scholarly journals The assembled structure of a complete tripartite bacterial multidrug efflux pump

2009 ◽  
Vol 106 (17) ◽  
pp. 7173-7178 ◽  
Author(s):  
Martyn F. Symmons ◽  
Evert Bokma ◽  
Eva Koronakis ◽  
Colin Hughes ◽  
Vassilis Koronakis
Keyword(s):  
Efflux Pump ◽  
Cell Envelope ◽  
Adaptor Protein ◽  
Drug Binding ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Inner Membrane ◽  
Multidrug Efflux Pumps ◽  
Docking Approach

Bacteria likeEscherichia coliandPseudomonas aeruginosaexpel drugs via tripartite multidrug efflux pumps spanning both inner and outer membranes and the intervening periplasm. In these pumps a periplasmic adaptor protein connects a substrate-binding inner membrane transporter to an outer membrane-anchored TolC-type exit duct. High-resolution structures of all 3 components are available, but a pump model has been precluded by the incomplete adaptor structure, because of the apparent disorder of its N and C termini. We reveal that the adaptor termini assemble a β-roll structure forming the final domain adjacent to the inner membrane. The completed structure enabled in vivo cross-linking to map intermolecular contacts between the adaptor AcrA and the transporter AcrB, defining a periplasmic interface between several transporter subdomains and the contiguous β-roll, β-barrel, and lipoyl domains of the adaptor. With short and long cross-links expressed as distance restraints, the flexible linear topology of the adaptor allowed a multidomain docking approach to model the transporter–adaptor complex, revealing that the adaptor docks to a transporter region of comparative stability distinct from those key to the proposed rotatory pump mechanism, putative drug-binding pockets, and the binding site of inhibitory DARPins. Finally, we combined this docking with our previous resolution of the AcrA hairpin–TolC interaction to develop a model of the assembled tripartite complex, satisfying all of the experimentally-derived distance constraints. This AcrA3-AcrB3-TolC3model presents a 610,000-Da, 270-Å-long efflux pump crossing the entire bacterial cell envelope.

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

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Zhao Wang ◽  
Guizhen Fan ◽  
Corey F Hryc ◽  
James N Blaza ◽  
Irina I Serysheva ◽  
...  
Keyword(s):  
Transport Mechanism ◽  
Drug Transport ◽  
Efflux Pump ◽  
Cell Envelope ◽  
Gram Negative Bacteria ◽  
Multidrug Efflux ◽  
Protein Assemblies ◽  
Multidrug Efflux Pump ◽  
Channel Opening ◽  
Activated State

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.

scholarly journals Control of Biofilm Formation in Healthcare: Recent Advances Exploiting Quorum-Sensing Interference Strategies and Multidrug Efflux Pump Inhibitors

Materials ◽  
2018 ◽  
Vol 11 (9) ◽  
pp. 1676 ◽  
Author(s):  
Bindu Subhadra ◽  
Dong Kim ◽  
Kyungho Woo ◽  
Surya Surendran ◽  
Chul Choi
Keyword(s):  
Quorum Sensing ◽  
Biofilm Formation ◽  
Efflux Pump ◽  
Financial Burden ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Persistent Infections ◽  
Healthcare Settings ◽  
Recent Advances ◽  
Multidrug Efflux Pumps

Biofilm formation in healthcare is an issue of considerable concern, as it results in increased morbidity and mortality, imposing a significant financial burden on the healthcare system. Biofilms are highly resistant to conventional antimicrobial therapies and lead to persistent infections. Hence, there is a high demand for novel strategies other than conventional antibiotic therapies to control biofilm-based infections. There are two approaches which have been employed so far to control biofilm formation in healthcare settings: one is the development of biofilm inhibitors based on the understanding of the molecular mechanism of biofilm formation, and the other is to modify the biomaterials which are used in medical devices to prevent biofilm formation. This review will focus on the recent advances in anti-biofilm approaches by interrupting the quorum-sensing cellular communication system and the multidrug efflux pumps which play an important role in biofilm formation. Research efforts directed towards these promising strategies could eventually lead to the development of better anti-biofilm therapies than the conventional treatments.

scholarly journals Dehydrozingerone Exhibits Synergistic Antifungal Activities in Combination with Dodecanol against Budding Yeast via the Restriction of Multidrug Resistance

2018 ◽  
Vol 5 (02) ◽  
pp. e61-e67
Author(s):  
Chika Yamawaki ◽  
Yoshihiro Yamaguchi ◽  
Akira Ogita ◽  
Toshio Tanaka ◽  
Ken-ichi Fujita
Keyword(s):  
Drug Resistance ◽  
Antifungal Activity ◽  
Efflux Pump ◽  
Fungal Infections ◽  
Zingiber Officinale ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Yeast Saccharomyces Cerevisiae ◽  
Multidrug Transporters ◽  
Multidrug Efflux Pumps

AbstractDrug resistance in fungal infections has been a more frequent occurrence with the increasing number of immunocompromised patients. In efforts to overcome the problem of fungal drug resistance, we focused on the phenolic compound dehydrozingerone, which is isolated from Zingiber officinale. The effectiveness of this compound on the model yeast Saccharomyces cerevisiae has not been reported. In our study, dehydrozingerone showed a weak antifungal activity against the yeast, but demonstrated a synergistic effect in combination with dodecanol, which typically only restricts cell growth transiently. Efflux of rhodamine 6G through the multidrug efflux pumps was significantly restricted by dehydrozingerone. The transcription level of PDR5, encoding a primary multidrug efflux pump in S. cerevisiae, was enhanced with dodecanol treatment, whereas the level was reduced by dehydrozingerone. These results suggest that dehydrozingerone may be effective for potentiating antifungal activity of other drugs that are expelled from fungi by multidrug transporters like Pdr5p.

scholarly journals CmeR Functions as a Pleiotropic Regulator and Is Required for Optimal Colonization of Campylobacter jejuni In Vivo

2008 ◽  
Vol 190 (6) ◽  
pp. 1879-1890 ◽  
Author(s):  
Baoqing Guo ◽  
Ying Wang ◽  
Feng Shi ◽  
Yi-Wen Barton ◽  
Paul Plummer ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Capsular Polysaccharide ◽  
Efflux Pump ◽  
Loss Of Function ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Reverse Transcription Pcr ◽  
Dicarboxylate Transport

ABSTRACT CmeR functions as a transcriptional repressor modulating the expression of the multidrug efflux pump CmeABC in Campylobacter jejuni. To determine if CmeR also regulates other genes in C. jejuni, we compared the transcriptome of the cmeR mutant with that of the wild-type strain using a DNA microarray. This comparison identified 28 genes that showed a ≥2-fold change in expression in the cmeR mutant. Independent real-time quantitative reverse transcription-PCR experiments confirmed 27 of the 28 differentially expressed genes. The CmeR-regulated genes encode membrane transporters, proteins involved in C4-dicarboxylate transport and utilization, enzymes for biosynthesis of capsular polysaccharide, and hypothetical proteins with unknown functions. Among the genes whose expression was upregulated in the cmeR mutant, Cj0561c (encoding a putative periplasmic protein) showed the greatest increase in expression. Subsequent experiments demonstrated that this gene is strongly repressed by CmeR. The presence of the known CmeR-binding site, an inverted repeat of TGTAAT, in the promoter region of Cj0561c suggests that CmeR directly inhibits the transcription of Cj0561c. Similar to expression of cmeABC, transcription of Cj0561c is strongly induced by bile compounds, which are normally present in the intestinal tracts of animals. Inactivation of Cj0561c did not affect the susceptibility of C. jejuni to antimicrobial compounds in vitro but reduced the fitness of C. jejuni in chickens. Loss-of-function mutation of cmeR severely reduced the ability of C. jejuni to colonize chickens. Together, these findings indicate that CmeR governs the expression of multiple genes with diverse functions and is required for Campylobacter adaptation in the chicken host.

scholarly journals Functional Cloning and Characterization of a Multidrug Efflux Pump, MexHI-OpmD, from a Pseudomonas aeruginosa Mutant

2003 ◽  
Vol 47 (9) ◽  
pp. 2990-2992 ◽  
Author(s):  
Hiroshi Sekiya ◽  
Takehiko Mima ◽  
Yuji Morita ◽  
Teruo Kuroda ◽  
Tohru Mizushima ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Ethidium Bromide ◽  
Rhodamine 6G ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Multidrug Efflux ◽  
Chromosomal Dna ◽  
Multidrug Efflux Pump ◽  
Multidrug Efflux Pumps

ABSTRACT We isolated mutant YM644, which showed elevated resistance to norfloxacin, ethidium bromide, acriflavine, and rhodamine 6G, from Pseudomonas aeruginosa YM64, a strain that lacks four major multidrug efflux pumps. The genes responsible for the resistance were mexHI-opmD. Elevated ethidium extrusion was observed with cells of YM644 and YM64 harboring a plasmid carrying the genes. Disruption of the genes in the chromosomal DNA of YM644 made the cells sensitive to the drugs.

Patupilone (epothilone B) inhibits growth and survival of multiple myeloma cells in vitro and in vivo

Blood ◽  
2005 ◽  
Vol 105 (1) ◽  
pp. 350-357 ◽  
Author(s):  
Boris Lin ◽  
Laurence Catley ◽  
Richard LeBlanc ◽  
Constantine Mitsiades ◽  
Renate Burger ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Growth Factor ◽  
Efflux Pump ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Growth And Survival ◽  
Epothilone B ◽  
Endothelial Growth Factor

Abstract In this study, we investigated the in vitro and in vivo efficacy of patupilone (epothilone B, EPO906), a novel nontaxane microtubule stabilizing agent, in treatment of multiple myeloma (MM). Patupilone directly inhibited growth and survival of MM cells, including those resistant to conventional chemotherapies, such as the taxane paclitaxel. Patupilone induced G2M arrest of MM cells, with subsequent apoptosis. Interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1), 2 known growth and survival factors for MM, did not protect MM.1S cells against patupilone-induced cell death. Proliferation of MM cells induced by adherence to bone marrow stromal cells (BMSCs) was also inhibited by patupilone and was paralleled by down-regulation of vascular endothelial growth factor (VEGF) secretion. Importantly, stimulation of cells from patients with MM, either with IL-6 or by adherence to BMSCs, enhanced the anti-proliferative and proapoptotic effects of patupilone. Moreover, patupilone was effective against MM cell lines that overexpress the MDR1/P-glycoprotein multidrug efflux pump. In addition, patupilone was effective in slowing tumor growth and prolonging median survival of mice that received orthotopical transplants with MM tumor cells. Taken together, these preclinical findings suggest that patupilone may be a safe and effective drug in the treatment of MM, providing the framework for clinical studies to improve patient outcome in MM. (Blood. 2005;105:350-357)

scholarly journals Multiresistance Genes of Rhizobium etli CFN42

2000 ◽  
Vol 13 (5) ◽  
pp. 572-577 ◽  
Author(s):  
Ramón González-Pasayo ◽  
Esperanza Martínez-Romero
Keyword(s):  
Wild Type Strain ◽  
Efflux Pump ◽  
Rhizobium Etli ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Enhanced Sensitivity ◽  
Multidrug Efflux Pumps ◽  
High Concentrations ◽  
Major Facilitator

Multidrug efflux pumps of bacteria are involved in the resistance to various antibiotics and toxic compounds. In Rhizobium etli, a mutualistic symbiont of Phaseolus vulgaris (bean), genes resembling multidrug efflux pump genes were identified and designated rmrA and rmrB. rmrA was obtained after the screening of transposon-generated fusions that are inducible by bean-root released flavonoids. The predicted gene products of rmrAB shared significant homology to membrane fusion and major facilitator proteins, respectively. Mutants of rmrA formed on average 40% less nodules in bean, while mutants of rmrA and rmrB had enhanced sensitivity to phytoalexins, flavonoids, and salicylic acid, compared with the wild-type strain. Multidrug resistance genes emrAB from Escherichia coli complemented an rmrA mutant from R. etli for resistance to high concentrations of naringenin.

scholarly journals Structural, Biochemical, and In Vivo Characterization of MtrR-Mediated Resistance to Innate Antimicrobials by the Human Pathogen Neisseria gonorrhoeae

2019 ◽  
Vol 201 (20) ◽  
Author(s):  
Grace A. Beggs ◽  
Yaramah M. Zalucki ◽  
Nicholas Gene Brown ◽  
Sheila Rastegari ◽  
Rebecca K. Phillips ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Bile Salts ◽  
Efflux Pump ◽  
Binding Modes ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Content Type ◽  
Multiple Drugs

ABSTRACT Neisseria gonorrhoeae responds to host-derived antimicrobials by inducing the expression of the mtrCDE-encoded multidrug efflux pump, which expels microbicides, such as bile salts, fatty acids, and multiple extrinsically administered drugs, from the cell. In the absence of these cytotoxins, the TetR family member MtrR represses the mtrCDE genes. Although antimicrobial-dependent derepression of mtrCDE is clear, the physiological inducers of MtrR are unknown. Here, we report the crystal structure of an induced form of MtrR. In the binding pocket of MtrR, we observed electron density that we hypothesized was N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), a component of the crystallization reagent. Using the MtrR-CAPS structure as an inducer-bound template, we hypothesized that bile salts, which bear significant chemical resemblance to CAPS, are physiologically relevant inducers. Indeed, characterization of MtrR-chenodeoxycholate and MtrR-taurodeoxycholate interactions, both in vitro and in vivo, revealed that these bile salts, but not glyocholate or taurocholate, bind MtrR tightly and can act as bona fide inducers. Furthermore, two residues, W136 and R176, were shown to be important in binding chenodeoxycholate but not taurodeoxycholate, suggesting different binding modes of the bile salts. These data provide insight into a crucial mechanism utilized by the pathogen to overcome innate human defenses. IMPORTANCE Neisseria gonorrhoeae causes a significant disease burden worldwide, and a meteoric rise in its multidrug resistance has reduced the efficacy of antibiotics previously or currently approved for therapy of gonorrheal infections. The multidrug efflux pump MtrCDE transports multiple drugs and host-derived antimicrobials from the bacterial cell and confers survival advantage on the pathogen within the host. Transcription of the pump is repressed by MtrR but relieved by the cytosolic influx of antimicrobials. Here, we describe the structure of induced MtrR and use this structure to identify bile salts as physiological inducers of MtrR. These findings provide a mechanistic basis for antimicrobial sensing and gonococcal protection by MtrR through the derepression of mtrCDE expression after exposure to intrinsic and clinically applied antimicrobials.

scholarly journals CmeABC Functions as a Multidrug Efflux System in Campylobacter jejuni

2002 ◽  
Vol 46 (7) ◽  
pp. 2124-2131 ◽  
Author(s):  
Jun Lin ◽  
Linda Overbye Michel ◽  
Qijing Zhang
Keyword(s):  
Campylobacter Jejuni ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Intrinsic Resistance ◽  
Multidrug Efflux Pump ◽  
Efflux Pump Inhibitor ◽  
Gram Negative ◽  
Multidrug Efflux Pumps

ABSTRACT Campylobacter jejuni, a gram-negative organism causing gastroenteritis in humans, is increasingly resistant to antibiotics. However, little is known about the drug efflux mechanisms in this pathogen. Here we characterized an efflux pump encoded by a three-gene operon (designated cmeABC) that contributes to multidrug resistance in C. jejuni 81-176. CmeABC shares significant sequence and structural homology with known tripartite multidrug efflux pumps in other gram-negative bacteria, and it consists of a periplasmic fusion protein (CmeA), an inner membrane efflux transporter belonging to the resistance-nodulation-cell division superfamily (CmeB), and an outer membrane protein (CmeC). Immunoblotting using CmeABC-specific antibodies demonstrated that cmeABC was expressed in wild-type 81-176; however, an isogenic mutant (9B6) with a transposon insertion in the cmeB gene showed impaired production of CmeB and CmeC. Compared to wild-type 81-176, 9B6 showed a 2- to 4,000-fold decrease in resistance to a range of antibiotics, heavy metals, bile salts, and other antimicrobial agents. Accumulation assays demonstrated that significantly more ethidium bromide and ciprofloxacin accumulated in mutant 9B6 than in wild-type 81-176. Addition of carbonyl cyanide m-chlorophenylhydrazone, an efflux pump inhibitor, increased the accumulation of ciprofloxacin in wild-type 81-176 to the level of mutant 9B6. PCR and immunoblotting analysis also showed that cmeABC was broadly distributed in various C. jejuni isolates and constitutively expressed in wild-type strains. Together, these findings formally establish that CmeABC functions as a tripartite multidrug efflux pump that contributes to the intrinsic resistance of C. jejuni to a broad range of structurally unrelated antimicrobial agents.

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