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Mapping the Dynamic Functions and Structural Features of AcrB Efflux Pump Transporter Using Accelerated Molecular Dynamics Simulations.

ABSTRACT: Multidrug efflux pumps confer resistance to their bacterial hosts by pumping out a diverse range of compounds, including most antibiotics. Being more familiar with the details of functional dynamics and conformations of these types of pumps could help in discovering approaches to stop them functioning properly. Computational approaches, particularly conventional molecular dynamics simulations followed by diverse post simulation analysis, are powerful methods that help researchers by opening a new window to study phenomena that are not detectable in as much detail in vitro or in vivo as they are in silico. In this study, accelerated molecular dynamics simulations were applied to study the dynamics of AcrB efflux pump transporters in interaction with PA?N and tetracycline as an inhibitor and a substrate, respectively, to compare the differences in the dynamics and consequently the mechanism of action of the pump. The different dynamics for PA?N -bound form of AcrB compared to the TET-bound form is likely to affect the rotating mechanism typically observed for AcrB transporter. This shows the dynamics of the active AcrB transporter is different in a substrate-bound state compared to an inhibitor-bound state. This advances our knowledge and helps to unravel the mechanism of tripartite efflux pumps.

SUBMITTER: Jamshidi S

PROVIDER: S-EPMC6041327 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Mapping the Dynamic Functions and Structural Features of AcrB Efflux Pump Transporter Using Accelerated Molecular Dynamics Simulations.

Jamshidi Shirin S Sutton J Mark JM Rahman Khondaker Miraz KM

Scientific reports 20180711 1

Multidrug efflux pumps confer resistance to their bacterial hosts by pumping out a diverse range of compounds, including most antibiotics. Being more familiar with the details of functional dynamics and conformations of these types of pumps could help in discovering approaches to stop them functioning properly. Computational approaches, particularly conventional molecular dynamics simulations followed by diverse post simulation analysis, are powerful methods that help researchers by opening a ne ...[more]

PMID: 29992991

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Project description:The continued challenges of the COVID-19 pandemic combined with the growing problem of antimicrobial-resistant bacterial infections has severely impacted global health. Specifically, the Gram-negative pathogen Klebsiella pneumoniae is one of the most prevalent causes of secondary bacterial infection in COVID-19 patients, with approximately an 83% mortality rate observed among COVID-19 patients with these bacterial coinfections. K. pneumoniae belongs to the ESKAPE group of pathogens, a group that commonly gives rise to severe infections that are often life-threatening. Recently, K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae has drawn wide public attention, as the mortality rate for this infection can be as high as 71%. The most predominant and clinically important multidrug efflux system in K. pneumoniae is the acriflavine resistance B (AcrB) multidrug efflux pump. This pump mediates resistance to different classes of structurally diverse antimicrobial agents, including quinolones, β-lactams, tetracyclines, macrolides, aminoglycosides, and chloramphenicol. We here report single-particle cryo-electron microscopy (cryo-EM) structures of K. pneumoniae AcrB, in both the absence and the presence of the antibiotic erythromycin. These structures allow us to elucidate specific pump-drug interactions and pinpoint exactly how this pump recognizes antibiotics. IMPORTANCE Klebsiella pneumoniae has emerged as one of the most problematic and highly antibiotic-resistant pathogens worldwide. It is the second most common causative agent involved in secondary bacterial infection in COVID-19 patients. K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae is a major concern in global public health because of the high mortality rate of this infection. Its drug resistance is due, in a significant part, to active efflux of these bactericides, a major mechanism that K. pneumoniae uses to resist to the action of multiple classes of antibiotics. Here, we report cryo-electron microscopy (cryo-EM) structures of the prevalent and clinically important K. pneumoniae AcrB multidrug efflux pump, in both the absence and the presence of the erythromycin antibiotic. These structures allow us to understand the action mechanism for drug recognition in this pump. Our studies will ultimately inform an era in structure-guided drug design to combat multidrug resistance in these Gram-negative pathogens.

Project description:Efflux is an important mechanism in Gram-negative bacteria conferring multidrug resistance. Inhibition of efflux is an encouraging strategy to restore the antibacterial activity of antibiotics. Chlorpromazine and amitriptyline have been shown to behave as efflux inhibitors. However, their mode of action is poorly understood. Exposure of Salmonella enterica serovar Typhimurium and Escherichia coli to chlorpromazine selected for mutations within genes encoding RamR and MarR, regulators of the multidrug tripartite efflux pump AcrAB-TolC. Further experiments with S. Typhimurium containing AcrB D408A (a nonfunctional efflux pump) and chlorpromazine or amitriptyline resulted in the reversion of the mutant acrB allele to the wild type. Together, this suggests these drugs are AcrB efflux substrates. Subsequent docking studies with AcrB from S. Typhimurium and E. coli, followed by molecular dynamics simulations and free energy calculations showed that chlorpromazine and amitriptyline bind at the hydrophobic trap, a preferred binding site for substrates and inhibitors within the distal binding pocket of AcrB. Based on these simulations, we suggest that chlorpromazine and amitriptyline inhibit AcrB-mediated efflux by interfering with substrate binding. Our findings provide evidence that these drugs are substrates and inhibitors of AcrB, yielding molecular details of their mechanism of action and informing drug discovery of new efflux inhibitors.IMPORTANCE 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.

Dataset Information

Mapping the Dynamic Functions and Structural Features of AcrB Efflux Pump Transporter Using Accelerated Molecular Dynamics Simulations.

Publications

Mapping the Dynamic Functions and Structural Features of AcrB Efflux Pump Transporter Using Accelerated Molecular Dynamics Simulations.

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