased over time during continued flow. The inhibition of contraction amplitude was dependent on the direction of the imposed flow, but independent of the magnitude of flow. Nitric oxide was partly but not completely responsible for the influence of flow on the mesenteric lymph pump. Exposure to NO mimicked the effects of flow, and inhibition of the NO synthase by N (G)-monomethyl-L-arginine attenuated but did not completely abolish the effects of flow.

BmeRABC5 is a multidrug efflux system that can confer metronidazole resistance in Bacteroides fragilis.

PubMed

Pumbwe, Lilian; Chang, Abraham; Smith, Rachel L; Wexler, Hannah M

2007-01-01

The RND-family efflux pump gene bmeB5 was previously shown to be overexpressed in metronidazole-resistant laboratory mutants of Bacteroides fragilis. In the present study, we characterized the bmeABC5 genes and an upstream putative TetR-family regulator gene (bmeR5). bmeR5 (645 bp) was located 51 bp upstream of bmeA5 and encoded a 24.9-kDa protein. Deletant strains lacking bmeB5 or bmeR5 were constructed from a wild-type B. fragilis strain ADB77. Strain antimicrobial susceptibility was determined and gene expression was quantified. bmeR5 was overexpressed in Escherichia coli using a 6x-His tag system; BmeR5-His6 was isolated from inclusion bodies and its binding to bmeABC5 promoter regions was determined. BmeR5-His6 bound specifically to the bmeR5-bmeC5 intergenic region (IT1). Deletion of bmeR5 (ADB77DeltabmeR5) resulted in a significant (p < 0.05) increase in expression of bmeA5, bmeB5, and bmeC5, and > two-fold increase in minimum inhibitory concentrations (MICs) of ampicillin, cefoxitin, cefoperazone, ciprofloxacin, imipenem, metronidazole, ethidium bromide, and sodium dodecyl sulfate (SDS). MICs were reduced by the efflux pump inhibitor carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The MICs of ampicillin, cefoperazone, metronidazole, and SDS were reduced by approximately two-fold in ADB77DeltabmeB5. A multidrug (metronidazole)-resistant, nim-negative B. fragilis clinical isolate overexpressed bmeABC5 genes, had a G-->T point mutation in IT1, and significantly reduced binding to BmeR5-His6. These data demonstrate that BmeR5 is a local repressor of bmeABC5 expression and that mutations in IT1 can lead to a derepression and resistance to multiple antimicrobial agents, including metronidazole.

Human multidrug resistance protein 8 (MRP8/ABCC11), an apical efflux pump for steroid sulfates, is an axonal protein of the CNS and peripheral nervous system.

PubMed

Bortfeld, M; Rius, M; König, J; Herold-Mende, C; Nies, A T; Keppler, D

2006-01-01

Dehydroepiandrosterone 3-sulfate and other neurosteroids are synthesized in the CNS and peripheral nervous system where they may modulate neuronal excitability by interacting with ligand-gated ion channels. For this modulatory activity, neurosteroids have to be locally released from either neurons or glial cells. We here identify the integral membrane protein ABCC11 (multidrug resistance protein 8) as an ATP-dependent efflux pump for steroid sulfates, including dehydroepiandrosterone 3-sulfate, and localize it to axons of the human CNS and peripheral nervous system. ABCC11 mRNA was detected in human brain by real-time polymerase chain reaction. Antibodies raised against ABCC11 served to detect the protein in brain by immunoblotting and immunofluorescence microscopy. ABCC11 was preferentially found in the white matter of the brain and co-localized with neurofilaments indicating that it is an axonal protein. Additionally, ABCC11 was localized to axons of the peripheral nervous system. For functional studies, ABCC11 was expressed in polarized Madin-Darby canine kidney cells where it was sorted to the apical membrane. This apical sorting is in accordance with the localization of ABCC11 to the axonal membrane of neurons. Inside-out plasma membrane vesicles containing recombinant ABCC11 mediated ATP-dependent transport of dehydroepiandrosterone 3-sulfate with a Km value of 21 microM. This transport function together with the localization of the ABCC11 protein in vicinity to GABAA receptors is consistent with a role of ABCC11 in dehydroepiandrosterone 3-sulfate release from neurons to sites of dehydroepiandrosterone 3-sulfate-mediated receptor modulation. Our findings may provide a basis for the characterization of mutations in the human ABCC11 gene and their linkage with neurological disorders.

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...

Chloride ion efflux regulates adherence, spreading, and respiratory burst of neutrophils stimulated by tumor necrosis factor-alpha (TNF) on biologic surfaces

PubMed Central

1996-01-01

Chloride ion efflux is an early event occurring after exposure of neutrophilic polymorphonuclear leukocytes (PMN) in suspension to several agonists, including cytokines such as tumor necrosis factor- alpha (TNF) and granulocyte/macrophage-colony stimulating factor (Shimizu, Y., R.H. Daniels, M.A. Elmore, M.J. Finnen, M.E. Hill, and J.M. Lackie. 1993. Biochem. Pharmacol. 9:1743-1751). We have studied TNF-induced Cl- movements in PMN residing on fibronectin (FN) (FN-PMN) and their relationships to adherence, spreading, and activation of the respiratory burst. Occupancy of the TNF-R55 and engagement of beta 2 integrins cosignaled for an early, marked, and prolonged Cl- efflux that was accompanied by a fall in intracellular chloride levels (Cl-i). A possible causal relationship between Cl- efflux, adherence, and respiratory burst was first suggested by kinetic studies, showing that TNF-induced Cl- efflux preceded both the adhesive and metabolic response, and was then confirmed by inhibition of all three responses by pretreating PMN with inhibitors of Cl- efflux, such as ethacrynic acid. Moreover, Cl- efflux induced by means other than TNF treatment, i.e., by using Cl(-)-free media, was followed by increased adherence, spreading, and metabolic activation, thus mimicking TNF effects. These studies provide the first evidence that a drastic decrease of Cl-i in FN-PMN may represent an essential step in the cascade of events leading to activation of proadhesive molecules, reorganization of the cytoskeleton network, and assembly of the O2(-)-forming NADPH oxidase. PMID:8896606

Single cell model for simultaneous drug delivery and efflux.

PubMed

Yi, C; Saidel, G M; Gratzl, M

1999-01-01

Multidrug resistance (MDR) of some cancer cells is a major challenge for chemotherapy of systemic cancers to overcome. To experimentally uncover the cellular mechanisms leading to MDR, it is necessary to quantitatively assess both drug influx into, and efflux from, the cells exposed to drug treatment. By using a novel molecular microdelivery system to enforce continuous and adjustable drug influx into single cells by controlled diffusion through a gel plug in a micropipet tip, drug resistance studies can now be performed on the single cell level. Our dynamic model of this scheme incorporates drug delivery, diffusive mixing, and accumulation inside the cytoplasm, and efflux by both passive and active membrane transport. Model simulations using available experimental information on these processes can assist in the design of MDR related experiments on single cancer cells which are expected to lead to a quantitative evaluation of mechanisms. Simulations indicate that drug resistance of a cancer cell can be quantified better by its dynamic response than by steady-state analysis.

V1-receptor mediated GSH efflux by vasopressin from rat hepatocytes.

PubMed

Sato, C; Liu, J H; Uchihara, M; Izumi, N; Yauchi, T; Sakaj, Y; Asahina, Y; Fukuma, T; Takano, T; Marumo, F

1992-01-01

Vasopression increases sinusoidal efflux of GSH in the perfused rat liver. The mechanism of this effect was studied in the perfused rat liver and in isolated rat hepatocytes. Vasopressin stimulated GSH efflux in both systems and a V1-receptor antagonist (OPC-21268) significantly inhibited the effect of vasopressin suggesting that vasopressin stimulates GSH efflux from rat hepatocytes via V1-receptor.

Tet38 Efflux Pump Affects Staphylococcus aureus Internalization by Epithelial Cells through Interaction with CD36 and Contributes to Bacterial Escape from Acidic and Nonacidic Phagolysosomes.

PubMed

Truong-Bolduc, Q C; Khan, N S; Vyas, J M; Hooper, D C

2017-02-01

We previously reported that the Tet38 efflux pump is involved in internalization of Staphylococcus aureus by A549 lung epithelial cells. A lack of tet38 reduced bacterial uptake by A549 cells to 36% of that of the parental strain RN6390. Using invasion assays coupled with confocal microscopy imaging, we studied the host cell receptor(s) responsible for bacterial uptake via interaction with Tet38. We also assessed the ability of S. aureus to survive following alkalinization of the phagolysosomes by chloroquine. Antibody to the scavenger receptor CD36 reduced the internalization of S. aureus RN6390 by A549 cells, but the dependence on CD36 was reduced in QT7 tet38, suggesting that an interaction between Tet38 and CD36 contributed to S. aureus internalization. Following fusion of the S. aureus-associated endosomes with lysosomes, alkalinization of the acidic environment with chloroquine led to a rapid increase in the number of S. aureus RN6390 bacteria in the cytosol, followed by a decrease shortly thereafter. This effect of chloroquine was not seen in the absence of intact Tet38 in mutant QT7. These data taken together suggest that Tet38 plays a role both in bacterial internalization via interaction with CD36 and in bacterial escape from the phagolysosomes. Copyright © 2017 American Society for Microbiology.

Tet38 Efflux Pump Affects Staphylococcus aureus Internalization by Epithelial Cells through Interaction with CD36 and Contributes to Bacterial Escape from Acidic and Nonacidic Phagolysosomes

PubMed Central

Truong-Bolduc, Q. C.; Khan, N. S.

2016-01-01

ABSTRACT We previously reported that the Tet38 efflux pump is involved in internalization of Staphylococcus aureus by A549 lung epithelial cells. A lack of tet38 reduced bacterial uptake by A549 cells to 36% of that of the parental strain RN6390. Using invasion assays coupled with confocal microscopy imaging, we studied the host cell receptor(s) responsible for bacterial uptake via interaction with Tet38. We also assessed the ability of S. aureus to survive following alkalinization of the phagolysosomes by chloroquine. Antibody to the scavenger receptor CD36 reduced the internalization of S. aureus RN6390 by A549 cells, but the dependence on CD36 was reduced in QT7 tet38, suggesting that an interaction between Tet38 and CD36 contributed to S. aureus internalization. Following fusion of the S. aureus-associated endosomes with lysosomes, alkalinization of the acidic environment with chloroquine led to a rapid increase in the number of S. aureus RN6390 bacteria in the cytosol, followed by a decrease shortly thereafter. This effect of chloroquine was not seen in the absence of intact Tet38 in mutant QT7. These data taken together suggest that Tet38 plays a role both in bacterial internalization via interaction with CD36 and in bacterial escape from the phagolysosomes. PMID:27956597

Hydrogen peroxide inhibits iodide influx and enhances iodide efflux in cultured FRTL-5 rat thyroid cells.

PubMed

Sugawara, M; Yamaguchi, D T; Lee, H Y; Yanagisawa, K; Murakami, S; Summer, C N; Johnson, D G; Levin, S R

1990-05-01

This study describes the effects of hydrogen peroxide on the two iodide transport systems, I influx and I efflux, in the cultured FRTL-5 rat thyroid cells. I influx was measured by the amount of I taken up by the cells during incubation with Na125I and NaI for 7 min, and I efflux was measured by calculating the rate of 125I release from the 125I-loaded cells in the presence and absence of 5 mmol/l H2O2. Exposure to greater than 100 mumol/l H2O2 for 40 min caused a significant inhibition of I influx; the inhibition was reversible and non-competitive with iodide. Thyroid Na+K+ ATPase activity, a major mechanism to drive I influx, decreased by 40% after the cells were exposed to 5 mmol/l H2O2 for 10 min. H2O2 enhanced I efflux only when Ca2+ was present in the medium. The mechanism of an enhanced I efflux by H2O2 appears to be mediated through the elevation of free cytosolic Ca2+ concentration. Our data indicate that H2O2 can affect I transport by inhibiting I influx and enhancing I efflux.

Enhancement of the Norfloxacin Antibiotic Activity by Gaseous Contact with the Essential Oil of Croton zehntneri

PubMed Central

Coutinho, HDM; Matias, EFF; Santos, KKA; Tintino, SR; Souza, CES; Guedes, GMM; Santos, FAD; Costa, JGM; Falcão-Silva, VS; Siqueira-Júnior, JP

2010-01-01

This is the first on the modulation of norfloxacin antibiotic activity by the volatile compounds of an essential oil. We report the chemical composition and antibiotic modifying activity of the essential oil extracted from the leaves of Croton zehntneri Pax et Hoffm (variety estragole), using the minimal inhibitory dose method and gaseous contact. The leaves of Croton zehntneri Pax et Hoffm (Euphorbiaceae) were subjected to hydrodistillation, and the essential oil extracted was examined with respect to the chemical composition, by gas chromatography-mass spectrometry (GC/MS), and to inhibitory activity of efflux pump by gaseous contact. The main component of the essential oil of C. zehntneri was estragole (76,8%). The gaseous components of the oil enhanced the inhibition zone of norfloxacin in 39,5%. This result shows that this oil influences the antibiotic activity of norfloxacin, possibly affecting the bacterial NorA efflux system, and may be used as an adjuvant in the antibiotic therapy of multidrug resistant pathogens. PMID:21264094

Ezetimibe Promotes Brush Border Membrane-to-Lumen Cholesterol Efflux in the Small Intestine

PubMed Central

Nakano, Takanari; Inoue, Ikuo; Takenaka, Yasuhiro; Ono, Hiraku; Katayama, Shigehiro; Awata, Takuya; Murakoshi, Takayuki

2016-01-01

Ezetimibe inhibits Niemann-Pick C1-like 1 (NPC1L1), an apical membrane cholesterol transporter of enterocytes, thereby reduces intestinal cholesterol absorption. This treatment also increases extrahepatic reverse cholesterol transport via an undefined mechanism. To explore this, we employed a trans-intestinal cholesterol efflux (TICE) assay, which directly detects circulation-to-intestinal lumen 3H-cholesterol transit in a cannulated jejunal segment, and found an increase of TICE by 45%. To examine whether such increase in efflux occurs at the intestinal brush border membrane(BBM)-level, we performed luminal perfusion assays, similar to TICE but the jejunal wall was labelled with orally-given 3H-cholesterol, and determined elevated BBM-to-lumen cholesterol efflux by 3.5-fold with ezetimibe. Such increased efflux probably promotes circulation-to-lumen cholesterol transit eventually; thus increases TICE. Next, we wondered how inhibition of NPC1L1, an influx transporter, resulted in increased efflux. When we traced orally-given 3H-cholesterol in mice, we found that lumen-to-BBM 3H-cholesterol transit was rapid and less sensitive to ezetimibe treatment. Comparison of the efflux and fractional cholesterol absorption revealed an inverse correlation, indicating the efflux as an opposite-regulatory factor for cholesterol absorption efficiency and counteracting to the naturally-occurring rapid cholesterol influx to the BBM. These suggest that the ezetimibe-stimulated increased efflux is crucial in reducing cholesterol absorption. Ezetimibe-induced increase in cholesterol efflux was approximately 2.5-fold greater in mice having endogenous ATP-binding cassette G5/G8 heterodimer, the major sterol efflux transporter of enterocytes, than the knockout counterparts, suggesting that the heterodimer confers additional rapid BBM-to-lumen cholesterol efflux in response to NPC1L1 inhibition. The observed framework for intestinal cholesterol fluxes may provide ways to modulate the flux

Ezetimibe Promotes Brush Border Membrane-to-Lumen Cholesterol Efflux in the Small Intestine.

PubMed

Nakano, Takanari; Inoue, Ikuo; Takenaka, Yasuhiro; Ono, Hiraku; Katayama, Shigehiro; Awata, Takuya; Murakoshi, Takayuki

2016-01-01

Ezetimibe inhibits Niemann-Pick C1-like 1 (NPC1L1), an apical membrane cholesterol transporter of enterocytes, thereby reduces intestinal cholesterol absorption. This treatment also increases extrahepatic reverse cholesterol transport via an undefined mechanism. To explore this, we employed a trans-intestinal cholesterol efflux (TICE) assay, which directly detects circulation-to-intestinal lumen 3H-cholesterol transit in a cannulated jejunal segment, and found an increase of TICE by 45%. To examine whether such increase in efflux occurs at the intestinal brush border membrane(BBM)-level, we performed luminal perfusion assays, similar to TICE but the jejunal wall was labelled with orally-given 3H-cholesterol, and determined elevated BBM-to-lumen cholesterol efflux by 3.5-fold with ezetimibe. Such increased efflux probably promotes circulation-to-lumen cholesterol transit eventually; thus increases TICE. Next, we wondered how inhibition of NPC1L1, an influx transporter, resulted in increased efflux. When we traced orally-given 3H-cholesterol in mice, we found that lumen-to-BBM 3H-cholesterol transit was rapid and less sensitive to ezetimibe treatment. Comparison of the efflux and fractional cholesterol absorption revealed an inverse correlation, indicating the efflux as an opposite-regulatory factor for cholesterol absorption efficiency and counteracting to the naturally-occurring rapid cholesterol influx to the BBM. These suggest that the ezetimibe-stimulated increased efflux is crucial in reducing cholesterol absorption. Ezetimibe-induced increase in cholesterol efflux was approximately 2.5-fold greater in mice having endogenous ATP-binding cassette G5/G8 heterodimer, the major sterol efflux transporter of enterocytes, than the knockout counterparts, suggesting that the heterodimer confers additional rapid BBM-to-lumen cholesterol efflux in response to NPC1L1 inhibition. The observed framework for intestinal cholesterol fluxes may provide ways to modulate the flux

CC-Chemokine Ligand 2 (CCL2) Suppresses High Density Lipoprotein (HDL) Internalization and Cholesterol Efflux via CC-Chemokine Receptor 2 (CCR2) Induction and p42/44 Mitogen-activated Protein Kinase (MAPK) Activation in Human Endothelial Cells.

PubMed

Sun, Run-Lu; Huang, Can-Xia; Bao, Jin-Lan; Jiang, Jie-Yu; Zhang, Bo; Zhou, Shu-Xian; Cai, Wei-Bin; Wang, Hong; Wang, Jing-Feng; Zhang, Yu-Ling

2016-09-09

High density lipoprotein (HDL) has been proposed to be internalized and to promote reverse cholesterol transport in endothelial cells (ECs). However, the mechanism underlying these processes has not been studied. In this study, we aim to characterize HDL internalization and cholesterol efflux in ECs and regulatory mechanisms. We found mature HDL particles were reduced in patients with coronary artery disease (CAD), which was associated with an increase in CC-chemokine ligand 2 (CCL2). In cultured primary human coronary artery endothelial cells and human umbilical vein endothelial cells, we determined that CCL2 suppressed the binding (4 °C) and association (37 °C) of HDL to/with ECs and HDL cellular internalization. Furthermore, CCL2 inhibited [(3)H]cholesterol efflux to HDL/apoA1 in ECs. We further found that CCL2 induced CC-chemokine receptor 2 (CCR2) expression and siRNA-CCR2 reversed CCL2 suppression on HDL binding, association, internalization, and on cholesterol efflux in ECs. Moreover, CCL2 induced p42/44 mitogen-activated protein kinase (MAPK) phosphorylation via CCR2, and p42/44 MAPK inhibition reversed the suppression of CCL2 on HDL metabolism in ECs. Our study suggests that CCL2 was elevated in CAD patients. CCL2 suppressed HDL internalization and cholesterol efflux via CCR2 induction and p42/44 MAPK activation in ECs. CCL2 induction may contribute to impair HDL function and form atherosclerosis in CAD. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

α-Synuclein Regulates Neuronal Cholesterol Efflux.

PubMed

Hsiao, Jen-Hsiang T; Halliday, Glenda M; Kim, Woojin Scott

2017-10-19

α-Synuclein is a neuronal protein that is at the center of focus in understanding the etiology of a group of neurodegenerative diseases called α-synucleinopathies, which includes Parkinson's disease (PD). Despite much research, the exact physiological function of α-synuclein is still unclear. α-Synuclein has similar biophysical properties as apolipoproteins and other lipid-binding proteins and has a high affinity for cholesterol. These properties suggest a possible role for α-synuclein as a lipid acceptor mediating cholesterol efflux (the process of removing cholesterol out of cells). To test this concept, we "loaded" SK-N-SH neuronal cells with fluorescently-labelled cholesterol, applied exogenous α-synuclein, and measured the amount of cholesterol removed from the cells using a classic cholesterol efflux assay. We found that α-synuclein potently stimulated cholesterol efflux. We found that the process was dose and time dependent, and was saturable at 1.0 µg/mL of α-synuclein. It was also dependent on the transporter protein ABCA1 located on the plasma membrane. We reveal for the first time a novel role of α-synuclein that underscores its importance in neuronal cholesterol regulation, and identify novel therapeutic targets for controlling cellular cholesterol levels.

Metabolic Targeting of Lactate Efflux by Malignant Glioma Inhibits Invasiveness and Induces Necrosis: An In Vivo Study1

PubMed Central

Colen, Chaim B; Shen, Yimin; Ghoddoussi, Farhad; Yu, Pingyang; Francis, Todd B; Koch, Brandon J; Monterey, Michael D; Galloway, Matthew P; Sloan, Andrew E; Mathupala, Saroj P

2011-01-01

Glioblastoma multiforme (GBM) are the most malignant among brain tumors. They are frequently refractory to chemotherapy and radiotherapy with mean patient survival of approximately 6 months, despite surgical intervention. The highly glycolytic nature of glioblastomas describes their propensity to metabolize glucose to lactic acid at an elevated rate. To survive, GBMs efflux lactic acid to the tumor microenvironment through transmembrane transporters denoted monocarboxylate transporters (MCTs). We hypothesized that inhibition of MCT function would impair the glycolytic metabolism and affect both glioma invasiveness and survival. We examined the effect on invasiveness with α-cyano-4-hydroxy-cinnamic acid (ACCA, 4CIN, CHCA), a small-molecule inhibitor of lactate transport, through Matrigel-based and organotypic (brain) slice culture invasive assays using U87-MG and U251-MG glioma cells. We then conducted studies in immunodeficient rats by stereotaxic intracranial implantation of the glioma cells followed by programmed orthotopic application of ACCA through osmotic pumps. Effect on the implanted tumor was monitored by small-animal magnetic resonance imaging. Our assays indicated that glioma invasion was markedly impaired when lactate efflux was inhibited. Convection-enhanced delivery of inhibitor to the tumor bed caused tumor necrosis, with 50% of the animals surviving beyond the experimental end points (3 months after inhibitor exhaustion). Most importantly, control animals did not display any adverse neurologic effects during orthotopic administration of ACCA to brain through programmed delivery. These results indicate the clinical potential of targeting lactate efflux in glioma through delivery of small-molecule inhibitors of MCTs either to the tumor bed or to the postsurgical resection cavity. PMID:21750656

Profound regulation of Na/K pump activity by transient elevations of cytoplasmic calcium in murine cardiac myocytes

PubMed Central

Lu, Fang-Min; Deisl, Christine; Hilgemann, Donald W

2016-01-01

Small changes of Na/K pump activity regulate internal Ca release in cardiac myocytes via Na/Ca exchange. We now show conversely that transient elevations of cytoplasmic Ca strongly regulate cardiac Na/K pumps. When cytoplasmic Na is submaximal, Na/K pump currents decay rapidly during extracellular K application and multiple results suggest that an inactivation mechanism is involved. Brief activation of Ca influx by reverse Na/Ca exchange enhances pump currents and attenuates current decay, while repeated Ca elevations suppress pump currents. Pump current enhancement reverses over 3 min, and results are similar in myocytes lacking the regulatory protein, phospholemman. Classical signaling mechanisms, including Ca-activated protein kinases and reactive oxygen, are evidently not involved. Electrogenic signals mediated by intramembrane movement of hydrophobic ions, such as hexyltriphenylphosphonium (C6TPP), increase and decrease in parallel with pump currents. Thus, transient Ca elevation and Na/K pump inactivation cause opposing sarcolemma changes that may affect diverse membrane processes. DOI: http://dx.doi.org/10.7554/eLife.19267.001 PMID:27627745

Comparison of surficial CO2 efflux to other measures of subsurface crude oil degradation.

PubMed

Warren, Ean; Sihota, Natasha J; Hostettler, Frances D; Bekins, Barbara A

2014-08-01

At a spill site near Bemidji, Minnesota, crude oil at the water table has been undergoing anaerobic biodegradation for over 30years. Previous work at this site has shown that methane produced from biodegradation of the oil migrates upward and is oxidized in a methanotrophic zone midway between the water table and the surface. To compare microbial activity measurement methods from multiple locations in the oil body, surficial carbon dioxide efflux, methanogen and methanotroph concentrations, and oil degradation state were collected. Carbon dioxide effluxes over the oil body averaged more than four times those at the background site. Methanotrophic bacteria concentrations measured using pmoA were over 10(5) times higher above the oil-contaminated sediments compared with the background site. Methanogenic archaea measured using mcrA ranged from 10(5) to over 10(7) in the oil and were below detection in the background. Methanogens correlated very well with methanotroph concentrations (r=0.99), n-alkylcyclohexane losses as a proxy for degradation state (r=-0.96), and somewhat less well with carbon dioxide efflux (r=0.92). Carbon dioxide efflux similarly correlated to methanotroph concentrations (r=0.90) and n-alkylcyclohexane losses (r=-0.91). Published by Elsevier B.V.

New saliva secretion model based on the expression of Na+-K+ pump and K+ channels in the apical membrane of parotid acinar cells.

PubMed

Almássy, János; Siguenza, Elias; Skaliczki, Marianna; Matesz, Klara; Sneyd, James; Yule, David I; Nánási, Péter P

2018-04-01

The plasma membrane of parotid acinar cells is functionally divided into apical and basolateral regions. According to the current model, fluid secretion is driven by transepithelial ion gradient, which facilitates water movement by osmosis into the acinar lumen from the interstitium. The osmotic gradient is created by the apical Cl - efflux and the subsequent paracellular Na + transport. In this model, the Na + -K + pump is located exclusively in the basolateral membrane and has essential role in salivary secretion, since the driving force for Cl - transport via basolateral Na + -K + -2Cl - cotransport is generated by the Na + -K + pump. In addition, the continuous electrochemical gradient for Cl - flow during acinar cell stimulation is maintained by the basolateral K + efflux. However, using a combination of single-cell electrophysiology and Ca 2+ -imaging, we demonstrate that photolysis of Ca 2+ close to the apical membrane of parotid acinar cells triggered significant K + current, indicating that a substantial amount of K + is secreted into the lumen during stimulation. Nevertheless, the K + content of the primary saliva is relatively low, suggesting that K + might be reabsorbed through the apical membrane. Therefore, we investigated the localization of Na + -K + pumps in acinar cells. We show that the pumps appear evenly distributed throughout the whole plasma membrane, including the apical pole of the cell. Based on these results, a new mathematical model of salivary fluid secretion is presented, where the pump reabsorbs K + from and secretes Na + to the lumen, which can partially supplement the paracellular Na + pathway.

Performance studies of Cryocooler based cryosorption pumps with indigenous activated carbons for fusion applications

NASA Astrophysics Data System (ADS)

Kasthurirengan, S.; Vivek, G. A.; Verma, Ravi; Behera, Upendra; Udgata, Swarup; Gangradey, Ranjana

2017-02-01

Cryosorption pumps are the only solution for pumping helium and hydrogen in fusion systems, due to their high pumping speeds and suitability in harsh environments. Their development requires the right Activated Carbons (ACs) and suitable adhesives to bind them to metallic panels with liquid helium (LHe) flow channels. However, their performance evaluation will require large quantities of LHe. Alternatively, these pumps can be built with small size panels adhered with ACs and cooled by a cryocooler. The paper describes the development of a cryopump using a commercial cryocooler (Sumitomo RDK415D), with 1.5W@4.2 K, integrated with small size AC panel mounted on 2nd stage, with the 1st stage acting as radiation shield. Under no load, the cryopump reaches the ultimate pressure of 2.1E-7 mbar. The pump is built using panels with different indigenously developed ACs such as granules, pellets, ACF-FK2 and activated carbon of knitted IPR cloth. We present the experimental results of pumping speeds for gases such as nitrogen, argon and helium using the procedures outlined by American Vacuum Society (AVS). These studies will enable to arrive at the right ACs and adhesives for the development of large scale cryosorption pumps with liquid helium flow.

Cholesterol binding, efflux, and a PDZ-interacting domain of scavenger receptor–BI mediate HDL-initiated signaling

PubMed Central

Assanasen, Chatchawin; Mineo, Chieko; Seetharam, Divya; Yuhanna, Ivan S.; Marcel, Yves L.; Connelly, Margery A.; Williams, David L.; de la Llera-Moya, Margarita; Shaul, Philip W.; Silver, David L.

2005-01-01

The binding of HDL to scavenger receptor–BI (SR-BI) mediates cholesterol movement. HDL also induces multiple cellular signals, which in endothelium occur through SR-BI and converge to activate eNOS. To determine the molecular basis of a signaling event induced by HDL, we examined the proximal mechanisms in HDL activation of eNOS. In endothelial cells, HDL and methyl-β-cyclodextrin caused comparable eNOS activation, whereas cholesterol-loaded methyl-β-cyclodextrin had no effect. Phosphatidylcholine-loaded HDL caused greater stimulation than native HDL, and blocking antibody against SR-BI, which prevents cholesterol efflux, prevented eNOS activation. In a reconstitution model in COS-M6 cells, wild-type SR-BI mediated eNOS activation by both HDL and small unilamellar vesicles (SUVs), whereas the SR-BI mutant AVI, which is incapable of efflux to SUV, transmitted signal by only HDL. In addition, eNOS activation by methyl-β-cyclodextrin was SR-BI dependent. Studies of mutant and chimeric class B scavenger receptors revealed that the C-terminal cytoplasmic PDZ-interacting domain and the C-terminal transmembrane domains of SR-BI are both necessary for HDL signaling. Furthermore, we demonstrated direct binding of cholesterol to the C-terminal transmembrane domain using a photoactivated derivative of cholesterol. Thus, HDL signaling requires cholesterol binding and efflux and C-terminal domains of SR-BI, and SR-BI serves as a cholesterol sensor on the plasma membrane. PMID:15841181

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

Biofilm formation ability of Salmonella enterica serovar Typhimurium acrAB mutants.

PubMed

Schlisselberg, Dov B; Kler, Edna; Kisluk, Guy; Shachar, Dina; Yaron, Sima

2015-10-01

Recent studies offer contradictory findings about the role of multidrug efflux pumps in bacterial biofilm development. Thus, the aim of this study was to investigate the involvement of the AcrAB efflux pump in biofilm formation by investigating the ability of AcrB and AcrAB null mutants of Salmonella enterica serovar Typhimurium to produce biofilms. Three models were used to compare the ability of S. Typhimurium wild-type and its mutants to form biofilms: formation of biofilm on polystyrene surfaces; production of biofilm (mat model) on the air/liquid interface; and expression of curli and cellulose on Congo red-supplemented agar plates. All three investigated genotypes formed biofilms with similar characteristics. However, upon exposure to chloramphenicol, formation of biofilms on solid surfaces as well as the production of curli were either reduced or were delayed more significantly in both mutants, whilst there was no visible effect on pellicle formation. It can be concluded that when no selective pressure is applied, S. Typhimurium is able to produce biofilms even when the AcrAB efflux pumps are inactivated, implying that the use of efflux pump inhibitors to prevent biofilm formation is not a general solution and that combined treatments might be more efficient. Other factors that affect the ability to produce biofilms depending on efflux pump activity are yet to be identified. Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Wood CO(2) efflux and foliar respiration for Eucalyptus in Hawaii and Brazil.

PubMed

Ryan, Michael G; Cavaleri, Molly A; Almeida, Auro C; Penchel, Ricardo; Senock, Randy S; Luiz Stape, José

2009-10-01

We measured CO(2) efflux from wood for Eucalyptus in Hawaii for 7 years and compared these measurements with those on three- and four-and-a-half-year-old Eucalyptus in Brazil. In Hawaii, CO(2) efflux from wood per unit biomass declined approximately 10x from age two to age five, twice as much as the decline in tree growth. The CO(2) efflux from wood in Brazil was 8-10x lower than that for comparable Hawaii trees with similar growth rates. Growth and maintenance respiration coefficients calculated from Hawaii wood CO(2) efflux declined with tree age and size (the growth coefficient declined from 0.4 mol C efflux mol C(-1) wood growth at age one to 0.1 mol C efflux mol C(-1) wood growth at age six; the maintenance coefficient from 0.006 to 0.001 micromol C (mol C biomass)(-1) s(-1) at 20 degrees C over the same time period). These results suggest interference with CO(2) efflux through bark that decouples CO(2) efflux from respiration. We also compared the biomass fractions and wood CO(2) efflux for the aboveground woody parts for 3- and 7-year-old trees in Hawaii to estimate how focusing measurements near the ground might bias the stand-level estimates of wood CO(2) efflux. Three-year-old Eucalyptus in Hawaii had a higher proportion of branches < 0.5 cm in diameter and a lower proportion of stem biomass than did 7-year-old trees. Biomass-specific CO(2) efflux measured at 1.4 m extrapolated to the tree could bias tree level estimates by approximately 50%, assuming no refixation from bark photosynthesis. However, the bias did not differ for the two tree sizes. Foliar respiration was identical per unit nitrogen for comparable treatments in Brazil and Hawaii (4.2 micromol C mol N(-1) s(-1) at 20 degrees C).

Analysis of sequence variation among smeDEF multi drug efflux pump genes and flanking DNA from defined 16S rRNA subgroups of clinical Stenotrophomonas maltophilia isolates.

PubMed

Gould, Virginia C; Okazaki, Aki; Howe, Robin A; Avison, Matthew B

2004-08-01

To determine the level of variation in the smeDEF efflux pump and smeT transcriptional regulator genes among three defined 16S rRNA sequence subgroups of clinical Stenotrophomonas maltophilia isolates. smeDEF sequencing used a PCR genome walking approach. Determination of the sequence surrounding smeDEF used a flanking primer PCR method and specific primers anchored in smeD or smeF together with random primers. smeDEF is chromosomal and located in the same position in the chromosome in all three subgroups of isolates. Flanking smeD is a gene, smeT, encoding a putative transcriptional repressor for smeDEF. Variation at these loci among the isolates is considerably lower (up to 10%) than at intrinsic beta-lactamase loci (up to 30%) in the same isolates, implying greater functional constraint. The smeD-smeT intergenic region contains a highly conserved section, which maps with previously predicted promoter/operator regions, and a hypervariable untranslated region, which can be used to subgroup clinical isolates. These data provide further evidence that it is possible to group clinical isolates of the inherently variable species, S. maltophilia, based on genotypic properties. Isolate D457, in which most work concerning smeDEF expression has been performed, does not fall into S. maltophilia subgroup A, which is the most typical.

Stimulation of accumbal GABAA receptors inhibits delta2-, but not delta1-, opioid receptor-mediated dopamine efflux in the nucleus accumbens of freely moving rats.

PubMed

Aono, Yuri; Kiguchi, Yuri; Watanabe, Yuriko; Waddington, John L; Saigusa, Tadashi

2017-11-15

The nucleus accumbens contains delta-opioid receptors that may reduce inhibitory neurotransmission. Reduction in GABA A receptor-mediated inhibition of accumbal dopamine release due to delta-opioid receptor activation should be suppressed by stimulating accumbal GABA A receptors. As delta-opioid receptors are divided into delta2- and delta1-opioid receptors, we analysed the effects of the GABA A receptor agonist muscimol on delta2- and delta1-opioid receptor-mediated accumbal dopamine efflux in freely moving rats using in vivo microdialysis. Drugs were administered intracerebrally through the dialysis probe. Doses of compounds indicate total amount administered (mol) during 25-50min infusions. The delta2-opioid receptor agonist deltorphin II (25.0nmol)- and delta1-opioid receptor agonist DPDPE (5.0nmol)-induced increases in dopamine efflux were inhibited by the delta2-opioid receptor antagonist naltriben (1.5nmol) and the delta1-opioid receptor antagonist BNTX (150.0pmol), respectively. Muscimol (250.0pmol) inhibited deltorphin II (25.0nmol)-induced dopamine efflux. The GABA A receptor antagonist bicuculline (50.0pmol), which failed to affect deltorphin II (25.0nmol)-induced dopamine efflux, counteracted the inhibitory effect of muscimol on deltorphin II-induced dopamine efflux. Neither muscimol (250.0pmol) nor bicuculline (50.0 and 500.0pmol) altered DPDPE (5.0nmol)-induced dopamine efflux. The present results show that reduction in accumbal GABA A receptor-mediated inhibition of dopaminergic activity is necessary to produce delta2-opioid receptor-induced increase in accumbal dopamine efflux. This study indicates that activation of delta2- but not delta1-opioid receptors on the cell bodies and/or terminals of accumbal GABAergic interneurons inhibits GABA release and, accordingly, decreases GABA A receptor-mediated inhibition of dopaminergic terminals, resulting in enhanced accumbal dopamine efflux. Copyright © 2017 Elsevier B.V. All rights reserved.

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. © The Author 2014. Published by Oxford University Press. All rights reserved.

Secondary multidrug efflux pump mutants alter Escherichia coli biofilm growth in the presence of cationic antimicrobial compounds.

PubMed

Bay, Denice C; Stremick, Carol A; Slipski, Carmine J; Turner, Raymond J

2017-04-01

Escherichia coli possesses many secondary active multidrug resistance transporters (MDTs) that confer overlapping substrate resistance to a broad range of antimicrobials via proton and/or sodium motive force. It is uncertain whether redundant MDTs uniquely alter cell survival when cultures grow planktonically or as biofilms. In this study, the planktonic and biofilm growth and antimicrobial resistance of 13 E. coli K-12 single MDT gene deletion strains in minimal and rich media were determined. Antimicrobial tolerance to tetracycline, tobramycin and benzalkonium were also compared for each ΔMDT strain. Four E. coli MDT families were represented in this study: resistance nodulation and cell division members acrA, acrB, acrD, acrE, acrF and tolC; multidrug and toxin extruder mdtK; major facilitator superfamily emrA and emrB; and small multidrug resistance members emrE, sugE, mdtI and mdtJ. Deletions of multipartite efflux system genes acrB, acrE and tolC resulted in significant reductions in both planktonic and biofilm growth phenotypes and enhanced antimicrobial susceptibilities. The loss of remaining MDT genes produced similar or enhanced (acrD, acrE, emrA, emrB, mdtK, emrE and mdtJ) biofilm growth and antimicrobial resistance. ΔMDT strains with enhanced antimicrobial tolerance also enhanced biofilm biomass. These findings suggest that many redundant MDTs regulate biofilm formation and drug tolerance. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Direct Comparison of Manganese Detoxification/Efflux Proteins and Molecular Characterization of ZnT10 Protein as a Manganese Transporter*

PubMed Central

Nishito, Yukina; Tsuji, Natsuko; Fujishiro, Hitomi; Takeda, Taka-aki; Yamazaki, Tomohiro; Teranishi, Fumie; Okazaki, Fumiko; Matsunaga, Ayu; Tuschl, Karin; Rao, Rajini; Kono, Satoshi; Miyajima, Hiroaki; Narita, Hiroshi; Himeno, Seiichiro; Kambe, Taiho

2016-01-01

Manganese homeostasis involves coordinated regulation of specific proteins involved in manganese influx and efflux. However, the proteins that are involved in detoxification/efflux have not been completely resolved nor has the basis by which they select their metal substrate. Here, we compared six proteins, which were reported to be involved in manganese detoxification/efflux, by evaluating their ability to reduce manganese toxicity in chicken DT40 cells, finding that human ZnT10 (hZnT10) was the most significant contributor. A domain swapping and substitution analysis between hZnT10 and the zinc-specific transporter hZnT1 showed that residue Asn43, which corresponds to the His residue constituting the potential intramembranous zinc coordination site in other ZnT transporters, is necessary to impart hZnT10's unique manganese mobilization activity; residues Cys52 and Leu242 in transmembrane domains II and V play a subtler role in controlling the metal specificity of hZnT10. Interestingly, the His → Asn reversion mutant in hZnT1 conferred manganese transport activity and loss of zinc transport activity. These results provide important information about manganese detoxification/efflux mechanisms in vertebrate cells as well as the molecular characterization of hZnT10 as a manganese transporter. PMID:27226609

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

2016-02-04

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. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Transintestinal transport of the anti-inflammatory drug 4F and the modulation of transintestinal cholesterol efflux[S

PubMed Central

Meriwether, David; Sulaiman, Dawoud; Wagner, Alan; Grijalva, Victor; Kaji, Izumi; Williams, Kevin J.; Yu, Liqing; Fogelman, Spencer; Volpe, Carmen; Bensinger, Steven J.; Anantharamaiah, G. M.; Shechter, Ishaiahu; Fogelman, Alan M.; Reddy, Srinivasa T.

2016-01-01

The site and mechanism of action of the apoA-I mimetic peptide 4F are incompletely understood. Transintestinal cholesterol efflux (TICE) is a process involved in the clearance of excess cholesterol from the body. While TICE is responsible for at least 30% of the clearance of neutral sterols from the circulation into the intestinal lumen, few pharmacological agents have been identified that modulate this pathway. We show first that circulating 4F selectively targets the small intestine (SI) and that it is predominantly transported into the intestinal lumen. This transport of 4F into the SI lumen is transintestinal in nature, and it is modulated by TICE. We also show that circulating 4F increases reverse cholesterol transport from macrophages and cholesterol efflux from lipoproteins via the TICE pathway. We identify the cause of this modulation of TICE either as 4F being a cholesterol acceptor with respect to enterocytes, from which 4F enhances cholesterol efflux, or as 4F being an intestinal chaperone with respect to TICE. Our results assign a novel role for 4F as a modulator of the TICE pathway and suggest that the anti-inflammatory functions of 4F may be a partial consequence of the codependent intestinal transport of both 4F and cholesterol. PMID:27199144

A new tetracycline efflux gene, tet(40), is located in tandem with tet(O/32/O) in a human gut firmicute bacterium and in metagenomic library clones.

PubMed

Kazimierczak, Katarzyna A; Rincon, Marco T; Patterson, Andrea J; Martin, Jennifer C; Young, Pauline; Flint, Harry J; Scott, Karen P

2008-11-01

The bacterium Clostridium saccharolyticum K10, isolated from a fecal sample obtained from a healthy donor who had received long-term tetracycline therapy, was found to carry three tetracycline resistance genes: tet(W) and the mosaic tet(O/32/O), both conferring ribosome protection-type resistance, and a novel, closely linked efflux-type resistance gene designated tet(40). tet(40) encodes a predicted membrane-associated protein with 42% amino acid identity to tetA(P). Tetracycline did not accumulate in Escherichia coli cells expressing the Tet(40) efflux protein, and resistance to tetracycline was reduced when cells were incubated with an efflux pump inhibitor. E. coli cells carrying tet(40) had a 50% inhibitory concentration of tetracycline of 60 microg/ml. Analysis of a transconjugant from a mating between donor strain C. saccharolyticum K10 and the recipient human gut commensal bacterium Roseburia inulinivorans suggested that tet(O/32/O) and tet(40) were cotransferred on a mobile element. Sequence analysis of a 37-kb insert identified on the basis of tetracycline resistance from a metagenomic fosmid library again revealed a tandem arrangement of tet(O/32/O) and tet(40), flanked by regions with homology to parts of the VanG operon previously identified in Enterococcus faecalis. At least 10 of the metagenomic inserts that carried tet(O/32/O) also carried tet(40), suggesting that tet(40), although previously undetected, may be an abundant efflux gene.

Evaluation of Ti-Zr-V (NEG) Thin Films for their pumping speed and pumping Capacity

NASA Astrophysics Data System (ADS)

Bansod, Tripti; Sindal, B. K.; Kumar, K. V. A. N. P. S.; Shukla, S. K.

2012-11-01

Deposition of NEG thin films onto the interior walls of the vacuum chambers is an advanced technique to convert a vacuum chamber from a gas source to an effective pump. These films offer considerably large pumping speed for reactive gases like CO, H2 etc. A UHV compatible pumping speed measurement system was developed in-house to measure the pumping speed of NEG coated chambers. To inject the fixed quantity of CO and H2 gas in pumping speed measurement set-up a calibrated leak was also developed. Stainless steel chambers were sputter coated with thin film of Ti-Zr-V getter material using varied parameters for different compositions and thickness. Pumping capacity which is a function of sorbed gas quantities was also studied at various activation temperatures. In order to optimize the activation temperature for maximum pumping speed for CO and H2, pumping speeds were measured at room temperature after activation at different temperatures. The experimental system detail, pumping performance of the NEG film at various activation temperatures and RGA analysis are presented.

In vitro and in vivo evaluation of drug-drug interaction between dabigatran and proton pump inhibitors.

PubMed

Ollier, Edouard; Hodin, Sophie; Basset, Thierry; Accassat, Sandrine; Bertoletti, Laurent; Mismetti, Patrick; Delavenne, Xavier

2015-12-01

To quantify the drug-drug interactions between dabigatran etexilate (DE) and proton pump inhibitors (PPI) and in particular the role of P-gp activity modulation. In the first part of the study, efflux ratios of DE were evaluated using the caco-2 cell line in the presence of pantoprazole, omeprazole, rabeprazole, lansoprazole and ciclosporin A (positive control). The two PPI that reduced the efflux ratio of dabigatran to the greatest and least extent, respectively, were used during the second part of the study, comprising a single-centre, randomised, open-label study with an incomplete Latin square design. Nine healthy volunteers received DE (150 mg) alone, DE (150 mg) with the first PPI and DE (150 mg) with the second PPI in randomised sequence. Dabigatran plasma concentration and thrombin time were measured in blood samples withdrawn at 11 time points after each treatment. Models were built using a nonlinear mixed-effect modelling approach. Omeprazole and rabeprazole were the two PPI that reduced the efflux ratio of DE least and most, respectively. The PK model was based on an inverse Gaussian absorption process with one compartment. The relationship between dabigatran concentration and thrombin time was considered linear. Some PK profiles had dramatically low concentration values due to poor absorption. These profiles were clustered using a between subject model mixture with interoccasion variability. The concomitant administration of PPI did not significantly change dabigatran pharmacokinetics. DE is subject to high absorption variability, precluding evaluation of the effect of PPI on its pharmacokinetics. © 2015 Société Française de Pharmacologie et de Thérapeutique.

Na(+)/K(+) pump interacts with the h-current to control bursting activity in central pattern generator neurons of leeches.

PubMed

Kueh, Daniel; Barnett, William H; Cymbalyuk, Gennady S; Calabrese, Ronald L

2016-09-02

The dynamics of different ionic currents shape the bursting activity of neurons and networks that control motor output. Despite being ubiquitous in all animal cells, the contribution of the Na(+)/K(+) pump current to such bursting activity has not been well studied. We used monensin, a Na(+)/H(+) antiporter, to examine the role of the pump on the bursting activity of oscillator heart interneurons in leeches. When we stimulated the pump with monensin, the period of these neurons decreased significantly, an effect that was prevented or reversed when the h-current was blocked by Cs(+). The decreased period could also occur if the pump was inhibited with strophanthidin or K(+)-free saline. Our monensin results were reproduced in model, which explains the pump's contributions to bursting activity based on Na(+) dynamics. Our results indicate that a dynamically oscillating pump current that interacts with the h-current can regulate the bursting activity of neurons and networks.

Peptides having reduced toxicity that stimulate cholesterol efflux

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bielicki, John K.; Johansson, Jan; Danho, Waleed

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 ABCA1 that parallels that of full-length apolipoproteins. Further, the peptides of the invention have little or no toxicity when administered at therapeutic and higher doses. 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.

Increased erythrocyte Na+ pump and NaK-ATPase activity during lithium therapy.

PubMed

Hokin-Neaverson, M; Burckhardt, W A; Jefferson, J W

1976-05-01

A significant mean increase of 18% in erythrocyte sodium pump activity (p less than 0.01, t test) was observed during lithium treatment, as compared with the activity before lithium treatment was started, in a group of 20 patients who were treated with lithium therapy for a variety of psychiatric conditions. The mean level of erythrocyte membrane ouabain-sensitive ATPase activity in a group of 35 subjects who were receiving lithium therapy was significantly higher than that of a different group of 38 subjects who were not receiving lithium therapy (p less than 0.005, t test). These observations may offer a biochemical mode of action for lithium in the treatment of bipolar affective disorder, since a deficiency of sodium pump activity has been shown to be associated with that disorder.

Inhibitory Effect of Flavonoids on the Efflux of N-Acetyl 5-Aminosalicylic Acid Intracellularly Formed in Caco-2 Cells

PubMed Central

Shin, Yoshimura; Kentaro, Kawano; Ryusuke, Matsumura; Narumi, Sugihara; Koji, Furuno

2009-01-01

N-acetyl 5-aminosalicylic acid (5-AcASA) that was intracellularly formed from 5-aminosalicylic acid (5-ASA) at 200 μM was discharged 5.3, 7.1, and 8.1-fold higher into the apical site than into the basolateral site during 1, 2, and 4-hour incubations, respectively, in Caco-2 cells grown in Transwells. The addition of flavonols (100 μM) such as fisetin and quercetin with 5-ASA remarkably decreased the apically directed efflux of 5-AcASA. When 5-ASA (200 μM) was added to Caco-2 cells grown in tissue culture dishes, the formation of 5-AcASA decreased, and, in addition, the formed 5-AcASA was found to be accumulated within the cells in the presence of such flavonols. Thus, the decrease in 5-AcASA efflux by such flavonols was attributed not only to the inhibition of N-acetyl-conjugation of 5-ASA but to the predominant cellular accumulation of 5-AcASA. Various flavonoids also had both of the effects with potencies that depend on their specific structures. The essential structure of flavonoids was an absence of a hydroxyl substitution at the C5 position on the A-ring of flavone structure for the inhibitory effect on the N-acetyl-conjugation of 5-ASA, and a presence of hydroxyl substitutions at the C3′ or C4′ position on the B-ring of flavone structure for the promoting effect on the cellular accumulation of 5-AcASA. Both the decrease in 5-AcASA apical efflux and the increase in 5-AcASA cellular accumulation were also caused by MK571 and indomethacin, inhibitors of MRPs, but not by quinidine, cyclosporin A, P-glycoprotein inhibitors, and mitoxantrone, a BCRP substrate. These results suggest that certain flavonoids suppress the apical efflux of 5-AcASA possibly by inhibiting MRPs pumps located on apical membranes in Caco-2 cells. PMID:19688110

Anti-bacterial activity of some Brazilian medicinal plants.

PubMed

de Lima, Maria Raquel Ferreira; de Souza Luna, Josiane; dos Santos, Aldenir Feitosa; de Andrade, Maria Cristina Caño; Sant'Ana, Antônio Euzébio Goulart; Genet, Jean-Pierre; Marquez, Béatrice; Neuville, Luc; Moreau, Nicole

2006-04-21

Extracts from various organs of 25 plants of Brazilian traditional medicine were assayed with respect to their anti-bacterial activities against Escherichia coli, a susceptible strain of Staphylococcus aureus and two resistant strains of Staphylococcus aureus harbouring the efflux pumps NorA and MsrA. Amongst the 49 extracts studied, 14 presented anti-bacterial activity against Staphylococcus aureus, including the ethanolic extracts from the rhizome of Jatropha elliptica, from the stem barks of Schinus terebinthifolius and Erythrina mulungu, from the stems and leaves of Caesalpinia pyramidalis and Serjania lethalis, and from the stem bark and leaves of Lafoensia pacari. The classes of compounds present in the active extracts were determined as a preliminary step towards their bioactivity-guided separation. No extracts were active against Escherichia coli.

[The participation of the transport-barrier functions of the plasma membrane in the development of fluoroquinolone (ciprofloxacin) resistance in Acholeplasma laidlawii].

PubMed

Abramycheva, N Iu; Govorun, V M

2000-01-01

The role of transport activity of Acholeplasma laidlawii plasmatic membrane in the development of resistance to ciprofloxacin was investigated. It was shown that ethidium bromide used as fluoroquinolone analogue in plasmatic membrane efflux pump was accumulated in ciprofloxacin-resistant cells in much less amount. It was estimated that ethidium bromide efflux depended on temperature, glucose and transmembrane electro-chemical proton potential. Inhibitors of efflux systems--reserpine and verapamil enhanced the ethidium bromide accumulation much more intensively in ciprofloxacin resistant cells. The results of investigation allowed to consider the existence of active efflux system for toxic agents in acholeplasma; in the case of ciprofloxacin-resistant strain these systems are inducible.

Active magnetic bearings: As applied to centrifugal pumps

NASA Technical Reports Server (NTRS)

Nelik, Lev; Cooper, Paul; Jones, Graham; Galecki, Dennis; Pinckney, Frank; Kirk, Gordon

1992-01-01

Application of magnetic bearings to boiler feed pumps presents various attractive features, such as longer bearing life, lower maintenance costs, and improved operability through control of the rotordynamics. Magnetic bearings were fitted to an eight-stage, 600 hp boiler feed pump, which generates 2600 ft of heat at 680 gpm and 3560 rpm. In addition to the varied and severe operating environment in steady state operation of this pump in a power plant, it is also subjected to transient loads during frequent starts and stops. These loads can now be measured by the in-built instrumentation of the magnetic bearings. Following site installation, a follow-up bearing tune-up was performed, and pump transient response testing was conducted. The bearing response was completely satisfactory, ensuring trouble-free pump operation even in the range of reduced load. The experience gained so far through design and testing proves feasibility of magnetic bearings for boiler feed pumps, which sets the stage for application of even higher energy centrifugal pumps equipped with magnetic bearings.

Active magnetic bearings: As applied to centrifugal pumps

NASA Astrophysics Data System (ADS)

Nelik, Lev; Cooper, Paul; Jones, Graham; Galecki, Dennis; Pinckney, Frank; Kirk, Gordon

1992-05-01

Application of magnetic bearings to boiler feed pumps presents various attractive features, such as longer bearing life, lower maintenance costs, and improved operability through control of the rotordynamics. Magnetic bearings were fitted to an eight-stage, 600 hp boiler feed pump, which generates 2600 ft of heat at 680 gpm and 3560 rpm. In addition to the varied and severe operating environment in steady state operation of this pump in a power plant, it is also subjected to transient loads during frequent starts and stops. These loads can now be measured by the in-built instrumentation of the magnetic bearings. Following site installation, a follow-up bearing tune-up was performed, and pump transient response testing was conducted. The bearing response was completely satisfactory, ensuring trouble-free pump operation even in the range of reduced load. The experience gained so far through design and testing proves feasibility of magnetic bearings for boiler feed pumps, which sets the stage for application of even higher energy centrifugal pumps equipped with magnetic bearings.

Acid sphingomyelinase-deficient macrophages have defective cholesterol trafficking and efflux.

PubMed

Leventhal, A R; Chen, W; Tall, A R; Tabas, I

2001-11-30

Cholesterol efflux from macrophage foam cells, a key step in reverse cholesterol transport, requires trafficking of cholesterol from intracellular sites to the plasma membrane. Sphingomyelin is a cholesterol-binding molecule that transiently exists with cholesterol in endosomes and lysosomes but is rapidly hydrolyzed by lysosomal sphingomyelinase (L-SMase), a product of the acid sphingomyelinase (ASM) gene. We therefore hypothesized that sphingomyelin hydrolysis by L-SMase enables cholesterol efflux by preventing cholesterol sequestration by sphingomyelin. Macrophages from wild-type and ASM knockout mice were incubated with [(3)H]cholesteryl ester-labeled acetyl-LDL and then exposed to apolipoprotein A-I or high density lipoprotein. In both cases, [(3)H]cholesterol efflux was decreased substantially in the ASM knockout macrophages. Similar results were shown for ASM knockout macrophages labeled long-term with [(3)H]cholesterol added directly to medium, but not for those labeled for a short period, suggesting defective efflux from intracellular stores but not from the plasma membrane. Cholesterol trafficking to acyl-coenzyme A:cholesterol acyltransferase (ACAT) was also defective in ASM knockout macrophages. Using filipin to probe cholesterol in macrophages incubated with acetyl-LDL, we found there was modest staining in the plasma membrane of wild-type macrophages but bright, perinuclear fluorescence in ASM knockout macrophages. Last, when wild-type macrophages were incubated with excess sphingomyelin to "saturate" L-SMase, [(3)H]cholesterol efflux was decreased. Thus, sphingomyelin accumulation due to L-SMase deficiency leads to defective cholesterol trafficking and efflux, which we propose is due to sequestration of cholesterol by sphingomyelin and possibly other mechanisms. This model may explain the low plasma high density lipoprotein found in ASM-deficient humans and may implicate L-SMase deficiency and/or sphingomyelin enrichment of lipoproteins as novel

[Antimicrobial activity of fosfomycin under various conditions against standard strains, beta-lactam resistant strains, and multidrug efflux system mutants].

PubMed

Mikuniya, Takeshi; Hiraishi, Toru; Maebashi, Kazunori; Ida, Takashi; Takata, Toshihiko; Hikida, Muneo; Yamada, Sakuo; Gotoh, Naomasa; Nishino, Takeshi

2005-04-01

The purpose of this study was to evaluate the possible benefit of fosfomycin (FOM) as prophylactic antibiotic in terms of antimicrobial activity and the potential of inducibility of beta-lactamase, compared with cefazolin, cefotiam, cefmetazole, and piperacillin that are commonly used as perioperative agents. The in vitro activity of FOM against aerobic Gram-negative bacteria using Mueller-Hinton agar or nutrient agar supplemented with glucose-6-phosphate (G6P) as tested medium increased within a range from 2 to 256 times the activity in the medium without G6P. However, the susceptibility of Gram-positive bacteria to FOM remained largely unchanged with or without G6P. There was no aerobic- or anaerobic-bacteria which changed susceptibility against beta-lactam antibiotics under various tested medium conditions. FOM demonstrated strong bactericidal activity against Escherichia coli and Pseudomonas aeruginosa in a dose dependent manner, and decreased viable cell counts of Staphylococcus aureus. In the case of P. aeruginosa, transmission electron micrographs study revealed that numerous lysed cells were present 2 hours after treatment with FOM at four times the MIC. First and second generation cephalosporins induced AmpC-type beta-lactamase in a dose dependent manner among beta-lactamase inducible strains of P. aeruginosa and Enterobacter cloacae. On the other hand, inducible activity of FOM on beta-lactamase production was less than 1/25 to 1/65 compared with those of cephalosporins. In addition, FOM maintained strong antimicrobial activity for over then 20 years after marketing, because of the excellent stability against various types of beta-lactamase produced by plasmid-carrying bacteria and clinical isolates. FOM was not extruded by four types of efflux systems, such as MexAB-OprM, MexCD-OprJ, MexXY/ OprM and MexEF-OprN, however beta-lactam antibiotics were substrates of MexAB-OprM and MexCD-OprJ. In conclusion, FOM provides adequate coverage for both aerobic Gram

Puerarin promotes ABCA1-mediated cholesterol efflux and decreases cellular lipid accumulation in THP-1 macrophages.

PubMed

Li, Cong-Hui; Gong, Duo; Chen, Ling-Yan; Zhang, Min; Xia, Xiao-Dan; Cheng, Hai-Peng; Huang, Chong; Zhao, Zhen-Wang; Zheng, Xi-Long; Tang, Xiao-Er; Tang, Chao-Ke

2017-09-15

It was reported that puerarin decreases the total cholesterol, low-density lipoprotein cholesterol (LDL-C), triglyceride (TG) and increases high-density lipoprotein cholesterol (HDL-C) level, but the underlying mechanism is unclear. This study was designed to determine whether puerarin decreased lipid accumulation via up-regulation of ABCA1-mediated cholesterol efflux in THP-1 macrophage-derived foam cells. Our results showed that puerarin significantly promoted the expression of ATP-binding cassette transporter A1 (ABCA1) mRNA and protein via the AMP-activated protein kinase (AMPK)-peroxisome proliferator-activated receptor gamma (PPARγ)-liver X receptor-alpha (LXR-α) pathway and decreased cellular lipid accumulation in human THP-1 macrophage-derived foam cells. The miR-7 directly targeted 3' untranslated region of STK11 (Serine/Threonine Kinase 11), which activated the AMPK pathway. Transfection with miR-7 mimic significantly reduced STK11 expression in puerarin-treated macrophages, decreased the phosphorylation of AMPK, down-regulated the expression of the PPARγ-LXR-α-ABCA1 expression. Additionally, treatment with miR-7 decreased cholesterol efflux and increased cholesterol levels in THP-1 macrophage-derived foam cells. Our study demonstrates that puerarin promotes ABCA1-mediated cholesterol efflux and decreases intracellular cholesterol levels through the pathway involving miR-7, STK11, and the AMPK-PPARγ-LXR-α-ABCA1 cascade. Copyright © 2017 Elsevier B.V. All rights reserved.

Copper Efflux Is Induced during Anaerobic Amino Acid Limitation in Escherichia coli To Protect Iron-Sulfur Cluster Enzymes and Biogenesis

PubMed Central

Fung, Danny Ka Chun; Lau, Wai Yin; Chan, Wing Tat

2013-01-01

Adaptation to changing environments is essential to bacterial physiology. Here we report a unique role of the copper homeostasis system in adapting Escherichia coli to its host-relevant environment of anaerobiosis coupled with amino acid limitation. We found that expression of the copper/silver efflux pump CusCFBA was significantly upregulated during anaerobic amino acid limitation in E. coli without the supplement of exogenous copper. Inductively coupled plasma mass spectrometry analysis of the total intracellular copper content combined with transcriptional assay of the PcusC-lacZ reporter in the presence of specific Cu(I) chelators indicated that anaerobic amino acid limitation led to the accumulation of free Cu(I) in the periplasmic space of E. coli, resulting in Cu(I) toxicity. Cells lacking cusCFBA and another copper transporter, copA, under this condition displayed growth defects and reduced ATP production during fumarate respiration. Ectopic expression of the Fe-S cluster enzyme fumarate reductase (Frd), or supplementation with amino acids whose biosynthesis involves Fe-S cluster enzymes, rescued the poor growth of ΔcusC cells. Yet, Cu(I) treatment did not impair the Frd activity in vitro. Further studies revealed that the alternative Fe-S cluster biogenesis system Suf was induced during the anaerobic amino acid limitation, and ΔcusC enhanced this upregulation, indicating the impairment of the Fe-S cluster assembly machinery and the increased Fe-S cluster demands under this condition. Taken together, we conclude that the copper efflux system CusCFBA is induced during anaerobic amino acid limitation to protect Fe-S cluster enzymes and biogenesis from the endogenously originated Cu(I) toxicity, thus facilitating the physiological adaptation of E. coli. PMID:23893112

Ibuprofen Potentiates the In Vivo Antifungal Activity of Fluconazole against Candida albicans Murine Infection

PubMed Central

Miranda, Isabel M.; Silva-Dias, Ana; Silva, Ana P.; Rodrigues, Acácio G.; Pina-Vaz, Cidália

2015-01-01

Candida albicans is the most prevalent cause of fungemia worldwide. Its ability to develop resistance in patients receiving azole antifungal therapy is well documented. In a murine model of systemic infection, we show that ibuprofen potentiates fluconazole antifungal activity against a fluconazole-resistant strain, drastically reducing the fungal burden and morbidity. The therapeutic combination of fluconazole with ibuprofen may constitute a new approach for the management of antifungal therapeutics to reverse the resistance conferred by efflux pump overexpression. PMID:25845879

Ascorbic acid efflux and re-uptake in endothelial cells: maintenance of intracellular ascorbate.

PubMed

May, James M; Qu, Zhi-chao

2009-05-01

Entry of vitamin C or ascorbate into most tissues requires its movement across the endothelial cell barrier of vessels. If trans-cellular ascorbate movement occurs, then it should be evident as ascorbate efflux from endothelial cells. Cultured EA.926 endothelial cells that had been loaded to about 3.5 mM intracellular ascorbate lost 70-80% of ascorbate to the medium over several hours at 37 degrees C via a non-saturable process that was insensitive to anion transport inhibitors and thiol reagents. Oxidation of this extracellular ascorbate by ascorbate oxidase or ferricyanide enhanced apparent ascorbate efflux, suggesting that efflux of the vitamin was countered in part by its re-uptake on ascorbate transporters. Although basal ascorbate efflux was not calcium-dependent, increased entry of calcium into the cells enhanced ascorbate release. These results support the hypothesis that ascorbate efflux reflects trans-endothelial cell ascorbate movement out of the blood vessel.

Design of New Antibacterial Enhancers Based on AcrB's Structure and the Evaluation of Their Antibacterial Enhancement Activity.

PubMed

Song, Yi; Qin, Rongxin; Pan, Xichun; Ouyang, Qin; Liu, Tianyu; Zhai, Zhaoxia; Chen, Yingchun; Li, Bin; Zhou, Hong

2016-11-18

Previously, artesunate (AS) and dihydroartemisinine 7 (DHA7) were found to have antibacterial enhancement activity against Escherichia coli via inhibition of the efflux pump AcrB. However, they were only effective against E. coli standard strains. This study aimed to develop effective antibacterial enhancers based on the previous work. Our results demonstrate that 86 new antibacterial enhancers were designed via 3D-SAR and molecular docking. Among them, DHA27 had the best antibacterial enhancement activity. It could potentiate the antibacterial effects of ampicillin against not only E. coli standard strain but also clinical strains, and of β-lactam antibiotics, not non-β-lactamantibiotics. DHA27 could increase the accumulation of daunomycin and nile red within E. coli ATCC 35218, but did not increase the bacterial membrane permeability. DHA27 reduced acrB 's mRNA expression of E. coli ATCC 35218 in a dose-dependent manner, and its antibacterial enhancement activity is related to the degree of acrB mRNA expression in E. coli clinical strains. The polypeptides from AcrB were obtained via molecular docking assay; the pre-incubated polypeptides could inhibit the activity of DHA27. Importantly, DHA27 had no cytotoxicity on cell proliferation. In conclusion, among newly designed antibacterial enhancers, DHA27 had favorable physical and pharmacological properties with no significant cytotoxicity at effective concentrations, and might serve as a potential efflux pump inhibitor in the future.

Inside out: efflux of carbon dioxide from leaves represents more than leaf metabolism.

PubMed

Stutz, Samantha S; Anderson, Jeremiah; Zulick, Rachael; Hanson, David T

2017-05-17

High concentrations of inorganic carbon in the xylem, produced from root, stem, and branch respiration, travel via the transpiration stream and eventually exit the plant through distant tissues as CO2. Unlike previous studies that focused on the efflux of CO2 from roots and woody tissues, we focus on efflux from leaves and the potential effect on leaf respiration measurements. We labeled transported inorganic carbon, spanning reported xylem concentrations, with 13C and then manipulated transpiration rates in the dark in order to vary the rates of inorganic carbon supply to cut leaves from Brassica napus and Populus deltoides. We used tunable diode laser absorbance spectroscopy to directly measure the rate of gross 13CO2 efflux, derived from inorganic carbon supplied from outside of the leaf, relative to gross 12CO2 efflux generated from leaf cells. These experiemnts showed that 13CO2 efflux was dependent upon the rate of inorganic carbon supply to the leaf and the rate of transpiration. Our data show that the gross leaf efflux of xylem-transported CO2 is likely small in the dark when rates of transpiration are low. However, gross leaf efflux of xylem-transported CO2 could approach half the rate of leaf respiration in the light when transpiration rates and branch inorganic carbon concentrations are high, irrespective of the grossly different petiole morphologies in our experiment. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Effects of ammonia and hepatic failure on the net efflux of endogenous glutamate, aspartate and taurine from rat cerebrocortical slices: modulation by elevated K+ concentrations.

PubMed

Zielińska, M; Hilgier, W; Law, R O; Gorynski, P; Albrecht, J

2002-01-01

Cerebrocortical minislices derived from control rats ("control slices") and from rats with thioacetamide (TAA)-induced hepatic failure showing moderate hyperammonemia and symptoms of hepatic encephalopathy (HE) ("HE slices"), were incubated with physiological saline in the absence or presence of 5 mM ammonium acetate ("ammonia"), at potassium ion (K+) concentrations ranging from 5 to 15 mM. The efflux of endogenous aspartate (Asp), glutamate (Glu) and taurine (Tau) to the incubation medium was assayed by HPLC. At 5 mM K+, perfusion of control slices with ammonia did not affect Glu and slightly depressed Asp efflux. Raising K+ concentrations in the incubation medium to 7.5 led to inhibition of Glu and Asp efflux by ammonia and the inhibitory effect was further potentiated at 10 mM K+. The inhibition was also significant at 15 mM K+. This suggests that, depression of excitatory neurotransmission associated with acute hyperammonemia is more pronounced under conditions of intense neuronal activity than in the resting state. HE moderately increased the efflux of Glu and Asp, and the stimulatory effect of HE on Glu and Asp efflux showed virtually no variation upon changing K+ concentration up to 15 mM. Ammonia strongly, and HE moderately, increased Tau efflux at 5 mM K+. However, both the ammonia- and HE-dependent Tau efflux decreased with increasing K+ concentration in the medium and was no longer significant at 10 mM concentration, indicating that intense neuronal activity obliterates the neuroprotective functions of this amino acid triggered by hyperammonemia.

Increased H+ efflux is sufficient to induce dysplasia and necessary for viability with oncogene expression

PubMed Central

Grillo-Hill, Bree K; Choi, Changhoon; Jimenez-Vidal, Maite; Barber, Diane L

2015-01-01

Intracellular pH (pHi) dynamics is increasingly recognized as an important regulator of a range of normal and pathological cell behaviors. Notably, increased pHi is now acknowledged as a conserved characteristic of cancers and in cell models is confirmed to increase proliferation and migration as well as limit apoptosis. However, the significance of increased pHi for cancer in vivo remains unresolved. Using Drosophila melanogaster, we show that increased pHi is sufficient to induce dysplasia in the absence of other transforming cues and potentiates growth and invasion with oncogenic Ras. Using a genetically encoded biosensor we also confirm increased pHi in situ. Moreover, in Drosophila models and clonal human mammary cells we show that limiting H+ efflux with oncogenic Raf or Ras induces acidosis and synthetic lethality. Further, we show lethality in invasive primary tumor cell lines with inhibiting H+ efflux. Synthetic lethality with reduced H+ efflux and activated oncogene expression could be exploited therapeutically to restrain cancer progression while limiting off-target effects. DOI: http://dx.doi.org/10.7554/eLife.03270.001 PMID:25793441

Ascorbic Acid Efflux and Re-uptake in Endothelial Cells: Maintenance of Intracellular Ascorbate

PubMed Central

May, James M.; Qu, Zhi-chao

2013-01-01

Entry of vitamin C or ascorbate into most tissues requires its movement across the endothelial cell barrier of vessels. If trans-cellular ascorbate movement occurs, then it should be evident as ascorbate efflux from endothelial cells. Cultured EA.926 endothelial cells that had been loaded to about 3.5 mM intracellular ascorbate lost 70–80% of ascorbate to the medium over several hours at 37 °C via a non-saturable process that was insensitive to anion transport inhibitors and thiol reagents. Oxidation of this extracellular ascorbate by ascorbate oxidase or ferricyanide enhanced apparent ascorbate efflux, suggesting that efflux of the vitamin was countered in part by its re-uptake on ascorbate transporters. Although basal ascorbate efflux was not calcium-dependent, increased entry of calcium into the cells enhanced ascorbate release. These results support the hypothesis that ascorbate efflux reflects trans-endothelial cell ascorbate movement out of the blood vessel. PMID:19148707

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.

Association of Cholesterol Efflux Capacity With Clinical Features of Metabolic Syndrome: Relevance to Atherosclerosis.

PubMed

Gall, Julie; Frisdal, Eric; Bittar, Randa; Le Goff, Wilfried; Bruckert, Eric; Lesnik, Philippe; Guerin, Maryse; Giral, Philippe

2016-11-23

The contribution of high-density lipoprotein to cardiovascular benefit is closely linked to its role in the cellular cholesterol efflux process; however, various clinical and biochemical variables are known to modulate the overall cholesterol efflux process. The aim of this study was to evaluate the extent to which clinical and biological anomalies associated with the establishment of the metabolic syndrome modulate cholesterol efflux capacity and contribute to development of atherosclerosis. This study involved patients (n=1202) displaying atherogenic dyslipidemia in primary prevention who were referred to our prevention center. Among these patients, 25% presented at least 3 criteria of the metabolic syndrome, as defined by the National Cholesterol Education Program Adult Treatment Panel III. We measured the capacity of 40-fold diluted serum to mediate cholesterol efflux from cholesterol-loaded human THP-1 macrophages. Cholesterol efflux capacity was reduced progressively by 4% to 11% (P<0.0001) as a function of the increasing number of coexisting criteria for the metabolic syndrome from 1 to 5. This observation was primarily related to reductions in scavenger receptor class B member 1 and ATP binding cassette subfamily G member 1-dependent efflux. Multivariate analyses indicate that serum efflux capacity was significantly associated with established metabolic syndrome (odds ratio 0.45; 95% CI 0.28-0.72; P=0.009) independent of age, low-density lipoprotein cholesterol, status with regard to lipid-lowering therapy, smoking status, and alcohol consumption. Our study revealed that individual criteria of metabolic syndrome are closely related synergistically to cholesterol efflux capacity. In addition, established metabolic syndrome and cholesterol efflux capacity were independently associated with clinical features of atherosclerosis. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Variability in soil CO2 efflux across distinct urban land cover types

NASA Astrophysics Data System (ADS)

Weissert, Lena F.; Salmond, Jennifer A.; Schwendenmann, Luitgard

2015-04-01

As a main source of greenhouse gases urban areas play an important role in the global carbon cycle. To assess the potential role of urban vegetation in mitigating carbon emissions we need information on the magnitude of biogenic CO2 emissions and its driving factors. We examined how urban land use types (urban forest, parklands, sportsfields) vary in their soil CO2 efflux. We measured soil CO2 efflux and its isotopic signature, soil temperature and soil moisture over a complete growing season in Auckland, New Zealand. Soil physical and chemical properties and vegetation characteristics were also measured. Mean soil CO2 efflux ranged from 4.15 to 12 μmol m-2 s-1. We did not find significant differences in soil CO2 efflux among land cover types due to high spatial variability in soil CO2 efflux among plots. Soil (soil carbon and nitrogen density, texture, soil carbon:nitrogen ratio) and vegetation characteristics (basal area, litter carbon density, grass biomass) were not significantly correlated with soil CO2 efflux. We found a distinct seasonal pattern with significantly higher soil CO2 efflux in autumn (Apr/May) and spring (Oct). In urban forests and sportsfields over 80% of the temporal variation was explained by soil temperature and soil water content. The δ13C signature of CO2 respired from parklands and sportsfields (-20 permil - -25 permil) were more positive compared to forest plots (-29 permil) indicating that parkland and sportsfields had a considerable proportion of C4 grasses. Despite the large intra-urban variability, our results compare to values reported from other, often climatically different cities, supporting the hypothesis of homogenization across urban areas as a result of human management practices.

Vertical gradients and seasonal variation in stem CO2 efflux within a Norway spruce stand.

PubMed

Tarvainen, Lasse; Räntfors, Mats; Wallin, Göran

2014-05-01

Stem CO2 efflux is known to vary seasonally and vertically along tree stems. However, annual tree- and stand-scale efflux estimates are commonly based on measurements made only a few times a year, during daytime and at breast height. In this study, the effect of these simplifying assumptions on annual efflux estimates and their influence on the estimates of the importance of stems in stand-scale carbon cycling are evaluated. In order to assess the strength of seasonal, diurnal and along-stem variability in CO2 efflux, half-hourly measurements were carried out at three heights on three mature Norway spruce (Picea abies (L.) Karst.) trees over a period of 3 years. Making the common assumption of breast height efflux rates being representative of the entire stem was found to result in underestimations of 10-17% in the annual tree-scale CO2 efflux. Upscaling using only daytime measurements from breast height increased the underestimation to 15-20%. Furthermore, the results show that the strength of the vertical gradient varies seasonally, being strongest in the early summer and non-existent during the cool months. The observed seasonality in the vertical CO2 efflux gradient could not be explained by variation in stem temperature, temperature response of the CO2 efflux (Q10), outer-bark permeability, CO2 transport in the xylem or CO2 release from the phloem. However, the estimated CO2 concentration immediately beneath the bark was considerably higher in the upper stem during the main period of diameter growth, coinciding with the strongest vertical efflux gradient. These results suggest that higher growth rates in the upper stem are the main cause for the observed vertical variation in the stem CO2 effluxes. Furthermore, the results indicate that accounting for the vertical efflux variation is essential for assessments of the importance of stems in stand-scale carbon cycling. © The Author 2014. Published by Oxford University Press. All rights reserved.

Temporal and Spatial Variations in Soil CO2 Effluxes of Different Ecosystems

NASA Astrophysics Data System (ADS)

Liang, N.; Kim, S.; Shimoyama, K.; Kim, Y.; Hirano, T.; Takagi, K.; Suto, H.; Fujinuma, Y.; Inoue, G.

2005-12-01

Regional networks for measuring carbon sequestration or loss by terrestrial ecosystems on a year round basis have been in operation since the mid-1990s. However, continuous measurements of soil CO2 efflux, the largest component of ecosystem respiration have only been reported over similar time scales at a few of the sites. Reasons include the lack of automated measurement systems that are commercially available, and the need for frequent servicing to ensure accurate measurements. We have developed a multichannel automated chamber system that can be used for continuous measuring soil CO2 efflux during snow-free seasons. We installed the chamber systems in boreal forest in Alaska, tundra in west Siberia, temperate and cool-temperate forests in Japan and Korea, tropical seasonal forest in Thailand, and tropical rainforest in Malaysia. Annual soil CO2 efflux were measured to be about 5-6 tC ha-1 y-1 in the boreal and cool-temperate forests, 10 tC ha-1 y-1 in the temperate forests, and 26 tC ha-1 y-1 in the tropical rainforests. Efflux showed significant seasonality in the boreal and temperate forest that corresponding with the seasonal soil temperature. However, the wavelike efflux rates in the tropical forests were correlated with the seasonality of soil moisture. Soil CO2 efflux of forest ecosystems showed large spatial variation and was correlated with vegetation type and the chamber size.

Cation activation of the pig kidney sodium pump: transmembrane allosteric effects of sodium.

PubMed Central

Karlish, S J; Stein, W D

1985-01-01

We have studied activation by Na or Rb ions of different transport modes of the Na-K pump, using phospholipid vesicles reconstituted with pig kidney Na-K-ATPase. The shape of the activation curves, sigmoid or quasi-hyperbolic, depends on the nature of the cation at the opposite surface and not on the specific mode of transport. ATP-dependent Na uptake into K-containing vesicles (Na-K exchange) is activated by cytoplasmic Na along a highly sigmoid curve in the absence of extracellular Na (Hill number, nH = 1.9). Activation displays progressively less-sigmoid curves as extracellular Na is raised to 150 mM (nH = 1.2). The maximal rate of the Na-K exchange is not affected. Na is not transported from the extracellular face by the pump in the presence of excess extracellular K, and the transmembrane effects of the extracellular Na are therefore 'allosteric' in nature. ATP-dependent Na-Na exchange (Lee & Blostein, 1980) and classical ATP-plus-ADP-dependent Na-Na exchange are activated by cytoplasmic Na along hyperbolic curves. ATP-dependent Na uptake into Tris-containing vesicles is activated by cytoplasmic Na along a somewhat sigmoidal curve. (ATP + Pi)-dependent Rb-Rb exchange is activated by cytoplasmic and extracellular Rb along strictly hyperbolic curves. The same applies for Rb-Rb exchange in the presence or absence of ATP or Pi alone. The presence of a high concentration of extracellular Na together with extracellular Rb induces a sigmoidal activation by cytoplasmic Rb of (ATP + Pi)-dependent Rb-Rb exchange (nH = 1.45) but does not affect the maximal rate of exchange. Slow passive Rb fluxes through the pump observed in the absence of other pump ligands (see Karlish & Stein, 1982 alpha) are activated by cytoplasmic Rb along a strictly hyperbolic curve with extracellular Rb, nH = 1.0 (Rb-Rb exchange), along a strongly sigmoid curve with extracellular Na, nH = 1.5 (Rb-Na exchange), and along less-sigmoid curves with extracellular Tris, nH = 1.24 (net Rb flux) or

Motuporamine Derivatives as Antimicrobial Agents and Antibiotic Enhancers against Resistant Gram-Negative Bacteria.