scholarly journals Molecular mechanism of proton-coupled ligand translocation by the bacterial efflux pump EmrE

2021 ◽  
Vol 17 (10) ◽  
pp. e1009454
Author(s):  
Jakub Jurasz ◽  
Maciej Bagiński ◽  
Jacek Czub ◽  
Miłosz Wieczór
Keyword(s):  
Structural Changes ◽  
Efflux Pump ◽  
Solvent Accessibility ◽  
Broad Class ◽  
Bound State ◽  
Multi Drug Resistance ◽  
Structural Level ◽  
Serious Infections ◽  
Alternating Access ◽  
Complex Drug

The current surge in bacterial multi-drug resistance (MDR) is one of the largest challenges to public health, threatening to render ineffective many therapies we rely on for treatment of serious infections. Understanding different factors that contribute to MDR is hence crucial from the global “one health” perspective. In this contribution, we focus on the prototypical broad-selectivity proton-coupled antiporter EmrE, one of the smallest known ligand transporters that confers resistance to aromatic cations in a number of clinically relevant species. As an asymmetric homodimer undergoing an “alternating access” protomer-swap conformational change, it serves as a model for the mechanistic understanding of more complex drug transporters. Here, we present a free energy and solvent accessibility analysis that indicates the presence of two complementary ligand translocation pathways that remain operative in a broad range of conditions. Our simulations show a previously undescribed desolvated apo state and anticorrelated accessibility in the ligand-bound state, explaining on a structural level why EmrE does not disrupt the pH gradient through futile proton transfer. By comparing the behavior of a number of model charged and/or aromatic ligands, we also explain the origin of selectivity of EmrE towards a broad class of aromatic cations. Finally, we explore unbiased pathways of ligand entry and exit to identify correlated structural changes implicated in ligand binding and release, as well as characterize key intermediates of occupancy changes.

scholarly journals Molecular mechanism of proton-coupled ligand translocation by the bacterial efflux pump EmrE

2021 ◽  
Author(s):  
Jakub Jurasz ◽  
Jacek Czub ◽  
Maciej Bagiński ◽  
Miłosz Wieczór
Keyword(s):  
Structural Changes ◽  
Efflux Pump ◽  
Solvent Accessibility ◽  
Broad Class ◽  
Bound State ◽  
Multi Drug Resistance ◽  
Structural Level ◽  
Serious Infections ◽  
Alternating Access ◽  
Complex Drug

AbstractThe current surge in bacterial multi-drug resistance (MDR) is one of the largest challenges to public health, threatening to render ineffective many therapies we rely on for treatment of serious infections. Understanding different factors that contribute to MDR is hence crucial from the global “one health” perspective. In this contribution, we focus on the prototypical broad-selectivity proton-coupled antiporter EmrE, one of the smallest known ligand transporters that confers resistance to aromatic cations in a number of clinically relevant species. As an asymmetric homodimer undergoing an “alternating access” protomer-swap conformational change, it serves as a model for the mechanistic understanding of more complex drug transporters. Here, we present a free energy and solvent accessibility analysis that indicates the presence of two complementary ligand translocation pathways that remain operative in a broad range of conditions. Our simulations show a previously undescribed desolvated apo state and anticorrelated accessibility in the ligand-bound state, explaining on a structural level why EmrE does not disrupt the pH gradient through futile proton transfer. By comparing the behavior of a number of model charged and/or aromatic ligands, we also explain the origin of selectivity of EmrE towards a broad class of aromatic cations. Finally, we explore unbiased pathways of ligand entry and exit to identify correlated structural changes implicated in ligand binding and release, as well as characterize key intermediates of occupancy changes.

scholarly journals Understanding the basis of antibiotic resistance: a platform for drug discovery

Microbiology ◽  
2014 ◽  
Vol 160 (11) ◽  
pp. 2366-2373 ◽  
Author(s):  
Laura J. V. Piddock
Keyword(s):  
Antibiotic Resistance ◽  
Salmonella Enterica ◽  
Efflux Pump ◽  
Antibiotic Resistance Genes ◽  
Multi Drug Resistance ◽  
Annual Conference ◽  
Altered Expression ◽  
Genetic Inactivation ◽  
Fluoroquinolone Antibiotics ◽  
Serovar Typhimurium

There are numerous genes in Salmonella enterica serovar Typhimurium that can confer resistance to fluoroquinolone antibiotics, including those that encode topoisomerase proteins, the primary targets of this class of drugs. However, resistance is often multifactorial in clinical isolates and it is not uncommon to also detect mutations in genes that affect the expression of proteins involved in permeability and multi-drug efflux. The latter mechanism, mediated by tripartite efflux systems, such as that formed by the AcrAB–TolC system, confers inherent resistance to many antibiotics, detergents and biocides. Genetic inactivation of efflux genes gives multi-drug hyper-susceptibility, and in the absence of an intact AcrAB–TolC system some chromosomal and transmissible antibiotic resistance genes no longer confer clinically relevant levels of resistance. Furthermore, a functional multi-drug resistance efflux pump, such as AcrAB–TolC, is required for virulence and the ability to form a biofilm. In part, this is due to altered expression of virulence and biofilm genes being sensitive to efflux status. Efflux pump expression can be increased, usually due to mutations in regulatory genes, and this confers resistance to clinically useful drugs such as fluoroquinolones and β-lactams. Here, I discuss some of the work my team has carried out characterizing the mechanisms of antibiotic resistance in Salmonella enterica serovar Typhimurium from the late 1980s to 2014. A video of this Prize Lecture, presented at the Society for General Microbiology Annual Conference 2014, can be viewed via this link: https://www.youtube.com/watch?v=MCRumMV99Yw.

scholarly journals Validation of Aztreonam-Avibactam Susceptibility Testing Using Digitally Dispensed Custom Panels

2020 ◽  
Vol 58 (4) ◽  
Author(s):  
Eric Ransom ◽  
Amelia Bhatnagar ◽  
Jean B. Patel ◽  
Maria-Jose Machado ◽  
Sandra Boyd ◽  
...  
Keyword(s):  
Susceptibility Testing ◽  
Internal Validation ◽  
Serious Infections ◽  
Control And Prevention ◽  
Approved Drugs ◽  
Multiple Experts ◽  
Fda Approved Drugs ◽  
The U.S ◽  
Complex Drug

ABSTRACT Aztreonam-avibactam is a combination antimicrobial agent with activity against carbapenemase-producing Enterobacteriaceae (CPE) with metallo-β-lactamases (MβLs). Although aztreonam-avibactam is not yet approved by the U.S. Food and Drug Administration (FDA), clinicians can administer this combination by using two FDA-approved drugs: aztreonam and ceftazidime-avibactam. This combination of drugs is recommended by multiple experts for treatment of serious infections caused by MβL-producing CPE. At present, in vitro antimicrobial susceptibility testing (AST) of aztreonam-avibactam is not commercially available; thus, most clinicians receive no laboratory-based guidance that can support consideration of aztreonam-avibactam for serious CPE infections. Here, we report our internal validation for aztreonam-avibactam AST by reference broth microdilution (BMD) according to Clinical and Laboratory Standards Institute (CLSI) guidelines. The validation was performed using custom frozen reference BMD panels prepared in-house at the Centers for Disease Control and Prevention (CDC). In addition, we took this opportunity to evaluate a new panel-making method using a digital dispenser, the Hewlett Packard (HP) D300e. Our studies demonstrate that the performance characteristics of digitally dispensed panels were equivalent to those of conventionally prepared frozen reference BMD panels for a number of drugs, including aztreonam-avibactam. We found the HP D300e digital dispenser to be easy to use and to provide the capacity to prepare complex drug panels. Our findings will help other clinical and public health laboratories implement susceptibility testing for aztreonam-avibactam.

From mixed sigma-2 receptor/P-glycoprotein targeting agents to selective P-glycoprotein modulators: Small structural changes address the mechanism of interaction at the efflux pump

2015 ◽  
Vol 89 ◽  
pp. 606-615 ◽  
Author(s):  
Carmen Abate ◽  
Maria Laura Pati ◽  
Marialessandra Contino ◽  
Nicola Antonio Colabufo ◽  
Roberto Perrone ◽  
...  
Keyword(s):  
Structural Changes ◽  
Efflux Pump ◽  
P Glycoprotein ◽  
Mechanism Of Interaction

scholarly journals Structural basis for the mechanism of ABC transporters

2015 ◽  
Vol 43 (5) ◽  
pp. 889-893 ◽  
Author(s):  
Konstantinos Beis
Keyword(s):  
Drug Resistance ◽  
Binding Site ◽  
Abc Transporter ◽  
Abc Transporters ◽  
Eukaryotic Cells ◽  
Structural Basis ◽  
Multi Drug Resistance ◽  
Hydrolysis Of ◽  
Transport Molecules ◽  
Alternating Access

The ATP-binding cassette (ABC) transporters are primary transporters that couple the energy stored in adenosine triphosphate (ATP) to the movement of molecules across the membrane. ABC transporters can be divided into exporters and importers; importers mediate the uptake of essential nutrients into cells and are found predominantly in prokaryotes whereas exporters transport molecules out of cells or into organelles and are found in all organisms. ABC exporters have been linked with multi-drug resistance in both bacterial and eukaryotic cells. ABC transporters are powered by the hydrolysis of ATP and transport their substrate via the alternating access mechanism, whereby the protein alternates between a conformation in which the substrate-binding site is accessible from the outside of the membrane, outward-facing and one in which it is inward-facing. In this mini-review, the structures of different ABC transporter types in different conformations are presented within the context of the alternating access mechanism and how they have shaped our current understanding of the mechanism of ABC transporters.

scholarly journals Impact of an Intervention to Control Imipenem-Resistant Acinetobacter baumannii and Its Resistance Mechanisms: An 8-Year Survey

2021 ◽  
Vol 11 ◽  
Author(s):  
Lida Chen ◽  
Pinghai Tan ◽  
Jianming Zeng ◽  
Xuegao Yu ◽  
Yimei Cai ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Acinetobacter Baumannii ◽  
Bacterial Resistance ◽  
Efflux Pump ◽  
Resistance Mechanism ◽  
Resistance Mechanisms ◽  
Multi Drug Resistance ◽  
Minimal Inhibitory Concentrations ◽  
Resistance Rates ◽  
The Impact

BackgroundThis study aimed to examine the impact of an intervention carried out in 2011 to combat multi-drug resistance and outbreaks of imipenem-resistant Acinetobacter baumannii (IRAB), and to explore its resistance mechanism.MethodsA total of 2572 isolates of A. baumannii, including 1673 IRAB isolates, were collected between 2007 and 2014. An intervention was implemented to control A. baumannii resistance and outbreaks. Antimicrobial susceptibility was tested by calculating minimal inhibitory concentrations (MICs), and outbreaks were typed using pulsed-field gel electrophoresis (PFGE). Resistance mechanisms were explored by polymerase chain reaction (PCR) and whole genome sequencing (WGS).ResultsFollowing the intervention in 2011, the resistance rates of A. baumannii to almost all tested antibiotics decreased, from 85.3 to 72.6% for imipenem, 100 to 80.8% for ceftriaxone, and 45.0 to 6.9% for tigecycline. The intervention resulted in a decrease in the number (seven to five), duration (8–3 months), and departments (five to three) affected by outbreaks; no outbreaks occurred in 2011. After the intervention, only blaAMPC (76.47 to 100%) and blaTEM–1 (75.74 to 96.92%) increased (P < 0.0001); whereas blaGES–1 (32.35 to 3.07%), blaPER–1 (21.32 to 1.54%), blaOXA–58 (60.29 to 1.54%), carO (37.50 to 7.69%), and adeB (9.56 to 3.08%) decreased (P < 0.0001). Interestingly, the frequency of class B β-lactamase genes decreased from 91.18% (blaSPM–1) and 61.03% (blaIMP–1) to 0%, while that of class D blaOXA–23 increased to 96.92% (P < 0.0001). WGS showed that the major PFGE types causing outbreaks each year (type 01, 11, 18, 23, 26, and 31) carried the same resistance genes (blaKPC–1, blaADC–25, blaOXA–66, and adeABC), AdeR-S mutations (G186V and A136V), and a partially blocked porin channel CarO. Meanwhile, plasmids harboring blaOXA–23 were found after the intervention.ConclusionThe intervention was highly effective in reducing multi-drug resistance of A. baumannii and IRAB outbreaks in the long term. The resistance mechanisms of IRAB may involve genes encoding β-lactamases, efflux pump overexpression, outer membrane porin blockade, and plasmids; in particular, clonal spread of blaOXA–23 was the major cause of outbreaks. Similar interventions may also help reduce bacterial resistance rates and outbreaks in other hospitals.

scholarly journals Distribution of carbapenemases and efflux pump in carbopenems-resistance Acinetobacter baumannii

2016 ◽  
Author(s):  
Zhu-Qiang Qiu ◽  
Li-Jing Zhu ◽  
Pan-Fei Hou
Keyword(s):  
Acinetobacter Baumannii ◽  
Efflux Pump ◽  
Polymyxin B ◽  
Clinical Specimens ◽  
Expression Difference ◽  
Serious Infections ◽  
The World ◽  
Agar Dilution ◽  
First Time ◽  
Antibiotic Genes

Acinetobacter baumannii has emerged as an important pathogen related to serious infections and nosocomial outbreaks around the world. The aim of this study was to detect the distribution of carbapenemases and efflux pump in carbopenems-resistance Acinetobacter baumannii(CRAB). In this study, 100 isolates of CRAB were collected from clinical specimens. Agar dilution was conducted to determine the minimum inhibitory concentrations (MICs) to 15 kinds of antibiotic. Genes of carbapenemases and efflux pumps were amplified by PCR. The expression difference of pump genes was also analyzed by real-time PCR between CRAB and carbopenems- sensitive Acinetobacter baumannii (CSAB). We found that most antibiotics, including aminoglycosides, fluoroquinolones and cephalosporins showed high MIC values in CRAB. While, all isolates were sensitive to polymyxin B. Among CRAB, 54, 32 and 16 isolates were positive for SHV-12，PER-1 and TEM-1, respectively. 86 isolates were positive for OXA-23. 55, 33 and 5 isolates carried adeB, adeJ and adeE genes. The expression level of adeB in CRAB was ten times higher than that in CSAB. Moreover, isolates with single adeE gene were detected for the first time in Acinetobacter baumannii.

scholarly journals Azithromycin resistance through interspecific acquisition of an epistasis dependent efflux pump component and transcriptional regulator in Neisseria gonorrhoeae

2018 ◽  
Author(s):  
Crista B. Wadsworth ◽  
Brian J. Arnold ◽  
Mohamad R. Abdul Sater ◽  
Yonatan H. Grad
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Promoter Region ◽  
Structural Changes ◽  
Population Genomics ◽  
Efflux Pump ◽  
Genetic Manipulation ◽  
Full Length ◽  
Mechanism Of Resistance ◽  
Promoter Mutations ◽  
Azithromycin Resistance

ABSTRACTMosaic interspecifically acquired alleles of the multiple transferable resistance (mtr) efflux pump operon correlate with reduced susceptibility to azithromycin in Neisseria gonorrhoeae in epidemiological studies. However, whether and how these alleles cause resistance is unclear. Here, we use population genomics, transformations, and transcriptional analyses to dissect the relationship between variant mtr alleles and azithromycin resistance. We find that the locus encompassing the mtrR transcriptional repressor and the mtrCDE pump is a hotspot of interspecific recombination introducing alleles from N. meningitidis and N. lactamica into N. gonorrhoeae, with multiple rare haplotypes in linkage disequilibrium at mtrD and the mtr promoter region. Transformations demonstrated that resistance is mediated through epistasis between these two loci and that the full length of the mosaic mtrD allele is required. Gene expression profiling revealed the mechanism of resistance in mosaics couples the novel mtrDalleles with promoter mutations enhancing expression of the pump. Overall, our results demonstrate that epistatic interactions at mtr gained from multiple Neisseria has contributed to azithromycin resistance in the gonococcal population.AUTHOR SUMMARYNeisseria gonorrhoeae is the sexually transmitted bacterial pathogen responsible for over 100 million cases of gonorrhea worldwide each year. The incidence of reduced susceptibility to the macrolide class antibiotic azithromycin has increased in the past decade; however, a large proportion of the genetic basis of resistance to this drug remains unexplained. Recently, resistance has been shown to be highly associated with mosaic alleles of the multiple transferable resistance (mtr) efflux pump, which have been gained via horizontal gene exchange with other Neisseria. However, if and how these alleles caused resistance was unknown. Here, we demonstrate that resistance has been gained through epistasis between mtrD and the mtr promoter region using evidence from both population genomics and experimental genetic manipulation. Epistasis also acts within the mtrD locus alone, requiring the full length of the gene for phenotypic resistance. Transcriptomic profiling indicates that the mechanism of resistance in mosaics is likely derived from both structural changes to mtrD, coupled with promoter mutations that result in regulatory changes to mtrCDE.

scholarly journals High resolution melting analysis and detection of Leishmania resistance: the role of multi drug resistance 1 gene

2021 ◽  
Vol 43 (1) ◽  
Author(s):  
Maryam Fekri Soofi Abadi ◽  
Alireza Moradabadi ◽  
Reza Vahidi ◽  
Saeedeh Shojaeepour ◽  
Sara Rostami ◽  
...  
Keyword(s):  
Drug Resistance ◽  
High Resolution ◽  
High Resolution Melting ◽  
Efflux Pump ◽  
Mdr1 Gene ◽  
Multi Drug Resistance ◽  
Sequencing Method ◽  
Melting Analysis ◽  
Resistance Protein

Abstract Background Pentavalent antimonial compounds are currently used to treat leishmaniasis and resistance to these drugs is a serious problem. Multidrug resistance protein is an efflux pump of the cell membrane that expels foreign compounds. This study designed to evaluate the mutations in the multi-drug resistance 1 (MDR1) gene, in biopsy specimens of Leishmania tropica, with high resolution melting (HRM) method. In this experimental study, genomic DNA was extracted from 130 patients with skin leishmaniasis. Then, nucleotide changes were investigated throughout the gene using HRM and sequencing methods. The samples categorized in 5 groups by differences in the melting temperature (Tm). Result The nucleotide changes analysis showed that 61% of the samples of different groups that were unresponsive to drug had mutations in the MDR1 gene, which were also confirmed by the sequencing method. These mutations can be one of the factors responsible for non-responsiveness to the treatment. Conclusion According to the findings, it seems that mutation in MDR1 gene could be responsible for drug resistance to pentavalent antimonial compounds. Furthermore, HRM method can be used to diagnose drug resistance in leishmaniasis. It is also recommended that further studies be done regarding the importance of drug resistance in the leishmania affected patients.

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