The Wild-type Structures of MexAB-OprM Multidrug Efflux Pump Revealed by 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.

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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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AcrAB Multidrug Efflux Pump Is Associated with Reduced Levels of Susceptibility to Tigecycline (GAR-936) in Proteus mirabilis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.2.665-669.2003 ◽

2003 ◽

Vol 47 (2) ◽

pp. 665-669 ◽

Cited By ~ 113

Author(s):

Melissa A. Visalli ◽

Ellen Murphy ◽

Steven J. Projan ◽

Patricia A. Bradford

Keyword(s):

Proteus Mirabilis ◽

Efflux Pump ◽

Wild Type ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Transposon Insertion ◽

Spectrum Activity ◽

Insertion Mutants ◽

Broad Spectrum Activity ◽

Increased Susceptibility

ABSTRACT Tigecycline has good broad-spectrum activity against many gram-positive and gram-negative pathogens with the notable exception of the Proteeae. A study was performed to identify the mechanism responsible for the reduced susceptibility to tigecycline in Proteus mirabilis. Two independent transposon insertion mutants of P. mirabilis that had 16-fold-increased susceptibility to tigecycline were mapped to the acrB gene homolog of the Escherichia coli AcrRAB efflux system. Wild-type levels of decreased susceptibility to tigecycline were restored to the insertion mutants by complementation with a clone containing a PCR-derived fragment from the parental wild-type acrRAB efflux gene cluster. The AcrAB transport system appears to be associated with the intrinsic reduced susceptibility to tigecycline in P. mirabilis.

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Cloning and Characterization of SmeT, a Repressor of the Stenotrophomonas maltophilia Multidrug Efflux Pump SmeDEF

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.11.3386-3393.2002 ◽

2002 ◽

Vol 46 (11) ◽

pp. 3386-3393 ◽

Cited By ~ 53

Author(s):

Patricia Sánchez ◽

Ana Alonso ◽

Jose L. Martinez

Keyword(s):

Stenotrophomonas Maltophilia ◽

Efflux Pump ◽

Wild Type ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

S1 Nuclease ◽

Cellular Factor ◽

S1 Nuclease Mapping ◽

Nuclease Mapping

ABSTRACT We report on the cloning of the gene smeT, which encodes the transcriptional regulator of the Stenotrophomonas maltophilia efflux pump SmeDEF. SmeT belongs to the TetR and AcrR family of transcriptional regulators. The smeT gene is located upstream from the structural operon of the pump genes smeDEF and is divergently transcribed from those genes. Experiments with S. maltophilia and the heterologous host Escherichia coli have demonstrated that SmeT is a transcriptional repressor. S1 nuclease mapping has demonstrated that expression of smeT is driven by a single promoter lying close to the 5′ end of the gene and that expression of smeDEF is driven by an unique promoter that overlaps with promoter PsmeT. The level of expression of smeT is higher in smeDEF-overproducing S. maltophilia strain D457R, which suggests that SmeT represses its own expression. Band-shifting assays have shown that wild-type strain S. maltophilia D457 contains a cellular factor(s) capable of binding to the intergenic smeT-smeD region. That cellular factor(s) was absent from smeDEF-overproducing S. maltophilia strain D457R. The sequence of smeT from D457R showed a point mutation that led to a Leu166Gln change within the SmeT protein. This change allowed overexpression of both smeDEF and smeT in D457R. It was noteworthy that expression of wild-type SmeT did not fully complement the smeT mutation in D457R. This suggests that the wild-type protein is not dominant over the mutant SmeT.

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Induction of mexCD-oprJ operon for a multidrug efflux pump by disinfectants in wild-type Pseudomonas aeruginosa PAO1

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkg173 ◽

2003 ◽

Vol 51 (4) ◽

pp. 991-994 ◽

Cited By ~ 60

Author(s):

Y. Morita

Keyword(s):

Pseudomonas Aeruginosa ◽

Efflux Pump ◽

Wild Type ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Pseudomonas Aeruginosa Pao1

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SAGA/ADA Complex Subunit Ada2 Is Required for Cap1- but Not Mrr1-Mediated Upregulation of the Candida albicans Multidrug Efflux PumpMDR1

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03065-14 ◽

2014 ◽

Vol 58 (9) ◽

pp. 5102-5110 ◽

Cited By ~ 16

Author(s):

Bernardo Ramírez-Zavala ◽

Selene Mogavero ◽

Eva Schöller ◽

Christoph Sasse ◽

P. David Rogers ◽

...

Keyword(s):

Transcription Factor ◽

Candida Albicans ◽

Efflux Pump ◽

Wild Type ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Gain Of Function ◽

Content Type ◽

Zinc Cluster ◽

Fluconazole Resistant

ABSTRACTOverexpression of the multidrug efflux pumpMDR1is one mechanism by which the pathogenic yeastCandida albicansdevelops resistance to the antifungal drug fluconazole. The constitutive upregulation ofMDR1in fluconazole-resistant, clinicalC. albicansisolates is caused by gain-of-function mutations in the zinc cluster transcription factor Mrr1. It has been suggested that Mrr1 activatesMDR1transcription by recruiting Ada2, a subunit of the SAGA/ADA coactivator complex. However,MDR1expression is also regulated by the bZIP transcription factor Cap1, which mediates the oxidative stress response inC. albicans. Here, we show that a hyperactive Mrr1 containing a gain-of-function mutation promotesMDR1overexpression independently of Ada2. In contrast, a C-terminally truncated, hyperactive Cap1 causedMDR1overexpression in a wild-type strain but only weakly in mutants lackingADA2. In the presence of benomyl or H2O2, compounds that induceMDR1expression in an Mrr1- and Cap1-dependent fashion,MDR1was upregulated with the same efficiency in wild-type andada2Δ cells. These results indicate that Cap1, but not Mrr1, recruits Ada2 to theMDR1promoter to induce the expression of this multidrug efflux pump and that Ada2 is not required forMDR1overexpression in fluconazole-resistantC. albicansstrains containing gain-of-function mutations in Mrr1.

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Multiresistance Genes of Rhizobium etli CFN42

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2000.13.5.572 ◽

2000 ◽

Vol 13 (5) ◽

pp. 572-577 ◽

Cited By ~ 71

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.

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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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Multidrug Efflux Pump AcrAB of Salmonella typhimuriumExcretes Only Those β-Lactam Antibiotics Containing Lipophilic Side Chains

Journal of Bacteriology ◽

10.1128/jb.180.17.4686-4692.1998 ◽

1998 ◽

Vol 180 (17) ◽

pp. 4686-4692 ◽

Cited By ~ 158

Author(s):

Hiroshi Nikaido ◽

Marina Basina ◽

Vy Nguyen ◽

Emiko Y. Rosenberg

Keyword(s):

Outer Membrane ◽

Cytoplasmic Membrane ◽

Efflux Pump ◽

Side Chains ◽

Wild Type ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Active Efflux ◽

Outer Leaflet ◽

Acrab Efflux Pump

ABSTRACT We found that the previously reported SS-B drug-supersusceptible mutant of Salmonella typhimurium (S. Sukupolvi, M. Vaara, I. M. Helander, P. Viljanen, and P. H. Mäkelä, J. Bacteriol. 159:704–712, 1984) had a mutation in the acrAB operon. Comparison of this mutant with its parent strain and with an AcrAB-overproducing strain showed that the activity of the AcrAB efflux pump often produced significant resistance to β-lactam antibiotics in the complete absence of β-lactamase. The effect of AcrAB activity on resistance was more pronounced with agents containing more lipophilic side chains, suggesting that such compounds were better substrates for this pump. This correlation is consistent with the hypothesis that only those molecules that become at least partially partitioned into the lipid bilayer of the cytoplasmic membrane are captured by the AcrAB pump. According to this mechanism, the pump successfully excretes even those β-lactams that fail to traverse the cytoplasmic membrane, because these compounds are likely to become partitioned into the outer leaflet of the bilayer. Even the compounds with lipophilic side chains were shown to penetrate across the outer membrane relatively rapidly, if the pump was inactivated genetically or physiologically. The exclusion of such compounds, exemplified by nafcillin, from cells of the wild-type S. typhimuriumwas previously interpreted as the result of poor diffusion across the outer membrane (H. Nikaido, Biochim. Biophys. Acta 433:118–132, 1976), but it is now recognized as the consequence of efficient pumping out of entering antibiotics by the active efflux process.

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NorA plasmid resistance to fluoroquinolones: role of copy number and norA frameshift mutations.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.7.1665 ◽

1996 ◽

Vol 40 (7) ◽

pp. 1665-1669 ◽

Cited By ~ 12

Author(s):

L Sun ◽

S Sreedharan ◽

K Plummer ◽

L M Fisher

Keyword(s):

Copy Number ◽

Efflux Pump ◽

Gene Promoter ◽

Fluoroquinolone Resistance ◽

Wild Type ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

E Coli ◽

Copy Numbers ◽

Mutational Activation

Staphylococcus aureus NorA protein is a transmembrane multidrug efflux pump that confers low-level resistance to hydrophilic fluoroquinolones. The norA gene promoter is active in Escherichia coli HB101. We have examined the genetic basis of norA-mediated resistance in E. coli by introducing a wild-type norA gene into HB101 in plasmid pCL1921, pBR322, or pUC18 exhibiting copy numbers that spanned a 22-fold range. Increased ciprofloxacin resistance correlated with norA transcript levels seen by Northern (RNA) analysis. Thus, contrary to some reports, a wild-type norA gene confers fluoroquinolone resistance in E. coli in a copy-number-dependent fashion and does not require mutational activation. Interestingly, a multicopy pUC19norA derivative gave transformants exhibiting a range of resistance phenotypes. The norA gene of one transformant carried a single base deletion (ATACAAT to AACAAT; the deleted base is underlined) in the putative--10 Pribnow box resulting in a promoter down-regulatory mutation; a second plasmid had acquired a frameshift producing a null mutation at codon 112. These mutations override the dual resistance-growth-inhibitory phenotype of high-copy-number norA plasmids. The results have implications for using the standard E. coli HB101 system to assess NorA function and potentially for plasmid-borne transmission of norA-mediated drug resistance.

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