scholarly journals Chlorpromazine and Amitriptyline Are Substrates and Inhibitors of the AcrB Multidrug Efflux Pump

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Elizabeth M. Grimsey ◽  
Chiara Fais ◽  
Robert L. Marshall ◽  
Vito Ricci ◽  
Maria Laura Ciusa ◽  
...  
Keyword(s):  
De Novo ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Inhibition Mechanism ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Content Type ◽  
Inhibitory Compounds ◽  
Serovar Typhimurium ◽  
Rnd Efflux Pumps

Efflux pumps of the resistance nodulation-cell division (RND) superfamily are major contributors to multidrug resistance for most of the Gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens. The development of inhibitors of these pumps would be highly desirable; however, several issues have thus far hindered all efforts at designing new efflux inhibitory compounds devoid of adverse effects. An alternative route to de novo design relies on the use of marketed drugs, for which side effects on human health have been already assessed. In this work, we provide experimental evidence that the antipsychotic drugs chlorpromazine and amitriptyline are inhibitors of the AcrB transporter, the engine of the major RND efflux pumps in Escherichia coli and Salmonella enterica serovar Typhimurium. Furthermore, in silico calculations have provided a molecular-level picture of the inhibition mechanism, allowing rationalization of experimental data and paving the way for similar studies with other classes of marketed compounds.

scholarly journals Molecular Mechanism of MBX2319 Inhibition of Escherichia coli AcrB Multidrug Efflux Pump and Comparison with Other Inhibitors

2014 ◽  
Vol 58 (10) ◽  
pp. 6224-6234 ◽  
Author(s):  
Attilio V. Vargiu ◽  
Paolo Ruggerone ◽  
Timothy J. Opperman ◽  
Son T. Nguyen ◽  
Hiroshi Nikaido
Keyword(s):  
Efflux Pump ◽  
Efflux Pumps ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Efflux Pump Inhibitor ◽  
Content Type ◽  
X Ray Crystallography ◽  
Resistance Nodulation Division ◽  
Hydrophobic Portion ◽  
Dynamics Simulations

ABSTRACTEfflux pumps of the resistance nodulation division (RND) superfamily, such as AcrB, make a major contribution to multidrug resistance in Gram-negative bacteria. The development of inhibitors of the RND pumps would improve the efficacy of current and next-generation antibiotics. To date, however, only one inhibitor has been cocrystallized with AcrB. Thus,in silicostructure-based analysis is essential for elucidating the interaction between other inhibitors and the efflux pumps. In this work, we used computer docking and molecular dynamics simulations to study the interaction between AcrB and the compound MBX2319, a novel pyranopyridine efflux pump inhibitor with potent activity against RND efflux pumps ofEnterobacteriaceaespecies, as well as other known inhibitors (D13-9001, 1-[1-naphthylmethyl]-piperazine, and phenylalanylarginine-β-naphthylamide) and the binding of doxorubicin to the efflux-defective F610A variant of AcrB. We also analyzed the binding of a substrate, minocycline, for comparison. Our results show that MBX2319 binds very tightly to the lower part of the distal pocket in the B protomer of AcrB, strongly interacting with the phenylalanines lining the hydrophobic trap, where the hydrophobic portion of D13-9001 was found to bind by X-ray crystallography. Additionally, MBX2319 binds to AcrB in a manner that is similar to the way in which doxorubicin binds to the F610A variant of AcrB. In contrast, 1-(1-naphthylmethyl)-piperazine and phenylalanylarginine-β-naphthylamide appear to bind to somewhat different areas of the distal pocket in the B protomer of AcrB than does MBX2319. However, all inhibitors (except D13-9001) appear to distort the structure of the distal pocket, impairing the proper binding of substrates.

scholarly journals The ABC-Type Efflux Pump MacAB Protects Salmonella enterica serovar Typhimurium from Oxidative Stress

mBio ◽  
2013 ◽  
Vol 4 (6) ◽  
Author(s):  
Lydia M. Bogomolnaya ◽  
Katharine D. Andrews ◽  
Marissa Talamantes ◽  
Aimee Maple ◽  
Yury Ragoza ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Salmonella Enterica ◽  
Efflux Pump ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Content Type ◽  
Serovar Typhimurium ◽  
Drug Efflux Pump

ABSTRACTMultidrug efflux pumps are integral membrane proteins known to actively excrete antibiotics. The macrolide-specific pump MacAB, the only ABC-type drug efflux pump inSalmonella, has previously been linked to virulence in mice. The molecular mechanism of this link betweenmacABand infection is unclear. We demonstrate thatmacABplays a role in the detoxification of reactive oxygen species (ROS), compounds that salmonellae are exposed to at various stages of infection.macABis induced upon exposure to H2O2and is critical for survival ofSalmonella entericaserovar Typhimurium in the presence of peroxide. Furthermore, we determined thatmacABis required for intracellular replication inside J774.A1 murine macrophages but is not required for survival in ROS-deficient J774.D9 macrophages.macABmutants also had reduced survival in the intestine in the mouse colitis model, a model characterized by a strong neutrophilic intestinal infiltrate where bacteria may experience the cytotoxic actions of ROS. Using an Amplex red-coupled assay,macABmutants appear to be unable to induce protection against exogenous H2O2in vitro, in contrast to the isogenic wild type. In mixed cultures, the presence of the wild-type organism, or media preconditioned by the growth of the wild-type organism, was sufficient to rescue themacABmutant from peroxide-mediated killing. Our data indicate that the MacAB drug efflux pump has functions beyond resistance to antibiotics and plays a role in the protection ofSalmonellaagainst oxidative stress. Intriguingly, our data also suggest the presence of a soluble anti-H2O2compound secreted bySalmonellacells through a MacAB-dependent mechanism.IMPORTANCEThe ABC-type multidrug efflux pump MacAB is known to be required forSalmonella entericaserovar Typhimurium virulence after oral infection in mice, yet the function of this pump during infection is unknown. We show that this pump is necessary for colonization of niches in infected mice where salmonellae encounter oxidative stress during infection. MacAB is required for growth in cultured macrophages that produce reactive oxygen species (ROS) but is not needed in macrophages that do not generate ROS. In addition, we show that MacAB is required to resist peroxide-mediated killingin vitroand for the inactivation of peroxide in the media. Finally, wild-type organisms, or supernatant from wild-type organisms grown in the presence of peroxide, rescue the growth defect ofmacABmutants in H2O2. MacAB appears to participate in the excretion of a compound that induces protection against ROS-mediated killing, revealing a new role for this multidrug efflux pump.

scholarly journals Amotosalen is a bacterial multidrug efflux pump substrate potentially affecting its pathogen inactivation activity

2021 ◽  
Author(s):  
Alex B. Green ◽  
Katelyn E. Zulauf ◽  
Katherine A. Truelson ◽  
Lucius Chiaraviglio ◽  
Meng Cui ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Multidrug Resistant ◽  
Pathogen Inactivation ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Minimal Inhibitory Concentrations ◽  
E Coli ◽  
Rnd Efflux Pumps

AbstractPathogen inactivation is a strategy to improve the safety of transfusion products. The Cerus Intercept technology makes use of a psoralen compound called amotosalen in combination with UVA light to inactivate bacteria, viruses and protozoa. Psoralens have structural similarity to bacterial multidrug-efflux pump substrates. As these efflux pumps are often overexpressed in multidrug-resistant pathogens and with recent reported outbreaks of transfusion-associated sepsis with Acinetobacter, we tested whether contemporary drug-resistant pathogens might show resistance to amotosalen and other psoralens based on multidrug efflux mechanisms through microbiological, biophysical and molecular modeling analysis. The main efflux systems in Enterobacterales and Acinetobacter baumannii, tripartite RND (resistance-nodulation-cell division) systems which span the inner and outer membranes of Gram-negative pathogens and expel antibiotics from the bacterial cytoplasm into the extracellular space, were specifically examined. We found that amotosalen was an efflux substrate for the TolC-dependent RND efflux pumps in E. coli and the AdeABC efflux pump from Acinetobacter baumannii, and that minimal inhibitory concentrations for contemporary bacterial isolates in vitro approached and exceeded the concentration of amotosalen used in the approved platelet and plasma inactivation procedures. These findings suggest that otherwise safe and effective inactivation methods should be further studied to exclude possible gaps in their ability to inactivate contemporary, multidrug-resistant bacterial pathogens.ImportancePathogen inactivation is a strategy to enhance the safety of transfused blood products. We identify the compound, amotosalen, widely used for pathogen inactivation, as a bacterial multidrug efflux substrate. Specifically, experiments suggest that amotosalen is pumped out of bacteria by the major TolC-dependent RND efflux pumps in E. coli and the AdeABC efflux pump in Acinetobacter baumannii. Such efflux pumps are often overexpressed in multidrug-resistant pathogens. Importantly, the minimal inhibitory concentrations for contemporary multidrug-resistant Enterobacterales, Acinetobacter baumannii, Pseudomonas aeruginosa, Burkholderia spp., and Stenotrophomonas maltophilia isolates approached or exceeded the amotosalen concentration used in approved platelet and plasma inactivation procedures, potentially as a result of efflux pump activity. Although there are important differences in methodology between our experiments and blood product pathogen inactivation, these findings suggest that otherwise safe and effective inactivation methods should be further studied to exclude possible gaps in their ability to inactivate contemporary, multidrug-resistant bacterial pathogens.

scholarly journals Magnitude of Voriconazole Resistance in Clinical and Environmental Isolates of Aspergillus flavus and Investigation into the Role of Multidrug Efflux Pumps

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Raees A. Paul ◽  
Shivaprakash M. Rudramurthy ◽  
Manpreet Dhaliwal ◽  
Pankaj Singh ◽  
Anup K. Ghosh ◽  
...  
Keyword(s):  
Aspergillus Flavus ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Azole Resistance ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Environmental Isolates ◽  
Content Type ◽  
Multidrug Efflux Pumps

ABSTRACT The magnitude of azole resistance in Aspergillus flavus and its underlying mechanism is obscure. We evaluated the frequency of azole resistance in a collection of clinical (n = 121) and environmental isolates (n = 68) of A. flavus by the broth microdilution method. Six (5%) clinical isolates displayed voriconazole MIC greater than the epidemiological cutoff value. Two of these isolates with non-wild-type MIC were isolated from same patient and were genetically distinct, which was confirmed by amplified fragment length polymorphism analysis. Mutations associated with azole resistance were not present in the lanosterol 14-α demethylase coding genes (cyp51A, cyp51B, and cyp51C). Basal and voriconazole-induced expression of cyp51A homologs and various efflux pump genes was analyzed in three each of non-wild-type and wild-type isolates. All of the efflux pump genes screened showed low basal expression irrespective of the azole susceptibility of the isolate. However, the non-wild-type isolates demonstrated heterogeneous overexpression of many efflux pumps and the target enzyme coding genes in response to induction with voriconazole (1 μg/ml). The most distinctive observation was approximately 8- to 9-fold voriconazole-induced overexpression of an ortholog of the Candida albicans ATP binding cassette (ABC) multidrug efflux transporter, Cdr1, in two non-wild-type isolates compared to those in the reference strain A. flavus ATCC 204304 and other wild-type strains. Although the dominant marker of azole resistance in A. flavus is still elusive, the current study proposes the possible role of multidrug efflux pumps, especially that of Cdr1B overexpression, in contributing azole resistance in A. flavus.

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 Multidrug Adaptive Resistance of Pseudomonas aeruginosa Swarming Cells

2019 ◽  
Vol 64 (3) ◽  
Author(s):  
Shannon R. Coleman ◽  
Travis Blimkie ◽  
Reza Falsafi ◽  
Robert E. W. Hancock
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Efflux Pump ◽  
Iron Acquisition ◽  
Polymyxin B ◽  
Rna Seq ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Content Type ◽  
Adaptive Resistance

ABSTRACT Swarming surface motility is a complex adaptation leading to multidrug antibiotic resistance and virulence factor production in Pseudomonas aeruginosa. Here, we expanded previous studies to demonstrate that under swarming conditions, P. aeruginosa PA14 is more resistant to multiple antibiotics, including aminoglycosides, β-lactams, chloramphenicol, ciprofloxacin, tetracycline, trimethoprim, and macrolides, than swimming cells, but is not more resistant to polymyxin B. We investigated the mechanism(s) of swarming-mediated antibiotic resistance by examining the transcriptomes of swarming cells and swarming cells treated with tobramycin by transcriptomics (RNA-Seq) and reverse transcriptase quantitative PCR (qRT-PCR). RNA-Seq of swarming cells (versus swimming) revealed 1,581 dysregulated genes, including 104 transcriptional regulators, two-component systems, and sigma factors, numerous upregulated virulence and iron acquisition factors, and downregulated ribosomal genes. Strain PA14 mutants in resistome genes that were dysregulated under swarming conditions were tested for their ability to swarm in the presence of tobramycin. In total, 41 mutants in genes dysregulated under swarming conditions were shown to be more resistant to tobramycin under swarming conditions, indicating that swarming-mediated tobramycin resistance was multideterminant. Focusing on two genes downregulated under swarming conditions, both prtN and wbpW mutants were more resistant to tobramycin, while the prtN mutant was additionally resistant to trimethoprim under swarming conditions; complementation of these mutants restored susceptibility. RNA-Seq of swarming cells treated with subinhibitory concentrations of tobramycin revealed the upregulation of the multidrug efflux pump MexXY and downregulation of virulence factors.

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.

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

2014 ◽  
Vol 58 (9) ◽  
pp. 5102-5110 ◽  
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.

scholarly journals A New Natural Product Analog of Blasticidin S Reveals Cellular Uptake Facilitated by the NorA Multidrug Transporter

2017 ◽  
Vol 61 (6) ◽  
Author(s):  
Jack R. Davison ◽  
Katheryn M. Lohith ◽  
Xiaoning Wang ◽  
Kostyantyn Bobyk ◽  
Sivakoteswara R. Mandadapu ◽  
...  
Keyword(s):  
Efflux Pump ◽  
Bacterial Strains ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Gene Encoding ◽  
Content Type ◽  
Bacterial Membranes ◽  
Development Of Resistance ◽  
Antibiotic Compounds ◽  
Blasticidin S

ABSTRACT The permeation of antibiotics through bacterial membranes to their target site is a crucial determinant of drug activity but in many cases remains poorly understood. During screening efforts to discover new broad-spectrum antibiotic compounds from marine sponge samples, we identified a new analog of the peptidyl nucleoside antibiotic blasticidin S that exhibited up to 16-fold-improved potency against a range of laboratory and clinical bacterial strains which we named P10. Whole-genome sequencing of laboratory-evolved strains of Staphylococcus aureus resistant to blasticidin S and P10, combined with genome-wide assessment of the fitness of barcoded Escherichia coli knockout strains in the presence of the antibiotics, revealed that restriction of cellular access was a key feature in the development of resistance to this class of drug. In particular, the gene encoding the well-characterized multidrug efflux pump NorA was found to be mutated in 69% of all S. aureus isolates resistant to blasticidin S or P10. Unexpectedly, resistance was associated with inactivation of norA, suggesting that the NorA transporter facilitates cellular entry of peptidyl nucleosides in addition to its known role in the efflux of diverse compounds, including fluoroquinolone antibiotics.

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