The pseudo-atomic structure of an RND-type tripartite multidrug efflux pump

2015 ◽  
Vol 396 (9-10) ◽  
pp. 1073-1082 ◽  
Author(s):  
Dijun Du ◽  
Jarrod Voss ◽  
Zhao Wang ◽  
Wah Chiu ◽  
Ben F. Luisi
Keyword(s):  
Antimicrobial Agents ◽  
Transmembrane Domain ◽  
Efflux Pump ◽  
Cell Envelope ◽  
Toxic Substances ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Transporter Family ◽  
Wide Range ◽  
Rnd Transporter

Abstract Microorganisms encode several classes of transmembrane molecular pumps that can expel a wide range of chemically distinct toxic substances. These machines contribute to the capacity of the organisms to withstand harsh environments, and they help to confer resistance against clinical antimicrobial agents. In Gram-negative bacteria, some of the pumps comprise tripartite assemblies that actively transport drugs and other harmful compounds across the cell envelope. We describe recent structural and functional data that have provided insights into the architecture and transport mechanism of the AcrA-AcrB-TolC pump of Escherichia coli. This multidrug efflux pump is powered by proton electrochemical gradients through the activity of AcrB, a member of the resistance/nodulation/cell division (RND) transporter family. Crystallographic data reveal how the small protein AcrZ binds to AcrB in a concave surface of the transmembrane domain, and we discuss how this interaction may affect the efflux activities of the transporter.

scholarly journals BpeAB-OprB, a Multidrug Efflux Pump in Burkholderia pseudomallei

2004 ◽  
Vol 48 (4) ◽  
pp. 1128-1135 ◽  
Author(s):  
Y. Y. Chan ◽  
T. M. C. Tan ◽  
Y. M. Ong ◽  
K. L. Chua
Keyword(s):  
Membrane Protein ◽  
Burkholderia Pseudomallei ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Efflux Pump Inhibitor ◽  
Membrane Fusion Protein ◽  
Wide Range ◽  
Inner Membrane Protein

ABSTRACT Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to a wide range of antimicrobial agents, including β-lactams, aminoglycosides, macrolides, and polymyxins. An operon, bpeR-bpeA-bpeB-oprB, which encodes a putative repressor, a membrane fusion protein, an inner membrane protein, and an outer membrane protein, respectively, of a multidrug efflux pump of the resistance-nodulation-division family was identified in B. pseudomallei. The divergently transcribed bpeR gene encodes a putative repressor protein of the TetR family which probably regulates the expression of the bpeAB-oprB gene cluster. Comparison of the MICs and minimal bactericidal concentrations of antimicrobials for bpeAB deletion mutant KHWΔbpeAB and its isogenic wild-type parent, KHW, showed that the B. pseudomallei BpeAB-OprB pump is responsible for the efflux of the aminoglycosides gentamicin and streptomycin, the macrolide erythromycin, and the dye acriflavine. Antibiotic efflux by the BpeAB-OprB pump was dependent on a proton gradient and differs from that by the AmrAB-OprA pump in that it did not efflux the aminoglycoside spectinomycin or the macrolide clarithromycin. The broad-spectrum efflux pump inhibitor MC-207,110 did not potentiate the effectiveness of the antimicrobials erythromycin and streptomycin in B. pseudomallei.

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 Cryo-EM Structures of a Gonococcal Multidrug Efflux Pump Illuminate a Mechanism of Drug Recognition and Resistance

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Meinan Lyu ◽  
Mitchell A. Moseng ◽  
Jennifer L. Reimche ◽  
Concerta L. Holley ◽  
Vijaya Dhulipala ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Drug Resistance ◽  
Neisseria Gonorrhoeae ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Transmitted Infection ◽  
Content Type ◽  
Cryo Electron Microscopy

ABSTRACT Neisseria gonorrhoeae is an obligate human pathogen and causative agent of the sexually transmitted infection (STI) gonorrhea. The most predominant and clinically important multidrug efflux system in N. gonorrhoeae is the multiple transferrable resistance (Mtr) pump, which mediates resistance to a number of different classes of structurally diverse antimicrobial agents, including clinically used antibiotics (e.g., β-lactams and macrolides), dyes, detergents and host-derived antimicrobials (e.g., cationic antimicrobial peptides and bile salts). Recently, it has been found that gonococci bearing mosaic-like sequences within the mtrD gene can result in amino acid changes that increase the MtrD multidrug efflux pump activity, probably by influencing antimicrobial recognition and/or extrusion to elevate the level of antibiotic resistance. Here, we report drug-bound solution structures of the MtrD multidrug efflux pump carrying a mosaic-like sequence using single-particle cryo-electron microscopy, with the antibiotics bound deeply inside the periplasmic domain of the pump. Through this structural approach coupled with genetic studies, we identify critical amino acids that are important for drug resistance and propose a mechanism for proton translocation. IMPORTANCE Neisseria gonorrhoeae has become a highly antimicrobial-resistant Gram-negative pathogen. Multidrug efflux is a major mechanism that N. gonorrhoeae uses to counteract the action of multiple classes of antibiotics. It appears that gonococci bearing mosaic-like sequences within the gene mtrD, encoding the most predominant and clinically important transporter of any gonococcal multidrug efflux pump, significantly elevate drug resistance and enhance transport function. Here, we report cryo-electron microscopy (EM) structures of N. gonorrhoeae MtrD carrying a mosaic-like sequence that allow us to understand the mechanism of drug recognition. Our work will ultimately inform structure-guided drug design for inhibiting these critical multidrug efflux pumps.

scholarly journals EfrAB, an ABC Multidrug Efflux Pump in Enterococcus faecalis

2003 ◽  
Vol 47 (12) ◽  
pp. 3733-3738 ◽  
Author(s):  
Eun-Woo Lee ◽  
M. Nazmul Huda ◽  
Teruo Kuroda ◽  
Tohru Mizushima ◽  
Tomofusa Tsuchiya
Keyword(s):  
Enterococcus Faecalis ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Multidrug Efflux ◽  
Chromosomal Dna ◽  
Multidrug Efflux Pump ◽  
E Coli ◽  
Dna Fragment

ABSTRACT A DNA fragment responsible for resistance to antimicrobial agents was cloned from the chromosomal DNA of Enterococcus faecalis ATCC 29212 by using drug-hypersensitive mutant Escherichia coli KAM32 as a host cell. Cells of E. coli KAM32 harboring a recombinant plasmid (pAEF82) carrying the DNA fragment became resistant to many structurally unrelated antimicrobial agents, such as norfloxacin, ciprofloxacin, doxycycline, acriflavine, 4′,6-diamidino-2-phenylindole, tetraphenylphosphonium chloride, daunorubicin, and doxorubicin. Since the sequence of the whole genome of E. faecalis is known, we sequenced several portions of the DNA insert in plasmid pAEF82 and identified two open reading frames within the insert. We designated the genes efrA and efrB. A search of the deduced amino acid sequences of EfrA and EfrB revealed that they are similar to each other and that they belong to the ATP-binding cassette (ABC) family of multidrug efflux transporters. Transformed E. coli KAM32 cells harboring efrAB showed energy-dependent efflux of acriflavine. The efflux activity was inhibited by reserpine, verapamil, and sodium-o-vanadate, known inhibitors of ABC efflux pumps.

scholarly journals SmdAB, a Heterodimeric ABC-Type Multidrug Efflux Pump, in Serratia marcescens

2007 ◽  
Vol 190 (2) ◽  
pp. 648-654 ◽  
Author(s):  
Taira Matsuo ◽  
Jing Chen ◽  
Yusuke Minato ◽  
Wakano Ogawa ◽  
Tohru Mizushima ◽  
...  
Keyword(s):  
Serratia Marcescens ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Amino Acid Sequences ◽  
Multidrug Efflux ◽  
Chromosomal Dna ◽  
Atpase Inhibitor ◽  
Multidrug Efflux Pump ◽  
Hoechst 33342 ◽  
E Coli

ABSTRACT We cloned genes, designated smdAB, that encode a multidrug efflux pump from the chromosomal DNA of clinically isolated Serratia marcescens NUSM8906. For cells of the drug-hypersensitive strain Escherichia coli KAM32 harboring a recombinant plasmid carrying smdAB, structurally unrelated antimicrobial agents such as norfloxacin, tetracycline, 4′,6-diamidino-2-phenylindole (DAPI), and Hoechst 33342 showed elevated MICs. The deduced amino acid sequences of both SmdA and SmdB exhibited similarities to the sequences of ATP-binding cassette (ABC)-type multidrug efflux pumps. The efflux of DAPI and Hoechst 33342 from E. coli cells expressing SmdAB was observed, and the efflux activities were inhibited by sodium o-vanadate, which is a well-known ATPase inhibitor. The introduction of smdA or smdB alone into E. coli KAM32 did not elevate the MIC of DAPI; thus, both SmdA and SmdB were required for function. These results indicate that SmdAB is probably a heterodimeric multidrug efflux pump of the ABC family in S. marcescens.

scholarly journals NorM of Vibrio parahaemolyticus Is an Na+-Driven Multidrug Efflux Pump

2000 ◽  
Vol 182 (23) ◽  
pp. 6694-6697 ◽  
Author(s):  
Yuji Morita ◽  
Atsuko Kataoka ◽  
Sumiko Shiota ◽  
Tohru Mizushima ◽  
Tomofusa Tsuchiya
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Efflux Pump ◽  
Sequence Similarity ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Significant Sequence Similarity ◽  
Wide Range ◽  
New Type ◽  
Biological World ◽  
Energy Dependent

ABSTRACT NorM of Vibrio parahaemolyticusapparently is a new type of multidrug efflux protein, with no significant sequence similarity to any known transport proteins. Based on the following experimental results, we conclude that NorM is an Na+-driven Na+/drug antiporter. (i) Energy-dependent ethidium efflux from cells possessing NorM was observed in the presence of Na+ but not of K+. (ii) An artificially imposed, inwardly directed Na+gradient elicited ethidium efflux from cells. (iii) The addition of ethidium to cells loaded with Na+ elicited Na+efflux. Thus, NorM is an Na+/drug antiporting multidrug efflux pump, the first to be found in the biological world. Judging from the similarity of the NorM sequence to those of putative proteins in sequence databases, it seems that Na+/drug antiporters are present not only in V. parahaemolyticus but also in a wide range of other organisms.

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.

scholarly journals Role of VltAB, an ABC Transporter Complex, in Viologen Tolerance inStreptococcus mutans

2011 ◽  
Vol 55 (4) ◽  
pp. 1460-1469 ◽  
Author(s):  
Saswati Biswas ◽  
Indranil Biswas
Keyword(s):  
Abc Transporter ◽  
Efflux Pump ◽  
Ammonium Compound ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Antibiotic Resistance Marker ◽  
Gram Positive ◽  
Wide Range ◽  
Insertion Mutants

ABSTRACTStreptococcus mutans, a Gram-positive organism, is the primary causative agent in the formation of dental caries in humans. To persist in the oral cavity,S. mutansmust be able to tolerate rapid environmental fluctuations and exposure to various toxic chemicals. However, the mechanisms underlying the ability of this cariogenic pathogen to survive and proliferate under harsh environmental conditions remain largely unknown. Here, we wanted to understand the mechanisms by whichS. mutanswithstands exposure to methyl viologen (MV), a quaternary ammonium compound (QAC) that generates superoxide radicals in the cell. To elucidate the essential genes for MV tolerance, screening of ∼3,500 mutants generated by ISS1mutagenesis, revealed 15 MV-sensitive mutants. Among them, five and four independent insertions had occurred in SMU.905 and SMU.906 genes, respectively. These two genes are appeared to be organized in an operon and encode a putative ABC transporter complex; we designated the genes asvltAandvltB, forviologentransporter. To verify our results,vltAwas deleted by using an antibiotic resistance marker; the mutant was just as sensitive to MV as the ISS1insertion mutants. Furthermore,vltAandvltBmutants were also sensitive to other viologen compounds such as benzyl and ethyl viologens. Complementation assays were also carried out to confirm the role of VltA and VltB in viologen tolerance. Sensitivity to various drugs, including a wide range of QACs, was evaluated. It appears that a functional VltA is also required for full resistance toward acriflavin, ethidium bromide, and safranin; all are well-known QACs. These results indicate that VltA/B constitute a heterodimeric multidrug efflux pump of the ABC family. BLAST-P analysis suggests that homologs of VltA/B are widely present in streptococci, enterococci, and other important Gram-positive pathogens.

scholarly journals Cross-Linked Complex between Oligomeric Periplasmic Lipoprotein AcrA and the Inner-Membrane-Associated Multidrug Efflux Pump AcrB from Escherichia coli

2000 ◽  
Vol 182 (15) ◽  
pp. 4264-4267 ◽  
Author(s):  
Helen I. Zgurskaya ◽  
Hiroshi Nikaido
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Intact Cells ◽  
Inner Membrane ◽  
Oligomeric Form ◽  
Chemical Cross Linking ◽  
Multidrug Efflux System

ABSTRACT In Escherichia coli, the intrinsic levels of resistance to multiple antimicrobial agents are produced through expression of the three-component multidrug efflux system AcrAB-TolC. AcrB is a proton-motive-force-dependent transporter located in the inner membrane, and AcrA and TolC are accessory proteins located in the periplasm and the outer membrane, respectively. In this study, these three proteins were expressed separately, and the interactions between them were analyzed by chemical cross-linking in intact cells. We show that AcrA protein forms oligomers, most probably trimers. In this oligomeric form, AcrA interacts specifically with AcrB transporter independently of substrate and TolC.

scholarly journals Covalently Linked Trimer of the AcrB Multidrug Efflux Pump Provides Support for the Functional Rotating Mechanism

2008 ◽  
Vol 191 (6) ◽  
pp. 1729-1737 ◽  
Author(s):  
Yumiko Takatsuka ◽  
Hiroshi Nikaido
Keyword(s):  
Conformational Changes ◽  
Transmembrane Domain ◽  
Efflux Pump ◽  
Polyacrylamide Gel Electrophoresis ◽  
Residual Activity ◽  
Salt Bridge ◽  
Relay Network ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Intact Cells

ABSTRACT Escherichia coli AcrB is a proton motive force-dependent multidrug efflux transporter that recognizes multiple toxic chemicals having diverse structures. Recent crystallographic studies of the asymmetric trimer of AcrB suggest that each protomer in the trimeric assembly goes through a cycle of conformational changes during drug export (functional rotation hypothesis). In this study, we devised a way to test this hypothesis by creating a giant gene in which three acrB sequences were connected together through short linker sequences. The “linked-trimer” AcrB was expressed well in the inner membrane fraction of ΔacrB ΔrecA strains, as a large protein of ∼300 kDa which migrated at the same rate as the wild-type AcrB trimer in native polyacrylamide gel electrophoresis. The strain expressing the linked-trimer AcrB showed resistance to some toxic compounds that was sometimes even higher than that of the cells expressing the monomeric AcrB, indicating that the linked trimer functions well in intact cells. When we inactivated only one of the three protomeric units in the linked trimer, either with mutations in the salt bridge/H-bonding network (proton relay network) in the transmembrane domain or by disulfide cross-linking of the external cleft in the periplasmic domain, the entire trimeric complex was inactivated. However, some residual activity was seen, presumably as a result of random recombination of monomeric fragments (produced by protease cleavage or by transcriptional/translational truncation). These observations provide strong biochemical evidence for the functionally rotating mechanism of AcrB pump action. The linked trimer will be useful for further biochemical studies of mechanisms of transport in the future.

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