Funnel-like Hexameric Assembly of the Periplasmic Adapter Protein in the Tripartite Multidrug Efflux Pump in Gram-negative Bacteria

SkQ1, a novel antibiotic targeting bacterial bioenergetics, is highly effective against both gram-positive and gram-negative bacteria. However, some gram-negative bacteria, such as Escherichia coli and Klebsiella pneumoniae, are highly resistant to it. In different gram-negative bacteria, this resistance is associated with the identity of their AcrB transporter protein sequence with the sequence of the AcrB protein from E. coli. SkQ1 is expelled from E. coli cells by the AcrAB-TolC multidrug efflux pump. In this study, we demonstrate that SkQ1 resistance in E. coli, in contrast to chloramphenicol resistance, does not depend on the presence of the multidrug efflux pump accessory protein AcrZ.

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The assembly and disassembly of the AcrAB-TolC three-component multidrug efflux pump

Biological Chemistry ◽

10.1515/hsz-2015-0150 ◽

2015 ◽

Vol 396 (9-10) ◽

pp. 1083-1089 ◽

Cited By ~ 12

Author(s):

Reinke Tobias Müller ◽

Klaas Martinus Pos

Keyword(s):

Escherichia Coli ◽

Efflux Pump ◽

Gram Negative Bacteria ◽

Electrochemical Gradient ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Gram Negative ◽

E Coli ◽

Pump Assembly ◽

Multiple Antibiotics

Abstract In Gram-negative bacteria, tripartite efflux pumps, like AcrAB-TolC from Escherichia coli, play a prominent role in the resistance against multiple antibiotics. Transport of the drugs across the outer membrane and its coupling to the electrochemical gradient is dependent on the presence of all three components. As the activity of the E. coli AcrAB-TolC efflux pump is dependent on both the concentration of substrates and the extent of the electrochemical gradient across the inner membrane, the dynamics of tripartite pump assembly and disassembly might be crucial for effective net transport of drugs towards the outside of the cell.

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Molecular Insights into an Antibiotic Enhancer Action of New Morpholine-Containing 5-Arylideneimidazolones in the Fight against MDR Bacteria

International Journal of Molecular Sciences ◽

10.3390/ijms22042062 ◽

2021 ◽

Vol 22 (4) ◽

pp. 2062

Author(s):

Aneta Kaczor ◽

Karolina Witek ◽

Sabina Podlewska ◽

Veronique Sinou ◽

Joanna Czekajewska ◽

...

Keyword(s):

Molecular Modelling ◽

Efflux Pump ◽

Gram Negative Bacteria ◽

Potential Mechanism ◽

Multidrug Efflux ◽

Aromatic Rings ◽

Allosteric Site ◽

Multidrug Efflux Pump ◽

Dual Action

In the search for an effective strategy to overcome antimicrobial resistance, a series of new morpholine-containing 5-arylideneimidazolones differing within either the amine moiety or at position five of imidazolones was explored as potential antibiotic adjuvants against Gram-positive and Gram-negative bacteria. Compounds (7–23) were tested for oxacillin adjuvant properties in the Methicillin-susceptible S. aureus (MSSA) strain ATCC 25923 and Methicillin-resistant S. aureus MRSA 19449. Compounds 14–16 were tested additionally in combination with various antibiotics. Molecular modelling was performed to assess potential mechanism of action. Microdilution and real-time efflux (RTE) assays were carried out in strains of K. aerogenes to determine the potential of compounds 7–23 to block the multidrug efflux pump AcrAB-TolC. Drug-like properties were determined experimentally. Two compounds (10, 15) containing non-condensed aromatic rings, significantly reduced oxacillin MICs in MRSA 19449, while 15 additionally enhanced the effectiveness of ampicillin. Results of molecular modelling confirmed the interaction with the allosteric site of PBP2a as a probable MDR-reversing mechanism. In RTE, the compounds inhibited AcrAB-TolC even to 90% (19). The 4-phenylbenzylidene derivative (15) demonstrated significant MDR-reversal “dual action” for β-lactam antibiotics in MRSA and inhibited AcrAB-TolC in K. aerogenes. 15 displayed also satisfied solubility and safety towards CYP3A4 in vitro.

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Correlation between the Antibiotic Resistance Genes and Susceptibility to Antibiotics among the Carbapenem-Resistant Gram-Negative Pathogens

Antibiotics ◽

10.3390/antibiotics10030255 ◽

2021 ◽

Vol 10 (3) ◽

pp. 255

Author(s):

Salma M. Abdelaziz ◽

Khaled M. Aboshanab ◽

Ibrahim S. Yahia ◽

Mahmoud A. Yassien ◽

Nadia A. Hassouna

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Efflux Pump ◽

Antibiotic Resistance Genes ◽

Multiple Drug ◽

P Value ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Gram Negative ◽

Carbapenem Resistant

In this study, the correlation between the antibiotic resistance genes and antibiotic susceptibility among the carbapenem-resistant Gram-negative pathogens (CRGNPs) recovered from patients diagnosed with acute pneumonia in Egypt was found. A total of 194 isolates including Klebsiella pneumoniae (89; 46%), Escherichia coli (47; 24%) and Pseudomonas aeruginosa (58; 30%) were recovered. Of these, 34 (18%) isolates were multiple drug resistant (MDR) and carbapenem resistant. For the K. pneumoniae MDR isolates (n = 22), blaNDM (14; 64%) was the most prevalent carbapenemase, followed by blaOXA-48 (11; 50%) and blaVIM (4; 18%). A significant association (p value < 0.05) was observed between the multidrug efflux pump (AcrA) and resistance to β-lactams and the aminoglycoside acetyl transferase gene (aac-6’-Ib) gene and resistance to ciprofloxacin, azithromycin and β-lactams (except for aztreonam). For P. aeruginosa, a significant association was noticed between the presence of the blaSHV gene and the multidrug efflux pump (MexA) and resistance to fluoroquinolones, amikacin, tobramycin, co-trimoxazole and β-lactams and between the aac-6’-Ib gene and resistance to aminoglycosides. All P. aeruginosa isolates (100%) harbored the MexAB-OprM multidrug efflux pump while 86% of the K. pneumoniae isolates harbored the AcrAB-TolC pump. Our results are of great medical importance for the guidance of healthcare practitioners for effective antibiotic prescription.

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

eLife ◽

10.7554/elife.24905 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 70

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.

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CmeABC Functions as a Multidrug Efflux System in Campylobacter jejuni

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.7.2124-2131.2002 ◽

2002 ◽

Vol 46 (7) ◽

pp. 2124-2131 ◽

Cited By ~ 332

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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Cryo-EM Determination of Eravacycline-Bound Structures of the Ribosome and the Multidrug Efflux Pump AdeJ of Acinetobacter baumannii

mBio ◽

10.1128/mbio.01031-21 ◽

2021 ◽

Vol 12 (3) ◽

Author(s):

Zhemin Zhang ◽

Christopher E. Morgan ◽

Robert A. Bonomo ◽

Edward W. Yu

Keyword(s):

Acinetobacter Baumannii ◽

Efflux Pump ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Antibiotic Resistant ◽

Gram Negative ◽

Content Type ◽

Bacterial Ribosome ◽

70S Ribosome ◽

Resistant Strains

ABSTRACT Antibiotic-resistant strains of the Gram-negative pathogen Acinetobacter baumannii have emerged as a significant global health threat. One successful therapeutic option to treat bacterial infections has been to target the bacterial ribosome. However, in many cases, multidrug efflux pumps within the bacterium recognize and extrude these clinically important antibiotics designed to inhibit the protein synthesis function of the bacterial ribosome. Thus, multidrug efflux within A. baumannii and other highly drug-resistant strains is a major cause of failure of drug-based treatments of infectious diseases. We here report the first structures of the Acinetobacter drug efflux (Ade)J pump in the presence of the antibiotic eravacycline, using single-particle cryo-electron microscopy (cryo-EM). We also describe cryo-EM structures of the eravacycline-bound forms of the A. baumannii ribosome, including the 70S, 50S, and 30S forms. Our data indicate that the AdeJ pump primarily uses hydrophobic interactions to bind eravacycline, while the 70S ribosome utilizes electrostatic interactions to bind this drug. Our work here highlights how an antibiotic can bind multiple bacterial targets through different mechanisms and potentially enables drug optimization by taking advantage of these different modes of ligand binding. IMPORTANCE Acinetobacter baumannii has developed into a highly antibiotic-resistant Gram-negative pathogen. The prevalent AdeJ multidrug efflux pump mediates resistance to different classes of antibiotics known to inhibit the function of the 70S ribosome. Here, we report the first structures of the A. baumannii AdeJ pump, both in the absence and presence of eravacycline. We also describe structures of the A. baumannii ribosome bound by this antibiotic. Our results indicate that AdeJ and the ribosome use very distinct binding modes for drug recognition. Our work will ultimately enable structure-based drug discovery to combat antibiotic-resistant A. baumannii infection.

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Cryo-Electron Microscopy Structure of an Acinetobacter baumannii Multidrug Efflux Pump

mBio ◽

10.1128/mbio.01295-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 9

Author(s):

Chih-Chia Su ◽

Christopher E. Morgan ◽

Sekhar Kambakam ◽

Malligarjunan Rajavel ◽

Harry Scott ◽

...

Keyword(s):

Electron Microscopy ◽

Acinetobacter Baumannii ◽

Efflux Pump ◽

Drug Efflux ◽

Bacterial Cells ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Gram Negative ◽

Content Type ◽

Cryo Electron Microscopy

ABSTRACT Resistance-nodulation-cell division multidrug efflux pumps are membrane proteins that catalyze the export of drugs and toxic compounds out of bacterial cells. Within the hydrophobe-amphiphile subfamily, these multidrug-resistant proteins form trimeric efflux pumps. The drug efflux process is energized by the influx of protons. Here, we use single-particle cryo-electron microscopy to elucidate the structure of the Acinetobacter baumannii AdeB multidrug efflux pump embedded in lipidic nanodiscs to a resolution of 2.98 Å. We found that each AdeB molecule within the trimer preferentially takes the resting conformational state in the absence of substrates. We propose that proton influx and drug efflux are synchronized and coordinated within the transport cycle. IMPORTANCE Acinetobacter baumannii is a successful human pathogen which has emerged as one of the most problematic and highly antibiotic-resistant Gram-negative bacteria worldwide. Multidrug efflux is a major mechanism that A. baumannii uses to counteract the action of multiple classes of antibiotics, such as β-lactams, tetracyclines, fluoroquinolones, and aminoglycosides. Here, we report a cryo-electron microscopy (cryo-EM) structure of the prevalent A. baumannii AdeB multidrug efflux pump, which indicates a plausible pathway for multidrug extrusion. Overall, our data suggest a mechanism for energy coupling that powers up this membrane protein to export antibiotics from bacterial cells. Our studies will ultimately inform an era in structure-guided drug design to combat multidrug resistance in these Gram-negative pathogens.

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Characterization of Posttranslationally Modified Multidrug Efflux Pumps Reveals an Unexpected Link between Glycosylation and Antimicrobial Resistance

mBio ◽

10.1128/mbio.02604-20 ◽

2020 ◽

Vol 11 (6) ◽

Author(s):

Sherif Abouelhadid ◽

John Raynes ◽

Tam Bui ◽

Jon Cuccui ◽

Brendan W. Wren

Keyword(s):

Antimicrobial Resistance ◽

Efflux Pump ◽

Multidrug Resistant ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Gram Negative ◽

Content Type ◽

Multidrug Efflux Pumps ◽

Pump Activity

ABSTRACT The substantial rise in multidrug-resistant bacterial infections is a current global imperative. Cumulative efforts to characterize antimicrobial resistance in bacteria has demonstrated the spread of six families of multidrug efflux pumps, of which resistance-nodulation-cell division (RND) is the major mechanism of multidrug resistance in Gram-negative bacteria. RND is composed of a tripartite protein assembly and confers resistance to a range of unrelated compounds. In the major enteric pathogen Campylobacter jejuni, the three protein components of RND are posttranslationally modified with N-linked glycans. The direct role of N-linked glycans in C. jejuni and other bacteria has long been elusive. Here, we present the first detailed account of the role of N-linked glycans and the link between N-glycosylation and antimicrobial resistance in C. jejuni. We demonstrate the multifunctional role of N-linked glycans in enhancing protein thermostability, stabilizing protein complexes and the promotion of protein-protein interaction, thus mediating antimicrobial resistance via enhancing multidrug efflux pump activity. This affirms that glycosylation is critical for multidrug efflux pump assembly. We present a generalized strategy that could be used to investigate general glycosylation system in Campylobacter genus and a potential target to develop antimicrobials against multidrug-resistant pathogens. IMPORTANCE Nearly all bacterial species have at least a single glycosylation system, but the direct effects of these posttranslational protein modifications are unresolved. Glycoproteome-wide analysis of several bacterial pathogens has revealed general glycan modifications of virulence factors and protein assemblies. Using Campylobacter jejuni as a model organism, we have studied the role of general N-linked glycans in the multidrug efflux pump commonly found in Gram-negative bacteria. We show, for the first time, the direct link between N-linked glycans and multidrug efflux pump activity. At the protein level, we demonstrate that N-linked glycans play a role in enhancing protein thermostability and mediating the assembly of the multidrug efflux pump to promote antimicrobial resistance, highlighting the importance of this posttranslational modification in bacterial physiology. Similar roles for glycans are expected to be found in other Gram-negative pathogens that possess general protein glycosylation systems.

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Cryoelectron Microscopy Structures of AdeB Illuminate Mechanisms of Simultaneous Binding and Exporting of Substrates

mBio ◽

10.1128/mbio.03690-20 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Christopher E. Morgan ◽

Przemyslaw Glaza ◽

Inga V. Leus ◽

Anhthu Trinh ◽

Chih-Chia Su ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Ethidium Bromide ◽

Efflux Pump ◽

Cryoelectron Microscopy ◽

Resistant Bacteria ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Antibiotic Resistant ◽

Gram Negative ◽

Content Type

ABSTRACT Acinetobacter baumannii is a Gram-negative pathogen that has emerged as one of the most highly antibiotic-resistant bacteria worldwide. Multidrug efflux within these highly drug-resistant strains and other opportunistic pathogens is a major cause of failure of drug-based treatments of infectious diseases. The best-characterized multidrug efflux system in A. baumannii is the prevalent Acinetobacter drug efflux B (AdeB) pump, which is a member of the resistance-nodulation-cell division (RND) superfamily. Here, we report six structures of the trimeric AdeB multidrug efflux pump in the presence of ethidium bromide using single-particle cryoelectron microscopy (cryo-EM). These structures allow us to directly observe various novel conformational states of the AdeB trimer, including the transmembrane region of trimeric AdeB can be associated with form a trimer assembly or dissociated into “dimer plus monomer” and “monomer plus monomer plus monomer” configurations. We also discover that a single AdeB protomer can simultaneously anchor a number of ethidium ligands and that different AdeB protomers can bind ethidium molecules simultaneously. Combined with molecular dynamics (MD) simulations, we reveal a drug transport mechanism that involves multiple multidrug-binding sites and various transient states of the AdeB membrane protein. Our data suggest that each AdeB protomer within the trimer binds and exports drugs independently. IMPORTANCE Acinetobacter baumannii has emerged as one of the most highly antibiotic-resistant Gram-negative pathogens. The prevalent AdeB multidrug efflux pump mediates resistance to a broad spectrum of clinically relevant antimicrobial agents. Here, we report six cryo-EM structures of the trimeric AdeB pump in the presence of ethidium bromide. We discover that a single AdeB protomer can simultaneously anchor a number of ligands, and different AdeB protomers can bind ethidium molecules simultaneously. The results indicate that each AdeB protomer within the trimer recognizes and extrudes drugs independently.

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