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Efflux Pumps (efflux + pump)
Kinds of Efflux Pumps Selected AbstractsCerulenin Analogues as Inhibitors of Efflux Pumps in Drug-resistant Candida albicansARCHIV DER PHARMAZIE, Issue 3 2009Florian Diwischek Abstract Overexpression of the ABC transporters Cdr1 and Cdr2 or the major facilitator Mdr1 causes multidrug resistance in the human fungal pathogen Candida albicans. The fatty acid synthesis inhibitor cerulenin and the structurally unrelated Golgi transport inhibitor brefeldin A are substrates for both types of efflux pumps in Candida albicans. In an effort to overcome efflux pump-mediated drug resistance in Candida albicans, cerulenin analogues were generated using a variety of synthesis pathways. The so obtained cerulenin derivatives were tested on multidrug-resistant Candida albicans isolates which constitutively overexpress either Mdr1 or Cdr1 and Cdr2. Some of these compounds were found to decrease Mdr1-mediated resistance to brefeldin A up to eightfold compared to the control. [source] The ttgGHI solvent efflux pump operon of Pseudomonas putida DOT-T1E is located on a large self-transmissible plasmidENVIRONMENTAL MICROBIOLOGY, Issue 6 2007José J. Rodríguez-Herva Summary Pseudomonas putida DOT-T1E is a solvent-tolerant strain able to grow in the presence of > 1% (v/v) toluene in the culture medium. A set of multidrug efflux pumps have been found to play a major role in the tolerance of this bacterium to organic solvents (Rojas et al., J Bacteriol 183: 3967,3973). In the course of studies of the mechanisms underlying solvent tolerance in DOT-T1E, we isolated a spontaneous solvent-sensitive mutant derivative which had lost the genes encoding the TtgGHI efflux pump, the most important extrusion element in quantitative terms. Genomic comparisons between the mutant and its parental strain by microarray analysis revealed that in addition to the ttgVW-ttgGHI gene cluster, another group of genes, highly similar to those found in the Tn4653A and ISPpu12 transposable elements of the TOL plasmid pWW0 from P. putida mt-2, were also absent from this strain. Further analysis demonstrated that strain DOT-T1E harboured a large plasmid (named pGRT1) that was lost from the solvent-sensitive mutant. Mapping analysis revealed that the ttgVW-ttgGHI genes and the Tn4653A -like transposon are borne by the pGRT1 plasmid. Plasmid pGRT1 is highly stable and its frequency of loss is below 10,8 per cell per generation under a variety of growth conditions, including nutritional and physical stresses. The pGRT1 plasmid is self-transmissible, and its acquisition by the toluene-sensitive P. putida KT2440 and Pseudomonas aeruginosa PAO1 increased the recipient's tolerance to toluene up to levels similar to those exhibited by P. putida DOT-T1E. We discuss the importance and potential benefits of this plasmid for the development of bacteria with enhanced solvent tolerance, and its potential impact for bioremediation and whole-cell biotransformations. [source] Functional role of the linker region in purified human P-glycoproteinFEBS JOURNAL, Issue 13 2009Tomomi Sato Human P-glycoprotein (P-gp), which conveys multidrug resistance, is an ATP-dependent drug efflux pump that transports a wide variety of structurally unrelated compounds out of cells. P-gp possesses a ,linker region' of , 75 amino acids that connects two homologous halves, each of which contain a transmembrane domain followed by a nucleotide-binding domain. To investigate the role of the linker region, purified human P-gp was cleaved by proteases at the linker region and then compared with native P-gp. Based on a verapamil-stimulated ATP hydrolase assay, size-exclusion chromatography analysis and a thermo-stability assay, cleavage of the P-gp linker did not directly affect the preservation of the overall structure or the catalytic process in ATP hydrolysis. However, linker cleavage increased the kcat values both with substrate (ksub) and without substrate (kbasal), but decreased the ksub/kbasal values of all 10 tested substrates. The former result indicates that cleaving the linker activates P-gp, while the latter result suggests that the linker region maintains the tightness of coupling between the ATP hydrolase reaction and substrate recognition. Inspection of structures of the P-gp homolog, MsbA, suggests that linker-cleaved P-gp has increased ATP hydrolase activity because the linker interferes with a conformational change that accompanies the ATP hydrolase reaction. Moreover, linker cleavage affected the specificity constants [ksub/Km(D)] for some substrates (i.e. linker cleavage probably shifts the substrate specificity profile of P-gp). Thus, this result also suggests that the linker region regulates the inherent substrate specificity of P-gp. [source] New insights into the P-glycoprotein-mediated effluxes of rhodaminesFEBS JOURNAL, Issue 3 2003Chatchanok Loetchutinat Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of the plasma drug transporter P-glycoprotein (P-gp). This protein is an active efflux pump for chemotherapeutic drugs, natural products and hydrophobic peptides. Despite the advances of recent years, we still have an unclear view of the molecular mechanism by which P-gp transports such a wide diversity of compounds across the membrane. Measurement of the kinetic characteristics of substrate transport is a powerful approach to enhancing our understanding of their function and mechanism. The aim of the present study was to further characterize the transport of several rhodamine analogues, either positively charged or zwitterionic. We took advantage of the intrinsic fluorescence of rhodamines and performed a flow-cytometric analysis of dye accumulation in the wild-type drug sensitive K562 that do not express P-gp and its MDR subline that display high levels of MDR. The measurements were made in real time using intact cells. The kinetic parameter, ka = VM/km, which is a measure of the efficiency of the P-gp-mediated efflux of a substrate was similar for almost all the rhodamine analogues tested. In addition these values were compared with those determined previously for the P-gp-mediated efflux of anthracycline. Our conclusion is that the compounds of these two classes of molecules, anthracyclines and rhodamines, are substrates of P-gp and that their pumping rates at limiting low substrate concentration are similar. The findings presented here are the first to show quantitative information about the kinetic parameters for P-gp-mediated efflux of rhodamine analogues in intact cells. [source] Functional interaction sites of OprM with MexAB in the Pseudomonas aeruginosa multidrug efflux pumpFEMS MICROBIOLOGY LETTERS, Issue 2 2009Eisaku Yoshihara Abstract Subunit-swapping between Pseudomonas aeruginosa MexAB-OprM and MexEF-OprN efflux pumps has shown that OprM can interact with MexEF to produce a functional efflux pump, but that OprN cannot functionally interact with MexAB. Taking advantage of this subunit selectivity, we carried out experiments using chimeric proteins composed of OprM and OprN to determine which regions of OprM are necessary for functional interaction with MexAB. We constructed two types of chimeric proteins: one with the N-terminal half of OprM and the C-terminal half of OprN (OprMN), and the second with these halves reversed (OprNM). Introduction of either of the chimeric protein genes into a mutant expressing MexEF alone restored the functionality of the efflux pump. However, expression of OprMN or OprNM in the presence of MexAB did not restore the pump functionality, indicating that the both the N- and C-terminal halves of OprM are necessary for a functional interaction with MexAB. [source] RESEARCH ARTICLE: RPD3 and ROM2 are required for multidrug resistance in Saccharomyces cerevisiaeFEMS YEAST RESEARCH, Issue 3 2008Silvia Borecka-Melkusova Abstract The PDR5 gene encodes the major multidrug resistance efflux pump in Saccharomyces cerevisiae. In drug-resistant cells, the hyperactive Pdr1p or Pdr3p transcriptional activators are responsible for the PDR5 upregulation. In this work, it is shown that the RPD3 gene encoding the histone deacetylase that functions as a transcriptional corepressor at many promoters and the ROM2 gene coding for the GDP/GTP exchange protein for Rho1p and Rho2p participating in signal transduction pathways are required for PDR5 transcription under cycloheximide-induced and noninduced conditions. Transposon insertion mutations in ROM2, RPD3 and some other genes encoding specific subunits of the large Rpd3L protein complex resulted in enhanced susceptibility of mutant cells to antifungals. In the rpd3, and rom2, mutants, the level of PDR5 mRNA and the rate of rhodamine 6G efflux were reduced. Unlike rpd3,, in rom2, mutant cells the drug hypersensitivity and the defect in PDR5 expression were suppressed by PDR1 or PDR3 overexpressed from heterologous promoters and by the hyperactive pdr3-9 mutant allele. The results indicate that Rpd3p histone deacetylase participating in chromatin remodeling and Rom2p participating in the cell integrity pathway are involved in the control of PDR5 expression and modulation of multidrug resistance in yeast. [source] Tacrolimus is a class II low-solubility high-permeability drug: The effect of P-glycoprotein efflux on regional permeability of tacrolimus in ratsJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 3 2002Shigeki Tamura Abstract The objective of this study is to investigate the role of P-glycoprotein (P-gp), a membrane efflux pump associated with multidrug resistance (MDR) and a known substrate for tacrolimus, in determining the regional intestinal permeability of tacrolimus in rats. Thus, isolated segments of rat jejunum, ileum, or colon were perfused with tacrolimus solutions containing polyethoxylated hydrogenated castor oil 60 surfactant, and with or without verapamil, a P-gp substrate used to reverse the MDR phenotype. The results indicated that the intrinsic permeability of tacrolimus in the jejunum, calculated on the basis of the concentration of non-micellized free tacrolimus, was quite high (,,1.4,×,10,4 cm/s). The apparent permeability (Papp) in the jejunum was unaffected by the presence of verapamil; however, the Papp in the ileum and the colon increased significantly in the presence of verapamil and were similar to the values observed in the jejunum. The results suggest that systemic absorption of tacrolimus from the gastrointestinal tract could be significantly affected by P-gp efflux mechanisms. It is also possible that differences in P-gp function at various intestinal sites in a subject or at a given intestinal site in various subjects could lead to large intra- and interindividual variability in bioavailability of tacrolimus following oral administration. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:719,729, 2002 [source] Effects of myricetin, an antioxidant, on the pharmacokinetics of losartan and its active metabolite, EXP-3174, in rats: possible role of cytochrome P450 3A4, cytochrome P450 2C9 and P-glycoprotein inhibition by myricetinJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 7 2010Dong-Hyun Choi Abstract Objectives, The effects of myricetin, a natural flavonoid, on the pharmacokinetics of losartan and its active metabolite, EXP-3174, were investigated in rats. Losartan and myricetin interact with cytochrome P450 (CYP) enzymes and P-glycoprotein, and the increase in the use of health supplements may result in myricetin being taken concomitantly with losartan as a combination therapy to treat or prevent cardiovascular diseases. Methods, The pharmacokinetic parameters of losartan and EXP-3174 were determined after oral administration of losartan (9 mg/kg) to rats in the presence or absence of myricetin (0.4, 2 and 8 mg/kg). The effects of myricetin on P-glycoprotein as well as CYP3A4 and CYP2C9 activity were also evaluated. Key findings, Myricetin inhibited CYP3A4 and CYP2C9 enzyme activity with a 50% inhibition concentration of 7.8 and 13.5 µm, respectively. In addition, myricetin significantly enhanced the cellular accumulation of rhodamine 123 in MCF-7/ADR cells overexpressing P-glycoprotein in a concentration-dependent manner. The pharmacokinetic parameters of losartan were significantly altered by myricetin compared with the control. The presence of myricetin (2 or 8 mg/kg) increased the area under the plasma concentration,time curve of losartan by 31.4,61.1% and peak plasma concentration of losartan by 31.8,50.2%. Consequently, the absolute bioavailability of losartan in the presence of myricetin increased significantly (P < 0.05, 2 mg/kg; P < 0.01, 8 mg/kg) compared with the control. There was no significant change in the time to reach the peak plasma concentration, apparent volume of distribution at steady state or terminal half-life of losartan in the presence of myricetin. Furthermore, concurrent use of myricetin (8 mg/kg) significantly decreased the metabolite,parent area under the plasma concentration,time curve ratio by 20%, implying that myricetin may inhibit the CYP-mediated metabolism of losartan to its active metabolite, EXP-3174. Conclusions, The enhanced bioavailability of losartan may be mainly due to inhibition of the CYP3A4- and CYP2C9-mediated metabolism of losartan in the small intestine or in the liver, and the P-glycoprotein efflux pump in the small intestine by myricetin. [source] Development of a PCR-based diagnostic test detecting a nt230(del4) MDR1 mutation in dogs: verification in a moxidectin-sensitive Australian ShepherdJOURNAL OF VETERINARY PHARMACOLOGY & THERAPEUTICS, Issue 1 2005J. GEYER A subpopulation of dogs of the Collie and Australian Shepherd breeds show increased sensitivity to central nervous actions of ivermectin, doramectin, loperamide, and probably several other drugs. The molecular background for this greater sensitivity is a nonsense mutation in the MDR1 efflux pump, which is part of the functional blood,brain barrier and normally limits drug penetration into the brain. This report describes a rapid PCR-based method for detection of this nt230(del4) MDR1 mutation using a small amount of genomic DNA from blood cells. Thereby, homozygous intact, homozygous mutated, and heterozygous mutated MDR1 genotypes can be clearly differentiated by high resolution polyacrylamide gel electrophoresis. Using this diagnostic test two Collies and one Australian Shepherd were screened for the nt230(del4) MDR1 mutation. The Collies had no history of altered drug sensitivity and showed homozygous intact and heterozygous mutated MDR1 alleles, respectively. However, the Australian Shepherd developed clear signs of neurotoxicity including ataxia, crawling, acoustic and tactile hyperexcitability, and miosis after a single dose of moxidectin (400 ,g/kg). For this dog two mutated MDR1 alleles were detected. This report describes for the first time moxidectin neurotoxicosis in a dog with a homozygous MDR1 mutation. [source] Indole and 7-hydroxyindole diminish Pseudomonas aeruginosa virulenceMICROBIAL BIOTECHNOLOGY, Issue 1 2009Jintae Lee Summary Indole is an extracellular biofilm signal for Escherichia coli, and many bacterial oxygenases readily convert indole to various oxidized compounds including 7-hydroxyindole (7HI). Here we investigate the impact of indole and 7HI on Pseudomonas aeruginosa PAO1 virulence and quorum sensing (QS)-regulated phenotypes; this strain does not synthesize these compounds but degrades them rapidly. Indole and 7HI both altered extensively gene expression in a manner opposite that of acylhomoserine lactones; the most repressed genes encode the mexGHI-opmD multidrug efflux pump and genes involved in the synthesis of QS-regulated virulence factors including pyocyanin (phz operon), 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) signal (pqs operon), pyochelin (pch operon) and pyoverdine (pvd operon). Corroborating these microarray results, indole and 7HI decreased production of pyocyanin, rhamnolipid, PQS and pyoverdine and enhanced antibiotic resistance. In addition, indole affected the utilization of carbon, nitrogen and phosphorus, and 7HI abolished swarming motility. Furthermore, 7HI reduced pulmonary colonization of P. aeruginosa in guinea pigs and increased clearance in lungs. Hence, indole-related compounds have potential as a novel antivirulence approach for the recalcitrant pathogen P. aeruginosa. [source] Role of the MexXY multidrug efflux pump in moderate aminoglycoside resistance in Pseudomonas aeruginosa isolates from Pseudomonas mastitisMICROBIOLOGY AND IMMUNOLOGY, Issue 8 2008Rungtip Chuanchuen ABSTRACT The contribution of the MexXY multidrug efflux system to aminoglycoside resistance was investigated in 18 clinical isolates of Pseudomonas aeruginosa obtained from dairy cows with Pseudomonas mastitis. All of the isolates expressed MexXY as determined by reverse transcription-PCR. The loss of mexXY resulted in increased susceptibility (two- to 16-fold decline in MIC) to aminoglycosides, confirming the contribution of this system in aminoglycoside resistance in these strains. As the impact of ,mexXY varied, overexpression of MexXY alone is not sufficient for aminoglycoside resistance. Expression of mexXY also varied and did not strictly correlate with aminoglycoside insusceptibility. Transcription levels of mexY were independent on mutations in mexZ, suggesting the existence of additional regulatory mechanisms other than mexZ. [source] PfCRT and the trans -vacuolar proton electrochemical gradient: regulating the access of chloroquine to ferriprotoporphyrin IXMOLECULAR MICROBIOLOGY, Issue 1 2006Patrick G. Bray Summary It is accepted that resistance of Plasmodium falciparum to chloroquine (CQ) is caused primarily by mutations in the pfcrt gene. However, a consensus has not yet been reached on the mechanism by which resistance is achieved. CQ-resistant (CQR) parasite lines accumulate less CQ than do CQ-sensitive (CQS) parasites. The CQR phenotype is complex with a component of reduced energy-dependent CQ uptake and an additional component that resembles energy-dependent CQ efflux. Here we show that the required energy input is in the form of the proton electrochemical gradient across the digestive vacuole (DV) membrane. Collapsing the DV proton gradient (or starving the parasites of glucose) results in similar levels of CQ accumulation in CQS and CQR lines. Under these conditions the accumulation of CQ is stimulated in CQR parasite lines but is reduced in CQS lines. Energy deprivation has no effect on the rate of CQ efflux from CQR lines implying that mutant PfCRT does not function as an efflux pump or active carrier. Using pfcrt -modified parasite lines we show that the entire CQ susceptibility phenotype is switched by the single K76T amino acid change in PfCRT. The efflux of CQ in CQR lines is not directly coupled to the energy supply, consistent with a model in which mutant PfCRT functions as a gated channel or pore, allowing charged CQ species to leak out of the DV. [source] Isopimaric acid from Pinus nigra shows activity against multidrug-resistant and EMRSA strains of Staphylococcus aureusPHYTOTHERAPY RESEARCH, Issue 6 2005Eileen Smith Abstract The diterpene isopimaric acid was extracted from the immature cones of Pinus nigra (Arnold) using bioassay-guided fractionation of a crude hexane extract. Isopimaric acid was assayed against multidrug-resistant (MDR) and methicillin-resistant Staphylococcus aureus (MRSA). The minimum inhibitory concentrations (MIC) were 32,64 µg/mL and compared with a commercially obtained resin acid, abietic acid, with MICs of 64 µg/mL. Resin acids are known to have antibacterial activity and are valued in traditional medicine for their antiseptic properties. These results show that isopimaric acid is active against MDR and MRSA strains of S. aureus which are becoming increasingly resistant to antibiotics. Both compounds were evaluated for modulation activity in combination with antibiotics, but did not potentiate the activity of the antibiotics tested. However, the compounds were also assayed in combination with the efflux pump inhibitor reserpine, to see if inhibition of the TetK or NorA efflux pump increased their activity. Interestingly, rather than a potentiation of activity by a reduction in MIC, a two to four-fold increase in MIC was seen. It may be that isopimaric acid and abietic acid are not substrates for these efflux pumps, but it is also possible that an antagonistic interaction with reserpine may render the antibiotics inactive. 1H-NMR of abietic acid and reserpine taken individually and in combination, revealed a shift in resonance of some peaks for both compounds when mixed together compared with the spectra of the compounds on their own. It is proposed that this may be due to complex formation between abietic acid and reserpine and that this complex formation is responsible for a reduction in activity and elevation of MIC. Copyright © 2005 John Wiley & Sons, Ltd. [source] Tissue Distribution of P-Glycoprotein in CatsANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 6 2009S. Van Der Heyden Summary Permeability glycoprotein (P-gp) is a membrane-bound efflux pump that exports various substances out of the cell. Variations in P-gp expression play an important role in susceptibility to toxic substances, drug efficacy and disease risk. In the present study, the distribution of the MDR1 -gene product P-gp was determined in normal tissues of domestic shorthair cats using immunohistochemistry. Two monoclonal antibodies C494 and C219 were used, recognizing a different epitope on the human P-gp molecule. A consistent positive immunolabelling was obtained. The tissue distribution and cellular locations with antibody C494 were similar to those in man and dogs; with liver, colon, adrenal cortex and brain capillaries being consistently and intensely labelled. However, the immunolabelling in the kidney was in contradiction to man and dogs. The C219 antibody seems to react with a specific form of P-gp, only expressed in feline tissues with a barrier function, i.e. endothelia of the brain, testes and ovaries, and intestinal epithelial cells in contact with the lumen. [source] Synthesis and Biological Evaluation of 2-Mercapto-1,3-benzothiazole Derivatives with Potential Antimicrobial ActivityARCHIV DER PHARMAZIE, Issue 10 2009Carlo Franchini Abstract The enhancement of bacterial resistance of pathogens to currently available antibiotics constitutes a serious public health threat. So, intensive efforts are underway worldwide to develop new antimicrobial agents. To identify compounds with a potent antimicrobial profile, we designed and synthesized low molecular weight 2-mercaptobenzothiazole derivatives 2a,2l and 3a,3l. Both series were screened for in-vitro antibacterial activity against the representative panel of Gram-positive and Gram-negative bacteria strains. The biological screening identified compounds 2e and 2l as the most active ones showing an interesting antibacterial activity with MIC values of 3.12 ,g/mL against Staphylococcus aureus and 25 ,g/mL against Escherichia coli, respectively. The replacement of the S-H by the S-Bn moiety resulted in considerable loss of the antibacterial action of the 3a,3l series. The antibiotic action of compounds 2e and 2l was also investigated by testing their activity against some clinical isolates with different antimicrobial resistance profile. Moreover, the involvement of the NorA efflux pump in the antibacterial activity of our molecules was evaluated. Finally, in this paper, we also describe the cytotoxic activity of the most interesting compounds by MTS assay against HeLa and MRC-5 cell lines. [source] Crystallization and preliminary X-ray crystallographic analysis of MacA from Actinobacillus actinomycetemcomitansACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2008Shunfu Piao Periplasmic membrane-fusion proteins (MFPs) are an essential component of the multidrug efflux pump in Gram-negative bacteria. They play a crucial role in bridging the outer membrane porin TolC and two distinct types of inner membrane transporters. The MFP MacA bridges the inner membrane ABC-type multidrug transporter MacB and the outer membrane porin TolC. MacA from the pathogenic bacterium Actinobacillus actinomycetemcomitans was expressed in Escherichia coli B834 (DE3) and the recombinant protein was purified using Ni,NTA affinity, Q anion-exchange and gel-filtration chromatography. The purified MacA protein was crystallized using the vapour-diffusion method. A MAD diffraction data set was collected to a resolution of 3.0,Ĺ at 100,K. The crystal belongs to space group P622, with unit-cell parameters a = b = 109.2, c = 255.4,Ĺ, , = , = 90, , = 120°, and contains one molecule in the asymmetric unit. [source] Iron acquisition within host cells and the pathogenicity of LeishmaniaCELLULAR MICROBIOLOGY, Issue 2 2008Chau Huynh Summary Iron is an essential cofactor for several enzymes and metabolic pathways, in both microbes and in their eukaryotic hosts. To avoid toxicity, iron acquisition is tightly regulated. This represents a particular challenge for pathogens that reside within the endocytic pathway of mammalian cells, because endosomes and lysosomes are gradually depleted in iron by host transporters. An important player in this process is Nramp1 (Slc11a1), a proton efflux pump that translocates Fe2+ and Mn2+ ions from macrophage lysosomes/phagolysosomes into the cytosol. Mutations in Nramp1 cause susceptibility to infection with the bacteria Salmonella and Mycobacteria and the protozoan Leishmania, indicating that an available pool of intraphagosomal iron is critical for the intracellular survival and replication of these pathogens. Salmonella and Mycobacteria are known to express iron transporter systems that effectively compete with host transporters for iron. Until recently, however, very little was known about the molecular strategy used by Leishmania for survival in the iron-poor environment of macrophage phagolysosomes. It is now clear that intracellular residence induces Leishmania amazonensis to express LIT1, a ZIP family membrane Fe2+ transporter that is required for intracellular growth and virulence. [source] Is plasmid-mediated quinolone resistance a clinically significant problem?CLINICAL MICROBIOLOGY AND INFECTION, Issue 4 2008L. Poirel Abstract Although resistance to quinolones is commonly chromosomally-encoded in Enterobacteriaceae, the emergence of plasmid-mediated quinolone resistance (PMQR) has also been reported, with at least three known resistance mechanisms to date, i.e., Qnr, aminoglycoside acetyltransferase AAC(6,)-Ib-cr and QepA. Qnr proteins protect target enzymes (DNA gyrase and type IV topoisomerase) from quinolone inhibition, the AAC(6,)-Ib-cr enzyme acetylates norfloxacin and ciprofloxacin, and the QepA efflux pump extrudes hydrophilic fluoroquinolones. Although these PMQR determinants confer only low-level resistance to quinolones and/or fluoroquinolones, they may provide a favourable background in which the selection of additional chromosomally-encoded quinolone resistance mechanisms can occur. [source] Activity of telithromycin compared with seven other agents against 1039 Streptococcus pyogenes pediatric isolates from ten centers in central and eastern EuropeCLINICAL MICROBIOLOGY AND INFECTION, Issue 7 2003B. Bozdogan In total, 1039 pediatric Streptococcus pyogenes isolates from Bulgaria, Croatia, the Czech Republic, Hungary, Latvia, Lithuania, Poland, Romania, Slovakia and Slovenia were studied. All strains were susceptible to penicillin G, levofloxacin, and quinupristin,dalfopristin, 91,100% to telithromycin, and 82,100% to erythromycin, azithromycin, and clarithromycin, and 90,100% to clindamycin. Macrolide resistance occurred mainly in Slovakia (25%), the Czech Republic (17.3%), and Croatia (15.8%). Overall, 9.7% of S. pyogenes isolates were erythromycin resistant due to erm(B) - or erm(A) -encoded methylases (72.3%) or to a mef(A) -encoded efflux pump (25.7%). One strain had alterations of both 23S rRNA (A2058G Escherichia coli numbering) and ribosomal protein L22 (G95D). [source] Evidence for efflux pumps, other than PmrA, associated with fluoroquinolone resistance in Streptococcus pneumoniaeCLINICAL MICROBIOLOGY AND INFECTION, Issue 2 2003N. P. Brenwald Fluoroquinolone resistance in pneumococci is known to be associated with the efflux pump, PmrA. However, there may be other efflux systems that also cause drug resistance. Two types of mutants were studied. The efflux phenotype from mutants selected by sub-MIC levofloxacin or gemifloxacin was transformed into R6. These transformants did not show increased pmrA transcripts in Northern blots; insertional inactivation of pmrA in the transformants did not abolish the efflux phenotype. A second set of efflux phenotype mutants was selected in R6:cat by ethidium bromide but not by norfloxacin; accumulation of ethidium bromide in the one among these mutants studied was reduced in comparison to its parent. This evidence suggests that systems other than PmrA can contribute to efflux-mediated resistance in pneumococci. [source] The ttgGHI solvent efflux pump operon of Pseudomonas putida DOT-T1E is located on a large self-transmissible plasmidENVIRONMENTAL MICROBIOLOGY, Issue 6 2007José J. Rodríguez-Herva Summary Pseudomonas putida DOT-T1E is a solvent-tolerant strain able to grow in the presence of > 1% (v/v) toluene in the culture medium. A set of multidrug efflux pumps have been found to play a major role in the tolerance of this bacterium to organic solvents (Rojas et al., J Bacteriol 183: 3967,3973). In the course of studies of the mechanisms underlying solvent tolerance in DOT-T1E, we isolated a spontaneous solvent-sensitive mutant derivative which had lost the genes encoding the TtgGHI efflux pump, the most important extrusion element in quantitative terms. Genomic comparisons between the mutant and its parental strain by microarray analysis revealed that in addition to the ttgVW-ttgGHI gene cluster, another group of genes, highly similar to those found in the Tn4653A and ISPpu12 transposable elements of the TOL plasmid pWW0 from P. putida mt-2, were also absent from this strain. Further analysis demonstrated that strain DOT-T1E harboured a large plasmid (named pGRT1) that was lost from the solvent-sensitive mutant. Mapping analysis revealed that the ttgVW-ttgGHI genes and the Tn4653A -like transposon are borne by the pGRT1 plasmid. Plasmid pGRT1 is highly stable and its frequency of loss is below 10,8 per cell per generation under a variety of growth conditions, including nutritional and physical stresses. The pGRT1 plasmid is self-transmissible, and its acquisition by the toluene-sensitive P. putida KT2440 and Pseudomonas aeruginosa PAO1 increased the recipient's tolerance to toluene up to levels similar to those exhibited by P. putida DOT-T1E. We discuss the importance and potential benefits of this plasmid for the development of bacteria with enhanced solvent tolerance, and its potential impact for bioremediation and whole-cell biotransformations. [source] Modulation of P-glycoprotein-mediated multidrug resistance by acceleration of passive drug permeation across the plasma membraneFEBS JOURNAL, Issue 23 2007Ronit Regev The drug concentration inside multidrug-resistant cells is the outcome of competition between the active export of drugs by drug efflux pumps, such as P-glycoprotein (Pgp), and the passive permeation of drugs across the plasma membrane. Thus, reversal of multidrug resistance (MDR) can occur either by inhibition of the efflux pumps or by acceleration of the drug permeation. Among the hundreds of established modulators of Pgp-mediated MDR, there are numerous surface-active agents potentially capable of accelerating drug transbilayer movement. The aim of the present study was to determine whether these agents modulate MDR by interfering with the active efflux of drugs or by allowing for accelerated passive permeation across the plasma membrane. Whereas Pluronic P85, Tween-20, Triton X-100 and Cremophor EL modulated MDR by inhibition of Pgp-mediated efflux, with no appreciable effect on transbilayer movement of drugs, the anesthetics chloroform, benzyl alcohol, diethyl ether and propofol modulated MDR by accelerating transbilayer movement of drugs, with no concomitant inhibition of Pgp-mediated efflux. At higher concentrations than those required for modulation, the anesthetics accelerated the passive permeation to such an extent that it was not possible to estimate Pgp activity. The capacity of the surface-active agents to accelerate passive drug transbilayer movement was not correlated with their fluidizing characteristics, measured as fluorescence anisotropy of 1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene. This compound is located among the headgroups of the phospholipids and does not reflect the fluidity in the lipid core of the membranes where the limiting step of drug permeation, namely drug flip-flop, occurs. [source] The transporters Pdr5p and Snq2p mediate diazaborine resistance and are under the control of the gain-of-function allele PDR1-12FEBS JOURNAL, Issue 6 2004Eva Wehrschütz-Sigl The spontaneous acquisition of resistance to a variety of unrelated cytotoxic compounds has important implications in medical treatment of infectious diseases and anticancer therapy. In the yeast Saccharomyces cerevisiae this phenomenon is caused by overexpression of membrane efflux pumps and is called pleiotropic drug resistance. We have found that allelic forms of the genes for the transcription activators Pdr1p and Pdr3p, designated PDR1-12 and PDR3-33, respectively, mediate resistance to diazaborine. Here we demonstrate that the transporters Pdr5p and Snq2p are involved in diazaborine detoxification. We report that in the PDR3-33 mutant diazaborine resistance is exerted mainly via overexpression of the PDR5 and SNQ2 genes, while in the PDR1-12 mutant, additional genes, i.e. the Yap1p target genes FLR1 and YCF1, are also involved in diazaborine detoxification. In addition, we show that in the presence of cycloheximide or diazaborine PDR5 can be activated by additional transcription factors beside Pdr1p and Pdr3p. [source] Permeation of tetracyclines through membranes of liposomes and Escherichia coliFEBS JOURNAL, Issue 2 2000Albrecht Sigler Uptake of tetracycline (tc), 2-tetracyclinonitrile (CN-tc), and 9-(N,N -dimethylglycylamido)-6-demethyl-6-deoxytetracycline (DMG-DMDOT) by liposomes containing Tet repressor (TetR) and by Escherichia coli cells overexpressing TetR was examined. TetR specifically binds to tetracyclines, enhances their fluorescence and thereby allows selective detection of tetracyclines that have crossed the membranes. Analysis of the diffusion of tc and DMG-DMDOT into liposomes yielded permeation coefficients of (2.4 ± 0.6) × 10,9 cm·s,1 and (3.3 ± 0.8) × 10,9 cm·s,1, respectively. Similar coefficients were obtained for uptake of these tetracyclines by E. coli, indicating that diffusion through the cytoplasmic membrane is the rate-limiting step. The permeation coefficients translate into half-equilibration times of approximately 35 ± 15 min and explain how efflux pumps can mediate resistance against tetracyclines. Furthermore, diffusion of CN-tc into liposomes was at least 400-fold slower than that of tc, indicating that the carboxamide group at position C2 is required for efficient permeation of tc through lipid membranes and thereby explaining the lack of antibiotic activity of CN-tc. [source] Functional interaction sites of OprM with MexAB in the Pseudomonas aeruginosa multidrug efflux pumpFEMS MICROBIOLOGY LETTERS, Issue 2 2009Eisaku Yoshihara Abstract Subunit-swapping between Pseudomonas aeruginosa MexAB-OprM and MexEF-OprN efflux pumps has shown that OprM can interact with MexEF to produce a functional efflux pump, but that OprN cannot functionally interact with MexAB. Taking advantage of this subunit selectivity, we carried out experiments using chimeric proteins composed of OprM and OprN to determine which regions of OprM are necessary for functional interaction with MexAB. We constructed two types of chimeric proteins: one with the N-terminal half of OprM and the C-terminal half of OprN (OprMN), and the second with these halves reversed (OprNM). Introduction of either of the chimeric protein genes into a mutant expressing MexEF alone restored the functionality of the efflux pump. However, expression of OprMN or OprNM in the presence of MexAB did not restore the pump functionality, indicating that the both the N- and C-terminal halves of OprM are necessary for a functional interaction with MexAB. [source] Bacterial silver resistance: molecular biology and uses and misuses of silver compoundsFEMS MICROBIOLOGY REVIEWS, Issue 2-3 2003Simon Silver Abstract Resistance to silver compounds as determined by bacterial plasmids and genes has been defined by molecular genetics. Silver resistance conferred by the Salmonella plasmid pMGH100 involves nine genes in three transcription units. A sensor/responder (SilRS) two-component transcriptional regulatory system governs synthesis of a periplasmic Ag(I)-binding protein (SilE) and two efflux pumps (a P-type ATPase (SilP) plus a three-protein chemiosmotic RND Ag(I)/H+ exchange system (SilCBA)). The same genes were identified on five of 19 additional IncH incompatibility class plasmids but thus far not on other plasmids. Of 70 random enteric isolates from a local hospital, isolates from catheters and other Ag-exposed sites, and total genomes of enteric bacteria, 10 have recognizable sil genes. The centrally located six genes are found and functional in the chromosome of Escherichia coli K-12, and also occur on the genome of E. coli O157:H7. The use of molecular epidemiological tools will establish the range and diversity of such resistance systems in clinical and non-clinical sources. Silver compounds are used widely as effective antimicrobial agents to combat pathogens (bacteria, viruses and eukaryotic microorganisms) in the clinic and for public health hygiene. Silver cations (Ag+) are microcidal at low concentrations and used to treat burns, wounds and ulcers. Ag is used to coat catheters to retard microbial biofilm development. Ag is used in hygiene products including face creams, ,alternative medicine' health supplements, supermarket products for washing vegetables, and water filtration cartridges. Ag is generally without adverse effects for humans, and argyria (irreversible discoloration of the skin resulting from subepithelial silver deposits) is rare and mostly of cosmetic concern. [source] Dynamics of in vitro acquisition of resistance by Candida parapsilosis to different azolesFEMS YEAST RESEARCH, Issue 4 2009Ana Teresa Pinto e Silva Abstract Candida parapsilosis is a common isolate from clinical fungal infectious episodes. Resistance of C. parapsilosis to azoles has been increasingly reported. To analyse the development of resistance in C. parapsilosis, four azole-susceptible clinical strains and one American Type Culture Collection type strain were cultured in the presence of fluconazole, voriconazole and posaconazole at different concentrations. The isolates developed variable degrees of azole resistance according to the antifungal used. Fluconazole was the fastest inducer while posaconazole was the slowest. Fluconazole and voriconazole induced resistance to themselves and each other, but not to posaconazole. Posaconazole induced resistance to all azoles. Developed resistance was stable; it could be confirmed after 30 days of subculture in drug-free medium. Azole-resistant isolates revealed a homogeneous population structure; the role of azole transporter efflux pumps was minor after evaluation by microdilution and cytometric assays with efflux pump blockers (verapamil, ibuprofen and carbonyl cyanide 3-chloro-phenylhydrazone). We conclude that the rapid development of azole resistance occurs by a mechanism that might involve mutation of genes responsible for ergosterol biosynthesis pathway, stressed by exposure to antifungals. [source] Hyaluronan oligosaccharides sensitize lymphoma resistant cell lines to vincristine by modulating P-glycoprotein activity and PI3K/Akt pathwayINTERNATIONAL JOURNAL OF CANCER, Issue 5 2008Rosalía I. Cordo Russo Abstract Multidrug resistance (MDR) is one of the main reasons for failure of cancer therapy. It may be mediated by overexpression of ATP-dependent efflux pumps or by alterations in survival or apoptotic pathways. Fragments generated by enzymatic degradation of hyaluronan (oHA) were able to modulate growth and cell survival and sensitize MDR breast cancer cells to cytotoxic drugs. In this work the relationship between oHA and MDR in lymphoid malignancies was analyzed using murine lymphoma cell lines resistant to doxorubicin (LBR-D160) or vincristine (LBR-V160) and a sensitive line (LBR-). After oHA treatment, higher apoptosis levels were observed in the resistant cell lines than in the sensitive one. Besides, oHA sensitized LBR-D160 and LBR-V160 to vincristine showing increased apoptosis induction when used in combination with vincristine. Native hyaluronan failed to increase apoptosis levels. As different survival factors could be modulated by hyaluronan, we investigated the PI3K/Akt pathway through PIP3 production and phosphorylated Akt (p-Akt) and survivin expression was also evaluated. Our results showed that oHA decreased p-Akt in the 3 cell lines while anti-CD44 treatment abolished this effect. Besides, survivin was downregulated only in LBR-V160 by oHA. When Pgp function was evaluated, we observed that oHA were able to inhibit Pgp efflux in murine and human resistant cell lines in a CD44-dependent way. In summary, we report for the first time that oHA per se modulate MDR in lymphoma cells by decreasing p-Akt as well as Pgp activity, thus suggesting that oHA could be useful in combination with classical chemotherapy in MDR hematological malignancies. © 2007 Wiley-Liss, Inc. [source] Differences in multidrug resistance phenotype and matrix metalloproteinases activity between endothelial cells from normal brain and gliomaJOURNAL OF NEUROCHEMISTRY, Issue 2 2003Anthony Régina Abstract Endothelial cells (ECs) are new targets for tumor therapy. In this work, we purified endothelial cells from intracerebral and subcutaneous experimental gliomas as well as from normal brain in order to define some of the phenotypical differences between angiogenic and quiescent brain vasculature. We show that the multidrug resistance genes encoding drug efflux pumps at the brain endothelium are expressed differently in normal and tumoral vasculature. We also show that ECs from gliomas present increased activity of gelatinase B (MMP9), key enzyme in the angiogenic process. Importantly, we observe a different phenotype between ECs in the intracerebral and subcutaneous models. Our results provide molecular evidence of phenotypic distinction between tumoral and normal brain vasculature and indicate that the EC phenotype depends on interactions both with tumor cells and also with the microenvironment. [source] Fluoroquinolone efflux mediated by ABC transportersJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 9 2008Ana I. Alvarez Abstract Quinolones and fluoroquinolones are broad spectrum bactericidal drugs, which are widely used in both human and veterinary medicine. These drugs can quite easily enter cells and are often used to treat intracellular pathogens. Some fluoroquinolones have been reported to undergo efflux, which could explain their low bioavailability. There is a growing need to understand resistance mechanisms to quinolones, involving for instance mutations or the action of efflux pumps. Several members of the ATP-binding cassette (ABC) drug efflux transporter family (MDR, MRP, ABCG2) significantly affect the pharmacokinetic disposition of quinolones. Active secretory mechanisms common to all fluoroquinolones have been suggested, as well as competition between fluoroquinolones at transporter sites. For grepafloxacin and its metabolites, MRP2 has been demonstrated to mediate biliary excretion. However, MDR1 is responsible for grepafloxacin intestinal secretion. Recently it has been shown that ciprofloxacin and enrofloxacin are efficiently transported ABCG2 substrates which are actively secreted into milk. It appears that multiple ABC transporters contribute to the overall secretion of fluoroquinolones. The objective of this work is to review the recent advances in insights into ABC transporters and their effects on fluoroquinolone disposition and resistance including data on drug secretion into milk. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:3483,3493, 2008 [source] | ||||||||||||||||