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.

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.

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.

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

PubMed

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

2017-02-23

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

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.

Analysis of resistance genes of clinical Pannonibacter phragmitetus strain 31801 by complete genome sequencing.

PubMed

Ming, De-Song; Chen, Qing-Qing; Chen, Xiao-Tin

2018-05-14

To clarify the resistance mechanisms of Pannonibacter phragmitetus 31801, isolated from the blood of a liver abscess patient, at the genomic level, we performed whole genomic sequencing using a PacBio RS II single-molecule real-time long-read sequencer. Bioinformatic analysis of the resulting sequence was then carried out to identify any possible resistance genes. Analyses included Basic Local Alignment Search Tool searches against the Antibiotic Resistance Genes Database, ResFinder analysis of the genome sequence, and Resistance Gene Identifier analysis within the Comprehensive Antibiotic Resistance Database. Prophages, clustered regularly interspaced short palindromic repeats (CRISPR), and other putative virulence factors were also identified using PHAST, CRISPRfinder, and the Virulence Factors Database, respectively. The circular chromosome and single plasmid of P. phragmitetus 31801 contained multiple antibiotic resistance genes, including those coding for three different types of β-lactamase [NPS β-lactamase (EC 3.5.2.6), β-lactamase class C, and a metal-dependent hydrolase of β-lactamase superfamily I]. In addition, genes coding for subunits of several multidrug-resistance efflux pumps were identified, including those targeting macrolides (adeJ, cmeB), tetracycline (acrB, adeAB), fluoroquinolones (acrF, ceoB), and aminoglycosides (acrD, amrB, ceoB, mexY, smeB). However, apart from the tripartite macrolide efflux pump macAB-tolC, the genome did not appear to contain the complete complement of subunit genes required for production of most of the major multidrug-resistance efflux pumps.

The Transcriptional Regulators NorG and MgrA Modulate Resistance to both Quinolones and β-Lactams in Staphylococcus aureus▿

PubMed Central

Truong-Bolduc, Que Chi; Hooper, David C.

2007-01-01

MgrA is a known regulator of the expression of several multidrug transporters in Staphylococcus aureus. We identified another regulator of multiple efflux pumps, NorG, by its ability, like that of MgrA, to bind specifically to the promoter of the gene encoding the NorA efflux pump. NorG is a member of the family of the GntR-like transcriptional regulators, and it binds specifically to the putative promoters of the genes encoding multidrug efflux pumps NorA, NorB, NorC, and AbcA. Overexpression of norG produces a threefold increase in norB transcripts associated with a fourfold increase in the level of resistance to quinolones. In contrast, disruption of norG produces no change in the level of transcripts of norA, norB, and norC but causes an increase of at least threefold in the transcript level of abcA, associated with a fourfold increase in resistance to methicillin, cefotaxime, penicillin G, and nafcillin. Overexpression of cloned abcA caused an 8- to 128-fold increase in the level of resistance to all four β-lactam antibiotics. Furthermore, MgrA and NorG have opposite effects on norB and abcA expression. MgrA acts as an indirect repressor for norB and a direct activator for abcA, whereas NorG acts as a direct activator for norB and a direct repressor for abcA. PMID:17277059

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.

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

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.

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

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

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

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.

A Pseudomonas putida efflux pump acts on short-chain alcohols.

PubMed

Basler, Georg; Thompson, Mitchell; Tullman-Ercek, Danielle; Keasling, Jay

2018-01-01

The microbial production of biofuels is complicated by a tradeoff between yield and toxicity of many fuels. Efflux pumps enable bacteria to tolerate toxic substances by their removal from the cells while bypassing the periplasm. Their use for the microbial production of biofuels can help to improve cell survival, product recovery, and productivity. However, no native efflux pump is known to act on the class of short-chain alcohols, important next-generation biofuels, and it was considered unlikely that such an efflux pump exists. We report that controlled expression of the RND-type efflux pump TtgABC from Pseudomonas putida DOT-T1E strongly improved cell survival in highly toxic levels of the next-generation biofuels n -butanol, isobutanol, isoprenol, and isopentanol. GC-FID measurements indicated active efflux of n -butanol when the pump is expressed. Conversely, pump expression did not lead to faster growth in media supplemented with low concentrations of n -butanol and isopentanol. TtgABC is the first native efflux pump shown to act on multiple short-chain alcohols. Its controlled expression can be used to improve cell survival and increase production of biofuels as an orthogonal approach to metabolic engineering. Together with the increased interest in P. putida for metabolic engineering due to its flexible metabolism, high native tolerance to toxic substances, and various applications of engineering its metabolism, our findings endorse the strain as an excellent biocatalyst for the high-yield production of next-generation biofuels.

Unexpected effects of azole transporter inhibitors on antifungal susceptibility in Candida glabrata and other pathogenic Candida species

PubMed Central

Nagayoshi, Yohsuke; Shimamura, Shintaro; Nakayama, Hironobu; Minematsu, Asuka; Yamauchi, Shunsuke; Takazono, Takahiro; Nakamura, Shigeki; Yanagihara, Katsunori; Kohno, Shigeru; Mukae, Hiroshi; Izumikawa, Koichi

2017-01-01

The pathogenic fungus Candida glabrata is often resistant to azole antifungal agents. Drug efflux through azole transporters, such as Cdr1 and Cdr2, is a key mechanism of azole resistance and these genes are under the control of the transcription factor Pdr1. Recently, the monoamine oxidase A (MAO-A) inhibitor clorgyline was shown to inhibit the azole efflux pumps, leading to increased azole susceptibility in C. glabrata. In the present study, we have evaluated the effects of clorgyline on susceptibility of C. glabrata to not only azoles, but also to micafungin and amphotericin B, using wild-type and several mutant strains. The addition of clorgyline to the culture media increased fluconazole susceptibility of a C. glabrata wild-type strain, whereas micafungin and amphotericin B susceptibilities were markedly decreased. These phenomena were also observed in other medically important Candida species, including Candida albicans, Candida parapsilosis, Candida tropicalis, and Candida krusei. Expression levels of CDR1, CDR2 and PDR1 mRNAs and an amount of Cdr1 protein in the C. glabrata wild-type strain were highly increased in response to the treatment with clorgyline. However, loss of Cdr1, Cdr2, Pdr1, and a putative clorgyline target (Fms1), which is an ortholog of human MAO-A, or overexpression of CDR1 did not affect the decreased susceptibility to micafungin and amphotericin B in the presence of clorgyline. The presence of other azole efflux pump inhibitors including milbemycin A4 oxime and carbonyl cyanide 3-chlorophenylhydrazone also decreased micafungin susceptibility in C. glabrata wild-type, Δcdr1, Δcdr2, and Δpdr1 strains. These findings suggest that azole efflux pump inhibitors increase azole susceptibility but concurrently induce decreased susceptibility to other classes of antifungals independent of azole transporter functions. PMID:28700656

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

PubMed Central

Piedade, Ana Paula; Morais, Paula V.

2015-01-01

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

Unexpected effects of azole transporter inhibitors on antifungal susceptibility in Candida glabrata and other pathogenic Candida species.

PubMed

Nagayoshi, Yohsuke; Miyazaki, Taiga; Shimamura, Shintaro; Nakayama, Hironobu; Minematsu, Asuka; Yamauchi, Shunsuke; Takazono, Takahiro; Nakamura, Shigeki; Yanagihara, Katsunori; Kohno, Shigeru; Mukae, Hiroshi; Izumikawa, Koichi

2017-01-01

The pathogenic fungus Candida glabrata is often resistant to azole antifungal agents. Drug efflux through azole transporters, such as Cdr1 and Cdr2, is a key mechanism of azole resistance and these genes are under the control of the transcription factor Pdr1. Recently, the monoamine oxidase A (MAO-A) inhibitor clorgyline was shown to inhibit the azole efflux pumps, leading to increased azole susceptibility in C. glabrata. In the present study, we have evaluated the effects of clorgyline on susceptibility of C. glabrata to not only azoles, but also to micafungin and amphotericin B, using wild-type and several mutant strains. The addition of clorgyline to the culture media increased fluconazole susceptibility of a C. glabrata wild-type strain, whereas micafungin and amphotericin B susceptibilities were markedly decreased. These phenomena were also observed in other medically important Candida species, including Candida albicans, Candida parapsilosis, Candida tropicalis, and Candida krusei. Expression levels of CDR1, CDR2 and PDR1 mRNAs and an amount of Cdr1 protein in the C. glabrata wild-type strain were highly increased in response to the treatment with clorgyline. However, loss of Cdr1, Cdr2, Pdr1, and a putative clorgyline target (Fms1), which is an ortholog of human MAO-A, or overexpression of CDR1 did not affect the decreased susceptibility to micafungin and amphotericin B in the presence of clorgyline. The presence of other azole efflux pump inhibitors including milbemycin A4 oxime and carbonyl cyanide 3-chlorophenylhydrazone also decreased micafungin susceptibility in C. glabrata wild-type, Δcdr1, Δcdr2, and Δpdr1 strains. These findings suggest that azole efflux pump inhibitors increase azole susceptibility but concurrently induce decreased susceptibility to other classes of antifungals independent of azole transporter functions.

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

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

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

PubMed

Soto, Sara M

2013-04-01

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

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

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

PubMed

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

2016-06-13

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

The major facilitator superfamily transporter Knq1p modulates boron homeostasis in Kluyveromyces lactis.

PubMed

Svrbicka, Alexandra; Toth Hervay, Nora; Gbelska, Yvetta

2016-03-01

Boron is an essential micronutrient for living cells, yet its excess causes toxicity. To date, the mechanisms of boron toxicity are poorly understood. Recently, the ScATR1 gene has been identified encoding the main boron efflux pump in Saccharomyces cerevisiae. In this study, we analyzed the ScATR1 ortholog in Kluyveromyces lactis--the KNQ1 gene, to understand whether it participates in boron stress tolerance. We found that the KNQ1 gene, encoding a permease belonging to the major facilitator superfamily, is required for K. lactis boron tolerance. Deletion of the KNQ1 gene led to boron sensitivity and its overexpression increased K. lactis boron tolerance. The KNQ1 expression was induced by boron and the intracellular boron concentration was controlled by Knq1p. The KNQ1 promoter contains two putative binding motifs for the AP-1-like transcription factor KlYap1p playing a central role in oxidative stress defense. Our results indicate that the induction of the KNQ1 expression requires the presence of KlYap1p and that Knq1p like its ortholog ScAtr1p in S. cerevisiae functions as a boron efflux pump providing boron resistance in K. lactis.

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

PubMed

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

2012-10-24

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

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

PubMed

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

2008-07-24

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

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

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

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.

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

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.

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.

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

PubMed

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

2016-01-18

In this study, efflux pump-mediated benzalkonium chloride (BC) resistance, including plasmid-encoded (Qac protein family and BcrABC) and chromosome-borne efflux pumps, was investigated in Listeria monocytogenes from retail food in China. Among the 59 L. monocytogenes strains, 13 (22.0%) strains were resistant to BC. The PCR results showed that bcrABC was harbored by 2 of 13 BC resistant strains. However, none of the qac genes were detected among the 59 strains. The bcrABC was absent in both of the plasmid cured strains, indicating that this BC resistance determinant was plasmid-encoded in the two bcrABC-positive strains. In the presence of reserpine, most of the bcrABC-negative strains had decreases in the MICs of BC, suggesting the existence of other efflux pumps and their role in BC resistance. After exposed to reserpine, the reduction in BC MICs was observed in the two cured strains, indicating that efflux pumps located on chromosome was also involved in BC resistance. Our findings suggest that food products may act as reservoirs for BC resistant isolates of L. monocytogenes and plasmid- and chromosome-encoded efflux pumps could mediate the BC resistance of L. monocytogenes, which is especially relevant to the adaption of this organism in food-related environments with frequent BC use. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

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.

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

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

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.

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

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

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

PubMed

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

2014-01-01

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

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 Efflux Systems in Microaerobic and Anaerobic Bacteria

PubMed Central

Xu, Zeling; Yan, Aixin

2015-01-01

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

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

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

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.

Quorum-sensing control of antibiotic resistance stabilizes cooperation in Chromobacterium violaceum.

PubMed

Evans, Kara C; Benomar, Saida; Camuy-Vélez, Lennel A; Nasseri, Ellen B; Wang, Xiaofei; Neuenswander, Benjamin; Chandler, Josephine R

2018-05-01

Many Proteobacteria use quorum sensing to regulate production of public goods, such as antimicrobials and proteases, that are shared among members of a community. Public goods are vulnerable to exploitation by cheaters, such as quorum sensing-defective mutants. Quorum sensing- regulated private goods, goods that benefit only producing cells, can prevent the emergence of cheaters under certain growth conditions. Previously, we developed a laboratory co-culture model to investigate the importance of quorum-regulated antimicrobials during interspecies competition. In our model, Burkholderia thailandensis and Chromobacterium violaceum each use quorum sensing-controlled antimicrobials to inhibit the other species' growth. Here, we show that C. violaceum uses quorum sensing to increase resistance to bactobolin, a B. thailandensis antibiotic, by increasing transcription of a putative antibiotic efflux pump. We demonstrate conditions where C. violaceum quorum-defective cheaters emerge and show that in these conditions, bactobolin restrains cheaters. We also demonstrate that bactobolin restrains quorum-defective mutants in our co-culture model, and the increase in antimicrobial-producing cooperators drives the C. violaceum population to become more competitive. Our results describe a mechanism of cheater restraint involving quorum control of efflux pumps and demonstrate that interspecies competition can reinforce cooperative behaviors by placing constraints on quorum sensing-defective mutants.

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.

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.

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.

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.

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

PubMed Central

2011-01-01

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

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.

Low Sensitivity of Listeria monocytogenes to Quaternary Ammonium Compounds

PubMed Central

Mereghetti, L.; Quentin, R.; Marquet-Van Der Mee, N.; Audurier, A.

2000-01-01

Ninety-seven epidemiologically unrelated strains of Listeria monocytogenes were investigated for their sensitivities to quaternary ammonium compounds (benzalkonium chloride and cetrimide). The MICs for seven serogroup 1/2 strains were high. Three came from the environment and four came from food; none were isolated from human or animal samples. All 97 strains carried the mdrL gene, which encodes a multidrug efflux pump, and the orfA gene, a putative transcriptional repressor of mdrL. The absence of plasmids in four of the seven resistant strains and the conservation of resistance after plasmid curing suggested that the resistance genes are not plasmid borne. Moreover, PCR amplification and Southern blot hybridization experiments failed to find genes phylogenetically related to the qacA and smr genes, encoding multidrug efflux systems previously described for the genus Staphylococcus. The high association between nontypeability by phages and the loss of sensitivity to quaternary ammonium compounds are suggestive of an intrinsic resistance due to modifications in the cell wall. PMID:11055967

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

PubMed

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

2012-03-08

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

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.

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

PubMed

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

2006-03-01

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

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

PubMed

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

2016-09-01

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

Engineering microbial biofuel tolerance and export using efflux pumps

PubMed Central

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

2011-01-01

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

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

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

Mukhopadhyay, Aindrila

2016-07-14

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

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

PubMed

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

2017-10-01

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

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.

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

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

PubMed Central

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

2003-01-01

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

Modified host cells with efflux pumps

DOEpatents

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

2016-08-30

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

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.

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

PubMed

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

2004-08-01

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

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

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

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

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

PubMed

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

2015-02-10

Multidrug efflux systems are a major cause of resistance to antimicrobials in bacteria, including those pathogenic to humans, animals, and plants. These proteins are ubiquitous in these pathogens, and five families of bacterial multidrug efflux systems have been identified to date. By using transcriptomic and biochemical analyses, we recently identified the novel AceI (Acinetobacter chlorhexidine efflux) protein from Acinetobacter baumannii that conferred resistance to the biocide chlorhexidine, via an active efflux mechanism. Proteins homologous to AceI are encoded in the genomes of many other bacterial species and are particularly prominent within proteobacterial lineages. In this study, we expressed 23 homologs of AceI and examined their resistance and/or transport profiles. MIC analyses demonstrated that, like AceI, many of the homologs conferred resistance to chlorhexidine. Many of the AceI homologs conferred resistance to additional biocides, including benzalkonium, dequalinium, proflavine, and acriflavine. We conducted fluorimetric transport assays using the AceI homolog from Vibrio parahaemolyticus and confirmed that resistance to both proflavine and acriflavine was mediated by an active efflux mechanism. These results show that this group of AceI homologs represent a new family of bacterial multidrug efflux pumps, which we have designated the proteobacterial antimicrobial compound efflux (PACE) family of transport proteins. Bacterial multidrug efflux pumps are an important class of resistance determinants that can be found in every bacterial genome sequenced to date. These transport proteins have important protective functions for the bacterial cell but are a significant problem in the clinical setting, since a single efflux system can mediate resistance to many structurally and mechanistically diverse antibiotics and biocides. In this study, we demonstrate that proteins related to the Acinetobacter baumannii AceI transporter are a new class of multidrug efflux systems which are very common in Proteobacteria: the proteobacterial antimicrobial compound efflux (PACE) family. This is the first new family of multidrug efflux pumps to be described in 15 years. Copyright © 2015 Hassan et al.

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

Zgurskaya, Helen; Smith, Jeremy

ORNL leveraged powerful supercomputing to support research led by University of Oklahoma scientists to identify chemicals that seek out and disrupt bacterial proteins called efflux pumps, known to be a major cause of antibiotic resistance. By running simulations on Titan, the team selected molecules most likely to target and potentially disable the assembly of efflux pumps found in E. coli bacteria cells.

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

2015-01-28

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

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

DOE PAGES

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

2017-06-26

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

Efflux drug transporters at the forefront of antimicrobial resistance.

PubMed

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

2017-10-01

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

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

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

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

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

The Role of the Transcription Factors MtrR and MtrA in the Fitness of the Pathogen Neisseria gonorrhoeae

DTIC Science & Technology

2007-10-19

MacA -MacB efflux pump is expressed at very low levels in N. gonorrhoeae; however, experimental alteration of the promoter sequence showed that the...specificity (132). The gonococcus expresses four efflux pump systems, namely MtrC-Mtr-D-MtrE (64), FarA-FarB-MtrE (97), NorM (158), and MacA -MacB (161...2005. Characterization of the MacA -MacB efflux system in Neisseria gonorrhoeae. The Journal of antimicrobial chemotherapy 56:856-860. 162. Rouquette, C

Transcriptional profiling suggests that multiple metabolic adaptations are required for effective proliferation of Pseudomonas aeruginosa in jet fuel.

PubMed

Gunasekera, Thusitha S; Striebich, Richard C; Mueller, Susan S; Strobel, Ellen M; Ruiz, Oscar N

2013-01-01

Fuel is a harsh environment for microbial growth. However, some bacteria can grow well due to their adaptive mechanisms. Our goal was to characterize the adaptations required for Pseudomonas aeruginosa proliferation in fuel. We have used DNA-microarrays and RT-PCR to characterize the transcriptional response of P. aeruginosa to fuel. Transcriptomics revealed that genes essential for medium- and long-chain n-alkane degradation including alkB1 and alkB2 were transcriptionally induced. Gas chromatography confirmed that P. aeruginosa possesses pathways to degrade different length n-alkanes, favoring the use of n-C11-18. Furthermore, a gamut of synergistic metabolic pathways, including porins, efflux pumps, biofilm formation, and iron transport, were transcriptionally regulated. Bioassays confirmed that efflux pumps and biofilm formation were required for growth in jet fuel. Furthermore, cell homeostasis appeared to be carefully maintained by the regulation of porins and efflux pumps. The Mex RND efflux pumps were required for fuel tolerance; blockage of these pumps precluded growth in fuel. This study provides a global understanding of the multiple metabolic adaptations required by bacteria for survival and proliferation in fuel-containing environments. This information can be applied to improve the fuel bioremediation properties of bacteria.

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

USDA-ARS?s Scientific Manuscript database

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

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

PubMed

Langevin, Ariel M; Dunlop, Mary J

2018-01-01

Stress tolerance studies are typically conducted in an all-or-none fashion. However, in realistic settings-such as in clinical or metabolic engineering applications-cells may encounter stresses at different rates. Therefore, how cells tolerate stress may depend on its rate of appearance. To address this, we studied how the rate of stress introduction affects bacterial stress tolerance by focusing on a key stress response mechanism. Efflux pumps, such as AcrAB-TolC of Escherichia coli , are membrane transporters well known for the ability to export a wide variety of substrates, including antibiotics, signaling molecules, and biofuels. Although efflux pumps improve stress tolerance, pump overexpression can result in a substantial fitness cost to the cells. We hypothesized that the ideal pump expression level would involve a rate-dependent trade-off between the benefit of pumps and the cost of their expression. To test this, we evaluated the benefit of the AcrAB-TolC pump under different rates of stress introduction, including a step, a fast ramp, and a gradual ramp. Using two chemically diverse stresses, the antibiotic chloramphenicol and the jet biofuel precursor pinene, we assessed the benefit provided by the pumps. A mathematical model describing these effects predicted the benefit as a function of the rate of stress introduction. Our findings demonstrate that as the rate of introduction is lowered, stress response mechanisms provide a disproportionate benefit to pump-containing strains, allowing cells to survive beyond the original inhibitory concentrations. IMPORTANCE Efflux pumps are ubiquitous in nature and provide stress tolerance in the cells of species ranging from bacteria to mammals. Understanding how pumps provide tolerance has far-reaching implications for diverse fields, from medicine to biotechnology. Here, we investigated how the rate of stressor appearance impacts tolerance. We focused on two distinct substrates of AcrAB-TolC efflux pumps, the antibiotic chloramphenicol and the biofuel precursor pinene. Interestingly, tolerance is highly dependent on the rate of stress introduction. Therefore, it is important to consider not only the total quantity of a stressor but also the rate at which it is applied. The implications of this work are significant because environments are rarely static; antibiotic concentrations change during dosing, and metabolic engineering processes change with time. Copyright © 2017 American Society for Microbiology.

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.

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

PubMed Central

Hong, Hyerim; Jung, Jaejoon; Park, Woojun

2014-01-01

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

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

DOE PAGES

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

2014-10-13

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

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

PubMed

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

2014-10-28

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

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.

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

PubMed

Hong, Hyerim; Jung, Jaejoon; Park, Woojun

2014-01-01

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

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

PubMed Central

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

2015-01-01

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

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

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 ...

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

PubMed

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

2011-04-22

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

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

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.

Supercomputer Simulations Help Develop New Approach to Fight Antibiotic Resistance

ScienceCinema

Zgurskaya, Helen; Smith, Jeremy

2018-06-13

ORNL leveraged powerful supercomputing to support research led by University of Oklahoma scientists to identify chemicals that seek out and disrupt bacterial proteins called efflux pumps, known to be a major cause of antibiotic resistance. By running simulations on Titan, the team selected molecules most likely to target and potentially disable the assembly of efflux pumps found in E. coli bacteria cells.

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

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

PubMed

Attaran, Bahareh; Falsafi, Tahereh; Ghorbanmehr, Nassim

2017-02-21

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

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

PubMed Central

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

2016-01-01

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

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

PubMed

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

2016-01-01

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

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.

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.

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

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.

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

PubMed

Kumar, Ayush; Worobec, Elizabeth A

2002-10-01

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

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

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

PubMed

Mombeshora, Molly; Mukanganyama, Stanley

2017-07-26

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

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

PubMed

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

2017-10-01

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

Xenon for tunnelling analysis of the efflux pump component OprN.

PubMed

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

2017-01-01

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

Xenon for tunnelling analysis of the efflux pump component OprN

PubMed Central

Garnier, Cyril; Ducruix, Arnaud; Broutin, Isabelle

2017-01-01

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

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.

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.

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

PubMed

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

2018-03-01

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

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

PubMed

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

2012-10-01

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

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.

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

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.

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

PubMed

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

2017-03-01

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

NorA efflux pump inhibitory activity of coumarins from Mesua ferrea.

PubMed

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

2013-10-01

The purpose of this investigation was to study the modulator and efflux pump inhibitor activity of coumarins isolated from Mesua ferrea against clinical strains as well as NorA-over expressed strain of Staphylococcus aureus 1199B. Seven coumarins were tested for modulator activity using ethidium bromide (EtBr) as a substrate. Compounds 1, 4-7 modulated the MIC of EtBr by ≥ 2 fold against wild type clinical strains of S. aureus 1199 and S. aureus 1199B, whereas compounds 4-7 modulated the MIC of EtBr by ≥ 16 fold against MRSA 831. Compounds 1, 4-7 also reduced the MIC of norfloxacin by ≥ 8 fold against S. aureus 1199B, and 4-6 reduced the MIC of norfloxacin by ≥ 8 fold against MRSA 831 at half of their MICs. Inhibition of EtBr efflux by NorA-overproducing S. aureus 1199B and MRSA 831 confirmed the role of compounds 4-6 as NorA efflux pump inhibitors (EPI). Dose-dependent activity at sub-inhibitory concentration (6.25 μg/mL) suggested that compounds 4 and 5 are promising EPI compared to verapamil against 1199B and MRSA 831 strains. © 2013.

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

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

PubMed

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

2016-04-01

In this study, we tested five compounds belonging to a novel series of piperazine arylideneimidazolones for the ability to inhibit the AcrAB-TolC efflux pump. The biphenylmethylene derivative (BM-19) and the fluorenylmethylene derivative (BM-38) were found to possess the strongest efflux pump inhibitor (EPI) activities in the AcrAB-TolC-overproducingEscherichia colistrain 3-AG100, whereas BM-9, BM-27, and BM-36 had no activity at concentrations of up to 50 μM in a Nile red efflux assay. MIC microdilution assays demonstrated that BM-19 at 1/4 MIC (intrinsic MIC, 200 μM) was able to reduce the MICs of levofloxacin, oxacillin, linezolid, and clarithromycin 8-fold. BM-38 at 1/4 MIC (intrinsic MIC, 100 μM) was able to reduce only the MICs of oxacillin and linezolid (2-fold). Both compounds markedly reduced the MIC of rifampin (BM-19, 32-fold; and BM-38, 4-fold), which is suggestive of permeabilization of the outer membrane as an additional mechanism of action. Nitrocefin hydrolysis assays demonstrated that in addition to their EPI activity, both compounds were in fact weak permeabilizers of the outer membrane. Moreover, it was found that BM-19, BM-27, BM-36, and BM-38 acted as near-infrared-emitting fluorescent membrane probes, which allowed for their use in a combined influx and efflux assay and thus for tracking of the transport of an EPI across the outer membrane by an efflux pump in real time. The EPIs BM-38 and BM-19 displayed the most rapid influx of all compounds, whereas BM-27, which did not act as an EPI, showed the slowest influx. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2017-01-01

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

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

PubMed Central

Al-Mohizea, Abdullah M.

2010-01-01

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

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

PubMed

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

2017-05-01

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

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

PubMed

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

2016-09-10

Among the mechanisms adopted by bacteria, efflux pumps (EPs) have been recognized as being significantly involved in contributing to resistance to commonly used antibacterial agents. However, little is known about their three-dimensional structures or the steric requirements for their inhibition. Lack of such knowledge includes NorA, one of the most studied Staphylococcus aureus EPs. In the present study, the use of two commercialized Cinchona alkaloid-based zwitterionic chiral stationary phases allowed the enantioseparation of four 2-((2-(4-propoxyphenyl)quinolin-4-yl)oxy)alkylamines 1-4 previously found to be potent S. aureus NorA efflux pump inhibitors when tested as racemates. In the identified optimal polar-ionic conditions (MeOH/THF/H2O-49/49/2 (v/v/v)+25mM formic acid, 12.5mM diethylamine), repeated consecutive injections of 1 allowed the isolation of sufficient amount of its enantiomers (2.6mg and 2.8mg, for (R)-1 and (S)-1, respectively) and then to evaluate their ability to inhibit the S. aureus NorA efflux pump. The biological evaluation highlighted the main contribution of the (R)-1 enantiomer to both the EtBr efflux inhibition and synergistic effect with against SA-1199B (norA+/A116E GrlA) respect to the racemate activity. The comparison between the experimental electronic circular dichroism and the time-dependent density functional theory calculations spectra of the two isolated enantiomeric fractions allowed for all compounds a clear and easy assignment of the enantiomeric elution order. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed

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

2017-01-12

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

Pseudomonas aeruginosa PumA acts on an endogenous phenazine to promote self-resistance.

PubMed

Sporer, Abigail J; Beierschmitt, Christopher; Bendebury, Anastasia; Zink, Katherine E; Price-Whelan, Alexa; Buzzeo, Marisa C; Sanchez, Laura M; Dietrich, Lars E P

2018-05-01

The activities of critical metabolic and regulatory proteins can be altered by exposure to natural or synthetic redox-cycling compounds. Many bacteria, therefore, possess mechanisms to transport or transform these small molecules. The opportunistic pathogen Pseudomonas aeruginosa PA14 synthesizes phenazines, redox-active antibiotics that are toxic to other organisms but have beneficial effects for their producer. Phenazines activate the redox-sensing transcription factor SoxR and thereby induce the transcription of a small regulon, including the operon mexGHI-opmD, which encodes an efflux pump that transports phenazines, and PA14_35160 (pumA), which encodes a putative monooxygenase. Here, we provide evidence that PumA contributes to phenazine resistance and normal biofilm development, particularly during exposure to or production of strongly oxidizing N-methylated phenazines. We show that phenazine resistance depends on the presence of residues that are conserved in the active sites of other putative and characterized monooxygenases found in the antibiotic producer Streptomyces coelicolor. We also show that during biofilm growth, PumA is required for the conversion of phenazine methosulfate to unique phenazine metabolites. Finally, we compare ∆mexGHI-opmD and ∆pumA strains in assays for colony biofilm morphogenesis and SoxR activation, and find that these deletions have opposing phenotypic effects. Our results suggest that, while MexGHI-OpmD-mediated efflux has the effect of making the cellular phenazine pool more reducing, PumA acts on cellular phenazines to make the pool more oxidizing. We present a model in which these two SoxR targets function simultaneously to control the biological activity of the P. aeruginosa phenazine pool.

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.

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

PubMed

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

2018-10-01

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

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

Motuporamine Derivatives as Antimicrobial Agents and Antibiotic Enhancers against Resistant Gram-Negative Bacteria.

PubMed

Borselli, Diane; Blanchet, Marine; Bolla, Jean-Michel; Muth, Aaron; Skruber, Kristen; Phanstiel, Otto; Brunel, Jean Michel

2017-02-01

Dihydromotuporamine C and its derivatives were evaluated for their in vitro antimicrobial activities and antibiotic enhancement properties against Gram-negative bacteria and clinical isolates. The mechanism of action of one of these derivatives, MOTU-N44, was investigated against Enterobacter aerogenes by using fluorescent dyes to evaluate outer-membrane depolarization and permeabilization. Its efficiency correlated with inhibition of dye transport, thus suggesting that these molecules inhibit drug transporters by de-energization of the efflux pump rather than by direct interaction of the molecule with the pump. This suggests that depowering the efflux pump provides another strategy to address antibiotic resistance. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

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

PubMed

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

2016-10-01

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

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

PubMed

Ahmad, Aijaz; Khan, Amber; Manzoor, Nikhat

2013-01-23

Thymol (THY) and carvacrol (CARV), the principal chemical components of thyme oil have long been known for their wide use in medicine due to antimicrobial and disinfectant properties. This study, however, draws attention to a possible synergistic antifungal effect of these monoterpenes with azole antimycotic-fluconazole. Resistance to azoles in Candida albicans involves over-expression of efflux-pump genes MDR1, CDR1, CDR2 or mutations and over-expression of target gene ERG11. The inhibition of drug efflux pumps is considered a feasible strategy to overcome clinical antifungal resistance. To put forward this approach, we investigated the combination effects of these monoterpenes and FLC against 38 clinically obtained FLC-sensitive, and eleven FLC-resistant Candida isolates. Synergism was observed with combinations of THY-FLC and CARV-FLC evaluated by checkerboard microdilution method and nature of the interactions was calculated by FICI. In addition, antifungal activity was assessed using agar-diffusion and time-kill curves. The drug efflux activity was determined using two dyes, Rhodamine6G (R6G) and fluorescent Hoechst 33342. No significant differences were observed in dye uptakes between FLC-susceptible and resistant isolates, incubated in glucose free buffer. However, a significantly higher efflux was recorded in FLC-resistant isolates when glucose was added. Both monoterpenes inhibited efflux by 70-90%, showing their high potency to block drug transporter pumps. Significant differences, in the expression levels of CDR1 and MDR1, induced by monoterpenes revealed reversal of FLC-resistance. The selectively fungicidal characteristics and ability to restore FLC susceptibility in resistant isolates signify a promising candidature of THY and CARV as antifungal agents in combinational treatments for candidiasis. Copyright © 2012 Elsevier B.V. All rights reserved.

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

PubMed Central

2011-01-01

Background Antimicrobial resistance mediated by efflux systems is still poorly characterized in Staphylococcus aureus, despite the description of several efflux pumps (EPs) for this bacterium. In this work we used several methodologies to characterize the efflux activity of 52 S. aureus isolates resistant to ciprofloxacin collected in a hospital in Lisbon, Portugal, in order to understand the role played by these systems in the resistance to fluoroquinolones. Results Augmented efflux activity was detected in 12 out of 52 isolates and correlated with increased resistance to fluoroquinolones. Addition of efflux inhibitors did not result in the full reversion of the fluoroquinolone resistance phenotype, yet it implied a significant decrease in the resistance levels, regardless of the type(s) of mutation(s) found in the quinolone-resistance determining region of grlA and gyrA genes, which accounted for the remaining resistance that was not efflux-mediated. Expression analysis of the genes coding for the main efflux pumps revealed increased expression only in the presence of inducing agents. Moreover, it showed that not only different substrates can trigger expression of different EP genes, but also that the same substrate can promote a variable response, according to its concentration. We also found isolates belonging to the same clonal type that showed different responses towards drug exposure, thus evidencing that highly related clinical isolates may diverge in the efflux-mediated response to noxious agents. The data gathered by real-time fluorometric and RT-qPCR assays suggest that S. aureus clinical isolates may be primed to efflux antimicrobial compounds. Conclusions The results obtained in this work do not exclude the importance of mutations in resistance to fluoroquinolones in S. aureus, yet they underline the contribution of efflux systems for the emergence of high-level resistance. All together, the results presented in this study show the potential role played by efflux systems in the development of resistance to fluoroquinolones in clinical isolates of S. aureus. PMID:22032541

Chemical composition and modulation of bacterial drug resistance of the essential oil from leaves of Croton grewioides.

PubMed

de Medeiros, Vivianne Marcelino; do Nascimento, Yuri Mangueira; Souto, Augusto Lopes; Madeiro, Sara Alves Lucena; Costa, Vicente Carlos de Oliveira; Silva, Suellen Maria P M; Falcão Silva, Vivyanne Dos Santos; Agra, Maria de Fátima; de Siqueira-Júnior, José Pinto; Tavares, Josean Fechine

2017-10-01

The essential oil from leaves of Croton grewioides Baill was obtained by hydrodistillation using Clevenger apparatus, and its chemical composition was analyzed by GC-MS, where 18 compounds were identified, mostly as monoterpenes (55.56%) and sesquiterpenes (44.44%), in which the major constituent was the α-pinene (47.43%). The essential oil of Croton grewioides (EOCg) and its major compound (α-pinene) were evaluated as modulators of antibiotic resistance in strain SA-1199B and IS-58 of Staphylococcus aureus that overexpresses efflux protein. The minimum inhibitory concentrations (MICs) of the antibiotics were determined by the microdilution assay in the absence and in the presence of sub-inhibitory concentration of EOCg and α-pinene. Although the EOCg and α-pinene did not indicate relevant antibacterial activity in vitro, they acted as antibiotic resistance modulators, i.e., EOCg in combination with norfloxacin, reducted its MIC, by 64× whereas in combination with tetracycline it was observed a reduction of 4×. Additionally, it was observed a MIC reduction of tetracycline by 32×, when combined with α-pinene. The results suggest that EOCg and α-pinene modulate or even reverse bacterial resistance as a putative efflux pump inhibitor. Copyright © 2017 Elsevier Ltd. All rights reserved.

The complete genome, comparative and functional analysis of Stenotrophomonas maltophilia reveals an organism heavily shielded by drug resistance determinants

PubMed Central

Crossman, Lisa C; Gould, Virginia C; Dow, J Maxwell; Vernikos, Georgios S; Okazaki, Aki; Sebaihia, Mohammed; Saunders, David; Arrowsmith, Claire; Carver, Tim; Peters, Nicholas; Adlem, Ellen; Kerhornou, Arnaud; Lord, Angela; Murphy, Lee; Seeger, Katharine; Squares, Robert; Rutter, Simon; Quail, Michael A; Rajandream, Mari-Adele; Harris, David; Churcher, Carol; Bentley, Stephen D; Parkhill, Julian; Thomson, Nicholas R; Avison, Matthew B

2008-01-01

Background Stenotrophomonas maltophilia is a nosocomial opportunistic pathogen of the Xanthomonadaceae. The organism has been isolated from both clinical and soil environments in addition to the sputum of cystic fibrosis patients and the immunocompromised. Whilst relatively distant phylogenetically, the closest sequenced relatives of S. maltophilia are the plant pathogenic xanthomonads. Results The genome of the bacteremia-associated isolate S. maltophilia K279a is 4,851,126 bp and of high G+C content. The sequence reveals an organism with a remarkable capacity for drug and heavy metal resistance. In addition to a number of genes conferring resistance to antimicrobial drugs of different classes via alternative mechanisms, nine resistance-nodulation-division (RND)-type putative antimicrobial efflux systems are present. Functional genomic analysis confirms a role in drug resistance for several of the novel RND efflux pumps. S. maltophilia possesses potentially mobile regions of DNA and encodes a number of pili and fimbriae likely to be involved in adhesion and biofilm formation that may also contribute to increased antimicrobial drug resistance. Conclusion The panoply of antimicrobial drug resistance genes and mobile genetic elements found suggests that the organism can act as a reservoir of antimicrobial drug resistance determinants in a clinical environment, which is an issue of considerable concern. PMID:18419807

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.

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.

Drug resistance is conferred on the model yeast Saccharomyces cerevisiae by expression of full-length melanoma-associated human ATP-binding cassette transporter ABCB5.

PubMed

Keniya, Mikhail V; Holmes, Ann R; Niimi, Masakazu; Lamping, Erwin; Gillet, Jean-Pierre; Gottesman, Michael M; Cannon, Richard D

2014-10-06

ABCB5, an ATP-binding cassette (ABC) transporter, is highly expressed in melanoma cells, and may contribute to the extreme resistance of melanomas to chemotherapy by efflux of anti-cancer drugs. Our goal was to determine whether we could functionally express human ABCB5 in the model yeast Saccharomyces cerevisiae, in order to demonstrate an efflux function for ABCB5 in the absence of background pump activity from other human transporters. Heterologous expression would also facilitate drug discovery for this important target. DNAs encoding ABCB5 sequences were cloned into the chromosomal PDR5 locus of a S. cerevisiae strain in which seven endogenous ABC transporters have been deleted. Protein expression in the yeast cells was monitored by immunodetection using both a specific anti-ABCB5 antibody and a cross-reactive anti-ABCB1 antibody. ABCB5 function in recombinant yeast cells was measured by determining whether the cells possessed increased resistance to known pump substrates, compared to the host yeast strain, in assays of yeast growth. Three ABCB5 constructs were made in yeast. One was derived from the ABCB5-β mRNA, which is highly expressed in human tissues but is a truncation of a canonical full-size ABC transporter. Two constructs contained full-length ABCB5 sequences: either a native sequence from cDNA or a synthetic sequence codon-harmonized for S. cerevisiae. Expression of all three constructs in yeast was confirmed by immunodetection. Expression of the codon-harmonized full-length ABCB5 DNA conferred increased resistance, relative to the host yeast strain, to the putative substrates rhodamine 123, daunorubicin, tetramethylrhodamine, FK506, or clorgyline. We conclude that full-length ABCB5 can be functionally expressed in S. cerevisiae and confers drug resistance.

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

PubMed

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

2009-05-01

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

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

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

PubMed

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

2017-06-01

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

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

PubMed

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

2015-08-01

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

In vitro characterization of pralidoxime transport and acetylcholinesterase reactivation across MDCK cells and stem cell-derived human brain microvascular endothelial cells (BC1-hBMECs).

PubMed

Gallagher, Erin; Minn, Il; Chambers, Janice E; Searson, Peter C

2016-07-11

Current therapies for organophosphate poisoning involve administration of oximes, such as pralidoxime (2-PAM), that reactivate the enzyme acetylcholinesterase. Studies in animal models have shown a low concentration in the brain following systemic injection. To assess 2-PAM transport, we studied transwell permeability in three Madin-Darby canine kidney (MDCKII) cell lines and stem cell-derived human brain microvascular endothelial cells (BC1-hBMECs). To determine whether 2-PAM is a substrate for common brain efflux pumps, experiments were performed in the MDCKII-MDR1 cell line, transfected to overexpress the P-gp efflux pump, and the MDCKII-FLuc-ABCG2 cell line, transfected to overexpress the BCRP efflux pump. To determine how transcellular transport influences enzyme reactivation, we developed a modified transwell assay where the inhibited acetylcholinesterase enzyme, substrate, and reporter are introduced into the basolateral chamber. Enzymatic activity was inhibited using paraoxon and parathion. The permeability of 2-PAM is about 2 × 10(-6) cm s(-1) in MDCK cells and about 1 × 10(-6) cm s(-1) in BC1-hBMECs. Permeability is not influenced by pre-treatment with atropine. In addition, 2-PAM is not a substrate for the P-gp or BCRP efflux pumps. The low permeability explains poor brain penetration of 2-PAM and therefore the slow enzyme reactivation. This elucidates one of the reasons for the necessity of sustained intravascular (IV) infusion in response to organophosphate poisoning.

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.

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

PubMed

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

2013-09-01

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

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.

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

PubMed Central

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

2011-01-01

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

Calcium Efflux Systems in Stress Signaling and Adaptation in Plants

PubMed Central

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

2011-01-01

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

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.

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

Evaluation of Four Calcium Channel Blockers as Fluconazole Resistance Inhibitors in Candida glabrata.

PubMed

Alnajjar, Lina M; Bulatova, Nailya R; Darwish, Rula M

2018-04-14

In this study we aimed to evaluate the ability of four calcium channel blockers, verapamil, diltiazem, nicardipine and nifedipine to enhance sensitivity of Candida glabrata strains to fluconazole. The synergistic antifungal effect was examined by checkerboard method; fractional inhibitory concentration index (FIC) was determined. Time-kill curve method was used for the most promising combination to further evaluate the synergetic effects. nicardipine showed additive effect with fluconazole against fluconazole-resistant and fluconazole-susceptible-dose-dependent strains (DSY565 and CBS138) known to express efflux pumps but not against fluconazole-sensitive strains. Nifedipine exhibited additive effect with fluconazole in both checkerboard (0.5< FIC <1) and time-kill curve methods (<2 log10 colony-forming units (CFU)/ml decrease in viable count). Additionally, nifedipine had own antifungal effect consistently against most of the strains used in this study with minimum inhibitory concentration of 8μg/ml. nicardipine showed additive effect with fluconazole in fluconazole-resistant strains of Candida glabrata-most probably via efflux pump inhibition as demonstrated selectively in fluconazole-resistant strains with known efflux pumps. Nifedipine displayed promising antifungal effect alone and additive effects with fluconazole. Copyright © 2018. Published by Elsevier Ltd.

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

PubMed

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

2015-04-28

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

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.

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.

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

PubMed

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

2018-06-06

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

Efflux systems in bacteria and their metabolic engineering applications.

PubMed

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

2015-11-01

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

Efflux pumps as antimicrobial resistance mechanisms.

PubMed

Poole, Keith

2007-01-01

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

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.

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

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

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

PubMed

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

2014-06-01

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

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

DOE PAGES

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

2018-02-09

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

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

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

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

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

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.

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.

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.

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

PubMed

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

2015-01-01

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

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

PubMed

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

2014-07-01

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

[The participation of the transport-barrier functions of the plasma membrane in the development of fluoroquinolone (ciprofloxacin) resistance in Acholeplasma laidlawii].

PubMed

Abramycheva, N Iu; Govorun, V M

2000-01-01

The role of transport activity of Acholeplasma laidlawii plasmatic membrane in the development of resistance to ciprofloxacin was investigated. It was shown that ethidium bromide used as fluoroquinolone analogue in plasmatic membrane efflux pump was accumulated in ciprofloxacin-resistant cells in much less amount. It was estimated that ethidium bromide efflux depended on temperature, glucose and transmembrane electro-chemical proton potential. Inhibitors of efflux systems--reserpine and verapamil enhanced the ethidium bromide accumulation much more intensively in ciprofloxacin resistant cells. The results of investigation allowed to consider the existence of active efflux system for toxic agents in acholeplasma; in the case of ciprofloxacin-resistant strain these systems are inducible.

Resistant mechanisms and molecular epidemiology of imipenem-resistant Acinetobacter baumannii.

PubMed

Xiao, Shu-Zhen; Chu, Hai-Qing; Han, Li-Zhong; Zhang, Zhe-Min; Li, Bing; Zhao, Lan; Xu, Liyun

2016-09-01

The aim of the study was to investigate the resistant mechanisms and homology of imipenem-resistant Acinetobacter baumannii (A. baumannii). A total of 46 non-duplicate imipenem‑resistant A. baumannii clinical isolates were collected from three tertiary hospitals between July, 2011 and June, 2012. The minimal inhibitory concentrations (MICs) of antimicrobial agents were determined using the agar dilution method. Phenylalanine‑arginine β-naphthylamide was used to detect the presence of the efflux pump-mediated resistant mechanism. Polymerase chain reaction was employed to amplify genes associated with drug resistance, including β‑lactamase genes, efflux pump genes and outer membrane protein gene CarO. A few amplicons were randomly selected and sequenced. Multilocus sequence analysis (MLST) was employed in typing A. baumanni. A. baumannii was resistant to imipenem, simultaneously showing resistance to several other antimicrobials. In addtition, 13 A. baumannii were found to mediate drug resistance through operation of the efflux pump. Of the various drug resistance genes tested, blaOXA‑51 was present in 46 isolates, blaOXA‑23 gene was present in 44 isolates and blaNDM gene was found in only one strain. Other drug resistant‑associated genes, including blaKPC, blaIMP, blaOXA-24, blaOXA‑58, blaSHV, blaGIM and blaVIM were not detected. Mutation of adeS and outer membrane protein gene CarO were found in a few of the imipenem‑resistant isolates. The MLST analysis revealed that all 46 clinical isolates were clustered into 11 genotypes and the most frequent genotype was ST208. In conclusion, β‑lactamase genes, genes involved in efflux pump and mutation of outer membrane protein encoding gene may be important in mediating imipenem resistance in A. baumannii. Of the 11 different genotypes, ST11 was shared by the majority of A. baumannii, which may be due to horizontal transfer of patients from hospitals.

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

Treesearch

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

2004-01-01

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

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.

Effects of thyroid hormone on Na sup + -K sup + transport in resting and stimulated rat skeletal muscle

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

Everts, M.E.; Clausen, T.

1988-11-01

The effects of hypothyroidism and 3,5,3{prime}-triiodothyronine (T{sub 3}) treatment on passive Na{sup +}-K{sup +} fluxes and Na{sup +}-K{sup +} pump concentration were investigated in isolated rat muscle. Within 12 h after a single dose of T{sub 3} (20 {mu}g/100 g body wt), K{sup +} efflux had increased by 21% in soleus and by 20% in extensor digitorum longus muscle. In the presence of ouabain, even larger effects were observed. These changes were associated with a 12% rise in amiloride-suppressible Na{sup +} influx but no significant increase in ({sup 3}H)ouabain binding site concentration. After 3 days of T{sub 3} treatment, themore » stimulating effect on K{sup +} efflux and Na{sup +} influx in soleus reached a plateau {approximately}80 and 40% above control levels, respectively, whereas the maximum increase in ({sup 3}H)ouabain binding site concentration (103%) was only fully developed after 8 days. Hypothyroidism decreased {sup 86}Rb efflux by 30%. The efflux of K{sup +} and the influx of Na{sup +} per contraction (both {approximately}7 nmol/g wet wt) as well as the net loss of K{sup +} induced by electrical stimulation were unaffected by T{sub 3} treatment. The rise in resting K{sup +} efflux after 12-24 h of T{sub 3} treatment could be partly blocked by dantrolene or trifluoroperazine, indicating that an increase in the cytoplasmic Ca{sup 2+} concentration may contribute to the early rise in K{sup +} efflux. It is concluded that the early rise in the resting passive leaks of Na{sup +} and K{sup +} induced by T{sub 3} is a major driving force for Na{sup +}-K{sup +} pump synthesis.« less

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

PubMed

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

2012-05-01

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

Transport and cytotoxicity of the anticancer drug 3-bromopyruvate in the yeast Saccharomyces cerevisiae.

PubMed

Lis, Paweł; Zarzycki, Marek; Ko, Young H; Casal, Margarida; Pedersen, Peter L; Goffeau, Andre; Ułaszewski, Stanisław

2012-02-01

We have investigated the cytotoxicity in Saccharomyces cerevisiae of the novel antitumor agent 3-bromopyruvate (3-BP). 3-BP enters the yeast cells through the lactate/pyruvate H(+) symporter Jen1p and inhibits cell growth at minimal inhibitory concentration of 1.8 mM when grown on non-glucose conditions. It is not submitted to the efflux pumps conferring Pleiotropic Drug Resistance in yeast. Yeast growth is more sensitive to 3-BP than Gleevec (Imatinib methanesulfonate) which in contrast to 3-BP is submitted to the PDR network of efflux pumps. The sensitivity of yeast to 3-BP is increased considerably by mutations or chemical treatment by buthionine sulfoximine that decrease the intracellular concentration of glutathione.

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.

Comparative effects of overproducing the AraC-type transcriptional regulators MarA, SoxS, RarA and RamA on antimicrobial drug susceptibility in Klebsiella pneumoniae.

PubMed

Jiménez-Castellanos, Juan-Carlos; Wan Ahmad Kamil, Wan Nur Ismah; Cheung, Ching Hei Phoebe; Tobin, Maryann S; Brown, James; Isaac, Sophie G; Heesom, Kate J; Schneiders, Thamarai; Avison, Matthew B

2016-07-01

In Klebsiella pneumoniae, overproduction of RamA and RarA leads to increased MICs of various antibiotics; MarA and SoxS are predicted to perform a similar function. We have compared the relative effects of overproducing these four AraC-type regulators on envelope permeability (a combination of outer membrane permeability and efflux), efflux pump and porin production, and antibiotic susceptibility in K. pneumoniae. Regulators were overproduced using a pBAD expression vector. Antibiotic susceptibility was measured using disc testing. Envelope permeability was estimated using a fluorescent dye accumulation assay. Porin and efflux pump production was quantified using proteomics and validated using real-time quantitative RT-PCR. Envelope permeability and antibiotic disc inhibition zone diameters both reduced during overproduction of RamA and to a lesser extent RarA or SoxS, but did not change following overproduction of MarA. These effects were associated with overproduction of the efflux pumps AcrAB (for RamA and SoxS) and OqxAB (for RamA and RarA) and the outer membrane protein TolC (for all regulators). Effects on porin production were strain specific. RamA is the most potent regulator of antibiotic permeability in K. pneumoniae, followed by RarA then SoxS, with MarA having very little effect. This observed relative potency correlates well with the frequency at which these regulators are reportedly overproduced in clinical isolates. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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.

Sublethal Exposure to Commercial Formulations of the Herbicides Dicamba, 2,4-Dichlorophenoxyacetic Acid, and Glyphosate Cause Changes in Antibiotic Susceptibility in Escherichia coli and Salmonella enterica serovar Typhimurium

PubMed Central

Kurenbach, Brigitta; Marjoshi, Delphine; Amábile-Cuevas, Carlos F.; Ferguson, Gayle C.; Godsoe, William; Gibson, Paddy

2015-01-01

ABSTRACT Biocides, such as herbicides, are routinely tested for toxicity but not for sublethal effects on microbes. Many biocides are known to induce an adaptive multiple-antibiotic resistance phenotype. This can be due to either an increase in the expression of efflux pumps, a reduced synthesis of outer membrane porins, or both. Exposures of Escherichia coli and Salmonella enterica serovar Typhimurium to commercial formulations of three herbicides—dicamba (Kamba), 2,4-dichlorophenoxyacetic acid (2,4-D), and glyphosate (Roundup)—were found to induce a changed response to antibiotics. Killing curves in the presence and absence of sublethal herbicide concentrations showed that the directions and the magnitudes of responses varied by herbicide, antibiotic, and species. When induced, MICs of antibiotics of five different classes changed up to 6-fold. In some cases the MIC increased, and in others it decreased. Herbicide concentrations needed to invoke the maximal response were above current food maximum residue levels but within application levels for all herbicides. Compounds that could cause induction had additive effects in combination. The role of soxS, an inducer of the AcrAB efflux pump, was tested in β-galactosidase assays with soxS-lacZ fusion strains of E. coli. Dicamba was a moderate inducer of the sox regulon. Growth assays with Phe-Arg β-naphtylamide (PAβN), an efflux pump inhibitor, confirmed a significant role of efflux in the increased tolerance of E. coli to chloramphenicol in the presence of dicamba and to kanamycin in the presence of glyphosate. Pathways of exposure with relevance to the health of humans, domestic animals, and critical insects are discussed. PMID:25805724

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

PubMed

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

2017-01-01

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

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.

The Ins and Outs of Cellular Ca2+ Transport

PubMed Central

Spalding, Edgar P.; Harper, Jeffrey F.

2011-01-01

The cytoplasmic Ca2+ signals that participate in nearly all aspects of plant growth and development encode information as binary switches or information-rich signatures. They are the result of influx (thermodynamically passive) and efflux (thermodynamically active) activities mediated by membrane transport proteins. On the influx side, confirming the molecular identities of Ca2+-permeable channels is still a major research topic. Cyclic nucleotide-gated channels and glutamate receptor-like channels are candidates well supported by evidence. On the efflux side, CAX antiporters and P-type ATPase pumps are the principal molecular entities. Both of these active transporters load Ca2+ into specific compartments and have the potential to reduce the magnitude and duration of a Ca2+ transient. Recent studies indicate calmodulin-activated Ca2+ pumps in endomembrane systems can dampen the magnitude and duration of a Ca2+ transient that could otherwise grow into a Ca2+ cell-death signature. An important challenge following molecular characterization of the influx and efflux pathways is to understand how they are coordinately regulated to produce a Ca2+ switch or encode specific information into a Ca2+ signature. PMID:21865080

Fluoroquinolone resistance in clinical and environmental isolates of Escherichia coli in Mexico City.

PubMed

Amábile-Cuevas, C F; Arredondo-García, J L; Cruz, A; Rosas, Irma

2010-01-01

To assess the different phenotypes and mechanisms of fluoroquinolone (FQ) resistance in clinical and environmental isolates of Escherichia coli. We compared FQ-resistant E. coli isolates, measuring minimal inhibitory concentrations (MIC) of ciprofloxacin, along with susceptibility to other antibiotics. We also searched for the presence of efflux pumps, using efflux inhibitors, and for plasmid-borne FQ-resistance by PCR. We found that, aside from the higher FQ-resistance prevalence among clinical strains, environmental ones resist much lower concentrations of ciprofloxacin. Efflux pumps mediate fluoroquinolone resistance as frequently among environmental isolates than in clinical strains. Plasmid-borne qnrA genes were not detected in any resistant strain. Environmental FQ-resistant strains may have a nonclinical origin and/or a selective pressure different from the clinical use of FQs. The identification of the source of low-level FQ-resistant strains (ciprofloxacin MIC c. 8 microg ml(-1)) in the environment could be important to curb the rapid emergence and spread of FQ-resistance in clinical settings, as these strains can easily become fully resistant to FQ concentrations achievable in fluids and tissues during therapy.

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

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

NASA Astrophysics Data System (ADS)

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

2015-04-01

TiO2 microparticles are widely used in food products, where they are added as a white food colouring agent. This food additive contains a significant amount of nanoscale particles; still the impact of TiO2 nanoparticles (TiO2-NPs) on gut cells is poorly documented. Our study aimed at evaluating the impact of rutile and anatase TiO2-NPs on the main functions of enterocytes, i.e. nutrient absorption driven by solute-liquid carriers (SLC transporters) and protection against other xenobiotics driven by efflux pumps from the ATP-binding cassette (ABC) family. We show that acute exposure of Caco-2 cells to both anatase (12 nm) and rutile (20 nm) TiO2-NPs induce early upregulation of a battery of efflux pumps and nutrient transporters. In addition they cause overproduction of reactive oxygen species and misbalance redox repair systems, without inducing cell mortality or DNA damage. Taken together, these data suggest that TiO2-NPs may increase the functionality of gut epithelial cells, particularly their property to form a protective barrier against exogenous toxicants and to absorb nutrients.TiO2 microparticles are widely used in food products, where they are added as a white food colouring agent. This food additive contains a significant amount of nanoscale particles; still the impact of TiO2 nanoparticles (TiO2-NPs) on gut cells is poorly documented. Our study aimed at evaluating the impact of rutile and anatase TiO2-NPs on the main functions of enterocytes, i.e. nutrient absorption driven by solute-liquid carriers (SLC transporters) and protection against other xenobiotics driven by efflux pumps from the ATP-binding cassette (ABC) family. We show that acute exposure of Caco-2 cells to both anatase (12 nm) and rutile (20 nm) TiO2-NPs induce early upregulation of a battery of efflux pumps and nutrient transporters. In addition they cause overproduction of reactive oxygen species and misbalance redox repair systems, without inducing cell mortality or DNA damage. Taken together, these data suggest that TiO2-NPs may increase the functionality of gut epithelial cells, particularly their property to form a protective barrier against exogenous toxicants and to absorb nutrients. Electronic supplementary information (ESI) available: Nanoparticle physico-chemical characterization: size distribution in exposure medium, as measured by DLS (Fig. S1), and X-ray diffraction patterns of A12 and R20 (Fig. S2); characterization of the protein corona on A12 and R20 (Table S1-S4 and experimental). See DOI: 10.1039/c5nr00505a

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

PubMed

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

2017-07-25

It is generally assumed that the acquisition of antibiotic resistance is associated with a fitness cost. We have shown that overexpression of the MexEF-OprN efflux pump does not decrease the fitness of a resistant Pseudomonas aeruginosa strain compared to its wild-type counterpart. This lack of fitness cost was associated with a metabolic rewiring that includes increased expression of the anaerobic nitrate respiratory chain when cells are growing under fully aerobic conditions. It was not clear whether this metabolic compensation was exclusive to strains overexpressing MexEF-OprN or if it extended to other resistant strains that overexpress similar systems. To answer this question, we studied a set of P. aeruginosa mutants that independently overexpress the MexAB-OprM, MexCD-OprJ, or MexXY efflux pumps. We observed increased expression of the anaerobic nitrate respiratory chain in all cases, with a concomitant increase in NO 3 consumption and NO production. These efflux pumps are proton/substrate antiporters, and their overexpression may lead to intracellular H + accumulation, which may in turn offset the pH homeostasis. Indeed, all studied mutants showed a decrease in intracellular pH under anaerobic conditions. The fastest way to eliminate the excess of protons is by increasing oxygen consumption, a feature also displayed by all analyzed mutants. Taken together, our results support metabolic rewiring as a general mechanism to avoid the fitness costs derived from overexpression of P. aeruginosa multidrug efflux pumps. The development of drugs that block this metabolic "reaccommodation" might help in reducing the persistence and spread of antibiotic resistance elements among bacterial populations. IMPORTANCE It is widely accepted that the acquisition of resistance confers a fitness cost in such a way that in the absence of antibiotics, resistant populations will be outcompeted by susceptible ones. Based on this assumption, antibiotic cycling regimes have been proposed in the belief that they will reduce the persistence and spread of resistance among bacterial pathogens. Unfortunately, trials testing this possibility have frequently failed, indicating that resistant microorganisms are not always outcompeted by susceptible ones. Indeed, some mutations do not result in a fitness cost, and in case they do, the cost may be compensated for by a secondary mutation. Here we describe an alternative nonmutational mechanism for compensating for fitness costs, which consists of the metabolic rewiring of resistant mutants. Deciphering the mechanisms involved in the compensation of fitness costs of antibiotic-resistant mutants may help in the development of drugs that will reduce the persistence of resistance by increasing said costs. Copyright © 2017 Olivares Pacheco et al.

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

PubMed Central

Beaugé, L. A.; Ortiz, Olga

1972-01-01

1. The efflux of labelled sodium as well as net sodium and lithium changes were studied in aged high sodium sartorius muscles of the South American frog Leptodactilus ocelatus. 2. In the presence of 2·5 mM potassium in the media, the replacement of external sodium with lithium or magnesium resulted in an increase in sodium efflux. The magnitude of such increase was always larger in lithium. 3. With the absence of potassium in the media, the response of sodium efflux to replacement of external sodium varied with the cation used as a substitute. In lithium Ringer there was always a noticeable increase, whereas in magnesium there was always a marked reduction. The same results were observed when calcium was substituted for magnesium. 4. The replacement of 60 mM external sodium with sucrose did not prevent the stimulating effect of 5 mM potassium on sodium efflux, nor the inhibitory action of 10-4 M ouabain. This indicates that neither sucrose by itself, nor the lowering of the ionic strength, modified to an appreciable extent the function of the sodium pump. 5. Net sodium extrusion took place against an electrochemical gradient in potassium-free — 50 mM sodium — mM lithium Ringer. About 75% of this efflux was ouabain sensitive. 6. Muscles made both sodium and lithium rich and incubated in potassium-free — 60 mM sodium — 50 mM lithium Ringer also showed net sodium extrusion against an electrochemical gradient, which was 85% ouabain sensitive. This extrusion took place even under conditions where the changes in free energy favouring lithium entry were always lower than the changes in free energy opposing sodium going out. This indicates that a sodium-lithium exchange by a counter-transport process is unlikely. 7. External potassium reduced the ouabain sensitive lithium influx in muscles incubated in lithium Ringer. The values found were 5·90 ± 0·39 μ-mole/g.hr and 2·66 ± 0·43 μmole/g.hr in potassium-free and 15 mM potassium respectively. At the same time potassium had no effect on the ouabain-insensitive lithium uptake. 8. Muscles incubated in potassium-free-magnesium Ringer had a residual sodium efflux which could not be accounted for by passive movement. About 40% of it was abolished by 10-4 M ouabain. This ouabain-sensitive part could be a consequence of some stimulation of the sodium pump by potassium leaking out of the cells. If this is correct it should be inhibited by external sodium and should not contribute to the total sodium efflux in potassium-free sodium media. 9. Magnesium was used as the reference cation to study the sodium-stimulated sodium efflux under potassium-free conditions. The total sodium efflux amounted to 0·668 hr-1 (rate constant) and was 71% ouabain sensitive. 10. The present experiments demonstrated that lithium ions have a direct stimulating effect on sodium efflux in high sodium skeletal muscle, and strongly support the notion that this effect is produced by an activation of the sodium pump through a potassium-like action. PMID:4637626

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

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.

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.

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.

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.

A new approach for pyrazinamide susceptibility testing in Mycobacterium tuberculosis.

PubMed

Zimic, Mirko; Loli, Sebastian; Gilman, Robert H; Gutierrez, Andrés; Fuentes, Patricia; Cotrina, Milagros; Kirwan, Daniela; Sheen, Patricia

2012-08-01

Pyrazinamide (PZA) is an important drug in the treatment of tuberculosis. Microbiological methods of PZA susceptibility testing are controversial and have low reproducibility. After conversion of PZA into pyrazinoic acid (POA) by the bacterial pyrazinamidase enzyme, the drug is expelled from the bacteria by an efflux pump. To evaluate the rate of POA extrusion from Mycobacterium tuberculosis as a parameter to detect PZA resistance. The rate of POA extrusion and PZA susceptibility determined by BACTEC 460 were measured for 34 strains in a previous study. PZA resistance was modeled in a logistic regression with the pyrazinoic efflux rate. POA efflux rate predicted PZA resistance with 70.83%-92.85% sensitivity and 100% specificity compared with BACTEC 460. POA efflux rate could be a useful tool for predicting PZA resistance in M. tuberculosis. Further exploration of this approach may lead to the development of new tools for diagnosing PZA resistance, which may be of public health importance.

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

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

PubMed

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

2013-11-01

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

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.

Mining Genomes of Marine Cyanobacteria for Elements of Zinc Homeostasis

PubMed Central

Barnett, James P.; Millard, Andrew; Ksibe, Amira Z.; Scanlan, David J.; Schmid, Ralf; Blindauer, Claudia Andrea

2012-01-01

Zinc is a recognized essential element for the majority of organisms, and is indispensable for the correct function of hundreds of enzymes and thousands of regulatory proteins. In aquatic photoautotrophs including cyanobacteria, zinc is thought to be required for carbonic anhydrase and alkaline phosphatase, although there is evidence that at least some carbonic anhydrases can be cambialistic, i.e., are able to acquire in vivo and function with different metal cofactors such as Co2+ and Cd2+. Given the global importance of marine phytoplankton, zinc availability in the oceans is likely to have an impact on both carbon and phosphorus cycles. Zinc concentrations in seawater vary over several orders of magnitude, and in the open oceans adopt a nutrient-like profile. Most studies on zinc handling by cyanobacteria have focused on freshwater strains and zinc toxicity; much less information is available on marine strains and zinc limitation. Several systems for zinc homeostasis have been characterized in the freshwater species Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803, but little is known about zinc requirements or zinc handling by marine species. Comparative metallo-genomics has begun to explore not only the putative zinc proteome, but also specific protein families predicted to have an involvement in zinc homeostasis, including sensors for excess and limitation (SmtB and its homologs as well as Zur), uptake systems (ZnuABC), putative intracellular zinc chaperones (COG0523) and metallothioneins (BmtA), and efflux pumps (ZiaA and its homologs). PMID:22514551

Antibiotic Resistance Mediated by the MacB ABC Transporter Family: A Structural and Functional Perspective.

PubMed

Greene, Nicholas P; Kaplan, Elise; Crow, Allister; Koronakis, Vassilis

2018-01-01

The MacB ABC transporter forms a tripartite efflux pump with the MacA adaptor protein and TolC outer membrane exit duct to expel antibiotics and export virulence factors from Gram-negative bacteria. Here, we review recent structural and functional data on MacB and its homologs. MacB has a fold that is distinct from other structurally characterized ABC transporters and uses a unique molecular mechanism termed mechanotransmission. Unlike other bacterial ABC transporters, MacB does not transport substrates across the inner membrane in which it is based, but instead couples cytoplasmic ATP hydrolysis with transmembrane conformational changes that are used to perform work in the extra-cytoplasmic space. In the MacAB-TolC tripartite pump, mechanotransmission drives efflux of antibiotics and export of a protein toxin from the periplasmic space via the TolC exit duct. Homologous tripartite systems from pathogenic bacteria similarly export protein-like signaling molecules, virulence factors and siderophores. In addition, many MacB-like ABC transporters do not form tripartite pumps, but instead operate in diverse cellular processes including antibiotic sensing, cell division and lipoprotein trafficking.

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

Transcriptomic Analyses Elucidate Adaptive Differences of Closely Related Strains of Pseudomonas aeruginosa in Fuel

PubMed Central

Gunasekera, Thusitha S.; Bowen, Loryn L.; Zhou, Carol E.; Howard-Byerly, Susan C.; Foley, William S.; Striebich, Richard C.; Dugan, Larry C.

2017-01-01

ABSTRACT Pseudomonas aeruginosa can utilize hydrocarbons, but different strains have various degrees of adaptation despite their highly conserved genome. P. aeruginosa ATCC 33988 is highly adapted to hydrocarbons, while P. aeruginosa strain PAO1, a human pathogen, is less adapted and degrades jet fuel at a lower rate than does ATCC 33988. We investigated fuel-specific transcriptomic differences between these strains in order to ascertain the underlying mechanisms utilized by the adapted strain to proliferate in fuel. During growth in fuel, the genes related to alkane degradation, heat shock response, membrane proteins, efflux pumps, and several novel genes were upregulated in ATCC 33988. Overexpression of alk genes in PAO1 provided some improvement in growth, but it was not as robust as that of ATCC 33988, suggesting the role of other genes in adaptation. Expression of the function unknown gene PA5359 from ATCC 33988 in PAO1 increased the growth in fuel. Bioinformatic analysis revealed that PA5359 is a predicted lipoprotein with a conserved Yx(FWY)xxD motif, which is shared among bacterial adhesins. Overexpression of the putative resistance-nodulation-division (RND) efflux pump PA3521 to PA3523 increased the growth of the ATCC 33988 strain, suggesting a possible role in fuel tolerance. Interestingly, the PAO1 strain cannot utilize n-C8 and n-C10. The expression of green fluorescent protein (GFP) under the control of alkB promoters confirmed that alk gene promoter polymorphism affects the expression of alk genes. Promoter fusion assays further confirmed that the regulation of alk genes was different in the two strains. Protein sequence analysis showed low amino acid differences for many of the upregulated genes, further supporting transcriptional control as the main mechanism for enhanced adaptation. IMPORTANCE These results support that specific signal transduction, gene regulation, and coordination of multiple biological responses are required to improve the survival, growth, and metabolism of fuel in adapted strains. This study provides new insight into the mechanistic differences between strains and helpful information that may be applied in the improvement of bacterial strains for resistance to biotic and abiotic factors encountered during bioremediation and industrial biotechnological processes. PMID:28314727

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.

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

PubMed

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

2014-02-01

The objective of the study was to evaluate the effect of hydroxypropyl-sulfobutyl-β-cyclodextrin (HP-SBE-βCD) on the bioavailability and intestinal absorption of edaravone, and identify its mechanism of action. We devised HP-SBE-βCD as a carrier and modulator of P-glycoprotein (Pgp) efflux pump, and edaravone as a model drug, and prepared edaravone/HP-SBE-βCD inclusion complex. HP-SBE-βCD improved the water solubility and enhanced the bioavailability of edaravone by 10.3-fold in rats. Then, in situ single-pass intestinal perfusion showed that HP-SBE-βCD had an effect of improving the permeability and inhibiting the efflux of edaravone. Furthermore, the effects of HP-SBE-βCD on Pgp were achieved through interfering with the lipid raft and depleting the cholesterol of enterocytes membrane. From the results, we presented the novel mechanisms. First, edaravone/HP-SBE-βCD had a lower release from the inclusion compound to protect edaravone from the low pH of the stomach. Then, HP-SBE-βCD modulated the membrane microenvironment of intestinal absorption epithelial cells. At last, the result was that HP-SBE-βCD enhanced the absorption of edaravone by interfering with Pgp. In conclusion, HP-SBE-βCD improves the bioavailability of drug not only because of its enhancing water solubility of the drug, but also because it modulates the Pgp-mediated efflux from enterocytes. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association.

Detection of ISAba1 in association with a novel allelic variant of the β-lactamase ADC-82 and class D β-lactamase genes mediating carbapenem resistance among the clinical isolates of MDR A. baumannii.

PubMed

Saranathan, Rajagopalan; Kumari, Rinki; Kalaivani, Ramakrishnan; Suresh, Sah; Rani, Anshu; Purty, Shashikala; Prashanth, K

2017-03-01

The objective of the present study is to investigate the diverse resistance determinants, their association with insertion sequence mobile elements and predilection of a particular clone for such associations in Acinetobacter baumannii. Fifty-four consecutive isolates collected during 2011-2012 from a tertiary care hospital were subjected to susceptibility testing followed by PCR screening of commonly reported β-lactamases and 16S rRNA methyltransferase encoding genes. The integrity of resistance-nodulation-cell division efflux pump-related genes in their respective operons was also investigated. β-Lactamase genes such as blaADC (100 %), blaOXA-23 (81 %), blaPER-1 (81 %), blaIMP-1 (31 %) and blaNDM-1 (15 %) were found to be present more frequently while blaVIM-2 and blaOXA-24 were not observed in our study population. ISAba1 was associated only with blaOXA-51-like like (30 %), blaOXA-23-like (55 %) and blaADC-like (33 %). armA was found in 87 % of isolates and ISAba1 linked with one novel variant of ADC, namely blaADC-82, which was identified to have 15 nucleotide differences with blaADC-79, and this finding is of much significance. In many isolates, efflux pump genes were not intact, resulting in severely altered effluxing functions. For the first time, we have identified ISAba1-mediated disruption of adeN among the isolates of ST 195B, which would have led to overexpression of AdeIJK efflux pump causing elevated resistance. Multilocus sequence typing revealed the predominance of CC 92B (IC-IIB) and CC 447B clonal complexes. High incidence of IC-II clones, novel resistance determinants (ADC-82) and elevated resistance mediated by ISAba1 reported here will be of enormous importance while assessing the emergence of extremely resistant A. baumannii in India.

Ion transport in broad bean leaf mesophyll under saline conditions.

PubMed

Percey, William J; Shabala, Lana; Breadmore, Michael C; Guijt, Rosanne M; Bose, Jayakumar; Shabala, Sergey

2014-10-01

Salt stress reduces the ability of mesophyll tissue to respond to light. Potassium outward rectifying channels are responsible for 84 % of Na (+) induced potassium efflux from mesophyll cells. Modulation in ion transport of broad bean (Vicia faba L.) mesophyll to light under increased apoplastic salinity stress was investigated using vibrating ion-selective microelectrodes (the MIFE technique). Increased apoplastic Na(+) significantly affected mesophyll cells ability to respond to light by modulating ion transport across their membranes. Elevated apoplastic Na(+) also induced a significant K(+) efflux from mesophyll tissue. This efflux was mediated predominately by potassium outward rectifying channels (84 %) and the remainder of the efflux was through non-selective cation channels. NaCl treatment resulted in a reduction in photosystem II efficiency in a dose- and time-dependent manner. In particular, reductions in Fv'/Fm' were linked to K(+) homeostasis in the mesophyll tissue. Increased apoplastic Na(+) concentrations induced vanadate-sensitive net H(+) efflux, presumably mediated by the plasma membrane H(+)-ATPase. It is concluded that the observed pump's activation is essential for the maintenance of membrane potential and ion homeostasis in the cytoplasm of mesophyll under salt stress.

Mannosylated thiolated polyethylenimine nanoparticles for the enhanced efficacy of antimonial drug against Leishmaniasis.

PubMed

Sarwar, Hafiz S; Ashraf, Sehreen; Akhtar, Sohail; Sohail, Muhammad F; Hussain, Syed Z; Rafay, Muhammad; Yasinzai, Masoom; Hussain, Irshad; Shahnaz, Gul

2018-01-01

Our aim was to inhibit trypanothione reductase (TR) and P-gp efflux pump of Leishmania by the use of thiolated polymers. Thus, increasing the intracellular accumulation and therapeutic effectiveness of antimonial compounds. Mannosylated thiolated chitosan and mannosylated thiolated chitosan-polyethyleneimine graft were synthesized and characterized. Meglumine antimoniate-loaded nanoparticles were prepared and evaluated for TR and P-gp efflux pump inhibition, biocompatibility, macrophage uptake and antileishmanial potential. Thiomers inhibited TR with Ki 2.021. The macrophage uptake was 33.7- and 18.9-fold higher with mannosylated thiolated chitosan-polyethyleneimine graft and mannosylated thiolated chitosan nanoparticles, respectively, as compared with the glucantime. Moreover, the in vitro antileishmanial activity showed 14.41- and 7.4-fold improved IC 50 for M-TCS-g-PEI and M-TCS, respectively as compared with glucantime. These results encouraged the concept that TR and P-gp inhibition by the use of thiomers improves the therapeutic efficacy of antimonial drugs.

Thiolated chitosans: useful excipients for oral drug delivery.

PubMed

Werle, Martin; Bernkop-Schnürch, Andreas

2008-03-01

To improve the bioavailability of orally administered drugs, formulations based on polymers are of great interest for pharmaceutical technologists. Thiolated chitosans are multifunctional polymers that exhibit improved mucoadhesive, cohesive and permeation-enhancing as well as efflux-pump-inhibitory properties. They can be synthesized by derivatization of the primary amino groups of chitosan with coupling reagents bearing thiol functions. Various data gained in-vitro as well as in-vivo studies clearly demonstrate the potential of thiolated chitosans for oral drug delivery. Within the current review, the synthesis and characterization of thiolated chitosans so far developed is summarized. Features of thiolated chitosans important for oral drug delivery are discussed as well. Moreover, different formulation approaches, such as matrix tablets and micro-/nanoparticles, as well as the applicability of thiolated chitosans for the oral delivery of various substance classes including peptides and efflux pump substrates, are highlighted.

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

PubMed

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

2018-04-25

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

Oleoyl Coenzyme A Regulates Interaction of Transcriptional Regulator RaaS (Rv1219c) with DNA in Mycobacteria*

PubMed Central

Turapov, Obolbek; Waddell, Simon J.; Burke, Bernard; Glenn, Sarah; Sarybaeva, Asel A.; Tudo, Griselda; Labesse, Gilles; Young, Danielle I.; Young, Michael; Andrew, Peter W.; Butcher, Philip D.; Cohen-Gonsaud, Martin; Mukamolova, Galina V.

2014-01-01

We have recently shown that RaaS (regulator of antimicrobial-assisted survival), encoded by Rv1219c in Mycobacterium tuberculosis and by bcg_1279c in Mycobacterium bovis bacillus Calmette-Guérin, plays an important role in mycobacterial survival in prolonged stationary phase and during murine infection. Here, we demonstrate that long chain acyl-CoA derivatives (oleoyl-CoA and, to lesser extent, palmitoyl-CoA) modulate RaaS binding to DNA and expression of the downstream genes that encode ATP-dependent efflux pumps. Moreover, exogenously added oleic acid influences RaaS-mediated mycobacterial improvement of survival and expression of the RaaS regulon. Our data suggest that long chain acyl-CoA derivatives serve as biological indicators of the bacterial metabolic state. Dysregulation of efflux pumps can be used to eliminate non-growing mycobacteria. PMID:25012658

Oleoyl coenzyme A regulates interaction of transcriptional regulator RaaS (Rv1219c) with DNA in mycobacteria.

PubMed

Turapov, Obolbek; Waddell, Simon J; Burke, Bernard; Glenn, Sarah; Sarybaeva, Asel A; Tudo, Griselda; Labesse, Gilles; Young, Danielle I; Young, Michael; Andrew, Peter W; Butcher, Philip D; Cohen-Gonsaud, Martin; Mukamolova, Galina V

2014-09-05

We have recently shown that RaaS (regulator of antimicrobial-assisted survival), encoded by Rv1219c in Mycobacterium tuberculosis and by bcg_1279c in Mycobacterium bovis bacillus Calmette-Guérin, plays an important role in mycobacterial survival in prolonged stationary phase and during murine infection. Here, we demonstrate that long chain acyl-CoA derivatives (oleoyl-CoA and, to lesser extent, palmitoyl-CoA) modulate RaaS binding to DNA and expression of the downstream genes that encode ATP-dependent efflux pumps. Moreover, exogenously added oleic acid influences RaaS-mediated mycobacterial improvement of survival and expression of the RaaS regulon. Our data suggest that long chain acyl-CoA derivatives serve as biological indicators of the bacterial metabolic state. Dysregulation of efflux pumps can be used to eliminate non-growing mycobacteria. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

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

PubMed

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

2018-04-01

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

An Arg-Gly-Asp peptide stimulates Ca2+ efflux from osteoclast precursors through a novel mechanism

NASA Technical Reports Server (NTRS)

Yamakawa, K.; Duncan, R.; Hruska, K. A.

1994-01-01

We examined the effect of a peptide containing the Arg-Gly-Asp (RGD) sequence on 45Ca2+ efflux from osteoclast precursors. 45Ca(2+)-loaded osteoclast precursors were treated with GRGDSP (170 microM) for 10 min after 30 min of basal perfusion with a bicarbonate-containing buffer. GRGDSP significantly increased fractional efflux of Ca2+ from treated cells compared with vehicle-treated cells (P < 0.01) or cells treated with up to 200 micrograms/ml of a control peptide containing GRGESP. The effect of RGD was sustained for 15 min after the peptide was removed from the perfusate, but control levels of Ca2+ efflux returned by 1 h. The Ca2+ efflux effect of GRGDSP was most likely due to activation of the plasma membrane Ca(2+)-adenosinetriphosphatase (Ca(2+)-ATPase) pump, as indicated by its inhibition with vanadate and a calmodulin antagonist, N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide, and the absence of an effect of Na+/Ca2+ exchange inhibition. An inhibitor of cyclic nucleotide-dependent protein kinases, N-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide (0.1 mM), failed to inhibit GRGDSP-stimulated Ca2+ efflux. However, genistein and herbimycin A, inhibitors of protein-tyrosine kinases, blocked Ca2+ efflux stimulated by GRGDSP. The results indicate that RGD sequences of matrix proteins may stimulate Ca2+ efflux from osteoclasts through activation of protein-tyrosine kinases and suggest that GRGDSP-stimulated Ca2+ efflux is mediated via the plasma membrane Ca(2+)-ATPase.

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

First report of qacG, qacH and qacJ genes in Staphylococcus haemolyticus human clinical isolates.

PubMed

Correa, J E; De Paulis, A; Predari, S; Sordelli, D O; Jeric, P E

2008-11-01

To investigate phenotypically and genotypically the presence of MDR efflux pumps in 21 clinical isolates of Staphylococcus haemolyticus collected over a period of 10 years. MICs of different antibiotics and biocides were determined by the broth dilution method in the presence/absence of carbonyl cyanide-m-chlorophenylhydrazone (CCCP), an efflux pump inhibitor. PCR followed by sequencing was performed to detect the qac genes that encode for antiseptic resistance. Clonal relationships were determined by PFGE SmaI patterns using a standard protocol. All the isolates were resistant to gentamicin, 15 to erythromycin, 18 to ciprofloxacin, 7 to chloramphenicol and 1 to tetracycline. They showed higher susceptibility to antibiotics when they were exposed to CCCP. The MICs of ethidium bromide, SDS and benzalkonium chloride were also decreased, whereas the MIC of triclosan was decreased in only four isolates in the presence CCCP. Of the 21 isolates, qacA/B was detected in 5 isolates, smr in all of the isolates, qacG in 11 isolates, qacH in 10 isolates and qacJ in 4 isolates. PFGE analysis of the 21 isolates clustered them into 14 clones at 90% similarity corresponding to differences of between 7 and 16 bands among the clones. The efflux mechanism seems to be an important mechanism to confer resistance to antibiotics and biocides through MDR pumps. It was observed that several qac genes coexist in some of the isolates and seem to act simultaneously in the removal of different compounds out of the bacterial cell. The qac genes are horizontally spread among different clones.

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.

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

PubMed

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

2017-03-17

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

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.

Resistance of Stenotrophomonas maltophilia to Fluoroquinolones: Prevalence in a University Hospital and Possible Mechanisms.

PubMed

Jia, Wei; Wang, Jiayuan; Xu, Haotong; Li, Gang

2015-05-13

The purpose of this study was to investigate the clinical distribution and genotyping of Stenotrophomonas maltophilia, its resistance to antimicrobial agents, and the possible mechanisms of this drug resistance. S. maltophilia isolates were collected from clinical specimens in a university hospital in Northwestern China during the period between 2010 and 2012, and were identified to the species level with a fully automated microbiological system. Antimicrobial susceptibility testing was performed for S. maltophilia with the Kirby-Bauer disc diffusion method. The minimal inhibitory concentrations (MICs) of norfloxacin, ofloxacin, chloramphenicol, minocycline, ceftazidime, levofloxacin and ciprofloxacin against S. maltophilia were assessed using the agar dilution method, and changes in the MIC of norfloxacin, ciprofloxacin and ofloxacin were observed after the addition of reserpine, an efflux pump inhibitor. Fluoroquinolone resistance genes were detected in S. maltophilia using a polymerase chain reaction (PCR) assay, and the expression of efflux pump smeD and smeF genes was determined using a quantitative fluorescent (QF)-PCR assay. Pulsed-field gel electrophoresis (PFGE) was employed to genotype identified S. maltophilia isolates. A total of 426 S. maltophilia strains were isolated from the university hospital from 2010 to 2012, consisting of 10.1% of total non-fermentative bacteria. The prevalence of norfloxacin, ciprofloxacin and ofloxacin resistance was 32.4%, 21.9% and 13.2% in the 114 S. maltophilia isolates collected from 2012, respectively. Following reserpine treatment, 19 S. maltophilia isolates positive for efflux pump were identified, and high expression of smeD and smeF genes was detected in two resistant isolates. gyrA, parC, smeD, smeE and smeF genes were detected in all 114 S. maltophilia isolates, while smqnr gene was found in 25.4% of total isolates. Glu-Lys mutation (GAA-AAA) was detected at the 151th amino acid of the gyrA gene, while Gly-Arg mutation (GGC-CGC) was found at the 37th amino acid of the parC gene. However, no significant difference was observed in the prevalence of gyrA or parC mutation between fluoroquinolone-resistant and -susceptible isolates (p> 0.05). The smqnr gene showed 92% to 99% heterogenicity among the 14 S. maltophilia clinical isolates. PFGE of 29 smqnr gene-positive S. maltophilia clinical isolates revealed 25 PFGE genotypes and 28 subgenotypes. Monitoring the clinical distribution and antimicrobial resistance of S. maltophilia is of great significance for the clinical therapy of bacterial infections. Reserpine is effective to inhibit the active efflux of norfloxacin, ciprofloxacin and ofloxacin on S. maltophilia and reduce MIC of fluoroquinolones against the bacteria. The expression of efflux pump smeD and smeF genes correlates with the resistance of S. maltophilia to fluoroquinolones.

Efflux inhibitor suppresses Streptococcus mutans virulence properties.

PubMed

Zeng, Huihui; Liu, Jia; Ling, Junqi

2017-04-01

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

A New Approach for Pyrazinamide Susceptibility Testing in Mycobacterium tuberculosis

PubMed Central

Loli, Sebastian; Gilman, Robert H.; Gutierrez, Andrés; Fuentes, Patricia; Cotrina, Milagros; Kirwan, Daniela; Sheen, Patricia

2012-01-01

Background: Pyrazinamide (PZA) is an important drug in the treatment of tuberculosis. Microbiological methods of PZA susceptibility testing are controversial and have low reproducibility. After conversion of PZA into pyrazinoic acid (POA) by the bacterial pyrazinamidase enzyme, the drug is expelled from the bacteria by an efflux pump. Objective: To evaluate the rate of POA extrusion from Mycobacterium tuberculosis as a parameter to detect PZA resistance. Methods: The rate of POA extrusion and PZA susceptibility determined by BACTEC 460 were measured for 34 strains in a previous study. PZA resistance was modeled in a logistic regression with the pyrazinoic efflux rate. Result: POA efflux rate predicted PZA resistance with 70.83%–92.85% sensitivity and 100% specificity compared with BACTEC 460. Conclusion: POA efflux rate could be a useful tool for predicting PZA resistance in M. tuberculosis. Further exploration of this approach may lead to the development of new tools for diagnosing PZA resistance, which may be of public health importance. PMID:22372927

Conjecture Regarding Posttranslational Modifications to the Arabidopsis Type I Proton-Pumping Pyrophosphatase (AVP1)

PubMed Central

Pizzio, Gaston A.; Hirschi, Kendal D.; Gaxiola, Roberto A.

2017-01-01

Agbiotechnology uses genetic engineering to improve the output and value of crops. Altering the expression of the plant Type I Proton-pumping Pyrophosphatase (H+-PPase) has already proven to be a useful tool to enhance crop productivity. Despite the effective use of this gene in translational research, information regarding the intracellular localization and functional plasticity of the pump remain largely enigmatic. Using computer modeling several putative phosphorylation, ubiquitination and sumoylation target sites were identified that may regulate Arabidopsis H+-PPase (AVP1- Arabidopsis Vacuolar Proton-pump 1) subcellular trafficking and activity. These putative regulatory sites will direct future research that specifically addresses the partitioning and transport characteristics of this pump. We posit that fine-tuning H+-PPases activity and cellular distribution will facilitate rationale strategies for further genetic improvements in crop productivity. PMID:28955362

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

PubMed

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

2017-05-01

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

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.

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.

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

PubMed

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

2011-06-01

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

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.

Mechanism of imidazolium ionic liquids toxicity in Saccharomyces cerevisiae and rational engineering of a tolerant, xylose-fermenting strain

DOE PAGES

Dickinson, Quinn; Bottoms, Scott; Hinchman, Li; ...

2016-01-20

In this study, imidazolium ionic liquids (IILs) underpin promising technologies that generate fermentable sugars from lignocellulose for future biorefineries. However, residual IILs are toxic to fermentative microbes such as Saccharomyces cerevisiae, making IIL-tolerance a key property for strain engineering. To enable rational engineering, we used chemical genomic profiling to understand the effects of IILs on S. cerevisiae. As a result, we found that IILs likely target mitochondria as their chemical genomic profiles closely resembled that of the mitochondrial membrane disrupting agent valinomycin. Further, several deletions of genes encoding mitochondrial proteins exhibited increased sensitivity to IIL. High-throughput chemical proteomics confirmed effectsmore » of IILs on mitochondrial protein levels. IILs induced abnormal mitochondrial morphology, as well as altered polarization of mitochondrial membrane potential similar to valinomycin. Deletion of the putative serine/threonine kinase PTK2 thought to activate the plasma-membrane proton efflux pump Pma1p conferred a significant IIL-fitness advantage. Conversely, overexpression of PMA1 conferred sensitivity to IILs, suggesting that hydrogen ion efflux may be coupled to influx of the toxic imidazolium cation. PTK2 deletion conferred resistance to multiple IILs, including [EMIM]Cl, [BMIM]Cl, and [EMIM]Ac. An engineered, xylose-converting ptk2Δ S. cerevisiae (Y133-IIL) strain consumed glucose and xylose faster and produced more ethanol in the presence of 1 % [BMIM]Cl than the wild-type PTK2 strain. We propose a model of IIL toxicity and resistance. In conclusion, this work demonstrates the utility of chemical genomics-guided biodesign for development of superior microbial biocatalysts for the ever-changing landscape of fermentation inhibitors.« less

Antibiotic Resistance Mediated by the MacB ABC Transporter Family: A Structural and Functional Perspective

PubMed Central

Greene, Nicholas P.; Kaplan, Elise; Crow, Allister; Koronakis, Vassilis

2018-01-01

The MacB ABC transporter forms a tripartite efflux pump with the MacA adaptor protein and TolC outer membrane exit duct to expel antibiotics and export virulence factors from Gram-negative bacteria. Here, we review recent structural and functional data on MacB and its homologs. MacB has a fold that is distinct from other structurally characterized ABC transporters and uses a unique molecular mechanism termed mechanotransmission. Unlike other bacterial ABC transporters, MacB does not transport substrates across the inner membrane in which it is based, but instead couples cytoplasmic ATP hydrolysis with transmembrane conformational changes that are used to perform work in the extra-cytoplasmic space. In the MacAB-TolC tripartite pump, mechanotransmission drives efflux of antibiotics and export of a protein toxin from the periplasmic space via the TolC exit duct. Homologous tripartite systems from pathogenic bacteria similarly export protein-like signaling molecules, virulence factors and siderophores. In addition, many MacB-like ABC transporters do not form tripartite pumps, but instead operate in diverse cellular processes including antibiotic sensing, cell division and lipoprotein trafficking. PMID:29892271

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.

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

A [Cu]rious Ribosomal Profiling Pattern Leads to the Discovery of Ribosomal Frameshifting in the Synthesis of a Copper Chaperone.

PubMed

Atkins, John F; Loughran, Gary; Baranov, Pavel V

2017-01-19

In many bacteria, separate genes encode a copper binding chaperone and a copper efflux pump, but in some the chaperone encoding gene has been elusive. In this issue of Molecular Cell, Meydan et al. (2017) report that ribosomes translating the ORF that encodes the copper pump frequently frameshift and terminate to produce the copper chaperone. Copyright © 2017 Elsevier Inc. All rights reserved.

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

PubMed Central

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

2014-01-01

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

Genes for all metals--a bacterial view of the periodic table. The 1996 Thom Award Lecture.

PubMed

Silver, S

1998-01-01

Bacterial chromosomes have genes for transport proteins for inorganic nutrient cations and oxyanions, such as NH4+, K+, Mg2+, Co2+, Fe3+, Mn2+, Zn2+ and other trace cations, and PO4(3-), SO4(2-) and less abundant oxyanions. Together these account for perhaps a few hundred genes in many bacteria. Bacterial plasmids encode resistance systems for toxic metal and metalloid ions including Ag+, AsO2-, AsO4(3-), Cd2+, Co2+, CrO4(2-), Cu2+, Hg2+, Ni2+, Pb2+, TeO3(2-), Tl+ and Zn2+. Most resistance systems function by energy-dependent efflux of toxic ions. A few involve enzymatic (mostly redox) transformations. Some of the efflux resistance systems are ATPases and others are chemiosmotic ion/proton exchangers. The Cd(2+)-resistance cation pump of Gram-positive bacteria is membrane P-type ATPase, which has been labeled with 32P from [gamma-32P]ATP and drives ATP-dependent Cd2+ (and Zn2+) transport by membrane vesicles. The genes defective in the human hereditary diseases of copper metabolism, Menkes syndrome and Wilson's disease, encode P-type ATPases that are similar to bacterial cadmium ATPases. The arsenic resistance system transports arsenite [As(III)], alternatively with the ArsB polypeptide functioning as a chemiosmotic efflux transporter or with two polypeptides, ArsB and ArsA, functioning as an ATPase. The third protein of the arsenic resistance system is an enzyme that reduces intracellular arsenate [As(V)] to arsenite [As(III)], the substrate of the efflux system. In Gram-negative cells, a three polypeptide complex functions as a chemiosmotic cation/protein exchanger to efflux Cd2+, Zn2+ and Co2+. This pump consists of an inner membrane (CzcA), an outer membrane (CzcC) and a membrane-spanning (CzcB) protein that function together.

Interplay of drug metabolizing enzymes with cellular transporters.

PubMed

Böhmdorfer, Michaela; Maier-Salamon, Alexandra; Riha, Juliane; Brenner, Stefan; Höferl, Martina; Jäger, Walter

2014-11-01

Many endogenous and xenobiotic substances and their metabolites are substrates for drug metabolizing enzymes and cellular transporters. These proteins may not only contribute to bioavailability of molecules but also to uptake into organs and, consequently, to overall elimination. The coordinated action of uptake transporters, metabolizing enzymes, and efflux pumps, therefore, is a precondition for detoxification and elimination of drugs. As the understanding of the underlying mechanisms is important to predict alterations in drug disposal, adverse drug reactions and, finally, drug-drug interactions, this review illustrates the interplay between selected uptake/efflux transporters and phase I/II metabolizing enzymes.

Complete genome sequence analysis of the fish pathogen Flavobacterium columnare provides insights into antibiotic resistance and pathogenicity related genes.

PubMed

Zhang, Yulei; Zhao, Lijuan; Chen, Wenjie; Huang, Yunmao; Yang, Ling; Sarathbabu, V; Wu, Zaohe; Li, Jun; Nie, Pin; Lin, Li

2017-10-01

We analyzed here the complete genome sequences of a highly virulent Flavobacterium columnare Pf1 strain isolated in our laboratory. The complete genome consists of a 3,171,081 bp circular DNA with 2784 predicted protein-coding genes. Among these, 286 genes were predicted as antibiotic resistance genes, including 32 RND-type efflux pump related genes which were associated with the export of aminoglycosides, indicating inducible aminoglycosides resistances in F. columnare. On the other hand, 328 genes were predicted as pathogenicity related genes which could be classified as virulence factors, gliding motility proteins, adhesins, and many putative secreted proteases. These genes were probably involved in the colonization, invasion and destruction of fish tissues during the infection of F. columnare. Apparently, our obtained complete genome sequences provide the basis for the explanation of the interactions between the F. columnare and the infected fish. The predicted antibiotic resistance and pathogenicity related genes will shed a new light on the development of more efficient preventional strategies against the infection of F. columnare, which is a major worldwide fish pathogen. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

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

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

PubMed Central

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

2013-01-01

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

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.

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

PubMed Central

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

2012-01-01

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

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

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.

Physiologically based pharmacokinetic modeling of deltamethrin: Development of a rat and human diffusion-limited model

EPA Science Inventory

Mirfazaelian et al. (2006) developed a physiologically based pharmacokinetic (PBPK) model for the pyrethroid pesticide deltamethrin in the rat. This model describes gastrointestinal tract absorption as a saturable process mediated by phase III efflux transporters which pump delta...

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

DOE PAGES

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

2015-04-01

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

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

PubMed Central

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

2015-01-01

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

Promethazine improves antibiotic efficacy and disrupts biofilms of Burkholderia pseudomallei.

PubMed

Sidrim, José Júlio Costa; Vasconcelos, David Caldas; Riello, Giovanna Barbosa; Guedes, Glaucia Morgana de Melo; Serpa, Rosana; Bandeira, Tereza de Jesus Pinheiro Gomes; Monteiro, André Jalles; Cordeiro, Rossana de Aguiar; Castelo-Branco, Débora de Souza Collares Maia; Rocha, Marcos Fábio Gadelha; Brilhante, Raimunda Sâmia Nogueira

2017-01-01

Efflux pumps are important defense mechanisms against antimicrobial drugs and maintenance of Burkholderia pseudomallei biofilms. This study evaluated the effect of the efflux pump inhibitor promethazine on the structure and antimicrobial susceptibility of B. pseudomallei biofilms. Susceptibility of planktonic cells and biofilms to promethazine alone and combined with antimicrobials was assessed by the broth microdilution test and biofilm metabolic activity was determined with resazurin. The effect of promethazine on 48 h-grown biofilms was also evaluated through confocal and electronic microscopy. The minimum inhibitory concentration (MIC) of promethazine was 780 mg l -1 , while the minimum biofilm elimination concentration (MBEC) was 780-3,120 mg l -1 . Promethazine reduced the MIC values for erythromycin, trimethoprim/sulfamethoxazole, gentamicin and ciprofloxacin and reduced the MBEC values for all tested drugs (p<0.05). Microscopic analyses demonstrated that promethazine altered the biofilm structure of B. pseudomallei, even at subinhibitory concentrations, possibly facilitating antibiotic penetration. Promethazine improves antibiotics efficacy against B. pseudomallei biofilms, by disrupting biofilm structure.

Combinatorial approaches with selected phytochemicals to increase antibiotic efficacy against Staphylococcus aureus biofilms.

PubMed

Abreu, Ana Cristina; Saavedra, Maria José; Simões, Lúcia C; Simões, Manuel

2016-10-01

Combinations of selected phytochemicals (reserpine, pyrrolidine, quinine, morin and quercetin) with antibiotics (ciprofloxacin, tetracycline and erythromycin) were tested on the prevention and control of Staphylococcus aureus biofilms. The phytochemicals were also studied for their ability to avoid antibiotic adaptation and to inhibit antibiotic efflux pumps. Morin, pyrrolidine and quercetin at subinhibitory concentrations had significant effects in biofilm prevention and/or control when applied alone and combined with antibiotics. Synergism between antibiotics and phytochemicals was found especially against biofilms of NorA overexpressing strain S. aureus SA1199B. This strain when growing with subinhibitory concentrations of ciprofloxacin developed increased tolerance to this antibiotic. However, this was successfully reversed by quinine and morin. In addition, reserpine and quercetin showed significant efflux pump inhibition. The overall results demonstrate the role of phytochemicals in co-therapies to promote more efficient treatments and decrease antimicrobial resistance to antibiotics, with substantial effects against S. aureus in both planktonic and biofilm states.

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

A bacterial view of the periodic table: genes and proteins for toxic inorganic ions.

PubMed

Silver, Simon; Phung, Le T

2005-12-01

Essentially all bacteria have genes for toxic metal ion resistances and these include those for Ag+, AsO2-, AsO4(3-), Cd2+ Co2+, CrO4(2-), Cu2+, Hg2+, Ni2+, Pb2+, TeO3(2-), Tl+ and Zn2+. The largest group of resistance systems functions by energy-dependent efflux of toxic ions. Fewer involve enzymatic transformations (oxidation, reduction, methylation, and demethylation) or metal-binding proteins (for example, metallothionein SmtA, chaperone CopZ and periplasmic silver binding protein SilE). Some of the efflux resistance systems are ATPases and others are chemiosmotic ion/proton exchangers. For example, Cd2+-efflux pumps of bacteria are either inner membrane P-type ATPases or three polypeptide RND chemiosmotic complexes consisting of an inner membrane pump, a periplasmic-bridging protein and an outer membrane channel. In addition to the best studied three-polypeptide chemiosmotic system, Czc (Cd2+, Zn2+, and Co2), others are known that efflux Ag+, Cu+, Ni2+, and Zn2+. Resistance to inorganic mercury, Hg2+ (and to organomercurials, such as CH3Hg+ and phenylmercury) involve a series of metal-binding and membrane transport proteins as well as the enzymes mercuric reductase and organomercurial lyase, which overall convert more toxic to less toxic forms. Arsenic resistance and metabolizing systems occur in three patterns, the widely-found ars operon that is present in most bacterial genomes and many plasmids, the more recently recognized arr genes for the periplasmic arsenate reductase that functions in anaerobic respiration as a terminal electron acceptor, and the aso genes for the periplasmic arsenite oxidase that functions as an initial electron donor in aerobic resistance to arsenite.

Data-based mathematical modeling of vectorial transport across double-transfected polarized cells.

PubMed

Bartholomé, Kilian; Rius, Maria; Letschert, Katrin; Keller, Daniela; Timmer, Jens; Keppler, Dietrich

2007-09-01

Vectorial transport of endogenous small molecules, toxins, and drugs across polarized epithelial cells contributes to their half-life in the organism and to detoxification. To study vectorial transport in a quantitative manner, an in vitro model was used that includes polarized MDCKII cells stably expressing the recombinant human uptake transporter OATP1B3 in their basolateral membrane and the recombinant ATP-driven efflux pump ABCC2 in their apical membrane. These double-transfected cells enabled mathematical modeling of the vectorial transport of the anionic prototype substance bromosulfophthalein (BSP) that has frequently been used to examine hepatobiliary transport. Time-dependent analyses of (3)H-labeled BSP in the basolateral, intracellular, and apical compartments of cells cultured on filter membranes and efflux experiments in cells preloaded with BSP were performed. A mathematical model was fitted to the experimental data. Data-based modeling was optimized by including endogenous transport processes in addition to the recombinant transport proteins. The predominant contributions to the overall vectorial transport of BSP were mediated by OATP1B3 (44%) and ABCC2 (28%). Model comparison predicted a previously unrecognized endogenous basolateral efflux process as a negative contribution to total vectorial transport, amounting to 19%, which is in line with the detection of the basolateral efflux pump Abcc4 in MDCKII cells. Rate-determining steps in the vectorial transport were identified by calculating control coefficients. Data-based mathematical modeling of vectorial transport of BSP as a model substance resulted in a quantitative description of this process and its components. The same systems biology approach may be applied to other cellular systems and to different substances.

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

Comparative antibacterial effects of moxifloxacin and levofloxacin on Streptococcus pneumoniae strains with defined mechanisms of resistance: impact of bacterial inoculum.

PubMed

Bowker, K E; Garvey, M I; Noel, A R; Tomaselli, S G; Macgowan, A P

2013-05-01

We aim to further define the impact of the mechanism of fluoroquinolone resistance and inoculum load on the pharmacodynamic effects of levofloxacin and moxifloxacin on Streptococcus pneumoniae. The antibacterial effects of and emergence of resistance (EoR) to moxifloxacin (400 mg once daily) or levofloxacin (750 mg once daily or 500 mg twice daily) were compared using five S. pneumoniae strains containing no known resistance mechanisms, efflux resistance mechanisms, a parC mutation or parC and gyrA mutations, at high (10(8) cfu/mL) and low (10(6) cfu/mL) inocula. An in vitro pharmacokinetic model was used and simulations were performed over 96 h. After drug exposure, isolates were tested for the presence of efflux pumps and mutations in the quinolone resistance-determining regions. A high inoculum diminished the antibacterial effect of moxifloxacin and levofloxacin. Levofloxacin at both dosages produced EoR with all strains. Levofloxacin regimens with AUC/MIC ratios <100 produced EoR. Moxifloxacin produced EoR with the parC strain only. Levofloxacin dosing regimens with low AUC/MIC ratios select for efflux pump overexpression, leading to fluoroquinolone resistance. Levofloxacin dosing may select for gyrA mutations, inducing moxifloxacin resistance. These data confirm that a fluoroquinolone AUC/MIC ratio of >100 is required for prevention of EoR.

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

PubMed Central

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

2016-01-01

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

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

PubMed Central

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

2011-01-01

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

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.

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.

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

PubMed Central

Venosa, R A

2003-01-01

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

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

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

PubMed

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

2016-05-01

P-Glycoprotein (P-gp) can protect cells by pumping out toxic compounds, and has been found widely expressed in fish tissues. Here, we illustrate the P-gp efflux ability for benzo(a)pyrene (BaP) in the hepatocytes of common carp (Cyprinus carpio) after exposing to fullerene aqueous suspension (nC60). The results revealed that nC60 increased the membrane fluidity by decreasing the ratio of saturated to unsaturated fatty acids, and increased the cholesterol contents. These findings, combined with 10-38% and 70-75% down-regulation of P-gp mRNA and protein respectively, suggested that nC60 caused inhibition on P-gp efflux transport system. Therefore, we further investigated the cellular efflux ability for BaP. Results showed unequivocally that nC60 is a potent P-gp inhibitor. The retaining BaP amounts after efflux were elevated by 1.7-2.8 fold during the 10 day exposure. Meanwhile, 5mg/L humic acid (one of the important fractions of natural organic matter, which is ubiquitous in aquatic environment) alleviated the nC60 damage to hepatocytes in terms of oxidative damage, cholesterol increment, and P-gp content reduction; and finally attenuated the suppressed P-gp efflux ability. Collectively, this study provides the first evidence of nC60 toxicity to P-gp functionality in fish and illustrates the possible mechanism of the suppressed P-gp efflux ability for BaP. Copyright © 2016 Elsevier B.V. All rights reserved.

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

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

Rincon, M.; Boss, W.F.

1986-04-01

Polyphosphoinositide breakdown plays an important role in signal transduction in animal cells (Berridge and Irvine, 1984, Nature, 312:315). Upon stimulation, phospholipase C hydrolyzes phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate (IP/sub 3/) and diacylglycerol both of which act as cellular second messengers. IP/sub 3/ mobilizes Ca/sup + +/ from internal stores, hence the cytosolic free Ca/sup + +/ concentration increases and those physiological activities regulated by Ca/sup + +/ are stimulated. To test if plant cells also responded to IP/sub 3/, Ca/sup + +/ efflux studies were done with fusogenic carrot protoplasts released in EGTA. The protoplasts were preloaded with /sup 45/Ca/supmore » + +/ placed in a Ca/sup + +/-free medium, and efflux determined as /sup 45/Ca/sup + +/ loss from the protoplasts. IP/sub 3/ (10-20..mu..M) caused enhanced /sup 45/Ca/sup + +/ efflux and the response was sustained for at least 15 min. In plants, as in animals, the observed IP/sub 3/-enhanced /sup 45/Ca/sup + +/ efflux suggested that IP/sub 3/ released Ca/sup + +/ from internal stores, and the increased free cytosolic Ca/sup + +/ activated Ca/sup + +/ pumping mechanisms which restored the Ca/sup + +/ concentration in the cytosol to the normal level.« less

Transcriptomic Analyses Elucidate Adaptive Differences of Closely Related Strains of Pseudomonas aeruginosa in Fuel.

PubMed

Gunasekera, Thusitha S; Bowen, Loryn L; Zhou, Carol E; Howard-Byerly, Susan C; Foley, William S; Striebich, Richard C; Dugan, Larry C; Ruiz, Oscar N

2017-05-15

Pseudomonas aeruginosa can utilize hydrocarbons, but different strains have various degrees of adaptation despite their highly conserved genome. P. aeruginosa ATCC 33988 is highly adapted to hydrocarbons, while P. aeruginosa strain PAO1, a human pathogen, is less adapted and degrades jet fuel at a lower rate than does ATCC 33988. We investigated fuel-specific transcriptomic differences between these strains in order to ascertain the underlying mechanisms utilized by the adapted strain to proliferate in fuel. During growth in fuel, the genes related to alkane degradation, heat shock response, membrane proteins, efflux pumps, and several novel genes were upregulated in ATCC 33988. Overexpression of alk genes in PAO1 provided some improvement in growth, but it was not as robust as that of ATCC 33988, suggesting the role of other genes in adaptation. Expression of the function unknown gene PA5359 from ATCC 33988 in PAO1 increased the growth in fuel. Bioinformatic analysis revealed that PA5359 is a predicted lipoprotein with a conserved Yx(FWY)xxD motif, which is shared among bacterial adhesins. Overexpression of the putative resistance-nodulation-division (RND) efflux pump PA3521 to PA3523 increased the growth of the ATCC 33988 strain, suggesting a possible role in fuel tolerance. Interestingly, the PAO1 strain cannot utilize n -C 8 and n -C 10 The expression of green fluorescent protein (GFP) under the control of alkB promoters confirmed that alk gene promoter polymorphism affects the expression of alk genes. Promoter fusion assays further confirmed that the regulation of alk genes was different in the two strains. Protein sequence analysis showed low amino acid differences for many of the upregulated genes, further supporting transcriptional control as the main mechanism for enhanced adaptation. IMPORTANCE These results support that specific signal transduction, gene regulation, and coordination of multiple biological responses are required to improve the survival, growth, and metabolism of fuel in adapted strains. This study provides new insight into the mechanistic differences between strains and helpful information that may be applied in the improvement of bacterial strains for resistance to biotic and abiotic factors encountered during bioremediation and industrial biotechnological processes. Copyright © 2017 American Society for Microbiology.

Crossing boundaries: the importance of cellular membranes in industrial biotechnology.

PubMed

Jezierska, Sylwia; Van Bogaert, Inge N A

2017-05-01

How small molecules cross cellular membranes is an often overlooked issue in an industrial microbiology and biotechnology context. This is to a large extent governed by the technical difficulties to study these transport systems or by the lack of knowledge on suitable efflux pumps. This review emphasizes the importance of microbial cellular membranes in industrial biotechnology by highlighting successful strategies of membrane engineering towards more resistant and hence better performing microorganisms, as well as transporter and other engineering strategies for increased efflux of primary and secondary metabolites. Furthermore, the benefits and limitations of eukaryotic subcellular compartmentalization are discussed, as well as the biotechnological potential of membrane vesicles.

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

PubMed

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

2007-03-01

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

SatR Is a Repressor of Fluoroquinolone Efflux Pump SatAB

PubMed Central

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

2013-01-01

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

Characterization of BcaA, a Putative Classical Autotransporter Protein in Burkholderia pseudomallei

PubMed Central

Campos, Cristine G.; Borst, Luke

2013-01-01

Burkholderia pseudomallei is a tier 1 select agent, and the causative agent of melioidosis, a disease with effects ranging from chronic abscesses to fulminant pneumonia and septic shock, which can be rapidly fatal. Autotransporters (ATs) are outer membrane proteins belonging to the type V secretion system family, and many have been shown to play crucial roles in pathogenesis. The open reading frame Bp1026b_II1054 (bcaA) in B. pseudomallei strain 1026b is predicted to encode a classical autotransporter protein with an approximately 80-kDa passenger domain that contains a subtilisin-related domain. Immediately 3′ to bcaA is Bp11026_II1055 (bcaB), which encodes a putative prolyl 4-hydroxylase. To investigate the role of these genes in pathogenesis, large in-frame deletion mutations of bcaA and bcaB were constructed in strain Bp340, an efflux pump mutant derivative of the melioidosis clinical isolate 1026b. Comparison of Bp340ΔbcaA and Bp340ΔbcaB mutants to wild-type B. pseudomallei in vitro demonstrated similar levels of adherence to A549 lung epithelial cells, but the mutant strains were defective in their ability to invade these cells and to form plaques. In a BALB/c mouse model of intranasal infection, similar bacterial burdens were observed after 48 h in the lungs and liver of mice infected with Bp340ΔbcaA, Bp340ΔbcaB, and wild-type bacteria. However, significantly fewer bacteria were recovered from the spleen of Bp340ΔbcaA-infected mice, supporting the idea of a role for this AT in dissemination or in survival in the passage from the site of infection to the spleen. PMID:23340315

Characterization of BcaA, a putative classical autotransporter protein in Burkholderia pseudomallei.

PubMed

Campos, Cristine G; Borst, Luke; Cotter, Peggy A

2013-04-01

Burkholderia pseudomallei is a tier 1 select agent, and the causative agent of melioidosis, a disease with effects ranging from chronic abscesses to fulminant pneumonia and septic shock, which can be rapidly fatal. Autotransporters (ATs) are outer membrane proteins belonging to the type V secretion system family, and many have been shown to play crucial roles in pathogenesis. The open reading frame Bp1026b_II1054 (bcaA) in B. pseudomallei strain 1026b is predicted to encode a classical autotransporter protein with an approximately 80-kDa passenger domain that contains a subtilisin-related domain. Immediately 3' to bcaA is Bp11026_II1055 (bcaB), which encodes a putative prolyl 4-hydroxylase. To investigate the role of these genes in pathogenesis, large in-frame deletion mutations of bcaA and bcaB were constructed in strain Bp340, an efflux pump mutant derivative of the melioidosis clinical isolate 1026b. Comparison of Bp340ΔbcaA and Bp340ΔbcaB mutants to wild-type B. pseudomallei in vitro demonstrated similar levels of adherence to A549 lung epithelial cells, but the mutant strains were defective in their ability to invade these cells and to form plaques. In a BALB/c mouse model of intranasal infection, similar bacterial burdens were observed after 48 h in the lungs and liver of mice infected with Bp340ΔbcaA, Bp340ΔbcaB, and wild-type bacteria. However, significantly fewer bacteria were recovered from the spleen of Bp340ΔbcaA-infected mice, supporting the idea of a role for this AT in dissemination or in survival in the passage from the site of infection to the spleen.

pncA gene expression and prediction factors on pyrazinamide resistance in Mycobacterium tuberculosis.

PubMed

Sheen, Patricia; Lozano, Katherine; Gilman, Robert H; Valencia, Hugo J; Loli, Sebastian; Fuentes, Patricia; Grandjean, Louis; Zimic, Mirko

2013-09-01

Mutations in the pyrazinamidase (PZAse) coding gene, pncA, have been considered as the main cause of pyrazinamide (PZA) resistance in Mycobacterium tuberculosis. However, recent studies suggest there is no single mechanism of resistance to PZA. The pyrazinoic acid (POA) efflux rate is the basis of the PZA susceptibility Wayne test, and its quantitative measurement has been found to be a highly sensitive and specific predictor of PZA resistance. Based on biological considerations, the POA efflux rate is directly determined by the PZAse activity, the level of pncA expression, and the efficiency of the POA efflux pump system. This study analyzes the individual and the adjusted contribution of PZAse activity, pncA expression and POA efflux rate on PZA resistance. Thirty M. tuberculosis strains with known microbiological PZA susceptibility or resistance were analyzed. For each strain, PZAse was recombinantly produced and its enzymatic activity measured. The level of pncA mRNA was estimated by quantitative RT-PCR, and the POA efflux rate was determined. Mutations in the pncA promoter were detected by DNA sequencing. All factors were evaluated by multiple regression analysis to determine their adjusted effects on the level of PZA resistance. Low level of pncA expression associated to mutations in the pncA promoter region was observed in pncA wild type resistant strains. POA efflux rate was the best predictor after adjusting for the other factors, followed by PZAse activity. These results suggest that tests which rely on pncA mutations or PZAse activity are likely to be less predictive of real PZA resistance than tests which measure the rate of POA efflux. This should be further analyzed in light of the development of alternate assays to determine PZA resistance. Copyright © 2013 Elsevier Ltd. All rights reserved.

pncA gene expression and prediction factors on pyrazinamide resistance in Mycobacterium tuberculosis

PubMed Central

Sheen, Patricia; Lozano, Katherine; Gilman, Robert H.; Valencia, Hugo J.; Loli, Sebastian; Fuentes, Patricia; Grandjean, Louis; Zimic, Mirko

2013-01-01

Summary Background Mutations in the pyrazinamidase (PZAse) coding gene, pncA, have been considered as the main cause of pyrazinamide (PZA) resistance in Mycobacterium tuberculosis. However, recent studies suggest there is no single mechanism of resistance to PZA. The pyrazinoic acid (POA) efflux rate is the basis of the PZA susceptibility Wayne test, and its quantitative measurement has been found to be a highly sensitive and specific predictor of PZA resistance. Based on biological considerations, the POA efflux rate is directly determined by the PZAse activity, the level of pncA expression, and the efficiency of the POA efflux pump system. Objective This study analyzes the individual and the adjusted contribution of PZAse activity, pncA expression and POA efflux rate on PZA resistance. Methods Thirty M. tuberculosis strains with known microbiological PZA susceptibility or resistance were analyzed. For each strain, PZAse was recombinantly produced and its enzymatic activity measured. The level of pncA mRNA was estimated by quantitative RT-PCR, and the POA efflux rate was determined. Mutations in the pncA promoter were detected by DNA sequencing. All factors were evaluated by multiple regression analysis to determine their adjusted effects on the level of PZA resistance. Findings Low level of pncA expression associated to mutations in the pncA promoter region was observed in pncA wild type resistant strains. POA efflux rate was the best predictor after adjusting for the other factors, followed by PZAse activity. These results suggest that tests which rely on pncA mutations or PZAse activity are likely to be less predictive of real PZA resistance than tests which measure the rate of POA efflux. This should be further analyzed in light of the development of alternate assays to determine PZA resistance. PMID:23867321

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

An adaptive response of Enterobacter aerogenes to imipenem: regulation of porin balance in clinical isolates.

PubMed

Lavigne, Jean-Philippe; Sotto, Albert; Nicolas-Chanoine, Marie-Hélène; Bouziges, Nicole; Pagès, Jean-Marie; Davin-Regli, Anne

2013-02-01

Imipenem (IPM) is a carbapenem antibiotic frequently used in severe hospital infections. Several reports have mentioned the emergence of resistant isolates exhibiting membrane modifications. A study was conducted between September 2005 and August 2007 to survey infections due to Enterobacter aerogenes in patients hospitalised in a French university hospital. Resistant E. aerogenes clinical isolates obtained from patients treated with IPM and collected during the 3 months following initiation of treatment were phenotypically and molecularly characterised for β-lactamases, efflux pumps activity and outer membrane proteins. Among the 339 patients infected with E. aerogenes during the study period, 41 isolates (12.1%) were resistant to extended-spectrum cephalosporins and 17 patients (5.0%) were treated with IPM. The isolates from these 17 patients presented TEM-24 and basal efflux expression. Following IPM treatment, an IPM-intermediate-susceptible (IPM-I) isolate emerged in 11 patients and an IPM-resistant (IPM-R) isolate in 6 patients. A change in the porin balance (Omp35/Omp36) was observed in IPM-I isolates exhibiting ertapenem resistance. Finally, a porin deficiency (Omp35 and Omp36 absence) was detected in IPM-R isolates associated with efflux pump expression. This study indicates that the alteration in porin expression, including the shift of porin expression and lack of porins, contribute to the E. aerogenes adaptive response to IPM treatment. Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

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

PubMed

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

2017-09-19

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

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

Advancements in the oral delivery of Docetaxel: challenges, current state-of-the-art and future trends

PubMed Central

Sohail, Muhammad Farhan; Rehman, Mubashar; Sarwar, Hafiz Shoaib; Naveed, Sara; Salman, Omer; Bukhari, Nadeem Irfan; Hussain, Irshad; Webster, Thomas J; Shahnaz, Gul

2018-01-01

The oral delivery of cancer chemotherapeutic drugs is challenging due to low bioavailability, gastrointestinal side effects, first-pass metabolism and P-glycoprotein efflux pumps. Thus, chemotherapeutic drugs, including Docetaxel, are administered via an intravenous route, which poses many disadvantages of its own. Recent advances in pharmaceutical research have focused on designing new and efficient drug delivery systems for site-specific targeting, thus leading to improved bioavailability and pharmacokinetics. A decent number of studies have been reported for the safe and effective oral delivery of Docetaxel. These nanocarriers, including liposomes, polymeric nanoparticles, metallic nanoparticles, hybrid nanoparticles, dendrimers and so on, have shown promising results in research papers and clinical trials. The present article comprehensively reviews the research efforts made so far in designing various advancements in the oral delivery of Docetaxel. Different strategies to improve oral bioavailability, prevent first-pass metabolism and inhibition of efflux pumping leading to improved pharmacokinetics and anticancer activity are discussed. The final portion of this review article presents key issues such as safety of nanomaterials, regulatory approval and future trends in nanomedicine research. PMID:29922053

Advancements in the oral delivery of Docetaxel: challenges, current state-of-the-art and future trends.

PubMed

Sohail, Muhammad Farhan; Rehman, Mubashar; Sarwar, Hafiz Shoaib; Naveed, Sara; Salman, Omer; Bukhari, Nadeem Irfan; Hussain, Irshad; Webster, Thomas J; Shahnaz, Gul

2018-01-01

The oral delivery of cancer chemotherapeutic drugs is challenging due to low bioavailability, gastrointestinal side effects, first-pass metabolism and P-glycoprotein efflux pumps. Thus, chemotherapeutic drugs, including Docetaxel, are administered via an intravenous route, which poses many disadvantages of its own. Recent advances in pharmaceutical research have focused on designing new and efficient drug delivery systems for site-specific targeting, thus leading to improved bioavailability and pharmacokinetics. A decent number of studies have been reported for the safe and effective oral delivery of Docetaxel. These nanocarriers, including liposomes, polymeric nanoparticles, metallic nanoparticles, hybrid nanoparticles, dendrimers and so on, have shown promising results in research papers and clinical trials. The present article comprehensively reviews the research efforts made so far in designing various advancements in the oral delivery of Docetaxel. Different strategies to improve oral bioavailability, prevent first-pass metabolism and inhibition of efflux pumping leading to improved pharmacokinetics and anticancer activity are discussed. The final portion of this review article presents key issues such as safety of nanomaterials, regulatory approval and future trends in nanomedicine research.

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

PubMed

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

2017-08-22

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

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

PubMed

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

2014-01-01

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

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

PubMed Central

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

2014-01-01

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

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

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.

Drug transporters, the blood–testis barrier, and spermatogenesis

PubMed Central

Su, Linlin; Mruk, Dolores D; Cheng, C Yan

2015-01-01

The blood–testis barrier (BTB), which is created by adjacent Sertoli cells near the basement membrane, serves as a ‘gatekeeper’ to prohibit harmful substances from reaching developing germ cells, most notably postmeiotic spermatids. The BTB also divides the seminiferous epithelium into the basal and adluminal (apical) compartment so that postmeiotic spermatid development, namely spermiogenesis, can take place in a specialized microenvironment in the apical compartment behind the BTB. The BTB also contributes, at least in part, to the immune privilege status of the testis, so that anti-sperm antibodies are not developed against antigens that are expressed transiently during spermatogenesis. Recent studies have shown that numerous drug transporters are expressed by Sertoli cells. However, many of these same drug transporters are also expressed by spermatogonia, spermatocytes, round spermatids, elongating spermatids, and elongated spermatids, suggesting that the developing germ cells are also able to selectively pump drugs ‘in’ and/or ‘out’ via influx or efflux pumps. We review herein the latest developments regarding the role of drug transporters in spermatogenesis. We also propose a model utilized by the testis to protect germ cell development from ‘harmful’ environmental toxicants and xenobiotics and/or from ‘therapeutic’ substances (e.g. anticancer drugs). We also discuss how drug transporters that are supposed to protect spermatogenesis can work against the testis in some instances. For example, when drugs (e.g. male contraceptives) that can perturb germ cell adhesion and/or maturation are actively pumped out of the testis or are prevented from entering the apical compartment, such as by efflux pumps. PMID:21134990

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.

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

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

Fermentative Production of Cysteine by Pantoea ananatis

PubMed Central

Takumi, Kazuhiro; Ziyatdinov, Mikhail Kharisovich; Samsonov, Viktor

2016-01-01

ABSTRACT Cysteine is a commercially important amino acid; however, it lacks an efficient fermentative production method. Due to its cytotoxicity, intracellular cysteine levels are stringently controlled via several regulatory modes. Managing its toxic effects as well as understanding and deregulating the complexities of regulation are crucial for establishing the fermentative production of cysteine. The regulatory modes include feedback inhibition of key metabolic enzymes, degradation, efflux pumps, and the transcriptional regulation of biosynthetic genes by a master cysteine regulator, CysB. These processes have been extensively studied using Escherichia coli for overproducing cysteine by fermentation. In this study, we genetically engineered Pantoea ananatis, an emerging host for the fermentative production of bio-based materials, to identify key factors required for cysteine production. According to this and our previous studies, we identified a major cysteine desulfhydrase gene, ccdA (formerly PAJ_0331), involved in cysteine degradation, and the cysteine efflux pump genes cefA and cefB (formerly PAJ_3026 and PAJ_p0018, respectively), which may be responsible for downregulating the intracellular cysteine level. Our findings revealed that ccdA deletion and cefA and cefB overexpression are crucial factors for establishing fermentative cysteine production in P. ananatis and for obtaining a higher cysteine yield when combined with genes in the cysteine biosynthetic pathway. To our knowledge, this is the first demonstration of cysteine production in P. ananatis, which has fundamental implications for establishing overproduction in this microbe. IMPORTANCE The efficient production of cysteine is a major challenge in the amino acid fermentation industry. In this study, we identified cysteine efflux pumps and degradation pathways as essential elements and genetically engineered Pantoea ananatis, an emerging host for the fermentative production of bio-based materials, to establish the fermentative production of cysteine. This study provides crucial insights into the design and construction of cysteine-producing strains, which may play central roles in realizing commercial basis production. PMID:28003193

Microenvironment acidity as a major determinant of tumor chemoresistance: Proton pump inhibitors (PPIs) as a novel therapeutic approach.

PubMed

Taylor, Sophie; Spugnini, Enrico Pierluigi; Assaraf, Yehuda G; Azzarito, Tommaso; Rauch, Cyril; Fais, Stefano

2015-11-01

Despite the major progresses in biomedical research and the development of novel therapeutics and treatment strategies, cancer is still among the dominant causes of death worldwide. One of the crucial challenges in the clinical management of cancer is primary (intrinsic) and secondary (acquired) resistance to both conventional and targeted chemotherapeutics. Multiple mechanisms have been identifiedthat underlie intrinsic and acquired chemoresistance: these include impaired drug uptake, increased drug efflux, deletion of receptors, altered drug metabolism, quantitative and qualitative alterations in drug targets, increased DNA damage repair and various mechanisms of anti-apoptosis. The fast efflux of anticancer drugs mediated by multidrug efflux pumps and the partial or complete reversibility of chemoresistance combined with the absence of genetic mutations suggests a multifactorial process. However, a growing body of recent evidence suggests that chemoresistance is often triggered by the highly acidic microenvironment of tumors. The vast majority of drugs, including conventional chemotherapeutics and more recent biological agents, are weak bases that are quickly protonated and neutralized in acidic environments, such as the extracellular microenvironment and the acidic organelles of tumor cells. It is therefore essential to develop new strategies to overcome the entrapment and neutralization of weak base drugs. One such strategy is the use of proton pump inhibitors which can enhance tumor chemosensitivity by increasing the pH of the tumor microenvironment. Recent clinical trials in animals with spontaneous tumors have indicated that patient alkalization is capable of reversing acquired chemoresistance in a large percentage of tumors that are refractory to chemotherapy. Of particular interest was the benefit of alkalization for patients undergoing metronomic regimens which are becoming more widely used in veterinary medicine. Overall, these results provide substantial new evidence that altering the acidic tumor microenvironment is an effective, well tolerated and low cost strategy for the overcoming of anticancer drug resistance. Copyright © 2015 Elsevier Ltd. All rights reserved.

Fermentative Production of Cysteine by Pantoea ananatis.

PubMed

Takumi, Kazuhiro; Ziyatdinov, Mikhail Kharisovich; Samsonov, Viktor; Nonaka, Gen

2017-03-01

Cysteine is a commercially important amino acid; however, it lacks an efficient fermentative production method. Due to its cytotoxicity, intracellular cysteine levels are stringently controlled via several regulatory modes. Managing its toxic effects as well as understanding and deregulating the complexities of regulation are crucial for establishing the fermentative production of cysteine. The regulatory modes include feedback inhibition of key metabolic enzymes, degradation, efflux pumps, and the transcriptional regulation of biosynthetic genes by a master cysteine regulator, CysB. These processes have been extensively studied using Escherichia coli for overproducing cysteine by fermentation. In this study, we genetically engineered Pantoea ananatis , an emerging host for the fermentative production of bio-based materials, to identify key factors required for cysteine production. According to this and our previous studies, we identified a major cysteine desulfhydrase gene, ccdA (formerly PAJ_0331), involved in cysteine degradation, and the cysteine efflux pump genes cefA and cefB (formerly PAJ_3026 and PAJ_p0018, respectively), which may be responsible for downregulating the intracellular cysteine level. Our findings revealed that ccdA deletion and cefA and cefB overexpression are crucial factors for establishing fermentative cysteine production in P. ananatis and for obtaining a higher cysteine yield when combined with genes in the cysteine biosynthetic pathway. To our knowledge, this is the first demonstration of cysteine production in P. ananatis , which has fundamental implications for establishing overproduction in this microbe. IMPORTANCE The efficient production of cysteine is a major challenge in the amino acid fermentation industry. In this study, we identified cysteine efflux pumps and degradation pathways as essential elements and genetically engineered Pantoea ananatis , an emerging host for the fermentative production of bio-based materials, to establish the fermentative production of cysteine. This study provides crucial insights into the design and construction of cysteine-producing strains, which may play central roles in realizing commercial basis production. Copyright © 2017 American Society for Microbiology.

Mechanisms of reduced susceptibility and genotypic prediction of antibiotic resistance in Prevotella isolated from cystic fibrosis (CF) and non-CF patients

PubMed Central

Sherrard, Laura J.; Schaible, Bettina; Graham, Kathryn A.; McGrath, Stef J.; McIlreavey, Leanne; Hatch, Joseph; Wolfgang, Matthew C.; Muhlebach, Marianne S.; Gilpin, Deirdre F.; Schneiders, Thamarai; Elborn, J. Stuart; Tunney, Michael M.

2014-01-01

Objectives To investigate mechanisms of reduced susceptibility to commonly used antibiotics in Prevotella cultured from patients with cystic fibrosis (CF), patients with invasive infection and healthy control subjects and to determine whether genotype can be used to predict phenotypic resistance. Methods The susceptibility of 157 Prevotella isolates to seven antibiotics was compared, with detection of resistance genes (cfxA-type gene, ermF and tetQ), mutations within the CfxA-type β-lactamase and expression of efflux pumps. Results Prevotella isolates positive for a cfxA-type gene had higher MICs of amoxicillin and ceftazidime compared with isolates negative for this gene (P < 0.001). A mutation within the CfxA-type β-lactamase (Y239D) was associated with ceftazidime resistance (P = 0.011). The UK CF isolates were 5.3-fold, 2.7-fold and 5.7-fold more likely to harbour ermF compared with the US CF, UK invasive and UK healthy control isolates, respectively. Higher concentrations of azithromycin (P < 0.001) and clindamycin (P < 0.001) were also required to inhibit the growth of the ermF-positive isolates compared with ermF-negative isolates. Furthermore, tetQ-positive Prevotella isolates had higher MICs of tetracycline (P = 0.001) and doxycycline (P < 0.001) compared with tetQ-negative isolates. Prevotella spp. were also shown, for the first time, to express resistance nodulation division (RND)-type efflux pumps. Conclusions This study has demonstrated that Prevotella isolated from various sources harbour a common pool of resistance genes and possess RND-type efflux pumps, which may contribute to tetracycline resistance. The findings indicate that antibiotic resistance is common in Prevotella spp., but the genotypic traits investigated do not reflect phenotypic antibiotic resistance in every instance. PMID:24917582

Genetic characterization of tigecycline resistance in clinical isolates of Enterobacter cloacae and Enterobacter aerogenes.

PubMed

Veleba, Mark; De Majumdar, Shyamasree; Hornsey, Michael; Woodford, Neil; Schneiders, Thamarai

2013-05-01

The intrinsically encoded ramA gene has been linked to tigecycline resistance through the up-regulation of efflux pump AcrAB in Enterobacter cloacae. The molecular basis for increased ramA expression in E. cloacae and Enterobacter aerogenes, as well as the role of AraC regulator rarA, has not yet been shown. To ascertain the intrinsic molecular mechanism(s) involved in tigecycline resistance in Enterobacter spp., we analysed the expression levels of ramA and rarA and corresponding efflux pump genes acrAB and oqxAB in Enterobacter spp. clinical isolates. The expression levels of ramA, rarA, oqxA and acrA were tested by quantitative real-time RT-PCR. The ramR open reading frames of the ramA-overexpressing strains were sequenced; strains harbouring mutations were transformed with wild-type ramR to study altered ramA expression and tigecycline susceptibility. Tigecycline resistance was mediated primarily by increased ramA expression in E. cloacae and E. aerogenes. Only the ramA-overexpressing E. cloacae isolates showed increased rarA and oqxA expression. Upon complementation with wild-type ramR, all Enterobacter spp. containing ramR mutations exhibited decreased ramA and acrA expression and increased tigecycline susceptibility. Exceptions were one E. cloacae strain and one E. aerogenes strain, where a decrease in ramA levels was not accompanied by lower acrA expression. Increased ramA expression due to ramR deregulation is the primary mediator of tigecycline resistance in clinical isolates of E. cloacae and E. aerogenes. However, some ramA-overexpressing isolates do not show changes in ramR, suggesting alternate pathways of ramA regulation; the rarA regulator and the oqxAB efflux pump may also play a role in tigecycline resistance in E. cloacae.

The consistent differential expression of genetic pathways following exposure of an industrial Pseudomonas aeruginosa strain to preservatives and a laundry detergent formulation.

PubMed

Green, Angharad E; Amézquita, Alejandro; Le Marc, Yvan; Bull, Matthew J; Connor, Thomas R; Mahenthiralingam, Eshwar

2018-05-01

Pseudomonas aeruginosa is a common contaminant associated with product recalls in the home and personal care industry. Preservation systems are used to prevent spoilage and protect consumers, but greater knowledge is needed of preservative resistance mechanisms used by P. aeruginosa contaminants. We aimed to identify genetic pathways associated with preservative exposure by using an industrial P. aeruginosa strain and implementing RNA-Seq to understand gene expression changes in response to industry relevant conditions. The consistent differential expression of five genetic pathways during exposure to multiple industrial growth conditions associated with benzisothiazolone (BIT) and phenoxyethanol (POE) preservatives, and a laundry detergent (LD) formulation, was observed. A MexPQ-OpmE Resistance Nodulation Division efflux pump system was commonly upregulated in response to POE, a combination of BIT and POE, and LD together with BIT. In response to all industry conditions, a putative sialic acid transporter and isoprenoid biosynthesis gnyRDBHAL operon demonstrated consistent upregulation. Two operons phnBA and pqsEDCBA involved in Pseudomonas quinolone signaling production and quorum-sensing were also consistently downregulated during exposure to all the industry conditions. The ability to identify consistently differentially expressed genetic pathways in P. aeruginosa can inform the development of future targeted preservation systems that maintain product safety and minimise resistance development.

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.

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

PubMed

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

2018-06-01

The aim of the present study was to investigate the correlation between the multi‑drug resistance of Shigella flexneri and the drug‑resistant gene cassette carried by integrons; in the meanwhile, to detect the associations between drug‑resistance and gene mutations of the active efflux pump acrAB‑tolC gene and its regulatory genes, including marOR, acrR and soxS. A total of 158 isolates were isolated from the stool samples of 1,026 children with diarrhoea aged 14 years old between May 2012 and October 2015 in Henan. The K‑B method was applied for the determination of drug resistance of Shigella flexneri, and polymerase chain reaction amplification was used for class 1, 2 and 3 integrase genes. Enzyme digestion and sequence analysis were performed for the variable regions of positive strains. Based on the drug sensitivity assessment, multi‑drug resistant strains that were resistant to five or more antibiotics, and sensitive strains were selected for amplification. Their active efflux pump genes, acrA and acrB, and regulatory genes, marOR, acrR and soxS, were selected for sequencing. The results revealed that 91.1% of the 158 strains were multi‑resistant to ampicillin, chloramphenicol, tetracycline and streptomycin, and 69.6% of the strains were multi‑resistant to sulfamethoxazole/trimethoprim. The resistance to ceftazidime, ciprofloxacin and levofloxacin was <32.9%. All strains (100%) were sensitive to cefoxitin, cefoperazone/sulbactam and imipenem. The rate of the class 1 integron positivity was 91.9% (144/158). Among these class 1 integron‑positive strains, 18 strains exhibited the resistance gene cassette dfrV in the variable region of the strain, four strains exhibited dfrA17‑aadA5 in the variable region and 140 strains exhibited blaOXA‑30‑aadA1 in the variable region. Four strains showed no resistance gene in the variable regions. The rate of class 2 integron positivity was 86.1% (136/158), and all positive strains harboured the dfrA1‑sat1‑aadA resistance gene cassette in the variable region. The class 3 integrase gene was not detected in these strains. The gene sequencing showed the deletion of base CATT in the 36, 37, 38, 39 site in the marOR gene, which is a regulatory gene of the active efflux pump, AcrAB‑TolC. Taken together, the multi‑drug resistance of Shigella flexneri was closely associated with gene mutations of class 1 and 2 integrons and the marOR gene.

Physiological and biochemical characteristics of adrenergic receptors and pathways in brown adipocytes

NASA Technical Reports Server (NTRS)

Horwitz, B. A.

1975-01-01

Mechanisms involved in the thermogenic response of brown adipose tissue (BAT) to sympathetic nervous stimulation (e.g., by cold exposure) and to norepinephrine (NE) release are investigated. Three effects appear to play a role in the increased oxygen consumption (and heat production) of the adipocytes: increased membrane permeability, activation of the beta-adrenergic pathway, and enhancement of Na(+)/K(+) membrane pump activity. Increased passive influx of Na(+) and efflux of K(+) due to greater permeability raise the energy demands of the Na/K pump; the pump is also stimulated by increased cyclic AMP synthesis resulting from activation by NE of membrane-bound adenyl cyclase. Studies with inhibitors such as propanolol, phentolamine, and ouabain support this hypothesis.

¹⁸FDG a PET tumor diagnostic tracer is not a substrate of the ABC transporter P-glycoprotein.

PubMed

Krasznai, Zoárd T; Trencsényi, György; Krasznai, Zoltán; Mikecz, Pál; Nizsalóczki, Enikő; Szalóki, Gábor; Szabó, Judit P; Balkay, László; Márián, Teréz; Goda, Katalin

2014-11-20

2-[(18)F]fluoro-2-deoxy-d-glucose ((18)FDG) is a tumor diagnostic radiotracer of great importance in both diagnosing primary and metastatic tumors and in monitoring the efficacy of the treatment. P-glycoprotein (Pgp) is an active transporter that is often expressed in various malignancies either intrinsically or appears later upon disease progression or in response to chemotherapy. Several authors reported that the accumulation of (18)FDG in P-glycoprotein (Pgp) expressing cancer cells (Pgp(+)) and tumors is different from the accumulation of the tracer in Pgp nonexpressing (Pgp(-)) ones, therefore we investigated whether (18)FDG is a substrate or modulator of Pgp pump. Rhodamine 123 (R123) accumulation experiments and ATPase assay were used to detect whether (18)FDG is substrate for Pgp. The accumulation and efflux kinetics of (18)FDG were examined in two different human gynecologic (A2780/A2780AD and KB-3-1/KB-V1) and a mouse fibroblast (3T3 and 3T3MDR1) Pgp(+) and Pgp(-) cancer cell line pairs both in cell suspension and monolayer cultures. We found that (18)FDG and its derivatives did not affect either the R123 accumulation in Pgp(+) cells or the basal and the substrate stimulated ATPase activity of Pgp supporting that they are not substrates or modulators of the pump. Measuring the accumulation and efflux kinetics of (18)FDG in different Pgp(+) and Pgp(-) cell line pairs, we have found that the Pgp(+) cells exhibited significantly higher (p⩽0.01) (18)FDG accumulation and slightly faster (18)FDG efflux kinetics compared to their Pgp(-) counterparts. The above data support the idea that expression of Pgp may increase the energy demand of cells resulting in higher (18)FDG accumulation and faster efflux. We concluded that (18)FDG and its metabolites are not substrates of Pgp. Copyright © 2014 Elsevier B.V. All rights reserved.

Analysis of the Fluoroquinolone Antibiotic Resistance Mechanism of Salmonella enterica Isolates.

PubMed

Kim, Soo-Young; Lee, Si-Kyung; Park, Myeong-Soo; Na, Hun-Taek

2016-09-28

Quinolone-resistant Salmonella strains were isolated from patient samples, and several quinolone-sensitive strains were used to analyze mutations in the quinolone resistance-determining region (QRDR) of gyrA, gyrB, parC, and parE and to screen for plasmid-mediated quinolone resistance. Among the 21 strains that showed resistance to nalidixic acid and ciprofloxacin (MIC 0.125-2.0 μg/ml), 17 strains had a mutation in QRDR codon 87 of gyrA, and 3 strains had a single mutation (Ser83 → Phe). Another cause of resistance, efflux pump regulation, was studied by examining the expression of acrB, ramA, marA, and soxS. Five strains, including Sal-KH1 and Sal-KH2, showed no increase in relative expression in an analysis using the qRT-PCR method (p < 0.05). In order to determine the genes involved in the resistance, the Sal-9 isolate that showed decreased susceptibility and did not contain a mutation in the gyrA QRDR was used to make the STM (MIC 8 μg/ml) and STH (MIC 16 μg/ml) ciprofloxacin-resistant mutants. The gyrA QRDR Asp87 → Gly mutation was identified in both the STM and STH mutants by mutation analysis. qRT-PCR analysis of the efflux transporter acrB of the AcrAB-TolC efflux system showed increased expression levels in both the STM (1.79-fold) and STH (2.0-fold) mutants. In addition, the expression of the transcriptional regulator marA was increased in both the STM (6.35-fold) and STH (21.73-fold) mutants. Moreover, the expression of soxS was increased in the STM (3.41-fold) and STH (10.05-fold) mutants (p < 0.05). Therefore, these results indicate that AcrAB-TolC efflux pump activity and the target site mutation in gyrA are involved in quinolone resistance.

The influence of the chloride gradient across red cell membranes on sodium and potassium movements

PubMed Central

Cotterrell, D.; Whittam, R.

1971-01-01

1. A study has been made to see whether active and passive movements of sodium and potassium in human red blood cells are influenced by changing the chloride gradient and hence the potential difference across the cell membrane. 2. Chloride distribution was measured between red cells and isotonic solutions with a range of concentrations of chloride and non-penetrating anions (EDTA, citrate, gluconate). The cell chloride concentration was greater than that outside with low external chloride, suggesting that the sign of the membrane potential was reversed. The chloride ratio (internal/external) was approximately equal to the inverse of the hydrogen ion ratio at normal and low external chloride, and inversely proportional to external pH. These results show that chloride is passively distributed, making it valid to calculate the membrane potential from the chloride ratio. 3. Ouabain-sensitive (pump) potassium influx and sodium efflux were decreased by not more than 20 and 40% respectively on reversing the chloride gradient, corresponding to a change in membrane potential from -9 to +30 mV. In contrast, passive (ouabain-insensitive) movements were reversibly altered — potassium influx was decreased about 60% and potassium efflux was increased some tenfold. Sodium influx was unaffected by the nature of the anion and depended only on the external sodium concentration, whereas ouabain-insensitive sodium efflux was increased about threefold. When external sodium was replaced by potassium there was a decrease in ouabain-insensitive sodium efflux with normal chloride, but an increase in low-chloride medium. 4. Net movements of sodium and potassium were roughly in accord with the unidirectional fluxes. 5. The results suggest that reversing the chloride gradient and, therefore, the sign of the membrane potential, had little effect on the sodium pump, but caused a marked increase in passive outward movements of both sodium and potassium ions. PMID:4996368

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

PubMed

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

2009-06-01

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

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.

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.

The autoinducer synthase LqsA and putative sensor kinase LqsS regulate phagocyte interactions, extracellular filaments and a genomic island of Legionella pneumophila.

PubMed

Tiaden, André; Spirig, Thomas; Sahr, Tobias; Wälti, Martin A; Boucke, Karin; Buchrieser, Carmen; Hilbi, Hubert

2010-05-01

The amoebae-resistant opportunistic pathogen Legionella pneumophila employs a biphasic life cycle to replicate in host cells and spread to new niches. Upon entering the stationary growth phase, the bacteria switch to a transmissive (virulent) state, which involves a complex regulatory network including the lqs gene cluster (lqsA-lqsR-hdeD-lqsS). LqsR is a putative response regulator that promotes host-pathogen interactions and represses replication. The autoinducer synthase LqsA catalyses the production of the diffusible signalling molecule 3-hydroxypentadecan-4-one (LAI-1) that is presumably recognized by the sensor kinase LqsS. Here, we analysed L. pneumophila strains lacking lqsA or lqsS. Compared with wild-type L. pneumophila, the DeltalqsS strain was more salt-resistant and impaired for the Icm/Dot type IV secretion system-dependent uptake by phagocytes. Legionella pneumophila strains lacking lqsS, lqsR or the alternative sigma factor rpoS sedimented more slowly and produced extracellular filaments. Deletion of lqsA moderately reduced the uptake of L. pneumophila by phagocytes, and the defect was complemented by expressing lqsA in trans. Unexpectedly, the overexpression of lqsA also restored the virulence defect and reduced filament production of L. pneumophila mutant strains lacking lqsS or lqsR, but not the phenotypes of strains lacking rpoS or icmT. These results suggest that LqsA products also signal through sensors not encoded by the lqs gene cluster. A transcriptome analysis of the DeltalqsA and DeltalqsS mutant strains revealed that under the conditions tested, lqsA regulated only few genes, whereas lqsS upregulated the expression of 93 genes at least twofold. These include 52 genes clustered in a 133 kb high plasticity genomic island, which is flanked by putative DNA-mobilizing genes and encodes multiple metal ion efflux pumps. Upon overexpression of lqsA, a cluster of 19 genes in the genomic island was also upregulated, suggesting that LqsA and LqsS participate in the same regulatory circuit.

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.

Synthetic organotelluride compounds induce the reversal of Pdr5p mediated fluconazole resistance in Saccharomyces cerevisiae.

PubMed

Reis de Sá, Leandro Figueira; Toledo, Fabiano Travanca; de Sousa, Bruno Artur; Gonçalves, Augusto César; Tessis, Ana Claudia; Wendler, Edison P; Comasseto, João V; Dos Santos, Alcindo A; Ferreira-Pereira, Antonio

2014-07-26

Resistance to fluconazole, a commonly used azole antifungal, is a challenge for the treatment of fungal infections. Resistance can be mediated by overexpression of ABC transporters, which promote drug efflux that requires ATP hydrolysis. The Pdr5p ABC transporter of Saccharomyces cerevisiae is a well-known model used to study this mechanism of antifungal resistance. The present study investigated the effects of 13 synthetic compounds on Pdr5p. Among the tested compounds, four contained a tellurium-butane group and shared structural similarities that were absent in the other tested compounds: a lateral hydrocarbon chain and an amide group. These four compounds were capable of inhibiting Pdr5p ATPase activity by more than 90%, they demonstrated IC50 values less than 2 μM and had an uncompetitive pattern of Pdr5p ATPase activity inhibition. These organotellurides did not demonstrate cytotoxicity against human erythrocytes or S. cerevisiae mutant strains (a strain that overexpress Pdr5p and a null mutant strain) even in concentrations above 100 μM. When tested at 100 μM, they could reverse the fluconazole resistance expressed by both the S. cerevisiae mutant strain that overexpress Pdr5p and a clinical isolate of Candida albicans. We have identified four organotellurides that are promising candidates for the reversal of drug resistance mediated by drug efflux pumps. These molecules will act as scaffolds for the development of more efficient and effective efflux pump inhibitors that can be used in combination therapy with available antifungals.

Synthetic organotelluride compounds induce the reversal of Pdr5p mediated fluconazole resistance in Saccharomyces cerevisiae

PubMed Central

2014-01-01

Background Resistance to fluconazole, a commonly used azole antifungal, is a challenge for the treatment of fungal infections. Resistance can be mediated by overexpression of ABC transporters, which promote drug efflux that requires ATP hydrolysis. The Pdr5p ABC transporter of Saccharomyces cerevisiae is a well-known model used to study this mechanism of antifungal resistance. The present study investigated the effects of 13 synthetic compounds on Pdr5p. Results Among the tested compounds, four contained a tellurium-butane group and shared structural similarities that were absent in the other tested compounds: a lateral hydrocarbon chain and an amide group. These four compounds were capable of inhibiting Pdr5p ATPase activity by more than 90%, they demonstrated IC50 values less than 2 μM and had an uncompetitive pattern of Pdr5p ATPase activity inhibition. These organotellurides did not demonstrate cytotoxicity against human erythrocytes or S. cerevisiae mutant strains (a strain that overexpress Pdr5p and a null mutant strain) even in concentrations above 100 μM. When tested at 100 μM, they could reverse the fluconazole resistance expressed by both the S. cerevisiae mutant strain that overexpress Pdr5p and a clinical isolate of Candida albicans. Conclusions We have identified four organotellurides that are promising candidates for the reversal of drug resistance mediated by drug efflux pumps. These molecules will act as scaffolds for the development of more efficient and effective efflux pump inhibitors that can be used in combination therapy with available antifungals. PMID:25062749

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

PubMed Central

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

2004-01-01

Blossoms are important sites of infection for Erwinia amylovora, the causal agent of fire blight of rosaceous plants. Before entering the tissue, the pathogen colonizes the stigmatic surface and has to compete for space and nutrient resources within the epiphytic community. Several epiphytes are capable of synthesizing antibiotics with which they antagonize phytopathogenic bacteria. Here, we report that a multidrug efflux transporter, designated NorM, of E. amylovora confers tolerance to the toxin(s) produced by epiphytic bacteria cocolonizing plant blossoms. According to sequence comparisons, the single-component efflux pump NorM is a member of the multidrug and toxic compound extrusion protein family. The corresponding gene is widely distributed among E. amylovora strains and related plant-associated bacteria. NorM mediated resistance to the hydrophobic cationic compounds norfloxacin, ethidium bromide, and berberine. A norM mutant was constructed and exhibited full virulence on apple rootstock MM 106. However, it was susceptible to antibiotics produced by epiphytes isolated from apple and quince blossoms. The epiphytes were identified as Pantoea agglomerans by 16S rRNA analysis and were isolated from one-third of all trees examined. The promoter activity of norM was twofold greater at 18°C than at 28°C. The lower temperature seems to be beneficial for host infection because of the availability of moisture necessary for movement of the pathogen to the infection sites. Thus, E. amylovora might employ NorM for successful competition with other epiphytic microbes to reach high population densities, particularly at a lower temperature. PMID:14766544

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

PubMed

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

2012-01-01

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

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

PubMed Central

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

2000-01-01

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

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

PubMed

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

2012-08-01

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

Intestinal P-glycoprotein inhibitors, benzoxanthone analogues.

PubMed

Chae, Song Wha; Lee, Jaeok; Park, Jung Hyun; Kwon, Youngjoo; Na, Younghwa; Lee, Hwa Jeong

2018-02-01

The inhibitors of P-glycoprotein (P-gp) which limits an access of exogenous compounds in the luminal membrane of the intestine have been studied to enhance the intestinal P-gp-mediated absorption of anticancer drugs. Inhibition of the efflux pump by synthesized benzoxanthone derivatives was investigated in vitro and in vivo. MCF-7/ADR cell line was used for cytotoxicity assay and [ 3 H]-daunomycin (DNM) accumulation/efflux study. Eight benzoxanthone analogues were tested for their effects on DNM cytotoxicity. Among them, three analogues were selected for the accumulation/efflux and P-gp ATPase studies. Paclitaxel (PTX), a P-gp substrate anticancer drug, was orally administered to rats with/without compound 1 (8,10-bis(thiiran-2-ylmethoxy)-7H-benzo[c]xanthen-7-one). The pharmacokinetic parameters of PTX in the presence/absence of compound 1 were evaluated from the plasma concentration-time profiles. Compound 1 increased the DNA accumulation to 6.5-fold and decreased the DNM efflux to approximately 1/2 in the overexpressing P-gp cell line. Relative bioavailability (RB) of PTX in rats was significantly increased up to 3.2-fold by compound 1 (0.5 or 2 mg/kg). Benzoxanthone analogue, compound 1 is strongly suggested to be a promising inhibitor of P-gp to improve an oral absorption of compounds for cancer therapy. © 2017 Royal Pharmaceutical Society.

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

PubMed

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

2016-12-01

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

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

Comparative Genomics Analysis of a New Exiguobacterium Strain from Salar de Huasco Reveals a Repertoire of Stress-Related Genes and Arsenic Resistance.

PubMed

Castro-Severyn, Juan; Remonsellez, Francisco; Valenzuela, Sandro L; Salinas, Cesar; Fortt, Jonathan; Aguilar, Pablo; Pardo-Esté, Coral; Dorador, Cristina; Quatrini, Raquel; Molina, Franck; Aguayo, Daniel; Castro-Nallar, Eduardo; Saavedra, Claudia P

2017-01-01

The Atacama Desert hosts diverse ecosystems including salt flats and shallow Andean lakes. Several heavy metals are found in the Atacama Desert, and microorganisms growing in this environment show varying levels of resistance/tolerance to copper, tellurium, and arsenic, among others. Herein, we report the genome sequence and comparative genomic analysis of a new Exiguobacterium strain, sp. SH31, isolated from an altiplanic shallow athalassohaline lake. Exiguobacterium sp. SH31 belongs to the phylogenetic Group II and its closest relative is Exiguobacterium sp. S17, isolated from the Argentinian Altiplano (95% average nucleotide identity). Strain SH31 encodes a wide repertoire of proteins required for cadmium, copper, mercury, tellurium, chromium, and arsenic resistance. Of the 34 Exiguobacterium genomes that were inspected, only isolates SH31 and S17 encode the arsenic efflux pump Acr3. Strain SH31 was able to grow in up to 10 mM arsenite and 100 mM arsenate, indicating that it is arsenic resistant. Further, expression of the ars operon and acr3 was strongly induced in response to both toxics, suggesting that the arsenic efflux pump Acr3 mediates arsenic resistance in Exiguobacterium sp. SH31.

Environmental forcing does not induce diel or synoptic variation in the carbon isotope content of forest soil respiration

DOE PAGES

Bowling, D. R.; Egan, J. E.; Hall, S. J.; ...

2015-08-31

Recent studies have examined temporal fluctuations in the amount and carbon isotope content (δ 13C) of CO 2 produced by the respiration of roots and soil organisms. These changes have been correlated with diel cycles of environmental forcing (e.g., sunlight and soil temperature) and with synoptic-scale atmospheric motion (e.g., rain events and pressure-induced ventilation). We used an extensive suite of measurements to examine soil respiration over 2 months in a subalpine forest in Colorado, USA (the Niwot Ridge AmeriFlux forest). Observations included automated measurements of CO 2 and δ 13C of CO 2 in the soil efflux, the soil gasmore » profile, and forest air. There was strong diel variability in soil efflux but no diel change in the δ 13C of the soil efflux (δ R) or the CO 2 produced by biological activity in the soil (δ J). Following rain, soil efflux increased significantly, but δ R and δ J did not change. Temporal variation in the δ 13C of the soil efflux was unrelated to measured environmental variables, and we failed to find an explanation for this unexpected result. Measurements of the δ 13C of the soil efflux with chambers agreed closely with independent observations of the isotopic composition of soil CO 2 production derived from soil gas well measurements. Deeper in the soil profile and at the soil surface, results confirmed established theory regarding diffusive soil gas transport and isotopic fractionation. Deviation from best-fit diffusion model results at the shallower depths illuminated a pump-induced ventilation artifact that should be anticipated and avoided in future studies. There was no evidence of natural pressure-induced ventilation of the deep soil. However, higher variability in δ 13C of the soil efflux relative to δ 13C of production derived from soil profile measurements was likely caused by transient pressure-induced transport with small horizontal length scales.« less

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.

Sublethal exposure to commercial formulations of the herbicides dicamba, 2,4-dichlorophenoxyacetic acid, and glyphosate cause changes in antibiotic susceptibility in Escherichia coli and Salmonella enterica serovar Typhimurium.

PubMed

Kurenbach, Brigitta; Marjoshi, Delphine; Amábile-Cuevas, Carlos F; Ferguson, Gayle C; Godsoe, William; Gibson, Paddy; Heinemann, Jack A

2015-03-24

Biocides, such as herbicides, are routinely tested for toxicity but not for sublethal effects on microbes. Many biocides are known to induce an adaptive multiple-antibiotic resistance phenotype. This can be due to either an increase in the expression of efflux pumps, a reduced synthesis of outer membrane porins, or both. Exposures of Escherichia coli and Salmonella enterica serovar Typhimurium to commercial formulations of three herbicides-dicamba (Kamba), 2,4-dichlorophenoxyacetic acid (2,4-D), and glyphosate (Roundup)-were found to induce a changed response to antibiotics. Killing curves in the presence and absence of sublethal herbicide concentrations showed that the directions and the magnitudes of responses varied by herbicide, antibiotic, and species. When induced, MICs of antibiotics of five different classes changed up to 6-fold. In some cases the MIC increased, and in others it decreased. Herbicide concentrations needed to invoke the maximal response were above current food maximum residue levels but within application levels for all herbicides. Compounds that could cause induction had additive effects in combination. The role of soxS, an inducer of the AcrAB efflux pump, was tested in β-galactosidase assays with soxS-lacZ fusion strains of E. coli. Dicamba was a moderate inducer of the sox regulon. Growth assays with Phe-Arg β-naphtylamide (PAβN), an efflux pump inhibitor, confirmed a significant role of efflux in the increased tolerance of E. coli to chloramphenicol in the presence of dicamba and to kanamycin in the presence of glyphosate. Pathways of exposure with relevance to the health of humans, domestic animals, and critical insects are discussed. Increasingly common chemicals used in agriculture, domestic gardens, and public places can induce a multiple-antibiotic resistance phenotype in potential pathogens. The effect occurs upon simultaneous exposure to antibiotics and is faster than the lethal effect of antibiotics. The magnitude of the induced response may undermine antibiotic therapy and substantially increase the probability of spontaneous mutation to higher levels of resistance. The combination of high use of both herbicides and antibiotics in proximity to farm animals and important insects, such as honeybees, might also compromise their therapeutic effects and drive greater use of antibiotics. To address the crisis of antibiotic resistance requires broadening our view of environmental contributors to the evolution of resistance. Copyright © 2015 Kurenbach et al.

Effect of glucose transport inhibitors on vincristine efflux in multidrug-resistant murine erythroleukaemia cells overexpressing the multidrug resistance-associated protein (MRP) and two glucose transport proteins, GLUT1 and GLUT3.

PubMed Central

Martell, R. L.; Slapak, C. A.; Levy, S. B.

1997-01-01

The relationship between mammalian facilitative glucose transport proteins (GLUT) and multidrug resistance was examined in two vincristine (VCR)-selected murine erythroleukaemia (MEL) PC4 cell lines. GLUT proteins, GLUT1 and GLUT3, were constitutively coexpressed in the parental cell line and also in the VCR-selected cell lines. Increased expression of the GLUT1 isoform was noted both in the PC-V40 (a non-P-glycoprotein, mrp-overexpressing subline) and in the more resistant PC-V160 (overexpressing mrp and mdr3) cell lines. Overexpression of GLUT3 was detected only in the PC-V160 subline. An increased rate of facilitative glucose transport (Vmax) and level of plasma membrane GLUT protein expression paralleled increased VCR resistance, active VCR efflux and decreased VCR steady-state accumulation in these cell lines. Glucose transport inhibitors (GTIs), cytochalasin B (CB) and phloretin blocked the active efflux and decreased steady-state accumulation of VCR in the PC-V40 subline. GTIs did not significantly affect VCR accumulation in the parental or PC-V160 cells. A comparison of protein sequences among GLUT1, GLUT3 and MRP revealed a putative cytochalasin B binding site in MRP, which displayed 44% sequence similarity/12% identity with that previously identified in GLUT1 and GLUT3; these regions also exhibited a similar hydropathy plot pattern. The findings suggested that CB bound to MRP and directly or indirectly lowered VCR efflux and/or CB bound to one or both GLUT proteins, which acted to lower the VCR efflux mediated by MRP. This is the first report of a non-neuronal murine cell line that expressed GLUT3. Images Figure 3 PMID:9010020

Fluconazole Resistance Associated with Drug Efflux and Increased Transcription of a Drug Transporter Gene, PDH1, in Candida glabrata

PubMed Central

Miyazaki, Haruko; Miyazaki, Yoshitsugu; Geber, Antonia; Parkinson, Tanya; Hitchcock, Christopher; Falconer, Derek J.; Ward, Douglas J.; Marsden, Katherine; Bennett, John E.

1998-01-01

Sequential Candida glabrata isolates were obtained from the mouth of a patient infected with human immunodeficiency virus type 1 who was receiving high doses of fluconazole for oropharyngeal thrush. Fluconazole-susceptible colonies were replaced by resistant colonies that exhibited both increased fluconazole efflux and increased transcripts of a gene which codes for a protein with 72.5% identity to Pdr5p, an ABC multidrug transporter in Saccharomyces cerevisiae. The deduced protein had a molecular mass of 175 kDa and was composed of two homologous halves, each with six putative transmembrane domains and highly conserved sequences of ATP-binding domains. When the earliest and most azole-susceptible isolate of C. glabrata from this patient was exposed to fluconazole, increased transcripts of the PDR5 homolog appeared, linking azole exposure to regulation of this gene. PMID:9661006

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

Increased cell membrane permeability to Na+ and K+ induced by thyroid hormone in rat skeletal muscle.

PubMed

Asano, Y

1978-01-01

Thyroid hormone (T3) increased Na+ dependent respiration accompanied by an increase in NaK-ATPase activity. Administration of T3 increased intracellular K+ concentration and Na/K ratio in thyroidectomized rats, and the Na+ efflux rate constant incubated in oxygenized Na+, K+-Ringers in euthyroid rats. However, the magnitude of the changes in intracellular K+ concentration was modest or invisible in comparison to the changes in QO2(t) and NaK-ATPase activity. The Na+ and K+ efflux rate constants in K+-free +ouabain Ringers were increased by T3 in both thyroidectomized and euthyroid rats. Thus, thyroid hormone stimulates not only Na pump but also the permeability of cell membrane to Na+ and K+. The both effects might contribute to the thyroid thermogenesis.

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

PubMed

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

2015-04-15

Six compounds (1-6), isolated from the methanol extract of the roots of the African medicinal plant Zanthoxylum capense Thunb. (Rutaceae), and seven ester derivatives (7-13) were evaluated for their antibacterial activities and modulatory effects on the MIC of antibiotics (erythromycin, oxacillin, and tetracycline) and ethidium bromide (EtBr) against a Staphylococcus aureus reference strain (ATCC 6538). Using the same model, compounds 1-13 were also assessed for their potential as efflux pump inhibitors by a fluorometric assay that measures the accumulation of the broad range efflux pump substrate EtBr. Compounds 8 and 11 were further evaluated for their antibacterial, modulatory and EtBr accumulation effects against four additional S. aureus strains, which included two clinical methicillin-resistant S. aureus (MRSA) strains. Compounds (1-13) have not shown antibacterial activity at the concentration ranges tested. When evaluated against S. aureus ATCC 6538, oxychelerythrine (1) a benzophenanthridine alkaloid, showed the highest modulatory activity enhancing the susceptibility of this strain to all the tested antibiotics from two to four-fold. Ailanthoidiol diacetate (8) and ailanthoidiol di-2-ethylbutanoate (11) were also good modulators when combined with EtBr, increasing the bacteria susceptibility by four and two-fold, respectively. In the EtBr accumulation assay, using ATCC 6538 strain, the phenylpropanoid (+)-ailanthoidiol (6) and most of its ester derivatives (8-11) exhibited higher activity than the positive control verapamil. The highest effects were found for compounds 8 and 11 that also increased the accumulation of EtBr, using S. aureus ATCC 25923 as model. Furthermore, both compounds (8, 11) were able to enhance the ciprofloxacin activity against the MRSA clinical strains tested, causing a reduction of the antibiotic MIC values from two to four-fold. The EtBr accumulation assay revealed that this modulation activity was not due to an inhibition of efflux pumps mechanism. These results suggested that Z. capense constituents may be valuable as leads for restoring antibiotic activity against MRSA strains. Copyright © 2015 Elsevier GmbH. All rights reserved.

Molecular identification of arsenic-resistant estuarine bacteria and characterization of their ars genotype.

PubMed

Sri Lakshmi Sunita, M; Prashant, S; Bramha Chari, P V; Nageswara Rao, S; Balaravi, Padma; Kavi Kishor, P B

2012-01-01

In the present study, 44 arsenic-resistant bacteria were isolated through serial dilutions on agar plate with concentrations ≥0.05 mM of sodium arsenite and ≥10 mM of sodium arsenate from Mandovi and Zuari--estuarine water systems. The ars genotype characterization in 36 bacterial isolates (resistant to 100 mM of sodium arsenate) revealed that only 17 isolates harboured the arsA (ATPase), B (arsenite permease) and C (arsenate reductase) genes on the plasmid DNA. The arsA, B and C genes were individually detected using PCR in 16, 9 and 13 bacterial isolates respectively. Molecular identification of the 17 isolates bearing the ars genotype was carried using 16S rDNA sequencing. A 1300 bp full length arsB gene encoding arsenite efflux pump and a 409 bp fragment of arsC gene coding for arsenate reductase were isolated from the genera Halomonas and Acinetobacter. Phylogenetic analysis of arsB and arsC genes indicated their close genetic relationship with plasmid borne ars genes of E. coli and arsenate reductase of plant origin. The putative arsenate reductase gene isolated from Acinetobacter species complemented arsenate resistance in E. coli WC3110 and JM109 validating its function. This study dealing with isolation of native arsenic-resistant bacteria and characterization of their ars genes might be useful to develop efficient arsenic detoxification strategies for arsenic contaminated aquifers.

The consistent differential expression of genetic pathways following exposure of an industrial Pseudomonas aeruginosa strain to preservatives and a laundry detergent formulation

PubMed Central

Amézquita, Alejandro; Le Marc, Yvan; Bull, Matthew J; Connor, Thomas R; Mahenthiralingam, Eshwar

2018-01-01

Abstract Pseudomonas aeruginosa is a common contaminant associated with product recalls in the home and personal care industry. Preservation systems are used to prevent spoilage and protect consumers, but greater knowledge is needed of preservative resistance mechanisms used by P. aeruginosa contaminants. We aimed to identify genetic pathways associated with preservative exposure by using an industrial P. aeruginosa strain and implementing RNA-Seq to understand gene expression changes in response to industry relevant conditions. The consistent differential expression of five genetic pathways during exposure to multiple industrial growth conditions associated with benzisothiazolone (BIT) and phenoxyethanol (POE) preservatives, and a laundry detergent (LD) formulation, was observed. A MexPQ-OpmE Resistance Nodulation Division efflux pump system was commonly upregulated in response to POE, a combination of BIT and POE, and LD together with BIT. In response to all industry conditions, a putative sialic acid transporter and isoprenoid biosynthesis gnyRDBHAL operon demonstrated consistent upregulation. Two operons phnBA and pqsEDCBA involved in Pseudomonas quinolone signaling production and quorum-sensing were also consistently downregulated during exposure to all the industry conditions. The ability to identify consistently differentially expressed genetic pathways in P. aeruginosa can inform the development of future targeted preservation systems that maintain product safety and minimise resistance development. PMID:29548026

A chromosome 4 trisomy contributes to increased fluconazole resistance in a clinical isolate of Candida albicans

PubMed Central

Anderson, Matthew Z.; Saha, Amrita; Haseeb, Abid

2017-01-01

Candida albicans is an important opportunistic fungal pathogen capable of causing both mucosal and disseminated disease. Infections are often treated with fluconazole, a front-line antifungal drug that targets the biosynthesis of ergosterol, a major component of the fungal cell membrane. Resistance to fluconazole can arise through a variety of mechanisms, including gain-of-function mutations, loss of heterozygosity events and aneuploidy. The clinical isolate P60002 was found to be highly resistant to azole-class drugs, yet lacked mutations or chromosomal rearrangements known to be associated with azole resistance. Transcription profiling suggested that increased expression of two putative drug efflux pumps, CDR11 and QDR1, might confer azole resistance. However, ectopic expression of the P60002 alleles of these genes in a drug-susceptible strain did not increase fluconazole resistance. We next examined whether the presence of three copies of chromosome 4 (Chr4) or chromosome 6 (Chr6) contributed to azole resistance in P60002. We established that Chr4 trisomy contributes significantly to fluconazole resistance, whereas Chr6 trisomy has no discernible effect on resistance. In contrast, a Chr4 trisomy did not increase fluconazole resistance when present in the standard SC5314 strain background. These results establish a link between Chr4 trisomy and elevated fluconazole resistance, and demonstrate the impact of genetic background on drug resistance phenotypes in C. albicans. PMID:28640746

Response to copper of Acidithiobacillus ferrooxidans ATCC 23270 grown in elemental sulfur.

PubMed

Almárcegui, Rodrigo J; Navarro, Claudio A; Paradela, Alberto; Albar, Juan Pablo; von Bernath, Diego; Jerez, Carlos A

2014-11-01

The response of Acidithiobacillus ferrooxidans ATCC 23270 to copper was analyzed in sulfur-grown cells by using quantitative proteomics. Forty-seven proteins showed altered levels in cells grown in the presence of 50 mM copper sulfate. Of these proteins, 24 were up-regulated and 23 down-regulated. As seen before in ferrous iron-grown cells, there was a notorious up-regulation of RND-type Cus systems and different RND-type efflux pumps, indicating that these proteins are very important in copper resistance. Copper also triggered the down-regulation of the major outer membrane porin of A. ferrooxidans in sulfur-grown bacteria, suggesting they respond to the metal by decreasing the influx of cations into the cell. On the contrary, copper in sulfur-grown cells caused an overexpression of putative TadA and TadB proteins known to be essential for biofilm formation in bacteria. Surprisingly, sulfur-grown microorganisms showed increased levels of proteins related with energy generation (rus and petII operons) in the presence of copper. Although rus operon is overexpressed mainly in cells grown in ferrous iron, the up-regulation of rusticyanin in sulfur indicates a possible role for this protein in copper resistance as well. Finally, copper response in A. ferrooxidans appears to be influenced by the substrate being oxidized by the microorganism. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Complete Sequence of a 184-Kilobase Catabolic Plasmid from Sphingomonas aromaticivorans F199†

PubMed Central

Romine, Margaret F.; Stillwell, Lisa C.; Wong, Kwong-Kwok; Thurston, Sarah J.; Sisk, Ellen C.; Sensen, Christoph; Gaasterland, Terry; Fredrickson, Jim K.; Saffer, Jeffrey D.

1999-01-01

The complete 184,457-bp sequence of the aromatic catabolic plasmid, pNL1, from Sphingomonas aromaticivorans F199 has been determined. A total of 186 open reading frames (ORFs) are predicted to encode proteins, of which 79 are likely directly associated with catabolism or transport of aromatic compounds. Genes that encode enzymes associated with the degradation of biphenyl, naphthalene, m-xylene, and p-cresol are predicted to be distributed among 15 gene clusters. The unusual coclustering of genes associated with different pathways appears to have evolved in response to similarities in biochemical mechanisms required for the degradation of intermediates in different pathways. A putative efflux pump and several hypothetical membrane-associated proteins were identified and predicted to be involved in the transport of aromatic compounds and/or intermediates in catabolism across the cell wall. Several genes associated with integration and recombination, including two group II intron-associated maturases, were identified in the replication region, suggesting that pNL1 is able to undergo integration and excision events with the chromosome and/or other portions of the plasmid. Conjugative transfer of pNL1 to another Sphingomonas sp. was demonstrated, and genes associated with this function were found in two large clusters. Approximately one-third of the ORFs (59 of them) have no obvious homology to known genes. PMID:10049392

In vivo and in vitro acquisition of resistance to voriconazole by Candida krusei.

PubMed

Ricardo, Elisabete; Miranda, Isabel M; Faria-Ramos, Isabel; Silva, Raquel M; Rodrigues, Acácio Gonçalves; Pina-Vaz, Cidália

2014-08-01

Candida krusei is an important agent of opportunistic infections that often displays resistance to several antifungals. We describe here the in vivo acquisition of resistance to voriconazole (VRC) by C. krusei isolates recovered from a leukemia patient during a long period of VRC therapy. In order to mimic the in vivo development of VRC resistance, a susceptible C. krusei isolate was exposed daily to 1 μg/ml of VRC in vitro. Interestingly, after 5 days of exposure to VRC, a MIC of 4 μg/ml was achieved; this value remained constant after 25 additional days of treatment with VRC and also after 30 consecutive days of incubation in VRC-free medium. Our objective was to determine the associated molecular resistance mechanisms, such as expression of efflux pump genes and ERG11 gene mutations, among the resistant strains. Synergistic effects between the efflux blocker tacrolimus (FK506) and VRC were found in all of the resistant strains. Moreover, ABC1 gene expression increased over time in both the in vivo- and in vitro-induced resistant strains, in contrast to the ABC2 and ERG11 genes, whose expression was invariably lower and constant. ERG11 gene sequencing showed two different types of mutations, i.e., heterozygosity at T1389T/C, corresponding to synonymous mutations, in C. krusei strains and a missense mutation at position T418C, resulting in a change from Tyr to His, among resistant C. krusei clinical isolates. This study highlights the relevance of ATP-dependent efflux pump (namely, Abc1p) activity in VRC resistance and describes new mutations in the ERG11 gene among resistant C. krusei clinical isolates. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Synthesis and P-glycoprotein induction activity of colupulone analogs.

PubMed

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

2015-05-21

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

Levofloxacin Efflux and smeD in Clinical Isolates of Stenotrophomonas maltophilia.

PubMed

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

2017-03-01

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

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

PubMed

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

2013-06-03

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

In vitro and in vivo evaluation of drug-drug interaction between dabigatran and proton pump inhibitors.

PubMed

Ollier, Edouard; Hodin, Sophie; Basset, Thierry; Accassat, Sandrine; Bertoletti, Laurent; Mismetti, Patrick; Delavenne, Xavier

2015-12-01

To quantify the drug-drug interactions between dabigatran etexilate (DE) and proton pump inhibitors (PPI) and in particular the role of P-gp activity modulation. In the first part of the study, efflux ratios of DE were evaluated using the caco-2 cell line in the presence of pantoprazole, omeprazole, rabeprazole, lansoprazole and ciclosporin A (positive control). The two PPI that reduced the efflux ratio of dabigatran to the greatest and least extent, respectively, were used during the second part of the study, comprising a single-centre, randomised, open-label study with an incomplete Latin square design. Nine healthy volunteers received DE (150 mg) alone, DE (150 mg) with the first PPI and DE (150 mg) with the second PPI in randomised sequence. Dabigatran plasma concentration and thrombin time were measured in blood samples withdrawn at 11 time points after each treatment. Models were built using a nonlinear mixed-effect modelling approach. Omeprazole and rabeprazole were the two PPI that reduced the efflux ratio of DE least and most, respectively. The PK model was based on an inverse Gaussian absorption process with one compartment. The relationship between dabigatran concentration and thrombin time was considered linear. Some PK profiles had dramatically low concentration values due to poor absorption. These profiles were clustered using a between subject model mixture with interoccasion variability. The concomitant administration of PPI did not significantly change dabigatran pharmacokinetics. DE is subject to high absorption variability, precluding evaluation of the effect of PPI on its pharmacokinetics. © 2015 Société Française de Pharmacologie et de Thérapeutique.

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.

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

PubMed

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

2012-06-01

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

A new tetracycline efflux gene, tet(40), is located in tandem with tet(O/32/O) in a human gut firmicute bacterium and in metagenomic library clones.

PubMed

Kazimierczak, Katarzyna A; Rincon, Marco T; Patterson, Andrea J; Martin, Jennifer C; Young, Pauline; Flint, Harry J; Scott, Karen P

2008-11-01

The bacterium Clostridium saccharolyticum K10, isolated from a fecal sample obtained from a healthy donor who had received long-term tetracycline therapy, was found to carry three tetracycline resistance genes: tet(W) and the mosaic tet(O/32/O), both conferring ribosome protection-type resistance, and a novel, closely linked efflux-type resistance gene designated tet(40). tet(40) encodes a predicted membrane-associated protein with 42% amino acid identity to tetA(P). Tetracycline did not accumulate in Escherichia coli cells expressing the Tet(40) efflux protein, and resistance to tetracycline was reduced when cells were incubated with an efflux pump inhibitor. E. coli cells carrying tet(40) had a 50% inhibitory concentration of tetracycline of 60 microg/ml. Analysis of a transconjugant from a mating between donor strain C. saccharolyticum K10 and the recipient human gut commensal bacterium Roseburia inulinivorans suggested that tet(O/32/O) and tet(40) were cotransferred on a mobile element. Sequence analysis of a 37-kb insert identified on the basis of tetracycline resistance from a metagenomic fosmid library again revealed a tandem arrangement of tet(O/32/O) and tet(40), flanked by regions with homology to parts of the VanG operon previously identified in Enterococcus faecalis. At least 10 of the metagenomic inserts that carried tet(O/32/O) also carried tet(40), suggesting that tet(40), although previously undetected, may be an abundant efflux gene.

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

PubMed

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

2015-11-01

Iron is an essential element for nearly all cells and limited iron availability often restricts growth. However, excess iron can also be deleterious, particularly when cells expressing high affinity iron uptake systems transition to iron rich environments. Bacillus subtilis expresses numerous iron importers, but iron efflux has not been reported. Here, we describe the B. subtilis PfeT protein (formerly YkvW/ZosA) as a P1B4 -type ATPase in the PerR regulon that serves as an Fe(II) efflux pump and protects cells against iron intoxication. Iron and manganese homeostasis in B. subtilis are closely intertwined: a pfeT mutant is iron sensitive, and this sensitivity can be suppressed by low levels of Mn(II). Conversely, a pfeT mutant is more resistant to Mn(II) overload. In vitro, the PfeT ATPase is activated by both Fe(II) and Co(II), although only Fe(II) efflux is physiologically relevant in wild-type cells, and null mutants accumulate elevated levels of intracellular iron. Genetic studies indicate that PfeT together with the ferric uptake repressor (Fur) cooperate to prevent iron intoxication, with iron sequestration by the MrgA mini-ferritin playing a secondary role. Protection against iron toxicity may also be a key role for related P1B4 -type ATPases previously implicated in bacterial pathogenesis. © 2015 John Wiley & Sons Ltd.

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

PubMed

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

2012-03-01

Clinical management of patients undergoing treatment of oropharyngeal candidiasis with azole antifungals can be impaired by azole resistance. High-level azole resistance is often caused by the overexpression of Candida albicans efflux pump Cdr1p. Inhibition of this pump therefore represents a target for combination therapies that reverse azole resistance. We assessed the therapeutic potential of the D-octapeptide derivative RC21v3, a Cdr1p inhibitor, in the treatment of murine oral candidiasis caused by either the azole-resistant C. albicans clinical isolate MML611 or its azole-susceptible parental strain MML610. RC21v3, fluconazole (FLC), or a combination of both drugs were administered orally to immunosuppressed ICR mice at 3, 24, and 27 h after oral inoculation with C. albicans. FLC protected the mice inoculated with MML610 from oral candidiasis, but was only partially effective in MML611-infected mice. The co-application of RC21v3 (0.02 μmol per dose) potentiated the therapeutic performance of FLC for mice infected with either strain. It caused a statistically significant decrease in C. albicans cfu isolated from the oral cavity of the infected mice and reduced oral lesions. RC21v3 also enhanced the therapeutic activity of itraconazole against MML611 infection. These results indicate that RC21v3 in combination with azoles has potential as a therapy against azole-resistant oral candidiasis. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Overcoming STC2 mediated drug resistance through drug and gene co-delivery by PHB-PDMAEMA cationic polyester in liver cancer cells.

PubMed

Cheng, Hongwei; Wu, Zhixian; Wu, Caisheng; Wang, Xiaoyuan; Liow, Sing Shy; Li, Zibiao; Wu, Yun-Long

2018-02-01

Stanniocalcin 2 (STC2) overexpression in hepatocellular carcinoma (HCC) could lead to poor prognosis, which might be due to its induced P-glycoprotein and Bcl-2 protein expression level increase. P-glycoprotein or membrane pump induced drug efflux and altered prosurvival Bcl-2 expression are key mechanisms for drug resistance leading to failure of chemotherapy in HCC. However, current strategy to overcome both P-glycoprotein and Bcl-2 protein induced drug resistance was rarely reported. In this work, we utilized an amphiphilic poly[(R)-3-hydroxybutyrate] (PHB)-b-poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) cationic polyester to encapsulate chemotherapeutic paclitaxel (PTX) in hydrophobic PHB domain and Bcl-2 convertor Nur77/ΔDBD gene (Nur77 without DNA binding domain for mitochondria localization) by formation of polyplex due to cationic PDMAEMA segment, to effectively inhibit the drug resistant HepG2/STC2 and SMCC7721/STC2 liver cancer cell growth. Thanks to the cationic nanoparticle complex formation ability and high transfection efficiency to express Bcl-2 conversion proteins, PHB-PDMAEMA/PTX@polyplex could partially impair P-glycoprotein induced PTX efflux and activate the apoptotic function of previous prosurvival Bcl-2 protein. This is the pioneer report of cationic amphiphilic polyester PHB-PDMAEMA to codeliver anticancer drug and therapeutic plasmid to overcome both pump and non-pump mediated chemotherapeutic resistance in liver cancer cells, which might be inspiring for the application of polyester in personalized cancer therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed

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

2011-04-15

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

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

PubMed Central

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

2011-01-01

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

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

Comparative Genomics Analysis of a New Exiguobacterium Strain from Salar de Huasco Reveals a Repertoire of Stress-Related Genes and Arsenic Resistance

PubMed Central

Castro-Severyn, Juan; Remonsellez, Francisco; Valenzuela, Sandro L.; Salinas, Cesar; Fortt, Jonathan; Aguilar, Pablo; Pardo-Esté, Coral; Dorador, Cristina; Quatrini, Raquel; Molina, Franck; Aguayo, Daniel; Castro-Nallar, Eduardo; Saavedra, Claudia P.

2017-01-01

The Atacama Desert hosts diverse ecosystems including salt flats and shallow Andean lakes. Several heavy metals are found in the Atacama Desert, and microorganisms growing in this environment show varying levels of resistance/tolerance to copper, tellurium, and arsenic, among others. Herein, we report the genome sequence and comparative genomic analysis of a new Exiguobacterium strain, sp. SH31, isolated from an altiplanic shallow athalassohaline lake. Exiguobacterium sp. SH31 belongs to the phylogenetic Group II and its closest relative is Exiguobacterium sp. S17, isolated from the Argentinian Altiplano (95% average nucleotide identity). Strain SH31 encodes a wide repertoire of proteins required for cadmium, copper, mercury, tellurium, chromium, and arsenic resistance. Of the 34 Exiguobacterium genomes that were inspected, only isolates SH31 and S17 encode the arsenic efflux pump Acr3. Strain SH31 was able to grow in up to 10 mM arsenite and 100 mM arsenate, indicating that it is arsenic resistant. Further, expression of the ars operon and acr3 was strongly induced in response to both toxics, suggesting that the arsenic efflux pump Acr3 mediates arsenic resistance in Exiguobacterium sp. SH31. PMID:28377753

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.

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.

Multixenobiotic resistance in Mytilus edulis: Molecular and functional characterization of an ABCG2- type transporter in hemocytes and gills.

PubMed

Ben Cheikh, Yosra; Xuereb, Benoit; Boulangé-Lecomte, Céline; Le Foll, Frank

2018-02-01

Among the cellular protection arsenal, ABC transporters play an important role in xenobiotic efflux in marine organisms. Two pumps belonging to B and C subfamily has been identified in Mytilus edulis. In this study, we investigated the presence of the third major subtype ABCG2/BCRP protein in mussel tissues. Transcript was expressed in hemocytes and with higher level in gills. Molecular characterization revealed that mussel ABCG2 transporter shares the sequence and organizational structure with mammalian and molluscan orthologs. Overall identity of the predicted amino acid sequence with corresponding homologs from other organisms was between 49% and 98%. Moreover, protein efflux activity was demonstrated using a combination of fluorescent allocrites and specific inhibitors. The accumulation of bodipy prazosin and pheophorbide A was heterogeneous in gills and hemocytes. Most of the used blockers enhanced probe accumulation at different levels, most significantly for bodipy prazosin. Moreover, Mrp classical blocker MK571 showed a polyspecificity. In conclusion, our data demonstrate that several ABC transporters contribute to MXR phenotype in the blue mussel including ABCG2 that forms an active pump in hemocytes and gills. Efforts are needed to distinguish between the different members and to explore their single function and specificity towards allocrites and chemosensitizers. Copyright © 2017 Elsevier B.V. All rights reserved.

Analysis of quinolone-resistance in commensal and diarrheagenic Escherichia coli isolates from infants in Lima, Peru

PubMed Central

Pons, Maria J.; Mosquito, S.; Gomes, C.; del Valle, L.J.; Ochoa, T.J.; Ruiz, J.

2014-01-01

Background Antibiotic resistance is an increasing problem, particularly in countries where antibiotic use is frequently not controlled. The aim of this study was to analyse the prevalence of the molecular mechanisms of quinolone-resistance in E. coli isolated from faeces of healthy Peruvian children or those presenting diarrhoea. Methods The presence of target mutations, transferable quinolone-resistance mechanisms and the role of Phe-Arg-β-Naphtylamyde inhibitible efflux pumps were studied in 96 Escherichia coli (46 diarrheogenic and 50 non-diarrheogenic) isolates exhibiting resistance or diminished susceptibility to quinolones. Results The most resistant phenotype, NalR and CipR, was most frequently present in isolates of healthy children. The distribution of quinolone resistance mechanisms between diarrheogenic (DEC) and commensal (non DEC) isolates was equitable, although the aac(6′)Ib-cr gene was mainly detected in DEC isolates: 17 (34%) vs non DEC isolates nine (20%). QnrB was present in five (10%) DEC vs three (6%) non DEC isolates. Conclusions Point mutations in gyrA and parC genes play a relevant role in quinolone resistance acquisition and highlight the role of efflux pumps also. This study provides knowledge about the molecular mechanisms involved in quinolone resistance in isolates in a non exposed population under high community antibiotic pressure. PMID:24306130

Whole genome and transcriptome analyses of environmental antibiotic sensitive and multi-resistant Pseudomonas aeruginosa isolates exposed to waste water and tap water

PubMed Central

Schwartz, Thomas; Armant, Olivier; Bretschneider, Nancy; Hahn, Alexander; Kirchen, Silke; Seifert, Martin; Dötsch, Andreas

2015-01-01

The fitness of sensitive and resistant Pseudomonas aeruginosa in different aquatic environments depends on genetic capacities and transcriptional regulation. Therefore, an antibiotic-sensitive isolate PA30 and a multi-resistant isolate PA49 originating from waste waters were compared via whole genome and transcriptome Illumina sequencing after exposure to municipal waste water and tap water. A number of different genomic islands (e.g. PAGIs, PAPIs) were identified in the two environmental isolates beside the highly conserved core genome. Exposure to tap water and waste water exhibited similar transcriptional impacts on several gene clusters (antibiotic and metal resistance, genetic mobile elements, efflux pumps) in both environmental P. aeruginosa isolates. The MexCD-OprJ efflux pump was overexpressed in PA49 in response to waste water. The expression of resistance genes, genetic mobile elements in PA49 was independent from the water matrix. Consistently, the antibiotic sensitive strain PA30 did not show any difference in expression of the intrinsic resistance determinants and genetic mobile elements. Thus, the exposure of both isolates to polluted waste water and oligotrophic tap water resulted in similar expression profiles of mentioned genes. However, changes in environmental milieus resulted in rather unspecific transcriptional responses than selected and stimuli-specific gene regulation. PMID:25186059

Strophanthidin-sensitive sodium fluxes in metabolically poisoned frog skeletal muscle

PubMed Central

1976-01-01

Strophanthidin-sensitive and insensitive unidirectional fluxes of Na were measured in fog sartorius muscles whose internal Na levels were elevated by overnight storage in the cold. ATP levels were lowered, and ADP levels raised, by metabolic poisoning with either 2,4- dinitrofluorobenzene or iodoacetamide. Strophanthidin-sensitive Na efflux and influx both increased after poisoning, while strophanthidin- insensitives fluxes did not. The increase in efflux did not require the presence of external K but was greatly attenuated when Li replaced Na as the major external cation. Membrane potential was not markedly altered by 2,4-dinitrofluorobenzene. These observations indicate that the sodium pump of frog skeletal muscle resembles that of squid giant axon and human erythrocyte in its ability to catalyze Na-Na exchange to an extent determined by intracellular ATP/ADP levels. PMID:1086888

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

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

PubMed

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

2011-10-01

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

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

PubMed

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

2014-09-01

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

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

PubMed Central

1995-01-01

We used microelectrodes to monitor the recovery (i.e., decrease) of intracellular pH (pHi) after using internal dialysis to load squid giant axons with alkali to pHi values of 7.7, 8.0, or 8.3. The dialysis fluid (DF) contained 400 mM K+ but was free of Na+ and Cl-. The artificial seawater (ASW) lacked Na+, K+, and Cl-, thereby eliminating effects of known acid-base transporters on pHi. Under these conditions, halting dialysis unmasked a slow pHi decrease caused at least in part by acid-base transport we refer to as "base efflux." Replacing K+ in the DF with either NMDG+ or TEA+ significantly reduced base efflux and made membrane voltage (Vm) more positive. Base efflux in K(+)-dialyzed axons was stimulated by decreasing the pH of the ASW (pHo) from 8 to 7, implicating transport of acid or base. Although postdialysis acidifications also occurred in axons in which we replaced the K+ in the DF with Li+, Na+, Rb+, or Cs+, only with Rb+ was base efflux stimulated by low pHo. Thus, the base effluxes supported by K+ and Rb+ appear to be unrelated mechanistically to those observed with Li+, Na+, or Cs+. The combination of 437 mM K+ and 12 mM HCO3- in the ASW, which eliminates the gradient favoring a hypothetical K+/HCO3- efflux, blocked pHi recovery in K(+)-dialyzed axons. However, the pHi recovery was not blocked by the combination of 437 mM Na+, veratridine, and CO2/HCO3- in the ASW, a treatment that inverts electrochemical gradients for H+ and HCO3- and would favor passive H+ and HCO3- fluxes that would have alkalinized the axon. Similarly, the recovery was not blocked by K+ alone or HCO3- alone in the ASW, nor was it inhibited by the K-H pump blocker Sch28080 nor by the Na-H exchange inhibitors amiloride and hexamethyleneamiloride. Our data suggest that a major component of base efflux in alkali-loaded axons cannot be explained by metabolism, a H+ or HCO3- conductance, or by a K-H exchanger. However, this component could be mediated by a novel K/HCO3- cotransporter. PMID:8648294

First biological evaluation of developed 3-benzyloxyfluorenes as novel class of MDR modulators.

PubMed

Krug, Martin; Voigt, Burkhardt; Baumert, Christiane; Lüpken, Ralf; Molnár, Joséf; Hilgeroth, Andreas

2010-06-01

A series of 3-benzyloxy-1-aza-9-oxafluorenes has been synthesized and biologically evaluated as novel MDR modulators. The concentration dependent inhibition of the efflux pump ABCB1 (P-glycoprotein) has been characterized and is discussed in relation to calculated lipophilicity data. Instead of the molecular lipophilicity the exact positioning of functional groups was found decisive for the biological activities. Copyright (c) 2010 Elsevier Masson SAS. All rights reserved.

Natural Polyphenol Disposition via Coupled Metabolic Pathways

PubMed Central

Liu, Zhongqiu; Hu, Ming

2009-01-01

A major challenge associated with the development of chemopreventive polyphenols is the lack of bioavailability in vivo, which are primarily the result of coupled metabolic activities of conjugating enzymes and efflux transporters. These coupling processes are present in most of tissues and organs in mammals and are efficient for the purposes of drug metabolism, elimination and detoxification. Therefore, it was expected that these coupling processes represent a significant barrier to the oral bioavailabilities of polyphenols. In various studies of this coupling process, it was identified that various conjugating enzymes such as UGT and SULT are capable of producing very hydrophilic metabolites of polyphenols, which cannot diffuse out of the cells and needs the action of efflux transporters to pump them out of the cells. Additional studies have shown that efflux transporters such as MRP2, BCRP and OAT appear to serve as the gate keeper when there is an excess capacity to metabolize the compounds. These efflux transporters may also act as the facilitator of metabolism when there is a product/metabolite inhibition. For polyphenols, these coupled processes enable a duo recycling scheme of enteric and enterohepatic recycling, which allows the polyphenols to be reabsorbed and results in longer than expected apparent plasma half-lives for these compounds and their conjugates. Since the vast majority of polyphenols in plasma are hydrophilic conjugates, more research is needed to determine if the metabolites are active or reactive, which will help explain their mechanism of actions. PMID:17539746

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

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

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

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

Dodecyltriphenylphosphonium inhibits multiple drug resistance in the yeast Saccharomyces cerevisiae.

PubMed

Knorre, Dmitry A; Markova, Olga V; Smirnova, Ekaterina A; Karavaeva, Iuliia E; Sokolov, Svyatoslav S; Severin, Fedor F

2014-08-08

Multiple drug resistance pumps are potential drug targets. Here we asked whether the lipophilic cation dodecyltriphenylphosphonium (C12TPP) can interfere with their functioning. First, we found that suppression of ABC transporter gene PDR5 increases the toxicity of C12TPP in yeast. Second, C12TPP appeared to prevent the efflux of rhodamine 6G - a fluorescent substrate of Pdr5p. Moreover, C12TPP increased the cytostatic effects of some other known Pdr5p substrates. The chemical nature of C12TPP suggests that after Pdr5p-driven extrusion the molecules return to the plasma membrane and then into the cytosol, thus effectively competing with other substrates of the pump. Copyright © 2014 Elsevier Inc. All rights reserved.

Ion transport studies and determination of the cell wall elastic modulus in the marine alga Halicystis parvula

PubMed Central

1976-01-01

Using cultured cells of the marine alga, Halicystis parvula, we measured the concentrations of 11 inorganic ions in the vacuolar sap and the electrical potential difference (PD) between the vacuole and the external solution. In normal cells under steady-state conditions a comparison of the electrochemical equilibrium (Nernst) potential for each ion with the PD of -82 mV (inside negative) indicates that Na+ and K+ are actively transported out of the vacuole whereas all anions are pumped into the cell. Although the [K+] in the vacuole is only 9 mM, the cytoplasmic [K+] is about 420 mM, which suggests that the outwardly directed pump is at the tonoplast. Using large Halicystis cells we perfused the vacuole with an artificial seawater and conducted a short- circuit analysis of ion transport. The short-circuit current (SCC) of 299 peq - cm-2-s-1 is not significantly different from the net influx of Cl-. There is a small, but statistically significant net efflux of K+ (less than 1 pmol-cm-2.-1), while the influx and efflux of Na+ are not significantly different. Therefore, the SCC is a good measure of the activity of the Cl- pump. Finally, we measured the volumetric elastic modulus (epsilon) of the cell wall by measuring the change in cell volume when the internal hydrostatic pressure was altered. The value of epsilon at applied pressures between 0 and 0.4 atm is about 0.6 atm, which is at least 100-fold lower than the values of epsilon for all other algae which have been studied. PMID:1271044

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.

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

PubMed

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

2011-01-14

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

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.

Antibiotic Resistance Genetic Markers and Integrons in White Soft Cheese: Aspects of Clinical Resistome and Potentiality of Horizontal Gene Transfer

PubMed Central

de Paula, Ana Caroline L.; Medeiros, Julliane D.; de Azevedo, Analice C.; Chagas, Jéssica M. de Assis; da Silva, Vânia L.

2018-01-01

Antibiotic resistance poses an important threat to global public health and has become a challenge to modern medicine. The occurrence of antibiotic-resistant bacteria in a broad range of foods has led to a growing concern about the impact that food may have as a reservoir of antibiotic resistance genes. Considering Minas Frescal Cheese (MFC)—a typical Brazilian white soft cheese—and its economic and cultural values, in this study, medically relevant antimicrobial-resistance genetic markers (AR genes) were screened, and the occurrence of integrons were evaluated in manufactured MFC using culture-independent approaches. Through a fingerprinting analysis, the tested MFCs were brand-clustered, indicating reproducibility along the production chain. A common core of resistance markers in all brands evaluated and related antimicrobials such as β-lactams, tetracyclines, quinolones, and sulfonamide was detected. Several other markers, including efflux pumps and aminoglycosides-resistance were distributed among brands. Class 1 and 2 integrons were observed, respectively, in 77% and 97% of the samples. The presence of AR genes is of special interest due to their clinical relevance. Taken together, the data may suggest that the production chain of MFC might contribute to the spread of putative drug-resistant bacteria, which could greatly impact human health. Furthermore, detection of class 1 and class 2 integrons in MFC has led to discussions about resistance gene spread in this traditional cheese, providing evidence of potential horizontal transfer of AR genes to human gut microbiota. PMID:29463055

Regulation of cation transport in Saccharomyces cerevisiae by the salt tolerance gene HAL3.

PubMed Central

Ferrando, A; Kron, S J; Rios, G; Fink, G R; Serrano, R

1995-01-01

Dynamic regulation of ion transport is essential for homeostasis as cells confront changes in their environment. The gene HAL3 encodes a novel component of this regulatory circuit in the yeast Saccharomyces cerevisiae. Overexpression of HAL3 improves growth of wild-type cells exposed to toxic concentrations of sodium and lithium and suppresses the salt sensitivity conferred by mutation of the calcium-dependent protein phosphatase calcineurin. Null mutants of HAL3 display salt sensitivity. The sequence of HAL3 gives little clue to its function. However, alterations in intracellular cation concentrations associated with changes in HAL3 expression suggest that HAL3 activity may directly increase cytoplasmic K+ and decrease Na+ and Li+. Cation efflux in S. cerevisiae is mediated by the P-type ATPase encoded by the ENA1/PMR24 gene, a putative plasma membrane Na+ pump whose expression is salt induced. Acting in concert with calcineurin, HAL3 is necessary for full activation of ENA1 expression. This functional complementarity is also reflected in the participation of both proteins in recovery from alpha-factor-induced growth arrest. Recently, HAL3 was isolated as a gene (named SIS2) which when overexpressed partially relieves loss of transcription of G1 cyclins in mutants lacking the protein phosphatase Sit4p. Therefore, HAL3 influences cell cycle control and ion homeostasis, acting in parallel to the protein phosphatases Sit4p and calcineurin. PMID:7565698

Antibiotic Resistance Genetic Markers and Integrons in White Soft Cheese: Aspects of Clinical Resistome and Potentiality of Horizontal Gene Transfer.

PubMed

de Paula, Ana Caroline L; Medeiros, Julliane D; de Azevedo, Analice C; de Assis Chagas, Jéssica M; da Silva, Vânia L; Diniz, Cláudio G

2018-02-19

Antibiotic resistance poses an important threat to global public health and has become a challenge to modern medicine. The occurrence of antibiotic-resistant bacteria in a broad range of foods has led to a growing concern about the impact that food may have as a reservoir of antibiotic resistance genes. Considering Minas Frescal Cheese (MFC)-a typical Brazilian white soft cheese-and its economic and cultural values, in this study, medically relevant antimicrobial-resistance genetic markers (AR genes) were screened, and the occurrence of integrons were evaluated in manufactured MFC using culture-independent approaches. Through a fingerprinting analysis, the tested MFCs were brand-clustered, indicating reproducibility along the production chain. A common core of resistance markers in all brands evaluated and related antimicrobials such as β-lactams, tetracyclines, quinolones, and sulfonamide was detected. Several other markers, including efflux pumps and aminoglycosides-resistance were distributed among brands. Class 1 and 2 integrons were observed, respectively, in 77% and 97% of the samples. The presence of AR genes is of special interest due to their clinical relevance. Taken together, the data may suggest that the production chain of MFC might contribute to the spread of putative drug-resistant bacteria, which could greatly impact human health. Furthermore, detection of class 1 and class 2 integrons in MFC has led to discussions about resistance gene spread in this traditional cheese, providing evidence of potential horizontal transfer of AR genes to human gut microbiota.

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

PubMed Central

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

2015-01-01

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

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

PubMed Central

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

2016-01-01

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

Characterization of carbapenem nonsusceptible Pseudomonas aeruginosa in Denmark: a nationwide, prospective study.

PubMed

Hansen, Frank; Johansen, Helle Krogh; Østergaard, Claus; Arpi, Magnus; Hansen, Dennis Schrøder; Littauer, Pia; Holm, Anette; Heltberg, Ole; Schumacher, Helga; Fuursted, Kurt; Lykke, Mari-Ann Domar; Tønning, Birgitte; Hammerum, Anette M; Justesen, Ulrik Stenz

2014-02-01

From January 1st 2011 through June 30th 2011, 116 nonreplicate, noncystic fibrosis-related Pseudomonas aeruginosa isolates with reduced carbapenem susceptibility were collected from 12 out of 13 Danish departments of clinical microbiology. The presence of acquired β-lactamases was assessed with combination tablet-diffusion methodology and polymerase chain reaction. In addition, antimicrobial susceptibility testing, an efflux pump inhibitor assay, and pulsed-field gel electrophoresis (PFGE) were performed. Isolates producing acquired β-lactamases were further investigated by serotyping and multi locus sequence typing. Eight isolates produced the metallo-β-lactamase (MBL) VIM-2, and one isolate produced OXA-10 and VEB-1-like extended-spectrum beta-lactamase (ESBL). Phenotypic indications of derepressed AmpC and efflux pump were seen in 56 and 43 isolates, respectively. Overall, the results indicate that mutational factors related to permeability--often combined with derepressed, chromosomal AmpC--is the main factor behind carbapenem nonsusceptibility in Danish P. aeruginosa isolates. The ESBL producer and all the VIM producers belonged to international clones. PFGE revealed that most of the isolates were unrelated, but clonal spread was seen; the 116 isolates distributed in 97 PFGE types, with the largest cluster consisting of 4 isolates (including three isolates from the same hospital with 100% similarity). Thirty-two isolates were pair-wise related, while the remaining isolates were clonally unrelated, as were all nine ESBL/MBL producers.

The combination of catechin and epicatechin callate from Fructus Crataegi potentiates beta-lactam antibiotics against methicillin-resistant staphylococcus aureus (MRSA) in vitro and in vivo.

PubMed

Qin, Rongxin; Xiao, Kangkang; Li, Bin; Jiang, Weiwei; Peng, Wei; Zheng, Jiang; Zhou, Hong

2013-01-16

Fructus crataegi (hawthorn) is the common name of all plant species in the genus Crataegus of the Rosaceae family. In the present study, three monomers of (+)-catechin (C), (-)-epicatechin gallate (ECg) and (-)-epigallocatechin (EGC) were isolated from the hawthorn under the guide of antibacterial sensitization activity. The bioactivity of the composite fraction in enhancing the antibacterial effect of oxacillin against methicillin-resistant Staphylococcus aureus (MRSA) was greater than that of the individual monomer of the hawthorn extract in vitro. Two-fold dilution and checkerboard methods were used to analyze antibacterial activity and screen for the combination and proportion of monomers with the best bioactivity. The result showed that C (128 mg/L) combined with ECg (16 mg/L) had the greatest effect and the combination also reduced the bacterial load in blood of septic mice challenged with a sublethal dose of MRSA, increased daunomycin accumulation within MRSA and down-regulated the mRNA expression of norA, norC and abcA, three important efflux pumps of MRSA. In summary, C and ECg enhanced the antibacterial effect of β-lactam antibiotics against MRSA in vitro and in vivo, which might be related to the increased accumulation of antibiotics within MRSA via suppression of important efflux pumps' gene expression.

Enhancement of geraniol resistance of Escherichia coli by MarA overexpression.

PubMed

Shah, Asad Ali; Wang, Chonglong; Chung, Young-Ryun; Kim, Jae-Yean; Choi, Eui-Sung; Kim, Seon-Won

2013-03-01

Improvement of a microorganism's tolerance against organic solvents is required for a microbial factory producing terpenoid based biofuels. The bacterial genes, marA, imp, cls and cti have been found to increase organic solvent tolerance. Thus, the tolerance against the following terpenoids (isopentenol, geraniol, myrcene, and farnesol) was studied with overexpression of marA, imp, cls and cti genes in Escherichia coli. The marA overexpression significantly enhanced the tolerance of E. coli against geraniol, whereas there was no tolerance improvement against the terpenoids by overexpression of cls and cti genes. The imp overexpression even yielded sensitive phenotype to the tested solvents. The colony forming efficiency of the marA overexpressing E. coli was increased by 10(4)-fold in plate overlay of geraniol compared to that of wild type E. coli and a two-fold decrease of intracellular geraniol accumulation was also observed in liquid culture of geraniol. Single knock-out mutations of marA, or one of the following genes (acrA, acrB and tolC) encoding AcrAB-TolC efflux pump made E. coli hypersensitive to geraniol. The geraniol tolerance conferred by marA overexpression was attributed to the AcrAB-TolC efflux pump that is activated by MarA. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Whole genome and transcriptome analyses of environmental antibiotic sensitive and multi-resistant Pseudomonas aeruginosa isolates exposed to waste water and tap water.

PubMed

Schwartz, Thomas; Armant, Olivier; Bretschneider, Nancy; Hahn, Alexander; Kirchen, Silke; Seifert, Martin; Dötsch, Andreas

2015-01-01

The fitness of sensitive and resistant Pseudomonas aeruginosa in different aquatic environments depends on genetic capacities and transcriptional regulation. Therefore, an antibiotic-sensitive isolate PA30 and a multi-resistant isolate PA49 originating from waste waters were compared via whole genome and transcriptome Illumina sequencing after exposure to municipal waste water and tap water. A number of different genomic islands (e.g. PAGIs, PAPIs) were identified in the two environmental isolates beside the highly conserved core genome. Exposure to tap water and waste water exhibited similar transcriptional impacts on several gene clusters (antibiotic and metal resistance, genetic mobile elements, efflux pumps) in both environmental P. aeruginosa isolates. The MexCD-OprJ efflux pump was overexpressed in PA49 in response to waste water. The expression of resistance genes, genetic mobile elements in PA49 was independent from the water matrix. Consistently, the antibiotic sensitive strain PA30 did not show any difference in expression of the intrinsic resistance determinants and genetic mobile elements. Thus, the exposure of both isolates to polluted waste water and oligotrophic tap water resulted in similar expression profiles of mentioned genes. However, changes in environmental milieus resulted in rather unspecific transcriptional responses than selected and stimuli-specific gene regulation. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

High prevalence of non-clonal imipenem-nonsusceptible Enterobacter spp. isolates in Korea and their association with porin down-regulation.

PubMed

Lee, Ji-Young; Hong, Yoon-Kyoung; Lee, Haejeong; Ko, Kwan Soo

2017-01-01

We investigated the prevalence and clonal distribution of imipenem-nonsusceptible Enterobacter clinical isolates from hospitals in Korea and the contributions of various mechanisms to imipenem nonsusceptibility. The in vitro antimicrobial susceptibility to imipenem of 357 non-duplicated Enterobacter isolates obtained from eight geographically distant tertiary care hospitals in Korea was evaluated. Imipenem-nonsusceptible Enterobacter isolates were genotyped. Additionally, β-lactamase genes were screened using PCR, and the expression of efflux pump and porin genes was investigated using quantitative RT-PCR. A total of 31 isolates (8.7%) were not susceptible to imipenem. Clonal diversity of 17 imipenem-nonsusceptible E. cloacae isolates was demonstrated by multilocus sequence typing. Fourteen imipenem-nonsusceptible E. aerogenes isolates were found to be distantly genetically related by an ERIC-PCR analysis. Expression levels of porin ompD and ompK35 genes were decreased in all imipenem-nonsusceptible E. cloacae and E. aerogenes isolates. However, only two isolates were found positive for bla IMP and bla VIM genes, and expression of the efflux pump gene, acrB, was not associated with reduced imipenem susceptibility. Imipenem resistance seems to have occurred independently in most of the imipenem-nonsusceptible isolates in this study, and decreased porin expression was found to be the main mechanism underlying this reduced susceptibility to imipenem. Copyright © 2016 Elsevier Inc. All rights reserved.

Thinking outside the "bug": a unique assay to measure intracellular drug penetration in gram-negative bacteria.

PubMed

Zhou, Ying; Joubran, Camil; Miller-Vedam, Lakshmi; Isabella, Vincent; Nayar, Asha; Tentarelli, Sharon; Miller, Alita

2015-04-07

Significant challenges are present in antibiotic drug discovery and development. One of these is the number of efficient approaches Gram-negative bacteria have developed to avoid intracellular accumulation of drugs and other cell-toxic species. In order to better understand these processes and correlate in vitro enzyme inhibition to whole cell activity, a better assay to evaluate a key factor, intracellular accumulation of the drug, is urgently needed. Here, we describe a unique liquid chromatography (LC)-mass spectrometry (MS) approach to measure the amount of cellular uptake of antibiotics by Gram-negative bacteria. This method, which measures the change of extracellular drug concentration, was evaluated by comparing the relative uptake of linezolid by Escherichia coli wild-type versus an efflux pump deficient strain. A higher dosage of the drug showed a higher accumulation in these bacteria in a dosing range of 5-50 ng/mL. The Escherichia coli efflux pump deficient strain had a higher accumulation of the drug than the wild-type strain as predicted. The approach was further validated by determining the relative meropenem uptake by Pseudomonas aeruginosa wild-type versus a mutant strain lacking multiple porins. These studies show great promise of being applied within antibiotic drug discovery, as a universal tool to aid in the search for compounds that can easily penetrate bacterial cells.

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.

Transport routes of metalloids into and out of the cell: a review of the current knowledge.

PubMed

Zangi, Ronen; Filella, Montserrat

2012-04-15

Except for their extra- and intra-cellular interfaces, cell membranes are hydrophobic and inhibit the transport of hydrophilic molecules. Metalloids in aqueous solutions form chemical species with oxygen and hydroxyl groups and, therefore, exist as hydrophilic neutral polar solutes or as hydrophilic anions. This characteristic of metalloids introduces a large barrier for their passage through the cell membrane via unaided diffusion. The necessity for an uptake mechanism for metalloids arises from the requirement of these species for the maintenance of life, such as the need of boron for plant cells. Conversely, the transport of these species out of the cell is necessary because some metalloids are toxic, such as arsenic and antimony, and their entrance into the cell is undesirable. The undesired uptake of these toxic species is possible via pathways designed for the uptake of other structurally and chemically similar essential compounds. Therefore, the extrusion of arsenic and antimony out of the cell is an example of a detoxification mechanism. As a consequence of the hydrophobic character of the cell membrane in all living systems, the main route for the uptake and efflux of metalloids is facilitated by transmembrane proteins, driven either by concentration gradients or by energy-fueled pumps. However, metalloids forming or embedded in nano-sized particles escape the need to cross the cell membrane because these particles can be taken into the cell by endocytosis. Here, we review the uptake and efflux pathways of boron, silicon, arsenic, and antimony through the cell membranes of different organisms and the protein channels involved in these processes. In particular, passive diffusion via aquaglyceroporins, active transport via primary and secondary ion pumps, extrusion into vacuoles of metalloid-thiol conjugates via ATP-binding cassette, the efflux of methylated metalloids, and endocytosis are summarized. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Dynamic relationship between neurostimulation and N-acetylaspartate metabolism in the human visual cortex: evidence that NAA functions as a molecular water pump during visual stimulation.

PubMed

Baslow, Morris H; Hrabe, Jan; Guilfoyle, David N

2007-01-01

N-acetyl-l-aspartic acid (NAA), an amino acid synthesized and stored primarily in neurons in the brain, has been proposed to be a molecular water pump (MWP) whose function is to rapidly remove water from neurons against a water gradient. In this communication, we describe the results of a functional (1)H proton magnetic resonance spectroscopy (fMRS) study, and provide evidence that in the human visual cortex, over a 10-min period of visual stimulation, there are stimulation-induced graded changes in the NAA MRS signal from that of a preceding 10-min baseline period with a decline in the NAA signal of 13.1% by the end of the 10-min stimulation period. Upon cessation of visual stimulation, the NAA signal gradually increases during a 10-min recovery period and once again approaches the baseline level. Because the NAA MRS signal reflects the NAA concentration, these changes indicate rapid focal changes in its concentration, and transient changes in its intercompartmental metabolism. These include its rates of synthesis and efflux from neurons and its hydrolysis by oligodendrocytes. During stimulation, the apparent rate of NAA efflux and hydrolysis increased 14.2 times, from 0.55 to 7.8 micromol g(-1) h(-1). During recovery, the apparent rate of synthesis increased 13.3 times, from 0.55 to 7.3 micromol g(-1) h(-1). The decline in the NAA signal during stimulation suggests that a rapid increase in the rate of NAA-obligated water release to extracellular fluid (ECF) is the initial and seminal event in response to neurostimulation. It is concluded that the NAA metabolic cycle in the visual cortex is intimately linked to rates of neuronal signaling, and that the functional cycle of NAA is associated with its release to ECF, thus supporting the hypothesis that an important function of the NAA metabolic cycle is that of an efflux MWP.

Analysis of Triclosan-Selected Salmonella enterica Mutants of Eight Serovars Revealed Increased Aminoglycoside Susceptibility and Reduced Growth Rates

PubMed Central

Rensch, Ulrike; Klein, Guenter; Kehrenberg, Corinna

2013-01-01

The biocide triclosan (TRC) is used in a wide range of household, personal care, veterinary, industrial and medical products to control microbial growth. This extended use raises concerns about a possible association between the application of triclosan and the development of antibiotic resistance. In the present study we determined triclosan mutant prevention concentrations (MPC) for Salmonella enterica isolates of eight serovars and investigated selected mutants for their mechanisms mediating decreased susceptibility to triclosan. MPCTRC values were 8 - 64-fold higher than MIC values and ranged between 1 - 16 µg/ml. The frequencies at which mutants were selected varied between 1.3 x 10-10 - 9.9 x 10-11. Even if MIC values of mutants decreased by 3-7 dilution steps in the presence of the efflux pump inhibitor Phe-Arg-β-naphtylamide, only minor changes were observed in the expression of genes encoding efflux components or regulators, indicating that neither the major multidrug efflux pump AcrAB-TolC nor AcrEF are up-regulated in triclosan-selected mutants. Nucleotide sequence comparisons confirmed the absence of alterations in the regulatory regions acrRA, soxRS, marORAB, acrSE and ramRA of selected mutants. Single bp and deduced Gly93→Val amino acid exchanges were present in fabI, the target gene of triclosan, starting from a concentration of 1 µg/ml TRC used for MPC determinations. The fabI genes were up to 12.4-fold up-regulated. Complementation experiments confirmed the contribution of Gly93→Val exchanges and fabI overexpression to decreased triclosan susceptibility. MIC values of mutants compared to parent strains were even equal or resulted in a more susceptible phenotype (1-2 dilution steps) for the aminoglycoside antibiotics kanamycin and gentamicin as well as for the biocide chlorhexidine. Growth rates of selected mutants were significantly lower and hence, might partly explain the rare occurrence of Salmonella field isolates exhibiting decreased susceptibility to triclosan. PMID:24205194

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.

Virulence determinants, drug resistance and mobile genetic elements of Laribacter hongkongensis: a genome-wide analysis

PubMed Central

2011-01-01

Background Laribacter hongkongensis is associated with community-acquired gastroenteritis and traveler's diarrhea. In this study, we performed an in-depth annotation of the genes in its genome related to the various steps in the infective process, drug resistance and mobile genetic elements. Results For acid and bile resistance, L. hongkongensis possessed a urease gene cassette, two arc gene clusters and bile salt efflux systems. For intestinal colonization, it possessed a putative adhesin of the autotransporter family homologous to those of diffusely adherent Escherichia coli (E. coli) and enterotoxigenic E. coli. To evade from host defense, it possessed superoxide dismutase and catalases. For lipopolysaccharide biosynthesis, it possessed the same set of genes that encode enzymes for synthesizing lipid A, two Kdo units and heptose units as E. coli, but different genes for its symmetrical acylation pattern, and nine genes for polysaccharide side chains biosynthesis. It contained a number of CDSs that encode putative cell surface acting (RTX toxin and hemolysins) and intracellular cytotoxins (patatin-like proteins) and enzymes for invasion (outer membrane phospholipase A). It contained a broad variety of antibiotic resistance-related genes, including genes related to β-lactam (n = 10) and multidrug efflux (n = 54). It also contained eight prophages, 17 other phage-related CDSs and 26 CDSs for transposases. Conclusions The L. hongkongensis genome possessed genes for acid and bile resistance, intestinal mucosa colonization, evasion of host defense and cytotoxicity and invasion. A broad variety of antibiotic resistance or multidrug resistance genes, a high number of prophages, other phage-related CDSs and CDSs for transposases, were also identified. PMID:21711902

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.

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

PubMed

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

2016-02-04

An innovative and straightforward synthesis of second-generation 2-arylbenzo[b]thiophenes as structural analogues of INF55 and the first generation of our laboratory-made molecules was developed. The synthesis of C2-arylated benzo[b]thiophene derivatives was achieved through a method involving direct arylation, followed by simple structural modifications. Among the 34 compounds tested, two of them were potent NorA pump inhibitors, which led to a 16-fold decrease in the ciprofloxacin minimum inhibitory concentration (MIC) against the SA-1199B strain at concentrations of 0.25 and 0.5 μg mL(-1) (1 and 1.5 μm, respectively). This is a promising result relative to that obtained for reserpine (MIC=20 μg mL(-1)), a reference compound amongst NorA pump inhibitors. These molecules thus represent promising candidates to be used in combination with ciprofloxacin against fluoroquinolone-resistant strains. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed

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

2017-09-01

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

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.

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

PubMed

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

2011-07-01

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

Ibuprofen Potentiates the In Vivo Antifungal Activity of Fluconazole against Candida albicans Murine Infection

PubMed Central

Miranda, Isabel M.; Silva-Dias, Ana; Silva, Ana P.; Rodrigues, Acácio G.; Pina-Vaz, Cidália

2015-01-01

Candida albicans is the most prevalent cause of fungemia worldwide. Its ability to develop resistance in patients receiving azole antifungal therapy is well documented. In a murine model of systemic infection, we show that ibuprofen potentiates fluconazole antifungal activity against a fluconazole-resistant strain, drastically reducing the fungal burden and morbidity. The therapeutic combination of fluconazole with ibuprofen may constitute a new approach for the management of antifungal therapeutics to reverse the resistance conferred by efflux pump overexpression. PMID:25845879

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.

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.

Mechanisms of resistance to quinolones: target alterations, decreased accumulation and DNA gyrase protection.

PubMed

Ruiz, Joaquim

2003-05-01

Quinolones are broad-spectrum antibacterial agents, commonly used in both clinical and veterinary medicine. Their extensive use has resulted in bacteria rapidly developing resistance to these agents. Two mechanisms of quinolone resistance have been established to date: alterations in the targets of quinolones, and decreased accumulation due to impermeability of the membrane and/or an overexpression of efflux pump systems. Recently, mobile elements have also been described, carrying the qnr gene, which confers resistance to quinolones.

Inhibitory Effect of Flavonoids on the Efflux of N-Acetyl 5-Aminosalicylic Acid Intracellularly Formed in Caco-2 Cells

PubMed Central

Shin, Yoshimura; Kentaro, Kawano; Ryusuke, Matsumura; Narumi, Sugihara; Koji, Furuno

2009-01-01

N-acetyl 5-aminosalicylic acid (5-AcASA) that was intracellularly formed from 5-aminosalicylic acid (5-ASA) at 200 μM was discharged 5.3, 7.1, and 8.1-fold higher into the apical site than into the basolateral site during 1, 2, and 4-hour incubations, respectively, in Caco-2 cells grown in Transwells. The addition of flavonols (100 μM) such as fisetin and quercetin with 5-ASA remarkably decreased the apically directed efflux of 5-AcASA. When 5-ASA (200 μM) was added to Caco-2 cells grown in tissue culture dishes, the formation of 5-AcASA decreased, and, in addition, the formed 5-AcASA was found to be accumulated within the cells in the presence of such flavonols. Thus, the decrease in 5-AcASA efflux by such flavonols was attributed not only to the inhibition of N-acetyl-conjugation of 5-ASA but to the predominant cellular accumulation of 5-AcASA. Various flavonoids also had both of the effects with potencies that depend on their specific structures. The essential structure of flavonoids was an absence of a hydroxyl substitution at the C5 position on the A-ring of flavone structure for the inhibitory effect on the N-acetyl-conjugation of 5-ASA, and a presence of hydroxyl substitutions at the C3′ or C4′ position on the B-ring of flavone structure for the promoting effect on the cellular accumulation of 5-AcASA. Both the decrease in 5-AcASA apical efflux and the increase in 5-AcASA cellular accumulation were also caused by MK571 and indomethacin, inhibitors of MRPs, but not by quinidine, cyclosporin A, P-glycoprotein inhibitors, and mitoxantrone, a BCRP substrate. These results suggest that certain flavonoids suppress the apical efflux of 5-AcASA possibly by inhibiting MRPs pumps located on apical membranes in Caco-2 cells. PMID:19688110

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

PubMed Central

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

2007-01-01

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

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

PubMed

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

2008-11-01

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

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

Ciprofloxacin degradation in anaerobic sulfate-reducing bacteria (SRB) sludge system: Mechanism and pathways.

PubMed

Jia, Yanyan; Khanal, Samir Kumar; Shu, Haoyue; Zhang, Huiqun; Chen, Guang-Hao; Lu, Hui

2018-06-01

Ciprofloxacin (CIP), a fluoroquinolone antibiotic, removal was examined for the first time, in an anaerobic sulfate-reducing bacteria (SRB) sludge system. About 28.0% of CIP was biodegraded by SRB sludge when the influent CIP concentration was 5000 μg/L. Some SRB genera with high tolerance to CIP (i.e. Desulfobacter), were enriched at CIP concentration of 5000 μg/L. The changes in antibiotic resistance genes (ARGs) of SRB sludge coupled with CIP biodegradation intermediates were used to understand the mechanism of CIP biodegradation for the first time. The percentage of efflux pump genes associated with ARGs increased, while the percentage of fluoroquinolone resistance genes that inhibit the DNA copy of bacteria decreased during prolonged exposure to CIP. It implies that some intracellular CIP was extruded into extracellular environment of microbial cells via efflux pump genes to reduce fluoroquinolone resistance genes accumulation caused by exposure to CIP. Additionally, the degradation products and the possible pathways of CIP biodegradation were also examined using the new method developed in this study. The results suggest that CIP was biodegraded intracellularly via desethylation reaction in piperazinyl ring and hydroxylation reaction catalyzed by cytochrome P450 enzymes. This study provides an insight into the mechanism and pathways of CIP biodegradation by SRB sludge, and opens-up a new opportunity for the treatment of CIP-containing wastewater using sulfur-mediated biological process. Copyright © 2018 Elsevier Ltd. All rights reserved.

Design of New Antibacterial Enhancers Based on AcrB's Structure and the Evaluation of Their Antibacterial Enhancement Activity.

PubMed

Song, Yi; Qin, Rongxin; Pan, Xichun; Ouyang, Qin; Liu, Tianyu; Zhai, Zhaoxia; Chen, Yingchun; Li, Bin; Zhou, Hong

2016-11-18

Previously, artesunate (AS) and dihydroartemisinine 7 (DHA7) were found to have antibacterial enhancement activity against Escherichia coli via inhibition of the efflux pump AcrB. However, they were only effective against E. coli standard strains. This study aimed to develop effective antibacterial enhancers based on the previous work. Our results demonstrate that 86 new antibacterial enhancers were designed via 3D-SAR and molecular docking. Among them, DHA27 had the best antibacterial enhancement activity. It could potentiate the antibacterial effects of ampicillin against not only E. coli standard strain but also clinical strains, and of β-lactam antibiotics, not non-β-lactamantibiotics. DHA27 could increase the accumulation of daunomycin and nile red within E. coli ATCC 35218, but did not increase the bacterial membrane permeability. DHA27 reduced acrB 's mRNA expression of E. coli ATCC 35218 in a dose-dependent manner, and its antibacterial enhancement activity is related to the degree of acrB mRNA expression in E. coli clinical strains. The polypeptides from AcrB were obtained via molecular docking assay; the pre-incubated polypeptides could inhibit the activity of DHA27. Importantly, DHA27 had no cytotoxicity on cell proliferation. In conclusion, among newly designed antibacterial enhancers, DHA27 had favorable physical and pharmacological properties with no significant cytotoxicity at effective concentrations, and might serve as a potential efflux pump inhibitor in the future.

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

PubMed

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

2017-02-01

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

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

PubMed

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

2015-07-01

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

Targeting intracellular Staphylococcus aureus to lower recurrence of orthopaedic infection.

PubMed

Dusane, Devendra H; Kyrouac, Douglas; Petersen, Iris; Bushrow, Luke; Calhoun, Jason H; Granger, Jeffrey F; Phieffer, Laura S; Stoodley, Paul

2018-04-01

Staphylococcus aureus is often found in orthopaedic infections and may be protected from commonly prescribed antibiotics by forming biofilms or growing intracellularly within osteoblasts. To investigate the effect of non-antibiotic compounds in conjunction with antibiotics to clear intracellular and biofilm forming S. aureus causing osteomyelitis. SAOS-2 osteoblast-like cell lines were infected with S. aureus BB1279. Antibiotics (vancomycin, VAN; and dicloxacillin, DICLOX), bacterial efflux pump inhibitors (piperine, PIP; carbonyl cyanide m-chlorophenyl hydrazone, CCCP), and bone morphogenetic protein (BMP-2) were evaluated individually and in combination to kill intracellular bacteria. We present direct evidence that after gentamicin killed extracellular planktonic bacteria and antibiotics had been stopped, seeding from the infected osteoblasts grew as biofilms. VAN was ineffective in treating the intracellular bacteria even at 10× MIC; however in presence of PIP or CCCP the intracellular S. aureus was significantly reduced. Bacterial efflux pump inhibitors (PIP and CCCP) were effective in enhancing permeability of antibiotics within the osteoblasts and facilitated killing of intracellular S. aureus. Confocal laser scanning microscopy (CLSM) showed increased uptake of propidium iodide within osteoblasts in presence of PIP and CCCP. BMP-2 had no effect on growth of S. aureus either alone or in combination with antibiotics. Combined application of antibiotics and natural agents could help in the treatment of osteoblast infected intracellular bacteria and biofilms associated with osteomyelitis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1086-1092, 2018. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Evaluation of Carbapenem Resistance Mechanisms and Its Association with Pseudomonas aeruginosa Infections in the Northwest of Iran.

PubMed

Akhi, Mohammad Taghi; Khalili, Younes; Ghotaslou, Reza; Yousefi, Saber; Kafil, Hossein Samadi; Naghili, Behrooz; Sheikhalizadeh, Vajihe

2018-03-01

The aims of this study were to determine carbapenem resistance mechanisms, molecular epidemiological relationship, clinical impact, and patient outcome of carbapenem-resistant Pseudomonas aeruginosa (CRPA) infections. A total of 42 nonduplicated CRPA were recovered from Urmia, Iran. Antimicrobial susceptibility tests were carried out using phenotypic methods. The carbapenem resistance mechanisms such as carbapenemase genes, efflux pump hyperexpression, AmpC overproduction, and OprD gene downregulation were determined by phenotypic and molecular methods. Eighteen metallo-β-lactamase (MBL) producer isolates were found to be sensitive to amikacin. Among the CRPA, 52.3%, 26.1%, 26.1%, and 59.5% were identified as carbapenemase, efflux pump hyperexpression, AmpC overproduction, and reduced expression OprD gene, respectively. Random Amplified Polymorphic DNA analysis yielded 25 distinct profiles. Most MBL-positive isolates were recovered from patients hospitalized in urology and internal wards with urinary tract infections. Most of the strains showed downregulation of porin. The clonal distribution of the strains was related to carbapenem resistance mechanisms (most of MBL producers belong to the same clones) and the same hospital wards where the isolates were collected. The study demonstrates that the main risk factor of MBL-related infections was hospitalization in non-intensive wards. Amikacin was considered a very efficient antibiotic to treatment of MBL-producing CRPA isolates. Our results showed that OprD downregulation and IMP-type MBL are the main carbapenem resistance mechanisms in CRPA isolates from northwest of Iran.

Entry into and Release of Solvents by Escherichia coli in an Organic-Aqueous Two-Liquid-Phase System and Substrate Specificity of the AcrAB-TolC Solvent-Extruding Pump

PubMed Central

Tsukagoshi, Norihiko; Aono, Rikizo

2000-01-01

Growth of Escherichia coli is inhibited upon exposure to a large volume of a harmful solvent, and there is an inverse correlation between the degree of inhibition and the log POW of the solvent, where POW is the partition coefficient measured for the partition equilibrium established between the n-octanol and water phases. The AcrAB-TolC efflux pump system is involved in maintaining intrinsic solvent resistance. We inspected the solvent resistance of ΔacrAB and/or ΔtolC mutants in the presence of a large volume of solvent. Both mutants were hypersensitive to weakly harmful solvents, such as nonane (log POW = 5.5). The ΔtolC mutant was more sensitive to nonane than the ΔacrAB mutant. The solvent entered the E. coli cells rapidly. Entry of solvents with a log POW higher than 4.4 was retarded in the parent cells, and the intracellular levels of these solvents were maintained at low levels. The ΔtolC mutant accumulated n-nonane or decane (log POW = 6.0) more abundantly than the parent or the ΔacrAB mutant. The AcrAB-TolC complex likely extrudes solvents with a log POW in the range of 3.4 to 6.0 through a first-order reaction. The most favorable substrates for the efflux system were considered to be octane, heptane, and n-hexane. PMID:10940021

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

PubMed

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

2009-10-01

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

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.

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

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

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).

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.

Active uptake of sodium in the gills of the hyperregulating shore crab Carcinus maenas

NASA Astrophysics Data System (ADS)

Siebers, D.; Lucu, Č.; Winkler, A.; Dalla Venezia, L.; Wille, H.

1986-03-01

Isolated posterior gills of shore crabs, Carcinus maenas, previously acclimated for at least 1 month to brackish water of 10 ‰ S, were connected with an artificial hemolymph circulation by means of thin polyethylene tubings. Gills were symmetrically perfused and bathed with 50 % sea water. Transepithelial potential differences (PDs) and fluxes of sodium between medium and blood were measured under control conditions and following reductions of PDs by means of 5 mM internal (blood side) ouabain, 0.5 mM internal and external (bathing medium) NaCN or by exhaustion of energy reserves along with a prolonged perfusion period of more than 9 h. In these experiments22Na was used as tracer. Each of the three modes of reducing transepithelial potential differences resulted in a decrease in sodium influxes from 500 1000 µmoles g-1 h-1 to 250 400 µmoles g-1 h-1. The findings suggest that sodium influx, which normally greatly exceeds efflux, was diminished by its active component. The remaining non-inhibitable influx equals efflux values. Our findings thus indicate that efflux is completely passive, while influx has — beside a passive component of efflux magnitudes — an additional active portion which is much larger than the passive component. Since ouabain is a specific inhibitor of the Na-K-ATPase, our results confirm previous findings (Siebers et al., 1985) that the basolaterally located Na-K-ATPase generates the transepithelial potential difference in the gills, which is inside negative by about 6 12 mV. Inhibition of the active portion of sodium influx by internal ouabain along with reduced PDs suggests that transepithelial PDs generated by the branchial sodium pump are the driving force for active sodium uptake in hyperregulating brackish water crabs.

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

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

PubMed Central

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

2011-01-01

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

Environmental adaptability and stress tolerance of Laribacter hongkongensis: a genome-wide analysis

PubMed Central

2011-01-01

Background Laribacter hongkongensis is associated with community-acquired gastroenteritis and traveler's diarrhea and it can reside in human, fish, frogs and water. In this study, we performed an in-depth annotation of the genes in its genome related to adaptation to the various environmental niches. Results L. hongkongensis possessed genes for DNA repair and recombination, basal transcription, alternative σ-factors and 109 putative transcription factors, allowing DNA repair and global changes in gene expression in response to different environmental stresses. For acid stress, it possessed a urease gene cassette and two arc gene clusters. For alkaline stress, it possessed six CDSs for transporters of the monovalent cation/proton antiporter-2 and NhaC Na+:H+ antiporter families. For heavy metals acquisition and tolerance, it possessed CDSs for iron and nickel transport and efflux pumps for other metals. For temperature stress, it possessed genes related to chaperones and chaperonins, heat shock proteins and cold shock proteins. For osmotic stress, 25 CDSs were observed, mostly related to regulators for potassium ion, proline and glutamate transport. For oxidative and UV light stress, genes for oxidant-resistant dehydratase, superoxide scavenging, hydrogen peroxide scavenging, exclusion and export of redox-cycling antibiotics, redox balancing, DNA repair, reduction of disulfide bonds, limitation of iron availability and reduction of iron-sulfur clusters are present. For starvation, it possessed phosphorus and, despite being asaccharolytic, carbon starvation-related CDSs. Conclusions The L. hongkongensis genome possessed a high variety of genes for adaptation to acid, alkaline, temperature, osmotic, oxidative, UV light and starvation stresses and acquisition of and tolerance to heavy metals. PMID:21711489

Factors Supporting Cysteine Tolerance and Sulfite Production in Candida albicans

PubMed Central

Hennicke, Florian; Grumbt, Maria; Lermann, Ulrich; Ueberschaar, Nico; Palige, Katja; Böttcher, Bettina; Jacobsen, Ilse D.; Staib, Claudia; Morschhäuser, Joachim; Monod, Michel; Hube, Bernhard; Hertweck, Christian

2013-01-01

The amino acid cysteine has long been known to be toxic at elevated levels for bacteria, fungi, and humans. However, mechanisms of cysteine tolerance in microbes remain largely obscure. Here we show that the human pathogenic yeast Candida albicans excretes sulfite when confronted with increasing cysteine concentrations. Mutant construction and phenotypic analysis revealed that sulfite formation from cysteine in C. albicans relies on cysteine dioxygenase Cdg1, an enzyme with similar functions in humans. Environmental cysteine induced not only the expression of the CDG1 gene in C. albicans, but also the expression of SSU1, encoding a putative sulfite efflux pump. Accordingly, the deletion of SSU1 resulted in enhanced sensitivity of the fungal cells to both cysteine and sulfite. To study the regulation of sulfite/cysteine tolerance in more detail, we screened a C. albicans library of transcription factor mutants in the presence of sulfite. This approach and subsequent independent mutant analysis identified the zinc cluster transcription factor Zcf2 to govern sulfite/cysteine tolerance, as well as cysteine-inducible SSU1 and CDG1 gene expression. cdg1Δ and ssu1Δ mutants displayed reduced hypha formation in the presence of cysteine, indicating a possible role of the newly proposed mechanisms of cysteine tolerance and sulfite secretion in the pathogenicity of C. albicans. Moreover, cdg1Δ mutants induced delayed mortality in a mouse model of disseminated infection. Since sulfite is toxic and a potent reducing agent, its production by C. albicans suggests diverse roles during host adaptation and pathogenicity. PMID:23417561

A Novel C2H2 Transcription Factor that Regulates gliA Expression Interdependently with GliZ in Aspergillus fumigatus

PubMed Central

Schoberle, Taylor J.; Nguyen-Coleman, C. Kim; Herold, Jennifer; Yang, Ally; Weirauch, Matt; Hughes, Timothy R.; McMurray, John S.; May, Gregory S.

2014-01-01

Secondary metabolites are produced by numerous organisms and can either be beneficial, benign, or harmful to humans. Genes involved in the synthesis and transport of these secondary metabolites are frequently found in gene clusters, which are often coordinately regulated, being almost exclusively dependent on transcription factors that are located within the clusters themselves. Gliotoxin, which is produced by a variety of Aspergillus species, Trichoderma species, and Penicillium species, exhibits immunosuppressive properties and has therefore been the subject of research for many laboratories. There have been a few proteins shown to regulate the gliotoxin cluster, most notably GliZ, a Zn2Cys6 binuclear finger transcription factor that lies within the cluster, and LaeA, a putative methyltransferase that globally regulates secondary metabolism clusters within numerous fungal species. Using a high-copy inducer screen in A. fumigatus, our lab has identified a novel C2H2 transcription factor, which plays an important role in regulating the gliotoxin biosynthetic cluster. This transcription factor, named GipA, induces gliotoxin production when present in extra copies. Furthermore, loss of gipA reduces gliotoxin production significantly. Through protein binding microarray and mutagenesis, we have identified a DNA binding site recognized by GipA that is in extremely close proximity to a potential GliZ DNA binding site in the 5′ untranslated region of gliA, which encodes an efflux pump within the gliotoxin cluster. Not surprisingly, GliZ and GipA appear to work in an interdependent fashion to positively control gliA expression. PMID:24784729

Stress induction in the bacteria Shewanella oneidensis and Deinococcus radiodurans in response to below-background ionizing radiation.

PubMed

Castillo, Hugo; Schoderbek, Donald; Dulal, Santosh; Escobar, Gabriela; Wood, Jeffrey; Nelson, Roger; Smith, Geoffrey

2015-01-01

The 'Linear no-threshold' (LNT) model predicts that any amount of radiation increases the risk of organisms to accumulate negative effects. Several studies at below background radiation levels (4.5-11.4 nGy h(-1)) show decreased growth rates and an increased susceptibility to oxidative stress. The purpose of our study is to obtain molecular evidence of a stress response in Shewanella oneidensis and Deinococcus radiodurans grown at a gamma dose rate of 0.16 nGy h(-1), about 400 times less than normal background radiation. Bacteria cultures were grown at a dose rate of 0.16 or 71.3 nGy h(-1) gamma irradiation. Total RNA was extracted from samples at early-exponential and stationary phases for the rt-PCR relative quantification (radiation-deprived treatment/background radiation control) of the stress-related genes katB (catalase), recA (recombinase), oxyR (oxidative stress transcriptional regulator), lexA (SOS regulon transcriptional repressor), dnaK (heat shock protein 70) and SOA0154 (putative heavy metal efflux pump). Deprivation of normal levels of radiation caused a reduction in growth of both bacterial species, accompanied by the upregulation of katB, recA, SOA0154 genes in S. oneidensis and the upregulation of dnaK in D. radiodurans. When cells were returned to background radiation levels, growth rates recovered and the stress response dissipated. Our results indicate that below-background levels of radiation inhibited growth and elicited a stress response in two species of bacteria, contrary to the LNT model prediction.

Contribution of Stress Responses to Antibiotic Tolerance in Pseudomonas aeruginosa Biofilms

PubMed Central

Franklin, Michael J.; Williamson, Kerry S.; Folsom, James P.; Boegli, Laura; James, Garth A.

2015-01-01

Enhanced tolerance of biofilm-associated bacteria to antibiotic treatments is likely due to a combination of factors, including changes in cell physiology as bacteria adapt to biofilm growth and the inherent physiological heterogeneity of biofilm bacteria. In this study, a transcriptomics approach was used to identify genes differentially expressed during biofilm growth of Pseudomonas aeruginosa. These genes were tested for statistically significant overlap, with independently compiled gene lists corresponding to stress responses and other putative antibiotic-protective mechanisms. Among the gene groups tested were those associated with biofilm response to tobramycin or ciprofloxacin, drug efflux pumps, acyl homoserine lactone quorum sensing, osmotic shock, heat shock, hypoxia stress, and stationary-phase growth. Regulons associated with Anr-mediated hypoxia stress, RpoS-regulated stationary-phase growth, and osmotic stress were significantly enriched in the set of genes induced in the biofilm. Mutant strains deficient in rpoS, relA and spoT, or anr were cultured in biofilms and challenged with ciprofloxacin and tobramycin. When challenged with ciprofloxacin, the mutant strain biofilms had 2.4- to 2.9-log reductions in viable cells compared to a 0.9-log reduction of the wild-type strain. Interestingly, none of the mutants exhibited a statistically significant alteration in tobramycin susceptibility compared to that with the wild-type biofilm. These results are consistent with a model in which multiple genes controlled by overlapping starvation or stress responses contribute to the protection of a P. aeruginosa biofilm from ciprofloxacin. A distinct and as yet undiscovered mechanism protects the biofilm bacteria from tobramycin. PMID:25870065

Diversity of the Tetracycline Mobilome within a Chinese Pig Manure Sample

PubMed Central

Leclercq, Sébastien Olivier; Wang, Chao; Zhu, Yaxin; Wu, Hai; Du, Xiaochen; Liu, Zhipei

2016-01-01

ABSTRACT Tetracycline antibiotics are widely used in livestock, and tetracycline resistance genes (TRG) are frequently reported in the manure of farmed animals. However, the diversity of TRG-carrying transposons in manure has still been rarely investigated. Using a culture-free functional metagenomic procedure, combined with large-insert library construction and sequencing, bioinformatic analyses, and functional experiments, we identified 17 distinct TRGs in a single pig manure sample, including two new tet genes: tet(59), encoding a tetracycline efflux pump, and tet(W/N/W), encoding mosaic ribosomal protection. Our study also revealed six new TRG-carrying putative nonconjugative transposons: Tn5706-like transposon Tn6298, IS200/605-related transposon Tn6303, Tn3 family transposon Tn6299, and three ISCR2-related transposons, Tn62300, Tn62301, and Tn62302. IMPORTANCE Fertilization of agricultural fields with animal manure is believed to play a major role in antibiotic resistance dissemination in the environment. There is growing concern for the possible spread of antibiotic resistance from the environment to humans since genetic resistance determinants may be located in transposons and other mobile genetic elements potentially transferable to pathogens. Among the various antibiotic resistance genes found in manure, tetracycline resistance genes (TRGs) are some of the most common. The present study provides a detailed snapshot of the tetracycline mobilome in a single pig manure sample, revealing an unappreciated diversity of TRGs and potential TRG mobility vectors. Our precise identification of the TRG-carrying units will enable us to investigate in more details their mobility effectiveness. PMID:27565618

Diversity of the Tetracycline Mobilome within a Chinese Pig Manure Sample.

PubMed

Leclercq, Sébastien Olivier; Wang, Chao; Zhu, Yaxin; Wu, Hai; Du, Xiaochen; Liu, Zhipei; Feng, Jie

2016-11-01

Tetracycline antibiotics are widely used in livestock, and tetracycline resistance genes (TRG) are frequently reported in the manure of farmed animals. However, the diversity of TRG-carrying transposons in manure has still been rarely investigated. Using a culture-free functional metagenomic procedure, combined with large-insert library construction and sequencing, bioinformatic analyses, and functional experiments, we identified 17 distinct TRGs in a single pig manure sample, including two new tet genes: tet(59), encoding a tetracycline efflux pump, and tet(W/N/W), encoding mosaic ribosomal protection. Our study also revealed six new TRG-carrying putative nonconjugative transposons: Tn5706-like transposon Tn6298, IS200/605-related transposon Tn6303, Tn3 family transposon Tn6299, and three ISCR2-related transposons, Tn62300, Tn62301, and Tn62302 IMPORTANCE: Fertilization of agricultural fields with animal manure is believed to play a major role in antibiotic resistance dissemination in the environment. There is growing concern for the possible spread of antibiotic resistance from the environment to humans since genetic resistance determinants may be located in transposons and other mobile genetic elements potentially transferable to pathogens. Among the various antibiotic resistance genes found in manure, tetracycline resistance genes (TRGs) are some of the most common. The present study provides a detailed snapshot of the tetracycline mobilome in a single pig manure sample, revealing an unappreciated diversity of TRGs and potential TRG mobility vectors. Our precise identification of the TRG-carrying units will enable us to investigate in more details their mobility effectiveness. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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.

Studies on the crystalline lens. XXI. Bidirectional carrier-mediated transport of lithium

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

Kinsey, V.E.; McLean, I.W.

1974-10-01

Movement of lithium both into and out of cultured rabbit lenses occurs by processes that obey Michaelis-Menten kinetics as well as by a nonsaturable process. The parameters describing the kinetics of transport are evaluated on the basis of a modified version of a pump-leak hypothesis that was shown previously to account for the fluxes of other alkali metal cations. Potassium is a potent competitive inhibitor of the carrier-mediated transport of lithium into the lens. Lithium weakly inhibits the influx of potassium by a noncompetitive process. The observation that lithium is a weak inhibitor of potassium transport (Ki = 70 mM)more » while having a moderate affinity for its carrier (Km = 4.0 mM) suggests that more than one site may be responsible for the transport of potassium and lithium into the lens. Active transport of lithium out of the lens does not appear to involve the sodium pump, since the rate of sodium efflux is unaffected by lithium. Both influx and efflux of lithium are inhibited by ouabain (10/sup -5/ M) but not by amiloride (10/sup -4/ M) or oxytocin (20 milliunits per milliliter). Lithium displaces proportionate amounts of sodium and potassium from intracellular fluid when lenses are cultured in the presence of this cation. The nonsaturable exchange of lithium is independent of observed differences in electric potential, indicating that lithium does not permeate the lenticular membranes by simple diffusion of lithium ions, but rather as a complex with either a free anion or a carrier within the membrane. The affinity of lithium for the carrier is essentially equal to that of cesium and much lower than that for rubidium, whereas the kd of both lithium and rubidium is much higher than that for cesium. These differences indicate that a common pathway cannot be responsible for the discrimination between cations by both the pump and the leak.« less

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.

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.

Draft genome sequence of a GES-5-producing Serratia marcescens isolated in southern Brazil.

PubMed

Nodari, Carolina Silva; Siebert, Marina; Matte, Ursula da Silveira; Barth, Afonso Luís

Serratia marcescens is a Gram-negative rod intrinsically resistant to polymyxins and usually associated with wound, respiratory and urinary tract infections. The whole genome of the first GES-5-producing S. marcescens isolated from a Brazilian patient was sequenced using Ion Torrent PGM System. Besides bla GES-5 , we were able to identify genes encoding for other β-lactamases, for aminoglycoside modifying enzymes and for an efflux pump to tetracyclines. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

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

PubMed

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

2003-09-01

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

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.

An overview of techniques for the measurement of calcium distribution, calcium fluxes, and cytosolic free calcium in mammalian cells.

PubMed Central

Borle, A B

1990-01-01

An array of techniques can be used to study cell calcium metabolism that comprises several calcium compartments and many types of transport systems such as ion channels, ATP-dependent pumps, and antiporters. The measurement of total cell calcium brings little information of value since 60 to 80% of total cell calcium is actually bound to the extracellular glycocalyx. Cell fractionation and differential centrifugation have been used to study intracellular Ca2+ compartmentalization, but the methods suffer from the possibility of Ca2+ loss or redistribution among cell fractions. Steady-state kinetic analyses of 45Ca uptake or desaturation curves have been used to study the distribution of Ca2+ among various kinetic pools in living cells and their rate of Ca2+ exchange, but the analyses are constrained by many limitations. Nonsteady-state tracer studies can provide information about rapid changes in calcium influx or efflux in and out of the cell. Zero-time kinetics of 45Ca uptake can detect instantaneous changes in calcium influx, while 45Ca fractional efflux ratio, can detect rapid stimulations or inhibitions of calcium efflux out of cells. Permeabilized cells have been successfully used to gauge the relative role of intracellular organelles in controlling [Ca2+]i. The measurement of the cytosolic ionized calcium ([Ca2+]i) is undoubtedly the most important and, physiologically, the most relevant method available. The choice of the appropriate calcium indicator, fluorescent, bioluminescent, metallochromic, or Ca2(+)-sensitive microelectrodes depends on the cell type and the magnitude and time constant of the event under study. Each probe has specific assets and drawbacks. The study of plasma membrane vesicles derived from baso-lateral or apical plasmalemma can also bring important information on the (Ca2(+)-Mg2+) ATPase-dependent calcium pump and on the kinetics and stoichiometry of the Na(+)-Ca2+ antiporter. The best strategy to study cell calcium metabolism is to use several different methods that focus on a specific problem from widely different angles. PMID:2190818

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

PubMed

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

2017-07-01

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

Plasmid-mediated quinolone resistance: Two decades on.

PubMed

Rodríguez-Martínez, José Manuel; Machuca, Jesús; Cano, María Eliecer; Calvo, Jorge; Martínez-Martínez, Luis; Pascual, Alvaro

2016-11-01

After two decades of the discovery of plasmid-mediated quinolone resistance (PMQR), three different mechanisms have been associated to this phenomenon: target protection (Qnr proteins, including several families with multiple alleles), active efflux pumps (mainly QepA and OqxAB pumps) and drug modification [AAC(6')-Ib-cr acetyltransferase]. PMQR genes are usually associated with mobile or transposable elements on plasmids, and, in the case of qnr genes, are often incorporated into sul1-type integrons. PMQR has been found in clinical and environmental isolates around the world and appears to be spreading. Although the three PMQR mechanisms alone cause only low-level resistance to quinolones, they can complement other mechanisms of chromosomal resistance to reach clinical resistance level and facilitate the selection of higher-level resistance, raising a threat to the treatment of infections by microorganisms that host these mechanisms. Copyright © 2016 Elsevier Ltd. 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.

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

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

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

2005-06-24

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

Increased Usage of Antiseptics Is Associated with Reduced Susceptibility in Clinical Isolates of Staphylococcus aureus

PubMed Central

Hardy, Katherine; Sunnucks, Katie; Gil, Hannah; Shabir, Sahida; Trampari, Eleftheria; Hawkey, Peter

2018-01-01

ABSTRACT Hospital-acquired infection is a major cause of morbidity and mortality, and regimes to prevent infection are crucial in infection control. These include the decolonization of vulnerable patients with methicillin-resistant Staphylococcus aureus (MRSA) carriage using antiseptics, including chlorhexidine and octenidine. Concern has been raised, however, regarding the possible development of biocide resistance. In this study, we assembled a panel of S. aureus isolates, including isolates collected before the development of chlorhexidine and octenidine and isolates, from a major hospital trust in the United Kingdom during a period when the decolonization regimes were altered. We observed significant increases in the MIC and minimum bactericidal concentration (MBC) of chlorhexidine in isolates from periods of high usage of chlorhexidine. Isolates with increased MICs and MBCs of octenidine rapidly emerged after octenidine was introduced in the trust. There was no apparent cross-resistance between the two biocidal agents. A combination of variable-number tandem repeat (VNTR) analysis, PCR for qac genes, and whole-genome sequencing was used to type isolates and examine possible mechanisms of resistance. There was no expansion of a single strain associated with decreased biocide tolerance, and biocide susceptibility did not correlate with carriage of qac efflux pump genes. Mutations within the NorA or NorB efflux pumps, previously associated with chlorhexidine export, were identified, however, suggesting that this may be an important mechanism of biocide tolerance. We present evidence that isolates are evolving in the face of biocide challenge in patients and that changes in decolonization regimes are reflected in changes in susceptibility of isolates. PMID:29844113

Risk factors associated with fluoroquinolone-resistant enterococcal urinary tract infections in a tertiary care university hospital in north India.

PubMed

Banerjee, Tuhina; Anupurba, Shampa

2016-10-01

Fluoroquinolone resistance in both Gram-positive and Gram-negative bacteria has increased with the widespread use of fluoroquinolones. Fluoroquinolone resistance in Gram-negative bacilli has been widely studied, though staphylococci and enterococci are also notably resistant. Enterococci being the second most common cause of healthcare-associated urinary tract infections (UTIs) fluoroquinolones are often the drug of choice. This study was undertaken to assess the risk factors associated with fluoroquinolone-resistant enterococcal UTI in a tertiary level health facility in north India. A total of 365 patients with UTI caused by enterococci were studied over a period of two years. Patients with ciprofloxacin-resistant and susceptible UTI were considered as cases and controls, respectively. Resistance profile of the isolates against common antibiotics was studied by minimum inhibitory concentration (MIC) determination. Mechanisms for fluoroquinolone resistance was studied by efflux pump inhibitor activity and multiplex PCR targeting the qnr genes. A total of 204 (55.89%) cases and 161 (44.1%) controls were identified. The fluoroquinolone-resistant isolates were significantly resistant to ampicillin, high strength aminoglycosides and vancomycin. The majority (78%) of the resistant isolates showed efflux pump activity. Treatment in indoor locations, presence of urinary catheters and pregnancy along with recent exposure to antibiotics especially fluoroquinolones, third generation cephalosporins and piperacillin-tazobactam were identified as independent risk factors. Our results showed that fluoroquinolone resistance in enterococcal UTI was largely associated with indoor usage of antibiotics and use of indwelling devices. Knowledge of risk factors is important to curb this emergence of resistance.

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.

A calcium-dependent protein kinase can inhibit a calmodulin-stimulated Ca2+ pump (ACA2) located in the endoplasmic reticulum of Arabidopsis

NASA Technical Reports Server (NTRS)

Hwang, I.; Sze, H.; Harper, J. F.; Evans, M. L. (Principal Investigator)

2000-01-01

The magnitude and duration of a cytosolic Ca(2+) release can potentially be altered by changing the rate of Ca(2+) efflux. In plant cells, Ca(2+) efflux from the cytoplasm is mediated by H(+)/Ca(2+)-antiporters and two types of Ca(2+)-ATPases. ACA2 was recently identified as a calmodulin-regulated Ca(2+)-pump located in the endoplasmic reticulum. Here, we show that phosphorylation of its N-terminal regulatory domain by a Ca(2+)-dependent protein kinase (CDPK isoform CPK1), inhibits both basal activity ( approximately 10%) and calmodulin stimulation ( approximately 75%), as shown by Ca(2+)-transport assays with recombinant enzyme expressed in yeast. A CDPK phosphorylation site was mapped to Ser(45) near a calmodulin binding site, using a fusion protein containing the N-terminal domain as an in vitro substrate for a recombinant CPK1. In a full-length enzyme, an Ala substitution for Ser(45) (S45/A) completely blocked the observed CDPK inhibition of both basal and calmodulin-stimulated activities. An Asp substitution (S45/D) mimicked phosphoinhibition, indicating that a negative charge at this position is sufficient to account for phosphoinhibition. Interestingly, prior binding of calmodulin blocked phosphorylation. This suggests that, once ACA2 binds calmodulin, its activation state becomes resistant to phosphoinhibition. These results support the hypothesis that ACA2 activity is regulated as the balance between the initial kinetics of calmodulin stimulation and CDPK inhibition, providing an example in plants for a potential point of crosstalk between two different Ca(2+)-signaling pathways.

CD147 Required for Corneal Endothelial Lactate Transport

PubMed Central

Li, Shimin; Nguyen, Tracy T.; Bonanno, Joseph A.

2014-01-01

Purpose. CD147/basigin is a chaperone for lactate:H+ cotransporters (monocarboxylate transporters) MCT1 and MCT4. We tested the hypothesis that MCT1 and -4 in corneal endothelium contribute to lactate efflux from stroma to anterior chamber and that silencing CD147 expression would cause corneal edema. Methods. CD147 was silenced via small interfering ribonucleic acid (siRNA) transfection of rabbit corneas ex vivo and anterior chamber lenti-small hairpin RNA (shRNA) pseudovirus in vivo. CD147 and MCT expression was examined by Western blot, RT-PCR, and immunofluorescence. Functional effects were examined by measuring lactate-induced cell acidification, corneal lactate efflux, [lactate], central cornea thickness (CCT), and Azopt (a carbonic anhydrase inhibitor) sensitivity. Results. In ex vivo corneas, 100 nM CD147 siRNA reduced CD147, MCT1, and MCT4 expression by 85%, 79%, and 73%, respectively, while MCT2 expression was unaffected. CD147 siRNA decreased lactate efflux from 3.9 ± 0.81 to 1.5 ± 0.37 nmol/min, increased corneal [lactate] from 19.28 ± 7.15 to 56.73 ± 8.97 nmol/mg, acidified endothelial cells (pHi = 6.83 ± 0.07 vs. 7.19 ± 0.09 in control), and slowed basolateral lactate-induced acidification from 0.0034 ± 0.0005 to 0.0012 ± 0.0005 pH/s, whereas apical acidification was unchanged. In vivo, CD147 shRNA increased CCT by 28.1 ± 0.9 μm at 28 days; Azopt increased CCT to 24.4 ± 3.12 vs. 12.0 ± 0.48 μm in control, and corneal [lactate] was 47.63 ± 6.29 nmol/mg in shCD147 corneas and 17.82 ± 4.93 nmol/mg in paired controls. Conclusions. CD147 is required for the expression of MCT1 and MCT4 in the corneal endothelium. Silencing CD147 slows lactate efflux, resulting in stromal lactate accumulation and corneal edema, consistent with lactate efflux as a significant component of the corneal endothelial pump. PMID:24970254

CD147 required for corneal endothelial lactate transport.

PubMed

Li, Shimin; Nguyen, Tracy T; Bonanno, Joseph A

2014-06-26

CD147/basigin is a chaperone for lactate:H(+) cotransporters (monocarboxylate transporters) MCT1 and MCT4. We tested the hypothesis that MCT1 and -4 in corneal endothelium contribute to lactate efflux from stroma to anterior chamber and that silencing CD147 expression would cause corneal edema. CD147 was silenced via small interfering ribonucleic acid (siRNA) transfection of rabbit corneas ex vivo and anterior chamber lenti-small hairpin RNA (shRNA) pseudovirus in vivo. CD147 and MCT expression was examined by Western blot, RT-PCR, and immunofluorescence. Functional effects were examined by measuring lactate-induced cell acidification, corneal lactate efflux, [lactate], central cornea thickness (CCT), and Azopt (a carbonic anhydrase inhibitor) sensitivity. In ex vivo corneas, 100 nM CD147 siRNA reduced CD147, MCT1, and MCT4 expression by 85%, 79%, and 73%, respectively, while MCT2 expression was unaffected. CD147 siRNA decreased lactate efflux from 3.9 ± 0.81 to 1.5 ± 0.37 nmol/min, increased corneal [lactate] from 19.28 ± 7.15 to 56.73 ± 8.97 nmol/mg, acidified endothelial cells (pHi = 6.83 ± 0.07 vs. 7.19 ± 0.09 in control), and slowed basolateral lactate-induced acidification from 0.0034 ± 0.0005 to 0.0012 ± 0.0005 pH/s, whereas apical acidification was unchanged. In vivo, CD147 shRNA increased CCT by 28.1 ± 0.9 μm at 28 days; Azopt increased CCT to 24.4 ± 3.12 vs. 12.0 ± 0.48 μm in control, and corneal [lactate] was 47.63 ± 6.29 nmol/mg in shCD147 corneas and 17.82 ± 4.93 nmol/mg in paired controls. CD147 is required for the expression of MCT1 and MCT4 in the corneal endothelium. Silencing CD147 slows lactate efflux, resulting in stromal lactate accumulation and corneal edema, consistent with lactate efflux as a significant component of the corneal endothelial pump. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

Dual activation of CFTR and CLCN2 by lubiprostone in murine nasal epithelia

PubMed Central

Schiffhauer, Eric S.; Vij, Neeraj; Kovbasnjuk, Olga; Kang, Po Wei; Walker, Doug; Lee, Seakwoo

2013-01-01

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. PMID:23316067

Scanning electrochemical microscopy of menadione-glutathione conjugate export from yeast cells

PubMed Central

Mauzeroll, Janine; Bard, Allen J.

2004-01-01

The uptake of menadione (2-methyl-1,4-naphthoquinone), which is toxic to yeast cells, and its expulsion as a glutathione complex were studied by scanning electrochemical microscopy. The progression of the in vitro reaction between menadione and glutathione was monitored electrochemically by cyclic voltammetry and correlated with the spectroscopic (UV–visible) behavior. By observing the scanning electrochemical microscope tip current of yeast cells suspended in a menadione-containing solution, the export of the conjugate from the cells with time could be measured. Similar experiments were performed on immobilized yeast cell aggregates stressed by a menadione solution. From the export of the menadione-glutathione conjugate detected at a 1-μm-diameter electrode situated 10 μm from the cells, a flux of about 30,000 thiodione molecules per second per cell was extracted. Numerical simulations based on an explicit finite difference method further revealed that the observation of a constant efflux of thiodione from the cells suggested the rate was limited by the uptake of menadione and that the efflux through the glutathione-conjugate pump was at least an order of magnitude faster. PMID:15148374

Phenotypic Characterization of a copA Mutant of Neisseria gonorrhoeae Identifies a Link between Copper and Nitrosative Stress

PubMed Central

Djoko, Karrera Y.; Franiek, Jessica A.; Edwards, Jennifer L.; Falsetta, Megan L.; Kidd, Stephen P.; Potter, Adam J.; Chen, Nathan H.; Apicella, Michael A.; Jennings, Michael P.

2012-01-01

NGO0579 is annotated copA in the Neisseria gonorrhoeae chromosome, suggesting that it encodes a cation-transporting ATPase specific for copper ions. Compared to wild-type cells, a copA mutant was more sensitive to killing by copper ions but not to other transition metals. The mutant also accumulated a greater amount of copper, consistent with the predicted role of CopA as a copper efflux pump. The copA mutant showed a reduced ability to invade and survive within human cervical epithelial cells, although its ability to form a biofilm on the surface of these cells was not significantly different from that of the wild type. In the presence of copper, the copA mutant exhibited increased sensitivity to killing by nitrite or nitric oxide. Therefore, we concluded that copper ion efflux catalyzed by CopA is linked to the nitrosative stress defense system of Neisseria gonorrhoeae. These observations suggest that copper may exert its effects as an antibacterial agent in the innate immune system via an interaction with reactive nitrogen species. PMID:22184419

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.

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 .

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.

Deciphering Multifactorial Resistance Phenotypes in Acinetobacter baumannii by Genomics and Targeted Label-free Proteomics.

PubMed

Cecchini, Tiphaine; Yoon, Eun-Jeong; Charretier, Yannick; Bardet, Chloé; Beaulieu, Corinne; Lacoux, Xavier; Docquier, Jean-Denis; Lemoine, Jerome; Courvalin, Patrice; Grillot-Courvalin, Catherine; Charrier, Jean-Philippe

2018-03-01

Resistance to β-lactams in Acinetobacter baumannii involves various mechanisms. To decipher them, whole genome sequencing (WGS) and real-time quantitative polymerase chain reaction (RT-qPCR) were complemented by mass spectrometry (MS) in selected reaction monitoring mode (SRM) in 39 clinical isolates. The targeted label-free proteomic approach enabled, in one hour and using a single method, the quantitative detection of 16 proteins associated with antibiotic resistance: eight acquired β-lactamases ( i.e. GES, NDM-1, OXA-23, OXA-24, OXA-58, PER, TEM-1, and VEB), two resident β-lactamases ( i.e. ADC and OXA-51-like) and six components of the two major efflux systems ( i.e. AdeABC and AdeIJK). Results were normalized using "bacterial quantotypic peptides," i.e. peptide markers of the bacterial quantity, to obtain precise protein quantitation (on average 8.93% coefficient of variation for three biological replicates). This allowed to correlate the levels of resistance to β-lactam with those of the production of acquired as well as resident β-lactamases or of efflux systems. SRM detected enhanced ADC or OXA-51-like production and absence or increased efflux pump production. Precise protein quantitation was particularly valuable to detect resistance mechanisms mediated by regulated genes or by overexpression of chromosomal genes. Combination of WGS and MS, two orthogonal and complementary techniques, allows thereby interpretation of the resistance phenotypes at the molecular level. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Genetic and physiological responses of Bacillus subtilis to metal ion stress.

PubMed

Moore, Charles M; Gaballa, Ahmed; Hui, Monica; Ye, Rick W; Helmann, John D

2005-07-01

Metal ion homeostasis is regulated principally by metalloregulatory proteins that control metal ion uptake, storage and efflux genes. We have used transcriptional profiling to survey Bacillus subtilis for genes that are rapidly induced by exposure to high levels of metal ions including Ag(I), Cd(II), Cu(II), Ni(II) and Zn(II) and the metalloid As(V). Many of the genes affected by metal stress were controlled by known metalloregulatory proteins (Fur, MntR, PerR, ArsR and CueR). Additional metal-induced genes are regulated by two newly defined metal-sensing ArsR/SmtB family repressors: CzrA and AseR. CzrA represses the CadA efflux ATPase and the cation diffusion facilitator CzcD and this repression is alleviated by Zn(II), Cd(II), Co(II), Ni(II) and Cu. CadA is the major determinant for Cd(II) resistance, while CzcD protects the cell against elevated levels of Zn(II), Cu, Co(II) and Ni(II). AseR negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. Our results extend the range of identified effectors for the As(III)-sensor ArsR to include Cd(II) and Ag(I) and for the Cu-sensor CueR to include Ag(I) and, weakly, Cd(II) and Zn(II). In addition to systems dedicated to metal homeostasis, specific metal stresses also strongly induced pathways related to cysteine, histidine and arginine metabolism.

Substance P is a functional neurotransmitter in the rat parotid gland.

PubMed

Gallacher, D V

1983-09-01

The technique of electrical field stimulation was employed to stimulate the intrinsic nerves of isolated rat parotid gland fragments. Responses to field stimulation were recorded as changes in enzyme secretion (amylase release), radiolabelled ion fluxes (86Rb efflux) and electrophysiological effects (changes in acinar cell membrane potential and input resistance). All effects of field stimulation were abolished by the neurotoxin, tetrodotoxin (TTX). Selective use of pharmacological antagonists revealed that both the sympathetic and parasympathetic nerves to this tissue were being excited by field stimulation. Importantly a significant component of the response to field stimulation persisted in the presence of combined autonomic receptor blockade by atropine, phentolamine and propranolol, i.e. due to release of a non-cholinergic, non-adrenergic neurotransmitter. The non-cholinergic, non-adrenergic neurotransmitter evoked amylase release, 86Rb efflux and electrophysiological effects seen as changes in acinar cell membrane potential and conductance, i.e. stimulus-permeability coupled. Two biologically active peptides, substance P (SP) and vasoactive intestinal polypeptide (VIP) were shown to evoke amylase release in the presence of combined autonomic blockade. VIP however did not evoke any increase in 86Rb efflux, i.e. not stimulus-permeability coupled. All the effects of the non-cholinergic, non-adrenergic transmitter were mimicked by substance P which evokes 86Rb efflux and electrophysiological effects in addition to amylase release. The non-cholinergic, non-adrenergic field stimulus effects on amylase release and 86Rb efflux were abolished or markedly attenuated in tissues which had been desensitized by prior exposure to exogenous substance P. In the presence of VIP, however, the non-cholinergic, non-adrenergic effects persisted and were apparently potentiated. Acute application of the neurotoxin capsaicin first stimulated a transient release of amylase and subsequently abolished the non-cholinergic, non-adrenergic field stimulus-evoked enzyme release. The putative substance P antagonist, D-Pro2, D-Trp7,9 substance P, reversibly blocked the response to both non-cholinergic, non-adrenergic nerve stimulation and exogenous substance P. It was demonstrated however that prolonged exposure to this antagonist is associated with non-reversible and, importantly, non-specific neurotoxic effects. It is concluded that substance P or a closely related peptide is a functional neurotransmitter in the rat parotid gland.

Furfural and hydroxymethylfurfural tolerance in Escherichia coli ΔacrR regulatory mutants.

PubMed

Luhe, Annette Lin; Lim, Chan Yuen; Gerken, Henri; Wu, Jinchuan; Zhao, Hua

2015-01-01

The presence of the highly toxic furfural and hydroxymethylfurfural (HMF) in the hydrolysate of lignocellulosic biomass prompted the investigation of the Escherichia coli ΔacrR regulatory mutant for higher tolerance to these compounds, to facilitate the production of biofuels and biochemicals, and further biocatalytic conversions. In comparison with the parental strain, the regulatory mutant with the upregulated efflux pump AcrAB-TolC produced moderately better growth and higher tolerance to concentrations of furfural and HMF between 1 and 2 g L(-1) . © 2014 International Union of Biochemistry and Molecular Biology, Inc.

A Novel Zn2-Cys6 Transcription Factor AtrR Plays a Key Role in an Azole Resistance Mechanism of Aspergillus fumigatus by Co-regulating cyp51A and cdr1B Expressions

PubMed Central

Shimizu, Kiminori; Paul, Sanjoy; Ohba, Ayumi; Gonoi, Tohru; Watanabe, Akira; Gomi, Katsuya

2017-01-01

Successful treatment of aspergillosis caused by Aspergillus fumigatus is threatened by an increasing incidence of drug resistance. This situation is further complicated by the finding that strains resistant to azoles, the major antifungal drugs for aspergillosis, have been widely disseminated across the globe. To elucidate mechanisms underlying azole resistance, we identified a novel transcription factor that is required for normal azole resistance in Aspergillus fungi including A. fumigatus, Aspergillus oryzae, and Aspergillus nidulans. This fungal-specific Zn2-Cys6 type transcription factor AtrR was found to regulate expression of the genes related to ergosterol biosynthesis, including cyp51A that encodes a target protein of azoles. The atrR deletion mutant showed impaired growth under hypoxic conditions and attenuation of virulence in murine infection model for aspergillosis. These results were similar to the phenotypes for a mutant strain lacking SrbA that is also a direct regulator for the cyp51A gene. Notably, AtrR was responsible for the expression of cdr1B that encodes an ABC transporter related to azole resistance, whereas SrbA was not involved in the regulation. Chromatin immunoprecipitation assays indicated that AtrR directly bound both the cyp51A and cdr1B promoters. In the clinically isolated itraconazole resistant strain that harbors a mutant Cyp51A (G54E), deletion of the atrR gene resulted in a hypersensitivity to the azole drugs. Together, our results revealed that AtrR plays a pivotal role in a novel azole resistance mechanism by co-regulating the drug target (Cyp51A) and putative drug efflux pump (Cdr1B). PMID:28052140

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

Na/Ca exchange in the basolateral membrane of the A6 cell monolayer: role in Cai homeostasis.

PubMed

Brochiero, E; Raschi, C; Ehrenfeld, J

1995-05-01

The presence of a Na/Ca exchanger in A6 cells was investigated by measuring intracellular calcium (Cai) fluctuations and the 45Ca fluxes through the basolateral membranes (blm) of the cell monolayer. Removal of Na+ from the medium produced a transient increase in Cai followed by a regulatory phase returning Cai to control levels in 3-4 min, this phase being greatly accelerated (< 60 s) by NaCl addition (apparent Km of approximately 5 mM Na+). The Cai increase was only found with the Na(+)-free medium on the basolateral side of the cell monolayer. A twofold increase in the 45Ca influx was observed under these conditions. In Ca(2+)- depleted cells, the initial Cai increase after Ca2+ addition to the medium was greater when the putative Na/Ca exchanger was not functioning (i.e. in a Na(+)-free medium). 45Ca effluxes through the blm of the monolayer were greatly and transiently increased by a Na(+)-free medium on the serosal side and blocked by orthovanadate (1 mM). The Cai increased induced by a hypo-osmotic shock was greater in cells bathed in a Na(+)-medium, conditions expected to block the activity of the Na/Ca exchanger. These findings support the hypothesis that a Na/Ca exchanger is present on the blm of A6 cells and affirm its role in Cai homeostasis in steady-state conditions and following osmotic shock. In addition, a Ca2+ pump also located on the blm and Ca2+ stores sensitive to inositol 1,4,5-trisphosphate were found to be implicated in Cai homeostasis.

The Insect Pathogen Serratia marcescens Db10 Uses a Hybrid Non-Ribosomal Peptide Synthetase-Polyketide Synthase to Produce the Antibiotic Althiomycin

PubMed Central

Challis, Gregory L.; Stanley-Wall, Nicola R.; Coulthurst, Sarah J.

2012-01-01

There is a continuing need to discover new bioactive natural products, such as antibiotics, in genetically-amenable micro-organisms. We observed that the enteric insect pathogen, Serratia marcescens Db10, produced a diffusible compound that inhibited the growth of Bacillis subtilis and Staphyloccocus aureus. Mapping the genetic locus required for this activity revealed a putative natural product biosynthetic gene cluster, further defined to a six-gene operon named alb1–alb6. Bioinformatic analysis of the proteins encoded by alb1–6 predicted a hybrid non-ribosomal peptide synthetase-polyketide synthase (NRPS-PKS) assembly line (Alb4/5/6), tailoring enzymes (Alb2/3) and an export/resistance protein (Alb1), and suggested that the machinery assembled althiomycin or a related molecule. Althiomycin is a ribosome-inhibiting antibiotic whose biosynthetic machinery had been elusive for decades. Chromatographic and spectroscopic analyses confirmed that wild type S. marcescens produced althiomycin and that production was eliminated on disruption of the alb gene cluster. Construction of mutants with in-frame deletions of specific alb genes demonstrated that Alb2–Alb5 were essential for althiomycin production, whereas Alb6 was required for maximal production of the antibiotic. A phosphopantetheinyl transferase enzyme required for althiomycin biosynthesis was also identified. Expression of Alb1, a predicted major facilitator superfamily efflux pump, conferred althiomycin resistance on another, sensitive, strain of S. marcescens. This is the first report of althiomycin production outside of the Myxobacteria or Streptomyces and paves the way for future exploitation of the biosynthetic machinery, since S. marcescens represents a convenient and tractable producing organism. PMID:23028578

Update of green tea interactions with cardiovascular drugs and putative mechanisms.

PubMed

Werba, José Pablo; Misaka, Shingen; Giroli, Monica Gianna; Shimomura, Kenju; Amato, Manuela; Simonelli, Niccolò; Vigo, Lorenzo; Tremoli, Elena

2018-04-01

Many patients treated with cardiovascular (CV) drugs drink green tea (GT), either as a cultural tradition or persuaded of its putative beneficial effects for health. Yet, GT may affect the pharmacokinetics and pharmacodynamics of CV compounds. Novel GT-CV drug interactions were reported for rosuvastatin, sildenafil and tacrolimus. Putative mechanisms involve inhibitory effects of GT catechins at the intestinal level on influx transporters OATP1A2 or OATP2B1 for rosuvastatin, on CYP3A for sildenafil and on both CYP3A and the efflux transporter p-glycoprotein for tacrolimus. These interactions, which add to those previously described with simvastatin, nadolol and warfarin, might lead, in some cases, to reduced drug efficacy or risk of drug toxicity. Oddly, available data on GT interaction with CV compounds with a narrow therapeutic index, such as warfarin and tacrolimus, derive from single case reports. Conversely, GT interactions with simvastatin, rosuvastatin, nadolol and sildenafil were documented through pharmacokinetic studies. In these, the effect of GT or GT derivatives on drug exposure was mild to moderate, but a high inter-individual variability was observed. Further investigations, including studies on the effect of the dose and the time of GT intake are necessary to understand more in depth the clinical relevance of GT-CV drug interactions. Copyright © 2018. Published by Elsevier B.V.

Enterobacter gergoviae membrane modifications are involved in the adaptive response to preservatives used in cosmetic industry.

PubMed

Périamé, Marina; Pagès, Jean-Marie; Davin-Regli, Anne

2015-01-01

The objective of this study was to understand the adaptive mechanisms in Enterobacter gergoviae which are involved in recurrent contaminations in cosmetic products that are incorporated with preservatives. Bacterial strains from two backgrounds were examined for a profound understanding of the mechanisms of adaptation against preservatives. It included a series of Ent. gergoviae strain-ATCC 33028 derivatives, isolated using increasing methylisothiazolinone-chloromethylisothiazolinone (MIT-CMIT) and triclosan concentrations. The other series was of Ent. gergoviae isolates from cosmetic products exhibiting MIT-CMIT and triclosan resistance. We evaluated the outer membrane protein modifications and efflux mechanisms activities responsible for the resistant trait via immunoblotting assays. Additionally, for understanding the efflux activity real-time efflux, experiments were performed. A cross-insusceptibility between preservatives and some disinfectants was observed in MIT-CMIT-resistant derivative isolates, but antibiotics susceptibility was not altered. Resistance to EDTA was significant in all preservatives insusceptible derivative strains, indicating modifications in the LPS layer. Furthermore, an array of real-time efflux assays indicated different activity levels while no variations were detected in porins and AcrAB-TolC pumps production. Overexpression of a specific flagellin-type protein was observed in one of the MIT-CMIT- and triclosan-resistant strains. Another candidate, a 25-kDa peroxiredoxin enzyme involved in oxidative detoxification, was identified to be overexpressed in MIT-CMIT derivative. A similar profile was also observed among strains isolated from cosmetic products. Our study highlights the existence of adaptive mechanisms such as overexpression of detoxifying enzymes, flagellin, modification of membrane structure/function in Ent. gergoviae. They might be involved in recurrent episodes of contaminations occurring in the cosmetic production lines. No cross-resistance could be observed with antibiotics when MICs to preservatives were increased; however, a decrease in the disinfectants bactericidal effects was confirmed in preservative-tolerant strains. This will impact industry disinfection strategies treatment against bacteria. © 2014 The Society for Applied Microbiology.

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

Newer systems for bacterial resistances to toxic heavy metals.

PubMed Central

Silver, S; Ji, G

1994-01-01

Bacterial plasmids contain specific genes for resistances to toxic heavy metal ions including Ag+, AsO2-, AsO4(3-), Cd2+, Co2+, CrO4(2-), Cu2+, Hg2+, Ni2+, Pb2+, Sb3+, and Zn2+. Recent progress with plasmid copper-resistance systems in Escherichia coli and Pseudomonas syringae show a system of four gene products, an inner membrane protein (PcoD), an outer membrane protein (PcoB), and two periplasmic Cu(2+)-binding proteins (PcoA and PcoC). Synthesis of this system is governed by two regulatory proteins (the membrane sensor PcoS and the soluble responder PcoR, probably a DNA-binding protein), homologous to other bacterial two-component regulatory systems. Chromosomally encoded Cu2+ P-type ATPases have recently been recognized in Enterococcus hirae and these are closely homologous to the bacterial cadmium efflux ATPase and the human copper-deficiency disease Menkes gene product. The Cd(2+)-efflux ATPase of gram-positive bacteria is a large P-type ATPase, homologous to the muscle Ca2+ ATPase and the Na+/K+ ATPases of animals. The arsenic-resistance system of gram-negative bacteria functions as an oxyanion efflux ATPase for arsenite and presumably antimonite. However, the structure of the arsenic ATPase is fundamentally different from that of P-type ATPases. The absence of the arsA gene (for the ATPase subunit) in gram-positive bacteria raises questions of energy-coupling for arsenite efflux. The ArsC protein product of the arsenic-resistance operons of both gram-positive and gram-negative bacteria is an intracellular enzyme that reduces arsenate [As(V)] to arsenite [As(III)], the substrate for the transport pump. Newly studied cation efflux systems for Cd2+, Zn2+, and Co2+ (Czc) or Co2+ and Ni2+ resistance (Cnr) lack ATPase motifs in their predicted polypeptide sequences. Therefore, not all plasmid-resistance systems that function through toxic ion efflux are ATPases. The first well-defined bacterial metallothionein was found in the cyanobacterium Synechococcus. Bacterial metallothionein is encoded by the smtA gene and contains 56 amino acids, including nine cysteine residues (fewer than animal metallothioneins). The synthesis of Synechococcus metallothionein is regulated by a repressor protein, the product of the adjacent but separately transcribed smtB gene. Regulation of metallothionein synthesis occurs at different levels; quickly by derepression of repressor activity, or over a longer time by deletion of the repressor gene at fixed positions and by amplification of the metallothionein DNA region leading to multiple copies of the gene. PMID:7843081

New Verapamil Analogs Inhibit Intracellular Mycobacteria without Affecting the Functions of Mycobacterium-Specific T Cells

PubMed Central

Ruminiski, Peter G.; Kumar, Malkeet; Singh, Kawaljit; Hamzabegovic, Fahreta; Hoft, Daniel F.; Eickhoff, Christopher S.; Selimovic, Asmir; Campbell, Mary; Chibale, Kelly

2015-01-01

There is a growing interest in repurposing mycobacterial efflux pump inhibitors, such as verapamil, for tuberculosis (TB) treatment. To aid in the design of better analogs, we studied the effects of verapamil on macrophages and Mycobacterium tuberculosis-specific T cells. Macrophage activation was evaluated by measuring levels of nitric oxide, tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and gamma interferon (IFN-γ). Since verapamil is a known autophagy inducer, the roles of autophagy induction in the antimycobacterial activities of verapamil and norverapamil were studied using bone marrow-derived macrophages from ATG5flox/flox (control) and ATG5flox/flox Lyz-Cre mice. Our results showed that despite the well-recognized effects of verapamil on calcium channels and autophagy, its action on intracellular M. tuberculosis does not involve macrophage activation or autophagy induction. Next, the effects of verapamil and norverapamil on M. tuberculosis-specific T cells were assessed using flow cytometry following the stimulation of peripheral blood mononuclear cells from TB-skin-test-positive donors with M. tuberculosis whole-cell lysate for 7 days in the presence or absence of drugs. We found that verapamil and norverapamil inhibit the expansion of M. tuberculosis-specific T cells. Additionally, three new verapamil analogs were found to inhibit intracellular Mycobacterium bovis BCG, and one of the three analogs (KSV21) inhibited intracellular M. tuberculosis replication at concentrations that did not inhibit M. tuberculosis-specific T cell expansion. KSV21 also inhibited mycobacterial efflux pumps to the same degree as verapamil. More interestingly, the new analog enhances the inhibitory activities of isoniazid and rifampin on intracellular M. tuberculosis. In conclusion, KSV21 is a promising verapamil analog on which to base structure-activity relationship studies aimed at identifying more effective analogs. PMID:26643325

Improving olefin tolerance and production in E. coli using native and evolved AcrB

DOE PAGES

Mingardon, Florence; Clement, Camille; Hirano, Kathleen; ...

2015-01-20

Microorganisms can be engineered for the production of chemicals utilized in the polymer industry. However many such target compounds inhibit microbial growth and might correspondingly limit production levels. Here, we focus on compounds that are precursors to bioplastics, specifically styrene and representative alpha-olefins; 1-hexene, 1-octene, and 1-nonene. We evaluated the role of the Escherichia coli efflux pump, AcrAB-TolC, in enhancing tolerance towards these olefin compounds. AcrAB-TolC is involved in the tolerance towards all four compounds in E. coli. Both styrene and 1-hexene are highly toxic to E. coli. Styrene is a model plastics precursor with an established route for productionmore » in E. coli (McKenna and Nielsen, 2011). Though our data indicates that AcrAB-TolC is important for its optimal production, we observed a strong negative selection against the production of styrene in E. coli. Thus we used 1-hexene as a model compound to implement a directed evolution strategy to further improve the tolerance phenotype towards this alpha-olefin. We focused on optimization of AcrB, the inner membrane domain known to be responsible for substrate binding, and found several mutations (A279T, Q584R, F617L, L822P, F927S, and F1033Y) that resulted in improved tolerance. Several of these mutations could also be combined in a synergistic manner. Our study shows efflux pumps to be an important mechanism in host engineering for olefins, and one that can be further improved using strategies such as directed evolution, to increase tolerance and potentially production.« less

Tetracycline Resistance and Presence of Tetracycline Resistance Determinants tet(V) and tap in Rapidly Growing Mycobacteria from Agricultural Soils and Clinical Isolates

PubMed Central

Kyselková, Martina; Chron̂áková, Alica; Volná, Lucie; Nêmec, Jan; Ulmann, Vít; Scharfen, Josef; Elhottová, Dana

2012-01-01

Rapidly growing mycobacteria (RGM) inhabit soil and water but certain strains represent a health risk for human and animals. Both clinical and soil RGM may be under selection pressure for resistance to tetracycline (TET) antibiotics, since tetracyclines are administrated to humans and farm animals, and TET residues enter soil through manuring; however, resistance to TET and the presence of TET-resistance genes have been assessed only in clinical isolates. We were therefore interested in comparing soil and clinical RGM in terms of TET resistance and the presence of TET-resistance genes. We used 44 RGM from grasslands with different exposure to animal manure, and 38 clinical RGM from Czech hospitals. There was no difference between the clinical and soil isolates in TET resistance, with >50% resistant isolates in both groups. otr(A), otr(B), tet(K), tet(L) or tet(M) were not detected in any soil or clinical isolate. In contrast, most isolates harbored tet(V) and tap, both encoding mycobacterial efflux pumps, including species where these genes have never been evidenced before. The phylogeny of tet(V) correlated with isolates’ BOX-PCR profiles, suggesting that this gene evolved along with mycobacterial genomes as a part of the intrinsic resistome. In certain cases, tet(V) and/or tap were found in TET-sensitive isolates, or inversely, were not found in resistant strains. Concluding, intrinsic efflux pumps may be more important for TET resistance than horizontally transferred genes in both soil and clinical RGM. Their simple presence, however, does not attest to resistance, and therefore their diversity, function and expression merit further research. PMID:22673307

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

Barnes, Charles Ashley

In Chapter 2 several experimental and data analysis methods used in this thesis are described. In Chapter 3 steady-state fluorescence spectroscopy was used to determine the concentration of the efflux pump inhibitors (EPIs), pheophorbide a and pyropheophorbide a, in the feces of animals and it was found that their levels far exceed those reported to be inhibitory to efflux pumps. In Chapter 4 the solvation dynamics of 6-Propionyl-2-(N,Ndimethyl) aminonaphthalene (PRODAN) was studied in reverse micelles. The two fluorescent states of PRODAN solvate on different time scales and as such care must be exercised in solvation dynamic studies involving it andmore » its analogs. In Chapter 5 we studied the experimental and theoretical solvation dynamics of coumarin 153 (C153) in wild-type (WT) and modified myoglobins. Based on the nuclear magnetic resonance (NMR) spectroscopy and time-resolved fluorescence studies, we have concluded that it is important to thoroughly characterize the structure of a protein and probe system before comparing the theoretical and experimental results. In Chapter 6 the photophysical and spectral properties of a derivative of the medically relevant compound curcumin called cyclocurcumin was studied. Based on NMR, fluorescence, and absorption studies, the ground- and excited-states of cyclocurcumin are complicated by the existence of multiple structural isomers. In Chapter 7 the hydrolysis of cellulose by a pure form of cellulase in an ionic liquid, HEMA, and its aqueous mixtures at various temperatures were studied with the goal of increasing the cellulose to glucose conversion for biofuel production. It was found that HEMA imparts an additional stability to cellulase and can allow for faster conversion of cellulose to glucose using a pre-treatment step in comparison to only buffer.« less

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.

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

Prevalence and characteristics of quinolone resistance in Escherichia coli in veal calves.

PubMed

Hordijk, Joost; Veldman, Kees; Dierikx, Cindy; van Essen-Zandbergen, Alieda; Wagenaar, Jaap A; Mevius, Dik

2012-04-23

Quinolone resistance is studied and reported increasingly in isolates from humans, food-producing animals and companion animals. Resistance can be caused by chromosomal mutations in topoisomerase genes, plasmid-mediated resistance genes, and active transport through efflux pumps. Cross sectional data on quinolone resistance mechanisms in non-pathogenic bacteria from healthy veal calves is limited. The purpose of this study was to determine the prevalence and characteristics of quinolone resistance mechanisms in Escherichia coli isolates from veal calves, after more than 20 years of quinolone usage in veal calves. MIC values were determined for all isolates collected as part of a national surveillance program on antimicrobial resistance in commensal bacteria in food-producing animals in The Netherlands. From the strains collected from veal calves in 2007 (n=175) all isolates with ciprofloxacin MIC ≥ 0.125 mg/L (n=25) were selected for this study, and screened for the presence of known quinolone resistance determinants. In this selection only chromosomal mutations in the topoisomerase type II and IV genes were detected. The number of mutations found per isolate correlated with an increasing ciprofloxacin MIC. No plasmid-mediated quinolone resistance genes were found. The contribution of efflux pumps varied from no contribution to a 16-fold increase in susceptibility. No correlation was found with the presence of resistance genes of other antimicrobial classes, even though all quinolone non-wild type isolates were resistant to 3 or more classes of antibiotics other than quinolones. Over twenty years of quinolone usage in veal calves in The Netherlands did not result in a widespread occurrence of plasmid-mediated quinolone resistance, limiting the transmission of quinolone resistance to clonal distribution. Copyright © 2011 Elsevier B.V. All rights reserved.

Detection of drug-resistance mechanism of Pseudomonas aeruginosa developing from a sensitive strain to a persister during carbapenem treatment.

PubMed

Shen, J L; Fang, Y P

2015-06-18

We explored the mechanism of the development from sensitivity to resistance to carbapenem in Pseudomonas aeruginosa. Two P. aeruginosa strains were collected during treatment with carbapenem. Strain homology was investigated using pulsed-field gel electrophoresis. Porin oprD2 expression was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The minimum inhibitory concentrations (MICs) of imipenem and meropenem with or without MC207110 were determined using the agar dilution method. The expression level of efflux pump mRNA was tested using real-time polymerase chain reaction. Metallo-lactamases (MBLs) were screened using the EDTA-disk synergy test. Genes encoding MBLs were amplified and then analyzed by DNA sequencing. The two treated strains belonged to the same pulsed-field gel electrophoresis type. The SDS-PAGE profile of the P. aeruginosa strains revealed that the 46-kDa membrane protein OprD2 of IMP(R)MEM(R) type strains was lost, whereas OprD2 of 1 IMP(S)MEM(S) strain was normal. With or without MC207110 treatment, the MIC of carbapenem-resistant P. aeruginosa decreased by 4-fold, while the MIC of carbapenem-sensitive P. aeruginosa did not. Compared with the carbapenem-sensitive strain, MexX mRNA expression in the carbapenem-resistant strain increased by 102.5-fold, while the mRNA expression of other efflux pumps did not markedly increase. Neither carbapenem-resistant nor carbapenem-sensitive P. aeruginosa produced MBL. The mechanism of development from sensitivity to resistance of P. aeruginosa to carbapenem during carbapenem treatment is due to porin oprD2 loss and an increased expression level of MexXY-OprM.

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

PubMed Central

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

2014-01-01

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