Development of Purine-Derived 18F-Labeled Pro-drug Tracers for Imaging of MRP1 Activity with PET

PubMed Central

2014-01-01

Multidrug resistance-associated protein 1 (MRP1) is a drug efflux transporter that has been implicated in the pathology of several neurological diseases and is associated with development of multidrug resistance. To enable measurement of MRP1 function in the living brain, a series of 6-halopurines decorated with fluorinated side chains have been synthesized and evaluated as putative pro-drug tracers. The tracers were designed to undergo conjugation with glutathione within the brain and hence form the corresponding MRP1 substrate tracers in situ. 6-Bromo-7-(2-[18F]fluoroethyl)purine showed good brain uptake and rapid metabolic conversion. Dynamic PET imaging demonstrated a marked difference in brain clearance rates between wild-type and mrp1 knockout mice, suggesting that the tracer can allow noninvasive assessment of MRP1 activity in vivo. PMID:24456310

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. Our study evaluates an often-underappreciated aspect of resistance-the impact of evolutionary selection. We evaluate selection on all annotated efflux genes in all sequenced ESKAPE pathogens, providing critical context for and insight into current and future development of efflux-targeting treatments for resistant bacterial infections.

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 developed to date. Our study evaluates an often-underappreciated aspect of resistance—the impact of evolutionary selection. We evaluate selection on all annotated efflux genes in all sequenced ESKAPE pathogens, providing critical context for and insight into current and future development of efflux-targeting treatments for resistant bacterial infections. PMID:29719870

Importance of Real-Time Assays To Distinguish Multidrug Efflux Pump-Inhibiting and Outer Membrane-Destabilizing Activities in Escherichia coli.

PubMed

Misra, Rajeev; Morrison, Keith D; Cho, Hyun Jae; Khuu, Thanh

2015-08-01

The constitutively expressed AcrAB multidrug efflux system of Escherichia coli shows a high degree of homology with the normally silent AcrEF system. Exposure of a strain with acrAB deleted to antibiotic selection pressure frequently leads to the insertion sequence-mediated activation of the homologous AcrEF system. In this study, we used strains constitutively expressing either AcrAB or AcrEF from their normal chromosomal locations to resolve a controversy about whether phenylalanylarginine β-naphthylamide (PAβN) inhibits the activities of AcrAB and AcrEF and/or acts synergistically with antibiotics by destabilizing the outer membrane permeability barrier. Real-time efflux assays allowed a clear distinction between the efflux pump-inhibiting activity of PAβN and the outer membrane-destabilizing action of polymyxin B nonapeptide (PMXBN). When added in equal amounts, PAβN, but not PMXBN, strongly inhibited the efflux activities of both AcrAB and AcrEF pumps. In contrast, when outer membrane destabilization was assessed by the nitrocefin hydrolysis assay, PMXBN exerted a much greater damaging effect than PAβN. Strong action of PAβN in inhibiting efflux activity compared to its weak action in destabilizing the outer membrane permeability barrier suggests that PAβN acts mainly by inhibiting efflux pumps. We concluded that at low concentrations, PAβN acts specifically as an inhibitor of both AcrAB and AcrEF efflux pumps; however, at high concentrations, PAβN in the efflux-proficient background not only inhibits efflux pump activity but also destabilizes the membrane. The effects of PAβN on membrane integrity are compounded in cells unable to extrude PAβN. The increase in multidrug-resistant bacterial pathogens at an alarming rate has accelerated the need for implementation of better antimicrobial stewardship, discovery of new antibiotics, and deeper understanding of the mechanism of drug resistance. The work carried out in this study highlights the importance of employing real-time fluorescence-based assays in differentiating multidrug efflux-inhibitory and outer membrane-destabilizing activities of antibacterial compounds. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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 preclinical development of lead EPIs. PMID:21470111

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

An allosteric transport mechanism for the AcrAB-TolC multidrug efflux pump

PubMed Central

Wang, Zhao; Fan, Guizhen; Hryc, Corey F; Blaza, James N; Serysheva, Irina I; Schmid, Michael F; Chiu, Wah; Luisi, Ben F; Du, Dijun

2017-01-01

Bacterial efflux pumps confer multidrug resistance by transporting diverse antibiotics from the cell. In Gram-negative bacteria, some of these pumps form multi-protein assemblies that span the cell envelope. Here, we report the near-atomic resolution cryoEM structures of the Escherichia coli AcrAB-TolC multidrug efflux pump in resting and drug transport states, revealing a quaternary structural switch that allosterically couples and synchronizes initial ligand binding with channel opening. Within the transport-activated state, the channel remains open even though the pump cycles through three distinct conformations. Collectively, our data provide a dynamic mechanism for the assembly and operation of the AcrAB-TolC pump. DOI: http://dx.doi.org/10.7554/eLife.24905.001 PMID:28355133

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

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 knowledge of the small molecule efflux pumps encoded by the B. cereus group and suggest the likely functions of numerous uncharacterised pumps. PMID:28472044

High-level fluoroquinolone-resistant clinical isolates of Escherichia coli overproduce multidrug efflux protein AcrA.

PubMed

Mazzariol, A; Tokue, Y; Kanegawa, T M; Cornaglia, G; Nikaido, H

2000-12-01

Immunoblotting with antibody against AcrA, an obligatory component of the AcrAB multidrug efflux system, showed that this protein was overexpressed by >/=170% in 9 of 10 clinical isolates of Esherichia coli with high-level ciprofloxacin resistance (MICs, >/=32 microg/ml) but not in any of the 15 isolates for which the MIC was

Interplay between three RND efflux pumps in doxycycline-selected strains of Burkholderia thailandensis.

PubMed

Biot, Fabrice Vincent; Lopez, Mélanie Monique; Poyot, Thomas; Neulat-Ripoll, Fabienne; Lignon, Sabrina; Caclard, Arnaud; Thibault, François Michel; Peinnequin, Andre; Pagès, Jean-Marie; Valade, Eric

2013-01-01

Efflux systems are involved in multidrug resistance in most Gram-negative non-fermentative bacteria. We have chosen Burkholderia thailandensis to dissect the development of multidrug resistance phenotypes under antibiotic pressure. We used doxycycline selection to obtain several resistant B. thailandensis variants. The minimal inhibitory concentrations of a large panel of structurally unrelated antibiotics were determined ± the efflux pump inhibitor phenylalanine-arginine ß-naphthylamide (PAßN). Membrane proteins were identified by proteomic method and the expressions of major efflux pumps in the doxycycline selected variants were compared to those of the parental strains by a quantitative RT-PCR analysis. Doxycycline selected variants showed a multidrug resistance in two major levels corresponding to the overproduction of two efflux pumps depending on its concentration: AmrAB-OprA and BpeEF-OprC. The study of two mutants, each lacking one of these pumps, indicated that a third pump, BpeAB-OprB, could substitute for the defective pump. Surprisingly, we observed antagonistic effects between PAßN and aminoglycosides or some ß-lactams. PAßN induced the overexpression of AmrAB-OprA and BpeAB-OprB pump genes, generating this unexpected effect. These results may account for the weak activity of PAßN in some Gram-negative species. We clearly demonstrated two antagonistic effects of this molecule on bacterial cells: the blocking of antibiotic efflux and an increase in efflux pump gene expression. Thus, doxycycline is a very efficient RND efflux pump inducer and PAßN may promote the production of some efflux pumps. These results should be taken into account when considering antibiotic treatments and in future studies on efflux pump inhibitors.

Sensitive and Specific Fluorescent Probes for Functional Analysis of the Three Major Types of Mammalian ABC Transporters

PubMed Central

Lebedeva, Irina V.; Pande, Praveen; Patton, Wayne F.

2011-01-01

An underlying mechanism for multi drug resistance (MDR) is up-regulation of the transmembrane ATP-binding cassette (ABC) transporter proteins. ABC transporters also determine the general fate and effect of pharmaceutical agents in the body. The three major types of ABC transporters are MDR1 (P-gp, P-glycoprotein, ABCB1), MRP1/2 (ABCC1/2) and BCRP/MXR (ABCG2) proteins. Flow cytometry (FCM) allows determination of the functional expression levels of ABC transporters in live cells, but most dyes used as indicators (rhodamine 123, DiOC2(3), calcein-AM) have limited applicability as they do not detect all three major types of ABC transporters. Dyes with broad coverage (such as doxorubicin, daunorubicin and mitoxantrone) lack sensitivity due to overall dimness and thus may yield a significant percentage of false negative results. We describe two novel fluorescent probes that are substrates for all three common types of ABC transporters and can serve as indicators of MDR in flow cytometry assays using live cells. The probes exhibit fast internalization, favorable uptake/efflux kinetics and high sensitivity of MDR detection, as established by multidrug resistance activity factor (MAF) values and Kolmogorov-Smirnov statistical analysis. Used in combination with general or specific inhibitors of ABC transporters, both dyes readily identify functional efflux and are capable of detecting small levels of efflux as well as defining the type of multidrug resistance. The assay can be applied to the screening of putative modulators of ABC transporters, facilitating rapid, reproducible, specific and relatively simple functional detection of ABC transporter activity, and ready implementation on widely available instruments. PMID:21799851

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.

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

Multidrug Efflux Pumps in Staphylococcus aureus: an Update.

PubMed

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

2013-01-01

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

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

Optimized efflux assay for the NorA multidrug efflux pump in Staphylococcus aureus.

PubMed

Zimmermann, Saskia; Tuchscherr, Lorena; Rödel, Jürgen; Löffler, Bettina; Bohnert, Jürgen A

2017-11-01

Real-time fluorescent efflux assays are commonly used for measuring the efflux of bacterial pumps. Here we describe an optimized protocol for the NorA efflux pump in S. aureus using DiOC 3 instead of ethidium bromide. Glucose and sodium formate were tested as energy carriers. This novel method is fast and reproducible. Copyright © 2017 Elsevier B.V. All rights reserved.

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

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

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

PubMed

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

2010-10-01

Enterobacter aerogenes predominates amongst Enterobacteriaceae species that are increasingly reported as producers of extended-spectrum beta-lactamases. Although this mechanism of resistance to beta-lactams is important, other mechanisms bestowing a multidrug-resistant (MDR) phenotype in this species are now well documented. Amongst these mechanisms is the overexpression of efflux pumps that extrude structurally unrelated antibiotics prior to their reaching their targets. Interestingly, although knowledge of the genetic background behind efflux pumps is rapidly advancing, few studies assess the physiological nature of the overall efflux pump system of this, or for that matter any other, bacterium. The study reported here evaluates physiologically the efflux pump system of an E. aerogenes ATCC reference as well as two strains whose MDR phenotypes are mediated by overexpressed efflux pumps. The activities of the efflux pumps in these strains are modulated by pH and glucose, although the effects of the latter are essentially restricted to pH 8, suggesting the presence of two general efflux pump systems, i.e. proton-motive force-dependent and ABC transporter types, respectively. Copyright 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

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

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.

Multidrug Resistance Protein 1 (MRP1, ABCC1), a “Multitasking” ATP-binding Cassette (ABC) Transporter*

PubMed Central

Cole, Susan P. C.

2014-01-01

The multidrug resistance protein 1 (MRP1) encoded by ABCC1 was originally discovered as a cause of multidrug resistance in tumor cells. However, it is now clear that MRP1 serves a broader role than simply mediating the ATP-dependent efflux of drugs from cells. The antioxidant GSH and the pro-inflammatory cysteinyl leukotriene C4 have been identified as key physiological organic anions effluxed by MRP1, and an ever growing body of evidence indicates that additional lipid-derived mediators are also substrates of this transporter. As such, MRP1 is a multitasking transporter that likely influences the etiology and progression of a host of human diseases. PMID:25281745

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.

Reviving Antibiotics: Efflux Pump Inhibitors That Interact with AcrA, a Membrane Fusion Protein of the AcrAB-TolC Multidrug Efflux Pump

DOE PAGES

Abdali, Narges; Parks, Jerry M.; Haynes, Keith M.; ...

2016-10-21

Antibiotic resistance is a major threat to human welfare. Inhibitors of multidrug efflux pumps (EPIs) are promising alternative therapeutics that could revive activities of antibiotics and reduce bacterial virulence. Identification of new druggable sites for inhibition is critical for developing effective EPIs, especially in light of constantly emerging resistance. We describe new EPIs that interact with and possibly inhibit the function of periplasmic membrane fusion proteins, critical components of efflux pumps that are responsible for the activation of the transporter and the recruitment of the outer-membrane channel. The discovered EPIs bind to AcrA, a component of the prototypical AcrAB-TolC pump,more » change its structure in vivo, inhibit efflux of fluorescent probes and potentiate the activities of antibiotics in Escherichia coli cells. These findings expand the chemical and mechanistic diversity of EPIs, suggest the mechanism for regulation of the efflux pump assembly and activity, and provide a promising path for reviving the activities of antibiotics in resistant bacteria.« less

Reviving Antibiotics: Efflux Pump Inhibitors That Interact with AcrA, a Membrane Fusion Protein of the AcrAB-TolC Multidrug Efflux Pump

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

Abdali, Narges; Parks, Jerry M.; Haynes, Keith M.

Antibiotic resistance is a major threat to human welfare. Inhibitors of multidrug efflux pumps (EPIs) are promising alternative therapeutics that could revive activities of antibiotics and reduce bacterial virulence. Identification of new druggable sites for inhibition is critical for developing effective EPIs, especially in light of constantly emerging resistance. We describe new EPIs that interact with and possibly inhibit the function of periplasmic membrane fusion proteins, critical components of efflux pumps that are responsible for the activation of the transporter and the recruitment of the outer-membrane channel. The discovered EPIs bind to AcrA, a component of the prototypical AcrAB-TolC pump,more » change its structure in vivo, inhibit efflux of fluorescent probes and potentiate the activities of antibiotics in Escherichia coli cells. These findings expand the chemical and mechanistic diversity of EPIs, suggest the mechanism for regulation of the efflux pump assembly and activity, and provide a promising path for reviving the activities of antibiotics in resistant bacteria.« less

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

PubMed

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

2016-08-01

During the early periods of antibiotic usage, bacterial infections were considered tamed. However, widespread antibiotic use has promoted the emergence of antibiotic-resistant pathogens, including multidrug resistant strains. Active efflux is a mechanism for bacterial resistance to inhibitory substances, known simply as drug efflux pumps. The bacterium Staphylococcus aureus is an important pathogenic bacterium responsible for an array of infections. The NorA efflux pump has been shown to be responsible for moderate fluoroquinolone resistance of S. aureus. The inhibition of the efflux pump was assayed using a sub-inhibitory concentration of standard efflux pump inhibitors and tannic acid (MIC/8), where its capacity to decrease the MIC of Ethidium bromide (EtBr) and antibiotics due to the possible inhibitory effect of these substances was observed. The MICs of EtBr and antibiotics were significantly reduced in the presence of tannic acid, indicating the inhibitory effect of this agent against the efflux pumps of both strains causing a three-fold reduction of the MIC when compared with the control. These results indicate the possible usage of tannic acid as an adjuvant in antibiotic therapy against multidrug resistant bacteria (MDR). Copyright © 2016 Elsevier Ltd. All rights reserved.

Bacterial resistance modifying tetrasaccharide agents from Ipomoea murucoides.

PubMed

Chérigo, Lilia; Pereda-Miranda, Rogelio; Gibbons, Simon

2009-01-01

As part of an ongoing project to identify oligosaccharides which modulate bacterial multidrug resistance, the CHCl(3)-soluble extract from flowers of a Mexican arborescent morning glory, Ipomoea murucoides, through preparative-scale recycling HPLC, yielded five lipophilic tetrasaccharide inhibitors of Staphylococcusaureus multidrug efflux pumps, murucoidins XII-XVI (1-5). The macrocyclic lactone-type structures for these linear hetero-tetraglycoside derivatives of jalapinolic acid were established by spectroscopic methods. These compounds were tested for in vitro antibacterial and resistance modifying activity against strains of Staphylococcus aureus possessing multidrug resistance efflux mechanisms. Only murucoidin XIV (3) displayed antimicrobial activity against SA-1199B (MIC 32microg/ml), a norfloxacin-resistant strain that over-expresses the NorA MDR efflux pump. The four microbiologically inactive (MIC>512microg/ml) tetrasaccharides increased norfloxacin susceptibility of this strain by 4-fold (8microg/ml from 32microg/ml) at concentrations of 25microg/ml, while murucoidin XIV (3) exerted the same potentiation effect at a concentration of 5microg/ml.

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.

A subset of virus-specific CD161+ T cells selectively express the multidrug transporter MDR1 and are resistant to chemotherapy in AML

PubMed Central

Alsuliman, Abdullah; Muftuoglu, Muharrem; Khoder, Ahmad; Ahn, Yong-Oon; Basar, Rafet; Verneris, Michael R.; Muranski, Pawel; Barrett, A. John; Liu, Enli; Li, Li; Stringaris, Kate; Armstrong-James, Darius; Shaim, Hila; Kondo, Kayo; Imahashi, Nobuhiko; Andersson, Borje; Marin, David; Champlin, Richard E.; Shpall, Elizabeth J.

2017-01-01

The establishment of long-lived pathogen-specific T cells is a fundamental property of the adaptive immune response. However, the mechanisms underlying long-term persistence of antigen-specific CD4+ T cells are not well-defined. Here we identify a subset of memory CD4+ T cells capable of effluxing cellular toxins, including rhodamine (Rho), through the multidrug efflux protein MDR1 (also known as P-glycoprotein and ABCB1). Drug-effluxing CD4+ T cells were characterized as CD161+CD95+CD45RA−CD127hiCD28+CD25int cells with a distinct chemokine profile and a Th1-polarized pro-inflammatory phenotype. CD4+CD161+Rho-effluxing T cells proliferated vigorously in response to stimulation with anti-CD3/CD28 beads and gave rise to CD161− progeny in vitro. These cells were also capable of self-renewal and maintained their phenotypic and functional characteristics when cultured with homeostatic cytokines. Multidrug-effluxing CD4+CD161+ T cells were enriched within the viral-specific Th1 repertoire of healthy donors and patients with acute myeloid leukemia (AML) and survived exposure to daunorubicin chemotherapy in vitro. Multidrug-effluxing CD4+CD161+ T cells also resisted chemotherapy-induced cytotoxicity in vivo and underwent significant expansion in AML patients rendered lymphopenic after chemotherapy, contributing to the repopulation of anti-CMV immunity. Finally, after influenza vaccination, the proportion of influenza-specific CD4+ T cells coexpressing CD161 was significantly higher after 2 years compared with 4 weeks after immunization, suggesting CD161 is a marker for long-lived antigen-specific memory T cells. These findings suggest that CD4+CD161+ T cells with rapid efflux capacity contribute to the maintenance of viral-specific memory T cells. These data provide novel insights into mechanisms that preserve antiviral immunity in patients undergoing chemotherapy and have implications for the development of novel immunotherapeutic approaches. PMID:27821506

Genomic introgression mapping of field-derived multiple-anthelmintic resistance in Teladorsagia circumcincta

PubMed Central

Hallsworth-Pepin, Kymberlie; Martin, John; Mitreva, Makedonka

2017-01-01

Preventive chemotherapy has long been practiced against nematode parasites of livestock, leading to widespread drug resistance, and is increasingly being adopted for eradication of human parasitic nematodes even though it is similarly likely to lead to drug resistance. Given that the genetic architecture of resistance is poorly understood for any nematode, we have analyzed multidrug resistant Teladorsagia circumcincta, a major parasite of sheep, as a model for analysis of resistance selection. We introgressed a field-derived multiresistant genotype into a partially inbred susceptible genetic background (through repeated backcrossing and drug selection) and performed genome-wide scans in the backcross progeny and drug-selected F2 populations to identify the major genes responsible for the multidrug resistance. We identified variation linking candidate resistance genes to each drug class. Putative mechanisms included target site polymorphism, changes in likely regulatory regions and copy number variation in efflux transporters. This work elucidates the genetic architecture of multiple anthelmintic resistance in a parasitic nematode for the first time and establishes a framework for future studies of anthelmintic resistance in nematode parasites of humans. PMID:28644839

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

Insertion and release of a solute into and from a vessel comprising biopolymers is a fundamental function in a biological system. A typical example is found in a multidrug efflux transporter. "Multidrug efflux" signifies that solutes such as drug molecules with diverse properties can be handled. In our view, the mechanism of the multidrug efflux is not chemically specific but rather has to be based on a physical factor. In earlier works, we showed that the spatial distribution of the solute-vessel potential of mean force (PMF) induced by the solvent plays imperative roles in the insertion/release process. The PMF can be decomposed into the energetic and entropic components. The entropic component, which originates from the translational displacement of solvent molecules, is rather insensitive to the solute-solvent and vessel inner surface-solvent affinities. This feature is not shared with the energetic component. When the vessel inner surface is neither solvophobic nor solvophilic, the solvents within the vessel cavity and in the bulk offer almost the same environment to any solute with solvophobicity or solvophilicity, and the energetic component becomes much smaller than the entropic component (i.e., the latter predominates over the former). Our idea is that the multidrug efflux can be realized if the insertion/release process is accomplished by the entropic component exhibiting the insensitivity to the solute properties. However, we have recently argued that the entropic release of the solute is not feasible as long as the vessel geometry is fixed. Here we consider a model of TolC, a cylindrical vessel possessing an entrance at one end and an exit at the other end for the solute. The spatial distribution of the PMF is calculated by employing the three-dimensional integral equation theory with rigid-body models in which the constituents interact only through hard-body potentials. Since the behavior of these models is purely entropic in origin, our analysis is 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.

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.

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

Solution Binding and Structural Analyses Reveal Potential Multidrug Resistance Functions for SAV2435 and CTR107 and Other GyrI-like Proteins.

PubMed

Moreno, Andrew; Froehlig, John R; Bachas, Sharrol; Gunio, Drew; Alexander, Teressa; Vanya, Aaron; Wade, Herschel

2016-08-30

Multidrug resistance (MDR) refers to the acquired ability of cells to tolerate a broad range of toxic compounds. One mechanism cells employ is to increase the level of expression of efflux pumps for the expulsion of xenobiotics. A key feature uniting efflux-related mechanisms is multidrug (MD) recognition, either by efflux pumps themselves or by their transcriptional regulators. However, models describing MD binding by MDR effectors are incomplete, underscoring the importance of studies focused on the recognition elements and key motifs that dictate polyspecific binding. One such motif is the GyrI-like domain, which is found in several MDR proteins and is postulated to have been adapted for small-molecule binding and signaling. Here we report the solution binding properties and crystal structures of two proteins containing GyrI-like domains, SAV2435 and CTR107, bound to various ligands. Furthermore, we provide a comparison with deposited crystal structures of GyrI-like proteins, revealing key features of GyrI-like domains that not only support polyspecific binding but also are conserved among GyrI-like domains. Together, our studies suggest that GyrI-like domains perform evolutionarily conserved functions connected to multidrug binding and highlight the utility of these types of studies for elucidating mechanisms of MDR.

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.

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

PubMed

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

2013-08-01

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

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.

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

PubMed Central

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

2013-01-01

It is known that bacteria showing a multi-drug resistance phenotype use several mechanisms to overcome the action of antibiotics. As a result, this phenotype can be a result of several mechanisms or a combination of thereof. The main mechanisms of antibiotic resistance are: mutations in target genes (such as DNA gyrase and topoisomerase IV); over-expression of efflux pumps; changes in the cell envelope; down regulation of membrane porins, and modified lipopolysaccharide component of the outer cell membrane (in the case of Gram-negative bacteria). In addition, adaptation to the environment, such as quorum sensing and biofilm formation can also contribute to bacterial persistence. Due to the rapid emergence and spread of bacterial isolates showing resistance to several classes of antibiotics, methods that can rapidly and efficiently identify isolates whose resistance is due to active efflux have been developed. However, there is still a need for faster and more accurate methodologies. Conventional methods that evaluate bacterial efflux pump activity in liquid systems are available. However, these methods usually use common efflux pump substrates, such as ethidium bromide or radioactive antibiotics and therefore, require specialized instrumentation, which is not available in all laboratories. In this review, we will report the results obtained with the Ethidium Bromide-agar Cartwheel method. This is an easy, instrument-free, agar based method that has been modified to afford the simultaneous evaluation of as many as twelve bacterial strains. Due to its simplicity it can be applied to large collections of bacteria to rapidly screen for multi-drug resistant isolates that show an over-expression of their efflux systems. The principle of the method is simple and relies on the ability of the bacteria to expel a fluorescent molecule that is substrate for most efflux pumps, ethidium bromide. In this approach, the higher the concentration of ethidium bromide required to produce fluorescence of the bacterial mass, the greater the efflux capacity of the bacterial cells. We have tested and applied this method to a large number of Gram-positive and Gram-negative bacteria to detect efflux activity among these multi-drug resistant isolates. The presumptive efflux activity detected by the Ethidium Bromide-agar Cartwheel method was subsequently confirmed by the determination of the minimum inhibitory concentration for several antibiotics in the presence and absence of known efflux pump inhibitors. PMID:23589748

ramR mutations affecting fluoroquinolone susceptibility in epidemic multidrug-resistant Salmonella enterica serovar Kentucky ST198

PubMed Central

Baucheron, Sylvie; Le Hello, Simon; Doublet, Benoît; Giraud, Etienne; Weill, François-Xavier; Cloeckaert, Axel

2013-01-01

A screening for non-target mutations affecting fluoroquinolone susceptibility was conducted in epidemic multidrug-resistant Salmonella enterica serovar Kentucky ST198. Among a panel of representative isolates (n = 27), covering the epidemic, only three showed distinct mutations in ramR resulting in enhanced expression of genes encoding the AcrAB-TolC efflux system and low increase in ciprofloxacin MIC. No mutations were detected in other regulatory regions of this efflux system. Ciprofloxacin resistance in serovar Kentucky ST198 is thus currently mainly due to multiple target gene mutations. PMID:23914184

Overcoming Multidrug Resistance in Human Cancer Cells by Natural Compounds

PubMed Central

Nabekura, Tomohiro

2010-01-01

Multidrug resistance is a phenomenon whereby tumors become resistant to structurally unrelated anticancer drugs. P-glycoprotein belongs to the large ATP-binding cassette (ABC) transporter superfamily of membrane transport proteins. P-glycoprotein mediates resistance to various classes of anticancer drugs including vinblastine, daunorubicin, and paclitaxel, by actively extruding the drugs from the cells. The quest for inhibitors of anticancer drug efflux transporters has uncovered natural compounds, including (-)-epigallocatechin gallate, curcumin, capsaicin, and guggulsterone, as promising candidates. In this review, studies on the effects of natural compounds on P-glycoprotein and anticancer drug efflux transporters are summarized. PMID:22069634

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

PubMed Central

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

2011-01-01

Background Traditional antibiotics are increasingly suffering from the emergence of multidrug resistance amongst pathogenic bacteria leading to a range of novel approaches to control microbial infections being investigated as potential alternative treatments. One plausible antimicrobial alternative could be the combination of conventional antimicrobial agents/antibiotics with small molecules which block multidrug efflux systems known as efflux pump inhibitors. Bioassay-driven purification and structural determination of compounds from plant sources have yielded a number of pump inhibitors which acted against gram positive bacteria. Methodology/Principal Findings In this study we report the identification and characterization of 4′,5′-O-dicaffeoylquinic acid (4′,5′-ODCQA) from Artemisia absinthium as a pump inhibitor with a potential of targeting efflux systems in a wide panel of Gram-positive human pathogenic bacteria. Separation and identification of phenolic compounds (chlorogenic acid, 3′,5′-ODCQA, 4′,5′-ODCQA) was based on hyphenated chromatographic techniques such as liquid chromatography with post column solid-phase extraction coupled with nuclear magnetic resonance spectroscopy and mass spectroscopy. Microbial susceptibility testing and potentiation of well know pump substrates revealed at least two active compounds; chlorogenic acid with weak antimicrobial activity and 4′,5′-ODCQA with pump inhibitory activity whereas 3′,5′-ODCQA was ineffective. These intitial findings were further validated with checkerboard, berberine accumulation efflux assays using efflux-related phenotypes and clinical isolates as well as molecular modeling methodology. Conclusions/Significance These techniques facilitated the direct analysis of the active components from plant extracts, as well as dramatically reduced the time needed to analyze the compounds, without the need for prior isolation. The calculated energetics of the docking poses supported the biological information for the inhibitory capabilities of 4′,5′-ODCQA and furthermore contributed evidence that CQAs show a preferential binding to Major Facilitator Super family efflux systems, a key multidrug resistance determinant in gram-positive bacteria. PMID:21483731

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.

pH-induced conformational changes of AcrA, the membrane fusion protein of Escherichia coli multidrug efflux system.

PubMed

Ip, Hermia; Stratton, Kelly; Zgurskaya, Helen; Liu, Jun

2003-12-12

The multidrug efflux system AcrA-AcrB-TolC of Escherichia coli expels a wide range of drugs directly into the external medium from the bacterial cell. The mechanism of the efflux process is not fully understood. Of an elongated shape, AcrA is thought to span the periplasmic space coordinating the concerted operation of the inner and outer membrane proteins AcrB and TolC. In this study, we used site-directed spin labeling (SDSL) EPR (electron paramagnetic resonance) spectroscopy to investigate the molecular conformations of AcrA in solution. Ten AcrA mutants, each with an alanine to cysteine substitution, were engineered, purified, and labeled with a nitroxide spin label. EPR analysis of spin-labeled AcrA variants indicates that the side chain mobilities are consistent with the predicted secondary structure of AcrA. We further demonstrated that acidic pH induces oligomerization and conformational change of AcrA, and that the structural changes are reversible. These results suggest that the mechanism of action of AcrA in drug efflux is similar to the viral membrane fusion proteins, and that AcrA actively mediates the efflux of substrates.

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

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

Strategies to potentiate antimicrobial photoinactivation by overcoming resistant phenotypes†

PubMed Central

Vera, D. Mariano A.; Haynes, Mark H; Ball, Anthony R.; Dai, D. Tianhong; Astrakas, Christos; Kelso, Michael J; Hamblin, Michael R; Tegos, George P.

2012-01-01

Conventional antimicrobial strategies have become increasingly ineffective due to the emergence of multidrug resistance among pathogenic microorganisms. The need to overcome these deficiencies has triggered the exploration of alternative treatments and unconventional approaches towards controlling microbial infections. Photodynamic therapy was originally established as an anti-cancer modality and is currently used in the treatment of age related macular degeneration. The concept of photodynamic inactivation requires cell exposure to light energy, typically wavelengths in the visible region that causes the excitation of photosensitizer molecules either exogenous or endogenous, which results in the production of reactive oxygen species. ROS produce cell inactivation and death through modification of intracellular components. The versatile characteristics of PDT prompted its investigation as an anti-infective discovery platform. Advances in understanding of microbial physiology have shed light on a series of pathways, and phenotypes that serve as putative targets for antimicrobial drug discovery. Investigations of these phenotypic elements in concert with PDT have been reported focused on multidrug efflux systems, biofilms, virulence and pathogenesis determinants. In many instances the results are promising but only preliminary and require further investigation. This review discusses the different antimicrobial PDT strategies and highlights the need for highly informative and comprehensive discovery approaches. PMID:22242675

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.

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

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

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.

Multidrug Transport Protein NorM from Vibrio cholerae Simultaneously Couples to Sodium- and Proton-Motive Force*

PubMed Central

Jin, Yoonhee; Nair, Asha; van Veen, Hendrik W.

2014-01-01

Membrane transporters belonging to the multidrug and toxic compound extrusion family mediate the efflux of unrelated pharmaceuticals from the interior of the cell in organisms ranging from bacteria to human. These proteins are thought to fall into two classes that couple substrate efflux to the influx of either Na+ or H+. We studied the energetics of drug extrusion by NorM from Vibrio cholerae in proteoliposomes in which purified NorM protein was functionally reconstituted in an inside-out orientation. We establish that NorM simultaneously couples to the sodium-motive force and proton-motive force, and biochemically identify protein regions and residues that play important roles in Na+ or H+ binding. As the positions of protons are not available in current medium and high-resolution crystal structures of multidrug and toxic compound extrusion transporters, our findings add a previously unrecognized parameter to mechanistic models based of these structures. PMID:24711447

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.

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 resistant strains of P. aeruginosa and E. coli. PMID:25025665

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 resistant strains of P. aeruginosa and E. coli.

Multidrug Resistance: Physiological Principles and Nanomedical Solutions

PubMed Central

Storm, Gert; Kiessling, Fabian; Lammers, Twan

2014-01-01

Multidrug (MDR) resistance is a pathophysiological phenomenon employed by cancer cells which limits the prolonged and effective use of chemotherapeutic agents. MDR is primarily based on the over-expression of drug efflux pumps in the cellular membrane. Prominent examples of such efflux pumps, which belong to the ATP-binding cassette (ABC) superfamily of proteins, are Pgp (P-glycoprotein) and MRP (multidrug resistance-associated protein), nowadays officially known as ABCB1 and ABCC1. Over the years, several strategies have been evaluated to overcome MDR, based not only on the use of low-molecular-weight MDR modulators, but also on the implementation of 1-100(0) nm-sized drug delivery systems. In the present manuscript, after introducing the most important physiological principles of MDR, we summarize prototypic nanomedical strategies to overcome multidrug resistance, including the use of carrier materials with intrinsic anti-MDR properties, the use of nanomedicines to modify the mode of cellular uptake, and the co-formulation of chemotherapeutic drugs together with low- and high-molecular-weight MDR inhibitors within a single drug delivery system. While certain challenges still need to be overcome before such constructs and concepts can be widely applied in the clinic, the insights obtained and the progress made strongly suggest that nanomedicine formulations hold significant potential for improving the treatment of multidrug-resistant malignancies. PMID:24120954

Reversal of multidrug resistance by surfactants.

PubMed Central

Woodcock, D. M.; Linsenmeyer, M. E.; Chojnowski, G.; Kriegler, A. B.; Nink, V.; Webster, L. K.; Sawyer, W. H.

1992-01-01

Cremophor EL, a pharmacologically inactive solubilising agent, has been shown to reverse multidrug resistance (MDR). Using flow cytometric evaluation of equilibrium intracellular levels of daunorubicin (DNR), we found that eight other surface active agents will also reverse MDR. All the active detergents contain polyethoxylated moieties but have no similarities in their hydrophobic components. The properties of three polyethoxylated surfactants that showed the lowest toxicities, Cremophor, Tween 80 and Solutol HS15, were examined in more detail. The concentrations of Tween 80 and Solutol required to reverse DNR exclusion were 10-fold lower than for Cremophor. However while concentrations greater than or equal to 1:10(2) of the former two surfactants resulted in breakdown of cells, even 1:10 of Cremophor did not lyse cells. Studies of the effects of Cremophor on the uptake and efflux of DNR in normal and MDR cell types showed that Cremophor increases intracellular DNR primarily by locking the rapid efflux from the cells. This blockage of drug efflux may be mediated by a substantial alteration in the fluidity of cell membranes induced by Cremophor, as shown by decreased fluorescence anisotropy of a membrane probe. Consistent with these data, coinjection of adriamycin plus Cremophor into mice carrying a multidrug resistant P388 transplantable tumour significantly increased the survival time of the mice compared with adriamycin treatment alone. PMID:1637678

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.

Coupling of UDP-glucuronosyltransferases and multidrug resistance-associated proteins is responsible for the intestinal disposition and poor bioavailability of emodin

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

Liu, Wei; Feng, Qian; Li, Ye

2012-12-15

Emodin is a poorly bioavailable but promising plant-derived anticancer drug candidate. The low oral bioavailability of emodin is due to its extensive glucuronidation in the intestine and liver. Caco-2 cell culture model was used to investigate the interplay between UDP-glucuronosyltransferases (UGTs) and efflux transporters in the intestinal disposition of emodin. Bidirectional transport assays of emodin at different concentrations were performed in the Caco-2 monolayers with or without multidrug resistance-associated protein (MRP) and breast cancer resistance protein (BCRP) efflux transporter chemical inhibitors. The bidirectional permeability of emodin and its glucuronide in the Caco-2 monolayers was determined. Emodin was rapidly metabolized tomore » emodin glucuronide in Caco-2 cells. LTC4, a potent inhibitor of MRP2, decreased the efflux of emodin glucuronide and also substantially increased the intracellular glucuronide level in the basolateral-to-apical (B–A) direction. MK-571, chemical inhibitor of MRP2, MRP3, and MRP4, significantly reduced the efflux of glucuronide in the apical-to-basolateral (A–B) and B–A directions in a dose-dependent manner. However, dipyridamole, a BCRP chemical inhibitor demonstrated no effect on formation and efflux of emodin glucuronide in Caco-2 cells. In conclusion, UGT is a main metabolic pathway for emodin in the intestine, and the MRP family is composed of major efflux transporters responsible for the excretion of emodin glucuronide in the intestine. The coupling of UGTs and MRP efflux transporters causes the extensive metabolism, excretion, and low bioavailability of emodin. -- Highlights: ► Glucuronidation is the main reason for the poor oral bioavailability of emodin. ► Efflux transporters are involved in the excretion of emodin glucuronide. ► The intestine is the main organ for metabolism of emodin.« less

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

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 ...

Effect of drug efflux transporters on placental transport of antiretroviral agent abacavir.

PubMed

Neumanova, Zuzana; Cerveny, Lukas; Greenwood, Susan L; Ceckova, Martina; Staud, Frantisek

2015-11-01

Abacavir is as a frequent part of combination antiretroviral therapy used in pregnant women. The aim of this study was to investigate, using in vitro, in situ and ex vivo experimental approaches, whether the transplacental pharmacokinetics of abacavir is affected by ATP-binding cassette (ABC) efflux transporters functionally expressed in the placenta: P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2), multidrug resistance-associated protein 2 (ABCC2) and multidrug resistance-associated protein 5 (ABCC5). In vitro transport assays revealed that abacavir is a substrate of human ABCB1 and ABCG2 transporters but not of ABCC2 or ABCC5. In addition, in situ experiments using dually perfused rat term placenta confirmed interactions of abacavir with placental Abcb1/Abcg2. In contrast, uptake studies in human placental villous fragments did not reveal any interaction of abacavir with efflux transporters suggesting a large contribution of passive diffusion and/or influx mechanisms to net transplacental abacavir transfer. Copyright © 2015 Elsevier Inc. All rights reserved.

An efflux pump (MexAB-OprM) of Pseudomonas aeruginosa is associated with antibacterial activity of Epigallocatechin-3-gallate (EGCG).

PubMed

Kanagaratnam, Rashmi; Sheikh, Rida; Alharbi, Fahad; Kwon, Dong H

2017-12-01

Pseudomonas aeruginosa is a notorious multidrug resistant nosocomial pathogen. An efflux pump (MexAB-OprM) is the main contributor to the multidrug resistance in clinical isolates of P. aeruginosa. Epigallocatechin-3-gallate (EGCG), a polyphenolic compound extracted from green tea, exhibits antibacterial activity. It is unclear that molecular details of the antibacterial activity of EGCG, EGCG-effect on antibiotic susceptibility, and clinical relevance of EGCG in bacteria. This study aimed to determine the roles of the efflux pump and an efflux pump inhibitor (phenylalanine-arginine β-naphthylamide; PAβN) in the antibacterial activity of EGCG and the EGCG-effect on antibiotic susceptibility. Twenty-two multidrug resistant clinical isolates of P. aeruginosa and a wild type P. aeruginosa PAO1 were used to determine antibacterial activity of EGCG and EGCG-effect on antibiotic susceptibility. An efflux pump (MexAB-OPrM) mutant strain, its complemented strain carrying an intact mexAB-oprM, and their parental strain were used to determine roles of MexAB-OprM in the antibacterial activity of EGCG and EGCG-mediated antibiotic susceptibility. PAβN was also used to evaluate EGCG as a possible efflux pump inhibitor. EGCG inhibited cellular growth and killed 100% of cells at 64-512 µg/ml and at 256-1024 µg/ml, respectively, in all tested 22 clinical isolates including the wild type strain. A subinhibitory concentration of EGCG significantly enhanced susceptibility to antibiotics, unexceptionally to chloramphenicol and tetracyclines (≥4-fold) of the clinical isolates. Both the antibacterial activity of EGCG and the EGCG-mediated antibiotic susceptibility were enhanced more in the efflux pump mutant strain (mexB::Gm) than the parental strain, suggesting additionally accumulated-EGCG produced the more antibacterial activity in the mutant strain. EGCG was synergistically interacted with PAβN with enhancing susceptibility to all tested antibiotics (up to >500-fold) at higher levels than either EGCG alone or PAβN alone, suggesting EGCG may also inhibit the efflux pump with additional accumulation of the antibiotics. The results demonstrate that EGCG exhibits antibacterial activity and enhances antibiotic effects against clinical isolates of P. aeruginosa. EGCG may inhibit the efflux pump (MexAB-OprM) through which are associated with the antibacterial activity of EGCG and the EGCG-mediated antibiotic susceptibility in P. aeruginosa. Copyright © 2017 Elsevier GmbH. All rights reserved.

Multidrug transport protein norM from vibrio cholerae simultaneously couples to sodium- and proton-motive force.

PubMed

Jin, Yoonhee; Nair, Asha; van Veen, Hendrik W

2014-05-23

Membrane transporters belonging to the multidrug and toxic compound extrusion family mediate the efflux of unrelated pharmaceuticals from the interior of the cell in organisms ranging from bacteria to human. These proteins are thought to fall into two classes that couple substrate efflux to the influx of either Na(+) or H(+). We studied the energetics of drug extrusion by NorM from Vibrio cholerae in proteoliposomes in which purified NorM protein was functionally reconstituted in an inside-out orientation. We establish that NorM simultaneously couples to the sodium-motive force and proton-motive force, and biochemically identify protein regions and residues that play important roles in Na(+) or H(+) binding. As the positions of protons are not available in current medium and high-resolution crystal structures of multidrug and toxic compound extrusion transporters, our findings add a previously unrecognized parameter to mechanistic models based of these structures. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Targeting multidrug resistance protein 1 (MRP1, ABCC1): past, present, and future.

PubMed

Cole, Susan P C

2014-01-01

The human ATP-binding cassette transporter multidrug resistance protein 1 (MRP1), encoded by ABCC1, was initially identified because of its ability to confer multidrug resistance in lung cancer cells. It is now established that MRP1 plays a role in protecting certain tissues from xenobiotic insults and that it mediates the cellular efflux of the proinflammatory cysteinyl leukotriene C4 as well as a vast array of other endo- and xenobiotic organic anions. Many of these are glutathione (GSH) or glucuronide conjugates, the products of Phase II drug metabolism. MRP1 also plays a role in the cellular efflux of the reduced and oxidized forms of GSH and thus contributes to the many physiological and pathophysiological processes influenced by these small peptides, including oxidative stress. In this review, the pharmacological and physiological aspects of MRP1 are considered in the context of the current status and future prospects of pharmacological and genetic modulation of MRP1 activity.

mar Operon Involved in Multidrug Resistance of Enterobacter aerogenes

PubMed Central

Chollet, Renaud; Bollet, Claude; Chevalier, Jacqueline; Malléa, Monique; Pagès, Jean-Marie; Davin-Regli, Anne

2002-01-01

We determined the sequence of the entire marRAB operon in Enterobacter aerogenes. It is functionally and structurally analogous to the Escherichia coli operon. The overexpression of E. aerogenes MarA induces a multidrug resistance phenotype in a susceptible strain, demonstrated by a noticeable resistance to various antibiotics, a decrease in immunodetected porins, and active efflux of norfloxacin. PMID:11897595

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.

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.

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.

MarA-Like Regulator of Multidrug Resistance in Yersinia pestis

PubMed Central

Udani, Rupa A.; Levy, Stuart B.

2006-01-01

MarA47Yp from Yersinia pestis, showing 47% identity to Escherichia coli MarA in its N terminus, caused resistance to antibiotics and to organic solvents when expressed in both E. coli and Y. pestis. Resistance was linked to increased expression of the AcrAB multidrug efflux pump. In four of five spontaneous multidrug-resistant mutants of Y. pestis independently selected by growth on tetracycline, the marA47Yp gene was overexpressed. The findings suggest that marA47Yp is a marA ortholog in Y. pestis. PMID:16940090

MarA-like regulator of multidrug resistance in Yersinia pestis.

PubMed

Udani, Rupa A; Levy, Stuart B

2006-09-01

MarA47(Yp) from Yersinia pestis, showing 47% identity to Escherichia coli MarA in its N terminus, caused resistance to antibiotics and to organic solvents when expressed in both E. coli and Y. pestis. Resistance was linked to increased expression of the AcrAB multidrug efflux pump. In four of five spontaneous multidrug-resistant mutants of Y. pestis independently selected by growth on tetracycline, the marA47(Yp) gene was overexpressed. The findings suggest that marA47(Yp) is a marA ortholog in Y. pestis.

Detection and characterisation of multi-drug resistance protein 1 (MRP-1) in human mitochondria.

PubMed

Roundhill, E A; Burchill, S A

2012-03-13

Overexpression of plasma membrane multi-drug resistance protein 1 (MRP-1) can lead to multidrug resistance. In this study, we describe for the first time the expression of mitochondrial MRP-1 in untreated human normal and cancer cells and tissues. MRP-1 expression and subcellular localisation in normal and cancer cells and tissues was examined by differential centrifugation and western blotting, and immunofluorescence microscopy. Viable mitochondria were isolated and MRP-1 efflux activity measured using the calcein-AM functional assay. MRP-1 expression was increased using retroviral infection and specific overexpression confirmed by RNA array. Cell viability was determined by trypan blue exclusion and annexin V-propidium iodide labelling of cells. MRP-1 was detected in the mitochondria of cancer and normal cells and tissues. The efflux activity of mitochondrial MRP-1 was more efficient (55-64%) than that of plasma membrane MRP-1 (11-22%; P<0.001). Induced MRP-1 expression resulted in a preferential increase in mitochondrial MRP-1, suggesting selective targeting to this organelle. Treatment with a non-lethal concentration of doxorubicin (0.85 nM, 8 h) increased mitochondrial and plasma membrane MRP-1, increasing resistance to MRP-1 substrates. For the first time, we have identified MRP-1 with efflux activity in human mitochondria. Mitochondrial MRP-1 may be an exciting new therapeutic target where historically MRP-1 inhibitor strategies have limited clinical success.

Antibacterial activity of exogenous glutathione and its synergism on antibiotics sensitize carbapenem-associated multidrug resistant clinical isolates of Acinetobacter baumannii.

PubMed

Alharbe, Roaa; Almansour, Ayidh; Kwon, Dong H

2017-10-01

A major clinical impact of A. baumannii is hospital-acquired infections including ventilator-associated pneumonia. The treatment of this pathogen is often difficult due to its innate and acquired resistance to almost all commercially available antibiotics. Infections with carbapenem-associated multidrug resistant A. baumannii is the most problematic. Glutathione is a tripeptide thiol-antioxidant and antibacterial activity of exogenous glutathione was reported in some bacteria. However, clinical relevance and molecular details of the antibacterial activity of glutathione are currently unclear. Seventy clinical isolates of A. baumannii including 63 carbapenem-associated multidrug resistant isolates and a type strain A. baumannii ATCC 19606 were used to determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Fractional inhibitory concentration (FIC) and time-killing activity with meropenem and/or glutathione were also determined in the carbapenem-associated multidrug resistant isolates. In addition, the roles of exogenous glutathione in multidrug efflux pumps and β-lactamase production were examined. Levels of MIC and MBC were ranged from 10 to 15mM of exogenous glutathione. All tested carbapenem-associated multidrug resistant isolates were sensitized by all tested antibiotics in combination with subinhibitory concentrations of glutathione. FIC levels of glutathione with carbapenem (meropenem) were all<0.5 and the carbapenem-associated multidrug resistant isolates were killed by subinhibitory concentrations of both glutathione and meropenem at>2log10 within 12h, suggesting glutathione synergistically interacts with meropenem. The roles of multidrug efflux pumps and β-lactamase production were excluded for the glutathione-mediated antibiotic susceptibility. Overall results demonstrate that the antibacterial activity of glutathione is clinically relevant and its synergism on antibiotics sensitizes clinical isolates of A. baumannii regardless of their resistance or susceptibility to antibiotics. This finding suggests that exogenous glutathione alone and/or in combination with existing antibiotics may be applicable to treat infections with carbapenem-associated multidrug resistant A. baumannii. Copyright © 2017 Elsevier GmbH. All rights reserved.

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 adjuvants of conventional therapy against E. coli and other Gram-negative bacteria, especially their multidrug resistant forms. Their major limitation is the high toxicity for human cells at the concentrations needed to be effective against bacteria. Their future molecular optimization to improve the efflux inhibitory properties and reduce relative toxicity will optimize their potential for clinical usage against multi-drug resistant bacterial infections due to efflux. PMID:28516003

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.

Co-formulation of P-glycoprotein Substrate and Inhibitor in Nanocarriers: An Emerging Strategy for Cancer Chemotherapy.

PubMed

Saneja, Ankit; Dubey, Ravindra Dhar; Alam, Noor; Khare, Vaibhav; Gupta, Prem N

2014-01-01

Scientific community is striving to understand the role of P-glycoprotein (P-gp) in drug discovery programs due to its impact on pharmacokinetic and multi-drug resistance (MDR) of anticancer drugs. A number of efforts to resolve the crystal structure and understanding the mechanism of P-gp mediated efflux have been made. Several generations of Pgp inhibitors have been developed to tackle this multi-specific efflux protein. Unfortunately, these inhibitors lack selectivity, exhibit poor solubility and severe pharmacokinetic interactions restricting their clinical use. The nanocarrier drug delivery systems (NDDS) are receiving increasing attention for P-gp modulating activity of pharmaceutical excipients which are used in their fabrication. In addition, NDDS can enhance the solubility and exhibited ability to bypass P-gp mediated efflux. The co-formulation of P-gp inhibitors and substrate anticancer drugs in single drug delivery system offers the advantage of bypassing P-gp mediated drug efflux as well as inhibiting the P-gp. Moreover, severe pharmacokinetic interactions between P-gp inhibitor and substrate anticancer drugs could be avoided by using this strategy. In this article we describe the co-formulation strategies using nanocarriers for modulation of pharmacokinetics as well as multi-drug resistance of anticancer drugs along with the challenges in this area.

EmrE, a multidrug transporter from Escherichia coli, transports monovalent and divalent substrates with the same stoichiometry.

PubMed

Rotem, Dvir; Schuldiner, Shimon

2004-11-19

Multidrug transporters recognize and transport substrates with apparently little common structural features. At times these substrates are neutral, negatively, or positively charged, and only limited information is available as to how these proteins deal with the energetic consequences of transport of substrates with different charges. Multidrug transporters and drug-specific efflux systems are responsible for clinically significant resistance to chemotherapeutic agents in pathogenic bacteria, fungi, parasites, and human cancer cells. Understanding how these efflux systems handle different substrates may also have practical implications in the development of strategies to overcome the resistance mechanisms mediated by these proteins. Here, we compare transport of monovalent and divalent substrates by EmrE, a multidrug transporter from Escherichia coli, in intact cells and in proteoliposomes reconstituted with the purified protein. The results demonstrated that whereas the transport of monovalent substrates involves charge movement (i.e. electrogenic), the transport of divalent substrate does not (i.e. electroneutral). Together with previous results, these findings suggest that an EmrE dimer exchanges two protons per substrate molecule during each transport cycle. In intact cells, under conditions where the only driving force is the electrical potential, EmrE confers resistance to monovalent substrates but not to divalent ones. In the presence of proton gradients, resistance to both types of substrates is detected. The finding that under some conditions EmrE does not remove certain types of drugs points out the importance of an in-depth understanding of mechanisms of action of multidrug transporters to devise strategies for coping with the problem of multidrug resistance.

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

Circumvention of the multidrug-resistance protein (MRP-1) by an antitumor drug through specific inhibition of gene transcription in breast tumor cells.

PubMed

Mansilla, Sylvia; Rojas, Marta; Bataller, Marc; Priebe, Waldemar; Portugal, José

2007-04-01

Multidrug-resistance protein 1 (MRP-1) confers resistance to a number of clinically important chemotherapeutic agents. The promoter of the mrp-1 gene contains an Sp1-binding site, which we targeted using the antitumor bis-anthracycline WP631. When MCF-7/VP breast cancer cells, which overexpress MRP-1 protein, were incubated with WP631 the expression of the multidrug-resistance protein gene decreased. Conversely, doxorubicin did not alter mrp-1 gene expression. The inhibition of gene expression was followed by a decrease in the activity of the MRP-1 protein. The IC(75) for WP631 (drug concentration required to inhibit cell growth by 75%) circumvented the drug-efflux pump, without addition of resistant modifiers. After treatment with WP631, MCF-7/VP cells were committed to die after entering mitosis (mitotic catastrophe), while treatment with doxorubicin did not affect cell growth. This is the first report on an antitumor drug molecule inhibiting the mrp-1 gene directly, rather than being simply a poor substrate for the transporter-mediated efflux. However, both situations appeared to coexist, thereby a superior cytotoxic effect was attained. Ours results suggest that WP631 offers great potential for the clinical treatment of tumors displaying a multidrug-resistance phenotype.

Crystallization and preliminary X-ray crystallographic analysis of MacA from Actinobacillus actinomycetemcomitans

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

Piao, Shunfu; Xu, Yongbin; Ha, Nam-Chul, E-mail: hnc@pusan.ac.kr

2008-05-01

A periplasmic membrane-fusion protein MacA from Actinobacillus actinomycetemcomitans, an essential component of the multidrug efflux pump in Gram-negative bacteria, was crystallized. 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 andmore » 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 Å at 100 K. The crystal belongs to space group P622, with unit-cell parameters a = b = 109.2, c = 255.4 Å, α = β = 90, γ = 120°, and contains one molecule in the asymmetric unit.« less

Draft genome sequence of a multidrug-resistant KPC-2-producing Enterobacter aerogenes isolated from a hospitalised patient in Brazil.

PubMed

Moura, Quézia; Fernandes, Miriam R; Cerdeira, Louise; Nhambe, Lúcia F; Ienne, Susan; Souza, Tiago A; Lincopan, Nilton

2017-09-01

Multidrug-resistant (MDR) Enterobacter aerogenes strains are frequently associated with nosocomial infections and high mortality rates, representing a serious public health problem. The aim of this study was to present the draft genome sequence of a MDR KPC-2-producing E. aerogenes isolated from a perineal swab of a hospitalised patient in Brazil. Genomic DNA was sequenced using an Illumina MiSeq platform. De novo genome assembly was carried out using the A5-Miseq pipeline, and whole-genome sequence analysis was performed using tools from the Center for Genomic Epidemiology. The strain harboured resistance genes to β-lactams, aminoglycosides, sulphonamides and trimethoprim in addition to genes encoding multidrug efflux system proteins, a quaternary ammonium transporter and heavy metal efflux system proteins. In addition, the strain harboured genes encoding diverse virulence factors. These data might allow a better understanding of the genetic basis of antimicrobial resistance and virulence in E. aerogenes strains. Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

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

Detection and characterisation of multi-drug resistance protein 1 (MRP-1) in human mitochondria

PubMed Central

Roundhill, E A; Burchill, S A

2012-01-01

Background: Overexpression of plasma membrane multi-drug resistance protein 1 (MRP-1) can lead to multidrug resistance. In this study, we describe for the first time the expression of mitochondrial MRP-1 in untreated human normal and cancer cells and tissues. Methods: MRP-1 expression and subcellular localisation in normal and cancer cells and tissues was examined by differential centrifugation and western blotting, and immunofluorescence microscopy. Viable mitochondria were isolated and MRP-1 efflux activity measured using the calcein-AM functional assay. MRP-1 expression was increased using retroviral infection and specific overexpression confirmed by RNA array. Cell viability was determined by trypan blue exclusion and annexin V-propidium iodide labelling of cells. Results: MRP-1 was detected in the mitochondria of cancer and normal cells and tissues. The efflux activity of mitochondrial MRP-1 was more efficient (55–64%) than that of plasma membrane MRP-1 (11–22% P<0.001). Induced MRP-1 expression resulted in a preferential increase in mitochondrial MRP-1, suggesting selective targeting to this organelle. Treatment with a non-lethal concentration of doxorubicin (0.85 n, 8 h) increased mitochondrial and plasma membrane MRP-1, increasing resistance to MRP-1 substrates. For the first time, we have identified MRP-1 with efflux activity in human mitochondria. Conclusion: Mitochondrial MRP-1 may be an exciting new therapeutic target where historically MRP-1 inhibitor strategies have limited clinical success. PMID:22353810

Mutation of Rv2887, a marR-like gene, confers Mycobacterium tuberculosis resistance to an imidazopyridine-based agent.

PubMed

Winglee, Kathryn; Lun, Shichun; Pieroni, Marco; Kozikowski, Alan; Bishai, William

2015-11-01

Drug resistance is a major problem in Mycobacterium tuberculosis control, and it is critical to identify novel drug targets and new antimycobacterial compounds. We have previously identified an imidazo[1,2-a]pyridine-4-carbonitrile-based agent, MP-III-71, with strong activity against M. tuberculosis. In this study, we evaluated mechanisms of resistance to MP-III-71. We derived three independent M. tuberculosis mutants resistant to MP-III-71 and conducted whole-genome sequencing of these mutants. Loss-of-function mutations in Rv2887 were common to all three MP-III-71-resistant mutants, and we confirmed the role of Rv2887 as a gene required for MP-III-71 susceptibility using complementation. The Rv2887 protein was previously unannotated, but domain and homology analyses suggested it to be a transcriptional regulator in the MarR (multiple antibiotic resistance repressor) family, a group of proteins first identified in Escherichia coli to negatively regulate efflux pumps and other mechanisms of multidrug resistance. We found that two efflux pump inhibitors, verapamil and chlorpromazine, potentiate the action of MP-III-71 and that mutation of Rv2887 abrogates their activity. We also used transcriptome sequencing (RNA-seq) to identify genes which are differentially expressed in the presence and absence of a functional Rv2887 protein. We found that genes involved in benzoquinone and menaquinone biosynthesis were repressed by functional Rv2887. Thus, inactivating mutations of Rv2887, encoding a putative MarR-like transcriptional regulator, confer resistance to MP-III-71, an effective antimycobacterial compound that shows no cross-resistance to existing antituberculosis drugs. The mechanism of resistance of M. tuberculosis Rv2887 mutants may involve efflux pump upregulation and also drug methylation. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Mutation of Rv2887, a marR-Like Gene, Confers Mycobacterium tuberculosis Resistance to an Imidazopyridine-Based Agent

PubMed Central

Winglee, Kathryn; Lun, Shichun; Pieroni, Marco; Kozikowski, Alan

2015-01-01

Drug resistance is a major problem in Mycobacterium tuberculosis control, and it is critical to identify novel drug targets and new antimycobacterial compounds. We have previously identified an imidazo[1,2-a]pyridine-4-carbonitrile-based agent, MP-III-71, with strong activity against M. tuberculosis. In this study, we evaluated mechanisms of resistance to MP-III-71. We derived three independent M. tuberculosis mutants resistant to MP-III-71 and conducted whole-genome sequencing of these mutants. Loss-of-function mutations in Rv2887 were common to all three MP-III-71-resistant mutants, and we confirmed the role of Rv2887 as a gene required for MP-III-71 susceptibility using complementation. The Rv2887 protein was previously unannotated, but domain and homology analyses suggested it to be a transcriptional regulator in the MarR (multiple antibiotic resistance repressor) family, a group of proteins first identified in Escherichia coli to negatively regulate efflux pumps and other mechanisms of multidrug resistance. We found that two efflux pump inhibitors, verapamil and chlorpromazine, potentiate the action of MP-III-71 and that mutation of Rv2887 abrogates their activity. We also used transcriptome sequencing (RNA-seq) to identify genes which are differentially expressed in the presence and absence of a functional Rv2887 protein. We found that genes involved in benzoquinone and menaquinone biosynthesis were repressed by functional Rv2887. Thus, inactivating mutations of Rv2887, encoding a putative MarR-like transcriptional regulator, confer resistance to MP-III-71, an effective antimycobacterial compound that shows no cross-resistance to existing antituberculosis drugs. The mechanism of resistance of M. tuberculosis Rv2887 mutants may involve efflux pump upregulation and also drug methylation. PMID:26303802

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

Structures of BmrR-Drug Complexes Reveal a Rigid Multidrug Binding Pocket And Transcription Activation Through Tyrosine Expulsion

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

Newberry, K.J.; Huffman, J.L.; Miller, M.C.

2009-05-22

BmrR is a member of the MerR family and a multidrug binding transcription factor that up-regulates the expression of the bmr multidrug efflux transporter gene in response to myriad lipophilic cationic compounds. The structural mechanism by which BmrR binds these chemically and structurally different drugs and subsequently activates transcription is poorly understood. Here, we describe the crystal structures of BmrR bound to rhodamine 6G (R6G) or berberine (Ber) and cognate DNA. These structures reveal each drug stacks against multiple aromatic residues with their positive charges most proximal to the carboxylate group of Glu-253 and that, unlike other multidrug binding pockets,more » that of BmrR is rigid. Substitution of Glu-253 with either alanine (E253A) or glutamine (E253Q) results in unpredictable binding affinities for R6G, Ber, and tetraphenylphosphonium. Moreover, these drug binding studies reveal that the negative charge of Glu-253 is not important for high affinity binding to Ber and tetraphenylphosphonium but plays a more significant, but unpredictable, role in R6G binding. In vitro transcription data show that E253A and E253Q are constitutively active, and structures of the drug-free E253A-DNA and E253Q-DNA complexes support a transcription activation mechanism requiring the expulsion of Tyr-152 from the multidrug binding pocket. In sum, these data delineate the mechanism by which BmrR binds lipophilic, monovalent cationic compounds and suggest the importance of the redundant negative electrostatic nature of this rigid drug binding pocket that can be used to discriminate against molecules that are not substrates of the Bmr multidrug efflux pump.« less

Occurrence of efflux mechanism and cephalosporinase variant in a population of Enterobacter aerogenes and Klebsiella pneumoniae isolates producing extended-spectrum beta-lactamases.

PubMed

Tran, Que-Tien; Dupont, Myrielle; Lavigne, Jean-Philippe; Chevalier, Jacqueline; Pagès, Jean-Marie; Sotto, Albert; Davin-Regli, Anne

2009-04-01

We investigated the occurrence of multidrug resistance in 44 Enterobacter aerogenes and Klebsiella pneumoniae clinical isolates. Efflux was involved in resistance in E. aerogenes isolates more frequently than in K. pneumoniae isolates (100 versus 38% of isolates) and was associated with the expression of phenylalanine arginine beta-naphthylamide-susceptible active efflux. AcrA-TolC overproduction in E. aerogenes isolates was noted. An analysis of four E. aerogenes isolates for which cefepime MICs were high revealed no modification in porin expression but a new specific mutation in the AmpC beta-lactamase.

Effect of bisphenol A on drug efflux in BeWo, a human trophoblast-like cell line.

PubMed

Jin, H; Audus, K L

2005-04-01

Bisphenol A (BPA) is a monomer of polycarbonate plastics that has estrogenic activities and has been shown to be a substrate for multidrug resistant efflux mechanisms, specifically, P-glycoprotein. Since the natural hormone estrogen reverses multidrug resistance in some cell types, we hypothesized that BPA might have a similar activity in trophoblasts. We have used BeWo cells as an in vitro model for human trophoblasts and calcein AM as a substrate for drug efflux mechanism to characterize BPA interactions with placental P-glycoprotein. We found that chronic exposure of BeWo cells to BPA did not alter intracellular calcein accumulation in a fashion that would be reflective of changes in P-glycoprotein expression. Immunoblots affirmed that BPA had small effects on P-glycoprotein expression. However, BeWo cells acutely exposed to BPA pretreatment were observed to have a significantly decreased calcein accumulation. Addition of cyclosporin A, a P-glycoprotein inhibitor and substrate, completely reversed BPA's effects on calcein accumulation and resulted in a net increase, relative to controls, in calcein accumulation by the BeWo cells. BPA was found not to stimulate P-gp ATPase or alter intracellular esterases mediating calcein release from calcein AM. Therefore, our results suggested that BPA stimulated drug efflux by BeWo cells probably by direct effects on P-glycoprotein.

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 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

TolC is required for pathogenicity of Xylella fastidiosa in Vitis vinifera grapevines.

PubMed

Reddy, Joseph D; Reddy, Stephanie L; Hopkins, Don L; Gabriel, Dean W

2007-04-01

Xylella fastidiosa infects a wide range of hosts and causes serious diseases on some of them. The complete genomic sequences of both a citrus variegated chlorosis (CVC) and a Pierce's disease (PD) strain revealed two type I protein secretion plus two multidrug resistance efflux systems, and all evidently were dependent on a single tolC homolog. Marker exchange mutagenesis of the single tolC gene in PD strain Temecula resulted in a total loss of pathogenicity on grape. Importantly, the tolC- mutant strains were not recovered after inoculation into grape xylem, strongly indicating that multidrug efflux is critical to survival of this fastidious pathogen. Both survival and pathogenicity were restored by complementation using tolC cloned in shuttle vector pBBR1MCS-5, which was shown to replicate autonomously, without selection, for 60 days in Temecula growing in planta. These results also demonstrate the ability to complement mutations in X. fastidiosa.

Inhibition of the NorA multi-drug transporter by oxygenated monoterpenes.

PubMed

Coêlho, Mayara Ladeira; Ferreira, Josie Haydée Lima; de Siqueira Júnior, José Pinto; Kaatz, Glenn W; Barreto, Humberto Medeiros; de Carvalho Melo Cavalcante, Ana Amélia

2016-10-01

The aim of this study was to investigate intrinsic antimicrobial activity of three monoterpenes nerol, dimethyl octanol and estragole, against bacteria and yeast strains, as well as, investigate if these compounds are able to inhibit the NorA efflux pump related to fluoroquinolone resistance in Staphylococcus aureus. Minimal inhibitory concentrations (MICs) of the monoterpenes against Staphylococcus aureus, Escherichia coli and Candida albicans strains were determined by micro-dilution assay. MICs of the norfloxacin against a S. aureus strain overexpressing the NorA protein were determined in the absence or in the presence of the monoterpenes at subinhibitory concentrations, aiming to verify the ability of this compounds act as efflux pump inhibitors. The monoterpenes were inactive against S. aureus however the nerol was active against E. coli and C. albicans. The addition of the compounds to growth media at sub-inhibitory concentrations enhanced the activity of norfloxacin against S. aureus SA1199-B. This result shows that bioactives tested, especially the nerol, are able to inhibit NorA efflux pump indicating a potential use as adjuvants of norfloxacin for therapy of infections caused by multi-drug resistant S. aureus strains. Copyright © 2016 Elsevier Ltd. All rights reserved.

The culturable soil antibiotic resistome: a community of multi-drug resistant bacteria.

PubMed

Walsh, Fiona; Duffy, Brion

2013-01-01

Understanding the soil bacterial resistome is essential to understanding the evolution and development of antibiotic resistance, and its spread between species and biomes. We have identified and characterized multi-drug resistance (MDR) mechanisms in the culturable soil antibiotic resistome and linked the resistance profiles to bacterial species. We isolated 412 antibiotic resistant bacteria from agricultural, urban and pristine soils. All isolates were multi-drug resistant, of which greater than 80% were resistant to 16-23 antibiotics, comprising almost all classes of antibiotic. The mobile resistance genes investigated, (ESBL, bla NDM-1, and plasmid mediated quinolone resistance (PMQR) resistance genes) were not responsible for the respective resistance phenotypes nor were they present in the extracted soil DNA. Efflux was demonstrated to play an important role in MDR and many resistance phenotypes. Clinically relevant Burkholderia species are intrinsically resistant to ciprofloxacin but the soil Burkholderia species were not intrinsically resistant to ciprofloxacin. Using a phenotypic enzyme assay we identified the antibiotic specific inactivation of trimethoprim in 21 bacteria from different soils. The results of this study identified the importance of the efflux mechanism in the soil resistome and variations between the intrinsic resistance profiles of clinical and soil bacteria of the same family.

Multidrug resistance in enteric and other gram-negative bacteria.

PubMed

George, A M

1996-05-15

In Gram-negative bacteria, multidrug resistance is a term that is used to describe mechanisms of resistance by chromosomal genes that are activated by induction or mutation caused by the stress of exposure to antibiotics in natural and clinical environments. Unlike plasmid-borne resistance genes, there is no alteration or degradation of drugs or need for genetic transfer. Exposure to a single drug leads to cross-resistance to many other structurally and functionally unrelated drugs. The only mechanism identified for multidrug resistance in bacteria is drug efflux by membrane transporters, even though many of these transporters remain to be identified. The enteric bacteria exhibit mostly complex multidrug resistance systems which are often regulated by operons or regulons. The purpose of this review is to survey molecular mechanisms of multidrug resistance in enteric and other Gram-negative bacteria, and to speculate on the origins and natural physiological functions of the genes involved.

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

Potential strategies for the eradication of multidrug-resistant Gram-negative bacterial infections.

PubMed

Huwaitat, Rawan; McCloskey, Alice P; Gilmore, Brendan F; Laverty, Garry

2016-07-01

Antimicrobial resistance is one of the leading threats to society. The increasing burden of multidrug-resistant Gram-negative infection is particularly concerning as such bacteria are demonstrating resistance to nearly all currently licensed therapies. Various strategies have been hypothesized to treat multidrug-resistant Gram-negative infections including: targeting the Gram-negative outer membrane; neutralization of lipopolysaccharide; inhibition of bacterial efflux pumps and prevention of protein folding. Silver and silver nanoparticles, fusogenic liposomes and nanotubes are potential strategies for extending the activity of licensed, Gram-positive selective, antibiotics to Gram-negatives. This may serve as a strategy to fill the current void in pharmaceutical development in the short term. This review outlines the most promising strategies that could be implemented to solve the threat of multidrug-resistant Gram-negative infections.

Novel insight into antimicrobial resistance and sensitivity phenotypes associated to qac and norA genotypes in Staphylococcus aureus.

PubMed

Marchi, Emmanuela; Furi, Leonardo; Arioli, Stefania; Morrissey, Ian; Di Lorenzo, Valeria; Mora, Diego; Giovannetti, Luciana; Oggioni, Marco Rinaldo; Viti, Carlo

2015-01-01

Staphylococcus aureus strains harboring QacA, QacB, QacC, QacG transporters and norA promoter up-regulating mutations were characterized by phenotype microarray (PM), standard methods for susceptibility testing, and ethidium bromide efflux assays, in order to increase knowledge on phenotypes associated to efflux pumps and their substrates. PM data and standard susceptibility testing lead to the identification of new potential efflux targets, such as guanidine hydrochloride or 8-hydroxyquinoline for QacA and QacC pumps, respectively. The identification of compounds to which the presence of efflux pumps induced increased susceptibility opens new perspectives for potential adjunct anti-resistance treatment (i.e. strains bearing QacB transporters showed increased susceptibility to thioridazine, amitriptyline and orphenadrine). Although the tested isolates were characterized by high degree of heterogeneity, a hallmark of clinical isolates, direct ethidium bromide efflux assays were effective in highlighting differences in efflux efficiency among strains. These data add to characterization of substrate specificity in the different classes of staphylococcal multidrug efflux systems conferring specific substrate profiles and efflux features to each of them. Copyright © 2014 Elsevier GmbH. All rights reserved.

Inhibition of bacterial multidrug resistance by celecoxib, a cyclooxygenase-2 inhibitor.

PubMed

Kalle, Arunasree M; Rizvi, Arshad

2011-01-01

Multidrug resistance (MDR) is a major problem in the treatment of infectious diseases and cancer. Accumulating evidence suggests that the cyclooxygenase-2 (COX-2)-specific inhibitor celecoxib would not only inhibit COX-2 but also help in the reversal of drug resistance in cancers by inhibiting the MDR1 efflux pump. Here, we demonstrate that celecoxib increases the sensitivity of bacteria to the antibiotics ampicillin, kanamycin, chloramphenicol, and ciprofloxacin by accumulating the drugs inside the cell, thus reversing MDR in bacteria.

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.

Molecular evidence and functional expression of multidrug resistance associated protein (MRP) in rabbit corneal epithelial cells.

PubMed

Karla, Pradeep K; Pal, Dananjay; Mitra, Ashim K

2007-01-01

Multidrug resistance associated protein (MRP) is a major family of efflux transporters involved in drug efflux leading to drug resistance. The objective of this study was to explore physical barriers for ocular drug absorption and to verify if the role of efflux transporters. MRP-2 is a major homologue of MRP family and found to express on the apical side of cell membrane. Cultured Rabbit Corneal Epithelial Cells (rCEC) were selected as an in vitro model for corneal epithelium. [14C]-erythromycin which is a proven substrate for MRP-2 was selected as a model drug for functional expression studies. MK-571, a known specific and potent inhibitor for MRP-2 was added to inhibit MRP mediated efflux. Membrane fraction of rCEC was used for western blot analysis. Polarized transport of [14C]-erythromycin was observed in rCEC and transport from B-->A was significantly high than from A-->B. Permeability's increased significantly from A-->B in the presence of MK-571 and ketoconozole. Uptake of [14C]-erythromycin in the presence of MK-571 was significantly higher than control in rCEC. RT-PCR analysis indicated a unique and distinct band at approximately 498 bp corresponding to MRP-2 in rCEC and MDCK11-MRP-2 cells. Immunoprecipitation followed by Western Blot analysis indicated a specific band at approximately 190 kDa in membrane fraction of rCEC and MDCK11-MRP-2 cells. For the first time we have demonstrated high expression of MRP-2 in rabbit corneal epithelium and its functional activity causing drug efflux. RT-PCR, immunoprecipitation followed by Western blot analysis further confirms the result.

Cross-functioning between the extraneuronal monoamine transporter and multidrug resistance protein 1 in the uptake of adrenaline and export of 5-(glutathion-S-yl)adrenaline in rat cardiomyocytes.

PubMed

Costa, Vera Marisa; Ferreira, Lusa Maria; Branco, Paula Srio; Carvalho, Flix; Bastos, Maria Lourdes; Carvalho, Rui Albuquerque; Carvalho, Mrcia; Remio, Fernando

2009-01-01

Isolated heart cells are highly susceptible to the toxicity of catecholamine oxidation products, namely, to catecholamine-glutathione adducts. Although cellular uptake and/or efflux of these products may constitute a crucial step, the knowledge about the involvement of transporters is still very scarce. This work aimed to contribute to the characterization of membrane transport mechanisms, namely, extraneuronal monoamine transporter (EMT), the multidrug resistant protein 1 (MRP1), and P-glycoprotein (P-gp) in freshly isolated cardiomyocytes from adult rats. These transporters may be accountable for uptake and/or efflux of adrenaline and an adrenaline oxidation product, 5-(glutathion-S-yl)adrenaline, in cardiomyocyte suspensions. Our results showed that 5-(glutathion-S-yl)adrenaline efflux was mediated by MRP1. Additionally, we demonstrated that the adduct formation occurs within the cardiomyocytes, since EMT inhibition reduced the intracellular adduct levels. The classical uptake2 transport in rat myocardial cells was inhibited by the typical EMT inhibitor, corticosterone, and surprisingly was also inhibited by low concentrations of another drug, a well-known P-gp inhibitor, GF120918. The P-gp activity was absent in the cells since P-gp-mediated efflux of quinidine was not blocked by GF120918. In conclusion, this work showed that freshly isolated cardiomyocytes from adult rats constitute a good model for the study of catecholamines and catecholamines metabolites membrane transport. The cardiomyocytes maintain EMT and MRP1 fully active, and these transporters contribute to the formation and efflux of 5-(glutathion-S-yl)adrenaline. In the present experimental conditions, P-gp activity is absent in the isolated cardiomyocytes.

Unlike Butylcycloheptylprodigiosin, Isolated Undecylprodigiosin from Streptomyces parvulus Is Not a MDR1 and BCRP Substrate in Multidrug-Resistant Cancers.

PubMed

Mirzaei, Seyed Abbas; Safari Kavishahi, Mansureh; Keshavarz, Zhila; Elahian, Fatemeh

2018-06-01

The search for new chemotherapeutics unaffected by efflux pumps would significantly increase life expectancy in patients with malignant cancers. In this study, butylcycloheptylprodigiosin and undecylprodigiosin were HPLC-purified and verified, using nuclear magnetic resonance spectroscopy. Cell cytotoxicity and transportation kinetics on multiple-drug resistance (MDR) cells were evaluated. Daunorubicin and butylcycloheptylprodigiosin were less toxic in the MDR1 overexpressing line, but undecylprodigiosin revealed potent toxicity toward MDR1 and BCRP expressing malignant cells. There was no noticeable change in MDR1 and BCRP transcripts during 3 days of treatment with prodiginines. While daunorubicin and mitoxantrone uptake from the cell environment significantly decreased with increasing multidrug resistance up to 46% and 62%, respectively, the accumulation of undecylprodigiosin and to a lesser extent butylcycloheptylprodigiosin in the resistance cells occurred cell- and dose-dependently via a passive diffusion process and were almost equally sensitive to the parent lines. The efflux of xenobiotics commenced immediately with different kinetics in various cells. A greater amount of daunorubicin and mitoxantrone were rapidly thrown out of their corresponding MDR cells in the absence of the specific inhibitor (3.01 and 1.81 dF/min, respectively) and represented functional efflux pumps. MDR pumps did not apparently influence undecylprodigiosin efflux patterns; but butylcycloheptylprodigiosin was partially removed from EPG85.257RDB cells at the rate of 2.66 and 1.41 dF/min in the absence and presence of verapamil, respectively.

Structural basis for the inhibition of bacterial multidrug exporters.

PubMed

Nakashima, Ryosuke; Sakurai, Keisuke; Yamasaki, Seiji; Hayashi, Katsuhiko; Nagata, Chikahiro; Hoshino, Kazuki; Onodera, Yoshikuni; Nishino, Kunihiko; Yamaguchi, Akihito

2013-08-01

The multidrug efflux transporter AcrB and its homologues are important in the multidrug resistance of Gram-negative pathogens. However, despite efforts to develop efflux inhibitors, clinically useful inhibitors are not available at present. Pyridopyrimidine derivatives are AcrB- and MexB-specific inhibitors that do not inhibit MexY; MexB and MexY are principal multidrug exporters in Pseudomonas aeruginosa. We have previously determined the crystal structure of AcrB in the absence and presence of antibiotics. Drugs were shown to be exported by a functionally rotating mechanism through tandem proximal and distal multisite drug-binding pockets. Here we describe the first inhibitor-bound structures of AcrB and MexB, in which these proteins are bound by a pyridopyrimidine derivative. The pyridopyrimidine derivative binds tightly to a narrow pit composed of a phenylalanine cluster located in the distal pocket and sterically hinders the functional rotation. This pit is a hydrophobic trap that branches off from the substrate-translocation channel. Phe 178 is located at the edge of this trap in AcrB and MexB and contributes to the tight binding of the inhibitor molecule through a π-π interaction with the pyridopyrimidine ring. The voluminous side chain of Trp 177 located at the corresponding position in MexY prevents inhibitor binding. The structure of the hydrophobic trap described in this study will contribute to the development of universal inhibitors of MexB and MexY in P. aeruginosa.

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.

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

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

Role of human placental apical membrane transporters in the efflux of glyburide, rosiglitazone, and metformin

PubMed Central

HEMAUER, Sarah J.; PATRIKEEVA, Svetlana L.; NANOVSKAYA, Tatiana N.; HANKINS, Gary D.V.; AHMED, Mahmoud S.

2010-01-01

Objective Substrates of placental efflux transporters could compete for a single transporter, which could result in an increase in the transfer of each substrate to the fetal circulation. Our aim was to determine the role of placental transporters in the biodisposition of oral hypoglycemic drugs that could be used as monotherapy or in combination therapy for gestational diabetes. Study design Inside-out brush border membrane vesicles from term placentas were used to determine the efflux of glyburide, rosiglitazone, and metformin by P-gp, Breast Cancer Resistance Protein (BCRP), and Multidrug Resistance Protein (MRP1). Results Glyburide was transported by MRP1 (43 ± 4%); BCRP (25 ± 5%); and P-gp (9 ± 5%). Rosiglitazone was transported predominantly by P-gp (71 ± 26%). Metformin was transported by P-gp (58 ± 20%) and BCRP (25 ± 14%). Conclusion Multiple placental transporters contribute to efflux of glyburide, rosiglitazone, and metformin. Administration of drug combinations could lead to their competition for efflux transporters. PMID:20350646

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 resistance to plant antimicrobials, maybe including flavonoids, which were identified as substrates of both pumps. Furthermore, we found that the mdtABC operon belongs to the regulon of the two-component regulator BaeR suggesting a role of this RND transporter in the cell envelope stress response of E. amylovora.

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 efflux pumps are involved in resistance to plant antimicrobials, maybe including flavonoids, which were identified as substrates of both pumps. Furthermore, we found that the mdtABC operon belongs to the regulon of the two-component regulator BaeR suggesting a role of this RND transporter in the cell envelope stress response of E. amylovora. PMID:25012600

Molecular basis of antimicrobial resistance in non-typable Haemophilus influenzae.

PubMed

Sánchez, L; Leranoz, S; Puig, M; Lorén, J G; Nikaido, H; Viñas, M

1997-09-01

Strains of the facultative anaerobe Haemophilus influenzae, both type b and non typable strains, are frequently multiresistant. The measurement of the antibiotic permeability of Haemophilus influenzae outer membrane (OM) shows that antibiotics can cross through the OM easily. Thus, enzymatic activity or efflux pumps could be responsible for multiresistance. An efflux system closely related to AcrAB of Escherichia coli is present in Haemophilus influenzae. However, their role in multiresistance seems irrelevant. Classical mechanisms such as plasmid exchange seems to be playing a major role in the multidrug resistance in Haemophilus influenzae.

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.

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.

Insights into the ion-coupling mechanism in the MATE transporter NorM-VC

NASA Astrophysics Data System (ADS)

Krah, Alexander; Zachariae, Ulrich

2017-08-01

Bacteria have developed a variety of different mechanisms to defend themselves from compounds that are toxic to them, such as antibiotics. One of these defence mechanisms is the expulsion of drugs or other noxious compounds by multidrug efflux pumps. Multidrug and toxic compound extrusion (MATE) transporters are efflux pumps that extrude metabolic waste and a variety of antibiotics out of the cell, using an ion gradient as energy source. They function via an alternating-access mechanism. When ions bind in the outward facing conformation, a large conformational change to the inward facing conformation is induced, from which the ion is released and the extruded chemical compound is bound. NorM proteins, which are usually coupled to a Na+ gradient, are members of the MATE family. However, for NorM-VC from Vibrio cholerae, it has been shown that this MATE transporter is additionally coupled to protons. How H+ and Na+ binding are coupled mechanistically to enable drug antiport is not well understood. In this study, we use molecular dynamics simulations to illuminate the sequence of ion binding events that enable efflux. Understanding this antiport mechanism is important to support the development of novel compounds that specifically inhibit the functional cycle of NorM transporters.

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

Nanodrug delivery in reversing multidrug resistance in cancer cells

PubMed Central

Kapse-Mistry, Sonali; Govender, Thirumala; Srivastava, Rohit; Yergeri, Mayur

2014-01-01

Different mechanisms in cancer cells become resistant to one or more chemotherapeutics is known as multidrug resistance (MDR) which hinders chemotherapy efficacy. Potential factors for MDR includes enhanced drug detoxification, decreased drug uptake, increased intracellular nucleophiles levels, enhanced repair of drug induced DNA damage, overexpression of drug transporter such as P-glycoprotein(P-gp), multidrug resistance-associated proteins (MRP1, MRP2), and breast cancer resistance protein (BCRP). Currently nanoassemblies such as polymeric/solid lipid/inorganic/metal nanoparticles, quantum dots, dendrimers, liposomes, micelles has emerged as an innovative, effective, and promising platforms for treatment of drug resistant cancer cells. Nanocarriers have potential to improve drug therapeutic index, ability for multifunctionality, divert ABC-transporter mediated drug efflux mechanism and selective targeting to tumor cells, cancer stem cells, tumor initiating cells, or cancer microenvironment. Selective nanocarrier targeting to tumor overcomes dose-limiting side effects, lack of selectivity, tissue toxicity, limited drug access to tumor tissues, high drug doses, and emergence of multiple drug resistance with conventional or combination chemotherapy. Current review highlights various nanodrug delivery systems to overcome mechanism of MDR by neutralizing, evading, or exploiting the drug efflux pumps and those independent of drug efflux pump mechanism by silencing Bcl-2 and HIF1α gene expressions by siRNA and miRNA, modulating ceramide levels and targeting NF-κB. “Theragnostics” combining a cytotoxic agent, targeting moiety, chemosensitizing agent, and diagnostic imaging aid are highlighted as effective and innovative systems for tumor localization and overcoming MDR. Physical approaches such as combination of drug with thermal/ultrasound/photodynamic therapies to overcome MDR are focused. The review focuses on newer drug delivery systems developed to overcome MDR in cancer cell. PMID:25071577

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.

Antibacterial activity of epigallocatechin-3-gallate (EGCG) and its synergism with β-lactam antibiotics sensitizing carbapenem-associated multidrug resistant clinical isolates of Acinetobacter baumannii.

PubMed

Lee, Spencer; Razqan, Ghaida Saleh Al; Kwon, Dong H

2017-01-15

Infections caused by Acinetobacter baumannii were responsive to conventional antibiotic therapy. However, recently, carbapenem-associated multidrug resistant isolates have been reported worldwide and present a major therapeutic challenge. Epigallocatechin-3-Gallate (EGCG) extracted from green tea exhibits antibacterial activity. We evaluated the antibacterial activity of EGCG and possible synergism with antibiotics in carbapenem-associated multidrug resistant A. baumannii. A potential mechanism for synergism was also explored. Seventy clinical isolates of A. baumannii collected from geographically different areas were analyzed by minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of EGCG. Checkerboard and time-killing assays were performed to exam the synergism between EGCG and antibiotics. The effects of EGCG on a multidrug efflux pump inhibitor (1-[1-naphthylmethyl] piperazine; NMP) and β-lactamase production were also examined in A. baumannii. Sixty-three of 70 clinical isolates of A. baumannii carried carbapenemase-encoding genes with carbapenem-associated multidrug resistance. Levels of MIC and MBC of EGCG ranged from 64 to 512µg/ml and from 128 to ≥1024µg/ml, respectively among the clinical isolates. MIC 90 and MBC 86 levels were 256µg/ml and 512µg/ml of EGCG, respectively. Subinhibitory concentration of EGCG in combination with all antibiotics tested, including carbapenem, sensitized (MICs fall≤1.0µg/ml) all carbapenem-associated multidrug resistant isolates. Checkerboard and time-killing assays showed synergism between EGCG and meropenem (or carbenicillin) counted as fractional inhibitory concentration of < 0.5 and cell numbers' decrease per ml of >2log10 within 12h, respectively. EGCG significantly increased the effect of NMP but was unrelated to β-lactamase production in A. baumannii, suggesting EGCG may be associated with inhibition of efflux pumps. Overall we suggest that EGCG-antibiotic combinations might provide an alternative approach to treat infections with A. baumannii regardless of antibiotic resistance. Copyright © 2016 Elsevier GmbH. All rights reserved.

Bacillus subtilis from Soybean Food Shows Antimicrobial Activity for Multidrug-Resistant Acinetobacter baumannii by Affecting the adeS Gene.

PubMed

Wang, Tieshan; Su, Jianrong

2016-12-28

Exploring novel antibiotics is necessary for multidrug-resistant pathogenic bacteria. Because the probiotics in soybean food have antimicrobial activities, we investigated their effects on multidrug-resistant Acinetobacter baumannii . Nineteen multidrug-resistant A. baumannii strains were clinifcally isolated as an experimental group and 11 multidrug-sensitive strains as controls. The growth rates of all bacteria were determined by using the analysis for xCELLigence Real-Time Cell. The combination of antibiotics showed synergistic effects on the strains in the control group but no effect on the strains in the experimental group. Efflux pump gene adeS was absent in all the strains from the control group, whereas it exists in all the strains from the experimental group. Furthermore, all the strains lost multidrug resistance when an adeS inhibitor was used. One strain of probiotics isolated from soybean food showed high antimicrobial activity for multidrug-resistant A. baumannii . The isolated strain belongs to Bacillus subtilis according to 16S RNA analysis. Furthermore, E. coli showed multidrug resistance when it was transformed with the adeS gene from A. baumannii whereas the resistant bacteria could be inhibited completely by isolated Bacillus subtilis . Thus, probiotics from soybean food provide potential antibiotics against multidrug-resistant pathogenic bacteria.

The Culturable Soil Antibiotic Resistome: A Community of Multi-Drug Resistant Bacteria

PubMed Central

Walsh, Fiona; Duffy, Brion

2013-01-01

Understanding the soil bacterial resistome is essential to understanding the evolution and development of antibiotic resistance, and its spread between species and biomes. We have identified and characterized multi-drug resistance (MDR) mechanisms in the culturable soil antibiotic resistome and linked the resistance profiles to bacterial species. We isolated 412 antibiotic resistant bacteria from agricultural, urban and pristine soils. All isolates were multi-drug resistant, of which greater than 80% were resistant to 16–23 antibiotics, comprising almost all classes of antibiotic. The mobile resistance genes investigated, (ESBL, bla NDM-1, and plasmid mediated quinolone resistance (PMQR) resistance genes) were not responsible for the respective resistance phenotypes nor were they present in the extracted soil DNA. Efflux was demonstrated to play an important role in MDR and many resistance phenotypes. Clinically relevant Burkholderia species are intrinsically resistant to ciprofloxacin but the soil Burkholderia species were not intrinsically resistant to ciprofloxacin. Using a phenotypic enzyme assay we identified the antibiotic specific inactivation of trimethoprim in 21 bacteria from different soils. The results of this study identified the importance of the efflux mechanism in the soil resistome and variations between the intrinsic resistance profiles of clinical and soil bacteria of the same family. PMID:23776501

Resin Glycosides from Ipomoea alba Seeds as Potential Chemosensitizers in Breast Carcinoma Cells.

PubMed

Cruz-Morales, Sara; Castañeda-Gómez, Jhon; Rosas-Ramírez, Daniel; Fragoso-Serrano, Mabel; Figueroa-González, Gabriela; Lorence, Argelia; Pereda-Miranda, Rogelio

2016-12-23

Multidrug resistance is the expression of one or more efflux pumps, such as P-glycoprotein, and is a major obstacle in cancer therapy. The use of new potent and noncytotoxic efflux pump modulators, coadministered with antineoplastic agents, is an alternative approach for increasing the success rate of therapy regimes with different drug combinations. This report describes the isolation and structure elucidation of six new resin glycosides from moon vine seeds (Ipomoea alba) as potential mammalian multidrug-resistance-modifying agents. Albinosides IV-IX (1-6), along with the known albinosides I-III (7-9), were purified from the CHCl 3 -soluble extract. Degradative chemical reactions in combination with NMR spectroscopy and mass spectrometry were used for their structural elucidation. Four new glycosidic acids, albinosinic acids D-G (10-13), were released by saponification of natural products 3-6. They were characterized as tetrasaccharides of either convolvulinolic (11S-hydroxytetradecanoic) or jalapinolic (11S-hydroxyhexadecanoic) acids. The potentiation of vinblastine susceptibility in multidrug-resistant human breast carcinoma cells of albinosides 1-6 was evaluated by modulation assays. The noncytotoxic albinosides VII (4) and VIII (5), at a concentration of 25 μg/mL, exerted the strongest potentiation of vinblastine susceptibility, with a reversal factor (RF MCF-7/Vin + ) of 201- and >2517-fold, respectively.

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).

Multidrug Resistance-associated Protein-1 (MRP-1)-dependent Glutathione Disulfide (GSSG) Efflux as a Critical Survival Factor for Oxidant-enriched Tumorigenic Endothelial Cells*

PubMed Central

Gordillo, Gayle M.; Biswas, Ayan; Khanna, Savita; Spieldenner, James M.; Pan, Xueliang; Sen, Chandan K.

2016-01-01

Endothelial cell tumors are the most common soft tissue tumors in infants. Tumor-forming endothelial (EOMA) cells are able to escape cell death fate despite excessive nuclear oxidant burden. Our previous work recognized perinuclear Nox-4 as a key contributor to EOMA growth. The objective of this work was to characterize the mechanisms by which EOMA cells evade oxidant toxicity and thrive. In EOMA cells, compared with in the cytosol, the nuclear GSSG/GSH ratio was 5-fold higher. Compared to the ratio observed in healthy murine aortic endothelial (MAE) cells, GSSG/GSH was over twice as high in EOMA cells. Multidrug resistance-associated protein-1 (MRP-1), an active GSSG efflux mechanism, showed 2-fold increased activity in EOMA compared with MAE cells. Hyperactive YB-1 and Ape/Ref-1 were responsible for high MRP-1 expression in EOMA. Proximity ligand assay demonstrated MRP-1 and YB-1 binding. Such binding enabled the nuclear targeting of MRP-1 in EOMA in a leptomycin-B-sensitive manner. MRP-1 inhibition as well as knockdown trapped nuclear GSSG, causing cell death of EOMA. Disulfide loading of cells by inhibition of GSSG reductase (bischoloronitrosourea) or thioredoxin reductase (auranofin) was effective in causing EOMA death as well. In sum, EOMA cells survive a heavy oxidant burden by rapid efflux of GSSG, which is lethal if trapped within the cell. A hyperactive MRP-1 system for GSSG efflux acts as a critical survival factor for these cells, making it a potential target for EOMA therapeutics. PMID:26961872

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.

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.

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

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.

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.

In Vivo Evolution of Bacterial Resistance in Two Cases of Enterobacter aerogenes Infections during Treatment with Imipenem

PubMed Central

Santini, Sébastien; Pinet, Elizabeth; Claverie, Jean-Michel; Davin-Régli, Anne-Véronique; Pagès, Jean-Marie; Masi, Muriel

2015-01-01

Infections caused by multidrug resistant (MDR) bacteria are a major concern worldwide. Changes in membrane permeability, including decreased influx and/or increased efflux of antibiotics, are known as key contributors of bacterial MDR. Therefore, it is of critical importance to understand molecular mechanisms that link membrane permeability to MDR in order to design new antimicrobial strategies. In this work, we describe genotype-phenotype correlations in Enterobacter aerogenes, a clinically problematic and antibiotic resistant bacterium. To do this, series of clinical isolates have been periodically collected from two patients during chemotherapy with imipenem. The isolates exhibited different levels of resistance towards multiple classes of antibiotics, consistently with the presence or the absence of porins and efflux pumps. Transport assays were used to characterize membrane permeability defects. Simultaneous genome-wide analysis allowed the identification of putative mutations responsible for MDR. The genome of the imipenem-susceptible isolate G7 was sequenced to closure and used as a reference for comparative genomics. This approach uncovered several loci that were specifically mutated in MDR isolates and whose products are known to control membrane permeability. These were omp35 and omp36, encoding the two major porins; rob, encoding a global AraC-type transcriptional activator; cpxA, phoQ and pmrB, encoding sensor kinases of the CpxRA, PhoPQ and PmrAB two-component regulatory systems, respectively. This report provides a comprehensive analysis of membrane alterations relative to mutational steps in the evolution of MDR of a recognized nosocomial pathogen. PMID:26398358

In Vivo Evolution of Bacterial Resistance in Two Cases of Enterobacter aerogenes Infections during Treatment with Imipenem.

PubMed

Philippe, Nadège; Maigre, Laure; Santini, Sébastien; Pinet, Elizabeth; Claverie, Jean-Michel; Davin-Régli, Anne-Véronique; Pagès, Jean-Marie; Masi, Muriel

2015-01-01

Infections caused by multidrug resistant (MDR) bacteria are a major concern worldwide. Changes in membrane permeability, including decreased influx and/or increased efflux of antibiotics, are known as key contributors of bacterial MDR. Therefore, it is of critical importance to understand molecular mechanisms that link membrane permeability to MDR in order to design new antimicrobial strategies. In this work, we describe genotype-phenotype correlations in Enterobacter aerogenes, a clinically problematic and antibiotic resistant bacterium. To do this, series of clinical isolates have been periodically collected from two patients during chemotherapy with imipenem. The isolates exhibited different levels of resistance towards multiple classes of antibiotics, consistently with the presence or the absence of porins and efflux pumps. Transport assays were used to characterize membrane permeability defects. Simultaneous genome-wide analysis allowed the identification of putative mutations responsible for MDR. The genome of the imipenem-susceptible isolate G7 was sequenced to closure and used as a reference for comparative genomics. This approach uncovered several loci that were specifically mutated in MDR isolates and whose products are known to control membrane permeability. These were omp35 and omp36, encoding the two major porins; rob, encoding a global AraC-type transcriptional activator; cpxA, phoQ and pmrB, encoding sensor kinases of the CpxRA, PhoPQ and PmrAB two-component regulatory systems, respectively. This report provides a comprehensive analysis of membrane alterations relative to mutational steps in the evolution of MDR of a recognized nosocomial pathogen.

In vitro and in vivo modulation of ABCG2 by functionalized aurones and structurally related analogs

PubMed Central

Sim, Hong-May; Wu, Chung-Pu; Ambudkar, Suresh V.; Go, Mei-Lin

2013-01-01

Over-expression of ABCG2 is linked to multidrug resistance in cancer chemotherapy. We have previously shown that functionalized aurones effectively reduced the efflux of pheophorbide A (an ABCG2 substrate) from ABCG2 over-expressing MDA-MB-231/R (“R”) cells. In the present report, we investigated the functional relevance of this observation and the mechanisms by which it occurs. Aurones and related analogs were investigated for re-sensitization of R cells to mitoxantrone (MX, a chemotherapeutic substrate of ABCG2) in cell-based assays, accumulation of intracellular MX by cell cytometry, interaction with ABCG2 by biochemical assays and in vivo efficacy in MX resistant nude mice xenografts. We found that methoxylated aurones interacted directly with ABCG2 to inhibit efflux activity, possibly by competing for occupancy of one of the substrate binding sites on ABCG2. The present evidence suggests that they are not transported by ABCG2 although they stimulate ABCG2-ATPase activity. Alteration of ABCG2 protein expression was also discounted. One member was found to re-sensitize R cells to MX in both in vitro and in vivo settings. Our study identified methoxylated aurones as promising compounds associated with low toxicities and potent modulatory effects on the ABCG2 efflux protein. Thus, they warrant further scrutiny as lead templates for development as reversal agents of multidrug resistance. PMID:21855533

SWEET sugar transporters for phloem transport and pathogen nutrition.

PubMed

Chen, Li-Qing

2014-03-01

Many intercellular solute transport processes require an apoplasmic step, that is, efflux from one cell and subsequent uptake by an adjacent cell. Cellular uptake transporters have been identified for many solutes, including sucrose; however, efflux transporters have remained elusive for a long time. Cellular efflux of sugars plays essential roles in many processes, such as sugar efflux as the first step in phloem loading, sugar efflux for nectar secretion, and sugar efflux for supplying symbionts such as mycorrhiza, and maternal efflux for filial tissue development. Furthermore, sugar efflux systems can be hijacked by pathogens for access to nutrition from hosts. Mutations that block recruitment of the efflux mechanism by the pathogen thus cause pathogen resistance. Until recently, little was known regarding the underlying mechanism of sugar efflux. The identification of sugar efflux carriers, SWEETs (Sugars Will Eventually be Exported Transporters), has shed light on cellular sugar efflux. SWEETs appear to function as uniporters, facilitating diffusion of sugars across cell membranes. Indeed, SWEETs probably mediate sucrose efflux from putative phloem parenchyma into the phloem apoplasm, a key step proceeding phloem loading. Engineering of SWEET mutants using transcriptional activator-like effector nuclease (TALEN)-based genomic editing allowed the engineering of pathogen resistance. The widespread expression of the SWEET family promises to provide insights into many other cellular efflux mechanisms.

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-function plasticity of the AcrB protein pertaining to its drug efflux activity. PMID:26240069

Multidrug Resistance Proteins (MRPs/ABCCs) in Cancer Chemotherapy and Genetic Diseases

PubMed Central

Chen, Zhe-Sheng; Tiwari, Amit K.

2011-01-01

The ATP-binding cassette (ABC) transporters are a superfamily of membrane proteins that are best known for their ability to transport a wide variety of exogenous and endogenous substances across membranes against a concentration gradient via ATP hydrolysis. There are seven subfamilies of human ABC transporters, one of the largest being the ‘C’ subfamily (gene symbol ABCC). Nine ABCC subfamily members, the so-called Multidrug Resistance Proteins (MRPs) 1-9, have been implicated in mediating multidrug resistance in tumor cells to varying degrees as the efflux extrude chemotherapeutic compounds (or their metabolites) from malignant cells. Some of the MRPs are also known to either influence drug disposition in normal tissues or modulate the elimination of drugs (or their metabolites) via hepatobiliary or renal excretory pathways. In addition, the cellular efflux of physiologically important organic anions such as leukotriene C4 and cAMP is mediated by one or more of the MRPs. Finally, mutations in several MRPs are associated with human genetic disorders. In this review article, the current biochemical and physiological knowledge of MRP1-MRP9 in cancer chemotherapy and human genetic disease is summarized. The mutations in MRP2/ABCC2 leading to conjugated hyperbilirubinemia (Dubin-Johnson syndrome) and in MRP6/ABCC6 leading to the connective tissue disorder Pseudoxanthoma elasticum are also discussed. PMID:21740521

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.

Nrf2 Regulates the Sensitivity of Mouse Keratinocytes to Nitrogen Mustard via Multidrug Resistance-Associated Protein 1 (Mrp1)

PubMed Central

Udasin, Ronald G.; Wen, Xia; Bircsak, Kristin M.; Aleksunes, Lauren M.; Shakarjian, Michael P.; Kong, Ah-Ng Tony; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

2016-01-01

Sulfur mustard and nitrogen mustard (mechlorethamine, HN2) are potent vesicants developed as chemical warfare agents. These electrophilic, bifunctional alkylating agents cause skin injury, including inflammation, edema, and blistering. HN2 covalently modifies macromolecules such as DNA, RNA, and proteins or is scavenged by glutathione, forming adducts that can contribute to toxicity. Multidrug resistance-associated protein 1 (Mrp1/MRP1) is a transmembrane ATPase known to efflux glutathione-conjugated electrophiles. In the present studies, we examined the effects of modulating Mrp1-mediated transport activity on the sensitivity of primary and PAM212 mouse keratinocytes to HN2. Primary keratinocytes, and to a lesser extent, PAM212 cells, express Mrp1 mRNA and protein and possess Mrp1 functional activity, as measured by calcein efflux. Sulforaphane, an activator of Nrf2, increased Mrp1 mRNA, protein, and functional activity in primary keratinocytes and PAM212 cells and decreased their sensitivity to HN2-induced growth inhibition (IC50 = 1.4 and 4.8 µM in primary keratinocytes and 1 and 13 µM in PAM212 cells, in the absence and presence of sulforaphane, respectively). The Mrp1 inhibitor, MK-571, reversed the effects of sulforaphane on HN2-induced growth inhibition in both primary keratinocytes and PAM212 cells. In primary keratinocytes from Nrf2−/− mice, sulforaphane had no impact on Mrp1 expression or activity, or on sensitivity to HN2, demonstrating that its effects depend on Nrf2. These data suggest that Mrp1-mediated efflux is important in regulating HN2-induced keratinocyte growth inhibition. Enhancing HN2 efflux from keratinocytes may represent a novel strategy for mitigating vesicant-induced cytotoxicity. PMID:26454883

Multidrug Resistance-associated Protein-1 (MRP-1)-dependent Glutathione Disulfide (GSSG) Efflux as a Critical Survival Factor for Oxidant-enriched Tumorigenic Endothelial Cells.

PubMed

Gordillo, Gayle M; Biswas, Ayan; Khanna, Savita; Spieldenner, James M; Pan, Xueliang; Sen, Chandan K

2016-05-06

Endothelial cell tumors are the most common soft tissue tumors in infants. Tumor-forming endothelial (EOMA) cells are able to escape cell death fate despite excessive nuclear oxidant burden. Our previous work recognized perinuclear Nox-4 as a key contributor to EOMA growth. The objective of this work was to characterize the mechanisms by which EOMA cells evade oxidant toxicity and thrive. In EOMA cells, compared with in the cytosol, the nuclear GSSG/GSH ratio was 5-fold higher. Compared to the ratio observed in healthy murine aortic endothelial (MAE) cells, GSSG/GSH was over twice as high in EOMA cells. Multidrug resistance-associated protein-1 (MRP-1), an active GSSG efflux mechanism, showed 2-fold increased activity in EOMA compared with MAE cells. Hyperactive YB-1 and Ape/Ref-1 were responsible for high MRP-1 expression in EOMA. Proximity ligand assay demonstrated MRP-1 and YB-1 binding. Such binding enabled the nuclear targeting of MRP-1 in EOMA in a leptomycin-B-sensitive manner. MRP-1 inhibition as well as knockdown trapped nuclear GSSG, causing cell death of EOMA. Disulfide loading of cells by inhibition of GSSG reductase (bischoloronitrosourea) or thioredoxin reductase (auranofin) was effective in causing EOMA death as well. In sum, EOMA cells survive a heavy oxidant burden by rapid efflux of GSSG, which is lethal if trapped within the cell. A hyperactive MRP-1 system for GSSG efflux acts as a critical survival factor for these cells, making it a potential target for EOMA therapeutics. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Candida Drug Resistance Protein 1, a Major Multidrug ATP Binding Cassette Transporter of Candida albicans, Translocates Fluorescent Phospholipids in a Reconstituted System†

PubMed Central

Shukla, Sudhanshu; Rai, Versha; Saini, Preeti; Banerjee, Dibyendu; Menon, Anant K.; Prasad, Rajendra

2008-01-01

Candida albicans drug resistance protein 1 (Cdr1p), an ATP-dependent drug efflux pump, contributes to multidrug resistance in Candida-infected immunocompromised patients. Previous cell-based assays suggested that Cdr1p also acts as a phospholipid translocator. To investigate this, we reconstituted purified Cdr1p into sealed membrane vesicles. Comparison of the ATPase activities of sealed and permeabilized proteoliposomes indicated that Cdr1p was asymmetrically reconstituted such that ~70% of the molecules had their ATP binding sites accessible to the extravesicular space. Fluorescent glycerophospholipids were incorporated into the outer leaflet of the proteoliposomes, and their transport into the inner leaflet was tracked with a quenching assay using membrane-impermeant dithionite. We observed ATP-dependent transport of the fluorescent lipids into the inner leaflet of the vesicles. With ~6 molecules of Cdr1p per vesicle on average, the half-time to reach the maximal extent of transport was ~15 min. Transport was reduced in vesicles reconstituted with Cdr1p variants with impaired ATPase activity and could be competed out to different levels by a molar excess of drugs such as fluconazole and miconazole that are known to be effluxed by Cdr1p. Transport was not affected by ampicillin, a compound that is not effluxed by Cdr1p. Our results suggest a direct link between the ability of Cdr1p to translocate fluorescent phospholipids and efflux drugs. We note that only a few members of the ABC superfamily of Candida have a well-defined role as drug exporters; thus, lipid translocation mediated by Cdr1p could reflect its cellular function. PMID:17924650

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.

The reversal effects of 3-bromopyruvate on multidrug resistance in vitro and in vivo derived from human breast MCF-7/ADR cells.

PubMed

Wu, Long; Xu, Jun; Yuan, Weiqi; Wu, Baojian; Wang, Hao; Liu, Guangquan; Wang, Xiaoxiong; Du, Jun; Cai, Shaohui

2014-01-01

P-glycoprotein mediated efflux is one of the main mechanisms for multidrug resistance in cancers, and 3-Bromopyruvate acts as a promising multidrug resistance reversal compound in our study. To test the ability of 3-Bromopyruvate to overcome P-glycoprotein-mediated multidrug resistance and to explore its mechanisms of multidrug resistance reversal in MCF-7/ADR cells, we evaluate the in vitro and in vivo modulatory activity of this compound. The in vitro and in vivo activity was determined using the MTT assay and human breast cancer xenograft models. The gene and protein expression of P-glycoprotein were determined using real-time polymerase chain reaction and the Western blotting technique, respectively. ABCB-1 bioactivity was tested by fluorescence microscopy, multi-mode microplate reader, and flow cytometry. The intracellular levels of ATP, HK-II, and ATPase activity were based on an assay kit according to the manufacturer's instructions. 3-Bromopyruvate treatment led to marked decreases in the IC50 values of selected chemotherapeutic drugs [e.g., doxorubicin (283 folds), paclitaxel (85 folds), daunorubicin (201 folds), and epirubicin (171 folds)] in MCF-7/ADR cells. 3-Bromopyruvate was found also to potentiate significantly the antitumor activity of epirubicin against MCF-7/ADR xenografts. The intracellular level of ATP decreased 44%, 46% in the presence of 12.5.25 µM 3-Bromopyruvate, whereas the accumulation of rhodamine 123 and epirubicin (two typical P-glycoprotein substrates) in cells was significantly increased. Furthermore, we found that the mRNA and the total protein level of P-glycoprotein were slightly altered by 3-Bromopyruvate. Moreover, the ATPase activity was significantly inhibited when 3-Bromopyruvate was applied. We demonstrated that 3-Bromopyruvate can reverse P-glycoprotein-mediated efflux in MCF-7/ADR cells. Multidrug resistance reversal by 3-Bromopyruvate occurred through at least three approaches, namely, a decrease in the intracellular level of ATP and HK-II bioactivity, the inhibition of ATPase activity, and the slight decrease in P-glycoprotein expression in MCF-7/ADR cells.

The Reversal Effects of 3-Bromopyruvate on Multidrug Resistance In Vitro and In Vivo Derived from Human Breast MCF-7/ADR Cells

PubMed Central

Yuan, Weiqi; Wu, Baojian; Wang, Hao; Liu, Guangquan; Wang, Xiaoxiong; Du, Jun; Cai, Shaohui

2014-01-01

Purpose P-glycoprotein mediated efflux is one of the main mechanisms for multidrug resistance in cancers, and 3-Bromopyruvate acts as a promising multidrug resistance reversal compound in our study. To test the ability of 3-Bromopyruvate to overcome P-glycoprotein-mediated multidrug resistance and to explore its mechanisms of multidrug resistance reversal in MCF-7/ADR cells, we evaluate the in vitro and in vivo modulatory activity of this compound. Methods The in vitro and in vivo activity was determined using the MTT assay and human breast cancer xenograft models. The gene and protein expression of P-glycoprotein were determined using real-time polymerase chain reaction and the Western blotting technique, respectively. ABCB-1 bioactivity was tested by fluorescence microscopy, multi-mode microplate reader, and flow cytometry. The intracellular levels of ATP, HK-II, and ATPase activity were based on an assay kit according to the manufacturer’s instructions. Results 3-Bromopyruvate treatment led to marked decreases in the IC50 values of selected chemotherapeutic drugs [e.g., doxorubicin (283 folds), paclitaxel (85 folds), daunorubicin (201 folds), and epirubicin (171 folds)] in MCF-7/ADR cells. 3-Bromopyruvate was found also to potentiate significantly the antitumor activity of epirubicin against MCF-7/ADR xenografts. The intracellular level of ATP decreased 44%, 46% in the presence of 12.5.25 µM 3-Bromopyruvate, whereas the accumulation of rhodamine 123 and epirubicin (two typical P-glycoprotein substrates) in cells was significantly increased. Furthermore, we found that the mRNA and the total protein level of P-glycoprotein were slightly altered by 3-Bromopyruvate. Moreover, the ATPase activity was significantly inhibited when 3-Bromopyruvate was applied. Conclusion We demonstrated that 3-Bromopyruvate can reverse P-glycoprotein-mediated efflux in MCF-7/ADR cells. Multidrug resistance reversal by 3-Bromopyruvate occurred through at least three approaches, namely, a decrease in the intracellular level of ATP and HK-II bioactivity, the inhibition of ATPase activity, and the slight decrease in P-glycoprotein expression in MCF-7/ADR cells. PMID:25372840

Multidrug Efflux Transporters Limit Accumulation of Inorganic, but Not Organic, Mercury in Sea Urchin Embryos

PubMed Central

Bošnjak, Ivana; Uhlinger, Kevin R.; Heim, Wesley; Smital, Tvrtko; Franekić-Čolić, Jasna; Coale, Kenneth; Epel, David; Hamdoun, Amro

2011-01-01

Mercuric compounds are persistent global pollutants that accumulate in marine organisms and in humans who consume them. While the chemical cycles and speciation of mercury in the oceans are relatively well described, the cellular mechanisms that govern which forms of mercury accumulate in cells and why they persist are less understood. In this study we examined the role of multidrug efflux transport in the differential accumulation of inorganic (HgCl2) and organic (CH3HgCl) mercury in sea urchin (Strongylocentrotus purpuratus) embryos. We found that inhibition of MRP/ABCC-type transporters increases intracellular accumulation of inorganic mercury but had no effect on accumulation of organic mercury. Similarly, pharmacological inhibition of metal conjugating enzymes by ligands GST/GSH significantly increases this antimitotic potency of inorganic mercury, but had no effect on the potency of organic mercury. Our results point to MRP-mediated elimination of inorganic mercury conjugates as a cellular basis for differences in the accumulation and potency of the two major forms of mercury found in marine environments. PMID:19924972

Reversal of multidrug resistance by morning glory resin glycosides in human breast cancer cells.

PubMed

Figueroa-González, Gabriela; Jacobo-Herrera, Nadia; Zentella-Dehesa, Alejandro; Pereda-Miranda, Rogelio

2012-01-27

Reversal of multidrug resistance (MDR) by thirty resin glycosides from the morning glory family (Convolvulaceae) was evaluated in vinblastine-resistant human breast carcinoma cells (MCF-7/Vin). The effects of these amphipathic compounds on the cytotoxicity and P-glycoprotein (P-gp)-mediated MDR were estimated with the sulforhodamine B colorimetric assay. Active noncytotoxic compounds exerted a potentiation effect of vinblastine susceptibility by 1- to over 1906-fold at tested concentrations of 5 and 25 μg/mL. Murucoidin V (1) enhanced vinblastine activity 255-fold when incorporated at 25 μg/mL and also, based on flow cytometry, significantly increased the intracellular accumulation of rhodamine 123 with the use of reserpine as a positive control for a MDR reversal agent. Incubation of MCF-7/Vin cells with 1 caused an increase in uptake and notably lowered the efflux rate of rhodamine 123. Decreased expression of P-glycoprotein by compound 1 was detected by immunofluorescence flow cytometry after incubation with an anti-P-gp monoclonal antibody. These results suggest that resin glycosides represent potential efflux pump inhibitors for overcoming MDR in cancer therapy.

Characterization of a xylose containing oligosaccharide, an inhibitor of multidrug resistance in Staphylococcus aureus, from Ipomoea pes-caprae.

PubMed

Escobedo-Martínez, Carolina; Cruz-Morales, Sara; Fragoso-Serrano, Mabel; Rahman, M Mukhlesur; Gibbons, Simon; Pereda-Miranda, Rogelio

2010-10-01

Pescaprein XVIII (1), a type of bacterial efflux pump inhibitor, was obtained from the CHCl(3)-soluble resin glycosides of beach morning glory (Ipomoea pes-caprae). The glycosidation sequence for pescaproside C, the glycosidic acid core of the lipophilic macrolactone 1 containing D-xylose and L-rhamnose, was characterized by means of several NMR techniques and FAB mass spectrometry. Recycling HPLC also yielded eight non-cytotoxic bacterial resistance modifiers, the two pescapreins XIX (2) and XX (3) as well as the known murucoidin VI (4), pecapreins II (6) and III (7), and stoloniferins III (5), IX (8) and X (9), all of which contain simonic acid B as their oligosaccharide core. Compounds 1-9 were tested for in vitro antibacterial and resistance-modifying activity against strains of Staphylococcus aureus possessing multidrug resistance efflux mechanisms. All of the pescapreins potentiated the action of norfloxacin against the NorA over-expressing strain by 4-fold (8 microg/mL from 32 microg/mL) at a concentration of 25 microg/mL. Copyright © 2010 Elsevier Ltd. All rights reserved.

In vitro studies on the putative function of N-acetylaspartate as an osmoregulator.

PubMed

Tranberg, Mattias; Abbas, Abdul-Karim; Sandberg, Mats

2007-07-01

Efflux and tissue content of N-acetylaspartate (NAA) and amino acids were evaluated from cultured and acutely prepared hippocampal slices in response to changes in osmolarity. The osmoregulator taurine, but not NAA, was lost from both types of slices after moderate reductions in extracellular osmolarity (-60 mOsm) for 10-48 h. Hypoosmotic shock (-166 mOsm) for 5 min resulted in unselective efflux of several amino acids from acutely prepared slices. Notably, the efflux of taurine, but not NAA, was prominent also after the shock. Efflux of NAA was markedly enhanced by NMDA and high K(+), in particular after the stimulation period. The high K(+)-mediated efflux was decreased by high extracellular osmolarity and a NMDA-receptor antagonist. The results indicate that NAA efflux can be induced by a sudden non-physiological decrease in extracellular osmolarity but not by prolonged more moderate changes in osmolarity. The mechanisms behind the efflux of NAA by high K(+) are complex and may involve both swelling and activation of NMDA-receptors.

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.

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.

The roles of organic anion permeases in aluminium resistance and mineral nutrition.

PubMed

Delhaize, Emmanuel; Gruber, Benjamin D; Ryan, Peter R

2007-05-25

Soluble aluminium (Al(3+)) is the major constraint to plant growth on acid soils. Plants have evolved mechanisms to tolerate Al(3+) and one type of mechanism relies on the efflux of organic anions that protect roots by chelating the Al(3+). Al(3+) resistance genes of several species have now been isolated and found to encode membrane proteins that facilitate organic anion efflux from roots. These proteins belong to the Al(3+)-activated malate transporter (ALMT) and multi-drug and toxin extrusion (MATE) families. We review the roles of these proteins in Al(3+) resistance as well as their roles in other aspects of mineral nutrition.

Multidrug resistance proteins restrain the intestinal absorption of trans-resveratrol in rats.

PubMed

Juan, M Emília; González-Pons, Eulalia; Planas, Joana M

2010-03-01

trans-Resveratrol, a natural antioxidant, has been described as a nutraceutic compound with important beneficial effects on health, but its low oral bioavailability hinders its therapeutic activity. Here, we studied the mechanisms of apical transport of trans-resveratrol in enterocytes and the role of ATP-binding cassette (ABC) transporters in the secretion of resveratrol glucuronide and sulfate resulting from the rapid intracellular metabolism. An intestinal perfusion method with recirculation in vivo was used in rats. Jejunal loops were perfused with increasing concentrations of trans-resveratrol and results showed that its uptake occurs by simple diffusion without the participation of a mediated transport. The apparent diffusion constant was 8.1 +/- 0.3 microL/(5 min.mg dry weight). The glycoprotein-P (Pgp, ABCB1), multidrug resistance-associated protein 2 (MRP2, ABCC2), and breast cancer resistance protein (BCRP, ABCG2) located in the apical membrane of enterocytes were investigated using specific inhibitors. The Pgp inhibitors verapamil (5 micromol/L) and cyclosporin A (5 micromol/L) did not affect the efflux of trans-resveratrol and its conjugates. The MRP2 inhibitors probenecid (2 mmol/L) and MK571 (10 micromol/L) reduced the efflux of glucuronide by 61 and 55%, respectively, and of sulfate by 43 and 28%, respectively. The BCRP inhibitor Ko143 (0.5 micromol/L) decreased the secretion of glucuronide by 64% and of sulfate by 46%. Our experiments identify MRP2 and BCRP as the 2 apical transporters involved in the efflux of resveratrol conjugates.

Computer simulations suggest direct and stable tip to tip interaction between the outer membrane channel TolC and the isolated docking domain of the multidrug RND efflux transporter AcrB.

PubMed

Schmidt, Thomas H; Raunest, Martin; Fischer, Nadine; Reith, Dirk; Kandt, Christian

2016-07-01

One way by which bacteria achieve antibiotics resistance is preventing drug access to its target molecule for example through an overproduction of multi-drug efflux pumps of the resistance nodulation division (RND) protein super family of which AcrAB-TolC in Escherichia coli is a prominent example. Although representing one of the best studied efflux systems, the question of how AcrB and TolC interact is still unclear as the available experimental data suggest that either both proteins interact in a tip to tip manner or do not interact at all but are instead connected by a hexamer of AcrA molecules. Addressing the question of TolC-AcrB interaction, we performed a series of 100 ns - 1 µs-molecular dynamics simulations of membrane-embedded TolC in presence of the isolated AcrB docking domain (AcrB(DD)). In 5/6 simulations we observe direct TolC-AcrB(DD) interaction that is only stable on the simulated time scale when both proteins engage in a tip to tip manner. At the same time we find TolC opening and closing freely on extracellular side while remaining closed at the inner periplasmic bottleneck region, suggesting that either the simulated time is too short or additional components are required to unlock TolC. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Zinc stress induces copper depletion in Acinetobacter baumannii.

PubMed

Hassan, Karl A; Pederick, Victoria G; Elbourne, Liam D H; Paulsen, Ian T; Paton, James C; McDevitt, Christopher A; Eijkelkamp, Bart A

2017-03-11

The first row transition metal ions zinc and copper are essential to the survival of many organisms, although in excess these ions are associated with significant toxicity. Here, we examined the impact of zinc and copper stress on Acinetobacter baumannii, a common opportunistic pathogen. We show that extracellular zinc stress induces a copper-specific depletion phenotype in A. baumannii ATCC 17978. Supplementation with copper not only fails to rescue this phenotype, but further exacerbates the copper depletion. Extensive analysis of the A. baumannii ATCC 17978 genome identified 13 putative zinc/copper resistance efflux pumps. Transcriptional analyses show that four of these transporters are responsive to zinc stress, five to copper stress and seven to the combination of zinc and copper stress, thereby revealing a likely foundation for the zinc-induced copper starvation in A. baumannii. In addition, we show that zinc and copper play crucial roles in management of oxidative stress and the membrane composition of A. baumannii. Further, we reveal that zinc and copper play distinct roles in macrophage-mediated killing of this pathogen. Collectively, this study supports the targeting of metal ion homeostatic mechanisms as an effective antimicrobial strategy against multi-drug resistant bacterial pathogens.

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

Therapeutic options and emerging alternatives for multidrug resistant staphylococcal infections.

PubMed

Magana, Maria; Ioannidis, Anastasios; Magiorkinis, Emmanouil; Ursu, Oleg; Bologa, Cristian G; Chatzipanagiotou, Stylianos; Hamblin, Michael R; Tegos, George P

2015-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) remains the single biggest challenge in infectious disease in the civilized world. Moreover, vancomycin resistance is also spreading, leading to fears of untreatable infections as were common in ancient times. Molecular microbiology and bioinformatics have revealed many of the mechanisms involved in resistance development. Mobile genetic elements, up-regulated virulence factors and multi-drug efflux pumps have been implicated. A range of approved antibiotics from the glycopeptide, lipopeptide, pleuromutilin, macrolide, oxazolidinone, lincosamide, aminoglycoside, tetracycline, steptogramin, and cephalosporin classes has been employed to treat MRSA infections. The upcoming pipeline of drugs for MRSA includes some new compounds from the above classes, together with fluoroquinolones, antibacterial peptide mimetics, aminomethylciclines, porphyrins, peptide deformylase inhibitors, oxadiazoles, and diaminopyrimidines. A range of non-drug alternative approaches has emerged for MRSA treatment. Bacteriophage-therapy including purified lysins has made a comeback after being discovered in the 1930s. Quorum-sensing inhibitors are under investigation. Small molecule inhibitors of multi-drug efflux pumps may potentiate existing antibiotics. The relative failure of staphylococcal vaccines is being revisited by efforts with multi-valent vaccines and improved adjuvants. Photodynamic therapy uses non-toxic photosensitizers and harmless visible light to produce reactive oxygen species that can nonspecifically destroy bacteria while preserving host cells. Preparation of nanoparticles can kill bacteria themselves, as well as improve the delivery of anti-bacterial drugs. Anti-MRSA drug discovery remains an exciting field with great promise for the future.

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 mitigating MDR strains of S. aureus.

CD44-engineered mesoporous silica nanoparticles for overcoming multidrug resistance in breast cancer

NASA Astrophysics Data System (ADS)

Wang, Xin; Liu, Ying; Wang, Shouju; Shi, Donghong; Zhou, Xianguang; Wang, Chunyan; Wu, Jiang; Zeng, Zhiyong; Li, Yanjun; Sun, Jing; Wang, Jiandong; Zhang, Longjiang; Teng, Zhaogang; Lu, Guangming

2015-03-01

Multidrug resistance is a major impediment for the successful chemotherapy in breast cancer. CD44 is over-expressed in multidrug resistant human breast cancer cells. CD44 monoclonal antibody exhibits anticancer potential by inhibiting proliferation and regulating P-glycoprotein-mediated drug efflux activity in multidrug resistant cells. Thereby, CD44 monoclonal antibody in combination with chemotherapeutic drug might be result in enhancing chemosensitivity and overcoming multidrug resistance. The purpose of this study is to investigate the effects of the CD44 monoclonal antibody functionalized mesoporous silica nanoparticles containing doxorubicin on human breast resistant cancer MCF-7 cells. The data showed that CD44-modified mesoporous silica nanoparticles increased cytotoxicity and enhanced the downregulation of P-glycoprotein in comparison to CD44 antibody. Moreover, CD44-engineered mesoporous silica nanoparticles provided active target, which promoted more cellular uptake of DOX in the resistant cells and more retention of DOX in tumor tissues than unengineered counterpart. Animal studies of the resistant breast cancer xenografts demonstrated that CD44-engineered drug delivery system remarkably induced apoptosis and inhibited the tumor growth. Our results indicated that the CD44-engineered mesoporous silica nanoparticle-based drug delivery system offers an effective approach to overcome multidrug resistance in human breast cancer.

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

Multidrug membrane transporters (efflux pumps) can selectively extrude a variety of structurally and functionally diverse substrates (e.g., chemotoxics, antibiotics), leading to multidrug resistance (MDR) and ineffective treatment of a wide variety of diseases. In this study, we have designed and constructed fusion gene (egfp-mexB) of N-terminal mexB with C-terminal egfp, inserted it into a plasmid vector (pMMB67EH), and successfully expressed it in ΔMexB (MexB deletion) strain of Pseudomonas aeruginosa to create a new strain that expresses MexA-(EGFP-MexB)-OprM. We characterized the fusion gene using gel electrophoresis and DNA sequencing, and determined their expression in live cells by measuring the fluorescence of EGFP in single live cells using fluorescence microscopy. Efflux function of the new strain was studied by measuring its accumulation kinetics of ethidium bromide (EtBr, a pump substrate) using fluorescence spectroscopy, which was compared with the cells (WT, ΔMexM, ΔABM, and nalB1) with various expression levels of MexAB-OprM. The new strain shows 6-fold lower accumulation rates of EtBr (15 μM) than ΔABM, 4-fold lower than ΔMexB, but only 1.1-fold higher than WT. As EtBr concentration increases to 40 μM, the new strain has nearly the same accumulation rate of EtBr as ΔMexB, but 1.4-fold higher than WT. We observed the nearly same level of inhibitory effect of CCCP (carbonyl cyanide-m-chlorophenylhydrazone) on the efflux of EtBr by the new strain and WT. Antibiotic susceptibility study shows that the minimum inhibitory concentrations (MICs) of aztreonam (AZT) and chloramphenicol (CP) for the new strain are 6-fold or 3-fold lower than WT, respectively, and 2-fold higher than those of ΔMexB. Taken together, the results suggest that the fusion protein partially retains the efflux function of MexAB-OprM. Modeled structure of the fusion protein shows that the position and orientation of the N-terminal fused EGFP domain may either partially block the translocation pore or restrict the movement of the individual pump domains, which leads to partially restrict efflux activity. PMID:24781334

Biology of Acinetobacter baumannii: Pathogenesis, Antibiotic Resistance Mechanisms, and Prospective Treatment Options

PubMed Central

Lee, Chang-Ro; Lee, Jung Hun; Park, Moonhee; Park, Kwang Seung; Bae, Il Kwon; Kim, Young Bae; Cha, Chang-Jun; Jeong, Byeong Chul; Lee, Sang Hee

2017-01-01

Acinetobacter baumannii is undoubtedly one of the most successful pathogens responsible for hospital-acquired nosocomial infections in the modern healthcare system. Due to the prevalence of infections and outbreaks caused by multi-drug resistant A. baumannii, few antibiotics are effective for treating infections caused by this pathogen. To overcome this problem, knowledge of the pathogenesis and antibiotic resistance mechanisms of A. baumannii is important. In this review, we summarize current studies on the virulence factors that contribute to A. baumannii pathogenesis, including porins, capsular polysaccharides, lipopolysaccharides, phospholipases, outer membrane vesicles, metal acquisition systems, and protein secretion systems. Mechanisms of antibiotic resistance of this organism, including acquirement of β-lactamases, up-regulation of multidrug efflux pumps, modification of aminoglycosides, permeability defects, and alteration of target sites, are also discussed. Lastly, novel prospective treatment options for infections caused by multi-drug resistant A. baumannii are summarized. PMID:28348979

Whole genome sequence to decipher the resistome of Shewanella algae, a multidrug-resistant bacterium responsible for pneumonia, Marseille, France.

PubMed

Cimmino, Teresa; Olaitan, Abiola Olumuyiwa; Rolain, Jean-Marc

2016-01-01

We characterize and decipher the resistome and the virulence factors of Shewanella algae MARS 14, a multidrug-resistant clinical strain using the whole genome sequencing (WGS) strategy. The bacteria were isolated from the bronchoalveolar lavage of a hospitalized patient in the Timone Hospital in Marseille, France who developed pneumonia after plunging into the Mediterranean Sea. The genome size of S. algae MARS 14 was 5,005,710 bp with 52.8% guanine cytosine content. The resistome includes members of class C and D beta-lactamases and numerous multidrug-efflux pumps. We also found the presence of several hemolysins genes, a complete flagellum system gene cluster and genes responsible for biofilm formation. Moreover, we reported for the first time in a clinical strain of Shewanella spp. the presence of a bacteriocin (marinocin). The WGS analysis of this pathogen provides insight into its virulence factors and resistance to antibiotics.

Geographical Distribution of MDR1 Expression in Leishmania Isolates, from Greece and Cyprus, Measured by the Rhodamine-123 Efflux Potential of the Isolates, Using Flow Cytometry

PubMed Central

Tsirigotakis, Nikolaos; Christodoulou, Vasiliki; Ntais, Pantelis; Mazeris, Apostolos; Koutala, Eleni; Messaritakis, Ippokratis; Antoniou, Maria

2016-01-01

Leishmaniasis, a neglected vector-borne disease caused by the protozoan parasite Leishmania, is encountered in 98 countries causing serious concerns to public health. The most alarming is the development of parasite drug resistance, a phenomenon increasingly encountered in the field rendering chemotherapy ineffective. Although resistance to drugs is a complex phenomenon, the rate of efflux of the fluorescent dye Rhodamine-123 from the parasite body, using flow cytometry, is an indication of the isolate's ability to efflux the drug, thus avoiding death. The rate of efflux measured 275 Leishmania strains, isolated from patients and dogs from Greece and Cyprus, was measured and mapped to study the geographical distribution of the multidrug resistance (MDR) gene expression as an indication of the drug resistance of the parasite. The map showed that out of the seven prefectures, where dogs presented high efflux rates, five also had patients with high efflux rates. In one, out of the 59 prefectures studied, the highest number of isolates with efflux slope α > 1, in both human and dog isolates, was found; a fact which may suggest that spread of drug resistance is taking place. The virulence of the Leishmania strains, assessed after infecting human macrophages of the THP-1 cell line, fluctuated from 1% to 59.3% with only 2.5% of the isolates showing infectivity > 50%. The most virulent strains were isolated from Attica and Crete. PMID:27001764

The emergence of clinical resistance to tigecycline.

PubMed

Sun, Yan; Cai, Yun; Liu, Xu; Bai, Nan; Liang, Beibei; Wang, Rui

2013-02-01

Tigecycline (TIG) exhibits broad-spectrum activity against many Gram-positive and Gram-negative pathogens. However, clinical resistance has emerged recently and has been detected following treatment with TIG. This observation suggests that long-term monotherapy may carry a high risk for TIG resistance. TIG resistance is observed most frequently in Acinetobacter baumannii and Enterobacteriaceae, especially in multidrug-resistant strains. Resistance-nodulation-cell division (RND)-type transporters and other efflux pumps may be factors for decreased sensitivity to TIG. Therefore, TIG should be cautiously used in the clinic, and efflux-mediated resistance should be closely monitored in order to prolong the lifespan of this useful antibiotic. Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Paclitaxel-loaded PLGA nanoparticles surface modified with transferrin and Pluronic((R))P85, an in vitro cell line and in vivo biodistribution studies on rat model.

PubMed

Shah, Neha; Chaudhari, Kiran; Dantuluri, Prudhviraju; Murthy, R S R; Das, Susobhan

2009-08-01

The development of multidrug resistance (due to drug efflux by P-glycoproteins) is a major drawback with the use of paclitaxel (PTX) in the treatment of cancer. The rationale behind this study is to prepare PTX nanoparticles (NPs) for the reversal of multidrug resistance based on the fact that PTX loaded into NPs is not recognized by P-glycoproteins and hence is not effluxed out of the cell. Also, the intracellular penetration of the NPs could be enhanced by anchoring transferrin (Tf) on the PTX-PLGA-NPs. PTX-loaded PLGA NPs (PTX-PLGA-NPs), Pluronic((R))P85-coated PLGA NPs (P85-PTX-PLGA-NPs), and Tf-anchored PLGA NPs (Tf-PTX-PLGA-NPs) were prepared and evaluted for cytotoxicity and intracellular uptake using C6 rat glioma cell line. A significant increase in cytotoxicity was observed in the order of Tf-PTX-PLGA-NPs > P85-PTX-PLGA-NPs > PTX-PLGA-NPs in comparison to drug solution. In vivo biodistribution on male Sprague-Dawley rats bearing C6 glioma (subcutaneous) showed higher tumor PTX concentrations in animals administered with PTX-NPs compared to drug solution.

[Opportunistic pathogen Candida glabrata and the mechanisms of its resistance to antifungal drugs].

PubMed

Berila, N; Subík, J

2010-04-01

Treatment of not only bacterial but also fungal infections is currently a growing concern. A major reason is the acquisition of multidrug resistance in both prokaryotic and human cells. The multidrug resistance phenotype is a cellular response to the presence of cytotoxic substances in the environment. The basic mechanism of multidrug resistance is overexpression of the membrane proteins involved in the extrusion of toxic substances outside the cell. The resistance mechanism based on the efflux of inhibitors as a result of the overproduction of transport proteins was also observed in some plant and animal pathogens and human tumour cells. The phenomenon of multidrug resistance associated with an excessive and long-term use of antifungals, in particular of azole derivatives, was also confirmed in the yeast Candida glabrata which is becoming a growing concern for health care professionals. Reduced susceptibility to azole derivatives in particular, a high potential for adapting to stressors, and multiple mechanisms of resistance to structurally and functionally unrelated antifungal drugs make the species C. glabrata a potential threat to hospital patients.

Reversal of P-glycoprotein-medicated multidrug resistance by LBM-A5 in vitro and a study of its pharmacokinetics in vivo.

PubMed

Zhao, Tianxiao; Song, Yun; Liu, Baomin; Qiu, Qianqian; Jiao, Lei; Li, Yunman; Huang, Wenlong; Qian, Hai

2015-01-01

The overexpression of P-glycoprotein (P-gp) in tumors leads to multidrug resistance (MDR), which is a significant obstacle in clinical cancer chemotherapy. The co-administration of anticancer drugs and MDR modulators is a promising strategy for overcoming this problem. Our study aimed to explore the reversal mechanism and safety of the MDR modulator LBM-A5 in vitro, and evaluate its pharmacokinetics and effects on doxorubicin metabolism in vivo. We evaluated an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay of anticancer agents mediated by LBM-A5, the effect of LBM-A5 on rhodamine123 intracellular accumulation, and the efflux in K562/DOX cells to investigate the reversal mechanisms of LBM-A5. The results showed that LBM-A5 inhibits rhodamine123 efflux and increases intracellular accumulation by inhibiting the efflux pump function of P-gp. Furthermore, the therapeutic index and CYP3A4 activity analysis in vitro suggested that LBM-A5 is reasonably safe to use. Also, LBM-A5 (10 mg/kg body mass) achieved the required plasma concentration in sufficient time to reverse MDR in vivo. Importantly, the LBM-A5 treatment group shared similar doxorubicin (DOX) pharmacokinetics with the free DOX group. Our results suggest that LBM-A5 effectively reverses MDR (EC50 = 483.6 ± 81.7 nmol·L(-1)) by inhibiting the function of P-gp, with relatively ideal pharmacokinetics and in a safe manner, and so may be a promising candidate for cancer chemotherapy research.

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.

Ram locus is a key regulator to trigger multidrug resistance in Enterobacter aerogenes.

PubMed

Molitor, Alexander; James, Chloë E; Fanning, Séamus; Pagès, Jean-Marie; Davin-Regli, Anne

2018-02-01

Several genetic regulators belonging to AraC family are involved in the emergence of MDR isolates of E. aerogenes due to alterations in membrane permeability. Compared with the genetic regulator Mar, RamA may be more relevant towards the emergence of antibiotic resistance. Focusing on the global regulators, Mar and Ram, we compared the amino acid sequences of the Ram repressor in 59 clinical isolates and laboratory strains of E. aerogenes. Sequence types were associated with their corresponding multi-drug resistance phenotypes and membrane protein expression profiles using MIC and immunoblot assays. Quantitative gene expression analysis of the different regulators and their targets (porins and efflux pump components) were performed. In the majority of the MDR isolates tested, ramR and a region upstream of ramA were mutated but marR or marA were unchanged. Expression and cloning experiments highlighted the involvement of the ram locus in the modification of membrane permeability. Overexpression of RamA lead to decreased porin production and increased expression of efflux pump components, whereas overexpression of RamR had the opposite effects. Mutations or deletions in ramR, leading to the overexpression of RamA predominated in clinical MDR E. aerogenes isolates and were associated with a higher-level of expression of efflux pump components. It was hypothesised that mutations in ramR, and the self-regulating region proximal to ramA, probably altered the binding properties of the RamR repressor; thereby producing the MDR phenotype. Consequently, mutability of RamR may play a key role in predisposing E. aerogenes towards the emergence of a MDR phenotype.

Nitrogen-containing ecdysteroid derivatives vs. multi-drug resistance in cancer: Preparation and antitumor activity of oximes, oxime ethers and a lactam.

PubMed

Vágvölgyi, Máté; Martins, Ana; Kulmány, Ágnes; Zupkó, István; Gáti, Tamás; Simon, András; Tóth, Gábor; Hunyadi, Attila

2018-01-20

Multidrug resistance is a widespread problem among various diseases and cancer is no exception. We had previously described the chemo-sensitizing activity of ecdysteroid derivatives with low polarity on drug susceptible and multi-drug resistant (MDR) cancer cells. We have also shown that these molecules have a marked selectivity towards the MDR cells. Recent studies on the oximation of various steroid derivatives indicated remarkable increase in their antitumor activity, but there is no related bioactivity data on ecdysteroid oximes. In our present study, 13 novel ecdysteroid derivatives (oximes, oxime ethers and a lactam) and one known compound were synthesized from 20-hydroxyecdysone 2,3;20,22-diacetonide and fully characterized by comprehensive NMR techniques revealing their complete 1 H and 13 C signal assignments. The compounds exerted moderate to strong in vitro antiproliferative activity on HeLa, SiHa, MCF-7 and MDA-MB-231 cell lines. Oxime and particularly oxime ether formation strongly increased their inhibitory activity on the efflux of rhodamine 123 by P-glycoprotein (P-gp), while the new ecdysteroid lactam did not interfere with the efflux function. All compounds exerted potent chemo-sensitizing activity towards doxorubicin on a mouse lymphoma cell line and on its MDR counterpart, and, on the latter, the lactam was found the most active. Because of its MDR-selective chemo-sensitizing activity with no functional effect on P-gp, this lactam is of high potential interest as a new lead for further antitumor studies. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Xenobiotic, Bile Acid, and Cholesterol Transporters: Function and Regulation

PubMed Central

Aleksunes, Lauren M.

2010-01-01

Transporters influence the disposition of chemicals within the body by participating in absorption, distribution, and elimination. Transporters of the solute carrier family (SLC) comprise a variety of proteins, including organic cation transporters (OCT) 1 to 3, organic cation/carnitine transporters (OCTN) 1 to 3, organic anion transporters (OAT) 1 to 7, various organic anion transporting polypeptide isoforms, sodium taurocholate cotransporting polypeptide, apical sodium-dependent bile acid transporter, peptide transporters (PEPT) 1 and 2, concentrative nucleoside transporters (CNT) 1 to 3, equilibrative nucleoside transporter (ENT) 1 to 3, and multidrug and toxin extrusion transporters (MATE) 1 and 2, which mediate the uptake (except MATEs) of organic anions and cations as well as peptides and nucleosides. Efflux transporters of the ATP-binding cassette superfamily, such as ATP-binding cassette transporter A1 (ABCA1), multidrug resistance proteins (MDR) 1 and 2, bile salt export pump, multidrug resistance-associated proteins (MRP) 1 to 9, breast cancer resistance protein, and ATP-binding cassette subfamily G members 5 and 8, are responsible for the unidirectional export of endogenous and exogenous substances. Other efflux transporters [ATPase copper-transporting β polypeptide (ATP7B) and ATPase class I type 8B member 1 (ATP8B1) as well as organic solute transporters (OST) α and β] also play major roles in the transport of some endogenous chemicals across biological membranes. This review article provides a comprehensive overview of these transporters (both rodent and human) with regard to tissue distribution, subcellular localization, and substrate preferences. Because uptake and efflux transporters are expressed in multiple cell types, the roles of transporters in a variety of tissues, including the liver, kidneys, intestine, brain, heart, placenta, mammary glands, immune cells, and testes are discussed. Attention is also placed upon a variety of regulatory factors that influence transporter expression and function, including transcriptional activation and post-translational modifications as well as subcellular trafficking. Sex differences, ontogeny, and pharmacological and toxicological regulation of transporters are also addressed. Transporters are important transmembrane proteins that mediate the cellular entry and exit of a wide range of substrates throughout the body and thereby play important roles in human physiology, pharmacology, pathology, and toxicology. PMID:20103563

Discovery of novel inhibitors of the NorA multidrug transporter of Staphylococcus aureus.

PubMed

Brincat, Jean Pierre; Carosati, Emanuele; Sabatini, Stefano; Manfroni, Giuseppe; Fravolini, Arnaldo; Raygada, Jose L; Patel, Diixa; Kaatz, Glenn W; Cruciani, Gabriele

2011-01-13

Four novel inhibitors of the NorA efflux pump of Staphylococcus aureus, discovered through a virtual screening process, are reported. The four compounds belong to different chemical classes and were tested for their in vitro ability to block the efflux of a well-known NorA substrate, as well as for their ability to potentiate the effect of ciprofloxacin (CPX) on several strains of S. aureus, including a NorA overexpressing strain. Additionally, the MIC values of each of the compounds individually are reported. A structure-activity relationship study was also performed on these novel chemotypes, revealing three new compounds that are also potent NorA inhibitors. The virtual screening procedure employed FLAP, a new methodology based on GRID force field descriptors.

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

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.

Kinetic Analysis of Rhodamines Efflux Mediated by the Multidrug Resistance Protein (MRP1)

PubMed Central

Saengkhae, Chantarawan; Loetchutinat, Chatchanok; Garnier-Suillerot, Arlette

2003-01-01

Characterization of rhodamine 123 as functional assay for MDR has been primarily focused on P-glycoprotein-mediated MDR. Several studies have suggested that Rh123 is also a substrate for MRP1. However, no quantitative studies of the MRP1-mediated efflux of rhodamines have, up to now, been performed. Measurement of the kinetic characteristics of substrate transport is a powerful approach to enhancing our understanding of their function and mechanism. In the present study, we have used a continuous fluorescence assay with four rhodamine dyes (rhodamine 6G, tetramethylrosamine, tetramethylrhodamine ethyl ester, and tetramethylrhodamine methyl ester) to quantify drug transport by MRP1 in living GLC4/ADR cells. The formation of a substrate concentration gradient was observed. MRP1-mediated transport of rhodamine was glutathione-dependent. The kinetics parameter, ka = VM/km, was very similar for the four rhodamine analogs but ∼10-fold less than the values of the same parameter determined previously for the MRP1-mediated efflux of anthracycline. The findings presented here are the first to show quantitative information about the kinetics parameters for MRP1-mediated efflux of rhodamine dyes. PMID:12944313

Bypassing P-Glycoprotein Drug Efflux Mechanisms: Possible Applications in Pharmacoresistant Schizophrenia Therapy

PubMed Central

Hoosain, Famida G.; Choonara, Yahya E.; Tomar, Lomas K.; Tyagi, Charu; du Toit, Lisa C.

2015-01-01

The efficient noninvasive treatment of neurodegenerative disorders is often constrained by reduced permeation of therapeutic agents into the central nervous system (CNS). A vast majority of bioactive agents do not readily permeate into the brain tissue due to the existence of the blood-brain barrier (BBB) and the associated P-glycoprotein efflux transporter. The overexpression of the MDR1 P-glycoprotein has been related to the occurrence of multidrug resistance in CNS diseases. Various research outputs have focused on overcoming the P-glycoprotein drug efflux transporter, which mainly involve its inhibition or bypassing mechanisms. Studies into neurodegenerative disorders have shown that the P-glycoprotein efflux transporter plays a vital role in the progression of schizophrenia, with a noted increase in P-glycoprotein function among schizophrenic patients, thereby reducing therapeutic outcomes. In this review, we address the hypothesis that methods employed in overcoming P-glycoprotein in cancer and other disease states at the level of the BBB and intestine may be applied to schizophrenia drug delivery system design to improve clinical efficiency of drug therapies. In addition, the current review explores polymers and drug delivery systems capable of P-gp inhibition and modulation. PMID:26491671

Genome-based insights into the resistome and mobilome of multidrug-resistant Aeromonas sp. ARM81 isolated from wastewater.

PubMed

Adamczuk, Marcin; Dziewit, Lukasz

2017-01-01

The draft genome of multidrug-resistant Aeromonas sp. ARM81 isolated from a wastewater treatment plant in Warsaw (Poland) was obtained. Sequence analysis revealed multiple genes conferring resistance to aminoglycosides, β-lactams or tetracycline. Three different β-lactamase genes were identified, including an extended-spectrum β-lactamase gene bla PER-1 . The antibiotic susceptibility was experimentally tested. Genome sequencing also allowed us to investigate the plasmidome and transposable mobilome of ARM81. Four plasmids, of which two carry phenotypic modules (i.e., genes encoding a zinc transporter ZitB and a putative glucosyltransferase), and 28 putative transposase genes were identified. The mobility of three insertion sequences (isoforms of previously identified elements ISAs12, ISKpn9 and ISAs26) was confirmed using trap plasmids.

Microbial responses to xenobiotic compounds. Identification of genes that allow Pseudomonas putida KT2440 to cope with 2,4,6‐trinitrotoluene

PubMed Central

Fernández, Matilde; Duque, Estrella; Pizarro‐Tobías, Paloma; Van Dillewijn, Pieter; Wittich, Rolf‐Michael; Ramos, Juan L.

2009-01-01

Summary Pseudomonas putida KT2440 grows in M9 minimal medium with glucose in the presence of 2,4,6‐trinitrotoluene (TNT) at a similar rate than in the absence of TNT, although global transcriptional analysis using DNA microarrays revealed that TNT exerts some stress. Response to TNT stress is regulated at the transcriptional level, as significant changes in the level of expression of 65 genes were observed. Of these genes, 39 appeared upregulated, and 26 were downregulated. The identity of upregulated genes suggests that P. putida uses two kinds of strategies to overcome TNT toxicity: (i) induction of genes encoding nitroreductases and detoxification‐related enzymes (pnrA, xenD, acpD) and (ii) induction of multidrug efflux pump genes (mexEF/oprN) to reduce intracellular TNT concentrations. Mutants of 13 up‐ and 7 downregulated genes were analysed with regards to TNT toxicity revealing the role of the MexE/MexF/OprN pump and a putative isoquinoline 1‐oxidoreductase in tolerance to TNT. The ORF PP1232 whose transcriptional level did not change in response to TNT affected growth in the presence of nitroaromatic compounds and it was found in a screening of 4000 randomly generated mutants. PMID:21261922

A light-driven proton pump from Haloterrigena turkmenica: Functional expression in Escherichia coli membrane and coupling with a H{sup +} co-transporter

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

Kamo, Naoki; Hashiba, Tsuyoshi; Kikukawa, Takashi

2006-03-10

A gene encoding putative retinal protein was cloned from Haloterrigena turkmenica (JCM9743). The deduced amino acid sequence was most closely related to that of deltarhodopsin, which functions as a light-driven H{sup +} pump and was identified in a novel strain Haloterrigena sp. arg-4 (K. Ihara, T. Uemura, I. Katagiri, T. Kitajima-Ihara, Y. Sugiyama, Y. Kimura, Y. Mukohata, Evolution of the archaeal rhodopsins: Evolution rate changes by gene duplication and functional differentiation, J. Mol. Biol. 285 (1999) 163-174. GenBank Accession No. AB009620). Thus, we called the present protein H. turkmenica deltarhodopsin (HtdR) in this report. Differing from the Halobacterium salinarum bacteriorhodopsinmore » (bR), functional expression of HtdR was achieved in Escherichia coli membrane with a high yield of 10-15mg protein/L culture. The photocycle of purified HtdR was similar to that of bR. The photo-induced electrogenic proton pumping activity of HtdR was verified. We co-expressed both HtdR and EmrE, a proton-coupled multi-drug efflux transporter in E. coli, and the cells successfully extruded ethidium, a substrate of EmrE, on illumination.« less

Natural Variation at the FRD3 MATE Transporter Locus Reveals Cross-Talk between Fe Homeostasis and Zn Tolerance in Arabidopsis thaliana

PubMed Central

Pineau, Christophe; Loubet, Stéphanie; Lefoulon, Cécile; Chalies, Claude; Fizames, Cécile; Lacombe, Benoit; Ferrand, Marina; Loudet, Olivier; Berthomieu, Pierre; Richard, Odile

2012-01-01

Zinc (Zn) is essential for the optimal growth of plants but is toxic if present in excess, so Zn homeostasis needs to be finely tuned. Understanding Zn homeostasis mechanisms in plants will help in the development of innovative approaches for the phytoremediation of Zn-contaminated sites. In this study, Zn tolerance quantitative trait loci (QTL) were identified by analyzing differences in the Bay-0 and Shahdara accessions of Arabidopsis thaliana. Fine-scale mapping showed that a variant of the Fe homeostasis-related FERRIC REDUCTASE DEFECTIVE3 (FRD3) gene, which encodes a multidrug and toxin efflux (MATE) transporter, is responsible for reduced Zn tolerance in A. thaliana. Allelic variation in FRD3 revealed which amino acids are necessary for FRD3 function. In addition, the results of allele-specific expression assays in F1 individuals provide evidence for the existence of at least one putative metal-responsive cis-regulatory element. Our results suggest that FRD3 works as a multimer and is involved in loading Zn into xylem. Cross-homeostasis between Fe and Zn therefore appears to be important for Zn tolerance in A. thaliana with FRD3 acting as an essential regulator. PMID:23236296

Assessment of berberine as a multi-target antimicrobial: a multi-omics study for drug discovery and repositioning.

PubMed

Karaosmanoglu, Kubra; Sayar, Nihat Alpagu; Kurnaz, Isil Aksan; Akbulut, Berna Sariyar

2014-01-01

Postgenomics drug development is undergoing major transformation in the age of multi-omics studies and drug repositioning. Rather than applications solely in personalized medicine, omics science thus additionally offers a better understanding of a broader range of drug targets and drug repositioning. Berberine is an isoquinoline alkaloid found in many medicinal plants. We report here a whole genome microarray study in tandem with proteomics techniques for mining the plethora of targets that are putatively involved in the antimicrobial activity of berberine against Escherichia coli. We found DNA replication/repair and transcription to be triggered by berberine, indicating that nucleic acids, in general, are among its targets. Our combined transcriptomics and proteomics multi-omics findings underscore that, in the presence of berberine, cell wall or cell membrane transport and motility-related functions are also specifically regulated. We further report a general decline in metabolism, as seen by repression of genes in carbohydrate and amino acid metabolism, energy production, and conversion. An involvement of multidrug efflux pumps, as well as reduced membrane permeability for developing resistance against berberine in E. coli was noted. Collectively, these findings offer original and significant leads for omics-guided drug discovery and future repositioning approaches in the postgenomics era, using berberine as a multi-omics case study.

Effect of β-elemene on the kinetics of intracellular transport of d-luciferin potassium salt (ABC substrate) in doxorubicin-resistant breast cancer cells and the associated molecular mechanism.

PubMed

Tang, Chao-Yuan; Zhu, Li-Xin; Yu, Jian-Dong; Chen, Zhi; Gu, Man-Cang; Mu, Chao-Feng; Liu, Qi; Xiong, Yang

2018-07-30

In order to explore the mechanism of the reversing multidrug resistance (MDR) phenotypes by β-elemene (β-ELE) in doxorubicin (DOX)-resistant breast cancer cells (MCF-7/DOX), both the functionality and quantity of the ABC transporters in MCF-7/DOX were studied. Bioluminescence imaging (BLI) was used to study the efflux of d-luciferin potassium salt, the substrate of ATP-binding cassette transporters (ABC transporters), in MCF-7/DOX cells treated by β-ELE. At the same time three major ABC transport proteins and genes-related MDR, P-glycoprotein (P-gp, ABCB1) and multidrug resistance-associated protein 1 (MRP, ABCC1) as well as breast cancer resistance protein (BCRP, ABCG2) were analyzed by q-PCR and Western blot. To investigate the efflux functionality of ABC transporters, MCF-7/DOX Fluc cell line with stably-overexpressed luciferase was established. BLI was then used to real-time monitor the efflux kinetics of d-luciferin potassium salt before and after MCF-7/DOX Fluc cells being treated with β-ELE or not. The results showed that the efflux of d-luciferin potassium salt from MCF-7/DOX Fluc was lessened when pretreated with β-ELE, which means that β-ELE may dampen the functionality of ABC transporters, thus decrease the efflux of d-fluorescein potassium or other chemotherapies which also serve as the substrates of ABC transporters. As the effect of β-ELE on the expression of ABC transporters, the results of q-PCR and Western blot showed that gene and protein expression of ABC transporters such as P-gp, MRP, and BCRP were down-regulated after the treatment of β-ELE. To verify the efficacy of β-ELE on reversing MDR, MCF-7/DOX cells were treated with the combination of DOX and β-ELE. MTT assay showed that β-ELE increased the inhibitory effect of DOX on the proliferation of MCF-7/DOX, and the IC 50 of the combination group was much lower than that of the single DOX or β-ELE treatment. In all, β-ELE may reverse MDR through the substrates of ABC transporters by two ways, to lessen the ABC protein efflux by weakening their functionality, or to reduce the quantity of ABC gene and protein expression. Copyright © 2018. Published by Elsevier B.V.

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.

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.

Increasing the cytotoxicity of doxorubicin in breast cancer MCF-7 cells with multidrug resistance using a mesoporous silica nanoparticle drug delivery system.

PubMed

Wang, Xin; Teng, Zhaogang; Wang, Haiyan; Wang, Chunyan; Liu, Ying; Tang, Yuxia; Wu, Jiang; Sun, Jin; Wang, Hai; Wang, Jiandong; Lu, Guangming

2014-01-01

Resistance to cytotoxic chemotherapy is the main cause of therapeutic failure and death in women with breast cancer. Overexpression of various members of the superfamily of adenosine triphosphate binding cassette (ABC)-transporters has been shown to be associated with multidrug resistance (MDR) phenotype in breast cancer cells. MDR1 protein promotes the intracellular efflux of drugs. A novel approach to address cancer drug resistance is to take advantage of the ability of nanocarriers to sidestep drug resistance mechanisms by endosomal delivery of chemotherapeutic agents. Doxorubicin (DOX) is an anthracycline antibiotic commonly used in breast cancer chemotherapy and a substrate for ABC-mediated drug efflux. In the present study, we developed breast cancer MCF-7 cells with overexpression of MDR1 and designed mesoporous silica nanoparticles (MSNs) which were used as a drug delivery system. We tested the efficacy of DOX in the breast cancer cell line MCF-7/MDR1 and in a MCF-7/MDR1 xenograft nude mouse model using the MSNs drug delivery system. Our data show that drug resistance in the human breast cancer cell line MCF-7/MDR1 can be overcome by treatment with DOX encapsulated within mesoporous silica nanoparticles.

A single-component multidrug transporter of the major facilitator superfamily is part of a network that protects E scherichia coli from bile salt stress

PubMed Central

Paul, Stephanie; Alegre, Kamela O; Holdsworth, Scarlett R; Rice, Matthew; Brown, James A; McVeigh, Paul; Kelly, Sharon M; Law, Christopher J

2014-01-01

Resistance to high concentrations of bile salts in the human intestinal tract is vital for the survival of enteric bacteria such as E scherichia coli. Although the tripartite AcrAB–TolC efflux system plays a significant role in this resistance, it is purported that other efflux pumps must also be involved. We provide evidence from a comprehensive suite of experiments performed at two different pH values (7.2 and 6.0) that reflect pH conditions that E . coli may encounter in human gut that MdtM, a single-component multidrug resistance transporter of the major facilitator superfamily, functions in bile salt resistance in E . coli by catalysing secondary active transport of bile salts out of the cell cytoplasm. Furthermore, assays performed on a chromosomal ΔacrB mutant transformed with multicopy plasmid encoding MdtM suggested a functional synergism between the single-component MdtM transporter and the tripartite AcrAB–TolC system that results in a multiplicative effect on resistance. Substrate binding experiments performed on purified MdtM demonstrated that the transporter binds to cholate and deoxycholate with micromolar affinity, and transport assays performed on inverted vesicles confirmed the capacity of MdtM to catalyse electrogenic bile salt/H+ antiport. PMID:24684269

Surface charge-specific interactions between polymer nanoparticles and ABC transporters in Caco-2 cells

NASA Astrophysics Data System (ADS)

Bhattacharjee, Sourav; van Opstal, Edward J.; Alink, Gerrit M.; Marcelis, Antonius T. M.; Zuilhof, Han; Rietjens, Ivonne M. C. M.

2013-06-01

The surface charge-dependent transport of polymeric nanoparticles (PNPs) across Caco-2 monolayers grown on transwell culture systems as an in vitro model for intestinal transport was tested. The transport of well-characterized, monodisperse, and fluorescent tri-block copolymer nanoparticles (TCNPs/size 45 nm) and polystyrene nanoparticles (PSNPs/size 50 nm), with different surface charges (positive and negative), was quantified. The positive PNPs showed a higher intracellular uptake and flux across the Caco-2 monolayers than the negative PNPs. Multidrug resistance/P-glycoprotein (MDR1/P-gp), a specific ATP-binding cassette (ABC) transporter, was found to play a major role in the cellular efflux of positive PNPs, whereas the multidrug resistance protein 1 took part in the efflux of negative PNPs from Caco-2 cells. The positive PNPs also caused an increased cellular uptake and apical to basolateral transport of the carcinogen PhIP across the Caco-2 monolayer. The flavonoid quercetin, which is known to interact with ABC transporters, promoted the intracellular uptake of different PNPs and interfered with the normal distribution patterns of PNPs in the transwell system. These results indicate that PNPs display surface charge-specific interactions with ABC transporters and can even affect the bioavailability of toxic food-borne compounds (like pro-carcinogens).

Ketoconazole and the modulation of multidrug resistance-mediated transport in Caco-2 and MDCKII-MDR1 drug transport models.

PubMed

Fan, Y; Rodriguez-Proteau, R

2008-02-01

The hypothesis tested was that ketoconazole can modulate P-glycoprotein, thereby altering cellular uptake and apparent permeability (P(app)) of multidrug-resistant substrates, such as cyclosporin A (CSA) and digoxin, across Caco-2, MDCKII-MDR1, and MDCKII wild-type cell transport models. (3)H-CSA/(3)H-digoxin transport experiments were performed with and without co-exposure to ketoconazole, and (3)H-ketoconzole transport experiments were performed with and without co-exposure to dietary flavonoids, epigallocatechin-3-gallate, and xanthohumol. Ketoconazole (3 microM) reduced the P(app) efflux of CSA and digoxin from 5.07 x 10(-6) to 2.91 x 10(-6) cm s(-1) and from 2.60 x 10(-6) to 1.41 x 10(-6) cm s(-1), respectively, in Caco-2 cells. In the MDCKII-MDR1 cells, ketoconazole reduced the P(app) efflux of CSA and increased the P(app) absorption of digoxin. Cellular uptake of ketoconazole in the Caco-2 cells was significantly inhibited by CSA and digoxin, whereas epigallocatechin-3-gallate and xanthohumol exhibited biphasic responses. In conclusion, ketoconazole modulates the P(app) of P-glycoprotein substrates by interacting with MDR1 protein. Epigallocatechin-3-gallate and xanthohumol modulate the transport and uptake of ketoconazole.

Self-assembled Multifunctional DNA Nanoflowers for the Circumvention of Multidrug Resistance in Targeted Anticancer Drug Delivery.

PubMed

Mei, Lei; Zhu, Guizhi; Qiu, Liping; Wu, Cuichen; Chen, Huapei; Liang, Hao; Cansiz, Sena; Lv, Yifan; Zhang, Xiaobing; Tan, Weihong

2015-11-01

Cancer chemotherapy has been impeded by side effects and multidrug resistance (MDR) partially caused by drug efflux from cancer cells, which call for targeted drug delivery systems additionally able to circumvent MDR. Here we report multifunctional DNA nanoflowers (NFs) for targeted drug delivery to both chemosensitive and MDR cancer cells and circumvent MDR in both leukemia and breast cancer cell models. NFs are self-assembled via liquid crystallization of DNA generated by Rolling Circle Replication, during which NFs are incorporated with aptamers for specific cancer cell recognition, fluorophores for bioimaging, and Doxorubicin (Dox)-binding DNA for drug delivery. NF sizes are tunable (down to ~200 nm in diameter), and the densely packed drug-binding motifs and porous intrastructures endow NFs with high drug loading capacity (71.4%, wt/wt). The Dox-loaded NFs (NF-Dox) are stable at physiological pH, yet drug release is facilitated in acidic or basic conditions. NFs deliver Dox into target chemosensitive and MDR cancer cells, preventing drug efflux and enhancing drug retention in MDR cells. Consequently, NF-Dox induces potent cytotoxicity in both target chemosensitive cells and MDR cells, but not nontarget cells, thus concurrently circumventing MDR and reducing side effects. Overall, these NFs are promising to circumvent MDR in targeted cancer therapy.

P-glycoprotein multidrug transporter in inflammatory bowel diseases: More questions than answers

PubMed Central

Cario, Elke

2017-01-01

The gastrointestinal barrier is constantly exposed to numerous environmental substrates that are foreign and potentially harmful. These xenobiotics can cause shifts in the intestinal microbiota composition, affect mucosal immune responses, disturb tissue integrity and impair regeneration. The multidrug transporter ABCB1/MDR1 p-glycoprotein (p-gp) plays a key role at the front line of host defence by efficiently protecting the gastrointestinal barrier from xenobiotic accumulation. This Editorial discusses how altered expression and function of ABCB1/MDR1 p-gp may contribute to the development and persistence of chronic intestinal inflammation in inflammatory bowel diseases (IBD). Recent evidence implies multiple interactions between intestinal microbiota, innate immunity and xenobiotic metabolism via p-gp. While decreased efflux activity may promote disease susceptibility and drug toxicity, increased efflux activity may confer resistance to therapeutic drugs in IBD. Mice deficient in MDR1A develop spontaneously chronic colitis, providing a highly valuable murine IBD model for the study of intestinal epithelial barrier function, immunoregulation, infectious co-triggers and novel therapeutic approaches. Possible associations of human ABCB1 gene polymorphisms with IBD susceptibility have been evaluated, but results are inconsistent. Future studies must focus on further elucidation of the pathophysiological relevance and immunological functions of p-gp and how its ambiguous effects could be therapeutically targeted in IBD. PMID:28321153

P-glycoprotein multidrug transporter in inflammatory bowel diseases: More questions than answers.

PubMed

Cario, Elke

2017-03-07

The gastrointestinal barrier is constantly exposed to numerous environmental substrates that are foreign and potentially harmful. These xenobiotics can cause shifts in the intestinal microbiota composition, affect mucosal immune responses, disturb tissue integrity and impair regeneration. The multidrug transporter ABCB1/MDR1 p-glycoprotein (p-gp) plays a key role at the front line of host defence by efficiently protecting the gastrointestinal barrier from xenobiotic accumulation. This Editorial discusses how altered expression and function of ABCB1/MDR1 p-gp may contribute to the development and persistence of chronic intestinal inflammation in inflammatory bowel diseases (IBD). Recent evidence implies multiple interactions between intestinal microbiota, innate immunity and xenobiotic metabolism via p-gp. While decreased efflux activity may promote disease susceptibility and drug toxicity, increased efflux activity may confer resistance to therapeutic drugs in IBD. Mice deficient in MDR1A develop spontaneously chronic colitis, providing a highly valuable murine IBD model for the study of intestinal epithelial barrier function, immunoregulation, infectious co-triggers and novel therapeutic approaches. Possible associations of human ABCB1 gene polymorphisms with IBD susceptibility have been evaluated, but results are inconsistent. Future studies must focus on further elucidation of the pathophysiological relevance and immunological functions of p-gp and how its ambiguous effects could be therapeutically targeted in IBD.

A New Natural Product Analog of Blasticidin S Reveals Cellular Uptake Facilitated by the NorA Multidrug Transporter

PubMed Central

Davison, Jack R.; Lohith, Katheryn M.; Wang, Xiaoning; Bobyk, Kostyantyn; Mandadapu, Sivakoteswara R.; Lee, Su-Lin; Cencic, Regina; Nelson, Justin; Simpkins, Scott; Frank, Karen M.; Pelletier, Jerry; Myers, Chad L.; Piotrowski, Jeff; Smith, Harold E.

2017-01-01

ABSTRACT The permeation of antibiotics through bacterial membranes to their target site is a crucial determinant of drug activity but in many cases remains poorly understood. During screening efforts to discover new broad-spectrum antibiotic compounds from marine sponge samples, we identified a new analog of the peptidyl nucleoside antibiotic blasticidin S that exhibited up to 16-fold-improved potency against a range of laboratory and clinical bacterial strains which we named P10. Whole-genome sequencing of laboratory-evolved strains of Staphylococcus aureus resistant to blasticidin S and P10, combined with genome-wide assessment of the fitness of barcoded Escherichia coli knockout strains in the presence of the antibiotics, revealed that restriction of cellular access was a key feature in the development of resistance to this class of drug. In particular, the gene encoding the well-characterized multidrug efflux pump NorA was found to be mutated in 69% of all S. aureus isolates resistant to blasticidin S or P10. Unexpectedly, resistance was associated with inactivation of norA, suggesting that the NorA transporter facilitates cellular entry of peptidyl nucleosides in addition to its known role in the efflux of diverse compounds, including fluoroquinolone antibiotics. PMID:28373194

A New Natural Product Analog of Blasticidin S Reveals Cellular Uptake Facilitated by the NorA Multidrug Transporter.

PubMed

Davison, Jack R; Lohith, Katheryn M; Wang, Xiaoning; Bobyk, Kostyantyn; Mandadapu, Sivakoteswara R; Lee, Su-Lin; Cencic, Regina; Nelson, Justin; Simpkins, Scott; Frank, Karen M; Pelletier, Jerry; Myers, Chad L; Piotrowski, Jeff; Smith, Harold E; Bewley, Carole A

2017-06-01

The permeation of antibiotics through bacterial membranes to their target site is a crucial determinant of drug activity but in many cases remains poorly understood. During screening efforts to discover new broad-spectrum antibiotic compounds from marine sponge samples, we identified a new analog of the peptidyl nucleoside antibiotic blasticidin S that exhibited up to 16-fold-improved potency against a range of laboratory and clinical bacterial strains which we named P10. Whole-genome sequencing of laboratory-evolved strains of Staphylococcus aureus resistant to blasticidin S and P10, combined with genome-wide assessment of the fitness of barcoded Escherichia coli knockout strains in the presence of the antibiotics, revealed that restriction of cellular access was a key feature in the development of resistance to this class of drug. In particular, the gene encoding the well-characterized multidrug efflux pump NorA was found to be mutated in 69% of all S. aureus isolates resistant to blasticidin S or P10. Unexpectedly, resistance was associated with inactivation of norA , suggesting that the NorA transporter facilitates cellular entry of peptidyl nucleosides in addition to its known role in the efflux of diverse compounds, including fluoroquinolone antibiotics. Copyright © 2017 American Society for Microbiology.

Zingiber officinale: Its antibacterial activity on Pseudomonas aeruginosa and mode of action evaluated by flow cytometry.

PubMed

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

2017-06-01

Biofilm formation, low membrane permeability and efflux activity developed by Pseudomonas aeruginosa, play an important role in the mechanism of infection and antimicrobial resistance. In the present study we evaluate the antibacterial effect of Zingiber officinale against multi-drug resistant strain of P. aeruginosa. The study explores antibacterial efficacy and time-kill study concomitantly the effect of herbal extract on bacterial cell physiology with the use of flow cytometry and inhibition of biofilm formation. Z. officinale was found to inhibit the growth of P. aeruginosa, significantly. A major decline in the Colony Forming Units was observed with 3 log 10  at 12 h of treatment. Also it is found to affect the membrane integrity of the pathogen, as 70.06 ± 2.009% cells were found to stain with Propidium iodide. In case of efflux activity 86.9 ± 2.08% cells were found in Ethidium bromide positive region. Biofilm formation inhibition ability was found in the range of 68.13 ± 4.11% to 84.86 ± 2.02%. Z.officinale is effective for killing Multi-Drug Resistant P. aeruginosa clinical isolate by affecting the cellular physiology and inhibiting the biofilm formation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Uncaria alkaloids reverse ABCB1-mediated cancer multidrug resistance

PubMed Central

Huang, Bao-Yuan; Zeng, Yu; Li, Ying-Jie; Huang, Xiao-Jun; Hu, Nan; Yao, Nan; Chen, Min-Feng; Yang, Zai-Gang; Chen, Zhe-Sheng; Zhang, Dong-Mei; Zeng, Chang-Qing

2017-01-01

The overexpression of ATP-binding cassette (ABC) transporters is the main cause of cancer multidrug resistance (MDR), which leads to chemotherapy failure. Uncaria alkaloids are the major active components isolated from uncaria, which is a common Chinese herbal medicine. In this study, the MDR-reversal activities of uncaria alkaloids, including rhynchophylline, isorhynchophylline, corynoxeine, isocorynoxeine (Icory), hirsutine and hirsuteine, were screened; they all exhibited potent reversal efficacy when combined with doxorubicin. Among them, Icory significantly sensitized ABCB1-overexpressing HepG2/ADM and MCF-7/ADR cells to vincristine, doxorubicin and paclitaxel, but not to the non-ABCB1 substrate cisplatin. Noteworthy, Icory selectively reversed ABCB1-overexpressing MDR cancer cells but not ABCC1- or ABCG2-mediated MDR. Further mechanistic study revealed that Icory increased the intracellular accumulation of doxorubicin in ABCB1-overexpressing cells by blocking the efflux function of ABCB1. Instead of inhibiting ABCB1 expression and localization, Icory acts as a substrate of the ABCB1 transporter by competitively binding to substrate binding sites. Collectively, these results indicated that Icory reversed ABCB1-mediated MDR by suppressing its efflux function, and it would be beneficial to increase the efficacy of these types of uncaria alkaloids and develop them to be selective ABCB1-mediated MDR-reversal agents. PMID:28534954

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 the evolution of mechanism in E. aerogenes.

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 in inhibitor-susceptible efflux throws light on a new step in the evolution of mechanism in E. aerogenes. PMID:18787654

In vitro, in vivo and ex vivo characterization of ibrutinib: a potent inhibitor of the efflux function of the transporter MRP1.

PubMed

Zhang, Hui; Patel, Atish; Ma, Shao-Lin; Li, Xiao Jie; Zhang, Yun-Kai; Yang, Pei-Qi; Kathawala, Rishil J; Wang, Yi-Jun; Anreddy, Nagaraju; Fu, Li-Wu; Chen, Zhe-Sheng

2014-12-01

The transporter, multidrug resistance protein 1 (MRP1, ABCC1), plays a critical role in the development of multidrug resistance (MDR). Ibrutinib is an inhibitor of Bruton's tyrosine kinase. Here we investigated the reversal effect of ibrutinib on MRP1-mediated MDR. Cytotoxicity was determined by MTT assay. The expression of protein was detected by Western blot. RT-PCR and Q-PCR were performed to detect the expression of MRP1 mRNA. The intracellular accumulation and efflux of substrates for MRP1 were measured by scintillation counter and flow cytometry. HEK293/MRP1 cell xenografts in nude mice were established to study the effects of ibrutinib in vivo. Ibrutinib significantly enhanced the cytotoxicity of MRP1 substrates in HEK293/MRP1 and HL60/Adr cells overexpressing MRP1. Furthermore, ibrutinib increased the accumulation of substrates in these MRP1-overexpressing cells by inhibiting the drug efflux function of MRP1. However, mRNA and protein expression of MRP1 remained unaltered after treatment with ibrutinib in MRP1-overexpressing cells. In vivo, ibrutinib enhanced the efficacy of vincristine to inhibit the growth of HEK293/MRP1 tumour xenografts in nude mice. Importantly, ibrutinib also enhances the cytotoxicity of vincristine in primary cultures of leukaemia blasts, derived from patients. Our results indicated that ibrutinib significantly increased the efficacy of the chemotherapeutic agents which were MRP1 substrates, in MRP1-overexpressing cells, in vitro, in vivo and ex vivo. These findings will lead to further studies on the effects of a combination of ibrutinib with chemotherapeutic agents in cancer patients overexpressing MRP1. © 2014 The British Pharmacological Society.

Interaction of dipeptide prodrugs of saquinavir with multidrug resistance protein-2 (MRP-2): evasion of MRP-2 mediated efflux.

PubMed

Jain, Ritesh; Agarwal, Sheetal; Mandava, Nanda Kishore; Sheng, Ye; Mitra, Ashim K

2008-10-01

Saquinavir (SQV), the first protease inhibitor approved by FDA to treat HIV-1 infection. This drug is a well-known substrate for multidrug resistance protein-2 (MRP-2). The objective of this study was to investigate whether derivatization of SQV to dipeptide prodrugs, valine-valine-saquinavir (Val-Val-SQV) and glycine-valine-saquinavir (Gly-Val-SQV), targeting peptide transporter can circumvent MRP-2 mediated efflux. Uptake and transport studies were carried out across MDCKII-MRP2 cell monolayers to investigate the interaction of SQV and its prodrugs with MRP-2. In situ single pass intestinal perfusion experiments in rat jejunum were performed to calculate intestinal absorption rate constants and permeabilities of SQV, Val-Val-SQV and Gly-Val-SQV. Uptake studies demonstrated that the prodrugs have significantly lower interaction with MRP-2 relative to SQV. Transepithelial transport of Val-Val-SQV and Gly-Val-SQV across MDCKII-MRP2 cells exhibited an enhanced absorptive flux and reduced secretory flux as compared to SQV. Intestinal perfusion studies revealed that synthesized prodrugs have higher intestinal permeabilities relative to SQV. Enhanced absorption of Val-Val-SQV and Gly-Val-SQV relative to SQV can be attributed to their translocation by the peptide transporter in the jejunum. In the presence of MK-571, a MRP family inhibitor, there was a significant increase in the permeabilities of SQV and Gly-Val-SQV indicating that these compounds are probably substrates for MRP-2. However, there was no change in the permeability of Val-Val-SQV with MK-571 indicating lack of any interaction of Val-Val-SQV with MRP-2. In conclusion, peptide transporter targeted prodrug modification of MRP-2 substrates may lead to shielding of these drug molecules from MRP-2 efflux pumps.

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.

In vitro, in vivo and ex vivo characterization of ibrutinib: a potent inhibitor of the efflux function of the transporter MRP1

PubMed Central

Zhang, Hui; Patel, Atish; Ma, Shao-Lin; Li, Xiao Jie; Zhang, Yun-Kai; Yang, Pei-Qi; Kathawala, Rishil J; Wang, Yi-Jun; Anreddy, Nagaraju; Fu, Li-Wu; Chen, Zhe-Sheng

2014-01-01

Background and Purpose The transporter, multidrug resistance protein 1 (MRP1, ABCC1), plays a critical role in the development of multidrug resistance (MDR). Ibrutinib is an inhibitor of Bruton's tyrosine kinase. Here we investigated the reversal effect of ibrutinib on MRP1-mediated MDR. Experimental Approach Cytotoxicity was determined by MTT assay. The expression of protein was detected by Western blot. RT-PCR and Q-PCR were performed to detect the expression of MRP1 mRNA. The intracellular accumulation and efflux of substrates for MRP1 were measured by scintillation counter and flow cytometry. HEK293/MRP1 cell xenografts in nude mice were established to study the effects of ibrutinib in vivo. Key Results Ibrutinib significantly enhanced the cytotoxicity of MRP1 substrates in HEK293/MRP1 and HL60/Adr cells overexpressing MRP1. Furthermore, ibrutinib increased the accumulation of substrates in these MRP1-overexpressing cells by inhibiting the drug efflux function of MRP1. However, mRNA and protein expression of MRP1 remained unaltered after treatment with ibrutinib in MRP1-overexpressing cells. In vivo, ibrutinib enhanced the efficacy of vincristine to inhibit the growth of HEK293/MRP1 tumour xenografts in nude mice. Importantly, ibrutinib also enhances the cytotoxicity of vincristine in primary cultures of leukaemia blasts, derived from patients. Conclusions and Implications Our results indicated that ibrutinib significantly increased the efficacy of the chemotherapeutic agents which were MRP1 substrates, in MRP1-overexpressing cells, in vitro, in vivo and ex vivo. These findings will lead to further studies on the effects of a combination of ibrutinib with chemotherapeutic agents in cancer patients overexpressing MRP1. PMID:25164592

Disulfide Cross-linking of a Multidrug and Toxic Compound Extrusion Transporter Impacts Multidrug Efflux*

PubMed Central

Radchenko, Martha; Nie, Rongxin; Lu, Min

2016-01-01

Multidrug and toxic compound extrusion (MATE) transporters contribute to multidrug resistance by extruding different drugs across cell membranes. The MATE transporters alternate between their extracellular and intracellular facing conformations to propel drug export, but how these structural changes occur is unclear. Here we combine site-specific cross-linking and functional studies to probe the movement of transmembrane helices in NorM from Neiserria gonorrheae (NorM-NG), a MATE transporter with known extracellular facing structure. We generated an active, cysteine-less NorM-NG and conducted pairwise cysteine mutagenesis on this variant. We found that copper phenanthroline catalyzed disulfide bond formation within five cysteine pairs and increased the electrophoretic mobility of the corresponding mutants. Furthermore, copper phenanthroline abolished the activity of the five paired cysteine mutants, suggesting that these substituted amino acids come in spatial proximity during transport, and the proximity changes are functionally indispensable. Our data also implied that the substrate-binding transmembrane helices move up to 10 Å in NorM-NG during transport and afforded distance restraints for modeling the intracellular facing transporter, thereby casting new light on the underlying mechanism. PMID:26975373

Molecular epidemiological survey of bacteremia by multidrug resistant Pseudomonas aeruginosa: the relevance of intrinsic resistance mechanisms

PubMed Central

Dantas, Raquel Cristina Cavalcanti; Silva, Rebecca Tavares e; Ferreira, Melina Lorraine; Gonçalves, Iara Rossi; Araújo, Bruna Fuga; de Campos, Paola Amaral; Royer, Sabrina; Batistão, Deivid William da Fonseca; Gontijo-Filho, Paulo Pinto; Ribas, Rosineide Marques

2017-01-01

The bacterial factors associated with bacteremia by multidrug-resistant and extensively drug-resistant P. aeruginosa, including overexpression of efflux pumps, AmpC overproduction, and loss/alteration of the OprD porin in isolates that are non-Metallo-β-Lactamase producing were analyzed in a retrospective study. Molecular analyses included strain typing by Pulsed Field Gel Electrophoresis and identification of key genes via qualitative and quantitative PCR-based assays. Previous use of carbapenems and tracheostomy was independently associated with the development of bacteremia by extensively drug-resistant and multidrug-resistant strains of P. aeruginosa. A high consumption of antimicrobials was observed, and 75.0% of the isolates contained amplicons with the blaSPM-1 and blaVIM genes. Of the 47 non-Metallo-β-Lactamase isolates, none had another type of carbapenemase. However, the isolates exhibited high rates of hyperproduction of AmpC, loss of the OprD porin (71.4%) and the presence of MexABOprM (57.1%) and MexXY (64.3%). This study suggests that in non-Metallo-β-Lactamase isolates, the association of intrinsic resistance mechanisms could contributes to the expression of multidrug-resistant/extensively drug-resistant phenotypes. PMID:28481953

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 hasF individual knockouts, we conclude that S. marcescens HasF is the sole outer-membrane component of the SdeAB pump. In addition, MIC data for sdeR-deficient and overexpressing strains confirm that SdeR is an activator of sdeAB and acts to enhance the overall multidrug resistance of S. marcescens.

Antibacterial activity of 4,5-dihydroxy-2-cyclopentan-1-one (DHCP) and cloning of a gene conferring DHCP resistance in Escherichia coli.

PubMed

Phadtare, S; Yamanaka, K; Kato, I; Inouye, M

2001-07-01

In the present study we report that 4,5-dihydroxy-2-cyclopentan-1-one (DHCP), which is derived from heat-treatment of uronic acid or its derivatives, has antibacterial activity against Escherichia coli. The compound causes complete growth inhibition at 350 microM concentration. We have cloned a gene from E. coli, which confers DHCP resistance when present in multicopy. The putative protein encoded by this gene (dep- DHCP efflux protein) is a transmembrane efflux protein with a high homology to other antibiotic-efflux proteins including those for chloramphenicol, bicyclomycin and tetracycline. However, the Dep protein does not confer cross-resistance to any of the antibiotics tested.

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.

Strategies to overcome or circumvent P-glycoprotein mediated multidrug resistance.

PubMed

Yuan, Hongyu; Li, Xun; Wu, Jifeng; Li, Jinpei; Qu, Xianjun; Xu, Wenfang; Tang, Wei

2008-01-01

Cancer patients who receive chemotherapy often experience intrinsic or acquired resistance to a broad spectrum of chemotherapeutic agents. The phenomenon, termed multidrug resistance (MDR), is often associated with the over-expression of P-glycoprotein, a transmembrane protein pump, which can enhance efflux of a various chemicals structurally unrelated at the expense of ATP depletion, resulting in decrease of the intracellular cytotoxic drug accumulation. The MDR has been a big threaten to the human health and the war fight for it continues. Although several other mechanisms for MDR are elucidated in recent years, considerable efforts attempting to inverse MDR are involved in exploring P-glycoprotein modulators and suppressing P-glycoprotein expression. In this review, we will report on the recent advances in various strategies for overcoming or circumventing MDR mediated by P-glycoprotein.

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.

A Truncated AdeS Kinase Protein Generated by ISAba1 Insertion Correlates with Tigecycline Resistance in Acinetobacter baumannii

PubMed Central

Sun, Jun-Ren; Perng, Cherng-Lih; Chan, Ming-Chin; Morita, Yuji; Lin, Jung-Chung; Su, Chih-Mao; Wang, Wei-Yao; Chang, Tein-Yao; Chiueh, Tzong-Shi

2012-01-01

Over-expression of AdeABC efflux pump stimulated continuously by the mutated AdeRS two component system has been found to result in antimicrobial resistance, even tigecycline (TGC) resistance, in multidrug-resistant Acinetobacter baumannii (MRAB). Although the insertion sequence, ISAba1, contributes to one of the AdeRS mutations, the detail mechanism remains unclear. In the present study we collected 130 TGC-resistant isolates from 317 carbapenem resistant MRAB (MRAB-C) isolates, and 38 of them were characterized with ISAba1 insertion in the adeS gene. The relationship between the expression of AdeABC efflux pump and TGC resistant was verified indirectly by successfully reducing TGC resistance with NMP, an efflux pump inhibitor. Further analysis showed that the remaining gene following the ISAba1 insertion was still transcribed to generate a truncated AdeS protein by the Pout promoter on ISAba1 instead of frame shift or pre-termination. Through introducing a series of recombinant adeRS constructs into a adeRS knockout strain, we demonstrated the truncated AdeS protein was constitutively produced and stimulating the expression of AdeABC efflux pump via interaction with AdeR. Our findings suggest a mechanism of antimicrobial resistance induced by an aberrant cytoplasmic sensor derived from an insertion element. PMID:23166700

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.

Disposition of Naringenin via Glucuronidation Pathway Is Affected by Compensating Efflux Transporters of Hydrophilic Glucuronides

PubMed Central

Xu, Haiyan; Kulkarni, Kaustubh H.; Singh, Rashim; Yang, Zhen; Wang, Stephen W.J.; Tam, Vincent H.; Hu, Ming

2010-01-01

The purposes of this study were to investigate how efflux transporters and UDP-glucuronosyltransferases (UGT) affect the disposition of naringenin. A rat intestinal perfusion model with bile duct cannulation was used along with rat intestinal and liver microsomes. In the intestinal perfusion model, both absorption and subsequent excretion of naringenin metabolites were rapid and site-dependent (p < 0.05). Naringenin was absorbed the most in colon and its glucuronides were excreted the most in duodenum. In metabolism studies, the intrinsic clearance value of naringenin glucuronidation was the highest in jejunum microsomes, followed by liver, ileal and colonic microsomes. The rapid metabolism in microsomes did not always translate into more efficient excretion in the rat perfusion model, however, because of presence of rate-limiting efflux transporters. When used separately, MK-571 (an inhibitor of multidrug resistance-related protein 2 or Mrp2) or dipyridamole (an inhibitor of breast cancer resistance protein or Bcrp1) did not affect excretion of naringenin glucuronides, but when used together, they significantly (p < 0.05) decreased intestinal and biliary excretion of naringenin glucuronides. In conclusion, efflux transporters Mrp2 and Bcrp1 are shown to compensate for each other and enable the intestinal excretion of flavonoid (i.e., naringenin) glucuronides. PMID:19736994

Effect of curcumin on human colon cancer multidrug resistance in vitro and in vivo.

PubMed

Lu, Wei-Dong; Qin, Yong; Yang, Chuang; Li, Lei; Fu, Zhong-Xue

2013-05-01

To determine whether curcumin reverses the multidrug resistance of human colon cancer cells in vitro and in vivo. In a vincristine-resistant cell line of human colon cancer, the cell viability of curcumin-treated cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Rhodamine123 efflux was evaluated to detect P-glycoprotein transporter activity, and expression of the multidrug resistance protein 1 and survivin genes was analyzed by reverse transcription polymerase chain reaction and western blotting. In addition, xenograft mouse tumors were grown and treated with curcumin. The morphology of the xenografts was investigated by hematoxylin-eosin staining. The in vivo expression of the multidrug resistance gene and P-glycoprotein and survivin genes and proteins was observed using reverse transcription-polymerase chain reaction and western blotting, respectively. Curcumin was not obviously toxic to the vincristine-resistant human colon cancer cells at concentrations less than 25 μM, but the growth of cells was significantly inhibited. At concentrations greater than 25 μM, curcumin was toxic in a concentration-dependent manner. The sensitivity of cells to vincristine, cisplatin, fluorouracil, and hydroxycamptothecin was enhanced, intracellular Rhodamine123 accumulation was increased (p<0.05), and the expression of the multidrug resistance gene and P-glycoprotein were significantly suppressed (p<0.05). The combination of curcumin and vincristine significantly inhibited xenograft growth. The expression of the multidrug resistance protein 1 and survivin genes was significantly reduced in xenografts of curcumin-treated mice and mice treated with both curcumin and vincristine relative to control mice. Curcumin has strong reversal effects on the multidrug resistance of human colon carcinoma in vitro and in vivo.

Effect of curcumin on human colon cancer multidrug resistance in vitro and in vivo

PubMed Central

Lu, Wei-Dong; Qin, Yong; Yang, Chuang; Li, Lei

2013-01-01

OBJECTIVE: To determine whether curcumin reverses the multidrug resistance of human colon cancer cells in vitro and in vivo. METHODS: In a vincristine-resistant cell line of human colon cancer, the cell viability of curcumin-treated cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Rhodamine123 efflux was evaluated to detect P-glycoprotein transporter activity, and expression of the multidrug resistance protein 1 and survivin genes was analyzed by reverse transcription polymerase chain reaction and western blotting. In addition, xenograft mouse tumors were grown and treated with curcumin. The morphology of the xenografts was investigated by hematoxylin-eosin staining. The in vivo expression of the multidrug resistance gene and P-glycoprotein and survivin genes and proteins was observed using reverse transcription-polymerase chain reaction and western blotting, respectively. RESULTS: Curcumin was not obviously toxic to the vincristine-resistant human colon cancer cells at concentrations less than 25 μM, but the growth of cells was significantly inhibited. At concentrations greater than 25 μM, curcumin was toxic in a concentration-dependent manner. The sensitivity of cells to vincristine, cisplatin, fluorouracil, and hydroxycamptothecin was enhanced, intracellular Rhodamine123 accumulation was increased (p<0.05), and the expression of the multidrug resistance gene and P-glycoprotein were significantly suppressed (p<0.05). The combination of curcumin and vincristine significantly inhibited xenograft growth. The expression of the multidrug resistance protein 1 and survivin genes was significantly reduced in xenografts of curcumin-treated mice and mice treated with both curcumin and vincristine relative to control mice. CONCLUSION: Curcumin has strong reversal effects on the multidrug resistance of human colon carcinoma in vitro and in vivo. PMID:23778405

Effect of bisphenol A on P-glycoprotein-mediated efflux and ultrastructure of the sea urchin embryo.

PubMed

Bošnjak, Ivana; Borra, Marco; Iamunno, Franco; Benvenuto, Giovanna; Ujević, Ivana; Bušelić, Ivana; Roje-Busatto, Romana; Mladineo, Ivona

2014-11-01

Usage of bisphenol A (BPA) in production of polycarbonate plastics has resulted in global distribution of BPA in the environment. These high concentrations cause numerous negative effects to the aquatic biota, among which the most known is the induction of endocrine disruption. The focus of this research was to determine the effects of two experimentally determined concentrations of BPA (100nM and 4μM) on cellular detoxification mechanisms during the embryonic development (2-cell, pluteus) of the rocky sea urchin (Paracentrotus lividus), primarily the potential involvement of multidrug efflux transport in the BPA intercellular efflux. The results of transport assay, measurements of the intracellular BPA and gene expression surveys, for the first time indicate the importance of P-glycoprotein (P-gp/ABCB1) in defense against BPA. Cytotoxic effects of BPA, validated by the immunohistochemistry (IHC) and the transmission electron microscopy (TEM), induced the aberrant karyokinesis, and consequently, the impairment of embryo development through the first cell division and retardation. Copyright © 2014 Elsevier B.V. All rights reserved.

Maize ZmALMT2 is a root anion transporter that mediates constitutive root malate efflux.

PubMed

Ligaba, Ayalew; Maron, Lyza; Shaff, Jon; Kochian, Leon; Piñeros, Miguel

2012-07-01

Root efflux of organic acid anions underlies a major mechanism of plant aluminium (Al) tolerance on acid soils. This efflux is mediated by transporters of the Al-activated malate transporter (ALMT) or the multi-drug and toxin extrusion (MATE) families. ZmALMT2 was previously suggested to be involved in Al tolerance based on joint association-linkage mapping for maize Al tolerance. In the current study, we functionally characterized ZmALMT2 by heterologously expressing it in Xenopus laevis oocytes and transgenic Arabidopsis. In oocytes, ZmALMT2 mediated an Al-independent electrogenic transport product of organic and inorganic anion efflux. Ectopic overexpression of ZmALMT2 in an Al-hypersensitive Arabidopsis KO/KD line lacking the Al tolerance genes, AtALMT1 and AtMATE, resulted in Al-independent constitutive root malate efflux which partially restored the Al tolerance phenotype. The lack of correlation between ZmALMT2 expression and Al tolerance (e.g., expression not localized to the root tip, not up-regulated by Al, and higher in sensitive versus tolerance maize lines) also led us to question ZmALMT2's role in Al tolerance. The functional properties of the ZmALMT2 transporter presented here, along with the gene expression data, suggest that ZmALMT2 is not involved in maize Al tolerance but, rather, may play a role in mineral nutrient acquisition and transport. Published 2011. This article is a U.S. Government work and is in the public domain in the USA.

The complete genome and proteome of Laribacter hongkongensis reveal potential mechanisms for adaptations to different temperatures and habitats.

PubMed

Woo, Patrick C Y; Lau, Susanna K P; Tse, Herman; Teng, Jade L L; Curreem, Shirly O T; Tsang, Alan K L; Fan, Rachel Y Y; Wong, Gilman K M; Huang, Yi; Loman, Nicholas J; Snyder, Lori A S; Cai, James J; Huang, Jian-Dong; Mak, William; Pallen, Mark J; Lok, Si; Yuen, Kwok-Yung

2009-03-01

Laribacter hongkongensis is a newly discovered Gram-negative bacillus of the Neisseriaceae family associated with freshwater fish-borne gastroenteritis and traveler's diarrhea. The complete genome sequence of L. hongkongensis HLHK9, recovered from an immunocompetent patient with severe gastroenteritis, consists of a 3,169-kb chromosome with G+C content of 62.35%. Genome analysis reveals different mechanisms potentially important for its adaptation to diverse habitats of human and freshwater fish intestines and freshwater environments. The gene contents support its phenotypic properties and suggest that amino acids and fatty acids can be used as carbon sources. The extensive variety of transporters, including multidrug efflux and heavy metal transporters as well as genes involved in chemotaxis, may enable L. hongkongensis to survive in different environmental niches. Genes encoding urease, bile salts efflux pump, adhesin, catalase, superoxide dismutase, and other putative virulence factors-such as hemolysins, RTX toxins, patatin-like proteins, phospholipase A1, and collagenases-are present. Proteomes of L. hongkongensis HLHK9 cultured at 37 degrees C (human body temperature) and 20 degrees C (freshwater habitat temperature) showed differential gene expression, including two homologous copies of argB, argB-20, and argB-37, which encode two isoenzymes of N-acetyl-L-glutamate kinase (NAGK)-NAGK-20 and NAGK-37-in the arginine biosynthesis pathway. NAGK-20 showed higher expression at 20 degrees C, whereas NAGK-37 showed higher expression at 37 degrees C. NAGK-20 also had a lower optimal temperature for enzymatic activities and was inhibited by arginine probably as negative-feedback control. Similar duplicated copies of argB are also observed in bacteria from hot springs such as Thermus thermophilus, Deinococcus geothermalis, Deinococcus radiodurans, and Roseiflexus castenholzii, suggesting that similar mechanisms for temperature adaptation may be employed by other bacteria. Genome and proteome analysis of L. hongkongensis revealed novel mechanisms for adaptations to survival at different temperatures and habitats.

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.

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.

Schiff base derived from thiosemicarbazone and anthracene showed high potential in overcoming multidrug resistance in vitro with low drug resistance index.

PubMed

Bai, Jie; Wang, Rui-Hui; Qiao, Yan; Wang, Aidong; Fang, Chen-Jie

2017-01-01

Multidrug resistance (MDR) is a huge obstacle in cancer chemotherapeutics. Overcoming MDR is a great challenge for anticancer drug discovery. Here, DNA binding and cytotoxicity of Schiff base L1 and L2 were explored to assess their efficiency in fighting cancer and overcoming the MDR. L1 and L2 could treat extremely chemoresistant MCF-7/ADR cell as drug-sensitive cell, with drug resistance index (DRI) <2.13, showing high potential in overcoming the MDR. The apoptotic ratio induced by L1 and L2 was low for both MCF-7 and MCF-7/ADR cells. L1 and L2 induced an impairment of cell cycle progression of MCF-7 and MCF-7/ADR cell lines and suppressed cell growth by perturbing progress through the G0/G1 phase, with L2 causing more profound effect, which might account for lower drug resistance after L2 treatment. The molecular docking revealed weak interaction between L1/L2 and P-glycoprotein (P-gp), the most important drug efflux pump and intracellular Rhodamine 123 accumulation indicated that the activity of P-gp was not inhibited by L1 and L2. Combined with the cellular uptake results, it implied that L1 and L2 could bypass P-gp efflux to exert anticancer activity.

Membrane microparticles mediate transfer of P-glycoprotein to drug sensitive cancer cells.

PubMed

Bebawy, M; Combes, V; Lee, E; Jaiswal, R; Gong, J; Bonhoure, A; Grau, G E R

2009-09-01

Multidrug resistance (MDR), a significant impediment to the successful treatment of cancer clinically, has been attributed to the overexpression of P-glycoprotein (P-gp), a plasma membrane multidrug efflux transporter. P-gp maintains sublethal intracellular drug concentrations by virtue of its drug efflux capacity. The cellular regulation of P-gp expression is currently known to occur at either pre- or post-transcriptional levels. In this study, we identify a 'non-genetic' mechanism whereby microparticles (MPs) serve as vectors in the acquisition and spread of MDR. MPs isolated from drug-resistant cancer cells (VLB(100)) were co-cultured with drug sensitive cells (CCRF-CEM) over a 4 h period to allow for MP binding and P-gp transfer. Presence of P-gp on MPs was established using flow cytometry (FCM) and western blotting. Whole-cell drug accumulation assays using rhodamine 123 and daunorubicin (DNR) were carried out to validate the transfer of functional P-gp after co-culture. We establish that MPs shed in vitro from drug-resistant cancer cells incorporate cell surface P-gp from their donor cells, effectively bind to drug-sensitive recipient cells and transfer functional P-gp to the latter. These findings serve to substantially advance our understanding of the molecular basis for the emergence of MDR in cancer clinically and lead to new treatment strategies which target and inhibit MP mediated transfer of P-gp during the course of treatment.

Combating multidrug-resistant Gram-negative bacterial infections.

PubMed

Xu, Ze-Qi; Flavin, Michael T; Flavin, John

2014-02-01

Multidrug-resistant (MDR) bacterial infections, especially those caused by Gram-negative pathogens, have emerged as one of the world's greatest health threats. The development of novel antibiotics to treat MDR Gram-negative bacteria has, however, stagnated over the last half century. This review provides an overview of recent R&D activities in the search for novel antibiotics against MDR Gram-negatives. It provides emphasis in three key areas. First, the article looks at new analogs of existing antibiotic molecules such as β-lactams, tetracyclines, and aminoglycoside as well as agents against novel bacterial targets such as aminoacyl-tRNA synthetase and peptide deformylase. Second, it also examines alternative strategies to conventional approaches including cationic antimicrobial peptides, siderophores, efflux pump inhibitors, therapeutic antibodies, and renewed interest in abandoned treatments or those with limited indications. Third, the authors aim to provide an update on the current clinical development status for each drug candidate. The traditional analog approach is insufficient to meet the formidable challenge brought forth by MDR superbugs. With the disappointing results of the genomics approach for delivering novel targets and drug candidates, alternative strategies to permeate the bacterial cell membrane, enhance influx, disrupt efflux, and target specific pathogens via therapeutic antibodies are attractive and promising. Coupled with incentivized business models, governmental policies, and a clarified regulatory pathway, it is hoped that the antibiotic pipeline will be filled with an effective armamentarium to safeguard global health.

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

PubMed

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

2016-06-28

Breast cancer is the most frequent malignancy in women. Multidrug resistance due to overexpression of ABC drug transporters is a common cause of chemotherapy failure and disease recurrence. Genistein (GNT) is a phytoestrogen present in soybeans and hormone supplements. We investigated the effect of GNT on the expression and function of ABC transporters in MCF-7 and MDA-MB-231 breast cancer cell lines. Results demonstrated an induction at the protein level of ABCC1 and ABCG2 and of ABCC1 in MCF-7 and MDA-MB-231, respectively. MCF-7 cells showed a concomitant increase in doxorubicin and mitoxantrone efflux and resistance, dependent on ABCG2 activity. ABCC1 induction by GNT in MDA-MB-231 cells modified neither drug efflux nor chemoresistance due to simultaneous acute inhibition of the transporter activity by GNT. All inductions took place at the translational level, as no increment in mRNA was observed and protein increase was prevented by cycloheximide. miR-181a, already demonstrated to inhibit ABCG2 translation, was down-regulated by GNT, explaining translational induction. Effects were independent of classical estrogen receptors. Results suggest potential nutrient-drug interactions that could threaten chemotherapy efficacy, especially in ABCG2-expressing tumors treated with substrates of this transporter. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Soybean NADP-Malic Enzyme Functions in Malate and Citrate Metabolism and Contributes to Their Efflux under Al Stress.

PubMed

Zhou, Ying; Yang, Zhenming; Xu, Yuezi; Sun, Haoran; Sun, Zhitao; Lin, Bao; Sun, Wenjing; You, Jiangfeng

2017-01-01

Malate accumulation has been suggested to balance Al-induced citrate synthesis and efflux in soybean roots. To test this hypothesis, characteristics of Al-induced accumulation and efflux of citrate and malate were compared between two soybean genotypes combining a functional analysis of GmME1 putatively encode a cytosolic NADP-malic enzyme. Similar amounts of citrate were released, and root elongation was equally inhibited before 8 h of Al treatment of Jiyu 70 and Jiyu 62 cultivars. Jiyu 70 began to secrete more citrate and exhibited higher Al resistance than did Jiyu 62 at 12 h. A sustained increase in internal malate and citrate concentrations was observed in Jiyu 70 at 24 h of Al treatment. However, Jiyu 62 decreased its malate concentration at 12 h and its citrate concentration at 24 h of Al treatment. GmME1 localized to the cytoplast and clustered closely with cytosolic malic enzymes AtME2 and SgME1 and was constitutively expressed in the roots. Al treatment induced higher NADP-malic enzyme activities and GmME1 expression levels in Jiyu 70 than in Jiyu 62 within 24 h. Compared with wild-type hairy roots, over-expressing GmME1 in hairy roots ( GmME1 -OE) produced higher expression levels of GmME1 but did not change the expression patterns of either of the putative citrate transporter genes GmAACT1 and GmFRDL or the malate transporter gene GmALMT1 , with or without Al treatment. GmME1 -OE showed a higher internal concentration and external efflux of both citrate and malate at 4 h of Al stress. Lighter hematoxylin staining and lower Al contents in root apices of GmME1 -OE hairy roots indicated greater Al resistance. Comprehensive experimental results suggest that sustaining Al-induced citrate efflux depends on the malate pool in soybean root apices. GmME1 encodes a cytosolic malic enzyme that contributes to increased internal malate and citrate concentrations and their external efflux to confer higher Al resistance.

Pseudoatomic Structure of the Tripartite Multidrug Efflux Pump AcrAB-TolC Reveals the Intermeshing Cogwheel-like Interaction between AcrA and TolC.

PubMed

Jeong, Hyeongseop; Kim, Jin-Sik; Song, Saemee; Shigematsu, Hideki; Yokoyama, Takeshi; Hyun, Jaekyung; Ha, Nam-Chul

2016-02-02

The resistance-nodulation-division type tripartite pump AcrAB-TolC and its homologs are responsible for multidrug resistance in Gram-negative bacteria by expelling a wide variety of toxic substrates. The three essential components, AcrA, AcrB, and TolC, must function in concert with each respective binding partner within the complex. In this study, we report an 8.2-Å resolution cryo-electron microscopy (cryo-EM) 3D reconstruction of the complex that consists of an AcrAB fusion protein and a chimeric TolC protein. The pseudoatomic structure derived from the cryo-EM reconstruction clearly demonstrates a model only compatible with the adaptor bridging mechanism, wherein the funnel-like AcrA hexamer forms an intermeshing cogwheel-like interaction with the α-barrel tip region of TolC. These observations provide a structural milestone for understanding multidrug resistance in pathogenic Gram-negative bacteria, and may also lead to the design of new antibacterial drugs. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

AST1306, a potent EGFR inhibitor, antagonizes ATP-binding cassette subfamily G member 2-mediated multidrug resistance.

PubMed

Zhang, Hui; Wang, Yi-Jun; Zhang, Yun-Kai; Wang, De-Shen; Kathawala, Rishil J; Patel, Atish; Talele, Tanaji T; Chen, Zhe-Sheng; Fu, Li-Wu

2014-08-01

AST1306, an inhibitor of EGFR and ErbB2, is currently in phase I of clinical trials. We evaluated the effect of AST306 on the reversal of multidrug resistance (MDR) induced by ATP-binding cassette (ABC) transporters. We found that AST1306 significantly sensitized the ABC subfamily G member 2 (ABCG2)-overexpressing cells to ABCG2 substrate chemotherapeutics. AST1306 significantly increased intracellular accumulation of [(3)H]-mitoxantrone in ABCG2-overexpressing cells by blocking ABCG2 efflux function. Moreover, AST1306 stimulated the ATPase activity of ABCG2. Homology modeling predicted the binding conformation of AST1306 to be within the transmembrane region of ABCG2. In conclusion, AST1306 could notably reverse ABCG2-mediated MDR. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

MRP1 and glucosylceramide are coordinately over expressed and enriched in rafts during multidrug resistance acquisition in colon cancer cells.

PubMed

Klappe, Karin; Hinrichs, John W J; Kroesen, Bart-Jan; Sietsma, Hannie; Kok, Jan Willem

2004-07-01

Previously we have described a novel multidrug-resistant cell line, HT29(col), which displayed over expression of the multidrug-resistance protein 1 (MRP1) and an altered sphingolipid composition, including enhanced levels of glucosylceramide (GlcCer; Kok JW, Veldman RJ, Klappe K, Koning H, Filipeanu C, Muller M. Int J Cancer 2000;87:172-8). In our study, long-term screening revealed that, during colchicine-induced acquisition of multidrug resistance in a new HT29(col) cell line, increases in GlcCer occurred concomitantly with upregulation of MRP1 expression. Both MRP1 and GlcCer were found enriched in Lubrol-insoluble membrane domains. The expression of MRP1 and GlcCer were tightly correlated, as indicated also by a reversal of both at the later stage of colchicine consolidation. Resistance to colchicine was determined by MRP1, while glucosylceramide synthase (GCS) did not contribute: 1). Resistance was fully inhibited by MK571. 2). GCS expression and activity were not upregulated in HT29(col) cells. 3). Inhibition of GCS did not affect MRP1-mediated efflux function or sensitivity to colchicine. Instead, overall sphingolipid metabolism was upregulated through an increased rate of ceramide biosynthesis. In conclusion, upregulation of MRP1 occurs in concert with upregulation of GlcCer during multidrug-resistance acquisition, and both are enriched in rafts. The increased GlcCer pool does not directly modulate MRP1 function and cell survival. Copyright 2004 Wiley-Liss, Inc.

Putative histidine kinase inhibitors with antibacterial effect against multi-drug resistant clinical isolates identified by in vitro and in silico screens

NASA Astrophysics Data System (ADS)

Velikova, Nadya; Fulle, Simone; Manso, Ana Sousa; Mechkarska, Milena; Finn, Paul; Conlon, J. Michael; Oggioni, Marco Rinaldo; Wells, Jerry M.; Marina, Alberto

2016-05-01

Novel antibacterials are urgently needed to address the growing problem of bacterial resistance to conventional antibiotics. Two-component systems (TCS) are widely used by bacteria to regulate gene expression in response to various environmental stimuli and physiological stress and have been previously proposed as promising antibacterial targets. TCS consist of a sensor histidine kinase (HK) and an effector response regulator. The HK component contains a highly conserved ATP-binding site that is considered to be a promising target for broad-spectrum antibacterial drugs. Here, we describe the identification of putative HK autophosphorylation inhibitors following two independent experimental approaches: in vitro fragment-based screen via differential scanning fluorimetry and in silico structure-based screening, each followed up by the exploration of analogue compounds as identified by ligand-based similarity searches. Nine of the tested compounds showed antibacterial effect against multi-drug resistant clinical isolates of bacterial pathogens and include three novel scaffolds, which have not been explored so far in other antibacterial compounds. Overall, putative HK autophosphorylation inhibitors were found that together provide a promising starting point for further optimization as antibacterials.

A Glycine Riboswitch in Streptococcus pyogenes Controls Expression of a Sodium:Alanine Symporter Family Protein Gene.

PubMed

Khani, Afsaneh; Popp, Nicole; Kreikemeyer, Bernd; Patenge, Nadja

2018-01-01

Regulatory RNAs play important roles in the control of bacterial gene expression. In this study, we investigated gene expression regulation by a putative glycine riboswitch located in the 5'-untranslated region of a sodium:alanine symporter family (SAF) protein gene in the group A Streptococcus pyogenes serotype M49 strain 591. Glycine-dependent gene expression mediated by riboswitch activity was studied using a luciferase reporter gene system. Maximal reporter gene expression was observed in the absence of glycine and in the presence of low glycine concentrations. Differences in glycine-dependent gene expression were not based on differential promoter activity. Expression of the SAF protein gene and the downstream putative cation efflux protein gene was investigated in wild-type bacteria by RT-qPCR transcript analyses. During growth in the presence of glycine (≥1 mM), expression of the genes were downregulated. Northern blot analyses revealed premature transcription termination in the presence of high glycine concentrations. Growth in the presence of 0.1 mM glycine led to the production of a full-length transcript. Furthermore, stability of the SAF protein gene transcript was drastically reduced in the presence of glycine. We conclude that the putative glycine riboswitch in S. pyogenes serotype M49 strain 591 represses expression of the SAF protein gene and the downstream putative cation efflux protein gene in the presence of high glycine concentrations. Sequence and secondary structure comparisons indicated that the streptococcal riboswitch belongs to the class of tandem aptamer glycine riboswitches.

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.

PKC phosphorylates residues in the N-terminal of the DA transporter to regulate amphetamine-induced DA efflux.

PubMed

Wang, Qiang; Bubula, Nancy; Brown, Jason; Wang, Yunliang; Kondev, Veronika; Vezina, Paul

2016-05-27

The DA transporter (DAT), a phosphoprotein, controls extracellular dopamine (DA) levels in the central nervous system through transport or reverse transport (efflux). Multiple lines of evidence support the claim that PKC significantly contributes to amphetamine-induced DA efflux. Other signaling pathways, involving CaMKII and ERK, have also been shown to regulate DAT mediated efflux. Here we assessed the contribution of putative PKC residues (S4, S7, S13) in the N-terminal of the DAT to amphetamine-induced DA efflux by transfecting DATs containing different serine to alanine (S-A) point mutations into DA pre-loaded HEK-293 cells and incubating these cells in amphetamine (2μM). The effects of a S-A mutation at the non-PKC residue S12 and a threonine to alanine (T-A) mutation at the ERK T53 residue were also assessed for comparison. WT-DATs were used as controls. In an initial experiment, we confirmed that inhibiting PKC with Go6976 (130nM) significantly reduced amphetamine-induced DA efflux. In subsequent experiments, cells transfected with the S4A, S12A, S13A, T53A and S4,7,13A mutants showed a reduction in amphetamine-induced DA efflux similar to that observed with Go6976. Interestingly, cells transfected with the S7A mutant, identified by some as a PKC-PKA residue, showed unperturbed WT-DAT levels of amphetamine-induced DA efflux. These results indicate that phosphorylation by PKC of select residues in the DAT N-terminal can regulate amphetamine-induced efflux. PKC can act either independently or in concert with other kinases such as ERK to produce this effect. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Bitter melon extracts enhance the activity of chemotherapeutic agents through the modulation of multiple drug resistance.

PubMed

Kwatra, Deep; Venugopal, Anand; Standing, David; Ponnurangam, Sivapriya; Dhar, Animesh; Mitra, Ashim; Anant, Shrikant

2013-12-01

Recently, we demonstrated that extracts of bitter melon (BME) can be used as a preventive/therapeutic agent in colon cancers. Here, we determined BME effects on anticancer activity and bioavailability of doxorubicin (DOX) in colon cancer cells. BME enhanced the effect of DOX on cell proliferation and sensitized the cells toward DOX upon pretreatment. Furthermore, there was both increased drug uptake and reduced drug efflux. We also observed a reduction in the expression of multidrug resistance conferring proteins (MDRCP) P-glycoprotein, MRP-2, and BCRP. Further BME suppressed DOX efflux in MDCK cells overexpressing the three efflux proteins individually, suggesting that BME is a potent inhibitor of MDR function. Next, we determined the effect of BME on PXR, a xenobiotic sensing nuclear receptor and a transcription factor that controls the expression of the three MDR genes. BME suppressed PXR promoter activity thereby suppressing its expression. Finally, we determined the effect of AMPK pathway on drug efflux because we have previously demonstrated that BME affects the pathway. However, inhibiting AMPK did not affect drug resistance, suggesting that BME may use different pathways for the anticancer and MDR modulating activities. Together, these results suggest that BME can enhance the bioavailability and efficacy of conventional chemotherapy. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

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

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.

Design of a large-scale femtoliter droplet array for single-cell analysis of drug-tolerant and drug-resistant bacteria.

PubMed

Iino, Ryota; Matsumoto, Yoshimi; Nishino, Kunihiko; Yamaguchi, Akihito; Noji, Hiroyuki

2013-01-01

Single-cell analysis is a powerful method to assess the heterogeneity among individual cells, enabling the identification of very rare cells with properties that differ from those of the majority. In this Methods Article, we describe the use of a large-scale femtoliter droplet array to enclose, isolate, and analyze individual bacterial cells. As a first example, we describe the single-cell detection of drug-tolerant persisters of Pseudomonas aeruginosa treated with the antibiotic carbenicillin. As a second example, this method was applied to the single-cell evaluation of drug efflux activity, which causes acquired antibiotic resistance of bacteria. The activity of the MexAB-OprM multidrug efflux pump system from Pseudomonas aeruginosa was expressed in Escherichia coli and the effect of an inhibitor D13-9001 were assessed at the single cell level.

Molecular mechanisms of gravity-dependent signaling in human melanocytic cells involve cyclic GMP

NASA Astrophysics Data System (ADS)

Ivanova, Krassimira; Lambers, Britta; Block, Ingrid; Bromeis, Birgit; Das, Pranab K.; Gerzer, Rupert

2005-08-01

Gravity alteration (micro- and hypergravity) is known to influence cell functions. As guanosine 3',5'-cyclic monophosphate (cGMP) is an important messenger in melanocyte signaling we have compared the regulation of cGMP levels in human melanocytes and melanoma cells with different metastatic potential under hypergravity conditions. We were able to demonstrate that long-term exposure to hypergravity stimulates cGMP efflux in cultured human melanocytes and non- metastatic melanoma cells, whereas highly metastatic melanoma cells appear to be insensitive to hypergravity, most probably, due to an up-regulated cGMP efflux at 1g. Here we report that these effects are associated with the expression of the multidrug resistance proteins 4 and 5 known to act as selective export pumps for amphiphilic anions like cGMP. Thus, an altered gravity vector may induce cGMP-dependent signaling events in melanocytic cells that could be important for malignant transformation.

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.

Multidrug-Resistant CTX-M-(15, 9, 2)- and KPC-2-Producing Enterobacter hormaechei and Enterobacter asburiae Isolates Possessed a Set of Acquired Heavy Metal Tolerance Genes Including a Chromosomal sil Operon (for Acquired Silver Resistance).

PubMed

Andrade, Leonardo N; Siqueira, Thiago E S; Martinez, Roberto; Darini, Ana Lucia C

2018-01-01

Bacterial resistance to antibiotics is concern in healthcare-associated infections. On the other hand, bacterial tolerance to other antimicrobials, like heavy metals, has been neglected and underestimated in hospital pathogens. Silver has long been used as an antimicrobial agent and it seems to be an important indicator of heavy metal tolerance. To explore this perspective, we searched for the presence of acquired silver resistance genes ( sil operon: silE, silS, silR, silC, silF, silB, silA , and silP ) and acquired extended-spectrum cephalosporin and carbapenem resistance genes ( bla CTX-M and bla KPC ) in Enterobacter cloacae Complex (EcC) ( n = 27) and Enterobacter aerogenes ( n = 8) isolated from inpatients at a general hospital. Moreover, the genetic background of the silA (silver-efflux pump) and the presence of other acquired heavy metal tolerance genes, pcoD (copper-efflux pump), arsB (arsenite-efflux pump), terF (tellurite resistance protein), and merA (mercuric reductase) were also investigated. Outstandingly, 21/27 (78%) EcC isolates harbored silA gene located in the chromosome. Complete sil operon was found in 19/21 silA -positive EcC isolates. Interestingly, 8/20 (40%) E. hormaechei and 5/6 (83%) E. asburiae co-harbored silA/pcoD genes and bla CTX-M-(15,2,or9) and/or bla KPC-2 genes. Frequent occurrences of arsB, terF , and merA genes were detected, especially in silA/pcoD -positive, multidrug-resistant (MDR) and/or CTX-M-producing isolates. Our study showed co-presence of antibiotic and heavy metal tolerance genes in MDR EcC isolates. In our viewpoint, there are few studies regarding to bacterial heavy metal tolerance and we call attention for more investigations and discussion about this issue in different hospital pathogens.

Multidrug-Resistant CTX-M-(15, 9, 2)- and KPC-2-Producing Enterobacter hormaechei and Enterobacter asburiae Isolates Possessed a Set of Acquired Heavy Metal Tolerance Genes Including a Chromosomal sil Operon (for Acquired Silver Resistance)

PubMed Central

Andrade, Leonardo N.; Siqueira, Thiago E. S.; Martinez, Roberto; Darini, Ana Lucia C.

2018-01-01

Bacterial resistance to antibiotics is concern in healthcare-associated infections. On the other hand, bacterial tolerance to other antimicrobials, like heavy metals, has been neglected and underestimated in hospital pathogens. Silver has long been used as an antimicrobial agent and it seems to be an important indicator of heavy metal tolerance. To explore this perspective, we searched for the presence of acquired silver resistance genes (sil operon: silE, silS, silR, silC, silF, silB, silA, and silP) and acquired extended-spectrum cephalosporin and carbapenem resistance genes (blaCTX−M and blaKPC) in Enterobacter cloacae Complex (EcC) (n = 27) and Enterobacter aerogenes (n = 8) isolated from inpatients at a general hospital. Moreover, the genetic background of the silA (silver-efflux pump) and the presence of other acquired heavy metal tolerance genes, pcoD (copper-efflux pump), arsB (arsenite-efflux pump), terF (tellurite resistance protein), and merA (mercuric reductase) were also investigated. Outstandingly, 21/27 (78%) EcC isolates harbored silA gene located in the chromosome. Complete sil operon was found in 19/21 silA-positive EcC isolates. Interestingly, 8/20 (40%) E. hormaechei and 5/6 (83%) E. asburiae co-harbored silA/pcoD genes and blaCTX−M−(15,2,or9) and/or blaKPC−2 genes. Frequent occurrences of arsB, terF, and merA genes were detected, especially in silA/pcoD-positive, multidrug-resistant (MDR) and/or CTX-M-producing isolates. Our study showed co-presence of antibiotic and heavy metal tolerance genes in MDR EcC isolates. In our viewpoint, there are few studies regarding to bacterial heavy metal tolerance and we call attention for more investigations and discussion about this issue in different hospital pathogens. PMID:29628916

Forced Expression of Heat Shock Protein 27 (Hsp27) Reverses P-Glycoprotein (ABCB1)-mediated Drug Efflux and MDR1 Gene Expression in Adriamycin-resistant Human Breast Cancer Cells*

PubMed Central

Kanagasabai, Ragu; Krishnamurthy, Karthikeyan; Druhan, Lawrence J.; Ilangovan, Govindasamy

2011-01-01

Mutant p53 accumulation has been shown to induce the multidrug resistance gene (MDR1) and ATP binding cassette (ABC)-based drug efflux in human breast cancer cells. In the present work, we have found that transcriptional activation of the oxidative stress-responsive heat shock factor 1 (HSF-1) and expression of heat shock proteins, including Hsp27, which is normally known to augment proteasomal p53 degradation, are inhibited in Adriamycin (doxorubicin)-resistant MCF-7 cells (MCF-7/adr). Such an endogenous inhibition of HSF-1 and Hsp27 in turn results in p53 mutation with gain of function in its transcriptional activity and accumulation in MCF-7/adr. Also, lack of HSF-1 enhances nuclear factor κB (NF-κB) DNA binding activity together with mutant p53 and induces MDR1 gene and P-glycoprotein (P-gp, ABCB1), resulting in a multidrug-resistant phenotype. Ectopic expression of Hsp27, however, significantly depleted both mutant p53 and NF-κB (p65), reversed the drug resistance by inhibiting MDR1/P-gp expression in MCF-7/adr cells, and induced cell death by increased G2/M population and apoptosis. We conclude from these results that HSF-1 inhibition and depletion of Hsp27 is a trigger, at least in part, for the accumulation of transcriptionally active mutant p53, which can either directly or NF-κB-dependently induce an MDR1/P-gp phenotype in MCF-7 cells. Upon Hsp27 overexpression, this pathway is abrogated, and the acquired multidrug resistance is significantly abolished so that MCF-7/adr cells are sensitized to Dox. Thus, clinical alteration in Hsp27 or NF-κB level will be a potential approach to circumvent drug resistance in breast cancer. PMID:21784846

Forced expression of heat shock protein 27 (Hsp27) reverses P-glycoprotein (ABCB1)-mediated drug efflux and MDR1 gene expression in Adriamycin-resistant human breast cancer cells.

PubMed

Kanagasabai, Ragu; Krishnamurthy, Karthikeyan; Druhan, Lawrence J; Ilangovan, Govindasamy

2011-09-23

Mutant p53 accumulation has been shown to induce the multidrug resistance gene (MDR1) and ATP binding cassette (ABC)-based drug efflux in human breast cancer cells. In the present work, we have found that transcriptional activation of the oxidative stress-responsive heat shock factor 1 (HSF-1) and expression of heat shock proteins, including Hsp27, which is normally known to augment proteasomal p53 degradation, are inhibited in Adriamycin (doxorubicin)-resistant MCF-7 cells (MCF-7/adr). Such an endogenous inhibition of HSF-1 and Hsp27 in turn results in p53 mutation with gain of function in its transcriptional activity and accumulation in MCF-7/adr. Also, lack of HSF-1 enhances nuclear factor κB (NF-κB) DNA binding activity together with mutant p53 and induces MDR1 gene and P-glycoprotein (P-gp, ABCB1), resulting in a multidrug-resistant phenotype. Ectopic expression of Hsp27, however, significantly depleted both mutant p53 and NF-κB (p65), reversed the drug resistance by inhibiting MDR1/P-gp expression in MCF-7/adr cells, and induced cell death by increased G(2)/M population and apoptosis. We conclude from these results that HSF-1 inhibition and depletion of Hsp27 is a trigger, at least in part, for the accumulation of transcriptionally active mutant p53, which can either directly or NF-κB-dependently induce an MDR1/P-gp phenotype in MCF-7 cells. Upon Hsp27 overexpression, this pathway is abrogated, and the acquired multidrug resistance is significantly abolished so that MCF-7/adr cells are sensitized to Dox. Thus, clinical alteration in Hsp27 or NF-κB level will be a potential approach to circumvent drug resistance in breast cancer.

Virtual screening of ABCC1 transporter nucleotidebinding domains as a therapeutic target in multidrug resistant cancer

PubMed Central

Rungsardthong, Kanin; Mares- Sámano, Sergio; Penny, Jeffrey

2012-01-01

ABCC1 is a member of the ATP-binding Cassette super family of transporters, actively effluxes xenobiotics from cells. Clinically, ABCC1 expression is linked to cancer multidrug resistance. Substrate efflux is energised by ATP binding and hydrolysis at the nucleotide-binding domains (NBDs) and inhibition of these events may help combat drug resistance. The aim of this study is to identify potential inhibitors of ABCC1 through virtual screening of National Cancer Institute (NCI) compounds. A threedimensional model of ABCC1 NBD2 was generated using MODELLER whilst the X-ray crystal structure of ABCC1 NBD1 was retrieved from the Protein Data Bank. A pharmacophore hypothesis was generated based on flavonoids known to bind at the NBDs using PHASE, and used to screen the NCI database. GLIDE was employed in molecular docking studies for all hit compounds identified by pharmacophore screening. The best potential inhibitors were identified as compounds possessing predicted binding affinities greater than ATP. Approximately 5% (13/265) of the hit compounds possessed lower docking scores than ATP in ABCC1 NBD1 (NSC93033, NSC662377, NSC319661, NSC333748, NSC683893, NSC226639, NSC94231, NSC55979, NSC169121, NSC166574, NSC73380, NSC127738, NSC115534), whereas approximately 7% (7/104) of docked NCI compounds were predicted to possess lower docking scores than ATP in ABCC1 NBD2 (NSC91789, NSC529483, NSC211168, NSC318214, NSC116519, NSC372332, NSC526974). Analyses of docking orientations revealed P-loop residues of each NBD and the aromatic amino acids Trp653 (NBD1) and Tyr1302 (NBD2) were key in interacting with high-affinity compounds. On the basis of docked orientation and docking score the compounds identified may be potential inhibitors of ABCC1 and require further pharmacological analysis. Abbreviations ABC - ATP-binding cassette, DHS - dehydrosilybin, MDR - multidrug resistance, NBD - nucleotide-binding domain, PDB - protein data bank. PMID:23144549

Antibacterial and antibiotic-potentiation activities of the methanol extract of some cameroonian spices against Gram-negative multi-drug resistant phenotypes

PubMed Central

2012-01-01

Background The present work was designed to evaluate the antibacterial properties of the methanol extracts of eleven selected Cameroonian spices on multi-drug resistant bacteria (MDR), and their ability to potentiate the effect of some common antibiotics used in therapy. Results The extract of Cinnamomum zeylanicum against Escherichia coli ATCC 8739 and AG100 strains showed the best activities, with the lowest minimal inhibitory concentration (MIC) of 64 μg/ml. The extract of Dorstenia psilurus was the most active when tested in the presence of an efflux pump inhibitor, phenylalanine Arginine-β- Naphtylamide (PAβN), a synergistic effect being observed in 56.25 % of the tested bacteria when it was combined with Erythromycin (ERY). Conclusion The present work evidently provides information on the role of some Cameroonian spices in the fight against multi-resistant bacteria. PMID:22709668

Reversion of the P-glycoprotein-mediated multidrug resistance of cancer cells by FK-506 derivatives.

PubMed

Jachez, B; Boesch, D; Grassberger, M A; Loor, F

1993-04-01

FK-506 is a resistance-modulating agent (RMA) for tumor cells whose multidrug resistance (MDR) involves a P-glycoprotein (Pgp)-mediated anti-cancer drug efflux. The family of FK-506 relatives and derivatives includes analogs which display a whole range of chemosensitizing strengths, from no detectable RMA activity to a complete reversion of the MDR phenotype. Similarly, FK-506 analogs display a whole range of immunosuppressive activities, including inactive ones. FK-506 was compared for RMA activity with 11 FK-506 analogs which were at least 20-fold less active than FK-506 for the inhibition of the bi-directional mixed lymphocyte reaction displayed the whole range of RMA activity. One such strong RMA derivative of FK-506 (SDZ 280-629) was further shown able to restore completely daunomycin retention by highly resistant MDR P388 tumor cells.

ESKAPEing the labyrinth of antibacterial discovery.

PubMed

Tommasi, Ruben; Brown, Dean G; Walkup, Grant K; Manchester, John I; Miller, Alita A

2015-08-01

Antimicrobial drug resistance is a growing threat to global public health. Multidrug resistance among the 'ESKAPE' organisms - encompassing Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. - is of particular concern because they are responsible for many serious infections in hospitals. Although some promising agents are in the pipeline, there is an urgent need for new antibiotic scaffolds. However, antibacterial researchers have struggled to identify new small molecules with meaningful cellular activity, especially those effective against multidrug-resistant Gram-negative pathogens. This difficulty ultimately stems from an incomplete understanding of efflux systems and compound permeation through bacterial membranes. This Opinion article describes findings from target-based and phenotypic screening efforts carried out at AstraZeneca over the past decade, discusses some of the subsequent chemistry challenges and concludes with a description of new approaches comprising a combination of computational modelling and advanced biological tools which may pave the way towards the discovery of new antibacterial agents.

Multidrug-Resistant Candida haemulonii and C. auris, Tel Aviv, Israel.

PubMed

Ben-Ami, Ronen; Berman, Judith; Novikov, Ana; Bash, Edna; Shachor-Meyouhas, Yael; Zakin, Shiri; Maor, Yasmin; Tarabia, Jalal; Schechner, Vered; Adler, Amos; Finn, Talya

2017-02-01

Candida auris and C. haemulonii are closely related, multidrug-resistant emerging fungal pathogens that are not readily distinguishable with phenotypic assays. We studied C. auris and C. haemulonii clinical isolates from 2 hospitals in central Israel. C. auris was isolated in 5 patients with nosocomial bloodstream infection, and C. haemulonii was found as a colonizer of leg wounds at a peripheral vascular disease clinic. Liberal use of topical miconazole and close contact among patients were implicated in C. haemulonii transmission. C. auris exhibited higher thermotolerance, virulence in a mouse infection model, and ATP-dependent drug efflux activity than C. haemulonii. Comparison of ribosomal DNA sequences found that C. auris strains from Israel were phylogenetically distinct from isolates from East Asia, South Africa and Kuwait, whereas C. haemulonii strains from different countries were closely interrelated. Our findings highlight the pathogenicity of C. auris and underscore the need to limit its spread.

Multidrug resistant bacteria are sensitive to Euphorbia prostrata and six others Cameroonian medicinal plants extracts.

PubMed

Voukeng, Igor K; Beng, Veronique P; Kuete, Victor

2017-07-25

Multidrug resistant (MDR) bacteria are responsible for therapeutic failure and there is an urgent need for novels compounds efficient on them. Eleven methanol extracts from seven Cameroonian medicinal plants were tested for their antibacterial activity using broth micro-dilution method against 36 MDR bacterial strains including Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae, Klebsiella pneumoniae, Providencia stuartii, Pseudomonas aeruginosa and Staphylococcus aureus. Euphorbia prostrata extract was found active against all the 36 tested bacteria including Gram-negative phenotypes over-expressing efflux pumps such as P. aeruginosa PA124, E. aerogenes CM64 and E. coli AG102. E. prostrata had minimal inhibitory concentrations values between 128 and 256 µg/mL on 55.55% of the studied microorganisms. Other plants extract displayed selective antibacterial activity. Results obtained in this study highlight the antibacterial potential of the tested plants and the possible use of E. prostrata to combat bacterial infections including MDR phenotypes.

Different efflux rates may determine the cellular accumulation of various bis(guanylhydrazones).

PubMed Central

Alhonen-Hongisto, L; Fagerström, R; Laine, R; Elo, H; Jänne, J

1984-01-01

Three bis(guanylhydrazones) (those of methylglyoxal, glyoxal and ethylglyoxal) were compared for their affinity for the putative polyamine carrier and for their cellular retention in L1210 mouse leukaemia cells. All the bis(guanylhydrazones) inhibited equally effectively the uptake of spermidine by the tumour cells, indicating that the compounds had roughly equal affinity for the polyamine carrier. The fact that methylglyoxal bis(guanylhydrazone) and glyoxal bis(guanylhydrazone) were much more effectively concentrated in the animal cells than was ethylglyoxal bis(guanylhydrazone) was obviously attributable to the finding that the efflux rate of ethylglyoxal bis(guanylhydrazone) greatly exceeded that of the other bis(guanylhydrazones). The rate of efflux of the drugs was slowed down if the tumour cells were treated with 2-difluoromethylornithine before exposure to the bis(guanylhydrazones). These results suggest that intracellular binding of the bis(guanylhydrazones) determines their cellular accumulation. PMID:6431972

Identification of the High-affinity Substrate-binding Site of the Multidrug and Toxic Compound Extrusion (MATE) Family Transporter from Pseudomonas stutzeri*

PubMed Central

Nie, Laiyin; Grell, Ernst; Malviya, Viveka Nand; Xie, Hao; Wang, Jingkang; Michel, Hartmut

2016-01-01

Multidrug and toxic compound extrusion (MATE) transporters exist in all three domains of life. They confer multidrug resistance by utilizing H+ or Na+ electrochemical gradients to extrude various drugs across the cell membranes. The substrate binding and the transport mechanism of MATE transporters is a fundamental process but so far not fully understood. Here we report a detailed substrate binding study of NorM_PS, a representative MATE transporter from Pseudomonas stutzeri. Our results indicate that NorM_PS is a proton-dependent multidrug efflux transporter. Detailed binding studies between NorM_PS and 4′,6-diamidino-2-phenylindole (DAPI) were performed by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and spectrofluorometry. Two exothermic binding events were observed from ITC data, and the high-affinity event was directly correlated with the extrusion of DAPI. The affinities are about 1 μm and 0.1 mm for the high and low affinity binding, respectively. Based on our homology model of NorM_PS, variants with mutations of amino acids that are potentially involved in substrate binding, were constructed. By carrying out the functional characterization of these variants, the critical amino acid residues (Glu-257 and Asp-373) for high-affinity DAPI binding were determined. Taken together, our results suggest a new substrate-binding site for MATE transporters. PMID:27235402

Whole genome sequencing for deciphering the resistome of Chryseobacterium indologenes, an emerging multidrug-resistant bacterium isolated from a cystic fibrosis patient in Marseille, France.

PubMed

Cimmino, T; Rolain, J-M

2016-07-01

We decipher the resistome of Chryseobacterium indologenes MARS15, an emerging multidrug-resistant clinical strain, using the whole genome sequencing strategy. The bacterium was isolated from the sputum of a hospitalized patient with cystic fibrosis in the Timone Hospital in Marseille, France. Genome sequencing was done with Illumina MiSeq using a paired-end strategy. The in silico analysis was done by RAST, the resistome by the ARG-ANNOT database and detection of polyketide synthase (PKS) by ANTISMAH. The genome size of C. indologenes MARS15 is 4 972 580 bp with 36.4% GC content. This multidrug-resistant bacterium was resistant to all β-lactams, including imipenem, and also to colistin. The resistome of C. indologenes MARS15 includes Ambler class A and B β-lactams encoding bla CIA and bla IND-2 genes and MBL (metallo-β-lactamase) genes, the CAT (chloramphenicol acetyltransferase) gene and the multidrug efflux pump AcrB. Specific features include the presence of an urease operon, an intact prophage and a carotenoid biosynthesis pathway. Interestingly, we report for the first time in C. indologenes a PKS cluster that might be responsible for secondary metabolite biosynthesis, similar to erythromycin. The whole genome sequence analysis provides insight into the resistome and the discovery of new details, such as the PKS cluster.

Dental plaque bacteria with reduced susceptibility to chlorhexidine are multidrug resistant.

PubMed

Saleem, Hafiz Ghulam Murtaza; Seers, Christine Ann; Sabri, Anjum Nasim; Reynolds, Eric Charles

2016-09-15

Chlorhexidine (CHX) is used in oral care products to help control dental plaque. In this study dental plaque bacteria were grown on media containing 2 μg/ml chlorhexidine gluconate to screen for bacteria with reduced CHX susceptibility. The isolates were characterized by 16S rRNA gene sequencing and antibiotic resistance profiles were determined using the disc diffusion method. The isolates were variably resistant to multiple drugs including ampicillin, kanamycin, gentamicin and tetracycline. Two species, Chryseobacterium culicis and Chryseobacterium indologenes were able to grow planktonically and form biofilms in the presence of 32 μg/ml CHX. In the CHX and multidrug resistant C. indologenes we demonstrated a 19-fold up-regulation of expression of the HlyD-like periplasmic adaptor protein of a tripartite efflux pump upon exposure to 16 μg/ml CHX suggesting that multidrug resistance may be mediated by this system. Exposure of biofilms of these resistant species to undiluted commercial CHX mouthwash for intervals from 5 to 60 s indicated that the mouthwash was unlikely to eliminate them from dental plaque in vivo. The study highlights the requirement for increased vigilance of the presence of multidrug resistant bacteria in dental plaque and raises a potential risk of long-term use of oral care products containing antimicrobial agents for the control of dental plaque.

AmgRS-mediated envelope stress-inducible expression of the mexXY multidrug efflux operon of Pseudomonas aeruginosa

PubMed Central

Lau, Calvin Ho-Fung; Krahn, Thomas; Gilmour, Christie; Mullen, Erin; Poole, Keith

2015-01-01

AmgRS is an envelope stress-responsive two-component system and aminoglycoside resistance determinant in Pseudomonas aeruginosa that is proposed to protect cells from membrane damage caused by aminoglycoside-generated mistranslated polypeptides. Consistent with this, a ΔamgR strain showed increased aminoglycoside-promoted membrane damage, damage that was largely absent in AmgRS-activated amgS-mutant strains. Intriguingly, one such mutation, V121G, while providing for enhanced resistance to aminoglycosides, rendered P. aeruginosa susceptible to several ribosome-targeting nonaminoglycoside antimicrobials that are inducers and presumed substrates of the MexXY-OprM multidrug efflux system. Surprisingly, the amgSV121G mutation increased mexXY expression threefold, suggesting that export of these nonaminoglycosides was compromised in the amgSV121G mutant. Nonetheless, a link was established between AmgRS activation and mexXY expression and this was confirmed in studies showing that aminoglycoside-promoted mexXY expression is dependent on AmgRS. While nonaminoglycosides also induced mexXY expression, this was not AmgRS-dependent, consistent with these agents not generating mistranslated polypeptides and not activating AmgRS. The aminoglycoside inducibility of mexXY was abrogated in a mutant lacking the AmgRS target genes htpX and PA5528, encoding a presumed cytoplasmic membrane-associated protease and a membrane protein of unknown function, respectively. Thus, aminoglycoside induction of mexXY is a response to membrane damage and activation of the AmgRS two-component system. PMID:25450797

De-Differentiation Confers Multidrug Resistance Via Noncanonical PERK-Nrf2 Signaling

PubMed Central

Del Vecchio, Catherine A.; Feng, Yuxiong; Sokol, Ethan S.; Tillman, Erik J.; Sanduja, Sandhya; Reinhardt, Ferenc; Gupta, Piyush B.

2014-01-01

Malignant carcinomas that recur following therapy are typically de-differentiated and multidrug resistant (MDR). De-differentiated cancer cells acquire MDR by up-regulating reactive oxygen species (ROS)–scavenging enzymes and drug efflux pumps, but how these genes are up-regulated in response to de-differentiation is not known. Here, we examine this question by using global transcriptional profiling to identify ROS-induced genes that are already up-regulated in de-differentiated cells, even in the absence of oxidative damage. Using this approach, we found that the Nrf2 transcription factor, which is the master regulator of cellular responses to oxidative stress, is preactivated in de-differentiated cells. In de-differentiated cells, Nrf2 is not activated by oxidation but rather through a noncanonical mechanism involving its phosphorylation by the ER membrane kinase PERK. In contrast, differentiated cells require oxidative damage to activate Nrf2. Constitutive PERK-Nrf2 signaling protects de-differentiated cells from chemotherapy by reducing ROS levels and increasing drug efflux. These findings are validated in therapy-resistant basal breast cancer cell lines and animal models, where inhibition of the PERK-Nrf2 signaling axis reversed the MDR of de-differentiated cancer cells. Additionally, analysis of patient tumor datasets showed that a PERK pathway signature correlates strongly with chemotherapy resistance, tumor grade, and overall survival. Collectively, these results indicate that de-differentiated cells up-regulate MDR genes via PERK-Nrf2 signaling and suggest that targeting this pathway could sensitize drug-resistant cells to chemotherapy. PMID:25203443

THE ROLE OF MULTIDRUG RESISTANCE ASSOCIATED PROTEIN (MRP) IN THE BLOOD-BRAIN BARRIER AND OPIOID ANALGESIA

PubMed Central

Su, Wendy; Pasternak, Gavril W.

2013-01-01

The blood brain barrier protects the brain from circulating compounds and drugs. The ATP-binding cassette (ABC) transporter P-glycoprotein (Pgp) is involved with the barrier, both preventing the influx of agent from the blood into the brain and facilitating the efflux of compounds from the brain into the blood, raising the possibility of a similar role for other transporters. Multidrug resistance associated protein (MRP), a 190 kDa protein similar to Pgp is also ABC transport that has been implicated in the blood brain barrier. The current study explores its role in opioid action. Immunohistochemically, it is localized in the choroid plexus in ratsand can be selectively downregulated by antisense treatment at both the level of mRNA, as shown by RT-PCR, and protein, as demonstrated immunohistochemically. Behaviorally, downregulation of MRP significantly enhances the analgesic potency of systemic morphine in MRP knockout mice and in antisense-treated rats by lowering the blood brain barrier. Following intracerebroventricular administration, a number of compounds, including some opioids, are rapidly secreted from the brain into the blood where they contribute to the overall analgesic effects by activating peripheral systems. MRP plays a role in this efflux. Downregulating MRP expression leads to a corresponding decrease in the transport and a diminished analgesic response from opioids administered intracerebroventricularly. Thus, the transporter protein MRP plays a role in maintaining the blood-brain barrier and modulates the activity of opioids. PMID:23508590

Doxorubicin loaded iron oxide nanoparticles overcome multidrug resistance in cancer in vitro

PubMed Central

Kievit, Forrest M.; Wang, Freddy Y.; Fang, Chen; Mok, Hyejung; Wang, Kui; Silber, John R.; Ellenbogen, Richard G.; Zhang, Miqin

2011-01-01

Multidrug resistance (MDR) is characterized by the overexpression of ATP-binding cassette (ABC) transporters that actively pump a broad class of hydrophobic chemotherapeutic drugs out of cancer cells. MDR is a major mechanism of treatment resistance in a variety of human tumors, and clinically applicable strategies to circumvent MDR remain to be characterized. Here we describe the fabrication and characterization of a drug-loaded iron oxide nanoparticle designed to circumvent MDR. Doxorubicin (DOX), an anthracycline antibiotic commonly used in cancer chemotherapy and substrate for ABC-mediated drug efflux, was covalently bound to polyethylenimine via a pH sensitive hydrazone linkage and conjugated to an iron oxide nanoparticle coated with amine terminated polyethylene glycol. Drug loading, physiochemical properties and pH lability of the DOX-hydrazone linkage were evaluated in vitro. Nanoparticle uptake, retention, and dose-dependent effects on viability were compared in wild-type and DOX-resistant ABC transporter over-expressing rat glioma C6 cells. We found that DOX release from nanoparticles was greatest at acidic pH, indicative of cleavage of the hydrazone linkage. DOX-conjugated nanoparticles were readily taken up by wild-type and drug-resistant cells. In contrast to free drug, DOX-conjugated nanoparticles persisted in drug-resistant cells, indicating that they were not subject to drug efflux. Greater retention of DOX-conjugated nanoparticles was accompanied by reduction of viability relative to cells treated with free drug. Our results suggest that DOX-conjugated nanoparticles could improve the efficacy of chemotherapy by circumventing MDR. PMID:21277920

Alpha-tocopheryl polyethylene glycol succinate-emulsified poly(lactic-co-glycolic acid) nanoparticles for reversal of multidrug resistance in vitro

NASA Astrophysics Data System (ADS)

Wang, Ying; Guo, Miao; Lu, Yu; Ding, Li-Ying; Ron, Wen-Ting; Liu, Ya-Qing; Song, Fei-Fei; Yu, Shu-Qin

2012-12-01

Multidrug resistance (MDR) is one of the factors in the failure of anticancer chemotherapy. In order to enhance the anticancer effect of P-glycoprotein (P-gp) substrates, inhibition of the P-gp efflux pump on MDR cells is a good tactic. We designed novel multifunctional drug-loaded alpha-tocopheryl polyethylene glycol succinate (TPGS)/poly(lactic-co-glycolic acid) (PLGA) nanoparticles (TPGS/PLGA/SN-38 NPs; SN-38 is 7-ethyl-10-hydroxy-camptothecin), with TPGS-emulsified PLGA NPs as the carrier and modulator of the P-gp efflux pump and SN-38 as the model drug. TPGS/PLGA/SN-38 NPs were prepared using a modified solvent extraction/evaporation method. Physicochemical characterizations of TPGS/PLGA/SN-38 NPs were in conformity with the principle of nano-drug delivery systems (nDDSs), including a diameter of about 200 nm, excellent spherical particles with a smooth surface, narrow size distribution, appropriate surface charge, and successful drug-loading into the NPs. The cytotoxicity of TPGS/PLGA/SN-38 NPs to MDR cells was increased by 3.56 times compared with that of free SN-38. Based on an intracellular accumulation study relative to the time-dependent uptake and efflux inhibition, we suggest novel mechanisms of MDR reversal of TPGS/PLGA NPs. Firstly, TPGS/PLGA/SN-38 NPs improved the uptake of the loaded drug by clathrin-mediated endocytosis in the form of unbroken NPs. Simultaneously, intracellular NPs escaped the recognition of P-gp by MDR cells. After SN-38 was released from TPGS/PLGA/SN-38 NPs in MDR cells, TPGS or/and PLGA may modulate the efflux microenvironment of the P-gp pump, such as mitochondria and the P-gp domain with an ATP-binding site. Finally, the controlled-release drug entered the nucleus of the MDR cell to induce cytotoxicity. The present study showed that TPGS-emulsified PLGA NPs could be functional carriers in nDDS for anticancer drugs that are also P-gp substrates. More importantly, to enhance the therapeutic effect of P-gp substrates, this work might provide a new insight into the design of pharmacologically inactive excipients that can serve as P-gp modulators instead of drugs that are P-gp inhibitors.

Osthole shows the potential to overcome P-glycoprotein‑mediated multidrug resistance in human myelogenous leukemia K562/ADM cells by inhibiting the PI3K/Akt signaling pathway.

PubMed

Wang, Hong; Jia, Xiu-Hong; Chen, Jie-Ru; Wang, Jian-Yong; Li, You-Jie

2016-06-01

P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) has been reported to play a pivotal role in tumor chemotherapy failure. Study after study has illustrated that the phosphoinositide 3-kinase (PI3K)/Akt signaling cascade is involved in the MDR phenotype and is correlated with P-gp expression in many human malignancies. In the present study, osthole, an O-methylated coumarin, exhibited potent reversal capability of MDR in myelogenous leukemia K562/ADM cells. Simultaneously, the uptake and efflux of Rhodamine-123 (Rh-123) and the accumulation of doxorubicin assays combined with flow cytometric analysis suggested that osthole could increase intracellular drug accumulation. Furthermore, osthole decreased the expression of multidrug resistance gene 1 (MDR1) at both the mRNA and protein levels. Further experiments elucidated that osthole could suppress P-gp expression by inhibiting the PI3K/Akt signaling pathway which might be the main mechanism accounting for the reversal potential of osthole in the MDR in K562/ADM cells. In conclusion, osthole combats MDR and could be a promising candidate for the development of novel MDR reversal modulators.

Design and Synthesis of 1-((1,5-Bis(4-chlorophenyl)-2-methyl-1H-pyrrol-3-yl)methyl)-4-methylpiperazine (BM212) and N-Adamantan-2-yl-N'-((E)-3,7-dimethylocta-2,6-dienyl)ethane-1,2-diamine (SQ109) Pyrrole Hybrid Derivatives: Discovery of Potent Antitubercular Agents Effective against Multidrug-Resistant Mycobacteria.

PubMed

Bhakta, Sanjib; Scalacci, Nicolò; Maitra, Arundhati; Brown, Alistair K; Dasugari, Saiprasad; Evangelopoulos, Dimitrios; McHugh, Timothy D; Mortazavi, Parisa N; Twist, Alexander; Petricci, Elena; Manetti, Fabrizio; Castagnolo, Daniele

2016-03-24

Novel pyrroles have been designed, synthesized, and evaluated against mycobacterial strains. The pyrroles have originally been designed as hybrids of the antitubercular drugs BM212 (1) and SQ109 (2), which showed common chemical features with very similar topological distribution. A perfect superposition of the structures of 1 and 2 revealed by computational studies suggested the introduction of bulky substituents at the terminal portion of the pyrrole C3 side chain and the removal of the C5 aryl moiety. Five compounds showed high activity toward Mycobacterium tuberculosis, while 9b and 9c were highly active also against multidrug-resistant clinical isolates. Compound 9c showed low eukaryotic cell toxicity, turning out to be an excellent lead candidate for preclinical trials. In addition, four compounds showed potent inhibition (comparable to that of verapamil) toward the whole-cell drug efflux pump activity of mycobacteria, thus turning out to be promising multidrug-resistance-reversing agents.

Induction of multixenobiotic defense mechanisms in resistant Daphnia magna clones as a general cellular response to stress.

PubMed

Jordão, Rita; Campos, Bruno; Lemos, Marco F L; Soares, Amadeu M V M; Tauler, Romà; Barata, Carlos

2016-06-01

Multixenobiotic resistance mechanisms (MXR) were recently identified in Daphnia magna. Previous results characterized gene transcripts of genes encoding and efflux activities of four putative ABCB1 and ABCC transporters that were chemically induced but showed low specificity against model transporter substrates and inhibitors, thus preventing us from distinguishing between activities of different efflux transporter types. In this study we report on the specificity of induction of ABC transporters and of the stress protein hsp70 in clones selected to be genetically resistant to ABCB1 chemical substrates. Clones resistant to mitoxantrone, ivermectin and pentachlorophenol showed distinctive transcriptional responses of transporter protein coding genes and of putative transporter dye activities. Expression of hsp70 proteins also varied across resistant clones. Clones resistant to mitoxantrone and pentachlorophenol showed high constitutive levels of hsp70. Transcriptional levels of the abcb1 gene transporter and of putative dye transporter activity were also induced to a greater extent in the pentachlorophenol resistant clone. Observed higher dye transporter activities in individuals from clones resistant to mitoxantrone and ivermectin were unrelated with transcriptional levels of the studied four abcc and abcb1 transporter genes. These findings suggest that Abcb1 induction in D. magna may be a part of a general cellular stress response. Copyright © 2016 Elsevier B.V. All rights reserved.

Role of Aspergillus niger acrA in Arsenic Resistance and Its Use as the Basis for an Arsenic Biosensor

PubMed Central

Choe, Se-In; Gravelat, Fabrice N.; Al Abdallah, Qusai; Lee, Mark J.; Gibbs, Bernard F.

2012-01-01

Arsenic contamination of groundwater sources is a major issue worldwide, since exposure to high levels of arsenic has been linked to a variety of health problems. Effective methods of detection are thus greatly needed as preventive measures. In an effort to develop a fungal biosensor for arsenic, we first identified seven putative arsenic metabolism and transport genes in Aspergillus niger, a widely used industrial organism that is generally regarded as safe (GRAS). Among the genes tested for RNA expression in response to arsenate, acrA, encoding a putative plasma membrane arsenite efflux pump, displayed an over 200-fold increase in gene expression in response to arsenate. We characterized the function of this A. niger protein in arsenic efflux by gene knockout and confirmed that AcrA was located at the cell membrane using an enhanced green fluorescent protein (eGFP) fusion construct. Based on our observations, we developed a putative biosensor strain containing a construct of the native promoter of acrA fused with egfp. We analyzed the fluorescence of this biosensor strain in the presence of arsenic using confocal microscopy and spectrofluorimetry. The biosensor strain reliably detected both arsenite and arsenate in the range of 1.8 to 180 μg/liter, which encompasses the threshold concentrations for drinking water set by the World Health Organization (10 and 50 μg/liter). PMID:22467499

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.

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

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.

The Impact of Efflux Pump Inhibitors on the Activity of Selected Non-Antibiotic Medicinal Products against Gram-Negative Bacteria.

PubMed

Laudy, Agnieszka E; Kulińska, Ewa; Tyski, Stefan

2017-01-11

The potential role of non-antibiotic medicinal products in the treatment of multidrug-resistant Gram-negative bacteria has recently been investigated. It is highly likely that the presence of efflux pumps may be one of the reasons for the weak activity of non-antibiotics, as in the case of some non-steroidal anti-inflammatory drugs (NSAIDs), against Gram-negative rods. The activity of eight drugs of potential non-antibiotic activity, active substance standards, and relevant medicinal products were analysed with and without of efflux pump inhibitors against 180 strains of five Gram-negative rod species by minimum inhibitory concentration (MIC) value determination in the presence of 1 mM MgSO₄. Furthermore, the influence of non-antibiotics on the susceptibility of clinical strains to quinolones with or without PAβN (Phe-Arg-β-naphthylamide) was investigated. The impacts of PAβN on the susceptibility of bacteria to non-antibiotics suggests that amitriptyline, alendronate, nicergoline, and ticlopidine are substrates of efflux pumps in Gram-negative rods. Amitriptyline/Amitriptylinum showed the highest direct antibacterial activity, with MICs ranging 100-800 mg/L against all studied species. Significant decreases in the MIC values of other active substances (acyclovir, atorvastatin, and famotidine) tested with pump inhibitors were not observed. The investigated non-antibiotic medicinal products did not alter the MICs of quinolones in the absence and in the presence of PAβN to the studied clinical strains of five groups of species.

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 mixture effect design with inhibitors was applied in transporter activity assays. • ABCB1- and ABCC-type efflux activities were distinguished in native gill tissue. • Inhibitory action of environmental chemicals targeted ABCB1-type efflux activity.« less

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

Studies on tridecaptin B(1), a lipopeptide with activity against multidrug resistant Gram-negative bacteria.

PubMed

Cochrane, Stephen A; Lohans, Christopher T; van Belkum, Marco J; Bels, Manon A; Vederas, John C

2015-06-07

Previously other groups had reported that Paenibacillus polymyxa NRRL B-30507 produces SRCAM 37, a type IIA bacteriocin with antimicrobial activity against Campylobacter jejuni. Genome sequencing and isolation of antimicrobial compounds from this P. polymyxa strain show that the antimicrobial activity is due to polymyxins and tridecaptin B1. The complete structural assignment, synthesis, and antimicrobial profile of tridecaptin B1 is reported, as well as the putative gene cluster responsible for its biosynthesis. This peptide displays strong activity against multidrug resistant Gram-negative bacteria, a finding that is timely to the current problem of antibiotic resistance.

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

PubMed

Jones, P M; George, A M

2005-04-30

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

Shigellosis in Bay of Bengal Islands, India: clinical and seasonal patterns, surveillance of antibiotic susceptibility patterns, and molecular characterization of multidrug-resistant Shigella strains isolated during a 6-year period from 2006 to 2011.

PubMed

Bhattacharya, D; Bhattacharya, H; Thamizhmani, R; Sayi, D S; Reesu, R; Anwesh, M; Kartick, C; Bharadwaj, A P; Singhania, M; Sugunan, A P; Roy, S

2014-02-01

This study aims to determine the clinical features and seasonal patterns associated with shigellosis, the antimicrobial resistance frequencies of the isolates obtained during the period 2006-2012 for 22 antibiotics, and the molecular characterization of multidrug-resistant strains isolated from endemic cases of shigellosis in the remote islands of India, with special reference to fluoroquinolone and third-generation cephalosporins resistance. During the period from January 2006 to December 2011, stool samples were obtained and processed to isolate Shigella spp. The isolates were evaluated with respect to their antibiotic resistance pattern and various multidrug resistance determinants, including resistance genes, quinolone resistance determinants, and extended-spectrum β-lactamase (ESBL) production. Morbidity for shigellosis was found to be 9.3 % among children in these islands. Cases of shigellosis occurred mainly during the rainy seasons and were found to be higher in the age group 2-5 years. A wide spectrum of resistance was observed among the Shigella strains, and more than 50 % of the isolates were multidrug-resistant. The development of multidrug-resistant strains was found to be associated with various drug-resistant genes, multiple mutations in the quinolone resistance-determining region (QRDR), and the presence of plasmid-mediated quinolone-resistant determinants and efflux pump mediators. This report represents the first presentation of the results of long-term surveillance and molecular characterization concerning antimicrobial resistances in clinical Shigella strains in these islands. Information gathered as part of the investigations will be instrumental in identifying emerging antimicrobial resistance, for developing treatment guidelines appropriate for that community, and to provide baseline data with which to compare outbreak strains in the future.

Upregulation of the PatAB Transporter Confers Fluoroquinolone Resistance to Streptococcus pseudopneumoniae

PubMed Central

Alvarado, María; Martín-Galiano, Antonio J.; Ferrándiz, María J.; Zaballos, Ángel; de la Campa, Adela G.

2017-01-01

We characterized the mechanism of fluoroquinolone-resistance in two isolates of Streptococcus pseudopneumoniae having fluoroquinolone-efflux as unique mechanism of resistance. Whole genome sequencing and genetic transformation experiments were performed together with phenotypic determinations of the efflux mechanism. The PatAB pump was identified as responsible for efflux of ciprofloxacin (MIC of 4 μg/ml), ethidium bromide (MICs of 8–16 μg/ml) and acriflavine (MICs of 4–8 μg/ml) in both isolates. These MICs were at least 8-fold lower in the presence of the efflux inhibitor reserpine. Complete genome sequencing indicated that the sequence located between the promoter of the patAB operon and the initiation codon of patA, which putatively forms an RNA stem-loop structure, may be responsible for the efflux phenotype. RT-qPCR determinations performed on RNAs of cultures treated or not treated with subinhibitory ciprofloxacin concentrations were performed. While no significant changes were observed in wild-type Streptococcus pneumoniae R6 strain, increases in transcription were detected in the ciprofloxacin-efflux transformants obtained with DNA from efflux-positive isolates, in the ranges of 1.4 to 3.4-fold (patA) and 2.1 to 2.9-fold (patB). Ciprofloxacin-induction was related with a lower predicted free energy for the stem-loop structure in the RNA of S. pseudopneumoniae isolates (−13.81 and −8.58) than for R6 (−15.32 kcal/mol), which may ease transcription. The presence of these regulatory variations in commensal S. pseudopneumoniae isolates, and the possibility of its transfer to Streptococcus pneumoniae by genetic transformation, could increase fluoroquinolone resistance in this important pathogen. PMID:29123510

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

Restoration of chemosensitivity in cancer cells with MDR phenotype by deoxyribozyme, compared with ribozyme.

PubMed

Xing, Ai-Yan; Shi, Duan-bo; Liu, Wei; Chen, Xu; Sun, Yan-Lin; Wang, Xiao; Zhang, Jian-ping; Gao, Peng

2013-06-01

One of the main mechanisms for multidrug resistance (MDR) involves multidrug resistance gene 1 (MDR1) which encodes P-glycoprotein (Pgp). Pgp acts as a drug efflux pump and exports chemotherapeutic agents from cancer cells. Specific inhibition of Pgp expression by gene therapy is considered a well-respective strategy having less innate toxicities. At present, the investigation of DRz in reversal MDR is scarce. In the study, phosphorothioate DRz that targets to the translation initiation codon AUG was synthesized and transfected into breast cancer cells and leukemia cells with MDR phenotype. ASODN (antisense oligonucleotide) and ribozyme targets to the same region were also synthesized for comparison analysis. Alterations in MDR1 mRNA and Pgp were determined by RT-PCR, Northern blot, flow cytometry and Rh123 retention tests. Chemosensitivity of the treated cells was determined by MTT assay. The results showed that DRz could significantly suppress expression of MDR1 mRNA and inhibit synthesis of Pgp. The efflux activity of Pgp was inhibited accordingly. Chemosensitivity assay showed that a 21-fold reduction in drug resistance for Adriamycin and a 45-fold reduction in drug resistance for Vinblastine were found in the treated cells 36h after transfection. These data suggest that DRz targeted to the translation initiation codon AUG can reverse MDR phenotype in cancer cells and restore their chemosensitivity. Moreover, the reversal efficiency of DRz is better than that of ribozyme and ASODN targets to the same region of MDR1 mRNA. Copyright © 2013 Elsevier Inc. All rights reserved.

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.

Differential chemosensitization of P-glycoprotein overexpressing K562/Adr cells by withaferin A and Siamois polyphenols

PubMed Central

2010-01-01

Background Multidrug resistance (MDR) is a major obstacle in cancer treatment and is often the result of overexpression of the drug efflux protein, P-glycoprotein (P-gp), as a consequence of hyperactivation of NFκB, AP1 and Nrf2 transcription factors. In addition to effluxing chemotherapeutic drugs, P-gp also plays a specific role in blocking caspase-dependent apoptotic pathways. One feature that cytotoxic treatments of cancer have in common is activation of the transcription factor NFκB, which regulates inflammation, cell survival and P-gp expression and suppresses the apoptotic potential of chemotherapeutic agents. As such, NFκB inhibitors may promote apoptosis in cancer cells and could be used to overcome resistance to chemotherapeutic agents. Results Although the natural withanolide withaferin A and polyphenol quercetin, show comparable inhibition of NFκB target genes (involved in inflammation, angiogenesis, cell cycle, metastasis, anti-apoptosis and multidrug resistance) in doxorubicin-sensitive K562 and -resistant K562/Adr cells, only withaferin A can overcome attenuated caspase activation and apoptosis in K562/Adr cells, whereas quercetin-dependent caspase activation and apoptosis is delayed only. Interestingly, although withaferin A and quercetin treatments both decrease intracellular protein levels of Bcl2, Bim and P-Bad, only withaferin A decreases protein levels of cytoskeletal tubulin, concomitantly with potent PARP cleavage, caspase 3 activation and apoptosis, at least in part via a direct thiol oxidation mechanism. Conclusions This demonstrates that different classes of natural NFκB inhibitors can show different chemosensitizing effects in P-gp overexpressing cancer cells with impaired caspase activation and attenuated apoptosis. PMID:20438634

Expression and activity of multidrug resistance protein 1 in a murine thymoma cell line

PubMed Central

Echevarria-Lima, Juliana; Kyle-Cezar, Fernanda; Leite, Daniela F P; Capella, Luiz; Capella, Márcia A M; Rumjanek, Vivian M

2005-01-01

Multidrug resistance proteins [MRPs and P-glycoprotein (Pgp)] are members of the family of ATP-binding cassette (ABC) transport proteins, originally described as being involved in the resistance against anti-cancer agents in tumour cells. These proteins act as ATP-dependent efflux pumps and have now been described in normal cells where they exert physiological roles. The aim of this work was to investigate the expression and activity of MRP and Pgp in the thymoma cell line, EL4. It was observed that EL4 cells expressed mRNA for MRP1, but not for MRP2, MRP3 or Pgp. The activity of ABC transport proteins was evaluated by using the efflux of the fluorescent probes carboxy-2′-7′-dichlorofluorescein diacetate (CFDA) and rhodamine 123 (Rho 123). EL4 cells did not retain CFDA intracellularly, and MRP inhibitors (probenecid, indomethacin and MK 571) decreased MRP1 activity in a concentration-dependent manner. As expected, EL4 cells accumulated Rho 123, and the presence of cyclosporin A and verapamil did not modify this accumulation. Most importantly, when EL4 cells were incubated in the presence of the MRP1 inhibitors indomethacin and MK 571 for 6 days, they started to express CD4 and CD8 molecules on their surface, producing double-positive cells and CD8 single-positive cells. Our results suggest that MRP activity is important for the maintenance of the undifferentiated state in this cell type. This finding might have implications in the physiological process of normal thymocyte maturation. PMID:15804283

LysoTracker and MitoTracker Red are transport substrates of P-glycoprotein: implications for anticancer drug design evading multidrug resistance.

PubMed

Zhitomirsky, Benny; Farber, Hodaya; Assaraf, Yehuda G

2018-04-01

LysoTracker and MitoTracker Red are fluorescent probes widely used for viable cell staining of lysosomes and mitochondria, respectively. They are utilized to study organelle localization and their resident proteins, assess organelle functionality and quantification of organelle numbers. The ATP-driven efflux transporter P-glycoprotein (P-gp) is expressed in normal and malignant tissues and extrudes structurally distinct endogenous and exogenous cytotoxic compounds. Thus, once aromatic hydrophobic compounds such as the above-mentioned fluorescent probes are recognized as transport substrates, efflux pumps including P-gp may abolish their ability to reach their cellular target organelles. Herein, we show that LysoTracker and MitoTracker Red are expelled from P-gp-overexpressing cancer cells, thus hindering their ability to fluorescently mark target organelles. We further demonstrate that tariquidar, a potent P-gp transport inhibitor, restores LysoTracker and MitoTracker Red cell entry. We conclude that LysoTracker and MitoTracker Red are P-gp transport substrates, and therefore, P-gp expression must be taken into consideration prior to cellular applications using these probes. Importantly, as MitoTracker was a superior P-gp substrate than LysoTracker Red, we discuss the implications for the future design of chemotherapeutics evading cancer multidrug resistance. Furthermore, restoration of MitoTracker Red fluorescence in P-gp-overexpressing cells may facilitate the identification of potent P-gp transport inhibitors (i.e. chemosensitizers). © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

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.

The ABC of Biofilm Drug Tolerance: the MerR-Like Regulator BrlR Is an Activator of ABC Transport Systems, with PA1874-77 Contributing to the Tolerance of Pseudomonas aeruginosa Biofilms to Tobramycin.

PubMed

Poudyal, Bandita; Sauer, Karin

2018-02-01

A hallmark of biofilms is their tolerance to killing by antimicrobial agents. In Pseudomonas aeruginosa , biofilm drug tolerance requires the c-di-GMP-responsive MerR transcriptional regulator BrlR. However, the mechanism by which BrlR mediates biofilm drug tolerance has not been elucidated. Here, we demonstrate that BrlR activates the expression of at least 7 ABC transport systems, including the PA1874-PA1875-PA1876-PA1877 (PA1874-77) operon, with chromatin immunoprecipitation and DNA binding assays confirming BrlR binding to the promoter region of PA1874-77. Insertional inactivation of the 7 ABC transport systems rendered P. aeruginosa PAO1 biofilms susceptible to tobramycin or norfloxacin. Susceptibility was linked to drug accumulation, with BrlR contributing to norfloxacin accumulation in a manner dependent on multidrug efflux pumps and the PA1874-77 ABC transport system. Inactivation of the respective ABC transport system, furthermore, eliminated the recalcitrance of biofilms to killing by tobramycin but not norfloxacin, indicating that drug accumulation is not linked to biofilm drug tolerance. Our findings indicate for the first time that BrlR, a MerR-type transcriptional activator, activates genes encoding several ABC transport systems, in addition to multiple multidrug efflux pump genes. Moreover, our data confirm a BrlR target contributing to drug tolerance, likely countering the prevailing dogma that biofilm tolerance arises from a multiplicity of factors. Copyright © 2018 American Society for Microbiology.

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

Genetic modification of haematopoietic cells for combined resistance to podophyllotoxins, other agents covered by MDR1-mediated efflux activity and nitrosoureas.

PubMed

Baum, C; Peinert, S; Carpinteiro, A; Eckert, H G; Fairbairn, L J

2000-05-01

Genetic transfer and expression of drug-resistance functions into haematopoietic stem and progenitor cells is a promising means to overcome both the acute and longterm side-effects of cytotoxic drugs in bone marrow. Here, we describe a functional analysis of a retroviral vector that co-expresses human cDNAs for multidrug resistance 1/P-glycoprotein (MDR1) and a double mutant of O(6)-alkylguanine-alkyltransferase (hATPA/GA) to high levels. The hATPA/GA protein contains two amino acid substitutions that render it resistant to compounds such as O(6)-benzylguanine that inhibit the wild-type protein which is often overexpressed in resistant tumour cells. Evidence for simultaneous drug resistance of genetically modified primary murine progenitor cells to colchicine or the podophyllotoxin etoposide, both covered by MDR1-mediated efflux activity, and the nitrosourea BCNU, which is counteracted by hATPA/GA, is presented using in vitro colony assays.

Copper Resistance of the Emerging Pathogen Acinetobacter baumannii

PubMed Central

Williams, Caitlin L.; Neu, Heather M.; Gilbreath, Jeremy J.; Michel, Sarah L. J.; Zurawski, Daniel V.

2016-01-01

ABSTRACT Acinetobacter baumannii is an important emerging pathogen that is capable of causing many types of severe infection, especially in immunocompromised hosts. Since A. baumannii can rapidly acquire antibiotic resistance genes, many infections are on the verge of being untreatable, and novel therapies are desperately needed. To investigate the potential utility of copper-based antibacterial strategies against Acinetobacter infections, we characterized copper resistance in a panel of recent clinical A. baumannii isolates. Exposure to increasing concentrations of copper in liquid culture and on solid surfaces resulted in dose-dependent and strain-dependent effects; levels of copper resistance varied broadly across isolates, possibly resulting from identified genotypic variation among strains. Examination of the growth-phase-dependent effect of copper on A. baumannii revealed that resistance to copper increased dramatically in stationary phase. Moreover, A. baumannii biofilms were more resistant to copper than planktonic cells but were still susceptible to copper toxicity. Exposure of bacteria to subinhibitory concentrations of copper allowed them to better adapt to and grow in high concentrations of copper; this copper tolerance response is likely achieved via increased expression of copper resistance mechanisms. Indeed, genomic analysis revealed numerous putative copper resistance proteins that share amino acid homology to known proteins in Escherichia coli and Pseudomonas aeruginosa. Transcriptional analysis revealed significant upregulation of these putative copper resistance genes following brief copper exposure. Future characterization of copper resistance mechanisms may aid in the search for novel antibiotics against Acinetobacter and other highly antibiotic-resistant pathogens. IMPORTANCE Acinetobacter baumannii causes many types of severe nosocomial infections; unfortunately, some isolates have acquired resistance to almost every available antibiotic, and treatment options are incredibly limited. Copper is an essential nutrient but becomes toxic at high concentrations. The inherent antimicrobial properties of copper give it potential for use in novel therapeutics against drug-resistant pathogens. We show that A. baumannii clinical isolates are sensitive to copper in vitro, both in liquid and on solid metal surfaces. Since bacterial resistance to copper is mediated though mechanisms of efflux and detoxification, we identified genes encoding putative copper-related proteins in A. baumannii and showed that expression of some of these genes is regulated by the copper concentration. We propose that the antimicrobial effects of copper may be beneficial in the development of future therapeutics that target multidrug-resistant bacteria. PMID:27520808

Novobiocin binding to NalD induces the expression of the MexAB-OprM pump in Pseudomonas aeruginosa.

PubMed

Chen, Weizhong; Wang, Dan; Zhou, Wenquan; Sang, Hong; Liu, Xichun; Ge, Zhiyun; Zhang, Jin; Lan, Lefu; Yang, Cai-Guang; Chen, Hao

2016-06-01

NalD was reported to be the secondary repressor of the MexAB-OprM multidrug efflux pump, the major system contributing to intrinsic multidrug resistance in Pseudomonas aeruginosa. Here, we show that novobiocin binds directly to NalD, which leads NalD to dissociate from the DNA promoter, and thus de-represses the expression of the MexAB-OprM pump. In addition, we have solved the crystal structure of NalD at a resolution of 2.90 Å. The structural alignment of NalD to its homologue TtgR reveals that the residues N129 and H167 in NalD are involved in its novobiocin-binding ability. We have confirmed the function of these two amino acids by EMSA and plate assay. The results presented here highlight the importance and diversity of regulatory mechanism in bacterial antibiotic resistance, and provide further insight for novel antimicrobial development. © 2016 John Wiley & Sons Ltd.

ω-3 Long Chain Polyunsaturated Fatty Acids as Sensitizing Agents and Multidrug Resistance Revertants in Cancer Therapy

PubMed Central

Corsetto, Paola Antonia; Kopecka, Joanna; Riganti, Chiara

2017-01-01

Chemotherapy efficacy is strictly limited by the resistance of cancer cells. The ω-3 long chain polyunsaturated fatty acids (ω-3 LCPUFAs) are considered chemosensitizing agents and revertants of multidrug resistance by pleiotropic, but not still well elucidated, mechanisms. Nowadays, it is accepted that alteration in gene expression, modulation of cellular proliferation and differentiation, induction of apoptosis, generation of reactive oxygen species, and lipid peroxidation are involved in ω-3 LCPUFA chemosensitizing effects. A crucial mechanism in the control of cell drug uptake and efflux is related to ω-3 LCPUFA influence on membrane lipid composition. The incorporation of docosahexaenoic acid in the lipid rafts produces significant changes in their physical-chemical properties affecting content and functions of transmembrane proteins, such as growth factors, receptors and ATP-binding cassette transporters. Of note, ω-3 LCPUFAs often alter the lipid compositions more in chemoresistant cells than in chemosensitive cells, suggesting a potential adjuvant role in the treatment of drug resistant cancers. PMID:29261109

Design, Synthesis and Evaluation of a Novel Series of Inhibitors Reversing P-Glycoprotein-Mediated Multidrug Resistance.

PubMed

Ghaleb, Hesham; Li, Huilan; Kairuki, Mutta; Qiu, Qianqian; Bi, Xinzhou; Liu, Chunxia; Liao, Chen; Li, Jieming; Hezam, Kamal; Huang, Wenlong; Qian, Hai

2018-05-22

Multidrug resistance (MDR) is still the main barrier to attaining effective results with chemotherapy. Discovery of new chemo-reversal agents is needed to overcome MDR. Our study focused on a better way to obtain novel drugs with triazole rings that have an MDR-reversal ability through click chemistry. Among 20 developed compounds, compound 19 had a minimal cytotoxic effect compared to tariquidar and verapamil (VRP) and showed a higher reversal activity than VRP through increased accumulation in K562/A02 cells. Compound 19 also played an important role in the P-gp efflux function of intracellular Rh123 and doxorubicin (DOX) accumulation in K562/A02 cells. Moreover, compound 19 exhibited a long lifetime of approximately 24 h. These results indicated that compound 19 is a potential lead compound for the design of new drugs to overcome cancer MDR. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Synthesis of Nanodiamond-Daunorubicin Conjugates to Overcome Multidrug Chemoresistance in Leukemia

PubMed Central

Man, Han B.; Kim, Hansung; Kim, Ho-Joong; Robinson, Erik; Liu, Wing Kam; Chow, Edward Kai-Hua; Ho, Dean

2013-01-01

Nanodiamonds (NDs) are promising candidates in nanomedicine, demonstrating significant potential as gene/drug delivery platforms for cancer therapy. We have synthesized ND vectors capable of chemotherapeutic loading and delivery with applications towards chemoresistant leukemia. The loading of Daunorubicin (DNR) onto NDs was optimized by adjusting reaction parameters such as acidity and concentration. The resulting conjugate, a novel therapeutic payload for NDs, was characterized extensively for size, surface charge, and loading efficiency. A K562 human myelogenous leukemia cell line, with multidrug resistance conferred by incremental DNR exposure, was used to demonstrate the efficacy enhancement resulting from ND-based delivery. While resistant K562 cells were able to overcome treatment from DNR alone, as compared with non-resistant K562 cells, NDs were able to improve DNR delivery into resistant K562 cells. By overcoming efflux mechanisms present in this resistant leukemia line, ND-enabled therapeutics have demonstrated the potential to improve cancer treatment efficacy, especially towards resistant strains. PMID:23916889

Optimization by Molecular Fine Tuning of Dihydro-β-agarofuran Sesquiterpenoids as Reversers of P-Glycoprotein-Mediated Multidrug Resistance.

PubMed

Callies, Oliver; Sánchez-Cañete, María P; Gamarro, Francisco; Jiménez, Ignacio A; Castanys, Santiago; Bazzocchi, Isabel L

2016-03-10

P-glycoprotein (P-gp) plays a crucial role in the development of multidrug resistance (MDR), a major obstacle for successful chemotherapy in cancer. Herein, we report on the development of a natural-product-based library of 81 dihydro-β-agarofuran sesquiterpenes (2-82) by optimization of the lead compound 1. The compound library was evaluated for its ability to inhibit P-gp-mediated daunomycin efflux in MDR cells. Selected analogues were further analyzed for their P-gp inhibition constant, intrinsic toxicity, and potency to reverse daunomycin and vinblastine resistances. Analogues 6, 24, 28, 59, and 66 were identified as having higher potency than compound 1 and verapamil, a first-generation P-gp modulator. SAR analysis revealed the size of the aliphatic chains and presence of nitrogen atoms are important structural characteristics to modulate reversal activity. The present study highlights the potential of these analogues as modulators of P-gp mediated MDR in cancer cells.

Collateral Sensitivity of Multidrug-Resistant Cells to the Orphan Drug Tiopronin

PubMed Central

Goldsborough, Andrew S.; Handley, Misty D.; Dulcey, Andrés E.; Pluchino, Kristen M.; Kannan, Pavitra; Brimacombe, Kyle R.; Hall, Matthew D.; Griffiths, Gary; Gottesman, Michael M.

2011-01-01

A major challenge in the treatment of cancer is multidrug resistance (MDR) that develops during chemotherapy. Here we demonstrate that tiopronin (1), a thiol-substituted N-propanoylglycine derivative, was selectively toxic to a series of cell lines expressing the drug efflux pump P-glycoprotein (P-gp, ABCB1) and MRP1 (ABCC1). Treatment of MDR cells with 1 led to instability of the ABCB1 mRNA and consequently a reduction in P-gp protein, despite functional assays demonstrating that tiopronin does not interact with P-gp. Long-term exposure of P-gp-expressing cells to 1 sensitized them to doxorubicin and taxol, both P-gp substrates. Treatment of MRP1-overexpressing cells with tiopronin led to a significant reduction in MRP1 protein. Synthesis and screening of analogs of tiopronin demonstrated that the thiol functional group was essential for collateral sensitivity, while substitution of the amino acid backbone altered but did not destroy specificity, pointing to future development of targeted analogs. PMID:21657271

Hsp27 promotes ABCA1 expression and cholesterol efflux through the PI3K/PKCζ/Sp1 pathway in THP-1 macrophages.

PubMed

Kuang, Hai-Jun; Zhao, Guo-Jun; Chen, Wu-Jun; Zhang, Min; Zeng, Gao-Feng; Zheng, Xi-Long; Tang, Chao-Ke

2017-09-05

Heat shock protein 27 (Hsp27) is a putative biomarker and therapeutic target in atherosclerosis. This study was to explore the potential mechanisms underlying Hsp27 effects on ATP-binding cassette transporter A1 (ABCA1) expression and cellular cholesterol efflux. THP-1 macrophage-derived foam cells were infected with adenovirus to express wild-type Hsp27, hyper-phosphorylated Hsp27 mimic (3D Hsp27), antisense Hsp27 or hypo-phosphorylated Hsp27 mimic (3A Hsp27). Wild-type and 3D Hsp27 were found to up-regulate ABCA1 mRNA and protein expression and increase cholesterol efflux from cells. Expression of antisense or 3A Hsp27 suppressed the expression of ABCA1 and cholesterol efflux. Furthermore, over-expression of wild-type and 3D Hsp27 significantly increased the levels of phosphorylated specificity protein 1 (Sp1), protein kinase C ζ (PKCζ) and phosphatidylinositol 3-kinase (PI3K). In addition, the up-regulation of ABCA1 expression and cholesterol efflux induced by 3D Hsp27 was suppressed by inhibition of Sp1, PKCζ and PI3K with specific kinase inhibitors. Taken together, our results revealed that Hsp27 may up-regulate the expression of ABCA1 and promotes cholesterol efflux through activation of the PI3K/PKCζ/Sp1 signal pathway in THP-1 macrophage-derived foam cells. Our findings may partly explain the mechanisms underlying the anti-atherogenic effect of Hsp27. Copyright © 2017 Elsevier B.V. All rights reserved.

Screening dietary flavonoids for the reversal of P-glycoprotein-mediated multidrug resistance in cancer

PubMed Central

Mohana, S; Ganesan, M; Agilan, B; Karthikeyan, R; Srithar, G; Beaulah Mary, R; Ananthakrishnan, D; Velmurugan, D; Rajendra Prasad, N; Ambudkar, Suresh V.

2016-01-01

P-glycoprotein (P-gp) serves as a therapeutic target for the development of inhibitors to overcome multidrug resistance in cancer cells. Although various approaches of virtual screening procedures have been practiced so far to develop first three generations of P-gp inhibitors, their toxicity and drug interaction profiles are still a matter of concern. To address the above important problem of developing safe and effective P-gp inhibitors, we have made systematic computational and experimental studies on the interaction of natural phytochemicals with human P-gp. Molecular docking and QSAR studies were carried out for 40 dietary phytochemicals in the drug-binding site of the transmembrane domains (TMDs) of P-gp. Dietary flavonoids exhibit better interactions with homology modeled human P-gp. Based on the computational analysis, selected flavonoids were tested for their inhibitory potential against P-gp transport function in drug resistant cell lines using calcein-AM and rhodamine 123 efflux assays. It has been found that quercetin and rutin were the highly desirable flavonoids for the inhibition of P-gp transport function and significantly reduced resistance in cytotoxicity assay to paclitaxel in P-gp overexpressing MDR cell lines. Hence, quercetin and rutin may be considered as potential chemosensitizing agents to overcome multidrug resistance in cancer. PMID:27216424

Screening dietary flavonoids for the reversal of P-glycoprotein-mediated multidrug resistance in cancer.

PubMed

Mohana, S; Ganesan, M; Agilan, B; Karthikeyan, R; Srithar, G; Beaulah Mary, R; Ananthakrishnan, D; Velmurugan, D; Rajendra Prasad, N; Ambudkar, Suresh V

2016-07-19

P-Glycoprotein (P-gp) serves as a therapeutic target for the development of inhibitors to overcome multidrug resistance in cancer cells. Although various screening procedures have been practiced so far to develop first three generations of P-gp inhibitors, their toxicity and drug interaction profiles are still a matter of concern. To address the above important problem of developing safe and effective P-gp inhibitors, we have made systematic computational and experimental studies on the interaction of natural phytochemicals with human P-gp. Molecular docking and QSAR studies were carried out for 40 dietary phytochemicals in the drug-binding site of the transmembrane domains (TMDs) of P-gp. Dietary flavonoids exhibit better interactions with homology modeled human P-gp. Based on the computational analysis, selected flavonoids were tested for their inhibitory potential against P-gp transport function in drug resistant cell lines using calcein-AM and rhodamine 123 efflux assays. It has been found that quercetin and rutin were the highly desirable flavonoids for the inhibition of P-gp transport function and they significantly reduced resistance in cytotoxicity assays to paclitaxel in P-gp overexpressing MDR cell lines. Hence, quercetin and rutin may be considered as potential chemosensitizing agents to overcome multidrug resistance in cancer.

Determination of multidrug resistance mechanisms in Clostridium perfringens type A isolates using RNA sequencing and 2D-electrophoresis.

PubMed

Ma, Yu-Hua; Ye, Gui-Sheng

2018-06-11

In this study, we screened differentially expressed genes in a multidrug-resistant isolate strain of Clostridium perfringens by RNA sequencing. We also separated and identified differentially expressed proteins (DEPs) in the isolate strain by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). The RNA sequencing results showed that, compared with the control strain, 1128 genes were differentially expressed in the isolate strain, and these included 227 up-regulated genes and 901 down-regulated genes. Bioinformatics analysis identified the following genes and gene categories that are potentially involved in multidrug resistance (MDR) in the isolate strain: drug transport, drug response, hydrolase activity, transmembrane transporter, transferase activity, amidase transmembrane transporter, efflux transmembrane transporter, bacterial chemotaxis, ABC transporter, and others. The results of the 2-DE showed that 70 proteins were differentially expressed in the isolate strain, 45 of which were up-regulated and 25 down-regulated. Twenty-seven DEPs were identified by MS and these included the following protein categories: ribosome, antimicrobial peptide resistance, and ABC transporter, all of which may be involved in MDR in the isolate strain of C. perfringens. The results provide reference data for further investigations on the drug resistant molecular mechanisms of C. perfringens.

Purification of a Multidrug Resistance Transporter for Crystallization Studies

PubMed Central

Alegre, Kamela O.; Law, Christopher J.

2015-01-01

Crystallization of integral membrane proteins is a challenging field and much effort has been invested in optimizing the overexpression and purification steps needed to obtain milligram amounts of pure, stable, monodisperse protein sample for crystallography studies. Our current work involves the structural and functional characterization of the Escherichia coli multidrug resistance transporter MdtM, a member of the major facilitator superfamily (MFS). Here we present a protocol for isolation of MdtM to increase yields of recombinant protein to the milligram quantities necessary for pursuit of structural studies using X-ray crystallography. Purification of MdtM was enhanced by introduction of an elongated His-tag, followed by identification and subsequent removal of chaperonin contamination. For crystallization trials of MdtM, detergent screening using size exclusion chromatography determined that decylmaltoside (DM) was the shortest-chain detergent that maintained the protein in a stable, monodispersed state. Crystallization trials of MdtM performed using the hanging-drop diffusion method with commercially available crystallization screens yielded 3D protein crystals under several different conditions. We contend that the purification protocol described here may be employed for production of high-quality protein of other multidrug efflux members of the MFS, a ubiquitous, physiologically and clinically important class of membrane transporters. PMID:27025617

ATP-binding cassette transporters in reproduction: a new frontier

PubMed Central

Bloise, E.; Ortiga-Carvalho, T.M.; Reis, F.M.; Lye, S.J.; Gibb, W.; Matthews, S.G.

2016-01-01

BACKGROUND The transmembrane ATP-binding cassette (ABC) transporters actively efflux an array of clinically relevant compounds across biological barriers, and modulate biodistribution of many physiological and pharmacological factors. To date, over 48 ABC transporters have been identified and shown to be directly and indirectly involved in peri-implantation events and fetal/placental development. They efflux cholesterol, steroid hormones, vitamins, cytokines, chemokines, prostaglandins, diverse xenobiotics and environmental toxins, playing a critical role in regulating drug disposition, immunological responses and lipid trafficking, as well as preventing fetal accumulation of drugs and environmental toxins. METHODS This review examines ABC transporters as important mediators of placental barrier functions and key reproductive processes. Expression, localization and function of all identified ABC transporters were systematically reviewed using PubMed and Google Scholar websites to identify relevant studies examining ABC transporters in reproductive tissues in physiological and pathophysiological states. Only reports written in English were incorporated with no restriction on year of publication. While a major focus has been placed on the human, extensive evidence from animal studies is utilized to describe current understanding of the regulation and function of ABC transporters relevant to human reproduction. RESULTS ABC transporters are modulators of steroidogenesis, fertilization, implantation, nutrient transport and immunological responses, and function as ‘gatekeepers’ at various barrier sites (i.e. blood-testes barrier and placenta) against potentially harmful xenobiotic factors, including drugs and environmental toxins. These roles appear to be species dependent and change as a function of gestation and development. The best-described ABC transporters in reproductive tissues (primarily in the placenta) are the multidrug transporters p-glycoprotein and breast cancer-related protein, the multidrug resistance proteins 1 through 5 and the cholesterol transporters ABCA1 and ABCG1. CONCLUSIONS The ABC transporters have various roles across multiple reproductive tissues. Knowledge of efflux direction, tissue distribution, substrate specificity and regulation of the ABC transporters in the placenta and other reproductive tissues is rapidly expanding. This will allow better understanding of the disposition of specific substrates within reproductive tissues, and facilitate development of novel treatments for reproductive disorders as well as improved approaches to protecting the developing fetus. PMID:26545808

[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-positive and Gram-negative bacteria causing postoperative infections. Further, FOM would not select/concentrate beta-lactamase producing bacteria in the clinical fields and would not be a substrate for multidrug efflux system of P. aeruginosa.

Secretome profile analysis of multidrug-resistant, monodrug-resistant and drug-susceptible Mycobacterium tuberculosis.

PubMed

Putim, Chanyanuch; Phaonakrop, Narumon; Jaresitthikunchai, Janthima; Gamngoen, Ratikorn; Tragoolpua, Khajornsak; Intorasoot, Sorasak; Anukool, Usanee; Tharincharoen, Chayada Sitthidet; Phunpae, Ponrut; Tayapiwatana, Chatchai; Kasinrerk, Watchara; Roytrakul, Sittiruk; Butr-Indr, Bordin

2018-03-01

The emergence of drug-resistant tuberculosis has generated great concern in the control of tuberculosis and HIV/TB patients have established severe complications that are difficult to treat. Although, the gold standard of drug-susceptibility testing is highly accurate and efficient, it is time-consuming. Diagnostic biomarkers are, therefore, necessary in discriminating between infection from drug-resistant and drug-susceptible strains. One strategy that aids to effectively control tuberculosis is understanding the function of secreting proteins that mycobacteria use to manipulate the host cellular defenses. In this study, culture filtrate proteins from Mycobacterium tuberculosis H37Rv, isoniazid-resistant, rifampicin-resistant and multidrug-resistant strains were gathered and profiled by shotgun-proteomics technique. Mass spectrometric analysis of the secreted proteome identified several proteins, of which 837, 892, 838 and 850 were found in M. tuberculosis H37Rv, isoniazid-resistant, rifampicin-resistant and multidrug-resistant strains, respectively. These proteins have been implicated in various cellular processes, including biological adhesion, biological regulation, developmental process, immune system process localization, cellular process, cellular component organization or biogenesis, metabolic process, and response to stimulus. Analysis based on STITCH database predicted the interaction of DNA topoisomerase I, 3-oxoacyl-(acyl-carrier protein) reductase, ESAT-6-like protein, putative prophage phiRv2 integrase, and 3-phosphoshikimate 1-carboxyvinyltransferase with isoniazid, rifampicin, pyrazinamide, ethambutol and streptomycin, suggesting putative roles in controlling the anti-tuberculosis ability. However, several proteins with no interaction with all first-line anti-tuberculosis drugs might be used as markers for mycobacterial identification.

The putative multidrug resistance protein MRP-7 inhibits methylmercury-associated animal toxicity and dopaminergic neurodegeneration in Caenorhabditis elegans

PubMed Central

VanDuyn, Natalia; Nass, Richard

2013-01-01

Parkinson’s disease (PD) is the most prevalent neurodegenerative motor disorder worldwide, and results in the progressive loss of dopamine (DA) neurons in the substantia nigra pars compacta. Gene-environment interactions are believed to play a significant role in the vast majority of PD cases, yet the toxicants and the associated genes involved in the neuropathology are largely ill-defined. Recent epidemiological and biochemical evidence suggests that methylmercury (MeHg) may be an environmental toxicant that contributes to the development of PD. Here we report that a gene coding for the putative multidrug resistance protein MRP-7 in Caenorhabditis elegans (C. elegans) modulates whole animal and DA neuron sensitivity to MeHg. In this study we demonstrate that genetic knockdown of MRP-7 results in a 2-fold increase in Hg levels and a dramatic increase in stress response proteins associated with the endoplasmic reticulum, golgi apparatus, and mitochondria, as well as an increase in MeHg-associated animal death. Chronic exposure to low concentrations of MeHg induces MRP-7 gene expression, while exposures in MRP-7 genetic knockdown animals results in a loss of DA neuron integrity without affecting whole animal viability. Furthermore, transgenic animals expressing a fluorescent reporter behind the endogenous MRP-7 promoter indicate that the transporter is expressed in DA neurons. These studies show for the first time that a multidrug resistance protein is expressed in DA neurons, and its expression inhibits MeHg-associated DA neuron pathology. PMID:24266639

The putative multidrug resistance protein MRP-7 inhibits methylmercury-associated animal toxicity and dopaminergic neurodegeneration in Caenorhabditis elegans.

PubMed

VanDuyn, Natalia; Nass, Richard

2014-03-01

Parkinson's disease (PD) is the most prevalent neurodegenerative motor disorder worldwide, and results in the progressive loss of dopamine (DA) neurons in the substantia nigra pars compacta. Gene-environment interactions are believed to play a significant role in the vast majority of PD cases, yet the toxicants and the associated genes involved in the neuropathology are largely ill-defined. Recent epidemiological and biochemical evidence suggests that methylmercury (MeHg) may be an environmental toxicant that contributes to the development of PD. Here, we report that a gene coding for the putative multidrug resistance protein MRP-7 in Caenorhabditis elegans modulates whole animal and DA neuron sensitivity to MeHg. In this study, we demonstrate that genetic knockdown of MRP-7 results in a twofold increase in Hg levels and a dramatic increase in stress response proteins associated with the endoplasmic reticulum, golgi apparatus, and mitochondria, as well as an increase in MeHg-associated animal death. Chronic exposure to low concentrations of MeHg induces MRP-7 gene expression, while exposures in MRP-7 genetic knockdown animals results in a loss of DA neuron integrity without affecting whole animal viability. Furthermore, transgenic animals expressing a fluorescent reporter behind the endogenous MRP-7 promoter indicate that the transporter is expressed in DA neurons. These studies show for the first time that a multidrug resistance protein is expressed in DA neurons, and its expression inhibits MeHg-associated DA neuron pathology. © 2013 International Society for Neurochemistry.

Tolerance to biodegraded crude oil in marine invertebrate embryos and larvae is associated with expression of a multixenobiotic resistance transporter.

PubMed

Hamdoun, Amro M; Griffin, Fred J; Cherr, Gary N

2002-11-13

The toxicity of water-soluble fractions of biodegraded crude oil (BWSF) to embryos and larvae of two marine invertebrates, the white sea urchin (Lytechinus anamesus) and the fat innkeeper (Urechis caupo), was studied. Santa Barbara Channel crude oil was artificially weathered and subjected to biodegradation using a mixed microbe culture obtained from natural oil seep sites. The degradation culture inoculated with seep sediment microbes accumulated 43.7 microg/l water-soluble hydrocarbons. In contrast water-soluble fractions from the non-degraded cultures (NWSF) only accumulated 3.05 microg/l. BWSF proved deleterious to Lytechinus embryo development at low concentrations (EC50 = 0.33 mg/l) but was essentially non-toxic to Urechis embryos/larvae up to 3.0 mg/l. An established mechanism for handling of a wide array of xenobiotics in Urechis embryos is the multixenobiotoic resistance transporter multixenobiotic response (MXR, also known as multidrug resistance, MDR). This mechanism is primarily mediated by ATP-dependent, efflux pumps that extrude a wide array of xenobiotic compounds. In this study, we show that Lytechinus larvae do not appear to express MXR efflux protein nor MXR mediated dye efflux capacity. In contrast, BWSF acts as a competitive inhibitor of MXR transport-mediated dye efflux in Urechis larvae. These results suggest that MXR may be an important mechanism for extrusion of the by-products of crude oil degradation by microbes, and that the level of its expression may determine the susceptibility of organisms to degraded oil hydrocarbons. Copyright 2002 Elsevier Science B.V.

Enhancing the aluminium tolerance of barley by expressing the citrate transporter genes SbMATE and FRD3

PubMed Central

Zhou, Gaofeng; Ryan, Peter R.

2014-01-01

Malate and citrate efflux from root apices is a mechanism of Al3+ tolerance in many plant species. Citrate efflux is facilitated by members of the MATE (multidrug and toxic compound exudation) family localized to the plasma membrane of root cells. Barley (Hordeum vulgare) is among the most Al3+-sensitive cereal species but the small genotypic variation in tolerance that is present is correlated with citrate efflux via a MATE transporter named HvAACT1. This study used a biotechnological approach to increase the Al3+ tolerance of barley by transforming it with two MATE genes that encode citrate transporters: SbMATE is the major Al3+-tolerance gene from sorghum whereas FRD3 is involved with Fe nutrition in Arabidopsis. Independent transgenic and null T3 lines were generated for both transgenes. Lines expressing SbMATE showed Al3+-activated citrate efflux from root apices and greater tolerance to Al3+ toxicity than nulls in hydroponic and short-term soil trials. Transgenic lines expressing FRD3 exhibited similar phenotypes except citrate release from roots occurred constitutively. The Al3+ tolerance of these lines was compared with previously generated transgenic barley lines overexpressing the endogenous HvAACT1 gene and the TaALMT1 gene from wheat. Barley lines expressing TaALMT1 showed significantly greater Al3+ tolerance than all lines expressing MATE genes. This study highlights the relative efficacy of different organic anion transport proteins for increasing the Al3+ tolerance of an important crop species. PMID:24692647

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.

Evaluating the Role of Multidrug Resistance Protein 3 (MDR3) Inhibition in Predicting Drug-Induced Liver Injury Using 125 Pharmaceuticals.

PubMed

Aleo, Michael D; Shah, Falgun; He, Kan; Bonin, Paul D; Rodrigues, A David

2017-05-15

The role of bile salt export protein (BSEP) inhibition in drug-induced liver injury (DILI) has been investigated widely, while inhibition of the canalicular multidrug resistant protein 3 (MDR3) has received less attention. This transporter plays a pivotal role in secretion of phospholipids into bile and functions coordinately with BSEP to mediate the formation of bile acid-containing biliary micelles. Therefore, inhibition of MDR3 in human hepatocytes was examined across 125 drugs (70 of Most-DILI-concern and 55 of No-DILI-concern). Of these tested, 41% of Most-DILI-concern and 47% of No-DILI-concern drugs had MDR3 IC 50 values of <50 μM. A better distinction across DILI classifications occurred when systemic exposure was considered where safety margins of 50-fold had low sensitivity (0.29), but high specificity (0.96). Analysis of physical chemical property space showed that basic compounds were twice as likely to be MDR3 inhibitors as acids, neutrals, and zwitterions and that inhibitors were more likely to have polar surface area (PSA) values of <100 Å 2 and cPFLogD values between 1.5 and 5. These descriptors, with different cutoffs, also highlighted a group of compounds that shared dual potency as MDR3 and BSEP inhibitors. Nine drugs classified as Most-DILI-concern compounds (four withdrawn, four boxed warning, and one liver injury warning in their approved label) had intrinsic potency features of <20 μM in both assays, thereby reinforcing the notion that multiple inhibitory mechanisms governing bile formation (bile acid and phospholipid efflux) may confer additional risk factors that play into more severe forms of DILI as shown by others for BSEP inhibitors combined with multidrug resistance-associated protein (MRP2, MRP3, MRP4) inhibitory properties. Avoiding physical property descriptors that highlight dual BSEP and MDR3 inhibition or testing drug candidates for inhibition of multiple efflux transporters (e.g., BSEP, MDR3, and MRPs) may be an effective strategy for prioritizing drug candidates with less likelihood of causing clinical DILI.

Transcriptional analysis reveals the critical role of RNA polymerase-binding transcription factor, DksA, in regulating multi-drug resistance of Escherichia coli.

PubMed

Wang, Jiawei; Cao, Li; Yang, Xiaowen; Wu, Qingmin; Lu, Lin; Wang, Zhen

2018-05-07

The objective of this study was to comprehensively identify the target genes regulated by the RNA polymerase-binding transcription factor DksA in Escherichia coli, and to clarify the role of DksA in multi-drug resistance. A clinical E. coli strain, E8, was selected to construct the dksA gene deletion mutant by using the Red recombination system. The minimum inhibitory concentrations (MICs) of 12 antibiotics in the E8ΔdksA (mutant) were markedly lower than those in the wild-type strain, E8. Genes differentially expressed in the wild-type and dksA mutant were detected using RNA-Seq and were validated by performing quantitative real-time PCR (qRT-PCR). In total, 168 differentially expressed genes were identified in E8ΔdksA, including 81 up-regulated and 87 down-regulated genes. Many of the genes identified are involved in metabolism, two-component systems, transcriptional regulators, and transport/membrane proteins. Interestingly, genes encoding the transcriptional regulator, MarR, which is known to repress the multiple drug resistance operon, marRAB; MdfA, a transport protein that exhibits multidrug efflux activities; oligopeptide transport system proteins OppA and OppD were among those differentially expressed, and could potentially contribute to the increased drug susceptibility of E8ΔdksA. In conclusion, DksA plays an important role in the multi-drug resistance of this E. coli strain, and directly or indirectly regulates the expression of several genes related to antibiotic resistance. Copyright © 2018. Published by Elsevier B.V.

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.

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.

The H2 receptor antagonist nizatidine is a P-glycoprotein substrate: characterization of its intestinal epithelial cell efflux transport.

PubMed

Dahan, Arik; Sabit, Hairat; Amidon, Gordon L

2009-06-01

The aim of this study was to elucidate the intestinal epithelial cell efflux transport processes that are involved in the intestinal transport of the H(2) receptor antagonist nizatidine. The intestinal epithelial efflux transport mechanisms of nizatidine were investigated and characterized across Caco-2 cell monolayers, in the concentration range 0.05-10 mM in both apical-basolateral (AP-BL) and BL-AP directions, and the transport constants of P-glycoprotein (P-gp) efflux activity were calculated. The concentration-dependent effects of various P-gp (verapamil, quinidine, erythromycin, ketoconazole, and cyclosporine A), multidrug resistant-associated protein 2 (MRP2; MK-571, probenecid, indomethacin, and p-aminohipuric acid), and breast cancer resistance protein (BCRP; Fumitremorgin C) inhibitors on nizatidine bidirectional transport were examined. Nizatidine exhibited 7.7-fold higher BL-AP than AP-BL Caco-2 permeability, indicative of net mucosal secretion. All P-gp inhibitors investigated displayed concentration-dependent inhibition on nizatidine secretion in both directions. The IC(50) of verapamil on nizatidine P-gp secretion was 1.2 x 10(-2) mM. In the absence of inhibitors, nizatidine displayed concentration-dependent secretion, with one saturable (J(max) = 5.7 x 10(-3) nmol cm(-2) s(-1) and K(m) = 2.2 mM) and one nonsaturable component (K(d) = 7 x 10(-4) microL cm(-2) s(-1)). Under complete P-gp inhibition, nizatidine exhibited linear secretory flux, with a slope similar to the nonsaturable component. V(max) and K(m) estimated for nizatidine P-gp-mediated secretion were 4 x 10(-3) nmol cm(-2) s(-1) and 1.2 mM, respectively. No effect was obtained with the MRP2 or the BCRP inhibitors. Being a drug commonly used in pediatrics, adults, and elderly, nizatidine susceptibility to efflux transport by P-gp revealed in this paper may be of significance in its absorption, distribution, and clearance, as well as possible drug-drug interactions.

AraC/XylS Family Stress Response Regulators Rob, SoxS, PliA, and OpiA in the Fire Blight Pathogen Erwinia amylovora

PubMed Central

Pletzer, Daniel; Schweizer, Gabriel

2014-01-01

Transcriptional regulators of the AraC/XylS family have been associated with multidrug resistance, organic solvent tolerance, oxidative stress, and virulence in clinically relevant enterobacteria. In the present study, we identified four homologous AraC/XylS regulators, Rob, SoxS, PliA, and OpiA, from the fire blight pathogen Erwinia amylovora Ea1189. Previous studies have shown that the regulators MarA, Rob, and SoxS from Escherichia coli mediate multiple-antibiotic resistance, primarily by upregulating the AcrAB-TolC efflux system. However, none of the four AraC/XylS regulators from E. amylovora was able to induce a multidrug resistance phenotype in the plant pathogen. Overexpression of rob led to a 2-fold increased expression of the acrA gene. However, the rob-overexpressing strain showed increased resistance to only a limited number of antibiotics. Furthermore, Rob was able to induce tolerance to organic solvents in E. amylovora by mechanisms other than efflux. We demonstrated that SoxS from E. amylovora is involved in superoxide resistance. A soxS-deficient mutant of Ea1189 was not able to grow on agar plates supplemented with the superoxide-generating agent paraquat. Furthermore, expression of soxS was induced by redox cycling agents. We identified two novel members of the AraC/XylS family in E. amylovora. PliA was highly upregulated during the early infection phase in apple rootstock and immature pear fruits. Multiple compounds were able to induce the expression of pliA, including apple leaf extracts, phenolic compounds, redox cycling agents, heavy metals, and decanoate. OpiA was shown to play a role in the regulation of osmotic and alkaline pH stress responses. PMID:24936054

In Vitro antibacterial and antibiotic-potentiation activities of four edible plants against multidrug-resistant gram-negative species

PubMed Central

2013-01-01

Background The present study was designed to investigate the antibacterial activities of the methanol extracts of four Cameroonian edible plants, locally used to treat microbial infections, and their synergistic effects with antibiotics against a panel of twenty nine Gram-negative bacteria including Multi-drug resistant (MDR) phenotypes expressing active efflux pumps. Methods The broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) of the extracts [alone and in the presence of the efflux pumps inhibitor (EPI) Phenylalanine-Arginine β-Naphtylamide (PAβN)], and those of antibiotics in association with the two of the most active ones, Piper nigrum and Telfairia occidentalis. The preliminary phytochemical screening of the extracts was conducted according to the standard phytochemical methods. Results Phytochemical analysis showed the presence of alkaloids and flavonoids in all studied extracts. Other chemical classes of secondary metabolites were selectively present in the extracts. The results of the MIC determination indicated that the crude extracts from P. nigrum and V. amygdalina were able to inhibit the growth of all the twenty nine studied bacteria within a concentration range of 32 to 1024 μg/mL. At a similar concentration range (32 to 1024 μg/mL) the extract from T. occidentalis inhibited the growth of 93.1% of the tested microorganisms. At MIC/2 and MIC/5, synergistic effects were noted between the extracts from P. nigrum and T. occidentalis and seven of the tested antibiotics on more than 70% of the tested bacteria. Conclusion The overall results of the present study provide information for the possible use of the studied edible plants extracts in the control of bacterial infections including MDR phenotypes. PMID:23885762

Synergistic effect of pH-responsive folate-functionalized poloxamer 407-TPGS-mixed micelles on targeted delivery of anticancer drugs.

PubMed

Butt, Adeel Masood; Mohd Amin, Mohd Cairul Iqbal; Katas, Haliza

2015-01-01

Doxorubicin (DOX), an anthracycline anticancer antibiotic, is used for treating various types of cancers. However, its use is associated with toxicity to normal cells and development of resistance due to overexpression of drug efflux pumps. Poloxamer 407 (P407) and vitamin E TPGS (D-α-tocopheryl polyethylene glycol succinate, TPGS) are widely used polymers as drug delivery carriers and excipients for enhancing the drug retention times and stability. TPGS reduces multidrug resistance, induces apoptosis, and shows selective anticancer activity against tumor cells. Keeping in view the problems, we designed a mixed micelle system encapsulating DOX comprising TPGS for its selective anticancer activity and P407 conjugated with folic acid (FA) for folate-mediated receptor targeting to cancer cells. FA-functionalized P407 was prepared by carbodiimide crosslinker chemistry. P407-TPGS/FA-P407-TPGS-mixed micelles were prepared by thin-film hydration method. Cytotoxicity of blank micelles, DOX, and DOX-loaded micelles was determined by alamarBlue(®) assay. The size of micelles was less than 200 nm with encapsulation efficiency of 85% and 73% for P407-TPGS and FA-P407-TPGS micelles, respectively. Intracellular trafficking study using nile red-loaded micelles indicated improved drug uptake and perinuclear drug localization. The micelles show minimal toxicity to normal human cell line WRL-68, enhanced cellular uptake of DOX, reduced drug efflux, increased DOX-DNA binding in SKOV3 and DOX-resistant SKOV3 human ovarian carcinoma cell lines, and enhanced in vitro cytotoxicity as compared to free DOX. FA-P407-TPGS-DOX micelles show potential as a targeted nano-drug delivery system for DOX due to their multiple synergistic factors of selective anticancer activity, inhibition of multidrug resistance, and folate-mediated selective uptake.

Synergistic effect of pH-responsive folate-functionalized poloxamer 407-TPGS-mixed micelles on targeted delivery of anticancer drugs

PubMed Central

Butt, Adeel Masood; Mohd Amin, Mohd Cairul Iqbal; Katas, Haliza

2015-01-01

Background Doxorubicin (DOX), an anthracycline anticancer antibiotic, is used for treating various types of cancers. However, its use is associated with toxicity to normal cells and development of resistance due to overexpression of drug efflux pumps. Poloxamer 407 (P407) and vitamin E TPGS (D-α-tocopheryl polyethylene glycol succinate, TPGS) are widely used polymers as drug delivery carriers and excipients for enhancing the drug retention times and stability. TPGS reduces multidrug resistance, induces apoptosis, and shows selective anticancer activity against tumor cells. Keeping in view the problems, we designed a mixed micelle system encapsulating DOX comprising TPGS for its selective anticancer activity and P407 conjugated with folic acid (FA) for folate-mediated receptor targeting to cancer cells. Methods FA-functionalized P407 was prepared by carbodiimide crosslinker chemistry. P407-TPGS/FA-P407-TPGS-mixed micelles were prepared by thin-film hydration method. Cytotoxicity of blank micelles, DOX, and DOX-loaded micelles was determined by alamarBlue® assay. Results The size of micelles was less than 200 nm with encapsulation efficiency of 85% and 73% for P407-TPGS and FA-P407-TPGS micelles, respectively. Intracellular trafficking study using nile red-loaded micelles indicated improved drug uptake and perinuclear drug localization. The micelles show minimal toxicity to normal human cell line WRL-68, enhanced cellular uptake of DOX, reduced drug efflux, increased DOX–DNA binding in SKOV3 and DOX-resistant SKOV3 human ovarian carcinoma cell lines, and enhanced in vitro cytotoxicity as compared to free DOX. Conclusion FA-P407-TPGS-DOX micelles show potential as a targeted nano-drug delivery system for DOX due to their multiple synergistic factors of selective anticancer activity, inhibition of multidrug resistance, and folate-mediated selective uptake. PMID:25709451

Chlorhexidine bathing for the prevention of colonization and infection with multidrug-resistant microorganisms in a hematopoietic stem cell transplantation unit over a 9-year period

PubMed Central

Mendes, Elisa Teixeira; Ranzani, Otavio T.; Marchi, Ana Paula; da Silva, Mariama Tomaz; Filho, José Ulysses Amigo; Alves, Tânia; Guimarães, Thais; Levin, Anna S.; Costa, Silvia Figueiredo

2016-01-01

Abstract Health care associated infections (HAIs) are currently among the major challenges to the care of hematopoietic stem cell transplantation (HSCT) patients. The objective of the present study was to evaluate the impact of 2% chlorhexidine (CHG) bathing on the incidence of colonization and infection with vancomycin-resistant Enterococcus (VRE), multidrug-resistant (MDR) gram-negative pathogens, and to evaluate their CHG minimum inhibitory concentration (MIC) after the intervention. A quasi-experimental study with duration of 9 years was conducted. VRE colonization and infection, HAI rates, and MDR gram-negative infection were evaluated by interrupted time series analysis. The antibacterial susceptibility profile and mechanism of resistance to CHG were analyzed in both periods by the agar dilution method in the presence or absence of the efflux pump inhibitor carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) and presence of efflux pumps (qacA/E, qacA, qacE, cepA, AdeA, AdeB, and AdeC) by polymerase chain reaction (PCR). The VRE colonization and infection rates were significantly reduced in the postintervention period (P = 0.001). However, gram-negative MDR rates in the unit increased in the last years of the study. The CHG MICs for VRE increased during the period of exposure to the antiseptic. A higher MIC at baseline period was observed in MDR gram-negative strains. The emergence of a monoclonal Pseudomonas aeruginosa clone was observed in the second period. Concluding, CHG bathing was efficient regarding VRE colonization and infection, whereas no similar results were found with MDR gram-negative bacteria. PMID:27861350

Structural Features Governing the Activity of Lactoferricin-Derived Peptides That Act in Synergy with Antibiotics against Pseudomonas aeruginosa In Vitro and In Vivo▿ †

PubMed Central

Sánchez-Gómez, Susana; Japelj, Bostjan; Jerala, Roman; Moriyón, Ignacio; Fernández Alonso, Mirian; Leiva, José; Blondelle, Sylvie E.; Andrä, Jörg; Brandenburg, Klaus; Lohner, Karl; Martínez de Tejada, Guillermo

2011-01-01

Pseudomonas aeruginosa is naturally resistant to many antibiotics, and infections caused by this organism are a serious threat, especially to hospitalized patients. The intrinsic low permeability of P. aeruginosa to antibiotics results from the coordinated action of several mechanisms, such as the presence of restrictive porins and the expression of multidrug efflux pump systems. Our goal was to develop antimicrobial peptides with an improved bacterial membrane-permeabilizing ability, so that they enhance the antibacterial activity of antibiotics. We carried out a structure activity relationship analysis to investigate the parameters that govern the permeabilizing activity of short (8- to 12-amino-acid) lactoferricin-derived peptides. We used a new class of constitutional and sequence-dependent descriptors called PEDES (peptide descriptors from sequence) that allowed us to predict (Spearman's ρ = 0.74; P < 0.001) the permeabilizing activity of a new peptide generation. To study if peptide-mediated permeabilization could neutralize antibiotic resistance mechanisms, the most potent peptides were combined with antibiotics, and the antimicrobial activities of the combinations were determined on P. aeruginosa strains whose mechanisms of resistance to those antibiotics had been previously characterized. A subinhibitory concentration of compound P2-15 or P2-27 sensitized P. aeruginosa to most classes of antibiotics tested and counteracted several mechanisms of antibiotic resistance, including loss of the OprD porin and overexpression of several multidrug efflux pump systems. Using a mouse model of lethal infection, we demonstrated that whereas P2-15 and erythromycin were unable to protect mice when administered separately, concomitant administration of the compounds afforded long-lasting protection to one-third of the animals. PMID:20956602

Structural features governing the activity of lactoferricin-derived peptides that act in synergy with antibiotics against Pseudomonas aeruginosa in vitro and in vivo.

PubMed

Sánchez-Gómez, Susana; Japelj, Bostjan; Jerala, Roman; Moriyón, Ignacio; Fernández Alonso, Mirian; Leiva, José; Blondelle, Sylvie E; Andrä, Jörg; Brandenburg, Klaus; Lohner, Karl; Martínez de Tejada, Guillermo

2011-01-01

Pseudomonas aeruginosa is naturally resistant to many antibiotics, and infections caused by this organism are a serious threat, especially to hospitalized patients. The intrinsic low permeability of P. aeruginosa to antibiotics results from the coordinated action of several mechanisms, such as the presence of restrictive porins and the expression of multidrug efflux pump systems. Our goal was to develop antimicrobial peptides with an improved bacterial membrane-permeabilizing ability, so that they enhance the antibacterial activity of antibiotics. We carried out a structure activity relationship analysis to investigate the parameters that govern the permeabilizing activity of short (8- to 12-amino-acid) lactoferricin-derived peptides. We used a new class of constitutional and sequence-dependent descriptors called PEDES (peptide descriptors from sequence) that allowed us to predict (Spearman's ρ = 0.74; P < 0.001) the permeabilizing activity of a new peptide generation. To study if peptide-mediated permeabilization could neutralize antibiotic resistance mechanisms, the most potent peptides were combined with antibiotics, and the antimicrobial activities of the combinations were determined on P. aeruginosa strains whose mechanisms of resistance to those antibiotics had been previously characterized. A subinhibitory concentration of compound P2-15 or P2-27 sensitized P. aeruginosa to most classes of antibiotics tested and counteracted several mechanisms of antibiotic resistance, including loss of the OprD porin and overexpression of several multidrug efflux pump systems. Using a mouse model of lethal infection, we demonstrated that whereas P2-15 and erythromycin were unable to protect mice when administered separately, concomitant administration of the compounds afforded long-lasting protection to one-third of the animals.

Molecular mechanisms of membrane impermeability in clinical isolates of Enterobacteriaceae exposed to imipenem selective pressure.

PubMed

Pavez, Monica; Vieira, Camila; de Araujo, Maria Rita; Cerda, Alvaro; de Almeida, Lara Mendes; Lincopan, Nilton; Mamizuka, Elsa Masae

2016-07-01

Intrinsic mechanisms leading to carbapenem-induced membrane impermeability and multidrug resistance are poorly understood in clinical isolates of Enterobacteriaceae. In this study, molecular behaviours during the establishment of membrane impermeability in members of the Enterobacteriaceae family under imipenem selective pressure were investigated. Clinical isolates (n = 22) exhibiting susceptibility to multiple antibiotics or characterised as extended-spectrum β-lactamase (ESBL)- or AmpC-producers were submitted to progressive passages on Mueller-Hinton agar plates containing subclinical concentrations of imipenem [0.5 × the minimum inhibitory concentration (MIC)]. Changes in outer membrane permeability were evaluated by determination of antimicrobial MICs, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and gene expression analysis related to membrane permeability (i.e. omp35-like, omp36-like and acrA) and regulatory mechanisms (i.e. marA and ompR) by quantitative reverse transcription PCR. Following imipenem induction, 73% of isolates showed increased carbapenem MICs by ≥2 doubling dilutions. At an early stage of treatment, imipenem modulated the expression of porins and efflux pump genes, represented by a reduction of 78% in omp36-like and a two-fold increase in acrA expression. Transcriptional factors marA and ompR were also affected by imipenem induction, increasing mRNA expression by 14- and 4-fold, respectively. High marA expression levels were associated with higher values of acrA expression. These results suggest that imipenem is an important factor in the development of an adaptive response to carbapenems by regulating key genes involved in the control of efflux pumps and porins, which could lead to a multidrug-resistant profile in clinical isolates, contributing to possible treatment failure. Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Asymmetric protonation of EmrE

PubMed Central

Morrison, Emma A.; Robinson, Anne E.; Liu, Yongjia

2015-01-01

The small multidrug resistance transporter EmrE is a homodimer that uses energy provided by the proton motive force to drive the efflux of drug substrates. The pKa values of its “active-site” residues—glutamate 14 (Glu14) from each subunit—must be poised around physiological pH values to efficiently couple proton import to drug export in vivo. To assess the protonation of EmrE, pH titrations were conducted with 1H-15N TROSY-HSQC nuclear magnetic resonance (NMR) spectra. Analysis of these spectra indicates that the Glu14 residues have asymmetric pKa values of 7.0 ± 0.1 and 8.2 ± 0.3 at 45°C and 6.8 ± 0.1 and 8.5 ± 0.2 at 25°C. These pKa values are substantially increased compared with typical pKa values for solvent-exposed glutamates but are within the range of published Glu14 pKa values inferred from the pH dependence of substrate binding and transport assays. The active-site mutant, E14D-EmrE, has pKa values below the physiological pH range, consistent with its impaired transport activity. The NMR spectra demonstrate that the protonation states of the active-site Glu14 residues determine both the global structure and the rate of conformational exchange between inward- and outward-facing EmrE. Thus, the pKa values of the asymmetric active-site Glu14 residues are key for proper coupling of proton import to multidrug efflux. However, the results raise new questions regarding the coupling mechanism because they show that EmrE exists in a mixture of protonation states near neutral pH and can interconvert between inward- and outward-facing forms in multiple different protonation states. PMID:26573622

Multidrug Resistant Pseudomonas aeruginosa Causing Prosthetic Valve Endocarditis: A Genetic-Based Chronicle of Evolving Antibiotic Resistance.

PubMed

Domitrovic, T Nicholas; Hujer, Andrea M; Perez, Federico; Marshall, Steven H; Hujer, Kristine M; Woc-Colburn, Laila E; Parta, Mark; Bonomo, Robert A

2016-10-01

Background.  Successful treatment of infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa is thwarted by the emergence of antibiotic resistance and biofilm formation on prosthetic devices. Our aims were to decipher the molecular basis of resistance in a unique case of prosthetic valve endocarditis (PVE) caused by MDR P. aeruginosa . Methods.  Five sequential MDR P. aeruginosa blood isolates collected during a 7-month period were recovered from a patient suffering from PVE previously exposed to β-lactam antibiotics. Minimum inhibitory concentrations (MICs) of several classes of antibiotics were used to indicate clinical resistance characteristics; relatedness of the isolates was determined using multilocus sequence typing and repetitive sequence-based polymerase chain reaction. Amplification and sequencing of regulatory and resistance genes was performed. Results.  All isolates belonged to ST 298, possessed bla PDC-16 , and were resistant to fluoroquinolones and carbapenems. In the course of therapy, we observed a >2-fold increase in cephalosporin resistance (4 µg/mL to >16 µg/mL). Sequencing of the AmpC regulator, amp R, revealed a D135N point mutation in cephalosporin-resistant isolates. Common carbapenemase genes were not identified. All isolates demonstrated a premature stop codon at amino acid 79 of the outer membrane protein OprD and mutations in the quinolone resistance-determining regions of gyr A and par C. Point mutations in nal C, an efflux pump regulator, were also observed. Conclusions.  In this analysis, we chart the molecular evolution of β-lactam resistance in a case of PVE. We show that mutations in regulatory genes controlling efflux and cephalosporinase production contributed to the MDR phenotype.

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.

Interference of flavonoids with fluorescent intracellular probes: methodological implications in the evaluation of the oxidative burst by flow cytometry.

PubMed

Peluso, Ilaria; Manafikhi, Husseen; Reggi, Raffaella; Palmery, Maura

2014-08-01

The evaluation of oxidative burst is particularly relevant in many pathological and subclinical conditions. Flow cytometry provides quick and accurate measures of the reactive oxygen species production by leukocytes in most situations. However, spurious results, related to probes' efflux may be observed in several instances. Many factors affect the evaluation of the oxidative burst with fluorescent probes that require intracellular deacetylation and could be substrate of the multidrug resistance proteins (MDR). After discussing the implications of the efflux of fluorophores in the normalization strategies in flow cytometry assays, we have pointed out the possible interference of flavonoids with fluorescet probes' staining and signal. We have also reviewed the results from human intervention studies regarding the evaluation of oxidative burst with these probes. In vitro, at concentrations close to post-ingestion circulating levels, some flavonoids and their metabolites could interfere with probes' staining and fluorescence signal through different mechanisms, such as the inhibition of esterases, the modulation of the MDR-mediate efflux of probe and the inhibition of the oxidation of probe. These effects may explain the contrasting results obtained by human intervention studies. Finally, also inflammatory state or the use of drugs substrate of MDR proteins could affect the evaluation of the oxidative burst with intracellular probes. © 2014 International Society for Advancement of Cytometry.

Crystal structure of an antigenic outer-membrane protein from Salmonella Typhi suggests a potential antigenic loop and an efflux mechanism.

PubMed

Guan, Hong-Hsiang; Yoshimura, Masato; Chuankhayan, Phimonphan; Lin, Chien-Chih; Chen, Nai-Chi; Yang, Ming-Chi; Ismail, Asma; Fun, Hoong-Kun; Chen, Chun-Jung

2015-11-13

ST50, an outer-membrane component of the multi-drug efflux system from Salmonella enterica serovar Typhi, is an obligatory diagnostic antigen for typhoid fever. ST50 is an excellent and unique diagnostic antigen with 95% specificity and 90% sensitivity and is used in the commercial diagnosis test kit (TYPHIDOT(TM)). The crystal structure of ST50 at a resolution of 2.98 Å reveals a trimer that forms an α-helical tunnel and a β-barrel transmembrane channel traversing the periplasmic space and outer membrane. Structural investigations suggest significant conformational variations in the extracellular loop regions, especially extracellular loop 2. This is the location of the most plausible antibody-binding domain that could be used to target the design of new antigenic epitopes for the development of better diagnostics or drugs for the treatment of typhoid fever. A molecule of the detergent n-octyl-β-D-glucoside is observed in the D-cage, which comprises three sets of Asp361 and Asp371 residues at the periplasmic entrance. These structural insights suggest a possible substrate transport mechanism in which the substrate first binds at the periplasmic entrance of ST50 and subsequently, via iris-like structural movements to open the periplasmic end, penetrates the periplasmic domain for efflux pumping of molecules, including poisonous metabolites or xenobiotics, for excretion outside the pathogen.

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.

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.

A Potato cDNA Encoding a Homologue of Mammalian Multidrug Resistant P-Glycoprotein

NASA Technical Reports Server (NTRS)

Wang, W.; Takezawa, D.; Poovaiah, B. W.

1996-01-01

A homologue of the multidrug resistance (MDR) gene was obtained while screening a potato stolon tip cDNA expression library with S-15-labeled calmodulin. The mammalian MDR gene codes for a membrane-bound P-glycoprotein (170-180 kDa) which imparts multidrug resistance to cancerous cells. The potato cDNA (PMDR1) codes for a polypeptide of 1313 amino acid residues (ca. 144 kDa) and its structural features are very similar to the MDR P-glycoprotein. The N-terminal half of the PMDR1-encoded protein shares striking homology with its C-terminal half, and each half contains a conserved ATP-binding site and six putative transmembrane domains. Southern blot analysis indicated that potato has one or two MDR-like genes. PMDR1 mRNA is constitutively expressed in all organs studied with higher expression in the stem and stolon tip. The PMDR1 expression was highest during tuber initiation and decreased during tuber development.

Tet(L) and Tet(K) Tetracycline-Divalent Metal/H+ Antiporters: Characterization of Multiple Catalytic Modes and a Mutagenesis Approach to Differences in Their Efflux Substrate and Coupling Ion Preferences

PubMed Central

Jin, Jie; Guffanti, Arthur A.; Bechhofer, David H.; Krulwich, Terry A.

2002-01-01

The Tet(L) protein encoded in the Bacillus subtilis chromosome and the closely related Tet(K) protein from Staphylococcus aureus plasmids are multifunctional antiporters that have three cytoplasmic efflux substrates: a tetracycline-divalent metal (TC-Me2+) complex that bears a net single positive charge, Na+, and K+. Tet(L) and Tet(K) had been shown to couple efflux of each of these substrates to influx of H+ as the coupling ion. In this study, competitive cross-inhibition between K+ and other cytoplasmic efflux substrates was demonstrated. Tet(L) and Tet(K) had also been shown to use K+ as an alternate coupling ion in support of Na+ or K+ efflux. Here they were shown to couple TC-Me2+ efflux to K+ uptake as well, exhibiting greater use of K+ as a coupling ion as the external pH increased. The substrate and coupling ion preferences of the two Tet proteins differed, especially in the higher preference of Tet(K) than Tet(L) for K+, both as a cytoplasmic efflux substrate and as an external coupling ion. Site-directed mutagenesis was employed to test the hypothesis that some feature of the putative “antiporter motif,” motif C, of Tet proteins would be involved in these characteristic preferences. Mutation of the A157 in Tet(L) to a hydroxyamino acid resulted in a more Tet(K)-like K+ preference both as coupling ion and efflux substrate. A reciprocal S157A mutant of Tet(K) exhibited reduced K+ preference. Competitive inhibition among substrates and the parallel effects of the single mutation upon K+ preference, as both an efflux substrate and coupling ion, are compatible with a model in which a single translocation pathway through the Tet(L) and Tet(K) transporters is used both for the cytoplasmic efflux substrates and for the coupling ions, in an alternating fashion. However, the effects of the A157 and other mutations of Tet(L) indicate that even if there are a shared binding site and translocation pathway, some elements of that pathway are used by all substrates and others are important only for particular substrates. PMID:12169596

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.

Molecular cloning, expression analysis and subcellular localization of a Transparent Testa 12 ortholog in brown cotton (Gossypium hirsutum L.).

PubMed

Gao, Jun-Shan; Wu, Nan; Shen, Zhi-Lin; Lv, Kai; Qian, Sen-He; Guo, Ning; Sun, Xu; Cai, Yong-Ping; Lin, Yi

2016-02-01

Transparent Testa 12 (TT12) is a kind of transmembrane transporter of proanthocyanidins (PAs), which belongs to a membrane-localized multidrug and toxin efflux (MATE) family, but the molecular basis of PAs transport is still poorly understood. Here, we cloned a full-length TT12 cDNA from the fiber of brown cotton (Gossypium hirsutum), named GhTT12 (GenBank accession No. KF240564), which comprised 1733 bp with an open reading frame (ORF) of 1503 bp and encoded a putative protein containing 500 amino acid residues with a typical MATE conserved domain. The GhTT12 gene had 96.8% similarity to AA genome in Gossypium arboretum. Quantitative RT-PCR analysis denoted that the relative expression of GhTT12 in brown cotton was 1-5 folds higher than that in white cotton. The mRNA level was the highest at 5 days post anthesis (DPA) and reduced gradually during the fiber development. Expressing GhTT12-fused green fluorescent protein (GFP) in Nicotiana tabacum showed that GhTT12-GFP was localized in the vacuole membrane. The content of PAs increased firstly and decreased afterwards, and reached the maximum at 15 DPA in brown cotton. But for white cotton, the content of PAs remained at a low level during the fiber development. We speculate that GhTT12 may participate in the transportation of PAs from the cytoplasmic matrix to the vacuole. Taken together, our data revealed that GhTT12 was functional as a PAs transmembrane transporter. Copyright © 2015 Elsevier B.V. All rights reserved.

Mutations of the central tyrosines of putative cholesterol recognition amino acid consensus (CRAC) sequences modify folding, activity, and sterol-sensing of the human ABCG2 multidrug transporter.

PubMed

Gál, Zita; Hegedüs, Csilla; Szakács, Gergely; Váradi, András; Sarkadi, Balázs; Özvegy-Laczka, Csilla

2015-02-01

Human ABCG2 is a plasma membrane glycoprotein causing multidrug resistance in cancer. Membrane cholesterol and bile acids are efficient regulators of ABCG2 function, while the molecular nature of the sterol-sensing sites has not been elucidated. The cholesterol recognition amino acid consensus (CRAC, L/V-(X)(1-5)-Y-(X)(1-5)-R/K) sequence is one of the conserved motifs involved in cholesterol binding in several proteins. We have identified five potential CRAC motifs in the transmembrane domain of the human ABCG2 protein. In order to define their roles in sterol-sensing, the central tyrosines of these CRACs (Y413, 459, 469, 570 and 645) were mutated to S or F and the mutants were expressed both in insect and mammalian cells. We found that mutation in Y459 prevented protein expression; the Y469S and Y645S mutants lost their activity; while the Y570S, Y469F, and Y645F mutants retained function as well as cholesterol and bile acid sensitivity. We found that in the case of the Y413S mutant, drug transport was efficient, while modulation of the ATPase activity by cholesterol and bile acids was significantly altered. We suggest that the Y413 residue within a putative CRAC motif has a role in sterol-sensing and the ATPase/drug transport coupling in the ABCG2 multidrug transporter. Copyright © 2014. Published by Elsevier B.V.

A Novel indole compound that inhibits Pseudomonas aeruginosa growth by targeting MreB is a substrate for MexAB-OprM.

PubMed

Robertson, Gregory T; Doyle, Timothy B; Du, Qun; Duncan, Leonard; Mdluli, Khisimuzi E; Lynch, A Simon

2007-10-01

Drug efflux systems contribute to the intrinsic resistance of Pseudomonas aeruginosa to many antibiotics and biocides and hamper research focused on the discovery and development of new antimicrobial agents targeted against this important opportunistic pathogen. Using a P. aeruginosa PAO1 derivative bearing deletions of opmH, encoding an outer membrane channel for efflux substrates, and four efflux pumps belonging to the resistance nodulation/cell division class including mexAB-oprM, we identified a small-molecule indole-class compound (CBR-4830) that is inhibitory to growth of this efflux-compromised strain. Genetic studies established MexAB-OprM as the principal pump for CBR-4830 and revealed MreB, a prokaryotic actin homolog, as the proximal cellular target of CBR-4830. Additional studies establish MreB as an essential protein in P. aeruginosa, and efflux-compromised strains treated with CBR-4830 transition to coccoid shape, consistent with MreB inhibition or depletion. Resistance genetics further suggest that CBR-4830 interacts with the putative ATP-binding pocket in MreB and demonstrate significant cross-resistance with A22, a structurally unrelated compound that has been shown to promote rapid dispersion of MreB filaments in vivo. Interestingly, however, ATP-dependent polymerization of purified recombinant P. aeruginosa MreB is blocked in vitro in a dose-dependent manner by CBR-4830 but not by A22. Neither compound exhibits significant inhibitory activity against mutant forms of MreB protein that bear mutations identified in CBR-4830-resistant strains. Finally, employing the strains and reagents prepared and characterized during the course of these studies, we have begun to investigate the ability of analogues of CBR-4830 to inhibit the growth of both efflux-proficient and efflux-compromised P. aeruginosa through specific inhibition of MreB function.

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.

Enhancing the aluminium tolerance of barley by expressing the citrate transporter genes SbMATE and FRD3.

PubMed

Zhou, Gaofeng; Pereira, Jorge F; Delhaize, Emmanuel; Zhou, Meixue; Magalhaes, Jurandir V; Ryan, Peter R

2014-06-01

Malate and citrate efflux from root apices is a mechanism of Al(3+) tolerance in many plant species. Citrate efflux is facilitated by members of the MATE (multidrug and toxic compound exudation) family localized to the plasma membrane of root cells. Barley (Hordeum vulgare) is among the most Al(3+)-sensitive cereal species but the small genotypic variation in tolerance that is present is correlated with citrate efflux via a MATE transporter named HvAACT1. This study used a biotechnological approach to increase the Al(3+) tolerance of barley by transforming it with two MATE genes that encode citrate transporters: SbMATE is the major Al(3+)-tolerance gene from sorghum whereas FRD3 is involved with Fe nutrition in Arabidopsis. Independent transgenic and null T3 lines were generated for both transgenes. Lines expressing SbMATE showed Al(3+)-activated citrate efflux from root apices and greater tolerance to Al(3+) toxicity than nulls in hydroponic and short-term soil trials. Transgenic lines expressing FRD3 exhibited similar phenotypes except citrate release from roots occurred constitutively. The Al(3+) tolerance of these lines was compared with previously generated transgenic barley lines overexpressing the endogenous HvAACT1 gene and the TaALMT1 gene from wheat. Barley lines expressing TaALMT1 showed significantly greater Al(3+) tolerance than all lines expressing MATE genes. This study highlights the relative efficacy of different organic anion transport proteins for increasing the Al(3+) tolerance of an important crop species. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Hypergravity differentially modulates cGMP efflux in human melanocytic cells stimulated by nitric oxide and natriuretic peptides

NASA Astrophysics Data System (ADS)

Ivanova, K.; Stieber, C.; Lambers, B.; Block, I.; Krieg, R.; Wellmann, A.; Gerzer, R.

Nitric oxide NO plays a key role in many patho physiologic processes including inflammation and skin cancer The diverse cellular effects of NO are mainly mediated by activation of the soluble guanylyl cyclase sGC isoform that leads to increases in intracellular cGMP levels whereas the membrane-bound isoforms serve as receptors for natriuretic peptides e g ANP In human skin epidermal melanocytes represent the principal cells for skin pigmentation by synthesizing the pigment melanin Melanin acts as a scavenger for free radicals that may arise during metabolic stress as a result of potentially harmful effects of the environment In previous studies we found that long-term exposure to hypergravity stimulated cGMP efflux in normal human melanocytes NHMs and non-metastatic melanoma cells at least partly by an enhanced expression of the multidrug resistance proteins MRP and cGMP transporters MRP4 5 The present study investigated whether hypergravity generated by centrifugal acceleration may modulate the cGMP efflux in NO-stimulated NHMs and melanoma cells MCs with different metastatic potential The NONOates PAPA-NO and DETA-NO were used as direct NO donors for cell stimulation In the presence of 0 1 mM DETA-NO t 1 2 sim 20 h long-term application of hypergravity up to 5 g for 24 h reduced intracellular cGMP levels by stimulating cGMP efflux in NHMs and non-metastatic MCs in comparison to 1 g whereas exposure to 5 g for 6 h in the presence of 0 1 mM PAPA-NO t 1 2 sim 30 min was not effective The hypergravity-stimulated

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

Constitutive androstane receptor upregulates Abcb1 and Abcg2 at the blood-brain barrier after CITCO activation.

PubMed

Lemmen, Julia; Tozakidis, Iasson E P; Bele, Prachee; Galla, Hans-Joachim

2013-03-21

ATP-driven efflux transporters are considered to be the major hurdle in the treatment of central nervous system (CNS) diseases. Abcb1 (P-glycoprotein) and Abcg2 (breast cancer resistance protein/brain multidrug resistance protein) belong to the best known ABC-transporters. These ABC-transporters limit the permeability of the blood-brain barrier and protect the brain against toxic compounds in the blood but on the other hand they also reduce the efficacy of CNS pharmacotherapy. Even after 40 years of extensive research, the regulatory mechanisms of these efflux transporters are still not completely understood. To unravel the efflux transporter regulation, we analyzed the effect of the nuclear receptor CAR (constitutive androstane receptor) on the expression of Abcb1 and Abcg2 in primary cultures of porcine brain capillary endothelial cells (PBCEC). CAR is a xenobiotic-activated transcription factor, which is, like the other important nuclear receptor pregnane X receptor (PXR), highly expressed in barrier tissue and known to be a positive regulator of ABC-transporters. We demonstrate that activation of porcine CAR by the human CAR (hCAR) ligand CITCO (6-(4-chlorophenyl)-imidazo[2,1-b]thiazole-5-carbaldehyde) leads to an up-regulation of both transporters, whereas the mouse-specific CAR ligand TCPOBOP (1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene) had no effect on transporter expression. The stimulation of PBCEC with CITCO caused a significant up-regulation of both efflux-transporters on RNA-level, protein level and transport level. Furthermore the additional application of a CAR inhibitor significantly decreased the transporter expression to control niveau. In conclusion our data prove CAR activation only by the human ligand CITCO leading to an increased ABC-transporter expression and transport activity. Copyright © 2013 Elsevier B.V. All rights reserved.

Evaluation of Reduced Susceptibility to Quaternary Ammonium Compounds and Bisbiguanides in Clinical Isolates and Laboratory-Generated Mutants of Staphylococcus aureus

PubMed Central

Furi, Leonardo; Ciusa, Maria Laura; Knight, Daniel; Di Lorenzo, Valeria; Tocci, Nadia; Cirasola, Daniela; Aragones, Lluis; Coelho, Joana Rosado; Freitas, Ana Teresa; Marchi, Emmanuela; Moce, Laura; Visa, Pilar; Northwood, John Blackman; Viti, Carlo; Borghi, Elisa; Orefici, Graziella

2013-01-01

The MICs and minimum bactericidal concentrations (MBCs) for the biocides benzalkonium chloride and chlorhexidine were determined against 1,602 clinical isolates of Staphylococcus aureus. Both compounds showed unimodal MIC and MBC distributions (2 and 4 or 8 mg/liter, respectively) with no apparent subpopulation with reduced susceptibility. To investigate further, all isolates were screened for qac genes, and 39 of these also had the promoter region of the NorA multidrug-resistant (MDR) efflux pump sequenced. The presence of qacA, qacB, qacC, and qacG genes increased the mode MIC, but not MBC, to benzalkonium chloride, while only qacA and qacB increased the chlorhexidine mode MIC. Isolates with a wild-type norA promoter or mutations in the norA promoter had similar biocide MIC distributions; notably, not all clinical isolates with norA mutations were resistant to fluoroquinolones. In vitro efflux mutants could be readily selected with ethidium bromide and acriflavine. Multiple passages were necessary to select mutants with biocides, but these mutants showed phenotypes comparable to those of mutants selected by dyes. All mutants showed changes in the promoter region of norA, but these were distinct from this region of the clinical isolates. Still, none of the in vitro mutants displayed fitness defects in a killing assay in Galleria mellonella larvae. In conclusion, our data provide an in-depth comparative overview on efflux in S. aureus mutants and clinical isolates, showing also that plasmid-encoded efflux pumps did not affect bactericidal activity of biocides. In addition, current in vitro tests appear not to be suitable for predicting levels of resistance that are clinically relevant. PMID:23669380

Genetic characterisation of tigecycline-resistant Enterobacter spp. in blood isolates causing bacteraemia.

PubMed

Cha, Min Kyeong; Kang, Cheol-In; Park, Ga Eun; Kim, So Hyun; Chung, Doo Ryeon; Peck, Kyong Ran; Song, Jae-Hoon

2018-01-05

Tigecycline (TIG) is one of the most important antimicrobial agents used to treat infections by multidrug-resistant bacteria. However, rates of TIG-resistant pathogens have increased recently. This study was conducted to identify the antimicrobial susceptibility profiles and to investigate the role of efflux pumps in high-level TIG-resistant Enterobacter spp. isolates causing bacteraemia. A total of 323 Enterobacter spp. causing bacteraemia were collected from eight hospitals in various regions of South Korea. Minimum inhibitory concentrations (MICs) were determined by the broth microdilution method and Etest. Expression levels of the efflux pump gene acrA and its regulators (ramA and rarA) were examined by quantitative real-time PCR. Isolate relatedness was determined by multilocus sequence typing (MLST). Among the 323 clinical isolates included in this study, 37 (11.5%) were TIG-non-susceptible, of which 8 isolates were highly resistant to TIG with MICs of 8mg/L (4 isolates) or 16mg/L (4 isolates). All high-level TIG-resistant isolates showed increased expression of acrA (0.93-13.3-fold) and ramA (1.4-8.2-fold). Isolates with a tigecycline MIC of 16mg/L also showed overexpression of rarA compared with TIG-susceptible isolates. In this study, overexpression of acrA, ramA and rarA was observed in high-level TIG-resistant Enterobacter spp. isolates. We suggest that rarA might be involved in the regulation of acrA overexpression in high-level TIG-resistant Enterobacter spp. isolates. Efflux pump-mediated resistance should be closely monitored because it could be indirectly attributed to the use of other antibiotics transported by the same efflux pump. Copyright © 2017. Published by Elsevier Ltd.

Antibacterial and Antibiotic-Modifying Activity of Methanol Extracts from Six Cameroonian Food Plants against Multidrug-Resistant Enteric Bacteria

PubMed Central

Dzotam, Joachim K.

2017-01-01

The present work was designed to investigate the antibacterial activities of methanol extracts from six Cameroonian edible plants and their synergistic effects with some commonly used antibiotics against multidrug-resistant (MDR) Gram-negative bacteria expressing active efflux pumps. The extracts were subjected to qualitative phytochemical screening and the microdilution broth method was used for antibacterial assays. The results of phytochemical tests indicate that all tested crude extracts contained polyphenols, flavonoids, triterpenes, and steroids. Extracts displayed selective antibacterial activities with the minimal inhibitory concentration (MIC) values ranging from 32 to 1024 μg/mL. The lowest MIC value (32 μg/mL) was recorded with Coula edulis extract against E. coli AG102 and K. pneumoniae K2 and with Mangifera indica bark extract against P. aeruginosa PA01 and Citrus sinensis extract against E. coli W3110 which also displayed the best MBC (256 μg/mL) value against E. coli ATCC8739. In combination with antibiotics, extracts from M. indica leaves showed synergistic effects with 75% (6/8) of the tested antibiotics against more than 80% of the tested bacteria. The findings of the present work indicate that the tested plants may be used alone or in combination in the treatment of bacterial infections including the multidrug-resistant bacteria. PMID:28904944

The cellular uptake mechanism, intracellular transportation, and exocytosis of polyamidoamine dendrimers in multidrug-resistant breast cancer cells.

PubMed

Zhang, Jie; Liu, Dan; Zhang, Mengjun; Sun, Yuqi; Zhang, Xiaojun; Guan, Guannan; Zhao, Xiuli; Qiao, Mingxi; Chen, Dawei; Hu, Haiyang

2016-01-01

Polyamidoamine dendrimers, which can deliver drugs and genetic materials to resistant cells, are attracting increased research attention, but their transportation behavior in resistant cells remains unclear. In this paper, we performed a systematic analysis of the cellular uptake, intracellular transportation, and efflux of PAMAM-NH2 dendrimers in multidrug-resistant breast cancer cells (MCF-7/ADR cells) using sensitive breast cancer cells (MCF-7 cells) as the control. We found that the uptake rate of PAMAM-NH2 was much lower and exocytosis of PAMAM-NH2 was much greater in MCF-7/ADR cells than in MCF-7 cells due to the elimination of PAMAM-NH2 from P-glycoprotein and the multidrug resistance-associated protein in MCF-7/ADR cells. Macropinocytosis played a more important role in its uptake in MCF-7/ADR cells than in MCF-7 cells. PAMAM-NH2 aggregated and became more degraded in the lysosomal vesicles of the MCF-7/ADR cells than in those of the MCF-7 cells. The endoplasmic reticulum and Golgi complex were found to participate in the exocytosis rather than endocytosis process of PAMAM-NH2 in both types of cells. Our findings clearly showed the intracellular transportation process of PAMAM-NH2 in MCF-7/ADR cells and provided a guide of using PAMAM-NH2 as a drug and gene vector in resistant cells.

Tetracycline improved the efficiency of other antimicrobials against Gram-negative multidrug-resistant bacteria.

PubMed

Mawabo, Isabelle K; Noumedem, Jaurès A K; Kuiate, Jules R; Kuete, Victor

2015-01-01

Treatment of infectious diseases with antimicrobials constituted a great achievement in the history of medicine. Unfortunately, the emergence of resistant strains of bacteria to all classes of antimicrobials limited their efficacy. The present study was aimed at evaluating the effect of combinations of antibiotics on multi-drug resistant Gram-negative (MDRGN) bacteria. A liquid micro-broth dilution method was used to evaluate the antibacterial activity of 10 different classes of antimicrobials on 20 bacterial strains belonging to six different species. The antimicrobials were associated with phenylalanine β-naphthylamide (PAβN), an efflux pump inhibitor, and with other antimicrobials at their sub-inhibitory concentrations. The effectiveness of each combination was monitored using the minimal inhibitory concentration (MIC) and the fractional inhibitory concentration (FIC). Most of the antimicrobials tested showed low antibacterial activity with a MIC value of 128 mg/L on a majority of the bacterial strains, justifying their multidrug-resistant (MDR) profile. Synergistic effects were mostly observed (FIC≤0.5) when ampicillin (AMP), cloxacillin (CLX), erythromycin (ERY), chloramphenicol (CHL), kanamycin (KAN) and streptomycin (STR) were combined with tetracycline (TET) at the sub-inhibitory concentration of MIC/5 or MIC/10. The results of the present work suggest that the association of several antimicrobials with TET could improve the fight against MDRGN bacterial species. Copyright © 2014 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.

Analysis of Tryptophan Residues in the Staphylococcal Multidrug Transporter QacA Reveals Long-Distance Functional Associations of Residues on Opposite Sides of the Membrane▿

PubMed Central

Hassan, Karl A.; Souhani, Talal; Skurray, Ronald A.; Brown, Melissa H.

2008-01-01

Tryptophan residues can possess a multitude of functions within a multidrug transport protein, e.g., mediating interactions with substrates or distal parts of the protein, or fulfilling a structural requirement, such as guiding the depth of membrane insertion. In this study, the nine tryptophan residues of the staphylococcal QacA multidrug efflux protein were individually mutated to alanine and phenylalanine, and the functional consequences of these changes were determined. Phenylalanine substitutions for each tryptophan residue were functionally tolerated. However, alanine modifications revealed an important functional role for three tryptophan residues, W58, W149, and W173, each of which is well conserved among QacA-related transport proteins in the major facilitator superfamily. The most functionally compromising mutation, an alanine substitution for W58, likely to be located at the extracellular interface of transmembrane segment 2, abolished all detectable QacA-mediated resistance and transport function. Second-site suppressor analyses identified several mutations that rescued the function of the W58A QacA mutant. Remarkably, all of these suppressor mutations were shown to be located in cytoplasmic loops between transmembrane helices 2 and 3 or 12 and 13, demonstrating novel functional associations between amino acid positions on opposite sides of the membrane and in distal N- and C-terminal regions of the QacA protein. PMID:18223078

Nanomedicine to Deal With Cancer Cell Biology in Multi-Drug Resistance.

PubMed

Tekchandani, Pawan; Kurmi, Balak Das; Paliwal, Shivani Rai

2017-01-01

Today Cancer still remains a major cause of mortality and death worldwide, in humans. Chemotherapy, a key treatment strategy in cancer, has significant hurdles such as the occurrence of chemoresistance in cancer, which is inherent unresponsiveness or acquired upon exposure to chemotherapeutics. The resistance of cancer cells to an antineoplastic agent accompanied to other chemotherapeutic drugs with different structures and mechanisms of action called multi-drug resistance (MDR) plays an important role in the failure of chemo- therapeutics. MDR is primarily based on the overexpression of drug efflux pumps in the cellular membrane, which belongs to the ATP-binding cassette (ABC) superfamily of proteins, are P-gp (P-glycoprotein) and multidrug resistance-associated protein (MRP). Over the years, various strategies have been evaluated to overcome MDR, based not only on the use of MDR modulators but also on the implementation an innovative approach and advanced nanosized drug delivery systems. Nanomedicine is an emerging tool of chemotherapy that focuses on alternative drug delivery for improvement of the treatment efficacy and reducing side effects to normal tissues. This review aims to focus on the details biology, reversal strategies option with the limitation of MDR and various advantages of the present medical science nanotechnology with intracellular delivery aspects for overcoming the significant potential for improving the treatment of MDR malignancies. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Antibacterial and Antibiotic-Modifying Activity of Methanol Extracts from Six Cameroonian Food Plants against Multidrug-Resistant Enteric Bacteria.

PubMed

Dzotam, Joachim K; Kuete, Victor

2017-01-01

The present work was designed to investigate the antibacterial activities of methanol extracts from six Cameroonian edible plants and their synergistic effects with some commonly used antibiotics against multidrug-resistant (MDR) Gram-negative bacteria expressing active efflux pumps. The extracts were subjected to qualitative phytochemical screening and the microdilution broth method was used for antibacterial assays. The results of phytochemical tests indicate that all tested crude extracts contained polyphenols, flavonoids, triterpenes, and steroids. Extracts displayed selective antibacterial activities with the minimal inhibitory concentration (MIC) values ranging from 32 to 1024  μ g/mL. The lowest MIC value (32  μ g/mL) was recorded with Coula edulis extract against E. coli AG102 and K. pneumoniae K2 and with Mangifera indica bark extract against P. aeruginosa PA01 and Citrus sinensis extract against E. coli W3110 which also displayed the best MBC (256  μ g/mL) value against E. coli ATCC8739. In combination with antibiotics, extracts from M. indica leaves showed synergistic effects with 75% (6/8) of the tested antibiotics against more than 80% of the tested bacteria. The findings of the present work indicate that the tested plants may be used alone or in combination in the treatment of bacterial infections including the multidrug-resistant bacteria.

Confinement-Induced Drug-Tolerance in Mycobacteria Mediated by an Efflux Mechanism

PubMed Central

Luthuli, Brilliant B.; Purdy, Georgiana E.; Balagaddé, Frederick K.

2015-01-01

Tuberculosis (TB) is the world’s deadliest curable disease, responsible for an estimated 1.5 million deaths annually. A considerable challenge in controlling this disease is the prolonged multidrug chemotherapy (6 to 9 months) required to overcome drug-tolerant mycobacteria that persist in human tissues, although the same drugs can sterilize genetically identical mycobacteria growing in axenic culture within days. An essential component of TB infection involves intracellular Mycobacterium tuberculosis bacteria that multiply within macrophages and are significantly more tolerant to antibiotics compared to extracellular mycobacteria. To investigate this aspect of human TB, we created a physical cell culture system that mimics confinement of replicating mycobacteria, such as in a macrophage during infection. Using this system, we uncovered an epigenetic drug-tolerance phenotype that appears when mycobacteria are cultured in space-confined bioreactors and disappears in larger volume growth contexts. Efflux mechanisms that are induced in space-confined growth environments contribute to this drug-tolerance phenotype. Therefore, macrophage-induced drug tolerance by mycobacteria may be an effect of confined growth among other macrophage-specific mechanisms. PMID:26295942

Enhanced gravi- and phototropism in plant mdr mutants mislocalizing the auxin efflux protein PIN1.

PubMed

Noh, Bosl; Bandyopadhyay, Anindita; Peer, Wendy Ann; Spalding, Edgar P; Murphy, Angus S

2003-06-26

Many aspects of plant growth and development are dependent on the flow of the hormone auxin down the plant from the growing shoot tip where it is synthesized. The direction of auxin transport in stems is believed to result from the basal localization within cells of the PIN1 membrane protein, which controls the efflux of the auxin anion. Mutations in two genes homologous to those encoding the P-glycoprotein ABC transporters that are especially abundant in multidrug-resistant tumour cells in animals were recently shown to block polar auxin transport in the hypocotyls of Arabidopsis seedlings. Here we show that the mdr mutants display faster and greater gravitropism and enhanced phototropism instead of the impaired curvature development expected in mutants lacking polar auxin transport. We find that these phenotypes result from a disruption of the normal accumulation of PIN1 protein along the basal end of hypocotyl cells associated with basipetal auxin flow. Lateral auxin conductance becomes relatively larger as a result, enhancing the growth differentials responsible for tropic responses.

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

Resistance-modifying Activity in Vinblastine-resistant Human Breast Cancer Cells by Oligosaccharides Obtained from Mucilage of Chia Seeds (Salvia hispanica).

PubMed

Rosas-Ramírez, Daniel G; Fragoso-Serrano, Mabel; Escandón-Rivera, Sonia; Vargas-Ramírez, Alba L; Reyes-Grajeda, Juan P; Soriano-García, Manuel

2017-06-01

The multidrug resistance (MDR) phenotype is considered as a major cause of the failure in cancer chemotherapy. The acquisition of MDR is usually mediated by the overexpression of drug efflux pumps of a P-glycoprotein. The development of compounds that mitigate the MDR phenotype by modulating the activity of these transport proteins is an important yet elusive target. Here, we screened the saponification and enzymatic degradation products from Salvia hispanica seed's mucilage to discover modulating compounds of the acquired resistance to chemotherapeutic in breast cancer cells. Preparative-scale recycling HPLC was used to purify the hydrolysis degradation products. All compounds were tested in eight different cancer cell lines and Vero cells. All compounds were noncytotoxic at the concentration tested against the drug-sensitive and multidrug-resistant cells (IC 50  > 29.2 μM). For the all products, a moderate vinblastine-enhancing activity from 4.55-fold to 6.82-fold was observed. That could be significant from a therapeutic perspective. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Design, synthesis and biological evaluation of LBM-A5 derivatives as potent P-glycoprotein-mediated multidrug resistance inhibitors.

PubMed

Wu, Yuxiang; Pan, Miaobo; Dai, Yuxuan; Liu, Baomin; Cui, Jian; Shi, Wei; Qiu, Qianqian; Huang, Wenlong; Qian, Hai

2016-05-15

A novel series of P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) inhibitors with triazol-N-phenethyl-tetrahydroisoquinoline or triazol-N-ethyl-tetrahydroisoquinoline scaffold were designed and synthesized via click chemistry. Most of the synthesized compounds showed higher reversal activity than verapamil (VRP). Among them, the most potent compound 4 showed a comparable activity with the known potent P-gp inhibitor WK-X-34 with lower cytotoxicity toward K562 cells (IC50>100μM). Compared with VRP, compound 4 exhibited more potency in increasing drug accumulation in K562/A02 MDR cells. Moreover, compound 4 could significantly reverse MDR in a dose-dependent manner and also persist longer chemo-sensitizing effect than VRP with reversibility. Further mechanism studies revealed that compound 4 could remarkably increase the intracellular accumulation of Adriamycin (ADM) in K562/A02 cells as well as inhibit rhodamine-123 (Rh123) efflux from the cells. These results suggested that compound 4 may represent a promising candidate for developing P-gp-mediated MDR inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

Influence of multidrug resistance and drug transport proteins on chemotherapy drug metabolism.

PubMed

Joyce, Helena; McCann, Andrew; Clynes, Martin; Larkin, Annemarie

2015-05-01

Chemotherapy involving the use of anticancer drugs remains an important strategy in the overall management of patients with metastatic cancer. Acquisition of multidrug resistance remains a major impediment to successful chemotherapy. Drug transporters in cell membranes and intracellular drug metabolizing enzymes contribute to the resistance phenotype and determine the pharmacokinetics of anticancer drugs in the body. ATP-binding cassette (ABC) transporters mediate the transport of endogenous metabolites and xenobiotics including cytotoxic drugs out of cells. Solute carrier (SLC) transporters mediate the influx of cytotoxic drugs into cells. This review focuses on the substrate interaction of these transporters, on their biology and what role they play together with drug metabolizing enzymes in eliminating therapeutic drugs from cells. The majority of anticancer drugs are substrates for the ABC transporter and SLC transporter families. Together, these proteins have the ability to control the influx and the efflux of structurally unrelated chemotherapeutic drugs, thereby modulating the intracellular drug concentration. These interactions have important clinical implications for chemotherapy because ultimately they determine therapeutic efficacy, disease progression/relapse and the success or failure of patient treatment.

Non-antibiotic antimicrobial triclosan induces multiple antibiotic resistance through genetic mutation.

PubMed

Lu, Ji; Jin, Min; Nguyen, Son Hoang; Mao, Likai; Li, Jie; Coin, Lachlan J M; Yuan, Zhiguo; Guo, Jianhua

2018-06-11

Antibiotic resistance poses a major threat to public health. Overuse and misuse of antibiotics are generally recognized as the key factors contributing to antibiotic resistance. However, whether non-antibiotic, anti-microbial (NAAM) chemicals can directly induce antibiotic resistance is unclear. We aim to investigate whether the exposure to a NAAM chemical triclosan (TCS) has an impact on inducing antibiotic resistance on Escherichia coli. Here, we report that at a concentration of 0.2 mg/L TCS induces multi-drug resistance in wild-type Escherichia coli after 30-day TCS exposure. The oxidative stress induced by TCS caused genetic mutations in genes such as fabI, frdD, marR, acrR and soxR, and subsequent up-regulation of the transcription of genes encoding beta-lactamases and multi-drug efflux pumps, together with down-regulation of genes related to membrane permeability. The findings advance our understanding of the potential role of NAAM chemicals in the dissemination of antibiotic resistance in microbes, and highlight the need for controlling biocide applications. Copyright © 2018 Elsevier Ltd. All rights reserved.

Marine Natural Products as Models to Circumvent Multidrug Resistance.

PubMed

Long, Solida; Sousa, Emília; Kijjoa, Anake; Pinto, Madalena M M

2016-07-08

Multidrug resistance (MDR) to anticancer drugs is a serious health problem that in many cases leads to cancer treatment failure. The ATP binding cassette (ABC) transporter P-glycoprotein (P-gp), which leads to premature efflux of drugs from cancer cells, is often responsible for MDR. On the other hand, a strategy to search for modulators from natural products to overcome MDR had been in place during the last decades. However, Nature limits the amount of some natural products, which has led to the development of synthetic strategies to increase their availability. This review summarizes the research findings on marine natural products and derivatives, mainly alkaloids, polyoxygenated sterols, polyketides, terpenoids, diketopiperazines, and peptides, with P-gp inhibitory activity highlighting the established structure-activity relationships. The synthetic pathways for the total synthesis of the most promising members and analogs are also presented. It is expected that the data gathered during the last decades concerning their synthesis and MDR-inhibiting activities will help medicinal chemists develop potential drug candidates using marine natural products as models which can deliver new ABC transporter inhibitor scaffolds.

Synthesis, activity and pharmacophore development for isatin-β-thiosemicarbazones with selective activity towards multidrug resistant cellsa

PubMed Central

Hall, Matthew D.; Salam, Noeris K.; Hellawell, Jennifer L.; Fales, Henry M.; Kensler, Caroline B.; Ludwig, Joseph A.; Szakacs, Gergely; Hibbs, David E.; Gottesman, Michael M.

2009-01-01

We have recently identified a new class of compounds that selectively kill cells that express P-glycoprotein (P-gp, MDR1), the ATPase efflux pump that confers multidrug resistance on cancer cells. Several isatin-β-thiosemicarbazones from our initial study have been validated, and a range of analogs synthesized and tested. A number demonstrated improved MDR1-selective activity over the lead, NSC73306 (1). Pharmacophores for cytotoxicity and MDR1-selectivity were generated to delineate the structural features required for activity. The MDR1-selective pharmacophore highlights the importance of aromatic/hydrophobic features at the N4 position of the thiosemicarbazone, and the reliance on the isatin moiety as key bioisosteric contributors. Additionally, a quantitative structure-activity relationship (QSAR) model that yielded a cross-validated correlation coefficient of 0.85 effectively predicts the cytotoxicty of untested thiosemicarbazones. Together, the models serve as effective approaches for predicting structures with MDR1-selective activity, and aid in directing the search for the mechanism of action of 1. PMID:19397322

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.

Masitinib antagonizes ATP-binding cassette subfamily G member 2-mediated multidrug resistance

PubMed Central

KATHAWALA, RISHIL J.; CHEN, JUN-JIANG; ZHANG, YUN-KAI; WANG, YI-JUN; PATEL, ATISH; WANG, DE-SHEN; TALELE, TANAJI T.; ASHBY, CHARLES R.; CHEN, ZHE-SHENG

2014-01-01

In this in vitro study, we determined whether masitinib could reverse multidrug resistance (MDR) in cells overexpressing the ATP binding cassette subfamily G member 2 (ABCG2) transporter. Masitinib (1.25 and 2.5 μM) significantly decreases the resistance to mitoxantrone (MX), SN38 and doxorubicin in HEK293 and H460 cells overexpressing the ABCG2 transporter. In addition, masitinib (2.5 μM) significantly increased the intracellular accumulation of [3H]-MX, a substrate for ABCG2, by inhibiting the function of ABCG2 and significantly decreased the efflux of [3H]-MX. However, masitinib (2.5 μM) did not significantly alter the expression of the ABCG2 protein. In addition, a docking model suggested that masitinib binds within the transmembrane region of a homology-modeled human ABCG2 transporter. Overall, our in vitro findings suggest that masitinib reverses MDR to various anti-neoplastic drugs in HEK293 and H460 cells overexpressing ABCG2 by inhibiting their transport activity as opposed to altering their levels of expression. PMID:24626598

Botrytis fragariae, a New Species Causing Gray Mold on Strawberries, Shows High Frequencies of Specific and Efflux-Based Fungicide Resistance.

PubMed

Rupp, Sabrina; Plesken, Cecilia; Rumsey, Sibylle; Dowling, Madeline; Schnabel, Guido; Weber, Roland W S; Hahn, Matthias

2017-05-01

Botrytis cinerea causes pre- and postharvest decay of many fruit and vegetable crops. A survey of German strawberry fields revealed Botrytis strains that differed from B. cinerea in diagnostic PCR markers and growth appearance. Phylogenetic analyses showed that these strains belong to an undescribed species in Botrytis clade 2, named Botrytis fragariae sp. nov. Isolates of B fragariae were detected in strawberry fields throughout Germany, sometimes at frequencies similar to those of B. cinerea , and in the southeastern United States. B fragariae was isolated from overwintering strawberry tissue but not from freshly infected fruit. B fragariae invaded strawberry tissues with an efficiency similar to or lower than that of B. cinerea but showed poor colonization of inoculated nonhost plant tissues. These data and the exclusive occurrence of this fungus on strawberry plants indicate that B fragariae is host specific and has a tissue preference different from that of B. cinerea Various fungicide resistance patterns were observed in B fragariae populations. Many B fragariae strains showed resistance to one or several chemical classes of fungicides and an efflux-based multidrug resistance (MDR1) phenotype previously described in B. cinerea Resistance-related mutations in B fragariae were identical or similar to those of B. cinerea for carbendazim (E198A mutation in tubA ), azoxystrobin (G143A in cytB ), iprodione (G367A+V368F in bos1 ), and MDR1 (gain-of-function mutations in the transcription factor mrr1 gene and overexpression of the drug efflux transporter gene atrB ). The widespread occurrence of B fragariae indicates that this species is adapted to fungicide-treated strawberry fields and may be of local importance as a gray mold pathogen alongside B. cinerea IMPORTANCE Gray mold is the most important fruit rot on strawberries worldwide and requires fungicide treatments for control. For a long time, it was believed to be caused only by Botrytis cinerea , a ubiquitous pathogen with a broad host range that quickly develops fungicide resistance. We report the discovery and description of a new species, named Botrytis fragariae , that is widely distributed in commercial strawberry fields in Germany and the southeastern United States. It was observed on overwintering tissue but not on freshly infected fruit and seems host specific on the basis of its occurrence and artificial infection tests. B fragariae has also developed resistance to several fungicides that is caused by mutations similar to those known in B. cinerea , including an efflux-based multidrug resistance. Our data indicate that B fragariae could be of practical importance as a strawberry pathogen in some regions where its abundance is similar to that of B. cinerea . Copyright © 2017 American Society for Microbiology.

Botrytis fragariae, a New Species Causing Gray Mold on Strawberries, Shows High Frequencies of Specific and Efflux-Based Fungicide Resistance

PubMed Central

Rupp, Sabrina; Plesken, Cecilia; Rumsey, Sibylle; Dowling, Madeline; Schnabel, Guido; Weber, Roland W. S.

2017-01-01

ABSTRACT Botrytis cinerea causes pre- and postharvest decay of many fruit and vegetable crops. A survey of German strawberry fields revealed Botrytis strains that differed from B. cinerea in diagnostic PCR markers and growth appearance. Phylogenetic analyses showed that these strains belong to an undescribed species in Botrytis clade 2, named Botrytis fragariae sp. nov. Isolates of B. fragariae were detected in strawberry fields throughout Germany, sometimes at frequencies similar to those of B. cinerea, and in the southeastern United States. B. fragariae was isolated from overwintering strawberry tissue but not from freshly infected fruit. B. fragariae invaded strawberry tissues with an efficiency similar to or lower than that of B. cinerea but showed poor colonization of inoculated nonhost plant tissues. These data and the exclusive occurrence of this fungus on strawberry plants indicate that B. fragariae is host specific and has a tissue preference different from that of B. cinerea. Various fungicide resistance patterns were observed in B. fragariae populations. Many B. fragariae strains showed resistance to one or several chemical classes of fungicides and an efflux-based multidrug resistance (MDR1) phenotype previously described in B. cinerea. Resistance-related mutations in B. fragariae were identical or similar to those of B. cinerea for carbendazim (E198A mutation in tubA), azoxystrobin (G143A in cytB), iprodione (G367A+V368F in bos1), and MDR1 (gain-of-function mutations in the transcription factor mrr1 gene and overexpression of the drug efflux transporter gene atrB). The widespread occurrence of B. fragariae indicates that this species is adapted to fungicide-treated strawberry fields and may be of local importance as a gray mold pathogen alongside B. cinerea. IMPORTANCE Gray mold is the most important fruit rot on strawberries worldwide and requires fungicide treatments for control. For a long time, it was believed to be caused only by Botrytis cinerea, a ubiquitous pathogen with a broad host range that quickly develops fungicide resistance. We report the discovery and description of a new species, named Botrytis fragariae, that is widely distributed in commercial strawberry fields in Germany and the southeastern United States. It was observed on overwintering tissue but not on freshly infected fruit and seems host specific on the basis of its occurrence and artificial infection tests. B. fragariae has also developed resistance to several fungicides that is caused by mutations similar to those known in B. cinerea, including an efflux-based multidrug resistance. Our data indicate that B. fragariae could be of practical importance as a strawberry pathogen in some regions where its abundance is similar to that of B. cinerea. PMID:28235878

The arabidopsis thaliana AGRAVITROPIC 1 gene encodes a component of the polar-auxin-transport efflux carrier

NASA Technical Reports Server (NTRS)

Chen, R.; Hilson, P.; Sedbrook, J.; Rosen, E.; Caspar, T.; Masson, P. H.

1998-01-01

Auxins are plant hormones that mediate many aspects of plant growth and development. In higher plants, auxins are polarly transported from sites of synthesis in the shoot apex to their sites of action in the basal regions of shoots and in roots. Polar auxin transport is an important aspect of auxin functions and is mediated by cellular influx and efflux carriers. Little is known about the molecular identity of its regulatory component, the efflux carrier [Estelle, M. (1996) Current Biol. 6, 1589-1591]. Here we show that mutations in the Arabidopsis thaliana AGRAVITROPIC 1 (AGR1) gene involved in root gravitropism confer increased root-growth sensitivity to auxin and decreased sensitivity to ethylene and an auxin transport inhibitor, and cause retention of exogenously added auxin in root tip cells. We used positional cloning to show that AGR1 encodes a putative transmembrane protein whose amino acid sequence shares homologies with bacterial transporters. When expressed in Saccharomyces cerevisiae, AGR1 promotes an increased efflux of radiolabeled IAA from the cells and confers increased resistance to fluoro-IAA, a toxic IAA-derived compound. AGR1 transcripts were localized to the root distal elongation zone, a region undergoing a curvature response upon gravistimulation. We have identified several AGR1-related genes in Arabidopsis, suggesting a global role of this gene family in the control of auxin-regulated growth and developmental processes.

Photochemical internalisation of chemotherapy potentiates killing of multidrug-resistant breast and bladder cancer cells.

PubMed

Adigbli, D K; Wilson, D G G; Farooqui, N; Sousi, E; Risley, P; Taylor, I; Macrobert, A J; Loizidou, M

2007-08-20

Multidrug resistance (MDR) is the major confounding factor in adjuvant solid tumour chemotherapy. Increasing intracellular amounts of chemotherapeutics to circumvent MDR may be achieved by a novel delivery method, photochemical internalisation (PCI). PCI consists of the co-administration of drug and photosensitiser; upon light activation the latter induces intracellular release of organelle-bound drug. We investigated whether co-administration of hypericin (photosensitiser) with mitoxantrone (MTZ, chemotherapeutic) plus illumination potentiates cytotoxicity in MDR cancer cells. We mapped the extent of intracellular co-localisation of drug/photosensitiser. We determined whether PCI altered drug-excreting efflux pump P-glycoprotein (Pgp) expression or function in MDR cells. Bladder and breast cancer cells and their Pgp-overexpressing MDR subclones (MGHU1, MGHU1/R, MCF-7, MCF-7/R) were given hypericin/MTZ combinations, with/without blue-light illumination. Pilot experiments determined appropriate sublethal doses for each. Viability was determined by the 3-[4,5-dimethylthiazolyl]-2,5-diphenyltetrazolium bromide assay. Intracellular localisation was mapped by confocal microscopy. Pgp expression was detected by immunofluorescence and Pgp function investigated by Rhodamine123 efflux on confocal microscopy. MTZ alone (0.1-0.2 microg ml(-1)) killed up to 89% of drug-sensitive cells; MDR cells exhibited less cytotoxicity (6-28%). Hypericin (0.1-0.2 microM) effects were similar for all cells; light illumination caused none or minimal toxicity. In combination, MTZ /hypericin plus illumination, potentiated MDR cell killing, vs hypericin or MTZ alone. (MGHU1/R: 38.65 and 36.63% increase, P<0.05; MCF-7/R: 80.2 and 46.1% increase, P<0.001). Illumination of combined MTZ/hypericin increased killing by 28.15% (P<0.05 MGHU1/R) compared to dark controls. Intracytoplasmic vesicular co-localisation of MTZ/hypericin was evident before illumination and at serial times post-illumination. MTZ was always found in sensitive cell nuclei, but not in dark resistant cell nuclei. In illuminated resistant cells there was some mobilisation of MTZ into the nucleus. Pgp expression remained unchanged, regardless of drug exposure. Pgp efflux was blocked by the Pgp inhibitor verapamil (positive control) but not impeded by hypericin. The increased killing of MDR cancer cells demonstrated is consistent with PCI. PCI is a promising technique for enhancing treatment efficacy.

Photochemical internalisation of chemotherapy potentiates killing of multidrug-resistant breast and bladder cancer cells

PubMed Central

Adigbli, D K; Wilson, D G G; Farooqui, N; Sousi, E; Risley, P; Taylor, I; MacRobert, A J; Loizidou, M

2007-01-01

Multidrug resistance (MDR) is the major confounding factor in adjuvant solid tumour chemotherapy. Increasing intracellular amounts of chemotherapeutics to circumvent MDR may be achieved by a novel delivery method, photochemical internalisation (PCI). PCI consists of the co-administration of drug and photosensitiser; upon light activation the latter induces intracellular release of organelle-bound drug. We investigated whether co-administration of hypericin (photosensitiser) with mitoxantrone (MTZ, chemotherapeutic) plus illumination potentiates cytotoxicity in MDR cancer cells. We mapped the extent of intracellular co-localisation of drug/photosensitiser. We determined whether PCI altered drug-excreting efflux pump P-glycoprotein (Pgp) expression or function in MDR cells. Bladder and breast cancer cells and their Pgp-overexpressing MDR subclones (MGHU1, MGHU1/R, MCF-7, MCF-7/R) were given hypericin/MTZ combinations, with/without blue-light illumination. Pilot experiments determined appropriate sublethal doses for each. Viability was determined by the 3-[4,5-dimethylthiazolyl]-2,5-diphenyltetrazolium bromide assay. Intracellular localisation was mapped by confocal microscopy. Pgp expression was detected by immunofluorescence and Pgp function investigated by Rhodamine123 efflux on confocal microscopy. MTZ alone (0.1–0.2 μg ml−1) killed up to 89% of drug-sensitive cells; MDR cells exhibited less cytotoxicity (6–28%). Hypericin (0.1–0.2 μM) effects were similar for all cells; light illumination caused none or minimal toxicity. In combination, MTZ /hypericin plus illumination, potentiated MDR cell killing, vs hypericin or MTZ alone. (MGHU1/R: 38.65 and 36.63% increase, P<0.05; MCF-7/R: 80.2 and 46.1% increase, P<0.001). Illumination of combined MTZ/hypericin increased killing by 28.15% (P<0.05 MGHU1/R) compared to dark controls. Intracytoplasmic vesicular co-localisation of MTZ/hypericin was evident before illumination and at serial times post-illumination. MTZ was always found in sensitive cell nuclei, but not in dark resistant cell nuclei. In illuminated resistant cells there was some mobilisation of MTZ into the nucleus. Pgp expression remained unchanged, regardless of drug exposure. Pgp efflux was blocked by the Pgp inhibitor verapamil (positive control) but not impeded by hypericin. The increased killing of MDR cancer cells demonstrated is consistent with PCI. PCI is a promising technique for enhancing treatment efficacy. PMID:17667930

Trans-Cinnamaldehyde and Eugenol Increase Acinetobacter baumannii Sensitivity to Beta-Lactam Antibiotics.

PubMed

Karumathil, Deepti P; Nair, Meera Surendran; Gaffney, James; Kollanoor-Johny, Anup; Venkitanarayanan, Kumar

2018-01-01

Multi-drug resistant (MDR) Acinetobacter baumannii is a major nosocomial pathogen causing a wide range of clinical conditions with significant mortality rates. A. baumannii strains are equipped with a multitude of antibiotic resistance mechanisms, rendering them resistant to most of the currently available antibiotics. Thus, there is a critical need to explore novel strategies for controlling antibiotic resistance in A. baumannii . This study investigated the efficacy of two food-grade, plant-derived antimicrobials (PDAs), namely trans -cinnamaldehyde (TC) and eugenol (EG) in decreasing A. baumannii 's resistance to seven β-lactam antibiotics, including ampicillin, methicillin, meropenem, penicillin, aztreonam, amoxicillin, and piperacillin. Two MDR A. baumannii isolates (ATCC 17978 and AB 251847) were separately cultured in tryptic soy broth (∼6 log CFU/ml) containing the minimum inhibitory concentration (MIC) of TC or EG with or without the MIC of each antibiotic at 37°C for 18 h. A. baumannii strains not exposed to the PDAs or antibiotics served as controls. Following incubation, A. baumannii counts were determined by broth dilution assay. In addition, the effect of PDAs on the permeability of outer membrane and efflux pumps in A. baumannii was measured. Further, the effect of TC and EG on the expression of A. baumannii genes encoding resistance to β-lactam antibiotics ( blaP ), efflux pumps ( adeABC ), and multi-drug resistant protein ( mdrp ) was studied using real-time quantitative PCR (RT-qPCR). The experiment was replicated three times with duplicate samples of each treatment and control. The results from broth dilution assay indicated that both TC and EG in combination with antibiotics increased the sensitivity of A. baumannii to all the tested antibiotics ( P < 0.05). The two PDAs inhibited the function of A. baumannii efflux pump, (AdeABC), but did not increase the permeability of its outer membrane. Moreover, RT-qPCR data revealed that TC and EG down-regulated the expression of majority of the genes associated with β-lactam antibiotic resistance, especially blaP and adeABC ( P < 0.05). The results suggest that TC and EG could potentially be used along with β-lactam antibiotics for controlling MDR A. baumannii infections; however, their clinical significance needs to be determined using in vivo studies.

Limited brain distribution of [3R,4R,5S]-4-acetamido-5-amino-3-(1-ethylpropoxy)-1-cyclohexene-1-carboxylate phosphate (Ro 64-0802), a pharmacologically active form of oseltamivir, by active efflux across the blood-brain barrier mediated by organic anion transporter 3 (Oat3/Slc22a8) and multidrug resistance-associated protein 4 (Mrp4/Abcc4).

PubMed

Ose, Atsushi; Ito, Mototsugu; Kusuhara, Hiroyuki; Yamatsugu, Kenzo; Kanai, Motomu; Shibasaki, Masakatsu; Hosokawa, Masakiyo; Schuetz, John D; Sugiyama, Yuichi

2009-02-01

[3R,4R,5S]-4-Acetamido-5-amino-3-(1-ethylpropoxy)-1-cyclohexene-1-carboxylate phosphate (Ro 64-0802) is a pharmacologically active form of the anti-influenza virus drug oseltamivir. Abnormal behavior is a suspected adverse effect of oseltamivir on the central nervous system. This study focused on the transport mechanisms of Ro 64-0802 across the blood-brain barrier (BBB). Ro 64-0802 was found to be a substrate of organic anion transporter 3 (OAT3/SLC22A8) and multidrug resistance-associated protein 4 (MRP4/ABCC4). Human embryonic kidney 293 cells expressing OAT3 exhibited a greater intracellular accumulation of Ro 64-0802 than mock-transfected cells (15 versus 1.2 microl/mg protein/10 min, respectively). The efflux of Ro 64-0802 was 3-fold greater when MRP4 was expressed in MDCKII cells and was significantly inhibited by indomethacin. After its microinjection into the cerebrum, the amount of Ro 64-0802 in brain was significantly greater in both Oat3(-/-) mice and Mrp4(-/-) mice compared with the corresponding wild-type mice (0.36 versus 0.080 and 0.32 versus 0.060 nmol at 120 min after injection, respectively). The brain/plasma concentration ratio (K(p,) (brain)) of Ro 64-0802, determined in wild-type mice after subcutaneous continuous infusion for 24 h, was close to the capillary volume (approximately 10 microl/g brain). Although the K(p,) (brain) of Ro 64-0802 was unchanged in Oat3(-/-) mice, it was significantly greater in Mrp4(-/-) mice (41 microl/g of brain). These results suggest that Ro 64-0802 can cross the BBB from the blood, but its brain distribution is limited by its active efflux by Mrp4 and Oat3 across the BBB. The transporter responsible for the brain uptake of Ro 64-0802 remains unknown, but Oat3 is a candidate transporter.

MRP2 mediated drug-drug interaction: indomethacin increases sulfasalazine absorption in the small intestine, potentially decreasing its colonic targeting.

PubMed

Dahan, Arik; Amidon, Gordon L

2010-02-15

We have recently shown that efflux transport, mediated by multidrug resistance-associated protein 2 (MRP2) and breast cancer resistance protein (BCRP), is responsible for sulfasalazine low-permeability in the small intestine, thereby enabling its colonic targeting and therapeutic action. The purpose of the present study was to evaluate the potential pharmacokinetic interaction between indomethacin and sulfasalazine, in the mechanism of efflux transporter competition. The concentration-dependent effects of indomethacin on sulfasalazine intestinal epithelial transport were investigated across Caco-2 cell monolayers, in both apical to basolateral (AP-BL) and BL-AP directions. The interaction was then investigated in the in situ single-pass rat jejunal perfusion model. Sulfasalazine displayed 30-fold higher BL-AP than AP-BL Caco-2 permeability, indicative of net mucosal secretion. Indomethacin significantly increased AP-BL and decreased BL-AP sulfasalazine Caco-2 transport, in a concentration-dependent manner, with IC(50) values of 75 and 196 microM respectively. In the rat model, higher sulfasalazine concentrations resulted in higher intestinal permeability, consistent with saturation of efflux transporter. Without indomethacin, sulfasalazine demonstrated low rat jejunal permeability (vs. metoprolol). Indomethacin significantly increased sulfasalazine P(eff), effectively shifting it from BCS (biopharmaceutics classification system) Class IV to II. In conclusion, the data indicate that concomitant intake of indomethacin and sulfasalazine may lead to increased absorption of sulfasalazine in the small intestine, thereby reducing its colonic concentration and potentially altering its therapeutic effect. Copyright 2009 Elsevier B.V. All rights reserved.

Total hepatocellular disposition profiling of rosuvastatin and pitavastatin in sandwich-cultured human hepatocytes.

PubMed

Kanda, Katsuhiro; Takahashi, Ryosuke; Yoshikado, Takashi; Sugiyama, Yuichi

2018-04-09

This study describes the total disposition profiling of rosuvastatin (RSV) and pitavastatin (PTV) using a single systematic procedure called D-PREX (Disposition Profile Exploration) in sandwich-cultured human hepatocytes (SCHH). The biliary excretion fractions of both statins were clearly observed, which were significantly decreased dependent on the concentration of Ko143, an inhibitor for breast cancer resistance protein (BCRP). Ko143 also decreased the basolateral efflux fraction of RSV, whereas that of PTV was not significantly affected. To understand these phenomena, effects of Ko143 on biliary excretion (BCRP and multidrug resistance-associated protein (MRP) 2) and basolateral efflux (MRP3 and MRP4) transporters were examined using transporter-expressing membrane vesicles. BCRP, MRP3 and MRP4-mediated transport of RSV was observed, and Ko143 inhibited these transporters except MRP3. BCRP and MRP4 also mediated the transport of PTV, but the Ko143-mediated inhibition was only clear for BCRP. These results might explain the Ko143-mediated complete and partial inhibition of the biliary excretion and the basolateral efflux of RSV, respectively, in SCHH. In conclusion, D-PREX with sequential sampling of supernatants prior to cell lysis enables the evaluation of total drug disposition profiles resulting from complex interplays of intracellular pathways, which would provide high-throughput evaluation of drug disposition during drug discovery. Copyright © 2018 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

PPAR-α, a lipid-sensing transcription factor, regulates blood–brain barrier efflux transporter expression

PubMed Central

More, Vijay R; Campos, Christopher R; Evans, Rebecca A; Oliver, Keith D; Chan, Gary NY; Miller, David S

2016-01-01

Lipid sensor peroxisome proliferator-activated receptor alpha (PPAR-α) is the master regulator of lipid metabolism. Dietary release of endogenous free fatty acids, fibrates, and certain persistent environmental pollutants, e.g. perfluoroalkyl fire-fighting foam components, are peroxisome proliferator-activated receptor alpha ligands. Here, we define a role for peroxisome proliferator-activated receptor alpha in regulating the expression of three ATP-driven drug efflux transporters at the rat and mouse blood–brain barriers: P-glycoprotein (Abcb1), breast cancer resistance protein (Bcrp/Abcg2), and multidrug resistance-associated protein 2 (Mrp2/Abcc2). Exposing isolated rat brain capillaries to linoleic acid, clofibrate, or PKAs increased the transport activity and protein expression of the three ABC transporters. These effects were blocked by the PPAR-α antagonist, GW6471. Dosing rats with 20 mg/kg or 200 mg/kg of clofibrate decreased the brain accumulation of the P-glycoprotein substrate, verapamil, by 50% (in situ brain perfusion; effects blocked by GW6471) and increased P-glycoprotein expression and activity in capillaries ex vivo. Fasting C57Bl/6 wild-type mice for 24 h increased both serum lipids and brain capillary P-glycoprotein transport activity. Fasting did not alter P-glycoprotein activity in PPAR-α knockout mice. These results indicate that hyperlipidemia, lipid-lowering fibrates and exposure to certain fire-fighting foam components activate blood–brain barrier peroxisome proliferator-activated receptor alpha, increase drug efflux transporter expression and reduce drug delivery to the brain. PMID:27193034

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

Tacrolimus is a class II low-solubility high-permeability drug: the effect of P-glycoprotein efflux on regional permeability of tacrolimus in rats.

PubMed

Tamura, Shigeki; Ohike, Atsuo; Ibuki, Rinta; Amidon, Gordon L; Yamashita, Shinji

2002-03-01

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 ( approximately 1.4 x 10(-4) cm/s). The apparent permeability (P(app)) in the jejunum was unaffected by the presence of verapamil; however, the P(app) 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. Copyright 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association .

Multidrug resistance: prospects for clinical management.

PubMed

Mansouri, A; Henle, K J; Nagle, W A

1992-01-01

Clinical success in the treatment of tumors with chemotherapy has significantly improved over the past several years. However, treatment failures due to drug resistance of cancer cells has remained a major problem. The classical form of multiple drug resistance is perhaps also the most common type of drug resistance, and represents the overexpression of a transmembrane glycoprotein pump (P-170) that mediates the efflux of a spectrum of structurally and functionally unrelated drugs. Here, we discuss recent evidence that support the concept that the total phenomenon of multiple drug resistance (MDR) involves several other mechanisms in addition to that underlying "classical" MDR. These include the action of other energy-dependent membrane efflux pumps, elevated levels of GSH for drug conjugation and detoxification to facilitate export, enhanced DNA repair facility, gene amplification and oncogene activation. The combination of mechanisms used by any particular cell line is variable and suggests that many of these mechanisms are independent. Successful reversal of drug resistance appears to require the identification of relevant operative resistance mechanisms. An example is the competitive inhibition of P-170 with verapamil, quinine and tamoxifen. A broadly successful strategy for killing drug-resistant cancer cells, however, could be based on either selective energy depletion of cancer cells or the permeabilization of tumor cells with an effective bypass of efflux pumps, since many mechanisms of drug resistance entail the energy-dependent export of toxins. The latter approach may be achieved via membrane lipid modifications or the introduction of membrane pores by biological or physical (electroporation) means.

Spontaneous and evolutionary changes in the antibiotic resistance of Burkholderia cenocepacia observed by global gene expression analysis.

PubMed

Sass, Andrea; Marchbank, Angela; Tullis, Elizabeth; Lipuma, John J; Mahenthiralingam, Eshwar

2011-07-22

Burkholderia cenocepacia is a member of the Burkholderia cepacia complex group of bacteria that cause infections in individuals with cystic fibrosis. B. cenocepacia isolate J2315 has been genome sequenced and is representative of a virulent, epidemic CF strain (ET12). Its genome encodes multiple antimicrobial resistance pathways and it is not known which of these is important for intrinsic or spontaneous resistance. To map these pathways, transcriptomic analysis was performed on: (i) strain J2315 exposed to sub-inhibitory concentrations of antibiotics and the antibiotic potentiator chlorpromazine, and (ii) on spontaneous mutants derived from J2315 and with increased resistance to the antibiotics amikacin, meropenem and trimethoprim-sulfamethoxazole. Two pan-resistant ET12 outbreak isolates recovered two decades after J2315 were also compared to identify naturally evolved gene expression changes. Spontaneous resistance in B. cenocepacia involved more gene expression changes and different subsets of genes than those provoked by exposure to sub inhibitory concentrations of each antibiotic. The phenotype and altered gene expression in the resistant mutants was also stable irrespective of the presence of the priming antibiotic. Both known and novel genes involved in efflux, antibiotic degradation/modification, membrane function, regulation and unknown functions were mapped. A novel role for the phenylacetic acid (PA) degradation pathway genes was identified in relation to spontaneous resistance to meropenem and glucose was found to repress their expression. Subsequently, 20 mM glucose was found to produce greater that 2-fold reductions in the MIC of multiple antibiotics against B. cenocepacia J2315. Mutation of an RND multidrug efflux pump locus (BCAM0925-27) and squalene-hopene cyclase gene (BCAS0167), both upregulated after chlorpromazine exposure, confirmed their role in resistance. The recently isolated outbreak isolates had altered the expression of multiple genes which mirrored changes seen in the antibiotic resistant mutants, corroborating the strategy used to model resistance. Mutation of an ABC transporter gene (BCAS0081) upregulated in both outbreak strains, confirmed its role in B. cenocepacia resistance. Global mapping of the genetic pathways which mediate antibiotic resistance in B. cenocepacia has revealed that they are multifactorial, identified potential therapeutic targets and also demonstrated that putative catabolite repression of genes by glucose can improve antibiotic efficacy.

Phosphoenolpyruvate metabolism in Jerusalem artichoke mitochondria.

PubMed

de Bari, Lidia; Valenti, Daniela; Pizzuto, Roberto; Atlante, Anna; Passarella, Salvatore

2007-04-01

We report here initial studies on phosphoenolpyruvate metabolism in coupled mitochondria isolated from Jerusalem artichoke tubers. It was found that: (1) phosphoenolpyruvate can be metabolized by Jerusalem artichoke mitochondria by virtue of the presence of the mitochondrial pyruvate kinase, shown both immunologically and functionally, located in the inner mitochondrial compartments and distinct from the cytosolic pyruvate kinase as shown by the different pH and inhibition profiles. (2) Jerusalem artichoke mitochondria can take up externally added phosphoenolpyruvate in a proton compensated manner, in a carrier-mediated process which was investigated by measuring fluorimetrically the oxidation of intramitochondrial pyridine nucleotide which occurs as a result of phosphoenolpyruvate uptake and alternative oxidase activation. (3) The addition of phosphoenolpyruvate causes pyruvate and ATP production, as monitored via HPLC, with their efflux into the extramitochondrial phase investigated fluorimetrically. Such an efflux occurs via the putative phosphoenolpyruvate/pyruvate and phosphoenolpyruvate/ATP antiporters, which differ from each other and from the pyruvate and the adenine nucleotide carriers, in the light of the different sensitivity to non-penetrant compounds. These carriers were shown to regulate the rate of efflux of both pyruvate and ATP. The appearance of citrate and oxaloacetate outside mitochondria was also found as a result of phosphoenolpyruvate addition.

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.

Co-delivery of docetaxel and verapamil by reduction-sensitive PEG-PLGA-SS-DTX conjugate micelles to reverse the multi-drug resistance of breast cancer.

PubMed

Guo, Yuanyuan; He, Wenxiu; Yang, Shengfeng; Zhao, Dujuan; Li, Zhonghao; Luan, Yuxia

2017-03-01

The clinical usage of docetaxel (DTX) has been blocked in the clinic because of its poor solubility and tumour multi-drug resistance (MDR). The dominating mechanism of MDR is the over-expression of p-gp on tumour cells. Traditional nano-medicines, such as nanoparticles and micelles, have been used to physically entrap DTX to improve their solubility, while the drug loading content was very low and the tumour resistance was neglected. In this study, the synthesized reduction-sensitive mPEG-PLGA-SS-DTX conjugate was utilized to load the p-gp inhibitor veraparmil (VRP) to prepare DTX and VRP co-delivered mPEG-PLGA-SS-DTX/VRP (PP-SS-DTX/VRP) multi-functional micelles to reverse MDR and enhance the anti-tumour effect of DTX. The micelles had a high drug loading content and showed an obvious reduction-sensitive release property for both DTX and VRP. In addition, an in vitro anti-tumour assay revealed that the micelles markedly inhibited the efflux activity of p-gp and accelerated cell apoptosis, resulting in the improvement of anti-tumour activity and reversal of MDR. The PP-SS-DTX micelles markedly enhanced the in vivo circulation time and increased the drug accumulation in tumour tissues. Therefore, the PP-SS-DTX/VRP micelle is a desirable drug delivery system for multi-drug resistance therapy of DTX and is very promising for clinical usage. Copyright © 2016 Elsevier B.V. All rights reserved.

Multifunctional QD-based co-delivery of siRNA and doxorubicin to HeLa cells for reversal of multidrug resistance and real-time tracking.

PubMed

Li, Jin-Ming; Wang, Yuan-Yuan; Zhao, Mei-Xia; Tan, Cai-Ping; Li, Yi-Qun; Le, Xue-Yi; Ji, Liang-Nian; Mao, Zong-Wan

2012-03-01

Co-delivery of siRNA and chemotherapeutic agents has been developed to combat multidrug resistance in cancer therapy. Recently, we developed a series of quantum dots (QDs) functionalized by β-cyclodextrin (β-CD) coupled to amino acids, some of which can be used to facilitate the delivery of siRNA. In this study, two CdSe/ZnSe QDs modified with β-CD coupled to L-Arg or L-His were used to simultaneously deliver doxorubicin (Dox) and siRNA targeting the MDR1 gene to reverse the multidrug resistance of HeLa cells. In this co-delivery system, Dox was firstly encapsulated into the hydrophobic cavities of β-CD, resulting in bypass of P-glycoprotein (P-gp)-mediated drug efflux. After complex formation of the mdr1 siRNA with Dox-loaded QDs via electrostatic interaction, significant down-regulation of mdr1 mRNA levels and P-gp expression was achieved as shown by RT-PCR and Western blotting experiments, respectively. The number of apoptotic HeLa cells after treatment with the complexes substantially exceeded the number of apoptotic cells induced by free Dox only. The intrinsic fluorescence of the QDs provided an approach to track the system by laser confocal microscopy. These multifunctional QDs are promising vehicles for the co-delivery of nucleic acids and chemotherapeutics and for real-time tracking of treatment. Copyright © 2011 Elsevier Ltd. All rights reserved.

Drug resistance-associated changes in sphingolipids and ABC transporters occur in different regions of membrane domains.

PubMed

Hinrichs, John W J; Klappe, Karin; van Riezen, Manon; Kok, Jan W

2005-11-01

We have recently shown that two ATP binding cassette (ABC) transporters are enriched in Lubrol-resistant noncaveolar membrane domains in multidrug-resistant human cancer cells [Hinrichs, J. W. J., K. Klappe, I. Hummel, and J. W. Kok. 2004. ATP-binding cassette transporters are enriched in non-caveolar detergent-insoluble glycosphingolipid-enriched membrane domains (DIGs) in human multidrug-resistant cancer cells. J. Biol. Chem. 279: 5734-5738]. Here, we show that aminophospholipids are relatively enriched in Lubrol-resistant membrane domains compared with Triton X-100-resistant membrane domains, whereas sphingolipids are relatively enriched in the latter. Moreover, Lubrol-resistant membrane domains contain more protein and lipid mass. Based on these results, we postulate a model for detergent-insoluble glycosphingolipid-enriched membrane domains consisting of a Lubrol-insoluble/Triton X-100-insoluble region and a Lubrol-insoluble/Triton X-100-soluble region. The latter region contains most of the ABC transporters as well as lipids known to be necessary for their efflux activity. Compared with drug-sensitive cells, the detergent-insoluble glycosphingolipid-enriched membrane domains (DIGs) in drug-resistant cells differ specifically in sphingolipid content and not in protein, phospholipid, or cholesterol content. In drug-resistant cells, sphingolipids with specific fatty acids (especially C24:1) are enriched in these membrane domains. Together, these data show that multidrug resistance-associated changes in both sphingolipids and ABC transporters occur in DIGs, but in different regions of these domains.

The reconstituted P-glycoprotein multidrug transporter is a flippase for glucosylceramide and other simple glycosphingolipids.

PubMed

Eckford, Paul D W; Sharom, Frances J

2005-07-15

The Pgp (P-glycoprotein) multidrug transporter, which is linked to multidrug resistance in human cancers, functions as an efflux pump for non-polar drugs, powered by the hydrolysis of ATP at its nucleotide binding domains. The drug binding sites of Pgp appear to be located within the cytoplasmic leaflet of the membrane bilayer, suggesting that Pgp may function as a 'flippase' for hydrophobic compounds. Pgp has been shown to translocate fluorescent phospholipids, and it has been suggested that it may also interact with GlcCer (glucosylceramide). Here we use a dithionite fluorescence quenching technique to show that reconstituted Pgp can flip several NBD (nitrobenzo-2-oxa-1,3-diazole)-labelled simple glycosphingolipids, including NBD-GlcCer, from one leaflet of the bilayer to the other in an ATP-dependent, vanadate-sensitive fashion. The rate of NBD-GlcCer flipping was similar to that observed for NBD-labelled PC (phosphatidylcholine). NBD-GlcCer flipping was inhibited in a concentration-dependent, saturable fashion by various Pgp substrates and modulators, and inhibition correlated well with the Kd for binding to the protein. The addition of a second sugar to the headgroup of the glycolipid to form NBD-lactosylceramide drastically reduced the rate of flipping compared with NBD-PC, probably because of the increased size and polarity contributed by the additional sugar residue. We conclude that Pgp functions as a broad-specificity outwardly-directed flippase for simple glycosphingolipids and membrane phospholipids.

Non-p-glycoprotein-mediated multidrug resistance in detransformed rat cells selected for resistance to methylglyoxal bis(guanylhydrazone).

PubMed

Weber, J M; Sircar, S; Horvath, J; Dion, P

1989-11-01

Three independent variants (G2, G4, G5), resistant to methylglyoxal bis(guanylhydrazone), an anticancer drug, have been isolated by single step selection from an adenovirus-transformed rat brain cell line (1). These variants display selective cross-resistance to several natural product drugs of dissimilar structure and action. Multidrug resistance has recently been shown to be caused by overexpression of the membrane-associated p-glycoprotein, most often caused by amplification of the mdr gene. Several types of experiments were conducted to determine whether the observed drug resistance in our cell lines could be due to changes at the mdr locus. The following results were obtained: (a) the mdr locus was not amplified; (b) transcription of the mdr gene and p-glycoprotein synthesis were not increased; (c) multidrug resistance cell lines, which carry an amplified mdr locus, were not cross-resistant to methylglyoxal bis(guanylhydrazone); (d) verapamil did not reverse the resistance of G cells or mdr cells to methylglyoxal bis(guanylhydrazone), nor that of G cells to vincristine; and (e) methylglyoxal bis(guanylhydrazone) resistance was recessive and depended on a block to drug uptake, as opposed to mdr cells which are dominant and express increased drug efflux. The results obtained suggest that the drug resistance in the G2, G4, and G5 cells was atypical and may be due to a mechanism distinct from that mediated by the mdr locus.

LCP crystallization and X-ray diffraction analysis of VcmN, a MATE transporter from Vibrio cholerae

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

Kusakizako, Tsukasa; Tanaka, Yoshiki; Hipolito, Christopher J.

A V. cholerae MATE transporter was crystallized using the lipidic cubic phase (LCP) method. X-ray diffraction data sets were collected from single crystals obtained in a sandwich plate and a sitting-drop plate to resolutions of 2.5 and 2.2 Å, respectively. Multidrug and toxic compound extrusion (MATE) transporters, one of the multidrug exporter families, efflux xenobiotics towards the extracellular side of the membrane. Since MATE transporters expressed in bacterial pathogens contribute to multidrug resistance, they are important therapeutic targets. Here, a MATE-transporter homologue from Vibrio cholerae, VcmN, was overexpressed in Escherichia coli, purified and crystallized in lipidic cubic phase (LCP). X-raymore » diffraction data were collected to 2.5 Å resolution from a single crystal obtained in a sandwich plate. The crystal belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 52.3, b = 93.7, c = 100.2 Å. As a result of further LCP crystallization trials, crystals of larger size were obtained using sitting-drop plates. X-ray diffraction data were collected to 2.2 Å resolution from a single crystal obtained in a sitting-drop plate. The crystal belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 61.9, b = 91.8, c = 100.9 Å. The present work provides valuable insights into the atomic resolution structure determination of membrane transporters.« less

Involvement of polyphosphate kinase in virulence and stress tolerance of uropathogenic Proteus mirabilis.

PubMed

Peng, Liang; Jiang, Qiao; Pan, Jia-Yun; Deng, Cong; Yu, Jing-Yi; Wu, Xiao-Man; Huang, Sheng-He; Deng, Xiao-Yan

2016-04-01

Proteus mirabilis (P. mirabilis), a gram-negative enteric bacterium, frequently causes urinary tract infections. Many virulence factors of uropathogenic P. mirabilis have been identified, including urease, flagella, hemolysin and fimbriae. However, the functions of polyphosphate kinase (PPK), which are related to the pathogenicity of many bacteria, remain entirely unknown in P. mirabilis. In this study, a ppk gene encoding the PPK insertional mutant in P. mirabilis strain HI4320 was constructed, and its biological functions were examined. The results of survival studies demonstrated that the ppk mutant was deficient in resistance to oxidative, hyperosmotic and heat stress. The swarming and biofilm formation abilities of P. mirabilis were also attenuated after the ppk interruption. In vitro and in vivo experiments suggested that ppk was required for P. mirabilis to invade the bladder. The negative phenotypes of the ppk mutant could be restored by ppk gene complementation. Furthermore, two-dimensional gel electrophoresis and liquid chromatography-mass spectrometry were used to analyze the proteomes of the wild-type strain and the ppk mutant. Compared with the wild-type strain, seven proteins including TonB-dependent receptor, universal stress protein G, major mannose-resistant/Proteus-like fimbrial protein (MR/P fimbriae), heat shock protein, flagellar capping protein, putative membrane protein and multidrug efflux protein were down-regulated, and four proteins including exported peptidase, repressor protein for FtsI, FKBP-type peptidyl-prolyl cis-trans isomerase and phosphotransferase were up-regulated in the ppk mutant. As a whole, these results indicate that PPK is an important regulator and plays a crucial role in stress tolerance and virulence in uropathogenic P. mirabilis.

Mechanisms of Increased Resistance to Chlorhexidine and Cross-Resistance to Colistin following Exposure of Klebsiella pneumoniae Clinical Isolates to Chlorhexidine

PubMed Central

Bock, Lucy J.; Bonney, Laura C.

2016-01-01

ABSTRACT Klebsiella pneumoniae is an opportunistic pathogen that is often difficult to treat due to its multidrug resistance (MDR). We have previously shown that K. pneumoniae strains are able to “adapt” (become more resistant) to the widely used bisbiguanide antiseptic chlorhexidine. Here, we investigated the mechanisms responsible for and the phenotypic consequences of chlorhexidine adaptation, with particular reference to antibiotic cross-resistance. In five of six strains, adaptation to chlorhexidine also led to resistance to the last-resort antibiotic colistin. Here, we show that chlorhexidine adaptation is associated with mutations in the two-component regulator phoPQ and a putative Tet repressor gene (smvR) adjacent to the major facilitator superfamily (MFS) efflux pump gene, smvA. Upregulation of smvA (10- to 27-fold) was confirmed in smvR mutant strains, and this effect and the associated phenotype were suppressed when a wild-type copy of smvR was introduced on plasmid pACYC. Upregulation of phoPQ (5- to 15-fold) and phoPQ-regulated genes, pmrD (6- to 19-fold) and pmrK (18- to 64-fold), was confirmed in phoPQ mutant strains. In contrast, adaptation of K. pneumoniae to colistin did not result in increased chlorhexidine resistance despite the presence of mutations in phoQ and elevated phoPQ, pmrD, and pmrK transcript levels. Insertion of a plasmid containing phoPQ from chlorhexidine-adapted strains into wild-type K. pneumoniae resulted in elevated expression levels of phoPQ, pmrD, and pmrK and increased resistance to colistin, but not chlorhexidine. The potential risk of colistin resistance emerging in K. pneumoniae as a consequence of exposure to chlorhexidine has important clinical implications for infection prevention procedures. PMID:27799211

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

Deregulation of Genes Associated with Alternate Drug Resistance Mechanisms in Mycobacterium tuberculosis.

PubMed

Sriraman, Kalpana; Nilgiriwala, Kayzad; Saranath, Dhananjaya; Chatterjee, Anirvan; Mistry, Nerges

2018-04-01

Alternate mechanisms of drug resistance involving intrinsic defense pathways play an important role in development of drug resistance. Deregulation of drug efflux, cellular metabolism, and DNA repair have been indicated to have effect on drug tolerance and persistence. Here we chose eight genes from these pathways to investigate their association with development of multidrug resistance (MDR). We generated mono drug resistant and MDR strains of rifampicin and isoniazid and examined the differential expression of genes belonging to efflux, DNA repair and cell wall lipid synthesis pathways. Rv1687c, recB, ppsD and embC genes showed significant (P <0.05) upregulation in mono-resistant (both rifampicin and isoniazid) as well as MDR strains. mmr showed significant upregulation with rifampicin resistance while Rv1457c showed significant upregulation only with mono-resistant strains. Highest expression change was observed with Rv1687c and ppsD. The study identified potential key genes that are significantly associated with development of drug resistance in vitro. These genes may help identify clinical strains predisposed to acquiring drug resistance in patients during the course of treatment or help in management of MDR forms of tuberculosis.

Extensive sphingolipid depletion does not affect lipid raft integrity or lipid raft localization and efflux function of the ABC transporter MRP1.

PubMed

Klappe, Karin; Dijkhuis, Anne-Jan; Hummel, Ina; van Dam, Annie; Ivanova, Pavlina T; Milne, Stephen B; Myers, David S; Brown, H Alex; Permentier, Hjalmar; Kok, Jan W

2010-09-15

We show that highly efficient depletion of sphingolipids in two different cell lines does not abrogate the ability to isolate Lubrol-based DRMs (detergent-resistant membranes) or detergent-free lipid rafts from these cells. Compared with control, DRM/detergent-free lipid raft fractions contain equal amounts of protein, cholesterol and phospholipid, whereas the classical DRM/lipid raft markers Src, caveolin-1 and flotillin display the same gradient distribution. DRMs/detergent-free lipid rafts themselves are severely depleted of sphingolipids. The fatty acid profile of the remaining sphingolipids as well as that of the glycerophospholipids shows several differences compared with control, most prominently an increase in highly saturated C(16) species. The glycerophospholipid headgroup composition is unchanged in sphingolipid-depleted cells and cell-derived detergent-free lipid rafts. Sphingolipid depletion does not alter the localization of MRP1 (multidrug-resistance-related protein 1) in DRMs/detergent-free lipid rafts or MRP1-mediated efflux of carboxyfluorescein. We conclude that extensive sphingolipid depletion does not affect lipid raft integrity in two cell lines and does not affect the function of the lipid-raft-associated protein MRP1.

Extensive sphingolipid depletion does not affect lipid raft integrity or lipid raft localization and efflux function of the ABC transporter MRP1

PubMed Central

Klappe, Karin; Dijkhuis, Anne-Jan; Hummel, Ina; vanDam, Annie; Ivanova, Pavlina T.; Milne, Stephen B.; Myers, David S.; Brown, H. Alex; Permentier, Hjalmar; Kok, Jan W.

2013-01-01

We show that highly efficient depletion of sphingolipids in two different cell lines does not abrogate the ability to isolate Lubrol-based DRMs (detergent-resistant membranes) or detergent-free lipid rafts from these cells. Compared with control, DRM/detergent-free lipid raft fractions contain equal amounts of protein, cholesterol and phospholipid, whereas the classical DRM/lipid raft markers Src, caveolin-1 and flotillin display the same gradient distribution. DRMs/detergent-free lipid rafts themselves are severely depleted of sphingolipids. The fatty acid profile of the remaining sphingolipids as well as that of the glycerophospholipids shows several differences compared with control, most prominently an increase in highly saturated C16 species. The glycerophospholipid headgroup composition is unchanged in sphingolipid-depleted cells and cell-derived detergent-free lipid rafts. Sphingolipid depletion does not alter the localization of MRP1 (multidrug-resistance-related protein 1) in DRMs/detergent-free lipid rafts or MRP1-mediated efflux of carboxyfluorescein. We conclude that extensive sphingolipid depletion does not affect lipid raft integrity in two cell lines and does not affect the function of the lipid-raft-associated protein MRP1. PMID:20604746

Multidrug resistance transporters Snq2p and Pdr5p mediate caffeine efflux in Saccharomyces cerevisiae.

PubMed

Tsujimoto, Yoshiyuki; Shimizu, Yoshihiro; Otake, Kazuya; Nakamura, Tatsuya; Okada, Ryutaro; Miyazaki, Toshitaka; Watanabe, Kunihiko

2015-01-01

SNQ2 was identified as a caffeine-resistance gene by screening a genomic library of Saccharomyces cerevisiae in a multicopy vector YEp24. SNQ2 encodes an ATP-binding cassette transporter and is highly homologous to PDR5. Multicopy of PDR5 also conferred resistance to caffeine, while its resistance was smaller than that of SNQ2. Residual caffeine contents were analyzed after transiently exposing cells to caffeine. The ratios of caffeine contents were 21.3 ± 8.8% (YEp24-SNQ2) and 81.9 ± 8.7% (YEp24-PDR5) relative to control (YEp24, 100%). In addition, multicopies of SNQ2 or PDR5 conferred resistance to rhodamine 6G (R6G), which was widely used as a substrate for transport assay. R6G was exported by both transporters, and their efflux activities were inhibited by caffeine with half-maximal inhibitory concentrations of 5.3 ± 1.9 (YEp24-SNQ2) and 17.2 ± 9.6 mM (YEp24-PDR5). These results demonstrate that Snq2p is a more functional transporter of caffeine than Pdr5p in yeast cells.

Intertissue Flow of Glutathione (GSH) as a Tumor Growth-promoting Mechanism

PubMed Central

Obrador, Elena; Benlloch, María; Pellicer, José A.; Asensi, Miguel; Estrela, José M.

2011-01-01

B16 melanoma F10 (B16-F10) cells with high glutathione (GSH) content show high metastatic activity in vivo. An intertissue flow of GSH, where the liver is the main reservoir, can increase GSH content in metastatic cells and promote their growth. We have studied here possible tumor-derived molecular signals that could activate GSH release from hepatocytes. GSH efflux increases in hepatocytes isolated from mice bearing liver or lung metastases, thus suggesting a systemic mechanism. Fractionation of serum-free conditioned medium from cultured B16-F10 cells and monoclonal antibody-induced neutralization techniques facilitated identification of interleukin (IL)-6 as a tumor-derived molecule promoting GSH efflux in hepatocytes. IL-6 activates GSH release through a methionine-sensitive/organic anion transporter polypeptide 1- and multidrug resistance protein 1-independent channel located on the sinusoidal site of hepatocytes. Specific siRNAs were used to knock down key factors in the main signaling pathways activated by IL-6, which revealed a STAT3-dependent mechanism. Our results show that IL-6 (mainly of tumor origin in B16-F10-bearing mice) may facilitate GSH release from hepatocytes and its interorgan transport to metastatic growing foci. PMID:21393247

Phytochemical Prospection and Modulation of Antibiotic Activity In Vitro by Lippia origanoides H.B.K. in Methicillin Resistant Staphylococcus aureus

PubMed Central

Medeiros Barreto, Humberto; Cerqueira Fontinele, Filipe; Pereira de Oliveira, Aldeídia; Arcanjo, Daniel Dias Rufino; Cavalcanti dos Santos, Bernadete Helena; de Abreu, Aislan Pereira Lira; Douglas Melo Coutinho, Henrique; Alves Carvalho da Silva, Romezio; Oliveira de Sousa, Taciana; Freire de Medeiros, Maria das Graças; Lopes Citó, Antonia Maria das Graças; Dantas Lopes, José Arimateia

2014-01-01

The Lippia origanoides H.B.K. ethanol extract (LOEE) and hexane (LOHEX), dichloromethane (LODCM), and ethyl acetate (LOEA) fractions were tested for their antimicrobial activity alone or in combination with antibiotics against a methicillin resistant Staphylococcus aureus (MRSA) strain. The natural products did not show antimicrobial activity against multidrug resistant strain at the clinically significant concentrations tested. However, a modulatory effect in the antibacterial activity of the neomycin and amikacin was verified when LOEE, LOHEX and LODCM were added to the growth medium at subinhibitory concentrations. A similar modulation was found when the natural products were changed for chlorpromazine, an inhibitor of bacterial efflux pumps, suggesting the involvement of resistance mediated by efflux system in the MRSA tested. The fractions LOHEX and LODCM showed a modulatory activity bigger than their majority compounds (carvacrol, thymol, and naringenin), indicating that this activity is not due to their majority compounds only, but it is probably due to a synergism between their chemical components. These results indicate that L. origanoides H.B.K. can be a source of phytochemicals able to modify the phenotype of resistance to aminoglycosides in MRSA. PMID:24683545

Interaction of Food Additives with Intestinal Efflux Transporters.

PubMed

Sjöstedt, Noora; Deng, Feng; Rauvala, Oskari; Tepponen, Tuomas; Kidron, Heidi

2017-11-06

Breast cancer resistance protein (BCRP), multidrug resistance associated protein 2 (MRP2) and P-glycoprotein (P-gp) are ABC transporters that are expressed in the intestine, where they are involved in the efflux of many drugs from enterocytes back into the intestinal lumen. The inhibition of BCRP, MRP2, and P-gp can result in enhanced absorption and exposure of substrate drugs. Food additives are widely used by the food industry to improve the stability, flavor, and consistency of food products. Although they are considered safe for consumption, their interactions with intestinal transporters are poorly characterized. Therefore, in this study, selected food additives, including preservatives, colorants, and sweeteners, were studied in vitro for their inhibitory effects on intestinal ABC transporters. Among the studied compounds, several colorants were able to inhibit BCRP and MRP2, whereas P-gp was fairly insensitive to inhibition. Additionally, one sweetener was identified as a potent inhibitor of BCRP. Dose-response studies revealed that the IC 50 values of the inhibitors were lower than the estimated intestinal concentrations after the consumption of beverages containing food colorants. This suggests that there is potential for previously unrecognized transporter-mediated food additive-drug interactions.

Masitinib Antagonizes ATP-Binding Cassette Subfamily C Member 10-Mediated Paclitaxel Resistance: A Preclinical Study

PubMed Central

Kathawala, Rishil J.; Sodani, Kamlesh; Chen, Kang; Patel, Atish; Abuznait, Alaa H.; Anreddy, Nagaraju; Sun, Yue-Li; Kaddoumi, Amal; Ashby, Charles R.; Chen, Zhe-Sheng

2014-01-01

Paclitaxel displays clinical activity against a wide variety of solid tumors. However, resistance to paclitaxel significantly attenuates the response to chemotherapy. The ABC transporter subfamily C member 10 (ABCC10), also known as multi-drug resistance protein 7 (MRP7) efflux transporter, is a major mediator of paclitaxel resistance. In this study, we show that masitinib, a small molecule stem-cell growth factor receptor (c-Kit) tyrosine kinase inhibitor, at non-toxic concentrations, significantly attenuates paclitaxel resistance in HEK293 cells transfected with ABCC10. Our in vitro studies indicated that masitinib (2.5 μM) enhanced the intracellular accumulation and decreased the efflux of paclitaxel by inhibiting the ABCC10 transport activity without altering the expression level of ABCC10 protein. Furthermore, masitinib, in combination with paclitaxel, significantly inhibited the growth of ABCC10-expressing tumors in nude athymic mice in vivo. Masitinib administration also resulted in a significant increase in the levels of paclitaxel in the plasma, tumors and lungs compared to paclitaxel alone. In conclusion, the combination of paclitaxel and masitinib could serve as a novel and useful therapeutic strategy to reverse paclitaxel resistance mediated by ABCC10. PMID:24431074

ATP-Binding Cassette Efflux Transporters in Human Placenta

PubMed Central

Ni, Zhanglin; Mao, Qingcheng

2010-01-01

Pregnant women are often complicated with diseases including viral or bacterial infections, epilepsy, hypertension, or pregnancy-induced conditions such as depression and gestational diabetes that require treatment with medication. In addition, substance abuse during pregnancy remains a major public health problem. Many drugs used by pregnant women are off label without the necessary dose, efficacy, and safety data required for rational dosing regimens of these drugs. Thus, a major concern arising from the widespread use of drugs by pregnant women is the transfer of drugs across the placental barrier, leading to potential toxicity to the developing fetus. Knowledge regarding the ATP-binding cassette (ABC) efflux transporters, which play an important role in drug transfer across the placental barrier, is absolutely critical for optimizing the therapeutic strategy to treat the mother while protecting the fetus during pregnancy. Such transporters include P-glycoprotein (P-gp, gene symbol ABCB1), the breast cancer resistance protein (BCRP, gene symbol ABCG2), and the multidrug resistance proteins (MRPs, gene symbol ABCCs). In this review, we summarize the current knowledge with respect to developmental expression and regulation, membrane localization, functional significance, and genetic polymorphisms of these ABC transporters in the placenta and their relevance to fetal drug exposure and toxicity. PMID:21118087

Physiological and Molecular Responses to Excess Boron in Citrus macrophylla W.

PubMed

Martínez-Cuenca, Mary-Rus; Martínez-Alcántara, Belén; Quiñones, Ana; Ruiz, Marta; Iglesias, Domingo J; Primo-Millo, Eduardo; Forner-Giner, M Ángeles

2015-01-01

This work provides insight into several mechanisms involved in boron (B) regulation pathway in response to high B conditions in Citrus. The study was carried out in Citrus macrophylla W. (Cm) seedlings cultured "in vitro" in media with 50 or 400 μM H3BO3 (control, Ct, and B-excess, +B, plants, respectively). Growth parameters, B concentration, leaf chlorophyll (Chl) concentration, the expression of the main putative genes involved in B transport and distribution, and leaf and root proline and malonaldehyde (MDA) concentrations, were assessed. Excess B led to high B concentration in +B plants (3.8- and 1.4-fold in leaves and roots, respectively) when compared with Ct ones. However, a minor effect was recorded in the plant (incipient visual symptoms, less than 27% reduction in root growth and 26% decrease in Chl b concentration). B toxicity down-regulated by half the expression level of putative B transporter genes NIP5 and PIP1. CmBOR1 gene was not repressed in +B plants and B accumulated in the shoots. High B level increased the transcripts of putative gene TIP5, involved in B transport across the tonoplast, by 3.3- and 2.4-fold in leaves and roots, respectively. The activity of V-PPiase proton pump, related with the electrochemical gradient in the vacuole, was also enhanced in +B organs. B toxicity up-regulated putative BOR4 gene (2.1- and 2.7-fold in roots and leaves, respectively), which codifies for an active efflux B transporter. Accordingly, B was located in +B plants preferently in an insoluble form on cell walls. Finally, excess B caused a significant rise in proline concentration (51% and 34% in roots and leaves, respectively), while the MDA level did not exceed 20%. In conclusion, Cm tolerance to a high B level is likely based on the synergism of several specific mechanisms against B toxicity, including: 1/ down-regulation of NIP5 and PIP1 boron transporters; 2/ activation of B efflux from cells due to the up-regulation of putative BOR4 gene; 3/ compartmentation of B in the vacuole through TIP5 transporter activation and the acidification of the organelle; 4/ insolubilisation of B and deposition in cell walls preventing from cytoplasm damage; and, 5/ induction of an efficient antioxidant system through proline accumulation.

Physiological and Molecular Responses to Excess Boron in Citrus macrophylla W

PubMed Central

Martínez-Cuenca, Mary-Rus; Martínez-Alcántara, Belén; Quiñones, Ana; Ruiz, Marta; Iglesias, Domingo J.; Primo-Millo, Eduardo; Forner-Giner, M. Ángeles

2015-01-01

This work provides insight into several mechanisms involved in boron (B) regulation pathway in response to high B conditions in Citrus. The study was carried out in Citrus macrophylla W. (Cm) seedlings cultured “in vitro” in media with 50 or 400 μM H3BO3 (control, Ct, and B-excess, +B, plants, respectively). Growth parameters, B concentration, leaf chlorophyll (Chl) concentration, the expression of the main putative genes involved in B transport and distribution, and leaf and root proline and malonaldehyde (MDA) concentrations, were assessed. Excess B led to high B concentration in +B plants (3.8- and 1.4-fold in leaves and roots, respectively) when compared with Ct ones. However, a minor effect was recorded in the plant (incipient visual symptoms, less than 27% reduction in root growth and 26% decrease in Chl b concentration). B toxicity down-regulated by half the expression level of putative B transporter genes NIP5 and PIP1. CmBOR1 gene was not repressed in +B plants and B accumulated in the shoots. High B level increased the transcripts of putative gene TIP5, involved in B transport across the tonoplast, by 3.3- and 2.4-fold in leaves and roots, respectively. The activity of V-PPiase proton pump, related with the electrochemical gradient in the vacuole, was also enhanced in +B organs. B toxicity up-regulated putative BOR4 gene (2.1- and 2.7-fold in roots and leaves, respectively), which codifies for an active efflux B transporter. Accordingly, B was located in +B plants preferently in an insoluble form on cell walls. Finally, excess B caused a significant rise in proline concentration (51% and 34% in roots and leaves, respectively), while the MDA level did not exceed 20%. In conclusion, Cm tolerance to a high B level is likely based on the synergism of several specific mechanisms against B toxicity, including: 1/ down-regulation of NIP5 and PIP1 boron transporters; 2/ activation of B efflux from cells due to the up-regulation of putative BOR4 gene; 3/ compartmentation of B in the vacuole through TIP5 transporter activation and the acidification of the organelle; 4/ insolubilisation of B and deposition in cell walls preventing from cytoplasm damage; and, 5/ induction of an efficient antioxidant system through proline accumulation. PMID:26225859

In Vitro Activity of Delafloxacin against Clinical Neisseria gonorrhoeae Isolates and Selection of Gonococcal Delafloxacin Resistance.

PubMed

Soge, Olusegun O; Salipante, Stephen J; No, David; Duffy, Erin; Roberts, Marilyn C

2016-05-01

We evaluated the in vitro activity of delafloxacin against a panel of 117 Neisseria gonorrhoeae strains, including 110 clinical isolates collected from 2012 to 2015 and seven reference strains, compared with the activities of seven antimicrobials currently or previously recommended for treatment of gonorrhea. We examined the potential for delafloxacin to select for resistant mutants in ciprofloxacin-susceptible and ciprofloxacin-resistant N. gonorrhoeae We characterized mutations in the gyrA, gyrB, parC, and parE genes and the multidrug-resistant efflux pumps (MtrC-MtrD-MtrE and NorM) by PCR and sequencing and by whole-genome sequencing. The MIC50, MIC90, and MIC ranges of delafloxacin were 0.06 μg/ml, 0.125 μg/ml, and ≤0.001 to 0.25 μg/ml, respectively. The frequency of spontaneous mutation ranged from 10(-7) to <10(-9) The multistep delafloxacin resistance selection of 30 daily passages resulted in stable resistant mutants. There was no obvious cross-resistance to nonfluoroquinolone comparator antimicrobials. A mutant with reduced susceptibility to ciprofloxacin (MIC, 0.25 μg/ml) obtained from the ciprofloxacin-susceptible parental strain had a novel Ser91Tyr alteration in the gyrA gene. We also identified new mutations in the gyrA and/or parC and parE genes and the multidrug-resistant efflux pumps (MtrC-MtrD-MtrE and NorM) of two mutant strains with elevated delafloxacin MICs of 1 μg/ml. Although delafloxacin exhibited potent in vitro activity against N. gonorrhoeae isolates and reference strains with diverse antimicrobial resistance profiles and demonstrated a low tendency to select for spontaneous mutants, it is important to establish the correlation between these excellent in vitro data and treatment outcomes through appropriate randomized controlled clinical trials. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

In Vitro Activity of Delafloxacin against Clinical Neisseria gonorrhoeae Isolates and Selection of Gonococcal Delafloxacin Resistance

PubMed Central

Salipante, Stephen J.; No, David; Duffy, Erin; Roberts, Marilyn C.

2016-01-01

We evaluated the in vitro activity of delafloxacin against a panel of 117 Neisseria gonorrhoeae strains, including 110 clinical isolates collected from 2012 to 2015 and seven reference strains, compared with the activities of seven antimicrobials currently or previously recommended for treatment of gonorrhea. We examined the potential for delafloxacin to select for resistant mutants in ciprofloxacin-susceptible and ciprofloxacin-resistant N. gonorrhoeae. We characterized mutations in the gyrA, gyrB, parC, and parE genes and the multidrug-resistant efflux pumps (MtrC-MtrD-MtrE and NorM) by PCR and sequencing and by whole-genome sequencing. The MIC50, MIC90, and MIC ranges of delafloxacin were 0.06 μg/ml, 0.125 μg/ml, and ≤0.001 to 0.25 μg/ml, respectively. The frequency of spontaneous mutation ranged from 10−7 to <10−9. The multistep delafloxacin resistance selection of 30 daily passages resulted in stable resistant mutants. There was no obvious cross-resistance to nonfluoroquinolone comparator antimicrobials. A mutant with reduced susceptibility to ciprofloxacin (MIC, 0.25 μg/ml) obtained from the ciprofloxacin-susceptible parental strain had a novel Ser91Tyr alteration in the gyrA gene. We also identified new mutations in the gyrA and/or parC and parE genes and the multidrug-resistant efflux pumps (MtrC-MtrD-MtrE and NorM) of two mutant strains with elevated delafloxacin MICs of 1 μg/ml. Although delafloxacin exhibited potent in vitro activity against N. gonorrhoeae isolates and reference strains with diverse antimicrobial resistance profiles and demonstrated a low tendency to select for spontaneous mutants, it is important to establish the correlation between these excellent in vitro data and treatment outcomes through appropriate randomized controlled clinical trials. PMID:26976873

Inhibition of vincristine binding to plasma membrane vesicles from daunorubicin-resistant Ehrlich ascites cells by multidrug resistance modulators.

PubMed Central

Sehested, M.; Jensen, P. B.; Skovsgaard, T.; Bindslev, N.; Demant, E. J.; Friche, E.; Vindeløv, L.

1989-01-01

The multidrug resistance (MDR) phenotype is presumed to be mostly dependent on changes in the resistant cell plasma membrane, notably the emergence of a 170 kDa glycoprotein called P-glycoprotein, which facilitate increased drug efflux. We have previously demonstrated that ATP-enhanced binding of vincristine (VCR) to plasma membrane vesicles is much greater in MDR than in wild type cells. The present study has shown that VCR binding to MDR Ehrlich ascites tumour cell plasma membrane vesicles is inhibited 50% most efficiently by quinidine (0.5 microM) followed by verapamil (4.1 microM) and trifluoperazine (23.2 microM). This is the reverse order of the effect on whole cells where a ranking of efficiency in terms of enhancement of VCR accumulation, inhibition of VCR efflux, DNA perturbation and modulation of resistance in a clonogenic assay, was trifluoperazine greater than or equal to verapamil much greater than quinidine. The detergent Tween 80 inhibited VCR binding to plasma membrane vesicles at 0.001% v/v which agreed with the level which modulated resistance and increased VCR accumulation in whole cells. No effect was observed on daunorubicin binding to MDR plasma membrane vesicles after incubation with either Tween 80 (up to 0.1% v/v) or verapamil (up to 25 microM). We conclude that the effect of a modulating drug in reversing resistance to VCR correlates with its ability to raise intracellular VCR levels but not with its capability to inhibit VCR binding to the plasma membrane. Thus, enhancement of VCR accumulation in MDR cells is hardly solely due to competition for a drug binding site on P-glycoprotein. Furthermore, the lack of a demonstrable effect on daunorubicin binding to the plasma membrane by modulators points to transport mechanisms which do not utilise specific drug binding to the plasma membrane. PMID:2605092

AraC/XylS family stress response regulators Rob, SoxS, PliA, and OpiA in the fire blight pathogen Erwinia amylovora.

PubMed

Pletzer, Daniel; Schweizer, Gabriel; Weingart, Helge

2014-09-01

Transcriptional regulators of the AraC/XylS family have been associated with multidrug resistance, organic solvent tolerance, oxidative stress, and virulence in clinically relevant enterobacteria. In the present study, we identified four homologous AraC/XylS regulators, Rob, SoxS, PliA, and OpiA, from the fire blight pathogen Erwinia amylovora Ea1189. Previous studies have shown that the regulators MarA, Rob, and SoxS from Escherichia coli mediate multiple-antibiotic resistance, primarily by upregulating the AcrAB-TolC efflux system. However, none of the four AraC/XylS regulators from E. amylovora was able to induce a multidrug resistance phenotype in the plant pathogen. Overexpression of rob led to a 2-fold increased expression of the acrA gene. However, the rob-overexpressing strain showed increased resistance to only a limited number of antibiotics. Furthermore, Rob was able to induce tolerance to organic solvents in E. amylovora by mechanisms other than efflux. We demonstrated that SoxS from E. amylovora is involved in superoxide resistance. A soxS-deficient mutant of Ea1189 was not able to grow on agar plates supplemented with the superoxide-generating agent paraquat. Furthermore, expression of soxS was induced by redox cycling agents. We identified two novel members of the AraC/XylS family in E. amylovora. PliA was highly upregulated during the early infection phase in apple rootstock and immature pear fruits. Multiple compounds were able to induce the expression of pliA, including apple leaf extracts, phenolic compounds, redox cycling agents, heavy metals, and decanoate. OpiA was shown to play a role in the regulation of osmotic and alkaline pH stress responses. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Multidrug resistance protein (MRP) 4 attenuates benzo[a]pyrene-mediated DNA-adduct formation in human bronchoalveolar H358 cells.

PubMed

Gelhaus, Stacy L; Gilad, Oren; Hwang, Wei-Ting; Penning, Trevor M; Blair, Ian A

2012-02-25

Multi-drug resistance protein (MRP) 4, an ATP-binding cassette (ABC) transporter, has broad substrate specificity. It facilitates the transport of bile salt conjugates, conjugated steroids, nucleoside analogs, eicosanoids, and cardiovascular drugs. Recent studies in liver carcinoma cells and hepatocytes showed that MRP4 expression is regulated by the aryl hydrocarbon receptor (AhR) and nuclear factor E2-related factor 2 (Nrf2). The AhR has particular importance in the lung and is most commonly associated with the up-regulation of cytochrome P-450 (CYP)-mediated metabolism of benzo[a]pyrene (B[a]P) to reactive intermediates. Treatment of H358, human bronchoalveolar, cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or (-)-benzo[a]pyrene-7,8-dihydro-7,8-diol (B[a]P-7,8-dihydrodiol), the proximate carcinogen of B[a]P, revealed that MRP4 expression was increased compared to control. This suggested that MRP4 expression might contribute to the paradoxical decrease in (+)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-2'-deoxyguanosine ((+)-anti-trans-B[a]PDE-dGuo) DNA-adducts observed in TCDD-treated H358 cells. We have now found that decreased MRP4 expression induced by a short hairpin RNA (shRNA), or chemical inhibition with probenecid, increased (+)-anti-trans-B[a]PDE-dGuo formation in cells treated with (-)-B[a]P-7,8-dihydrodiol, but not the ultimate carcinogen (+)-anti-trans-B[a]PDE. Thus, up-regulation of MRP4 increased cellular efflux of (-)-B[a]P-7,8-dihydrodiol, which attenuated DNA-adduct formation. This is the first report identifying a specific MRP efflux transporter that decreases DNA damage arising from an environmental carcinogen. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Emergence of carbapenem non-susceptible multidrug resistant Acinetobacter baumannii strains of clonal complexes 103(B) and 92(B) harboring OXA-type carbapenemases and metallo-β-lactamases in Southern India.

PubMed

Saranathan, Rajagopalan; Vasanth, Vaidyanathan; Vasanth, Thamodharan; Shabareesh, Pidathala Raghavendra Venkata; Shashikala, P; Devi, Chandrakesan Sheela; Kalaivani, Ramakrishnan; Asir, Johny; Sudhakar, Pagal; Prashanth, K

2015-05-01

The molecular epidemiology and carbapenem resistance mechanisms of clinical isolates of Acinetobacter baumannii obtained from a south Indian tertiary care hospital were investigated by repetitive extragenic palindromic sequence PCR (REP-PCR) and multi-locus sequence typing (MLST). Analysis of resistant determinants was achieved by PCR screening for the presence of genes encoding OXA-carbapenemases, metallo-β-lactamases (MBLs) and efflux pumps. REP-PCR generated around eight clusters of high heterogeneity; of these, two major clusters (I and V) appeared to be clonal in origin. Analysis of representative isolates from different clusters by MLST revealed that most of the isolates belonged to sequence type 103 of CC103(B) . Second most prevalent ST belonged to clonal complex (CC) 92(B) which is also referred to as international clone II. Most of the isolates were multi-drug resistant, being susceptible only to polymyxin-B and newer quinolones. Class D β-lactamases such as blaOXA-51-like (100%), blaOXA-23-like (56.8%) and blaOXA-24-like (14.8%) were found to be predominant, followed by a class B β-lactamase, namely blaIMP-1 (40.7%); none of the isolates had blaOXA-58 like, blaNDM-1 or blaSIM-1 . Genes of efflux-pump adeABC were predominant, most of isolates being biofilm producers that were PCR-positive for autoinducer synthase gene (>94%). Carbapenem non-susceptible isolates were highly diverse and present throughout the hospital irrespective of type of ward or intensive care unit. Although previous reports have documented diverse resistant mechanisms in A. baumannii, production of MBL and OXA-type of carbapenamases were found to be the predominant mechanism(s) of carbapenem resistance identified in strains isolated from Southern India. © 2015 The Societies and Wiley Publishing Asia Pty Ltd.

Phenotypic and molecular antibiotic resistance profile of Enterococcus faecalis and Enterococcus faecium isolated from different traditional fermented foods.

PubMed

Sánchez Valenzuela, Antonio; Lavilla Lerma, Leyre; Benomar, Nabil; Gálvez, Antonio; Pérez Pulido, Rubén; Abriouel, Hikmate

2013-02-01

A collection of 55 enterococci (41 Enterococcus faecium and 14 E. faecalis strains) isolated from various traditional fermented foodstuffs of both animal and vegetable origins, and water was evaluated for resistance against 15 antibiotics. Lower incidence of resistance was observed with gentamicin, ampicillin, penicillin and teicoplanin. However, a high incidence of antibiotic resistance was detected for rifampicin (12 out of 14 of isolates), ciprofloxacin (9/14), and quinupristin/dalfopristin (8/14) in E. faecalis strains. Enterococcus faecium isolates were resistant to rifampicin (25/41), ciprofloxacin (23/41), erythromycin (18/41), levofloxacin (16/41), and nitrofurantoin (15/41). One Enterococcus faecalis and two E. faecium strains were resistant to vancomycin (MIC>16 μg/mL). Among 55 isolates, 27 (19 E. faecium and eight E. faecalis) were resistant to at least three antibiotics. High level of multidrug resistance to clinically important antibiotics was detected in E. faecalis strains (57% of E. faecalis versus 46% of E. faecium), which showed resistance to six to seven antibiotics, especially those isolated from foods of animal origin. So, it is necessary to re-evaluate the use of therapeutic antibiotics in stock farms at both regional and international levels due to the high number of multiple resistant (MR) bacteria. Fifty-six MR E. faecalis and E. faecium strains selected from this and previous studies (Valenzuela et al., 2008, 2010) were screened by polymerase chain reaction for antibiotic resistance genes, revealing the presence of tet(L), tet(M), ermB, cat, efrA, efrB, mphA, or msrA/B genes. The ABC Multidrug Efflux Pump EfrAB was detected in 96% of E. faecalis strains and also in 13% of E. faecium strains; this is the first report describing EfrAB in this enterococcal species. The efflux pump-associated msrA/B gene was detected in 66.66% of E. faecium strains, but not in E. faecalis strains.

Abundance of Drug Transporters in the Human Kidney Cortex as Quantified by Quantitative Targeted Proteomics

PubMed Central

Prasad, Bhagwat; Johnson, Katherine; Billington, Sarah; Lee, Caroline; Chung, Git W.; Brown, Colin D.A.; Kelly, Edward J.; Himmelfarb, Jonathan

2016-01-01

Protein expression of renal uptake and efflux transporters was quantified by quantitative targeted proteomics using the surrogate peptide approach. Renal uptake transporters assessed in this study included organic anion transporters (OAT1–OAT4), organic cation transporter 2 (OCT2), organic/carnitine cation transporters (OCTN1 and OCTN2), and sodium-glucose transporter 2 (SGLT2); efflux transporters included P-glycoprotein, breast cancer resistance protein, multidrug resistance proteins (MRP2 and MRP4), and multidrug and toxin extrusion proteins (MATE1 and MATE2-K). Total membrane was isolated from the cortex of human kidneys (N = 41). The isolated membranes were digested by trypsin and the digest was subjected to liquid chromatography–tandem mass spectrometry analysis. The mean expression of surrogate peptides was as follows (given with the standard deviation, in picomoles per milligram of total membrane protein): OAT1 (5.3 ± 1.9), OAT2 (0.9 ± 0.3), OAT3 (3.5 ± 1.6), OAT4 (0.5 ± 0.2), OCT2 (7.4 ± 2.8), OCTN1 (1.3 ± 0.6), OCTN2 (0.6 ± 0.2), P-glycoprotein (2.1 ± 0.8), MRP2 (1.4 ± 0.6), MRP4 (0.9 ± 0.6), MATE1 (5.1 ± 2.3), and SGLT2 (3.7 ± 1.8). Breast cancer resistance protein (BCRP) and MATE2-K proteins were detectable but were below the lower limit of quantification. Interestingly, the protein expression of OAT1 and OAT3 was significantly correlated (r > 0.8). A significant correlation was also observed between expression of multiple other drug transporters, such as OATs/OCT2 or OCTN1/OCTN2, and SGLT2/OCTNs, OCT, OATs, and MRP2. These renal transporter data should be useful in deriving in vitro to in vivo scaling factors to accurately predict renal clearance and kidney epithelial cell exposure to drugs or their metabolites. PMID:27621205

The naphthoquinones, vitamin K3 and its structural analogue plumbagin, are substrates of the multidrug resistance linked ATP binding cassette drug transporter ABCG2.

PubMed

Shukla, Suneet; Wu, Chung-Pu; Nandigama, Krishnamachary; Ambudkar, Suresh V

2007-12-01

Vitamin K3 (menadione; 2-methyl-1,4-naphthoquinone) is a structural precursor of vitamins K1 and K2, which are essential for blood clotting. The naturally occurring structural analogue of this vitamin, plumbagin (5-hydroxy-menadione), is known to modulate cellular proliferation, apoptosis, carcinogenesis, and radioresistance. We here report that both vitamin K3 and plumbagin are substrates of the multidrug resistance-linked ATP binding cassette drug transporter, ABCG2. Vitamin K3 and plumbagin specifically inhibited the ABCG2-mediated efflux of mitoxantrone but did not have any effect on the ABCB1-mediated efflux of rhodamine 123. This inhibition of ABCG2 function was due to their interaction at the substrate-binding site(s). Vitamin K3 and plumbagin inhibited the binding of [(125)I]iodoarylazidoprazosin, a substrate of ABCG2, to this transporter in a concentration-dependent manner with IC(50) values of 7.3 and 22.6 micromol/L, respectively, but had no effect on the binding of the photoaffinity analogue to ABCB1. Both compounds stimulated ABCG2-mediated ATP hydrolysis and also inhibited the mitoxantrone-stimulated ATPase activity of the ABCG2 transporter, but did not have any significant effect on the ATPase activity of ABCB1. In a cytotoxicity assay, ABCG2-expressing HEK cells were 2.8- and 2.3-fold resistant to plumbagin and vitamin K3, respectively, compared with the control cells, suggesting that they are substrates of this transporter. Collectively, these data show for the first time that vitamin K3 is a substrate of the ABCG2 transporter. Thus, ABCG2 may have a role in the regulation of vitamin K3 levels in the body. In addition, vitamin K3 and its structural derivative, plumbagin, could potentially be used to modulate ABCG2 function.

The naphthoquinones, vitamin K3 and its structural analog plumbagin, are substrates of the multidrug resistance-linked ABC drug transporter ABCG2

PubMed Central

Shukla, Suneet; Wu, Chung-Pu; Nandigama, Krishnamachary; Ambudkar, Suresh V.

2008-01-01

Vitamin K3 (Menadione; 2-methyl-1,4-naphthoquinone) is a structural precursor of vitamins K1 and K2 which are essential for blood clotting. The naturally occurring structural analog of this vitamin, plumbagin (5-hydroxy-menadione), is known to modulate cellular proliferation, apoptosis, carcinogenesis, and radioresistance. We, here, report that both vitamin K3 and plumbagin are substrates of the multidrug resistance-linked ATP binding cassette (ABC) drug transporter, ABCG2. Vitamin K3 and plumbagin specifically inhibited the ABCG2-mediated efflux of mitoxantrone, but did not have any effect on the ABCB1-mediated efflux of rhodamine 123. This inhibition of ABCG2 function was due to their interaction at the substrate-binding site(s). They inhibited the binding of [125I]-Iodoarylazidoprazosin (IAAP), a substrate of ABCG2, to this transporter in a concentration-dependent manner with IC50 values of 7.3 and 22.6 μM, respectively, but had no effect on the binding of this photoaffinity analog to ABCB1. Both compounds stimulated ABCG2-mediated ATP hydrolysis and also inhibited the mitoxantrone-stimulated ATPase activity of this transporter, but did not have any significant effect on the ATPase activity of ABCB1. In a cytotoxicity assay, ABCG2-expressing HEK cells were 2.8- and 2.3-fold resistant to plumbagin and vitamin K3, respectively, compared to the control cells, suggesting that they are substrates of this transporter. Collectively, these data demonstrate for the first time that vitamin K3 is a substrate of the ABCG2 transporter. Thus, ABCG2 may have a role in the regulation of vitamin K3 levels in the body. In addition, vitamin K3 and its structural derivative, plumbagin, could potentially be used to modulate ABCG2 function. PMID:18065489

Review and phylogenetic analysis of qac genes that reduce susceptibility to quaternary ammonium compounds in Staphylococcus species

PubMed Central

Ussery, David; Nielsen, Lene N.; Ingmer, Hanne

2015-01-01

The qac genes of Staphylococcus species encode multidrug efflux pumps: membrane proteins that export toxic molecules and thus increase tolerance to a variety of compounds such as disinfecting agents, including quaternary ammonium compounds (for which they are named), intercalating dyes and some antibiotics. In Stapylococcus species, six different plasmid-encoded Qac efflux pumps have been described, and they belong to two major protein families. QacA and QacB are members of the Major Facilitator Superfamily, while QacC, QacG, QacH, and QacJ all belong to the Small Multidrug Resistance (SMR) family. Not all SMR proteins are called Qac and the reverse is also true, which has caused confusion in the literature and in gene annotations. The discovery of qac genes and their presence in various staphylococcal populations is briefly reviewed. A sequence comparison revealed that some of the PCR primers described in the literature for qac detection may miss particular qac genes due to lack of DNA conservation. Despite their resemblance in substrate specificity, the Qac proteins belonging to the two protein families have little in common. QacA and QacB are highly conserved in Staphylococcus species, while qacA was also detected in Enterococcus faecalis, suggesting that these plasmid-born genes have spread across bacterial genera. Nevertheless, these qacA and qacB genes are quite dissimilar to their closest homologues in other organisms. In contrast, SMR-type Qac proteins display considerable sequence variation, despite their short length, even within the Staphylococcus genus. Phylogenetic analysis of these genes identified similarity to a large number of other SMR members, found in staphylococci as well as in other genera. A number of phylogenetic trees of SMR Qac proteins are presented here, starting with genes present in S. aureus and S. epidermidis, and extending this to related genes found in other species of this genus, and finally to genes found in other genera. PMID:25883793

Review and phylogenetic analysis of qac genes that reduce susceptibility to quaternary ammonium compounds in Staphylococcus species.

PubMed

Wassenaar, Trudy M; Ussery, David; Nielsen, Lene N; Ingmer, Hanne

2015-03-01

The qac genes of Staphylococcus species encode multidrug efflux pumps: membrane proteins that export toxic molecules and thus increase tolerance to a variety of compounds such as disinfecting agents, including quaternary ammonium compounds (for which they are named), intercalating dyes and some antibiotics. In Stapylococcus species, six different plasmid-encoded Qac efflux pumps have been described, and they belong to two major protein families. QacA and QacB are members of the Major Facilitator Superfamily, while QacC, QacG, QacH, and QacJ all belong to the Small Multidrug Resistance (SMR) family. Not all SMR proteins are called Qac and the reverse is also true, which has caused confusion in the literature and in gene annotations. The discovery of qac genes and their presence in various staphylococcal populations is briefly reviewed. A sequence comparison revealed that some of the PCR primers described in the literature for qac detection may miss particular qac genes due to lack of DNA conservation. Despite their resemblance in substrate specificity, the Qac proteins belonging to the two protein families have little in common. QacA and QacB are highly conserved in Staphylococcus species, while qacA was also detected in Enterococcus faecalis, suggesting that these plasmid-born genes have spread across bacterial genera. Nevertheless, these qacA and qacB genes are quite dissimilar to their closest homologues in other organisms. In contrast, SMR-type Qac proteins display considerable sequence variation, despite their short length, even within the Staphylococcus genus. Phylogenetic analysis of these genes identified similarity to a large number of other SMR members, found in staphylococci as well as in other genera. A number of phylogenetic trees of SMR Qac proteins are presented here, starting with genes present in S. aureus and S. epidermidis, and extending this to related genes found in other species of this genus, and finally to genes found in other genera.

The multidrug resistance-associated protein 1 transports methoxychlor and protects the seminiferous epithelium from injury.

PubMed

Tribull, Tiffany E; Bruner, Richard H; Bain, Lisa J

2003-04-30

We examined the ability of the multidrug resistance-associated protein 1 (MRP1/ABCC1) to transport pesticides, as this transporter mediates the cellular efflux of a variety of xenobiotics, typically as glucuronide, sulfate, or glutathione conjugates. NIH3T3 cells stably expressing MRP1 were 3.37-fold more resistant to the toxicity of fenitrothion, 3.12-fold more resistant to chlorpropham, and 2.5-fold more resistant to methoxychlor, a pesticide with estrogenic and anti-androgenic metabolites. The cells expressing MRP1 also eliminated methoxychlor two times more rapidly than their mock-transfected counterparts. We then examined whether mrp1 expression could alter the toxicity of methoxychlor in vivo using male FVB/mrp1 knockout mice (FVB/mrp1-/-). Both control and knockout mice were fed 25 mg/kg methoxychlor in honey for 39 days, and its effects on testicular morphology were examined. Methoxychlor treatment did not significantly affect testicular morphology in the FVB mice, but markedly reduced the number of developing spermatocytes in the FVB/mrp1-/- mice. These results suggest that MRPI may play a role in protecting the seminiferous tubules from methoxychlor-induced damage.

Novel function of N,N-bis(2-chloroethyl)docos-13-enamide for reversal of multidrug resistance in tongue cancer.

PubMed

Qin, Qing; Ma, Peng-Fei; Kuang, Xiao-Cong; Gao, Ming-Xing; Mo, De-Huan; Xia, Shuang; Jin, Ning; Xia, Jun-Jie; Qi, Zhong-Quan; Lin, Cui-Wu

2013-12-05

Multidrug resistance (MDR) is a key element in the failure of chemotherapies, and development of agents to overcome MDR is crucial to improving cancer treatments. The overexpression of glutathione-S-transferases (GSTs) is one of the major mechanisms of MDR. Because some agents used in traditional Chinese medicine have strong antitumor effects coupled with low toxicity; we investigated the ability of N,N-bis(2-chloroethyl)docos-13-enamide (compound J), the synthesized analog of a highly unsaturated fatty acid from Isatis tinctoria L., to reverse the MDR induced by adriamycin (ADM) in TCA8113/ADM cells. We found that compound J significantly increased the cytotoxicity of ADM in TCA8113/ADM cells, with a reversal fold of 2.461. Analysis of the mechanisms through which compound J reversed MDR indicated that compound J significantly decreased the activity of GSTs and enhanced the depletion of GSH in TCA8113/ADM cells, but did not affect the P-glycoprotein (P-gp) efflux. Taken together, our data suggested that compound J was an excellent candidate for reversing MDR in cancer therapy. © 2013 Published by Elsevier B.V.

Mutation-Induced Population Shift in the MexR Conformational Ensemble Disengages DNA Binding: A Novel Mechanism for MarR Family Derepression.

PubMed

Anandapadamanaban, Madhanagopal; Pilstål, Robert; Andresen, Cecilia; Trewhella, Jill; Moche, Martin; Wallner, Björn; Sunnerhagen, Maria

2016-08-02

MexR is a repressor of the MexAB-OprM multidrug efflux pump operon of Pseudomonas aeruginosa, where DNA-binding impairing mutations lead to multidrug resistance (MDR). Surprisingly, the crystal structure of an MDR-conferring MexR mutant R21W (2.19 Å) presented here is closely similar to wild-type MexR. However, our extended analysis, by molecular dynamics and small-angle X-ray scattering, reveals that the mutation stabilizes a ground state that is deficient of DNA binding and is shared by both mutant and wild-type MexR, whereas the DNA-binding state is only transiently reached by the more flexible wild-type MexR. This population shift in the conformational ensemble is effected by mutation-induced allosteric coupling of contact networks that are independent in the wild-type protein. We propose that the MexR-R21W mutant mimics derepression by small-molecule binding to MarR proteins, and that the described allosteric model based on population shifts may also apply to other MarR family members. Copyright © 2016 Elsevier Ltd. All rights reserved.

P-glycoprotein, but not Multidrug Resistance Protein 4, Plays a Role in the Systemic Clearance of Irinotecan and SN-38 in Mice

PubMed Central

Tagen, Michael; Zhuang, Yanli; Zhang, Fan; Harstead, K. Elaine; Shen, Jun; Schaiquevich, Paula; Fraga, Charles H.; Panetta, John C.; Waters, Christopher M.; Stewart, Clinton F.

2015-01-01

The ATP-binding cassette transporters P-glycoprotein (ABCB1, MDR1) and multidrug resistance protein 4 (MRP4) efflux irinotecan and its active metabolite SN-38 in vitro, and thus may contribute to system clearance of these compounds. Mdr1a/b−/−, Mrp4−/−, and wild-type mice were administered 20 or 40 mg/kg irinotecan, and plasma samples were collected for 6 hours. Irinotecan and SN-38 lactone and carboxylate were quantitated and data were analyzed with nonlinear mixed-effects modeling. Mdr1a/b genotype was a significant covariate for the clearance of both irinotecan lactone and SN-38 lactone. Exposures to irinotecan lactone and SN-38 lactone after a 40 mg/kg dose were 1.6-fold higher in Mdr1a/b−/− mice compared to wild-type mice. Plasma concentrations of irinotecan lactone, irinotecan carboxylate, and SN-38 lactone in Mrp4−/− mice were similar to the wild-type controls. These results suggest that P-gp plays a role in irinotecan and SN-38 elimination, but Mrp4 does not affect irinotecan or SN-38 plasma pharmacokinetics. PMID:20583968

Inhibition of the multidrug resistance P-glycoprotein: time for a change of strategy?

PubMed

Callaghan, Richard; Luk, Frederick; Bebawy, Mary

2014-04-01

P-glycoprotein (P-gp) is a key player in the multidrug-resistant phenotype in cancer. The protein confers resistance by mediating the ATP-dependent efflux of an astonishing array of anticancer drugs. Its broad specificity has been the subject of numerous attempts to inhibit the protein and restore the efficacy of anticancer drugs. The general strategy has been to develop compounds that either compete with anticancer drugs for transport or act as direct inhibitors of P-gp. Despite considerable in vitro success, there are no compounds currently available to "block" P-gp-mediated resistance in the clinic. The failure may be attributed to toxicity, adverse drug interaction, and numerous pharmacokinetic issues. This review provides a description of several alternative approaches to overcome the activity of P-gp in drug-resistant cells. These include 1) drugs that specifically target resistant cells, 2) novel nanotechnologies to provide high-dose, targeted delivery of anticancer drugs, 3) compounds that interfere with nongenomic transfer of resistance, and 4) approaches to reduce the expression of P-gp within tumors. Such approaches have been developed through the pursuit of greater understanding of resistance mediators such as P-gp, and they show considerable potential for further application.

Multidrug and toxin extrusion proteins as transporters of antimicrobial drugs.

PubMed

Nies, Anne T; Damme, Katja; Schaeffeler, Elke; Schwab, Matthias

2012-12-01

Antimicrobial drugs are essential in the treatment of infectious diseases. A better understanding of transport processes involved in drug disposition will improve the predictability of drug-drug interactions with consequences for drug response. Multidrug And Toxin Extrusion (MATE; SLC47A) proteins are efflux transporters mediating the excretion of several antimicrobial drugs as well as other organic compounds into bile and urine, thereby contributing to drug disposition. This review summarizes current knowledge of the structural and molecular features of human MATE transporters including their functional role in drug transport with a specific focus on antimicrobial drugs. The PubMed database was searched using the terms "MATE1," "MATE-2K," "MATE2," "SLC47A1," "SLC47A2," and "toxin extrusion protein" (up to June 2012). MATE proteins have been recognized as important transporters mediating the final excretion step of cationic drugs into bile and urine. These include the antiviral drugs acyclovir, amprenavir, and ganciclovir, the antibiotics cephalexin, cephradine and levofloxacin, as well as the antimalarial agents chloroquine and quinine. It is therefore important to enhance our understanding of the role of MATEs in drug extrusion with particular emphasis on the functional consequences of genetic variants on disposition of these antimicrobial drugs.

ABC transporters affect the elimination and toxicity of CdTe quantum dots in liver and kidney cells

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

Chen, Mingli; Yin, Huancai; Bai, Pengli

This paper aimed to investigate the role of adenosine triphosphate-binding cassette (ABC) transporters on the efflux and the toxicity of nanoparticles in liver and kidney cells. In this study, we synthesized CdTe quantum dots (QDs) that were monodispersed and emitted green fluorescence (maximum peak at 530 nm). Such QDs tended to accumulate in human hepatocellular carcinoma cells (HepG2), human kidney cells 2 (HK-2), and Madin-Darby canine kidney (MDCK) cells, and cause significant toxicity in all the three cell lines. Using specific inhibitors and inducers of P-glycoprotein (Pgp) and multidrug resistance associated proteins (Mrps), the cellular accumulation and subsequent toxicity ofmore » QDs in HepG2 and HK-2 cells were significantly affected, while only slight changes appeared in MDCK cells, corresponding well with the functional expressions of ABC transporters in cells. Moreover, treatment of QDs caused concentration- and time- dependent induction of ABC transporters in HepG2 and HK-2 cells, but such phenomenon was barely found in MDCK cells. Furthermore, the effects of CdTe QDs on ABC transporters were found to be greater than those of CdCl{sub 2} at equivalent concentrations of cadmium, indicating that the effects of QDs should be a combination of free Cd{sup 2+} and specific properties of QDs. Overall, these results indicated a strong dependence between the functional expressions of ABC transporters and the efflux of QDs, which could be an important reason for the modulation of QDs toxicity by ABC transporters. - Highlights: • ABC transporters contributed actively to the cellular efflux of CdTe quantum dots. • ABC transporters affected the cellular toxicity of CdTe quantum dots. • Treatment of CdTe quantum dots induced the gene expression of ABC transporters. • Free Cd{sup 2+} should be partially involved in the effects of QDs on ABC transporters. • Cellular efflux of quantum dots could be an important modulator for its toxicity.« less

Vaborbactam: Spectrum of Beta-Lactamase Inhibition and Impact of Resistance Mechanisms on Activity in Enterobacteriaceae

PubMed Central

Sun, Dongxu; Rubio-Aparicio, Debora; Nelson, Kirk; Tsivkovski, Ruslan; Griffith, David C.; Dudley, Michael N.

2017-01-01

ABSTRACT Vaborbactam (formerly RPX7009) is a new beta-lactamase inhibitor based on a cyclic boronic acid pharmacophore. The spectrum of beta-lactamase inhibition by vaborbactam and the impact of bacterial efflux and permeability on its activity were determined using a panel of strains with beta-lactamases cloned from various classes and a panel of Klebsiella pneumoniae carbapenemase 3 (KPC-3)-producing isogenic strains with various combinations of efflux and porin mutations. Vaborbactam is a potent inhibitor of class A carbapenemases, such as KPC, as well as an inhibitor of other class A (CTX-M, SHV, TEM) and class C (P99, MIR, FOX) beta-lactamases. Vaborbactam does not inhibit class D or class B carbapenemases. When combined with meropenem, vaborbactam had the highest potency compared to the potencies of vaborbactam in combination with other antibiotics against strains producing the KPC beta-lactamase. Consistent with broad-spectrum beta-lactamase inhibition, vaborbactam reduced the meropenem MICs for engineered isogenic strains of K. pneumoniae with increased meropenem MICs due to a combination of extended-spectrum beta-lactamase production, class C beta-lactamase production, and reduced permeability due to porin mutations. Vaborbactam crosses the outer membrane of K. pneumoniae using both OmpK35 and OmpK36, but OmpK36 is the preferred porin. Efflux by the multidrug resistance efflux pump AcrAB-TolC had a minimal impact on vaborbactam activity. Investigation of the vaborbactam concentration necessary for restoration of meropenem potency showed that vaborbactam at 8 μg/ml results in meropenem MICs of ≤2 μg/ml in the most resistant engineered strains containing multiple mutations. Vaborbactam is a highly active beta-lactamase inhibitor that restores the activity of meropenem and other beta-lactam antibiotics in beta-lactamase-producing bacteria, particularly KPC-producing carbapenem-resistant Enterobacteriaceae. PMID:28848018

Glycyrrhetinic Acid Accelerates the Clearance of Triptolide through P-gp In Vitro.

PubMed

Li, Zhihua; Yan, Miao; Cao, Lingjuan; Fang, Pingfei; Guo, Zhaohui; Hou, Zhenyan; Zhang, Bikui

2017-07-01

Triptolide (TP) is an active ingredient isolated from Tripterygium wilfordii Hook. f. (TWHF), which is a traditional herbal medicine widely used for the treatment of rheumatoid arthritis and autoimmune disease in the clinic. However, its adverse reactions of hepatotoxicity and nephrotoxicity have been frequently reported which limited its clinical application. The aim of this study was to investigate the mechanism of glycyrrhetinic acid (GA) effecting on the elimination of TP in HK-2 cells and the role of the efflux transporters of P-gp and multidrug resistance-associated proteins (MRPs) in this process. An ultra performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS) analytical method was established to determine the intracellular concentration of TP. In order to study the role of efflux transporters of P-gp and MRPs in GA impacting on the accumulation of TP, the inhibitors of efflux transporters (P-gp: verapamil; MRPs: MK571) were used in this study. The results showed that GA could enhance the elimination of TP and reduce the TP accumulation in HK-2 cells. Verapamil and MK571 could increase the intracellular concentration of TP; in addition, GA co-incubation with verapamil significantly increased the TP cellular concentration compared with the control group. In conclusion, GA could reduce the accumulation of TP in HK-2 cells, which was related to P-gp. This is probably one of the mechanisms that TP combined with GA to detoxify its toxicity. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

The analysis of the antibiotic resistome offers new opportunities for therapeutic intervention.

PubMed

Corona, Fernando; Blanco, Paula; Alcalde-Rico, Manuel; Hernando-Amado, Sara; Lira, Felipe; Bernardini, Alejandra; Sánchez, María B; Martínez, José L

2016-06-01

Most efforts in the development of antimicrobials have focused on the screening of lethal targets. Nevertheless, the constant expansion of antimicrobial resistance makes the antibiotic resistance determinants themselves suitable targets for finding inhibitors to be used in combination with antibiotics. Among them, inhibitors of antibiotic inactivating enzymes and of multidrug efflux pumps are suitable candidates for improving the efficacy of antibiotics. In addition, the application of systems biology tools is helping to understand the changes in bacterial physiology associated to the acquisition of resistance, including the increased susceptibility to other antibiotics displayed by some antibiotic-resistant mutants. This information is useful for implementing novel strategies based in metabolic interventions or combination of antibiotics for improving the efficacy of antibacterial therapy.

Repositioning of Tyrosine Kinase Inhibitors as Antagonists of ATP-Binding Cassette Transporters in Anticancer Drug Resistance

PubMed Central

Wang, Yi-Jun; Zhang, Yun-Kai; Kathawala, Rishil J.; Chen, Zhe-Sheng

2014-01-01

The phenomenon of multidrug resistance (MDR) has attenuated the efficacy of anticancer drugs and the possibility of successful cancer chemotherapy. ATP-binding cassette (ABC) transporters play an essential role in mediating MDR in cancer cells by increasing efflux of drugs from cancer cells, hence reducing the intracellular accumulation of chemotherapeutic drugs. Interestingly, small-molecule tyrosine kinase inhibitors (TKIs), such as AST1306, lapatinib, linsitinib, masitinib, motesanib, nilotinib, telatinib and WHI-P154, have been found to have the capability to overcome anticancer drug resistance by inhibiting ABC transporters in recent years. This review will focus on some of the latest and clinical developments with ABC transporters, TKIs and anticancer drug resistance. PMID:25268163

Characterisation of SalRAB a Salicylic Acid Inducible Positively Regulated Efflux System of Rhizobium leguminosarum bv viciae 3841

PubMed Central

Tett, Adrian J.; Karunakaran, Ramakrishnan; Poole, Philip S.

2014-01-01

Salicylic acid is an important signalling molecule in plant-microbe defence and symbiosis. We analysed the transcriptional responses of the nitrogen fixing plant symbiont, Rhizobium leguminosarum bv viciae 3841 to salicylic acid. Two MFS-type multicomponent efflux systems were induced in response to salicylic acid, rmrAB and the hitherto undescribed system salRAB. Based on sequence similarity salA and salB encode a membrane fusion and inner membrane protein respectively. salAB are positively regulated by the LysR regulator SalR. Disruption of salA significantly increased the sensitivity of the mutant to salicylic acid, while disruption of rmrA did not. A salA/rmrA double mutation did not have increased sensitivity relative to the salA mutant. Pea plants nodulated by salA or rmrA strains did not have altered nodule number or nitrogen fixation rates, consistent with weak expression of salA in the rhizosphere and in nodule bacteria. However, BLAST analysis revealed seventeen putative efflux systems in Rlv3841 and several of these were highly differentially expressed during rhizosphere colonisation, host infection and bacteroid differentiation. This suggests they have an integral role in symbiosis with host plants. PMID:25133394

Plasmid Profile Analysis and bla VIM Gene Detection of Metalo β-lactamase (MBL) Producing Pseudomonas aeruginosa Isolates from Clinical Samples

PubMed Central

M, Jeya

2014-01-01

Introduction:Pseudomonas aeruginosa is a frequent colonizer of hospitalized patients. They are responsible for serious infections such as meningitis, urological infections, septicemia and pneumonia. Carbapenem resistance of Pseudomonas aeruginosa is currently increasingly reported which is often mediated by production of metallo-β-lactamase (MBL). Multidrug resistant Pseudomonas aeruginosa isolates may involve reduced cell wall permeability, production of chromosomal and plasmid mediated β lactamases, aminoglycosides modifying enzymes and an active multidrug efflux mechanism. Objective: This study is aimed to detect the presence and the nature of plasmids among metallo-β-lactamase producing Pseudomonas aeruginosa isolates. Also to detect the presence of bla VIM gene from these isolates. Materials and Methods: Clinical isolates of Pseudomonas aeruginosa showing the metalo-β-lactamase enzyme (MBL) production were isolated. The MBL production was confirmed by three different methods. From the MBL producing isolates plasmid extraction was done by alkaline lysis method. Plasmid positive isolates were subjected for blaVIM gene detection by PCR method. Results: Two thousand seventy six clinical samples yielded 316 (15.22%) Pseudomonas aeruginosa isolates, out of which 141 (44.62%) were multidrug resistant. Among them 25 (17.73%) were metallo-β-lactamase enzyme producers. Plasmids were extracted from 18 out of 25 isolates tested. Five out of 18 isolates were positive for the blaVIM gene detection by the PCR amplification. Conclusion: The MBL producers were susceptible to polymyxin /colistin with MIC ranging from 0.5 – 2μg/ml. Molecular detection of specific genes bla VIM were positive among the carbapenem resistant isolates. PMID:25120980

Mesoporous silica nanoparticles loading doxorubicin reverse multidrug resistance: performance and mechanism

NASA Astrophysics Data System (ADS)

Shen, Jianan; He, Qianjun; Gao, Yu; Shi, Jianlin; Li, Yaping

2011-10-01

Multidrug resistance (MDR) is one of the major obstacles for successful chemotherapy in cancer. One of the effective approaches to overcome MDR is to use nanoparticle-mediated drug delivery to increase drug accumulation in drug resistant cancer cells. In this work, we first report that the performance and mechanism of an inorganic engineered delivery system based on mesoporous silica nanoparticles (MSNs) loading doxorubicin (DMNs) to overcome the MDR of MCF-7/ADR (a DOX-resistant and P-glycoprotein (P-gp) over-expression cancer cell line). The experimental results showed that DMNs could enhance the cellular uptake of doxorubicin (DOX) and increase the cell proliferation suppression effect of DOX against MCF-7/ADR cells. The IC50 of DMNs against MCF-7/ADR cells was 8-fold lower than that of free DOX. However, an improved effect of DOX in DMNs against MCF-7 cells (a DOX-sensitive cancer cell line) was not found. The increased cellular uptake and nuclear accumulation of DOX delivered by DMNs in MCF-7/ADR cells was confirmed by confocal laser scanning microscopy, and could result from the down-regulation of P-gp and bypassing the efflux action by MSNs themselves. The cellular uptake mechanism of DMNs indicated that the macropinocytosis was one of the pathways for the uptake of DMNs by MCF-7/ADR cells. The in vivo biodistribution showed that DMNs induced a higher accumulation of DOX in drug resistant tumors than free DOX. These results suggested that MSNs could be an effective delivery system to overcome multidrug resistance.

Plasmid Profile Analysis and bla VIM Gene Detection of Metalo β-lactamase (MBL) Producing Pseudomonas aeruginosa Isolates from Clinical Samples.

PubMed

S, Jayanthi; M, Jeya

2014-06-01

Pseudomonas aeruginosa is a frequent colonizer of hospitalized patients. They are responsible for serious infections such as meningitis, urological infections, septicemia and pneumonia. Carbapenem resistance of Pseudomonas aeruginosa is currently increasingly reported which is often mediated by production of metallo-β-lactamase (MBL). Multidrug resistant Pseudomonas aeruginosa isolates may involve reduced cell wall permeability, production of chromosomal and plasmid mediated β lactamases, aminoglycosides modifying enzymes and an active multidrug efflux mechanism. This study is aimed to detect the presence and the nature of plasmids among metallo-β-lactamase producing Pseudomonas aeruginosa isolates. Also to detect the presence of bla VIM gene from these isolates. Clinical isolates of Pseudomonas aeruginosa showing the metalo-β-lactamase enzyme (MBL) production were isolated. The MBL production was confirmed by three different methods. From the MBL producing isolates plasmid extraction was done by alkaline lysis method. Plasmid positive isolates were subjected for blaVIM gene detection by PCR method. Two thousand seventy six clinical samples yielded 316 (15.22%) Pseudomonas aeruginosa isolates, out of which 141 (44.62%) were multidrug resistant. Among them 25 (17.73%) were metallo-β-lactamase enzyme producers. Plasmids were extracted from 18 out of 25 isolates tested. Five out of 18 isolates were positive for the blaVIM gene detection by the PCR amplification. The MBL producers were susceptible to polymyxin /colistin with MIC ranging from 0.5 - 2μg/ml. Molecular detection of specific genes bla VIM were positive among the carbapenem resistant isolates.

Two members of the mouse mdr gene family confer multidrug resistance with overlapping but distinct drug specificities.

PubMed Central

Devault, A; Gros, P

1990-01-01

We report the cloning and functional analysis of a complete clone for the third member of the mouse mdr gene family, mdr3. Nucleotide and predicted amino acid sequence analyses showed that the three mouse mdr genes encode highly homologous membrane glycoproteins, which share the same length (1,276 residues), the same predicted functional domains, and overall structural arrangement. Regions of divergence among the three proteins are concentrated in discrete segments of the predicted polypeptides. Sequence comparison indicated that the three mouse mdr genes were created from a common ancestor by two independent gene duplication events, the most recent one producing mdr1 and mdr3. When transfected and overexpressed in otherwise drug-sensitive cells, the mdr3 gene, like mdr1 and unlike mdr2, conferred multidrug resistance to these cells. In independently derived transfected cell clones expressing similar amounts of either MDR1 or MDR3 protein, the drug resistance profile conferred by mdr3 was distinct from that conferred by mdr1. Cells transfected with and expressing MDR1 showed a marked 7- to 10-fold preferential resistance to colchicine and Adriamycin compared with cells expressing equivalent amounts of MDR3. Conversely, cells transfected with and expressing MDR3 showed a two- to threefold preferential resistance to actinomycin D over their cellular counterpart expressing MDR1. These results suggest that MDR1 and MDR3 are membrane-associated efflux pumps which, in multidrug-resistant cells and perhaps normal tissues, have overlapping but distinct substrate specificities. Images PMID:1969610

Comparative Genomics of Klebsiella pneumoniae Strains with Different Antibiotic Resistance Profiles▿†

PubMed Central

Kumar, Vinod; Sun, Peng; Vamathevan, Jessica; Li, Yong; Ingraham, Karen; Palmer, Leslie; Huang, Jianzhong; Brown, James R.

2011-01-01

There is a global emergence of multidrug-resistant (MDR) strains of Klebsiella pneumoniae, a Gram-negative enteric bacterium that causes nosocomial and urinary tract infections. While the epidemiology of K. pneumoniae strains and occurrences of specific antibiotic resistance genes, such as plasmid-borne extended-spectrum β-lactamases (ESBLs), have been extensively studied, only four complete genomes of K. pneumoniae are available. To better understand the multidrug resistance factors in K. pneumoniae, we determined by pyrosequencing the nearly complete genome DNA sequences of two strains with disparate antibiotic resistance profiles, broadly drug-susceptible strain JH1 and strain 1162281, which is resistant to multiple clinically used antibiotics, including extended-spectrum β-lactams, fluoroquinolones, aminoglycosides, trimethoprim, and sulfamethoxazoles. Comparative genomic analysis of JH1, 1162281, and other published K. pneumoniae genomes revealed a core set of 3,631 conserved orthologous proteins, which were used for reconstruction of whole-genome phylogenetic trees. The close evolutionary relationship between JH1 and 1162281 relative to other K. pneumoniae strains suggests that a large component of the genetic and phenotypic diversity of clinical isolates is due to horizontal gene transfer. Using curated lists of over 400 antibiotic resistance genes, we identified all of the elements that differentiated the antibiotic profile of MDR strain 1162281 from that of susceptible strain JH1, such as the presence of additional efflux pumps, ESBLs, and multiple mechanisms of fluoroquinolone resistance. Our study adds new and significant DNA sequence data on K. pneumoniae strains and demonstrates the value of whole-genome sequencing in characterizing multidrug resistance in clinical isolates. PMID:21746949

Small-molecule inhibitors of multidrug resistance-associated protein 1 and related processes: A historic approach and recent advances.

PubMed

Stefan, Sven Marcel; Wiese, Michael

2018-05-29

Multidrug resistance-associated protein 1 (MRP1, ABCC1) is an ATP-binding cassette (ABC) transport protein. This efflux pump uses the energy of ATP hydrolysis to export structurally diverse antineoplastic agents in human cancers. The upregulation of MRP1 (either inherent or acquired) is one major reason for the occurrence of the phenomenon called multidrug resistance (MDR). MDR is characterized by a reduced outcome of chemotherapy due to the active intracellular clearance of cytostatic drugs below the necessary effect concentration. Much effort has been made to overcome MDR, which implied high-throughput screenings of already known pharmacological and natural compounds, modification of intrinsic substrates, as well as design and synthesis of new inhibitors. This review is meant not only to summarize the most recent results over the past 10 years, but also to highlight major achievements regarding reversal of MRP1-mediated MDR, from the time of its discovery until today. The focus lies on small-molecule compounds that feature either direct MRP1 inhibition/transport blockage, toxicity against MRP1-overexpressing cells, inhibition/modification of intracellular processes necessary for MRP1 function, or modification of MRP1-related metabolic and genomic mechanisms. Considering all aspects, this review might be useful to (re)consider possible strategies to overcome MRP1-mediated MDR. Furthermore, it may be the basis for developing new, even better, highly potent, less toxic, and selective (as well as broad-spectrum) MRP1 inhibitors that will enter clinical evaluations in different malignancies and finally conduce to overcome MDR in general. © 2018 Wiley Periodicals, Inc.

Celastraceae sesquiterpenes as a new class of modulators that bind specifically to human P-glycoprotein and reverse cellular multidrug resistance.

PubMed

Muñoz-Martínez, Francisco; Lu, Peihua; Cortés-Selva, Fernando; Pérez-Victoria, José María; Jiménez, Ignacio A; Ravelo, Angel G; Sharom, Frances J; Gamarro, Francisco; Castanys, Santiago

2004-10-01

Overexpression of ABCB1 (MDR1) P-glycoprotein, a multidrug efflux pump, is one mechanism by which tumor cells may develop multidrug resistance (MDR), preventing the successful chemotherapeutic treatment of cancer. Sesquiterpenes from Celastraceae family are natural compounds shown previously to reverse MDR in several human cancer cell lines and Leishmania strains. However, their molecular mechanism of reversion has not been characterized. In the present work, we have studied the ability of 28 dihydro-beta-agarofuran sesquiterpenes to reverse the P-glycoprotein-dependent MDR phenotype and elucidated their molecular mechanism of action. Cytotoxicity assays using human MDR1-transfected NIH-3T3 cells allowed us to select the most potent sesquiterpenes reversing the in vitro resistance to daunomycin and vinblastine. Flow cytometry experiments showed that the above active compounds specifically inhibited drug transport activity of P-glycoprotein in a saturable, concentration-dependent manner (K(i) down to 0.24 +/- 0.01 micromol/L) but not that of ABCC1 (multidrug resistance protein 1; MRP1), ABCC2 (MRP2), and ABCG2 (breast cancer resistance protein; BCRP) transporters. Moreover, sesquiterpenes inhibited at submicromolar concentrations the P-glycoprotein-mediated transport of [(3)H]colchicine and tetramethylrosamine in plasma membrane from CH(R)B30 cells and P-glycoprotein-enriched proteoliposomes, supporting that P-glycoprotein is their molecular target. Photoaffinity labeling in plasma membrane and fluorescence spectroscopy experiments with purified protein suggested that sesquiterpenes interact with transmembrane domains of P-glycoprotein. Finally, sesquiterpenes modulated P-glycoprotein ATPase-activity in a biphasic, concentration-dependent manner: they stimulated at very low concentrations but inhibited ATPase activity as noncompetitive inhibitors at higher concentrations. Sesquiterpenes from Celastraceae are promising P-glycoprotein modulators with potential applications in cancer chemotherapy because of their MDR reversal potency and specificity for P-glycoprotein.

Reversal effect and mechanism of Ginkgo biloba exocarp extracts in multidrug resistance of mice S180 tumor cells

PubMed Central

Hu, Bi-Yuan; Gu, Yun-Hao; Cao, Chen-Jie; Wang, Jun; Han, Dong-Dong; Tang, Ying-Chao; Chen, Hua-Sheng; Xu, Aihua

2016-01-01

The aim of the present study was to investigate the reversal effect and its related mechanism of Ginkgo biloba exocarp extracts (GBEEs) in obtained multidrug resistance (MDR) of mice S180 tumor cells in vitro and in vivo. In order to simulate the clinical PFC [cis-dichlorodiamineplatinum, cisplatin (DDP) + fluorouracil (FU), FU+cyclophosphamide and cyclophosphamide] scheme, a gradually increasing dose was administered in a phased induction in order to induce S180 cells in vivo and to make them obtain multidrug resistance. The results in vitro demonstrated that GBEE could significantly increase the IC50 of DDP on S180 MDR cells, increase the accumulation of Adriamycin (ADR) and rhodamine 123 (Rho 123), and reduce the efflux of Rho 123 of S180 MDR cells. The results from the in vivo treatment with a combination of GBEE and DDP to S180 MDR ascites tumor in mice demonstrated that each dose of GBEE could effectively reverse the drug-resistance of S180 MDR cells to DDP in order to extend the survival time of mice with ascite tumors and inhibit tumor growth in solid tumor mice. In addition, GBEE effectively inhibited the expression of MDR-1 mRNA and multidrug resistance-associated protein-1 mRNA in S180 MDR cells of ascites tumor in mice and improved the expression levels of cytokines, including interleukin (IL)-3, IL-18 and interferon-γ in the blood serum of S180 MDR tumor-bearing mice. The present study showed that the mechanism of GBEE reversal of MDR may be associated with the inhibition of the functional activity of P-glycoprotein, the downregulation of drug resistance related gene expression of S180 MDR cells and the improvement of the production of related serum cytokines of S180 MDR tumor mice. PMID:27698692

Jatrophane diterpenoids from Euphorbia sororia as potent modulators against P-glycoprotein-based multidrug resistance.

PubMed

Hu, Rui; Gao, Jie; Rozimamat, Rushangul; Aisa, Haji Akber

2018-02-25

Five new (1-5) and ten known (6-15) jatrophane diterpenoids were isolated from the fructus of Euphorbia sororia and their structures were elucidated by extensive spectroscopic analysis. The absolute configurations of compounds 1 and 4 were confirmed by X-ray crystallographic analysis. Cytotoxicity and anti-multidrug resistance effects of these jatrophane diterpenoids were evaluated in multidrug-resistant MCF-7/ADR breast cancer cells with an overexpression of P-glycoprotein (P-gp). Eight compounds (1, 2, 4, 6, 8, 10, 11, and 15) showed promising chemoreversal abilities compared to verapamil (VRP). The most potent compound, Euphosorophane A (1), possessed many advantages, including (1) high potency (EC 50  = 92.68 ± 18.28 nM) in reversing P-gp-mediated resistance to doxorubicin (DOX), low cytotoxicity, and a high therapeutic index, (2) potency in reversing resistance to other cytotoxic agents associated with MDR, and (3) inhibition of P-gp-mediated Rhodamine123 (Rh123) efflux function in MCF-7/ADR cells. The results of the Western blot analysis indicated that the multidrug resistance (MDR) reversal induced by 1 was not due to the inhibiton of P-gp expression. Compound 1 stimulated P-gp-ATPase activity and caused the dose-dependent inhibition of DOX transport activity. Lineweaver-Burk and Dixon plots implied that 1 was a competitive inhibitor to DOX in the binding site of P-gp with a Ki of 0.49-0.50 μM. Our data suggested that 1 had a high binding affinity toward the DOX recognition site of P-gp. This resulted in inhibiting DOX transport, increasing intracellular DOX concentration, and finally resensitizing MCF-7/ADR to DOX. In addition, we discussed some added contents in the structure-activity relationship (SAR) of jatrophane diterpenoids. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Whole-Genome Survey of the Putative ATP-Binding Cassette Transporter Family Genes in Vitis vinifera

PubMed Central

Çakır, Birsen; Kılıçkaya, Ozan

2013-01-01

The ATP-binding cassette (ABC) protein superfamily constitutes one of the largest protein families known in plants. In this report, we performed a complete inventory of ABC protein genes in Vitis vinifera, the whole genome of which has been sequenced. By comparison with ABC protein members of Arabidopsis thaliana, we identified 135 putative ABC proteins with 1 or 2 NBDs in V. vinifera. Of these, 120 encode intrinsic membrane proteins, and 15 encode proteins missing TMDs. V. vinifera ABC proteins can be divided into 13 subfamilies with 79 “full-size,” 41 “half-size,” and 15 “soluble” putative ABC proteins. The main feature of the Vitis ABC superfamily is the presence of 2 large subfamilies, ABCG (pleiotropic drug resistance and white-brown complex homolog) and ABCC (multidrug resistance-associated protein). We identified orthologs of V. vinifera putative ABC transporters in different species. This work represents the first complete inventory of ABC transporters in V. vinifera. The identification of Vitis ABC transporters and their comparative analysis with the Arabidopsis counterparts revealed a strong conservation between the 2 species. This inventory could help elucidate the biological and physiological functions of these transporters in V. vinifera. PMID:24244377

The Essential Oil from Origanum vulgare L. and Its Individual Constituents Carvacrol and Thymol Enhance the Effect of Tetracycline against Staphylococcus aureus.

PubMed

Cirino, Isis Caroline S; Menezes-Silva, Suellen Maria P; Silva, Helena Tainá D; de Souza, Evandro Leite; Siqueira-Júnior, José P

2014-01-01

In an ongoing project to evaluate essential oils as modulators of antibiotic resistance, the essential oil from Origanum vulgare L. (OVEO), as well as its individual constituents carvacrol (CAR) and thymol (THY), were investigated using Staphylococcus aureus strains possessing efflux mechanisms of resistance to norfloxacin, erythromycin and tetracycline. The minimum inhibitory concentration (MIC) values of the antibiotics were determined by agar dilution method, in the absence and in the presence of subinhibitory concentrations of OVEO, CAR or THY. Along with relevant antistaphylococcal activity, OVEO, CAR and THY modulated the activity of tetracycline, i.e. in combination with antibiotics a reduction in the MIC was observed (up to fourfold). The results presented here represent, as far as we know, the first report of OVEO, CAR and THY as putative efflux pump inhibitors. Broadly, these findings indicate that essential oils could serve as potential sources of compounds capable of modulating drug resistance. © 2015 S. Karger AG, Basel.

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.

A Different Microbiome Gene Repertoire in the Airways of Cystic Fibrosis Patients with Severe Lung Disease

PubMed Central

Bacci, Giovanni; Fiscarelli, Ersilia; Taccetti, Giovanni; Dolce, Daniela; Paganin, Patrizia; Morelli, Patrizia; Tuccio, Vanessa; De Alessandri, Alessandra; Lucidi, Vincenzina

2017-01-01

In recent years, next-generation sequencing (NGS) was employed to decipher the structure and composition of the microbiota of the airways in cystic fibrosis (CF) patients. However, little is still known about the overall gene functions harbored by the resident microbial populations and which specific genes are associated with various stages of CF lung disease. In the present study, we aimed to identify the microbial gene repertoire of CF microbiota in twelve patients with severe and normal/mild lung disease by performing sputum shotgun metagenome sequencing. The abundance of metabolic pathways encoded by microbes inhabiting CF airways was reconstructed from the metagenome. We identified a set of metabolic pathways differently distributed in patients with different pulmonary function; namely, pathways related to bacterial chemotaxis and flagellar assembly, as well as genes encoding efflux-mediated antibiotic resistance mechanisms and virulence-related genes. The results indicated that the microbiome of CF patients with low pulmonary function is enriched in virulence-related genes and in genes encoding efflux-mediated antibiotic resistance mechanisms. Overall, the microbiome of severely affected adults with CF seems to encode different mechanisms for the facilitation of microbial colonization and persistence in the lung, consistent with the characteristics of multidrug-resistant microbial communities that are commonly observed in patients with severe lung disease. PMID:28758937

Hepatic Disposition of Gemfibrozil and Its Major Metabolite Gemfibrozil 1-O-β-Glucuronide.

PubMed

Kimoto, Emi; Li, Rui; Scialis, Renato J; Lai, Yurong; Varma, Manthena V S

2015-11-02

Gemfibrozil (GEM), which decreases serum triglycerides and low density lipoprotein, perpetrates drug-drug interactions (DDIs) with several drugs. These DDIs are primarily attributed to the inhibition of drug transporters and metabolic enzymes, particularly cytochrome P450 (CYP) 2C8 by the major circulating metabolite gemfibrozil 1-O-β-glucuronide (GG). Here, we characterized the transporter-mediated hepatic disposition of GEM and GG using sandwich-cultured human hepatocytes (SCHH) and transporter-transfect systems. Significant active uptake was noted in SCHH for the metabolite. GG, but not GEM, showed substrate affinity to organic anion transporting polypeptide (OATP) 1B1, 1B3, and 2B1. In SCHH, glucuronidation was characterized affinity constants (Km) of 7.9 and 61.4 μM, and biliary excretion of GG was observed. Furthermore, GG showed active basolateral efflux from preloaded SCHH and ATP-dependent uptake into membrane vesicles overexpressing multidrug resistance-associated protein (MRP) 2, MRP3, and MRP4. A mathematical model was developed to estimate hepatic uptake and efflux kinetics of GEM and GG based on SCHH studies. Collectively, the hepatic transporters play a key role in the disposition and thus determine the local concentrations of GEM and more so for GG, which is the predominant inhibitory species against CYP2C8 and OATP1B1.

Antibiotic resistance in Staphylococcus aureus. Current status and future prospects.

PubMed

Foster, Timothy J

2017-05-01

The major targets for antibiotics in staphylococci are (i) the cell envelope, (ii) the ribosome and (iii) nucleic acids. Several novel targets emerged from recent targeted drug discovery programmes including the ClpP protease and FtsZ from the cell division machinery. Resistance can either develop by horizontal transfer of resistance determinants encoded by mobile genetic elements viz plasmids, transposons and the staphylococcal cassette chromosome or by mutations in chromosomal genes. Horizontally acquired resistance can occur by one of the following mechanisms: (i) enzymatic drug modification and inactivation, (ii) enzymatic modification of the drug binding site, (iii) drug efflux, (iv) bypass mechanisms involving acquisition of a novel drug-resistant target, (v) displacement of the drug to protect the target. Acquisition of resistance by mutation can result from (i) alteration of the drug target that prevents the inhibitor from binding, (ii) derepression of chromosomally encoded multidrug resistance efflux pumps and (iii) multiple stepwise mutations that alter the structure and composition of the cell wall and/or membrane to reduce drug access to its target. This review focuses on development of resistance to currently used antibiotics and examines future prospects for new antibiotics and informed use of drug combinations. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Competitive Growth Enhances Conditional Growth Mutant Sensitivity to Antibiotics and Exposes a Two-Component System as an Emerging Antibacterial Target in Burkholderia cenocepacia.

PubMed

Gislason, April S; Choy, Matthew; Bloodworth, Ruhi A M; Qu, Wubin; Stietz, Maria S; Li, Xuan; Zhang, Chenggang; Cardona, Silvia T

2017-01-01

Chemogenetic approaches to profile an antibiotic mode of action are based on detecting differential sensitivities of engineered bacterial strains in which the antibacterial target (usually encoded by an essential gene) or an associated process is regulated. We previously developed an essential-gene knockdown mutant library in the multidrug-resistant Burkholderia cenocepacia by transposon delivery of a rhamnose-inducible promoter. In this work, we used Illumina sequencing of multiplex-PCR-amplified transposon junctions to track individual mutants during pooled growth in the presence of antibiotics. We found that competition from nontarget mutants magnified the hypersensitivity of a clone underexpressing gyrB to novobiocin by 8-fold compared with hypersensitivity measured during clonal growth. Additional profiling of various antibiotics against a pilot library representing most categories of essential genes revealed a two-component system with unknown function, which, upon depletion of the response regulator, sensitized B. cenocepacia to novobiocin, ciprofloxacin, tetracycline, chloramphenicol, kanamycin, meropenem, and carbonyl cyanide 3-chlorophenylhydrazone, but not to colistin, hydrogen peroxide, and dimethyl sulfoxide. We named the gene cluster esaSR for enhanced sensitivity to antibiotics sensor and response regulator. Mutational analysis and efflux activity assays revealed that while esaS is not essential and is involved in antibiotic-induced efflux, esaR is an essential gene and regulates efflux independently of antibiotic-mediated induction. Furthermore, microscopic analysis of cells stained with propidium iodide provided evidence that depletion of EsaR has a profound effect on the integrity of cell membranes. In summary, we unraveled a previously uncharacterized two-component system that can be targeted to reduce antibiotic resistance in B. cenocepacia. Copyright © 2016 American Society for Microbiology.

Emodin As an Effective Agent in Targeting Cancer Stem-Like Side Population Cells of Gallbladder Carcinoma

PubMed Central

Li, Xin-xing; Dong, Ying; Wang, Wei; Wang, Hao-lu; Chen, Yu-ying; Shi, Gui-ying; Yi, Jing

2013-01-01

Side population (SP) cells are previously identified from bone marrow based on their capacity to efflux of the fluorescent dye Hoechst 33342. Recent studies demonstrate that SP cells isolated from various cancer cell lines and primary tumors possess stem-cell-like properties. Thus, targeting tumor SP cells may provide new strategies for treatment in clinic. We previously showed that 1,3,8-trihydroxy-6-methylanthraquinone (emodin), a reactive oxygen species (ROS) generator, enhanced sensitivity of gallbladder cancer SGC-996 cells to cisplatin (CDDP) via generation of ROS and downregulation of multidrug-resistance-associated protein 1 (MRP1). To determine whether emodin also acts effectively on cancer stem cells of gallbladder carcinoma, we use SP cells as a model of cancer stem-cell-like cells. Here, we found that emodin, via ROS-related mechanism and suppressing the function of ATP-binding cassette super-family G member (ABCG2), which is known to be associated with Hoechst dye efflux activity of SP cells, not only reduced the ratio, inhibited clone formation, and eliminated sphere formation of SP cells effectively, but also promoted obviously the intracellular accumulation of doxorubicin, the main substrate of the efflux pump ABCG2. In addition, emodin could sensitize CDDP, via inhibition of expression of ABCG2, to overcome chemoresistance of SP cells. Importantly, similar to the experiment in vitro, emodin/CDDP co-treatment in vivo suppressed the tumor growth derived from SP cells through downregulating ABCG2 expression. Our results suggest that emodin is an effective agent targeting cancer stem-like SP cells of gallbladder carcinoma, either alone or acts as a chemotherapy enhancer. PMID:22974371

Bardoxolone methyl modulates efflux transporter and detoxifying enzyme expression in cisplatin-induced kidney cell injury.

PubMed

Atilano-Roque, Amandla; Aleksunes, Lauren M; Joy, Melanie S

2016-09-30

Cisplatin is prescribed for the treatment of solid tumors and elicits toxicity to kidney tubules, which limits its clinical use. Nuclear factor erythroid 2-related factor 2 (Nrf2, NFE2L2) is a critical transcription factor that has been shown to protect against kidney injury through activation of antioxidant mechanisms. We aimed to evaluate the ability of short-term treatment with the Nrf2 activator bardoxolone methyl (CDDO-Me) to protect against cisplatin-induced kidney cell toxicity. Cell viability was assessed in human kidney proximal tubule epithelial cells (hPTCs) exposed to low, intermediate, and high cisplatin concentrations in the presence and absence of CDDO-Me, administered either prior to or after cisplatin. Treatment with cisplatin alone resulted in reductions in hPTC viability, while CDDO-Me administered prior to or after cisplatin exposure yielded significantly higher cell viability (17%-71%). Gene regulation (mRNA expression) studies revealed the ability of CDDO-Me to modify protective pathways including Nrf2 induced detoxifying genes [GCLC (increased 1.9-fold), NQO1 (increased 9.3-fold)], and an efflux transporter [SLC47A1 (increased 4.5-fold)] at 12h. Protein assessments were in agreement with gene expression. Immunofluorescence revealed localization of GCLC and NQO1 to the nucleus and cytosol, respectively, with CDDO-Me administered prior to or after cisplatin exposure. The findings of enhanced cell viability and increased expression of detoxifying enzymes (GCLC and NQO1) and the multidrug and toxin extrusion protein 1 (MATE1) efflux transporter (SLC47A1) in hPTCs exposed to CDDO-Me, suggest that intermittent treatment with CDDO-Me prior to or after cisplatin exposure may be a promising approach to mitigate acute kidney injury. Copyright © 2016. Published by Elsevier Ireland Ltd.

Efflux transport of chrysin and apigenin sulfates in HEK293 cells overexpressing SULT1A3: The role of multidrug resistance-associated protein 4 (MRP4/ABCC4).

PubMed

Li, Wan; Sun, Hua; Zhang, Xingwang; Wang, Huan; Wu, Baojian

2015-11-01

Efflux transport is a critical determinant to the pharmacokinetics of sulfate conjugates. Here we aimed to establish SULT1A3 stably transfected HEK293 cells, and to determine the contributions of BCRP and MRP transporters to excretion of chrysin and apigenin sulfates. The cDNA of SULT1A3 was stably introduced into HEK293 cells using a lentiviral vector, generating a sulfonation active cell line (i.e., SULT293 cells). Identification of sulfate transporters was achieved through chemical inhibition (using chemical inhibitors) and biological inhibition (using short-hairpin RNAs (shRNAs)) methods. Sulfated metabolites were rapidly generated and excreted upon incubation of SULT293 cells with chrysin and apigenin. Ko143 (a selective BCRP inhibitor) did not show inhibitory effects on sulfate disposition, whereas the pan-MRP inhibitor MK-571 caused significant reductions (38.5-64.3%, p<0.001) in sulfate excretion and marked elevations (160-243%, p<0.05) in sulfate accumulation. Further, two efflux transporters (BCRP and MRP4) expressed in the cells were knocked-down by shRNA-mediated silencing. Neither sulfate excretion nor sulfate accumulation was altered in BCRP knocked-down cells as compared to scramble cells. By contrast, MRP4 knock-down led to moderate decreases (17.1-20.6%, p<0.05) in sulfate excretion and increases (125-135%, p<0.05) in sulfate accumulation. In conclusion, MRP4 was identified as an exporter for chrysin and apigenin sulfates. The SULT1A3 modified HEK293 cells were an appropriate tool to study SULT1A3-mediated sulfonation and to characterize BCRP/MRP4-mediated sulfate transport. Copyright © 2015 Elsevier Inc. All rights reserved.

Improvement of Transmembrane Transport Mechanism Study of Imperatorin on P-Glycoprotein-Mediated Drug Transport.

PubMed

Liao, Zheng-Gen; Tang, Tao; Guan, Xue-Jing; Dong, Wei; Zhang, Jing; Zhao, Guo-Wei; Yang, Ming; Liang, Xin-Li

2016-11-24

P-glycoprotein (P-gp) affects the transport of many drugs; including puerarin and vincristine. Our previous study demonstrated that imperatorin increased the intestinal absorption of puerarin and vincristine by inhibiting P-gp-mediated drug efflux. However; the underlying mechanism was not known. The present study investigated the mechanism by which imperatorin promotes P-gp-mediated drug transport. We used molecular docking to predict the binding force between imperatorin and P-gp and the effect of imperatorin on P-gp activity. P-gp efflux activity and P-gp ATPase activity were measured using a rhodamine 123 (Rh-123) accumulation assay and a Pgp-Glo™ assay; respectively. The fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to assess cellular membrane fluidity in MDCK-MDR1 cells. Western blotting was used to analyze the effect of imperatorin on P-gp expression; and P-gp mRNA levels were assessed by qRT-PCR. Molecular docking results demonstrated that the binding force between imperatorin and P-gp was much weaker than the force between P-gp and verapamil (a P-gp substrate). Imperatorin activated P-gp ATPase activity; which had a role in the inhibition of P-gp activity. Imperatorin promoted Rh-123 accumulation in MDCK-MDR1 cells and decreased cellular membrane fluidity. Western blotting demonstrated that imperatorin inhibited P-gp expression; and qRT-PCR revealed that imperatorin down-regulated P-gp (MDR1) gene expression. Imperatorin decreased P-gp-mediated drug efflux by inhibiting P-gp activity and the expression of P-gp mRNA and protein. Our results suggest that imperatorin could down-regulate P-gp expression to overcome multidrug resistance in tumors.

Anethole potentiates dodecanol's fungicidal activity by reducing PDR5 expression in budding yeast.

PubMed

Fujita, Ken-Ichi; Ishikura, Takayuki; Jono, Yui; Yamaguchi, Yoshihiro; Ogita, Akira; Kubo, Isao; Tanaka, Toshio

2017-02-01

trans-Anethole (anethole), a major component of anise oil, has a broad antimicrobial spectrum and a weaker antimicrobial potency than other available antibiotics. When combined with polygodial, nagilactone E, and n-dodecanol, anethole has been shown to exhibit synergistic antifungal activity against a budding yeast, Saccharomyces cerevisiae, and a human opportunistic pathogenic yeast, Candida albicans. However, the mechanism underlying this synergistic effect of anethole has not been characterized. We studied this mechanism using dodecanol-treated S. cerevisiae cells and focusing on genes related to multidrug efflux. Although dodecanol transiently reduced the number of colony forming units, this recovered to levels similar to those of untreated cells with continued incubation beyond 24h. Reverse transcription polymerase chain reaction analysis revealed overexpression of an ATP-binding cassette (ABC) transporter gene, PDR5, in addition to a slight increase in PDR11, PDR12, and PDR15 transcriptions in dodecanol-treated cells. In the presence of anethole, these effects were attenuated and the fungicidal activity of dodecanol was extended. Dodecanol showed longer lasting fungicidal activity against a Δpdr5. In addition, Δpdr3 and Δlge1, lack transcription factors of PDR5 and PDR3, were partly and completely susceptible to dodecanol, respectively. Furthermore, combination of anethole with fluconazole was also found to exhibit synergy on C. albicans. These results indicated that although anethole reduced the transcription of several transporters, PDR5 expression was particularly relevant to dodecanol efflux. Anethole is expected to be a promising candidate drug for the inhibition of efflux by reducing the transcription of several ABC transporters. Copyright © 2016 Elsevier B.V. All rights reserved.

Determination of P-glycoprotein surface expression and functional ability after in vitro treatment with darunavir or raltegravir in lymphocytes of healthy donors.

PubMed

Tempestilli, Massimo; Gentilotti, Elisa; Tommasi, Chiara; Nicastri, Emanuele; Martini, Federico; De Nardo, Pasquale; Narciso, Pasquale; Pucillo, Leopoldo P

2013-08-01

It has been shown that P-glycoprotein (P-gp) can greatly affect the cell uptake of antiretroviral drugs, thus hampering their access to HIV-1 replication sites. Lymphocytes are important sites of replication of HIV and target of other drugs, modification on these cells of P-gp could have an effect on pharmacokinetic of antiretrovirals and drug substrates. Blood samples from 16 healthy volunteers were used to determine the expression of P-gp on total, T and T helper lymphocytes after exposure to darunavir, a second generation protease inhibitor, and raltegravir, the first approved integrase inhibitor. Moreover, the effect of the drugs on P-gp functional activity was also studied by the rhodamine-123 efflux test. Darunavir, but not raltegravir, exposure caused a moderate, dose-dependent increment in P-gp expression in total, T and T helper lymphocytes, as demonstrated by the relative frequency of P-gp+ cells and by the amount of P-gp molecules present on cell surface. Functionally, incubation with darunavir led to a marked inhibition of P-gp activity measured by the efflux of rhodamine-123 similar to that observed by verapamil, a specific P-gp inhibitor. Raltegravir was not able to modify the efflux of rhodamine-123 level. Data show that darunavir, unlike raltegravir, may modify the expression and functionality of P-gp on human lymphocytes, thus leading to potential changes in intracellular concentrations of darunavir in patients treated with other drugs substrate of P-gp and vice versa. Our study highlights the need for studies on drug interactions via the P-gp modulation mechanism, especially with the current multi-drug regimens. Copyright © 2013 Elsevier B.V. All rights reserved.

Sub-minimum inhibitory concentrations of colistin and polymyxin B promote Acinetobacter baumannii biofilm formation

PubMed Central

Unno, Yuka; Ubagai, Tsuneyuki; Ono, Yasuo

2018-01-01

We investigated the numbers of planktonic and biofilm cells and the expression levels of genes encoding efflux pumps and biofilm-related proteins in 10 clinical isolates of multi-drug resistant Acinetobacter baumannii (MDRA) as well as in its standard strain ATCC 19606 in the presence of colistin (CST), polymyxin B (PMB), minomycin (MIN), and tigecycline (TGC) at their respective sub-MICs. The number of planktonic and biofilm cells of ATCC 19606 decreased in the presence of all aforementioned antibiotics in a dose-dependent manner. Cell number also decreased in two representative MDRA strains, R2 and R3, in the presence of MIN and TGC in a dose-dependent manner. In contrast, the number of biofilm cells in these two strains increased in the presence of CST, while they increased significantly in the presence of PMB in R2 only. Pearson correlation analysis revealed that the number of biofilm cells was positively and significantly correlated with the mRNA levels of genes encoding efflux pumps (adeB and adeG) and autoinducer synthase (abaI) in strain R2 and adeB, adeG, adeJ, poly-acetyl-glucosamine-porin (pgaA), and abaI in strain R3 in the presence of CST. It was positively and significantly correlated with the mRNA levels of genes encoding adeB in strain R2 and an outer membrane protein A (ompA) and biofilm-associated protein (bap) in strain R3 in the presence of PMB. These results provide valuable insights into the biofilm formation potency of clinical isolates of MDRA that depends on efflux pumps and biofilm-related genes and its regulation by antibiotics. PMID:29554105

Development and Validation of a Computational Model Ensemble for the Early Detection of BCRP/ABCG2 Substrates during the Drug Design Stage.

PubMed

Gantner, Melisa E; Peroni, Roxana N; Morales, Juan F; Villalba, María L; Ruiz, María E; Talevi, Alan

2017-08-28

Breast Cancer Resistance Protein (BCRP) is an ATP-dependent efflux transporter linked to the multidrug resistance phenomenon in many diseases such as epilepsy and cancer and a potential source of drug interactions. For these reasons, the early identification of substrates and nonsubstrates of this transporter during the drug discovery stage is of great interest. We have developed a computational nonlinear model ensemble based on conformational independent molecular descriptors using a combined strategy of genetic algorithms, J48 decision tree classifiers, and data fusion. The best model ensemble consists in averaging the ranking of the 12 decision trees that showed the best performance on the training set, which also demonstrated a good performance for the test set. It was experimentally validated using the ex vivo everted rat intestinal sac model. Five anticonvulsant drugs classified as nonsubstrates for BRCP by the model ensemble were experimentally evaluated, and none of them proved to be a BCRP substrate under the experimental conditions used, thus confirming the predictive ability of the model ensemble. The model ensemble reported here is a potentially valuable tool to be used as an in silico ADME filter in computer-aided drug discovery campaigns intended to overcome BCRP-mediated multidrug resistance issues and to prevent drug-drug interactions.

Tangeretin, a citrus pentamethoxyflavone, antagonizes ABCB1-mediated multidrug resistance by inhibiting its transport function.

PubMed

Feng, Sen-Ling; Yuan, Zhong-Wen; Yao, Xiao-Jun; Ma, Wen-Zhe; Liu, Liang; Liu, Zhong-Qiu; Xie, Ying

2016-08-01

Multidrug resistance (MDR) and tumor metastasis are the main causes of chemotherapeutic treatment failure and mortality in cancer patients. In this study, at achievable nontoxic plasma concentrations, citrus flavonoid tangeretin has been shown to reverse ABCB1-mediated cancer resistance to a variety of chemotherapeutic agents effectively. Co-treatment of cells with tangeretin and paclitaxel activated apoptosis as well as arrested cell cycle at G2/M-phase. Tangeretin profoundly inhibited the ABCB1 transporter activity since it significantly increased the intracellular accumulation of doxorubicin, and flutax-2 in A2780/T cells and decreased the efflux of ABCB1 substrates in Caco2 cells without altering the expression of ABCB1. Moreover, it stimulated the ATPase activity and inhibited verapamil-stimulated ATPase activity in a concentration-dependent manner, indicating a direct interaction with the transporter. The molecular docking results indicated a favorable binding of tangeretin with the transmemberane region site 1 of homology modeled ABCB1 transporter. The overall results demonstrated that tangeretin could sensitize ABCB1-overexpressing cancer cells to chemotherapeutical agents by directly inhibiting ABCB1 transporter function, which encouraged further animal and clinical studies in the treatment of resistant cancers. Copyright © 2016 Elsevier Ltd. All rights reserved.

Schistosoma mansoni P-glycoprotein levels increase in response to praziquantel exposure and correlate with reduced praziquantel susceptibility.

PubMed

Messerli, Shanta M; Kasinathan, Ravi S; Morgan, William; Spranger, Stefani; Greenberg, Robert M

2009-09-01

One potential physiological target for new antischistosomals is the parasite's system for excretion of wastes and xenobiotics. P-glycoprotein (Pgp), a member of the ATP-binding-cassette superfamily of proteins, is an ATP-dependent efflux pump involved in transport of toxins and xenobiotics from cells. In vertebrates, increased expression of Pgp is associated with multidrug resistance in tumor cells. Pgp may also play a role in drug resistance in helminths. In this report, we examine the relationship between praziquantel (PZQ), the current drug of choice against schistosomiasis, and Pgp expression in Schistosoma mansoni. We show that levels of RNA for SMDR2, a Pgp homolog from S. mansoni, increase transiently in adult male worms following exposure to sub-lethal concentrations (100-500 nM) of PZQ. A corresponding, though delayed, increase in anti-Pgp immunoreactive protein expression occurs in adult males following exposure to PZQ. The level of anti-Pgp immunoreactivity in particular regions of adult worms also increases in response to PZQ. Adult worms from an Egyptian S. mansoni isolate with reduced sensitivity to PZQ express increased levels of SMDR2 RNA and anti-Pgp-immunoreactive protein, perhaps indicating a role for multidrug resistance proteins in development or maintenance of PZQ resistance.

Schistosoma mansoni P-glycoprotein levels increase in response to praziquantel exposure and correlate with reduced praziquantel susceptibility

PubMed Central

Messerli, Shanta M.; Kasinathan, Ravi S.; Morgan, William; Spranger, Stefani; Greenberg, Robert M.

2009-01-01

One potential physiological target for new antischistosomals is the parasite’s system for excretion of wastes and xenobiotics. P-glycoprotein (Pgp), a member of the ATP-binding cassette superfamily of proteins, is an ATP-dependent efflux pump involved in transport of toxins and xenobiotics from cells. In vertebrates, increased expression of Pgp is associated with multidrug resistance in tumor cells. Pgp may also play a role in drug resistance in helminths. In this report, we examine the relationship between praziquantel (PZQ), the current drug of choice against schistosomiasis, and Pgp expression in Schistosoma mansoni. We show that levels of RNA for SMDR2, a Pgp homolog from S. mansoni, increase transiently in adult male worms following exposure to sublethal concentrations (100 – 500 nM) of PZQ. A corresponding, though delayed, increase in anti-Pgp immunoreactive protein expression occurs in adult males following exposure to PZQ. The level of anti-Pgp immunoreactivity in particular regions of adult worms also increases in response to PZQ. Adult worms from an Egyptian S. mansoni isolate with reduced sensitivity to PZQ express increased levels of SMDR2 RNA and anti-Pgp-immunoreactive protein, perhaps indicating a role for multidrug resistance proteins in development or maintenance of PZQ resistance. PMID:19406169

Pseudomonas Aeruginosa: Resistance to the Max

PubMed Central

Poole, Keith

2011-01-01

Pseudomonas aeruginosa is intrinsically resistant to a variety of antimicrobials and can develop resistance during anti-pseudomonal chemotherapy both of which compromise treatment of infections caused by this organism. Resistance to multiple classes of antimicrobials (multidrug resistance) in particular is increasingly common in P. aeruginosa, with a number of reports of pan-resistant isolates treatable with a single agent, colistin. Acquired resistance in this organism is multifactorial and attributable to chromosomal mutations and the acquisition of resistance genes via horizontal gene transfer. Mutational changes impacting resistance include upregulation of multidrug efflux systems to promote antimicrobial expulsion, derepression of ampC, AmpC alterations that expand the enzyme's substrate specificity (i.e., extended-spectrum AmpC), alterations to outer membrane permeability to limit antimicrobial entry and alterations to antimicrobial targets. Acquired mechanisms contributing to resistance in P. aeruginosa include β-lactamases, notably the extended-spectrum β-lactamases and the carbapenemases that hydrolyze most β-lactams, aminoglycoside-modifying enzymes, and 16S rRNA methylases that provide high-level pan-aminoglycoside resistance. The organism's propensity to grow in vivo as antimicrobial-tolerant biofilms and the occurrence of hypermutator strains that yield antimicrobial resistant mutants at higher frequency also compromise anti-pseudomonal chemotherapy. With limited therapeutic options and increasing resistance will the untreatable P. aeruginosa infection soon be upon us? PMID:21747788

New structure–activity relationships of chalcone inhibitors of breast cancer resistance protein: polyspecificity toward inhibition and critical substitutions against cytotoxicity

PubMed Central

Rangel, Luciana Pereira; Winter, Evelyn; Gauthier, Charlotte; Terreux, Raphaël; Chiaradia-Delatorre, Louise D; Mascarello, Alessandra; Nunes, Ricardo J; Yunes, Rosendo A; Creczynski-Pasa, Tania B; Macalou, Sira; Lorendeau, Doriane; Baubichon-Cortay, Hélène; Ferreira-Pereira, Antonio; Di Pietro, Attilio

2013-01-01

Adenosine triphosphate-binding cassette subfamily G member 2 (ABCG2) plays a major role in cancer cell multidrug resistance, which contributes to low eifficacy of chemotherapy. Chalcones were recently found to be potent and specific inhibitors, but unfortunately display a significant cytotoxicity. A cellular screening against ABCG2-mediated mitoxantrone efflux was performed here by flow cytometry on 54 chalcone derivatives from three different series with a wide panel of substituents. The identified leads, with submicromolar IC50 (half maximal inhibitory concentration) values, showed that the previously identified 2′-OH-4′,6′-dimethoxyphenyl, as A-ring, could be efficiently replaced by a 2′-naphthyl group, or a 3′,4′-methylenedioxyphenyl with lower affinity. Such a structural variability indicates 3polyspecificity of the multidrug transporter for inhibitors. At least two methoxyl groups were necessary on B-ring for optimal inhibition, but substitution at positions 3, 4, and 5 induced cytotoxicity. The presence of a large O-benzyl substituent at position 4 and a 2′-naphthyl as A-ring markedly decreased the cytotoxicity, giving a high therapeutic ratio, which constitutes a critical requirement for future in-vivo assays in animal models. PMID:24109177

Hepatocyte SLAMF3 reduced specifically the multidrugs resistance protein MRP-1 and increases HCC cells sensitization to anti-cancer drugs

PubMed Central

Eugenio, Mélanie Simoes; Demey, Baptiste; Singh, Amrathlal Rabbind; Ossart, Christèle; Bagami, Mohammed Al; Regimbeau, Jean-Marc; Nguyen-Khac, Eric; Naassila, Mickael

2016-01-01

Multidrug resistance MDR proteins (MRPs) are members of the C family of a group of proteins named ATP binding cassette (ABC) transporters. MRPs can transport drugs including anticancer drugs, nucleoside analogs, antimetabolites and tyrosine kinase inhibitors. Drugs used in HCC therapy, such as tyrosine kinase inhibitor sorafenib, are substrates of uptake and/or efflux transporters. Variable expression of MRPs at the plasma membrane of tumor cells may contribute to drug resistance and subsequent clinical response. Recently, we reported that the hepatocyte SLAMF3 expression (Signaling Lymphocytic Activation Molecule Family member 3) was reduced in tumor cells from hepatocellular carcinoma (HCC) compared to its high expression in adjacent tissues. In the present study, we make a strong correlation between induced SLAMF3 overexpression and the specific loss of MRP-1 expression and its functionalities as a drugs resistance transporter. No changes were observed on expression of ABCG2 and MDR. More importantly, we highlight a strong inverse correlation between MRP-1 and SLAMF3 expression in patients with HCC. We propose that the SLAMF3 overexpression in cancerous cells could represent a potential therapeutic strategy to improve the drugs sensibility of resistant cells and thus control the therapeutic failure in HCC patients. PMID:27081035

Hepatocyte SLAMF3 reduced specifically the multidrugs resistance protein MRP-1 and increases HCC cells sensitization to anti-cancer drugs.

PubMed

Fouquet, Grégory; Debuysscher, Véronique; Ouled-Haddou, Hakim; Eugenio, Mélanie Simoes; Demey, Baptiste; Singh, Amrathlal Rabbind; Ossart, Christèle; Al Bagami, Mohammed; Regimbeau, Jean-Marc; Nguyen-Khac, Eric; Naassila, Mickael; Marcq, Ingrid; Bouhlal, Hicham

2016-05-31

Multidrug resistance MDR proteins (MRPs) are members of the C family of a group of proteins named ATP binding cassette (ABC) transporters. MRPs can transport drugs including anticancer drugs, nucleoside analogs, antimetabolites and tyrosine kinase inhibitors. Drugs used in HCC therapy, such as tyrosine kinase inhibitor sorafenib, are substrates of uptake and/or efflux transporters. Variable expression of MRPs at the plasma membrane of tumor cells may contribute to drug resistance and subsequent clinical response. Recently, we reported that the hepatocyte SLAMF3 expression (Signaling Lymphocytic Activation Molecule Family member 3) was reduced in tumor cells from hepatocellular carcinoma (HCC) compared to its high expression in adjacent tissues. In the present study, we make a strong correlation between induced SLAMF3 overexpression and the specific loss of MRP-1 expression and its functionalities as a drugs resistance transporter. No changes were observed on expression of ABCG2 and MDR. More importantly, we highlight a strong inverse correlation between MRP-1 and SLAMF3 expression in patients with HCC. We propose that the SLAMF3 overexpression in cancerous cells could represent a potential therapeutic strategy to improve the drugs sensibility of resistant cells and thus control the therapeutic failure in HCC patients.

Coarse-grained Simulations of Substrate Export through Multidrug Efflux Transporter AcrB

NASA Astrophysics Data System (ADS)

Jewel, Yead; Dutta, Prashanta; Liu, Jin

2017-11-01

The treatment of bacterial infectious diseases hampered by the overexpression of multidrug resistance (MDR) systems. The MDR system actively pumps the antibiotic drugs as well as other toxic compounds out of the cells. During the pumping, AcrB (one of the key MDR components) undergoes a series of large-scale proton/substrate dependent conformational changes. In this work, we implement a hybrid coarse-grained PACE force field that couples the united-atom protein model with the coarse-grained MARTINI water/lipid, to investigate the conformational changes of AcrB. We first develop the substrate force field which is compatible with PACE, then we implement the force field to explore large scale structural changes of AcrB in microsecond simulations. The effects of the substrate and the protonation states of two key residues: Asp407 and Asp408, are investigated. Our results show that the drug export through AcrB is proton as well as substrate dependent. Our simulations explain molecular mechanisms of substrate transport through AcrB complex, as well as provide valuable insights for designing proper antibiotic drugs. Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R01GM122081.

Effect of the F610A mutation on substrate extrusion in the AcrB transporter: explanation and rationale by molecular dynamics simulations.

PubMed

Vargiu, Attilio V; Collu, Francesca; Schulz, Robert; Pos, Klaas M; Zacharias, Martin; Kleinekathöfer, Ulrich; Ruggerone, Paolo

2011-07-20

The tripartite efflux pump AcrAB-TolC is responsible for the intrinsic and acquired multidrug resistance in Escherichia coli. Its active part, the homotrimeric transporter AcrB, is in charge of the selective binding of substrates and energy transduction. The mutation F610A has been shown to significantly reduce the minimum inhibitory concentration of doxorubicin and many other substrates, although F610 does not appear to interact strongly with them. Biochemical study of transport kinetics in AcrB is not yet possible, except for some β-lactams, and other techniques should supply this important information. Therefore, in this work, we assess the impact of the F610A mutation on the functionality of AcrB by means of computational techniques, using doxorubicin as substrate. We found that the compound slides deeply inside the binding pocket after mutation, increasing the strength of the interaction. During subsequent conformational alterations of the transporter, doxorubicin was either not extruded from the binding site or displaced along a direction other than the one associated with extrusion. Our study indicates how subtle interactions determine the functionality of multidrug transporters, since decreased transport might not be simplistically correlated to decreased substrate binding affinity.

A co-delivery nanosystem of chemotherapeutics and DNAzyme overcomes cancer drug resistance and metastasis

NASA Astrophysics Data System (ADS)

Sun, Shu-Pin; Liu, Ching-Ping; Huang, I.-Ping; Chu, Chia-Hui; Chung, Ming-Fang; Cheng, Shih-Hsun; Lin, Shu-Yi; Lo, Leu-Wei

2017-12-01

Multidrug resistance (MDR) constitutes a major problem in the management of cancer and cancer metastasized from primary-source tumor causes cancer-related deaths. Our new approach is the co-delivery of chemotherapy drugs with a transcription-factor-targeting genetic agent to simultaneously inhibit the growth and metastasis of cancer cells. C-Jun is a transcription factor that regulates multidrug resistance-associated protein 1 (MRP1) pump efflux transcription and tumor metastasis. In this work, we reported that mesoporous silica nanoparticles (MSNs) can be functionalized to co-deliver doxorubicin (Dox) and DNAzyme (Dz) to increase cancer cell killing in an additive fashion. The MSNs were sequentially conjugated with Dox into the MSNs’ nanochannels and Dz onto the MSNs’ outermost surface to target c-Jun as the Dox@MSN-Dz co-delivery system. The Dox-resistant PC-3 cells treated with Dox@MSN-Dz efficiently enhanced the intracellular Dox concentration due to the abrogation of Dox-induced MRP1 expression through the downregulation of c-Jun expression by Dz. Additionally, significant reductions in invasion and migration related to metastasis were also observed in cells treated with Dox@MSN-Dz. Therefore, our results contribute new insight to the treatment of MDR combined metastatic cancer cells, worthwhile for studying its potential for development in clinical translation.

Effect of Chlorine Exposure on the Survival and Antibiotic Gene Expression of Multidrug Resistant Acinetobacter baumannii in Water

PubMed Central

Karumathil, Deepti Prasad; Yin, Hsin-Bai; Kollanoor-Johny, Anup; Venkitanarayanan, Kumar

2014-01-01

Acinetobacter baumannii is a multidrug resistant pathogen capable of causing a wide spectrum of clinical conditions in humans. Acinetobacter spp. is ubiquitously found in different water sources. Chlorine being the most commonly used disinfectant in water, the study investigated the effect of chlorine on the survival of A. baumannii in water and transcription of genes conferring antibiotic resistance. Eight clinical isolates of A. baumannii, including a fatal meningitis isolate (ATCC 17978) (~108 CFU/mL) were separately exposed to free chlorine concentrations (0.2, 1, 2, 3 and 4 ppm) with a contact time of 30, 60, 90 and 120 second. The surviving pathogen counts at each specified contact time were determined using broth dilution assay. In addition, real-time quantitative PCR (RT-qPCR) analysis of the antibiotic resistance genes (efflux pump genes and those encoding resistance to specific antibiotics) of three selected A. baumannii strains following exposure to chlorine was performed. Results revealed that all eight A. baumannii isolates survived the tested chlorine levels during all exposure times (p > 0.05). Additionally, there was an up-regulation of all or some of the antibiotic resistance genes in A. baumannii, indicating a chlorine-associated induction of antibiotic resistance in the pathogen. PMID:24514427

Inhibition of Glutathione Peroxidase Mediates the Collateral Sensitivity of Multidrug-resistant Cells to Tiopronin*

PubMed Central

Hall, Matthew D.; Marshall, Travis S.; Kwit, Alexandra D. T.; Miller Jenkins, Lisa M.; Dulcey, Andrés E.; Madigan, James P.; Pluchino, Kristen M.; Goldsborough, Andrew S.; Brimacombe, Kyle R.; Griffiths, Gary L.; Gottesman, Michael M.

2014-01-01

Multidrug resistance (MDR) is a major obstacle to the successful chemotherapy of cancer. MDR is often the result of overexpression of ATP-binding cassette transporters following chemotherapy. A common ATP-binding cassette transporter that is overexpressed in MDR cancer cells is P-glycoprotein, which actively effluxes drugs against a concentration gradient, producing an MDR phenotype. Collateral sensitivity (CS), a phenomenon of drug hypersensitivity, is defined as the ability of certain compounds to selectively target MDR cells, but not the drug-sensitive parent cells from which they were derived. The drug tiopronin has been previously shown to elicit CS. However, unlike other CS agents, the mechanism of action was not dependent on the expression of P-glycoprotein in MDR cells. We have determined that the CS activity of tiopronin is mediated by the generation of reactive oxygen species (ROS) and that CS can be reversed by a variety of ROS-scavenging compounds. Specifically, selective toxicity of tiopronin toward MDR cells is achieved by inhibition of glutathione peroxidase (GPx), and the mode of inhibition of GPx1 by tiopronin is shown in this report. Why MDR cells are particularly sensitive to ROS is discussed, as is the difficulty in exploiting this hypersensitivity to tiopronin in the clinic. PMID:24930045

Antifungal activity of ionic liquids based on (-)-menthol: a mechanism study.

PubMed

Suchodolski, Jakub; Feder-Kubis, Joanna; Krasowska, Anna

2017-04-01

The mechanism of toxicity of chiral ionic liquids with (1R,2S,5R)-(-)-menthol [C n -Am-Men][Cl] (n=10, 11 or 12) in the fungus Candida albicans is reported here. Ionic liquids were more toxic towards Candida strain lacking all identified multidrug resistance efflux pumps. Moreover, the compounds tested inhibited C. albicans filamentation at the concentration at which detached fungal cells also adhered to the plastic surface. Our results showed the high activity of all the tested chiral ionic liquids in the permeabilization of C. albicans' membranes and in the digestion and interruption of the cell wall. The investigated ionic liquids thus have potential as disinfectants because besides their antifungal and antiadhesive action these compounds do not cause hemolysis. Copyright © 2017 Elsevier GmbH. All rights reserved.

The CopRS Two-Component System Is Responsible for Resistance to Copper in the Cyanobacterium Synechocystis sp. PCC 68031[C][W][OA

PubMed Central

Giner-Lamia, Joaquín; López-Maury, Luis; Reyes, José C.; Florencio, Francisco J.

2012-01-01

Photosynthetic organisms need copper for cytochrome oxidase and for plastocyanin in the fundamental processes of respiration and photosynthesis. However, excess of free copper is detrimental inside the cells and therefore organisms have developed homeostatic mechanisms to tightly regulate its acquisition, sequestration, and efflux. Herein we show that the CopRS two-component system (also known as Hik31-Rre34) is essential for copper resistance in Synechocystis sp. PCC 6803. It regulates expression of a putative heavy-metal efflux-resistance nodulation and division type copper efflux system (encoded by copBAC) as well as its own expression (in the copMRS operon) in response to the presence of copper in the media. Mutants in this two-component system or the efflux system render cells more sensitive to the presence of copper in the media and accumulate more intracellular copper than the wild type. Furthermore, CopS periplasmic domain is able to bind copper, suggesting that CopS could be able to detect copper directly. Both operons (copMRS and copBAC) are also induced by the photosynthetic inhibitor 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone but this induction requires the presence of copper in the media. The reduced response of two mutant strains to copper, one lacking plastocyanin and a second one impaired in copper transport to the thylakoid, due to the absence of the PI-type ATPases PacS and CtaA, suggests that CopS can detect intracellular copper. In addition, a tagged version of CopS with a triple HA epitope localizes to both the plasma and the thylakoid membranes, suggesting that CopS could be involved in copper detection in both the periplasm and the thylakoid lumen. PMID:22715108

The CopRS two-component system is responsible for resistance to copper in the cyanobacterium Synechocystis sp. PCC 6803.

PubMed

Giner-Lamia, Joaquín; López-Maury, Luis; Reyes, José C; Florencio, Francisco J

2012-08-01

Photosynthetic organisms need copper for cytochrome oxidase and for plastocyanin in the fundamental processes of respiration and photosynthesis. However, excess of free copper is detrimental inside the cells and therefore organisms have developed homeostatic mechanisms to tightly regulate its acquisition, sequestration, and efflux. Herein we show that the CopRS two-component system (also known as Hik31-Rre34) is essential for copper resistance in Synechocystis sp. PCC 6803. It regulates expression of a putative heavy-metal efflux-resistance nodulation and division type copper efflux system (encoded by copBAC) as well as its own expression (in the copMRS operon) in response to the presence of copper in the media. Mutants in this two-component system or the efflux system render cells more sensitive to the presence of copper in the media and accumulate more intracellular copper than the wild type. Furthermore, CopS periplasmic domain is able to bind copper, suggesting that CopS could be able to detect copper directly. Both operons (copMRS and copBAC) are also induced by the photosynthetic inhibitor 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone but this induction requires the presence of copper in the media. The reduced response of two mutant strains to copper, one lacking plastocyanin and a second one impaired in copper transport to the thylakoid, due to the absence of the P(I)-type ATPases PacS and CtaA, suggests that CopS can detect intracellular copper. In addition, a tagged version of CopS with a triple HA epitope localizes to both the plasma and the thylakoid membranes, suggesting that CopS could be involved in copper detection in both the periplasm and the thylakoid lumen.

Membrane Fusion Proteins of Type I Secretion System and Tripartite Efflux Pumps Share a Binding Motif for TolC in Gram-Negative Bacteria

PubMed Central

Yoon, Bo-Young; Song, Saemee; Lee, Kangseok; Ha, Nam-Chul

2012-01-01

The Hly translocator complex of Escherichia coli catalyzes type I secretion of the toxin hemolysin A (HlyA). In this complex, HlyB is an inner membrane ABC (ATP Binding Cassette)-type transporter, TolC is an outer membrane channel protein, and HlyD is a periplasmic adaptor anchored in the inner membrane that bridges HlyB to TolC. This tripartite organization is reminiscent of that of drug efflux systems such as AcrA-AcrB-TolC and MacA-MacB-TolC of E. coli. We have previously shown the crucial role of conserved residues located at the hairpin tip region of AcrA and MacA adaptors during assembly of their cognate systems. In this study, we investigated the role of the putative tip region of HlyD using HlyD mutants with single amino acid substitutions at the conserved positions. In vivo and in vitro data show that all mutations abolished HlyD binding to TolC and resulted in the absence of HlyA secretion. Together, our results suggest that, similarly to AcrA and MacA, HlyD interacts with TolC in a tip-to-tip manner. A general model in which these conserved interactions induce opening of TolC during drug efflux and type I secretion is discussed. PMID:22792337

MdPIN1b encodes a putative auxin efflux carrier and has different expression patterns in BC and M9 apple rootstocks.

PubMed

Gan, Zengyu; Wang, Yi; Wu, Ting; Xu, Xuefeng; Zhang, Xinzhong; Han, Zhenhai

2018-03-01

Lower promoter activity is closely associated with lower MdPIN1b expression in the M9 interstem, which might contribute to the dwarfing effect in apple trees. Apple trees grafted onto dwarfing rootstock Malling 9 (M9) produce dwarfing tree architecture with high yield and widely applying in production. Previously, we have reported that in Malus 'Red Fuji' (RF) trees growing on M9 interstem and Baleng Crab (BC) rootstock, IAA content was relatively higher in bark tissue of M9 interstem than that in scion or rootstock. As IAA polar transportation largely depends on the PIN-FORMED (PIN) auxin efflux carrier. Herein, we identify two putative auxin efflux carrier genes in Malus genus, MdPIN1a and MdPIN1b, which were closely related to the AtPIN1. We found that MdPIN1b was expressed preferentially in BC and M9, and the expression of MdPIN1b was significantly lower in the phloem of M9 interstem than that in the scion and rootstock. The distinct expression of MdPIN1b and IAA content were concentrated in the cambium and adjacent xylem or phloem, and MdPIN1b protein was localized on cell plasma membrane in onion epidermal cells transiently expressing 35S:MdPIN1b-GFP fusion protein. Interestingly, an MdPIN1b mutant allele in the promoter region upstream of M9 exhibited decreased MdPIN1b expression compared to BC. MdPIN1b over-expressing interstem in tobacco exhibited increased polar auxin transport. It is proposed that natural allelic differences decreased promoter activity is closely associated with lower MdPIN1b expression in the M9 interstem, which might limit the basipetal transport of auxin, and in turn might contribute to the dwarfing effect. Taken together, these results reveal allelic variation underlying an important apple rootstock trait, and specifically a novel molecular genetic mechanism underlying dwarfing mechanism.

High-throughput screening identifies Ceefourin 1 and Ceefourin 2 as highly selective inhibitors of multidrug resistance protein 4 (MRP4).

PubMed

Cheung, Leanna; Flemming, Claudia L; Watt, Fujiko; Masada, Nanako; Yu, Denise M T; Huynh, Tony; Conseil, Gwenaëlle; Tivnan, Amanda; Polinsky, Alexander; Gudkov, Andrei V; Munoz, Marcia A; Vishvanath, Anasuya; Cooper, Dermot M F; Henderson, Michelle J; Cole, Susan P C; Fletcher, Jamie I; Haber, Michelle; Norris, Murray D

2014-09-01

Multidrug resistance protein 4 (MRP4/ABCC4), a member of the ATP-binding cassette (ABC) transporter superfamily, is an organic anion transporter capable of effluxing a wide range of physiologically important signalling molecules and drugs. MRP4 has been proposed to contribute to numerous functions in both health and disease; however, in most cases these links remain to be unequivocally established. A major limitation to understanding the physiological and pharmacological roles of MRP4 has been the absence of specific small molecule inhibitors, with the majority of established inhibitors also targeting other ABC transporter family members, or inhibiting the production, function or degradation of important MRP4 substrates. We therefore set out to identify more selective and well tolerated inhibitors of MRP4 that might be used to study the many proposed functions of this transporter. Using high-throughput screening, we identified two chemically distinct small molecules, Ceefourin 1 and Ceefourin 2, that inhibit transport of a broad range of MRP4 substrates, yet are highly selective for MRP4 over other ABC transporters, including P-glycoprotein (P-gp), ABCG2 (Breast Cancer Resistance Protein; BCRP) and MRP1 (multidrug resistance protein 1; ABCC1). Both compounds are more potent MRP4 inhibitors in cellular assays than the most widely used inhibitor, MK-571, requiring lower concentrations to effect a comparable level of inhibition. Furthermore, Ceefourin 1 and Ceefourin 2 have low cellular toxicity, and high microsomal and acid stability. These newly identified inhibitors should be of great value for efforts to better understand the biological roles of MRP4, and may represent classes of compounds with therapeutic application. Copyright © 2014 Elsevier Inc. All rights reserved.

Modulation of human multidrug-resistance MDR-1 gene by natural curcuminoids

PubMed Central

Limtrakul, Pornngarm; Anuchapreeda, Songyot; Buddhasukh, Duang

2004-01-01

Background Multidrug resistance (MDR) is a phenomenon that is often associated with decreased intracellular drug accumulation in patient's tumor cells resulting from enhanced drug efflux. It is related to the overexpression of a membrane protein, P-glycoprotein (Pgp-170), thereby reducing drug cytotoxicity. A variety of studies have tried to find MDR modulators which increase drug accumulation in cancer cells. Methods In this study, natural curcuminoids, pure curcumin, demethoxycurcumin and bisdemethoxycurcumin, isolated from turmeric (Curcuma longa Linn), were compared for their potential ability to modulate the human MDR-1 gene expression in multidrug resistant human cervical carcinoma cell line, KB-V1 by Western blot analysis and RT-PCR. Results Western blot analysis and RT-PCR showed that all the three curcuminoids inhibited MDR-1 gene expression, and bisdemethoxycurcumin produced maximum effect. In additional studies we found that commercial grade curcuminoid (approximately 77% curcumin, 17% demethoxycurcumin and 3% bisdemthoxycurcumin) decreased MDR-1 gene expression in a dose dependent manner and had about the same potent inhibitory effect on MDR-1 gene expression as our natural curcuminoid mixtures. Conclusion These results indicate that bisdemethoxycurcumin is the most active of the curcuminoids present in turmeric for modulation of MDR-1 gene. Treatment of drug resistant KB-V1 cells with curcumin increased their sensitivity to vinblastine, which was consistent with a decreased MDR-1 gene product, a P-glycoprotein, on the cell plasma membrane. Although many drugs that prevent the P-glycoprotein function have been reported, this report describes the inhibition of MDR-1 expression by a phytochemical. The modulation of MDR-1 expression may be an attractive target for new chemosensitizing agents. PMID:15090070

Effects of Zuccagnia punctata extracts and their flavonoids on the function and expression of ABCB1/P-glycoprotein multidrug transporter.

PubMed

Chieli, Elisabetta; Romiti, Nadia; Catiana Zampini, Iris; Garrido, Gabino; Inés Isla, María

2012-12-18

Zuccagnia punctata extracts (ZpE) are used in ethnomedicine as antimicrobial and anti-inflammatory drugs. The pharmacological properties of ZpE and their polyphenolic components suggest that they may be used as potential modulators on the P-glycoprotein (P-gp) multidrug transporter. P-gp is well known for its role in the acquired drug resistance by tumors following chemotherapy, causing a low drug bioavailability by extruding them out of the cells. To evaluate the effects of ZpE and three of their phenolic components: 7-hydroxyflavanone (HF), 3,7-dihydroxyflavone (DHF) and 2',4'-dihydroxychalcone (DHC) on P-gp activity and expression. The effects of natural products on ABCB1/P-gp function and expression were evaluated by R-123 accumulation assay and western blot analysis using HK-2 cells as experimental model. The ABCB1 mRNA content was determined by SQRT-PCR. The accumulation of R-123 in HK-2 cells was significantly increased by ZpE and DHF, and to a lesser extent by DHC, indicating their roles on the efflux transporter activity. However, HF did not show any effect. HK-2 cells maintained in the presence of ZpE or DHF for 72 h, showed an increase in P-gp expression whereas activity was unchanged or decreased. No changes were observed in ABCB1 mRNA content. Furthermore, in these assay conditions, more sensibility of HK-2 cells to the cytotoxic action of cyclosporine A (P-gp substrate) was observed. These results may suggest an impact of Zuccagnia punctata and some of its components on the pharmacokinetics of drugs that are P-gp substrates, as well as a potential role on multidrug resistance modulation. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Overcoming the heterologous bias: an in vivo functional analysis of multidrug efflux transporter, CgCdr1p in matched pair clinical isolates of Candida glabrata.

PubMed

Puri, Nidhi; Manoharlal, Raman; Sharma, Monika; Sanglard, Dominique; Prasad, Rajendra

2011-01-07

We have taken advantage of the natural milieu of matched pair of azole sensitive (AS) and azole resistant (AR) clinical isolates of Candida glabrata for expressing its major ABC multidrug transporter, CgCdr1p for structure and functional analysis. This was accomplished by tagging a green fluorescent protein (GFP) downstream of ORF of CgCDR1 and integrating the resultant fusion protein at its native chromosomal locus in AS and AR backgrounds. The characterization confirmed that in comparison to AS isolate, CgCdr1p-GFP was over-expressed in AR isolates due to its hyperactive native promoter and the GFP tag did not affect its functionality in either construct. We observed that in addition to Rhodamine 6 G (R6G) and Fluconazole (FLC), a recently identified fluorescent substrate of multidrug transporters Nile Red (NR) could also be expelled by CgCdr1p. Competition assays with these substrates revealed the presence of overlapping multiple drug binding sites in CgCdr1p. Point mutations employing site directed mutagenesis confirmed that the role played by unique amino acid residues critical to ATP catalysis and localization of ABC drug transporter proteins are well conserved in C. glabrata as in other yeasts. This study demonstrates a first in vivo novel system where over-expression of GFP tagged MDR transporter protein can be driven by its own hyperactive promoter of AR isolates. Taken together, this in vivo system can be exploited for the structure and functional analysis of CgCdr1p and similar proteins wherein the artefactual concerns encountered in using heterologous systems are totally excluded. Copyright © 2010 Elsevier Inc. All rights reserved.

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 silver nanoparticles are effective in eliminating MDR Enterobacter cloacae isolates and their action was not inhibited by AcrAB-TolC efflux protein expression. As such, the above nanoparticles have strong potential to be used as effective and alternate therapeutic candidates to combat MDR gram-negative Enterobacterial pathogens.

The Elementary Mass Action Rate Constants of P-gp Transport for a Confluent Monolayer of MDCKII-hMDR1 Cells

PubMed Central

Tran, Thuy Thanh; Mittal, Aditya; Aldinger, Tanya; Polli, Joseph W.; Ayrton, Andrew; Ellens, Harma; Bentz, Joe

2005-01-01

The human multi-drug resistance membrane transporter, P-glycoprotein, or P-gp, has been extensively studied due to its importance to human health and disease. Thus far, the kinetic analysis of P-gp transport has been limited to steady-state Michaelis-Menten approaches or to compartmental models, neither of which can prove molecular mechanisms. Determination of the elementary kinetic rate constants of transport will be essential to understanding how P-gp works. The experimental system we use is a confluent monolayer of MDCKII-hMDR1 cells that overexpress P-gp. It is a physiologically relevant model system, and transport is measured without biochemical manipulations of P-gp. The Michaelis-Menten mass action reaction is used to model P-gp transport. Without imposing the steady-state assumptions, this reaction depends upon several parameters that must be simultaneously fitted. An exhaustive fitting of transport data to find all possible parameter vectors that best fit the data was accomplished with a reasonable computation time using a hierarchical algorithm. For three P-gp substrates (amprenavir, loperamide, and quinidine), we have successfully fitted the elementary rate constants, i.e., drug association to P-gp from the apical membrane inner monolayer, drug dissociation back into the apical membrane inner monolayer, and drug efflux from P-gp into the apical chamber, as well as the density of efflux active P-gp. All three drugs had overlapping ranges for the efflux active P-gp, which was a benchmark for the validity of the fitting process. One novel finding was that the association to P-gp appears to be rate-limited solely by drug lateral diffusion within the inner monolayer of the plasma membrane for all three drugs. This would be expected if P-gp structure were open to the lipids of the apical membrane inner monolayer, as has been suggested by recent structural studies. The fitted kinetic parameters show how P-gp efflux of a wide range of xenobiotics has been maximized. PMID:15501934

Multidrug Resistance-Associated Protein 4 (MRP4/ABCC4) Controls Efflux Transport of Hesperetin Sulfates in Sulfotransferase 1A3-Overexpressing Human Embryonic Kidney 293 Cells.

PubMed

Sun, Hua; Wang, Xiao; Zhou, Xiaotong; Lu, Danyi; Ma, Zhiguo; Wu, Baojian

2015-10-01

Sulfonation is an important metabolic pathway for hesperetin. However, the mechanisms for the cellular disposition of hesperetin and its sulfate metabolites are not fully established. In this study, disposition of hesperetin via the sulfonation pathway was investigated using human embryonic kidney (HEK) 293 cells overexpressing sulfotransferase 1A3. Two monosulfates, hesperetin-3'-O-sulfate (H-3'-S) and hesperetin-7-O-sulfate (H-7-S), were rapidly generated and excreted into the extracellular compartment upon incubation of the cells with hesperetin. Regiospecific sulfonation of hesperetin by the cell lysate followed the substrate inhibition kinetics (Vmax = 0.66 nmol/min per mg, Km = 12.9 μM, and Ksi= 58.1 μM for H-3'-S; Vmax = 0.29 nmol/min per mg, Km = 14.8 μM, and Ksi= 49.1 μM for H-7-S). The pan-multidrug resistance-associated protein (MRP) inhibitor MK-571 at 20 μM essentially abolished cellular excretion of both H-3'-S and H-7-S (the excretion activities were only 6% of the control), whereas the breast cancer resistance protein-selective inhibitor Ko143 had no effects on sulfate excretion. In addition, knockdown of MRP4 led to a substantial reduction (>47.1%; P < 0.01) in sulfate excretion. Further, H-3'-S and H-7-S were good substrates for transport by MRP4 according to the vesicular transport assay. Moreover, sulfonation of hesperetin and excretion of its metabolites were well characterized by a two-compartment pharmacokinetic model that integrated drug uptake and sulfonation with MRP4-mediated sulfate excretion. In conclusion, the exporter MRP4 controlled efflux transport of hesperetin sulfates in HEK293 cells. Due to significant expression in various organs/tissues (including the liver and kidney), MRP4 should be a determining factor for the elimination and body distribution of hesperetin sulfates. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Involvement of P-glycoprotein and multidrug resistance associated protein 1 in the transport of tanshinone IIB, a primary active diterpenoid quinone from the roots of Salvia miltiorrhiza, across the blood-brain barrier.

PubMed

Zhou, Zhi-Wei; Chen, Xiao; Liang, Jun; Yu, Xi-Yong; Wen, Jing-Yuan; Zhou, Shu-Feng

2007-08-01

Tanshinone IIB (TSB) is a major constituent of Salvia miltiorrhiza, which is widely used in treatment of cardiovascular and central nervous system (CNS) diseases such as coronary heart disease and stroke. This study aimed to investigate the role of various drug transporters in the brain penetration of TSB using several in vitro and in vivo mouse and rat models. The uptake and efflux of TSB in rat primary microvascular endothelial cells (RBMVECs) were ATP-dependent and significantly altered in the presence of a P-glycoprotein (P-gp) or multidrug resistance associated protein (Mrp1/2) inhibitor. A polarized transport of TSB was found in RBMVEC monolayers with facilitated efflux from the abluminal to luminal side. Addition of a P-gp inhibitor (e.g. verapamil) in both abluminal and luminal sides attenuated the polarized transport. In an in situ rat brain perfusion model, TSB crossed the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier at a greater rate than that for sucrose, and the brain penetration was increased in the presence of a P-gp or Mrp1/2 inhibitor. The brain levels of TSB were only about 30% of that in the plasma and it could be increased to up to 72% of plasma levels when verapamil, quinidine, or probenecid was co-administered in rats. The entry of TSB to CNS increased by 67-97% in rats subjected to middle cerebral artery occlusion or treatment with the neurotoxin, quinolinic acid, compared to normal rats. Furthermore, The brain levels of TSB in mdr1a(-/-) and mrp1(-/-) mice were 28- to 2.6-fold higher than those in the wild-type mice. TSB has limited brain penetration through the BBB due to the contribution of P-gp and to a lesser extent of Mrp1 in rodents. Further studies are needed to confirm whether these corresponding transporters in humans are involved in limiting the penetration of TSB across the BBB and the clinical relevance.

Characterization and Genomic Study of Phage vB_EcoS-B2 Infecting Multidrug-Resistant Escherichia coli

PubMed Central

Xu, Yue; Yu, Xinyan; Gu, Yu; Huang, Xu; Liu, Genyan; Liu, Xiaoqiu

2018-01-01

The potential of bacteriophage as an alternative antibacterial agent has been reconsidered for control of pathogenic bacteria due to the widespread occurrence of multi-drug resistance bacteria. More and more lytic phages have been isolated recently. In the present study, we isolated a lytic phage named vB_EcoS-B2 from waste water. VB_EcoS-B2 has an icosahedral symmetry head and a long tail without a contractile sheath, indicating that it belongs to the family Siphoviridae. The complete genome of vB_EcoS-B2 is composed of a circular double stranded DNA of 44,283 bp in length, with 54.77% GC content. vB_EcoS-B2 is homologous to 14 relative phages (such as Escherichia phage SSL-2009a, Escherichia phage JL1, and Shigella phage EP23), but most of these phages exhibit different gene arrangement. Our results serve to extend our understanding toward phage evolution of family Siphoviridae of coliphages. Sixty-five putative open reading frames were predicted in the complete genome of vB_EcoS-B2. Twenty-one of proteins encoded by vB_EcoS-B2 were determined in phage particles by Mass Spectrometry. Bacteriophage genome and proteome analysis confirmed the lytic nature of vB_EcoS-B2, namely, the absence of toxin-coding genes, islands of pathogenicity, or genes through lysogeny or transduction. Furthermore, vB_EcoS-B2 significantly reduced the growth of E. coli MG1655 and also inhibited the growth of several multi-drug resistant clinical stains of E. coli. Phage vB_EcoS-B2 can kill some of the MRD E. coli entirely, strongly indicating us that it could be one of the components of phage cocktails to treat multi-drug resistant E. coli. This phage could be used to interrupt or reduce the spread of multi-drug resistant E. coli. PMID:29780362

Overcoming the heterologous bias: An in vivo functional analysis of multidrug efflux transporter, CgCdr1p in matched pair clinical isolates of Candida glabrata

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

Puri, Nidhi; Manoharlal, Raman; Sharma, Monika

2011-01-07

Research highlights: {yields} First report to demonstrate an in vivo expression system of an ABC multidrug transporter CgCdr1p of C. glabrata. {yields} First report on the structure and functional characterization of CgCdr1p. {yields} Functional conservation of divergent but typical residues of CgCdr1p. {yields} CgCdr1p elicits promiscuity towards substrates and has a large drug binding pocket with overlapping specificities. -- Abstract: We have taken advantage of the natural milieu of matched pair of azole sensitive (AS) and azole resistant (AR) clinical isolates of Candida glabrata for expressing its major ABC multidrug transporter, CgCdr1p for structure and functional analysis. This was accomplishedmore » by tagging a green fluorescent protein (GFP) downstream of ORF of CgCDR1 and integrating the resultant fusion protein at its native chromosomal locus in AS and AR backgrounds. The characterization confirmed that in comparison to AS isolate, CgCdr1p-GFP was over-expressed in AR isolates due to its hyperactive native promoter and the GFP tag did not affect its functionality in either construct. We observed that in addition to Rhodamine 6 G (R6G) and Fluconazole (FLC), a recently identified fluorescent substrate of multidrug transporters Nile Red (NR) could also be expelled by CgCdr1p. Competition assays with these substrates revealed the presence of overlapping multiple drug binding sites in CgCdr1p. Point mutations employing site directed mutagenesis confirmed that the role played by unique amino acid residues critical to ATP catalysis and localization of ABC drug transporter proteins are well conserved in C. glabrata as in other yeasts. This study demonstrates a first in vivo novel system where over-expression of GFP tagged MDR transporter protein can be driven by its own hyperactive promoter of AR isolates. Taken together, this in vivo system can be exploited for the structure and functional analysis of CgCdr1p and similar proteins wherein the arte-factual concerns encountered in using heterologous systems are totally excluded.« less

Genome Sequence of Cronobacter sakazakii BAA-894 and Comparative Genomic Hybridization Analysis with Other Cronobacter Species

PubMed Central

Kucerova, Eva; Clifton, Sandra W.; Xia, Xiao-Qin; Long, Fred; Porwollik, Steffen; Fulton, Lucinda; Fronick, Catrina; Minx, Patrick; Kyung, Kim; Warren, Wesley; Fulton, Robert; Feng, Dongyan; Wollam, Aye; Shah, Neha; Bhonagiri, Veena; Nash, William E.; Hallsworth-Pepin, Kymberlie; Wilson, Richard K.

2010-01-01

Background The genus Cronobacter (formerly called Enterobacter sakazakii) is composed of five species; C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, and C. dublinensis. The genus includes opportunistic human pathogens, and the first three species have been associated with neonatal infections. The most severe diseases are caused in neonates and include fatal necrotizing enterocolitis and meningitis. The genetic basis of the diversity within the genus is unknown, and few virulence traits have been identified. Methodology/Principal Findings We report here the first sequence of a member of this genus, C. sakazakii strain BAA-894. The genome of Cronobacter sakazakii strain BAA-894 comprises a 4.4 Mb chromosome (57% GC content) and two plasmids; 31 kb (51% GC) and 131 kb (56% GC). The genome was used to construct a 387,000 probe oligonucleotide tiling DNA microarray covering the whole genome. Comparative genomic hybridization (CGH) was undertaken on five other C. sakazakii strains, and representatives of the four other Cronobacter species. Among 4,382 annotated genes inspected in this study, about 55% of genes were common to all C. sakazakii strains and 43% were common to all Cronobacter strains, with 10–17% absence of genes. Conclusions/Significance CGH highlighted 15 clusters of genes in C. sakazakii BAA-894 that were divergent or absent in more than half of the tested strains; six of these are of probable prophage origin. Putative virulence factors were identified in these prophage and in other variable regions. A number of genes unique to Cronobacter species associated with neonatal infections (C. sakazakii, C. malonaticus and C. turicensis) were identified. These included a copper and silver resistance system known to be linked to invasion of the blood-brain barrier by neonatal meningitic strains of Escherichia coli. In addition, genes encoding for multidrug efflux pumps and adhesins were identified that were unique to C. sakazakii strains from outbreaks in neonatal intensive care units. PMID:20221447

Essential Oils and Their Components as Modulators of Antibiotic Activity against Gram-Negative Bacteria

PubMed Central

Aelenei, Petruta; Miron, Anca; Trifan, Adriana; Bujor, Alexandra; Gille, Elvira; Aprotosoaie, Ana Clara

2016-01-01

Gram-negative bacteria cause infections that are difficult to treat due to the emergence of multidrug resistance. This review summarizes the current status of the studies investigating the capacity of essential oils and their components to modulate antibiotic activity against Gram-negative bacteria. Synergistic interactions are particularly discussed with reference to possible mechanisms by which essential oil constituents interact with antibiotics. Special emphasis is given to essential oils and volatile compounds that inhibit efflux pumps, thus reversing drug resistance in Gram-negative bacteria. In addition, indifference and antagonism between essential oils/volatile compounds and conventional antibiotics have also been reported. Overall, this literature review reveals that essential oils and their purified components enhance the efficacy of antibiotics against Gram-negative bacteria, being promising candidates for the development of new effective formulations against Gram-negative bacteria. PMID:28930130

eUnaG: a new ligand-inducible fluorescent reporter to detect drug transporter activity in live cells

PubMed Central

Yeh, Johannes T.-H.; Nam, Kwangho; Yeh, Joshua T.-H.; Perrimon, Norbert

2017-01-01

The absorption, distribution, metabolism and excretion (ADME) of metabolites and toxic organic solutes are orchestrated by the ATP-binding cassette (ABC) transporters and the organic solute carrier family (SLC) proteins. A large number of ABC and SLC transpoters exist; however, only a small number have been well characterized. To facilitate the analysis of these transporters, which is important for drug safety and physiological studies, we developed a sensitive genetically encoded bilirubin (BR)-inducible fluorescence sensor (eUnaG) to detect transporter-coupled influx/efflux of organic compounds. This sensor can be used in live cells to measure transporter activity, as excretion of BR depends on ABC and SLC transporters. Applying eUnaG in functional RNAi screens, we characterize l(2)03659 as a Drosophila multidrug resistant-associated ABC transporter. PMID:28176814

ATP-binding cassette (ABC) proteins in aquatic invertebrates: Evolutionary significance and application in marine ecotoxicology.

PubMed

Jeong, Chang-Bum; Kim, Hui-Su; Kang, Hye-Min; Lee, Jae-Seong

2017-04-01

The ATP-binding cassette (ABC) protein superfamily is known to play a fundamental role in biological processes and is highly conserved across animal taxa. The ABC proteins function as active transporters for multiple substrates across the cellular membrane by ATP hydrolysis. As this superfamily is derived from a common ancestor, ABC genes have evolved via lineage-specific duplications through the process of adaptation. In this review, we summarized information about the ABC gene families in aquatic invertebrates, considering their evolution and putative functions in defense mechanisms. Phylogenetic analysis was conducted to examine the evolutionary significance of ABC gene families in aquatic invertebrates. Particularly, a massive expansion of multixenobiotic resistance (MXR)-mediated efflux transporters was identified in the absence of the ABCG2 (BCRP) gene in Ecdysozoa and Platyzoa, suggesting that a loss of Abcg2 gene occurred sporadically in these species during divergence of Protostome to Lophotrochozoa. Furthermore, in aquatic invertebrates, the ecotoxicological significance of MXR is discussed while considering the role of MXR-mediated efflux transporters in response to various environmental pollutants. Copyright © 2017 Elsevier B.V. All rights reserved.

Streptomyces venezuelae ISP5230 Maintains Excretion of Jadomycin upon Disruption of the MFS Transporter JadL Located within the Natural Product Biosynthetic Gene Cluster

PubMed Central

Forget, Stephanie M.; McVey, Jennifer; Vining, Leo C.

2017-01-01

JadL was identified as a Major Facilitator Superfamily (MFS) transporter (T.C. 2.A.1) through sequence homology. The protein is encoded by jadL, situated within the jadomycin biosynthetic gene cluster. JadL has, therefore, been assigned a putative role in host defense by exporting its probable substrates, the jadomycins, a family of secondary metabolites produced by Streptomyces venezuelae ISP5230. Herein, we evaluate this assumption through the construction and analysis of a jadL disrupted mutant, S. venezuelae VS678 (ΔjadL::aac(3)IV). Quantitative determination of jadomycin production with the jadL disrupted mutant did not show a significant decrease in production in comparison to the wildtype strain, as determined by HPLC and by tandem mass spectrometry. These results suggest that efflux of jadomycin occurs upon disruption of jadL, or that JadL is not involved in jadomycin efflux. Potentially, other transporters within S. venezuelae ISP5230 may adopt this role upon inactivation of JadL to export jadomycins. PMID:28377749

Enhanced transport of P-glycoprotein substrate saquinavir in presence of thiolated chitosan.

PubMed

Föger, Florian; Kafedjiiski, Krum; Hoyer, Herbert; Loretz, Brigitta; Bernkop-Schnürch, Andreas

2007-02-01

It was the aim of this study to investigate the effect of chitosan-4-thiobutylamidine (Ch-TBA) and reduced glutathione (GSH) on the absorption of P-glycoprotein (P-gp) and multidrug resistance protein (MRP) substrate saquinavir in vitro and in vivo. Bidirectional transport studies were performed with Caco-2 cell monolayers and additionally with freshly excised rat small intestinal mucosa mounted in Ussing type chambers. Furthermore, a delivery system based on Ch-TBA and GSH was evaluated in vivo in rats. The functional activity of the efflux pumps in Caco-2 cells and rat intestinal mucosa during the experiment was proven by the efflux ratio of saquinavir, which was 6.4 for Caco-2 cells and 2.1 for rat intestinal mucosa, respectively. Ch-TBA and particularly the combination of Ch-TBA with GSH enhanced apical (AP) absorption and decreased the secretory transport of saquinavir. In presence of 0.5% Ch-TBA and 0.5% GSH, the uptake of saquinavir was 1.6-fold improved in Caco-2 monolayer and 2.1-fold improved in rat intestinal mucosa. In vivo, the area under the plasma concentration time curve (AUC) of saquinavir was 1.4-fold and Cmax 1.6-fold increased, in comparison with control. Results of this study showed that Ch-TBA in combination with GSH can be an interesting tool for increasing the oral bioavailability of actively secreted compounds.

In vitro screening of dual flavonoid combinations for reversing P-glycoprotein-mediated multidrug resistance: Focus on antiepileptic drugs.

PubMed

Ferreira, Ana; Santos, Adriana O; Falcão, Amílcar; Alves, Gilberto

2018-01-01

The combined use of different P-glycoprotein (P-gp) inhibitors may be a relevant approach to the synergistic and safer inhibition of the P-gp-mediated drug efflux. Herein, we aimed to explore dual combinations of the flavonoids baicalein, (-)-epigallocatechin gallate, kaempferol, quercetin and silymarin to reverse the interference of P-gp on the intracellular accumulation of antiepileptic drugs (AEDs). The intracellular accumulation of rhodamine 123 (a classic P-gp substrate) and of several commonly used AEDs (carbamazepine, phenytoin, oxcarbazepine) or their metabolites (carbamazepine-10,11-epoxide and licarbazepine) was evaluated in MDCK-MDR1 cells in the presence and absence of individual flavonoids and their combinations. A selected flavonoid combination [(-)-epigallocatechin gallate/silymarin] was also evaluated in transepithelial transport experiments using licarbazepine (active metabolite of oxcarbazepine) as a model compound. Most flavonoid combinations increased rhodamine 123 intracellular uptake in a greater extent than their additive individual effects at similar concentrations. Moreover, selected (-)-epigallocatechin gallate/silymarin and kaempferol/baicalein combinations also enhanced the intracellular accumulation of all AEDs and metabolites. Overall, the combination of (-)-epigallocatechin gallate/silymarin was the most promising one. Thus, dual flavonoid combinations may be useful to overcome the P-gp-mediated efflux of AEDs and their metabolites, making their association to AED therapy a potentially valuable approach to circumvent pharmacoresistance in epilepsy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Listeria monocytogenes Strains Selected on Ciprofloxacin or the Disinfectant Benzalkonium Chloride Exhibit Reduced Susceptibility to Ciprofloxacin, Gentamicin, Benzalkonium Chloride, and Other Toxic Compounds▿

PubMed Central

Rakic-Martinez, Mira; Drevets, Douglas A.; Dutta, Vikrant; Katic, Vera; Kathariou, Sophia

2011-01-01

Listeria monocytogenes is a leading agent for severe food-borne illness and death in the United States and other nations. Even though drug resistance has not yet threatened therapeutic interventions for listeriosis, selective pressure associated with exposure to antibiotics and disinfectants may result in reduced susceptibility to these agents. In this study, selection of several L. monocytogenes strains on either ciprofloxacin (2 μg/ml) or the quaternary ammonium disinfectant benzalkonium chloride (BC; 10 μg/ml) led to derivatives with increased MICs not only to these agents but also to several other toxic compounds, including gentamicin, the dye ethidium bromide, and the chemotherapeutic drug tetraphenylphosphonium chloride. The spectrum of compounds to which these derivatives exhibited reduced susceptibility was the same regardless of whether they were selected on ciprofloxacin or on BC. Inclusion of strains harboring the large plasmid pLM80 revealed that MICs to ciprofloxacin and gentamicin did not differ between the parental and plasmid-cured strains. However, ciprofloxacin-selected derivatives of pLM80-harboring strains had higher MICs than those derived from the plasmid-cured strains. Susceptibility to the antimicrobials was partially restored in the presence of the potent efflux inhibitor reserpine. Taken together, these data suggest that mutations in efflux systems are responsible for the multidrug resistance phenotype of strains selected on ciprofloxacin or BC. PMID:22003016

Biofilms in vitro and in vivo: do singular mechanisms imply cross-resistance?

PubMed

Gilbert, P; Allison, D G; McBain, A J

2002-01-01

Microbial biofilm has become inexorably linked with man's failure to control them by antibiotic and biocide regimes that are effective against suspended bacteria. This failure relates to a localized concentration of biofilm bacteria, and their extracellular products (exopolymers and extracellular enzymes), that moderates the access of the treatment agent and starves the more deeply placed cells. Biofilms, therefore, typically present gradients of physiology and concentration for the imposed treatment agent, which enables the less susceptible clones to survive. Such clones might include efflux mutants in addition to genotypes with modifications in single gene products. Clonal expansion following subeffective treatment would, in the case of many antibiotics, lead to the emergence of a resistant population. This tends not to occur for biocidal treatments where the active agent exhibits multiple pharmacological activity towards a number of specific cellular targets. Whilst resistance development towards biocidal agents is highly unlikely, subeffective exposure will lead to the selection of less susceptible clones, modified either in efflux or in their most susceptible target. The latter might also confer resistance to antibiotics where the target is shared. Thus, recent reports have demonstrated that sublethal concentrations of the antibacterial and antifungal agent triclosan can select for resistant mutants in Escherichia coli and that this agent specifically targets the enzyme enoyl reductase that is involved in lipid biosynthesis. Triclosan may, therefore, select for mutants in a target that is shared with the anti-E. coli diazaborine compounds and the antituberculosis drug isoniazid. Although triclosan may be a uniquely specific biocide, sublethal concentrations of less specific antimicrobial agents may also select for mutations within their most sensitive targets, some of which might be common to therapeutic agents. Sublethal treatment with chemical antimicrobial agents has also been demonstrated to induce the expression of multidrug efflux pumps and efflux mutants. Whilst efflux does not confer protection against use concentrations of biocidal products it is sufficient to confer protection against therapeutic doses of many antibiotics. It has, therefore, been widely speculated that biocide misuse may have an insidious effect, contributing to the evolution and persistence of drug resistance within microbial communities. Whilst such notions are supported by laboratory studies that utilize pure cultures, recent evidence has strongly refuted such linkage within the general environment where complex, multispecies biofilms predominate and where biocidal products are routinely deployed. In such situations the competition, for nutrients and space, between community members of disparate sensitivities far outweighs any potential benefits bestowed by the changes in an individual's antimicrobial susceptibility.

The effects of gill remodeling on transepithelial sodium fluxes and the distribution of presumptive sodium-transporting ionocytes in goldfish (Carassius auratus).

PubMed

Bradshaw, Julia C; Kumai, Yusuke; Perry, Steve F

2012-04-01

Goldfish, Carassius auratus, adaptively remodel their gills in response to changes in ambient oxygen and temperature, altering the functional lamellar surface area to balance the opposing requirements for respiration and osmoregulation. In this study, the effects of thermal- and hypoxia-mediated gill remodeling on branchial Na(+) fluxes and the distribution of putative Na(+)-transporting ionocytes in goldfish were assessed. When assessed either in vitro (isolated gill arches) or in vivo at a common water temperature, the presence of an interlamellar cell mass (ILCM) in fish acclimated to 7°C clearly decreased Na(+) efflux across the gill relative to fish maintained at 25°C and lacking an ILCM. However, loss of the ILCM in 7°C-acclimated fish exposed to hypoxia led to a decrease in Na(+) efflux (assessed under hypoxic conditions) despite the apparent large increases in functional lamellar surface area. Goldfish possessing an ILCM were able to sustain Na(+) uptake, albeit at a lower rate matched to efflux, owing to the re-distribution of ionocytes expressing genes thought to be involved in Na(+) uptake [Na(+)/H(+) exchanger isoform 3 (NHE3) and V- type H(+)-ATPase] to the edge of the ILCM where they can establish contact with the surrounding environment. NHE-expressing cells co-localized with Na(+)/K(+)-ATPase expression, suggesting a role for NHE in Na(+)-uptake in the goldfish. Implications of the ILCM on ion fluxes in the goldfish are discussed.

Dual activation of CFTR and CLCN2 by lubiprostone in murine nasal epithelia.

PubMed

Schiffhauer, Eric S; Vij, Neeraj; Kovbasnjuk, Olga; Kang, Po Wei; Walker, Doug; Lee, Seakwoo; Zeitlin, Pamela L

2013-03-01

Multiple sodium and chloride channels on the apical surface of nasal epithelial cells contribute to periciliary fluid homeostasis, a function that is disrupted in patients with cystic fibrosis (CF). Among these channels is the chloride channel CLCN2, which has been studied as a potential alternative chloride efflux pathway in the absence of CFTR. The object of the present study was to use the nasal potential difference test (NPD) to quantify CLCN2 function in an epithelial-directed TetOn CLCN2 transgenic mouse model (TGN-K18rtTA-hCLCN2) by using the putative CLCN2 pharmacological agonist lubiprostone and peptide inhibitor GaTx2. Lubiprostone significantly increased chloride transport in the CLCN2-overexpressing mice following activation of the transgene by doxycycline. This response to lubiprostone was significantly inhibited by GaTx2 after CLCN2 activation in TGN-CLCN2 mice. Cftr(-/-) and Clc2(-/-) mice showed hyperpolarization indicative of chloride efflux in response to lubiprostone, which was fully inhibited by GaTx2 and CFTR inhibitor 172 + GlyH-101, respectively. Our study reveals lubiprostone as a pharmacological activator of both CFTR and CLCN2. Overexpression and activation of CLCN2 leads to improved mouse NPD readings, suggesting it is available as an alternative pathway for epithelial chloride secretion in murine airways. The utilization of CLCN2 as an alternative chloride efflux channel could provide clinical benefit to patients with CF, especially if the pharmacological activator is administered as an aerosol.

Role of the Caenorhabditis elegans multidrug resistance gene, mrp-4, in gut granule differentiation.

PubMed

Currie, Erin; King, Brian; Lawrenson, Andrea L; Schroeder, Lena K; Kershner, Aaron M; Hermann, Greg J

2007-11-01

Caenorhabditis elegans gut granules are lysosome-related organelles with birefringent contents. mrp-4, which encodes an ATP-binding cassette (ABC) transporter homologous to mammalian multidrug resistance proteins, functions in the formation of gut granule birefringence. mrp-4(-) embryos show a delayed appearance of birefringent material in the gut granule but otherwise appear to form gut granules properly. mrp-4(+) activity is required for the extracellular mislocalization of birefringent material, body-length retraction, and NaCl sensitivity, phenotypes associated with defective gut granule biogenesis exhibited by embryos lacking the activity of GLO-1/Rab38, a putative GLO-1 guanine nucleotide exchange factor GLO-4, and the AP-3 complex. Multidrug resistance protein (MRP)-4 localizes to the gut granule membrane, consistent with it playing a direct role in the transport of molecules that compose and/or facilitate the formation of birefringent crystals within the gut granule. However, MRP-4 is also present in oocytes and early embryos, and our genetic analyses indicate that its site of action in the formation of birefringent material may not be limited to just the gut granule in embryos. In a search for genes that function similarly to mrp-4(+), we identified WHT-2, another ABC transporter that acts in parallel to MRP-4 for the formation of birefringent material in the gut granule.

Population Genetics Study of Isoniazid Resistance Mutations and Evolution of Multidrug-Resistant Mycobacterium tuberculosis†

PubMed Central

Hazbón, Manzour Hernando; Brimacombe, Michael; Bobadilla del Valle, Miriam; Cavatore, Magali; Guerrero, Marta Inírida; Varma-Basil, Mandira; Billman-Jacobe, Helen; Lavender, Caroline; Fyfe, Janet; García-García, Lourdes; León, Clara Inés; Bose, Mridula; Chaves, Fernando; Murray, Megan; Eisenach, Kathleen D.; Sifuentes-Osornio, José; Cave, M. Donald; Ponce de León, Alfredo; Alland, David

2006-01-01

The molecular basis for isoniazid resistance in Mycobacterium tuberculosis is complex. Putative isoniazid resistance mutations have been identified in katG, ahpC, inhA, kasA, and ndh. However, small sample sizes and related potential biases in sample selection have precluded the development of statistically valid and significant population genetic analyses of clinical isoniazid resistance. We present the first large-scale analysis of 240 alleles previously associated with isoniazid resistance in a diverse set of 608 isoniazid-susceptible and 403 isoniazid-resistant clinical M. tuberculosis isolates. We detected 12 mutant alleles in isoniazid-susceptible isolates, suggesting that these alleles are not involved in isoniazid resistance. However, mutations in katG, ahpC, and inhA were strongly associated with isoniazid resistance, while kasA mutations were associated with isoniazid susceptibility. Remarkably, the distribution of isoniazid resistance-associated mutations was different in isoniazid-monoresistant isolates from that in multidrug-resistant isolates, with significantly fewer isoniazid resistance mutations in the isoniazid-monoresistant group. Mutations in katG315 were significantly more common in the multidrug-resistant isolates. Conversely, mutations in the inhA promoter were significantly more common in isoniazid-monoresistant isolates. We tested for interactions among mutations and resistance to different drugs. Mutations in katG, ahpC, and inhA were associated with rifampin resistance, but only katG315 mutations were associated with ethambutol resistance. There was also a significant inverse association between katG315 mutations and mutations in ahpC or inhA and between mutations in kasA and mutations in ahpC. Our results suggest that isoniazid resistance and the evolution of multidrug-resistant strains are complex dynamic processes that may be influenced by interactions between genes and drug-resistant phenotypes. PMID:16870753

Emodin reverses leukemia multidrug resistance by competitive inhibition and downregulation of P-glycoprotein

PubMed Central

Min, Hongping; Niu, Miaomiao; Zhang, Weilin; Yan, Jia; Li, Jiachang; Tan, Xiying; Li, Bo; Su, Mengxiang; Di, Bin; Yan, Fang

2017-01-01

Development of multidrug resistance (MDR) is a continuous clinical challenge partially due to the overexpression of P-glycoprotein (P-gp) for chronic myelogenous leukemia (CML) patients. Herein, we evaluated the inhibitory potency of emodin, a natural anthraquinone derivative isolated from Rheum palmatum L, on P-gp in P-gp positive K562/ADM cells. Competition experiments combined with molecular docking analysis were utilized to investigate the binding modes between emodin and binding sites of P-gp. Emodin reversed adriamycin resistance in K562/ADM cells accompanied with the decrease of P-gp protein expression, further increasing the uptake of rhodamine123 in both K562/ADM and Caco-2 cells, indicating the inhibition of P-gp efflux function. Moreover, when incubated with emodin under different conditions where P-gp was inhibited, K562/ADM cells displayed increasing intracellular uptake of emodin, suggesting that emodin may be the potential substrate of P-gp. Importantly, rhodamine 123 could increase the Kintrinsic (Ki) value of emodin linearly, whereas, verapamil could not, implying that emodin competitively bound to the R site of P-gp and noncompetition existed between emodin and verapamil at the M site, in a good accordance with the results of molecular docking that emodin bound to the R site of P-gp with higher affinity. Based on our results, we suggest that emodin might be used to modulate P-gp function and expression. PMID:29121121

Emodin reverses leukemia multidrug resistance by competitive inhibition and downregulation of P-glycoprotein.

PubMed

Min, Hongping; Niu, Miaomiao; Zhang, Weilin; Yan, Jia; Li, Jiachang; Tan, Xiying; Li, Bo; Su, Mengxiang; Di, Bin; Yan, Fang

2017-01-01

Development of multidrug resistance (MDR) is a continuous clinical challenge partially due to the overexpression of P-glycoprotein (P-gp) for chronic myelogenous leukemia (CML) patients. Herein, we evaluated the inhibitory potency of emodin, a natural anthraquinone derivative isolated from Rheum palmatum L, on P-gp in P-gp positive K562/ADM cells. Competition experiments combined with molecular docking analysis were utilized to investigate the binding modes between emodin and binding sites of P-gp. Emodin reversed adriamycin resistance in K562/ADM cells accompanied with the decrease of P-gp protein expression, further increasing the uptake of rhodamine123 in both K562/ADM and Caco-2 cells, indicating the inhibition of P-gp efflux function. Moreover, when incubated with emodin under different conditions where P-gp was inhibited, K562/ADM cells displayed increasing intracellular uptake of emodin, suggesting that emodin may be the potential substrate of P-gp. Importantly, rhodamine 123 could increase the Kintrinsic (Ki) value of emodin linearly, whereas, verapamil could not, implying that emodin competitively bound to the R site of P-gp and noncompetition existed between emodin and verapamil at the M site, in a good accordance with the results of molecular docking that emodin bound to the R site of P-gp with higher affinity. Based on our results, we suggest that emodin might be used to modulate P-gp function and expression.

Tamoxifen reduces P-gp-mediated multidrug resistance via inhibiting the PI3K/Akt signaling pathway in ER-negative human gastric cancer cells.

PubMed

Mao, Zonglei; Zhou, Jin; Luan, Junwei; Sheng, Weihua; Shen, Xiaochun; Dong, Xiaoqiang

2014-03-01

Multidrug resistance (MDR), mediated by overexpression of drug efflux transporters such as P-glycoprotein (P-gp), is a major problem limiting successful chemotherapy of gastric cancer. Tamoxifen (TAM), a triphenylethylene nonsteroidal antiestrogen agent, shows broad-spectrum antitumor properties. Emerging studies demonstrated that TAM could significantly reduce the MDR in a variety of human cancers. Here we investigated the effects and possible underlying mechanisms of action of TAM on the reversion of MDR in ER-negative human gastric cancer cells. Our results demonstrated that in MDR phenotype SGC7901/CDDP gastric cancer cells TAM dramatically lowered the IC50 of CDDP, 5-FU and ADM, increased the intracellular Rhodamine123 accumulation and induced G0/G1 phase arrest, while G2/M phase decreased accordingly. Furthermore, at the molecular level, TAM substantially decreased the expression of P-gp, p-Akt and the Akt-regulated downstream effectors such as p-GSK-3β, p-BAD, Bcl-XL and cyclinD1 proteins without affecting the expression of t-Akt, t-GSK-3β, t-BAD proteins in SGC7901/CDDP cells. Thus, our findings demonstrate that TAM reverses P-gp-mediated gastric cancer cell MDR via inhibiting the PI3K/Akt signaling pathway. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Multi-Drug Resistance Transporter 2 Regulates Mucosal Inflammation by Facilitating the Synthesis of Hepoxilin A3

PubMed Central

Pazos, Michael; Siccardi, Dario; Mumy, Karen L.; Bien, Jeffrey D.; Louie, Steve; Shi, Hai Ning; Gronert, Karsten; Mrsny, Randall J.; McCormick, Beth A.

2008-01-01

Neutrophil transmigration across mucosal surfaces contributes to dysfunction of epithelial barrier properties, a characteristic underlying many mucosal inflammatory diseases. Thus, insight into the directional movement of neutrophils across epithelial barriers will provide important information relating to the mechanisms of such inflammatory disorders. The eicosanoid hepoxilin A3, an endogenous product of 12-lipoxygenase activity, is secreted from the apical surface of the epithelial barrier and establishes a chemotatic gradient to guide neutrophils from the submucosa, across epithelia to the luminal site of an inflammatory stimulus - the final step in neutrophil recruitment. Currently, little is known regarding how hepoxilin A3 is secreted from the intestinal epithelium during an inflammatory insult. In this study we reveal that hepoxilin A3 is a substrate for the apical efflux ABC transporter, multi-drug resistance protein 2 (MRP2). Moreover, using multiple in vitro and in vivo models we show that induction of intestinal inflammation profoundly up-regulates apical expression of MRP2, and that interfering with hepoxilin A3 synthesis and/or inhibition of MRP2 function results in a marked reduction in inflammation and severity of disease. Lastly, examination of inflamed intestinal epithelia in human biopsies revealed up-regulation of MRP2. Thus, blocking hepoxilin A3 synthesis and/or inhibiting MRP2 may lead to the development of new therapeutic strategies for the treatment of epithelial-associated inflammatory conditions. PMID:19017997

Quercetin-glutamic acid conjugate with a non-hydrolysable linker; a novel scaffold for multidrug resistance reversal agents through inhibition of P-glycoprotein.

PubMed

Kim, Mi Kyoung; Kim, Yunyoung; Choo, Hyunah; Chong, Youhoon

2017-02-01

Previously, we have reported remarkable effect of a quercetin-glutamic acid conjugate to reverse multidrug resistance (MDR) of cancer cells to a broad spectrum of anticancer agents through inhibition of P-glycoprotein (Pgp)-mediated drug efflux. Due to the hydrolysable nature, MDR-reversal activity of the quercetin conjugate was attributed to its hydrolysis product, quercetin. However, several lines of evidence demonstrated that the intact quercetin-glutamic acid conjugate has stronger MDR-reversal activity than quercetin. In order to evaluate this hypothesis and to identify a novel scaffold for MDR-reversal agents, we prepared quercetin conjugates with a glutamic acid attached at the 7-O position via a non-hydrolysable linker. Pgp inhibition assay, Pgp ATPase assay, and MDR-reversal activity assay were performed, and the non-hydrolysable quercetin conjugates showed significantly higher activities compared with those of quercetin. Unfortunately, the quercetin conjugates were not as effective as verapamil in Pgp-inhibition and thereby reversing MDR, but it is worth to note that the structurally modified quercetin conjugates with a non-cleavable linker showed significantly improved MDR-reversal activity compared with quercetin. Taken together, the quercetin conjugates with appropriate structural modifications were shown to have a potential to serve as a scaffold for the design of novel MDR-reversal agents. Copyright © 2016 Elsevier Ltd. All rights reserved.