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The Structural and Functional Study of Efflux Pumps Belonging to...
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The Structural and Functional Study of Efflux Pumps Belonging to the RND Transporters Family from Gram-Negative Bacteria

A special issue of Antibiotics (ISSN 2079-6382). This special issue belongs to the section "Mechanism and Evolution of Antibiotic Resistance".

Deadline for manuscript submissions: closed (30 November 2021) | Viewed by 34240

Printed Edition Available!
A printed edition of this Special Issue is available here.

Special Issue Editors

Dr. Isabelle Broutin

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Guest Editor
Faculty of Pharmacy, Université de Paris, CNRS, laboratory CiTCoM
Interests: efflux pumps;structure;antibiotic resistance;membrane proteins
Prof. Attilio V Vargiu

E-Mail Website
Guest Editor
Physics, University of Cagliari, Cagliari, Italy
Interests: efflux pumps;molecular dynamics;enhanced-sampling methods;molecular docking;hydrogels
Prof. Dr. Henrietta Venter

E-Mail Website
Guest Editor
School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, Australia
Interests: antimicrobial resistance; drug efflux proteins; novel antibiotics; mechanisms of resistance; multidrug resistance
Special Issues, Collections and Topics in MDPI journals
Dr. Gilles Phan

E-Mail Website
Guest Editor
Université de Paris, CNRS, laboratory CiTCoM
Interests: bacterial efflux pumps; secretion systems; two-components systems

Special Issue Information

Dear Colleagues,

Antimicrobial-resistant bacterial infections are a major and costly public health concern. Several pathogens are already pan-resistant, representing a major cause of mortality in patients suffering from nosocomial infections. Drug efflux pumps, which remove compounds from the bacterial cell, thereby lowering antimicrobial concentration to subtoxic levels, play a major role in multidrug resistance.

Gram-negative bacteria are particularly resistant, and some are identified by the World Health Organization as the most urgent priority of pathogens in need of new antimicrobial drug discovery. The clinically most relevant efflux pumps in Gram-negative bacteria belong to the RND (resistance nodulation cell division) family that forms a tripartite macromolecular assembly spanning both membranes and the periplasmic space of Gram-negative organisms. Along with functional studies and in silico approaches, many structures of the individual components, as well as that of the fully assembled pumps from several pathogens, have been solved. Nevertheless, a lot of questions concerning the assembly and the mechanism of efflux remain, and there are still no inhibitors of efflux pumps available in clinical treatment. 

In this Special Issue, we would like to present up-to-date knowledge of the mechanism of these efflux pumps, the identification and characterization of RND efflux pumps from emerging pathogens and their role in antimicrobial resistance, as well as progress made on the development of specific inhibitors (including the development of in vitro or in vivo tools for inhibitor selection). This collection of data could serve as a basis for antimicrobial drug discovery aimed at inhibiting drug efflux pumps to reverse resistance in some of the most resistant pathogens.

Dr. Isabelle Broutin
Prof. Attilio V Vargiu
Prof. Dr. Henrietta Venter
Dr. Gilles Phan
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Antibiotics is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2900 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • Antimicrobial resistance
  • RND transporters
  • Drug transport
  • Efflux pump
  • Structural biology
  • Molecular dynamics
  • Liposome
  • In cellulo measurement
  • Efflux pump inhibitor

Published Papers (10 papers)

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Editorial

Jump to: Research, Review

3 pages, 185 KiB  
Editorial
The Structural and Functional Study of Efflux Pumps Belonging to the RND Transporters Family from Gram-Negative Bacteria
by Attilio Vittorio Vargiu, Gilles Phan, Henrietta Venter and Isabelle Broutin
Antibiotics 2022, 11(4), 429; https://0-doi-org.brum.beds.ac.uk/10.3390/antibiotics11040429 - 23 Mar 2022
Cited by 1 | Viewed by 1709
Abstract
Antimicrobial-resistant bacterial infections are a major and costly public health concern [...] Full article
(This article belongs to the Special Issue The Structural and Functional Study of Efflux Pumps Belonging to the RND Transporters Family from Gram-Negative Bacteria)

Research

Jump to: Editorial, Review

15 pages, 3629 KiB  
Article
Molecular Determinants for OMF Selectivity in Tripartite RND Multidrug Efflux Systems
by Esther Boyer, Jean Dessolin, Margaux Lustig, Marion Decossas, Gilles Phan, Quentin Cece, Grégory Durand, Véronique Dubois, Joris Sansen, Jean-Christophe Taveau, Isabelle Broutin, Laetitia Daury and Olivier Lambert
Antibiotics 2022, 11(2), 126; https://0-doi-org.brum.beds.ac.uk/10.3390/antibiotics11020126 - 18 Jan 2022
Cited by 5 | Viewed by 2750
Abstract
Tripartite multidrug RND efflux systems made of an inner membrane transporter, an outer membrane factor (OMF) and a periplasmic adaptor protein (PAP) form a canal to expel drugs across Gram-negative cell wall. Structures of MexA–MexB–OprM and AcrA–AcrB–TolC, from Pseudomonas aeruginosa and Escherichia coli [...] Read more.
Tripartite multidrug RND efflux systems made of an inner membrane transporter, an outer membrane factor (OMF) and a periplasmic adaptor protein (PAP) form a canal to expel drugs across Gram-negative cell wall. Structures of MexA–MexB–OprM and AcrA–AcrB–TolC, from Pseudomonas aeruginosa and Escherichia coli, respectively, depict a reduced interfacial contact between OMF and PAP, making unclear the comprehension of how OMF is recruited. Here, we show that a Q93R mutation of MexA located in the α-hairpin domain increases antibiotic resistance in the MexAQ93R–MexB–OprM-expressed strain. Electron microscopy single-particle analysis reveals that this mutation promotes the formation of tripartite complexes with OprM and non-cognate components OprN and TolC. Evidence indicates that MexAQ93R self-assembles into a hexameric form, likely due to interprotomer interactions between paired R93 and D113 amino acids. C-terminal deletion of OprM prevents the formation of tripartite complexes when mixed with MexA and MexB components but not when replacing MexA with MexAQ93R. This study reveals the Q93R MexA mutation and the OprM C-terminal peptide as molecular determinants modulating the assembly process efficacy with cognate and non-cognate OMFs, even though they are outside the interfacial contact. It provides insights into how OMF selectivity operates during the formation of the tripartite complex. Full article
(This article belongs to the Special Issue The Structural and Functional Study of Efflux Pumps Belonging to the RND Transporters Family from Gram-Negative Bacteria)
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21 pages, 4122 KiB  
Article
A Model for Allosteric Communication in Drug Transport by the AcrAB-TolC Tripartite Efflux Pump
by Anya Webber, Malitha Ratnaweera, Andrzej Harris, Ben F. Luisi and Véronique Yvette Ntsogo Enguéné
Antibiotics 2022, 11(1), 52; https://0-doi-org.brum.beds.ac.uk/10.3390/antibiotics11010052 - 01 Jan 2022
Cited by 6 | Viewed by 2407
Abstract
RND family efflux pumps are complex macromolecular machines involved in multidrug resistance by extruding antibiotics from the cell. While structural studies and molecular dynamics simulations have provided insights into the architecture and conformational states of the pumps, the path followed by conformational changes [...] Read more.
RND family efflux pumps are complex macromolecular machines involved in multidrug resistance by extruding antibiotics from the cell. While structural studies and molecular dynamics simulations have provided insights into the architecture and conformational states of the pumps, the path followed by conformational changes from the inner membrane protein (IMP) to the periplasmic membrane fusion protein (MFP) and to the outer membrane protein (OMP) in tripartite efflux assemblies is not fully understood. Here, we investigated AcrAB-TolC efflux pump’s allostery by comparing resting and transport states using difference distance matrices supplemented with evolutionary couplings data and buried surface area measurements. Our analysis indicated that substrate binding by the IMP triggers quaternary level conformational changes in the MFP, which induce OMP to switch from the closed state to the open state, accompanied by a considerable increase in the interface area between the MFP subunits and between the OMPs and MFPs. This suggests that the pump’s transport-ready state is at a more favourable energy level than the resting state, but raises the puzzle of how the pump does not become stably trapped in a transport-intermediate state. We propose a model for pump allostery that includes a downhill energetic transition process from a proposed ‘activated’ transport state back to the resting pump. Full article
(This article belongs to the Special Issue The Structural and Functional Study of Efflux Pumps Belonging to the RND Transporters Family from Gram-Negative Bacteria)
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12 pages, 2200 KiB  
Article
Characterization and Molecular Determinants for β-Lactam Specificity of the Multidrug Efflux Pump AcrD from Salmonella typhimurium
by Jenifer Cuesta Bernal, Jasmin El-Delik, Stephan Göttig and Klaas M. Pos
Antibiotics 2021, 10(12), 1494; https://0-doi-org.brum.beds.ac.uk/10.3390/antibiotics10121494 - 06 Dec 2021
Cited by 4 | Viewed by 2577
Abstract
Gram-negative Tripartite Resistance Nodulation and cell Division (RND) superfamily efflux pumps confer various functions, including multidrug and bile salt resistance, quorum-sensing, virulence and can influence the rate of mutations on the chromosome. Multidrug RND efflux systems are often characterized by a wide substrate [...] Read more.
Gram-negative Tripartite Resistance Nodulation and cell Division (RND) superfamily efflux pumps confer various functions, including multidrug and bile salt resistance, quorum-sensing, virulence and can influence the rate of mutations on the chromosome. Multidrug RND efflux systems are often characterized by a wide substrate specificity. Similarly to many other RND efflux pump systems, AcrAD-TolC confers resistance toward SDS, novobiocin and deoxycholate. In contrast to the other pumps, however, it in addition confers resistance against aminoglycosides and dianionic β-lactams, such as sulbenicillin, aztreonam and carbenicillin. Here, we could show that AcrD from Salmonella typhimurium confers resistance toward several hitherto unreported AcrD substrates such as temocillin, dicloxacillin, cefazolin and fusidic acid. In order to address the molecular determinants of the S. typhimurium AcrD substrate specificity, we conducted substitution analyses in the putative access and deep binding pockets and in the TM1/TM2 groove region. The variants were tested in E. coli ΔacrBΔacrD against β-lactams oxacillin, carbenicillin, aztreonam and temocillin. Deep binding pocket variants N136A, D276A and Y327A; access pocket variant R625A; and variants with substitutions in the groove region between TM1 and TM2 conferred a sensitive phenotype and might, therefore, be involved in anionic β-lactam export. In contrast, lower susceptibilities were observed for E. coli cells harbouring deep binding pocket variants T139A, D176A, S180A, F609A, T611A and F627A and the TM1/TM2 groove variant I337A. This study provides the first insights of side chains involved in drug binding and transport for AcrD from S. typhimurium. Full article
(This article belongs to the Special Issue The Structural and Functional Study of Efflux Pumps Belonging to the RND Transporters Family from Gram-Negative Bacteria)
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17 pages, 2309 KiB  
Article
Insight into the AcrAB-TolC Complex Assembly Process Learned from Competition Studies
by Prasangi Rajapaksha, Isoiza Ojo, Ling Yang, Ankit Pandeya, Thilini Abeywansha and Yinan Wei
Antibiotics 2021, 10(7), 830; https://0-doi-org.brum.beds.ac.uk/10.3390/antibiotics10070830 - 08 Jul 2021
Cited by 8 | Viewed by 3869
Abstract
The RND family efflux pump AcrAB-TolC in E. coli and its homologs in other Gram-negative bacteria are major players in conferring multidrug resistance to the cells. While the structure of the pump complex has been elucidated with ever-increasing resolution through crystallography and Cryo-EM [...] Read more.
The RND family efflux pump AcrAB-TolC in E. coli and its homologs in other Gram-negative bacteria are major players in conferring multidrug resistance to the cells. While the structure of the pump complex has been elucidated with ever-increasing resolution through crystallography and Cryo-EM efforts, the dynamic assembly process remains poorly understood. Here, we tested the effect of overexpressing functionally defective pump components in wild type E. coli cells to probe the pump assembly process. Incorporation of a defective component is expected to reduce the efflux efficiency of the complex, leading to the so called “dominant negative” effect. Being one of the most intensively studied bacterial multidrug efflux pumps, many AcrA and AcrB mutations have been reported that disrupt efflux through different mechanisms. We examined five groups of AcrB and AcrA mutants, defective in different aspects of assembly and substrate efflux. We found that none of them demonstrated the expected dominant negative effect, even when expressed at concentrations many folds higher than their genomic counterpart. The assembly of the AcrAB-TolC complex appears to have a proof-read mechanism that effectively eliminated the formation of futile pump complex. Full article
(This article belongs to the Special Issue The Structural and Functional Study of Efflux Pumps Belonging to the RND Transporters Family from Gram-Negative Bacteria)
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15 pages, 2408 KiB  
Article
RND Efflux Systems Contribute to Resistance and Virulence of Aliarcobacter butzleri
by Cristiana Mateus, Ana Rita Nunes, Mónica Oleastro, Fernanda Domingues and Susana Ferreira
Antibiotics 2021, 10(7), 823; https://0-doi-org.brum.beds.ac.uk/10.3390/antibiotics10070823 - 06 Jul 2021
Cited by 8 | Viewed by 2390
Abstract
Aliarcobacter butzleri is an emergent enteropathogen that can be found in a range of environments. This bacterium presents a vast repertoire of efflux pumps, such as the ones belonging to the resistance nodulation cell division family, which may be associated with bacterial resistance, [...] Read more.
Aliarcobacter butzleri is an emergent enteropathogen that can be found in a range of environments. This bacterium presents a vast repertoire of efflux pumps, such as the ones belonging to the resistance nodulation cell division family, which may be associated with bacterial resistance, as well as virulence. Thus, this work aimed to evaluate the contribution of three RND efflux systems, AreABC, AreDEF and AreGHI, in the resistance and virulence of A. butzleri. Mutant strains were constructed by inactivation of the gene that encodes the inner membrane protein of these systems. The bacterial resistance profile of parental and mutant strains to several antimicrobials was assessed, as was the intracellular accumulation of the ethidium bromide dye. Regarding bacterial virulence, the role of these three efflux pumps on growth, strain fitness, motility, biofilm formation ability, survival in adverse conditions (oxidative stress and bile salts) and human serum and in vitro adhesion and invasion to Caco-2 cells was evaluated. We observed that the mutants from the three efflux pumps were more susceptible to several classes of antimicrobials than the parental strain and presented an increase in the accumulation of ethidium bromide, indicating a potential role of the efflux pumps in the extrusion of antimicrobials. The mutant strains had no bacterial growth defects; nonetheless, they presented a reduction in relative fitness. For the three mutants, an increase in the susceptibility to oxidative stress was observed, while only the mutant for AreGHI efflux pump showed a relevant role in bile stress survival. All the mutant strains showed an impairment in biofilm formation ability, were more susceptible to human serum and were less adherent to intestinal epithelial cells. Overall, the results support the contribution of the efflux pumps AreABC, AreDEF and AreGHI of A. butzleri to antimicrobial resistance, as well as to bacterial virulence. Full article
(This article belongs to the Special Issue The Structural and Functional Study of Efflux Pumps Belonging to the RND Transporters Family from Gram-Negative Bacteria)
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8 pages, 926 KiB  
Communication
Exploring the Contribution of the AcrB Homolog MdtF to Drug Resistance and Dye Efflux in a Multidrug Resistant E. coli Isolate
by Sabine Schuster, Martina Vavra, Ludwig Greim and Winfried V. Kern
Antibiotics 2021, 10(5), 503; https://0-doi-org.brum.beds.ac.uk/10.3390/antibiotics10050503 - 28 Apr 2021
Cited by 9 | Viewed by 1990
Abstract
In Escherichia coli, the role of RND-type drug transporters other than the major efflux pump AcrB has largely remained undeciphered (particularly in multidrug resistant pathogens), because genetic engineering in such isolates is challenging. The present study aimed to explore the capability of [...] Read more.
In Escherichia coli, the role of RND-type drug transporters other than the major efflux pump AcrB has largely remained undeciphered (particularly in multidrug resistant pathogens), because genetic engineering in such isolates is challenging. The present study aimed to explore the capability of the AcrB homolog MdtF to contribute to the extrusion of noxious compounds and to multidrug resistance in an E. coli clinical isolate with demonstrated expression of this efflux pump. An mdtF/acrB double-knockout was engineered, and susceptibility changes with drugs from various classes were determined in comparison to the parental strain and its acrB and tolC single-knockout mutants. The potential of MdtF to participate in the export of agents with different physicochemical properties was additionally assessed using accumulation and real-time efflux assays with several fluorescent dyes. The results show that there was limited impact to the multidrug resistant phenotype in the tested E. coli strain, while the RND-type transporter remarkably contributes to the efflux of all tested dyes. This should be considered when evaluating the efflux phenotype of clinical isolates via dye accumulation assays. Furthermore, the promiscuity of MdtF should be taken into account when developing new antibiotic agents. Full article
(This article belongs to the Special Issue The Structural and Functional Study of Efflux Pumps Belonging to the RND Transporters Family from Gram-Negative Bacteria)
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Review

Jump to: Editorial, Research

23 pages, 584 KiB  
Review
Clinical Status of Efflux Resistance Mechanisms in Gram-Negative Bacteria
by Anne Davin-Regli, Jean-Marie Pages and Aurélie Ferrand
Antibiotics 2021, 10(9), 1117; https://0-doi-org.brum.beds.ac.uk/10.3390/antibiotics10091117 - 16 Sep 2021
Cited by 17 | Viewed by 4524
Abstract
Antibiotic efflux is a mechanism that is well-documented in the phenotype of multidrug resistance in bacteria. Efflux is considered as an early facilitating mechanism in the bacterial adaptation face to the concentration of antibiotics at the infectious site, which is involved in the [...] Read more.
Antibiotic efflux is a mechanism that is well-documented in the phenotype of multidrug resistance in bacteria. Efflux is considered as an early facilitating mechanism in the bacterial adaptation face to the concentration of antibiotics at the infectious site, which is involved in the acquirement of complementary efficient mechanisms, such as enzymatic resistance or target mutation. Various efflux pumps have been described in the Gram-negative bacteria most often encountered in infectious diseases and, in healthcare-associated infections. Some are more often involved than others and expel virtually all families of antibiotics and antibacterials. Numerous studies report the contribution of these pumps in resistant strains previously identified from their phenotypes. The authors characterize the pumps involved, the facilitating antibiotics and those mainly concerned by the efflux. However, today no study describes a process for the real-time quantification of efflux in resistant clinical strains. It is currently necessary to have at hospital level a reliable and easy method to quantify the efflux in routine and contribute to a rational choice of antibiotics. This review provides a recent overview of the prevalence of the main efflux pumps observed in clinical practice and provides an idea of the prevalence of this mechanism in the multidrug resistant Gram-negative bacteria. The development of a routine diagnostic tool is now an emergency need for the proper application of current recommendations regarding a rational use of antibiotics. Full article
(This article belongs to the Special Issue The Structural and Functional Study of Efflux Pumps Belonging to the RND Transporters Family from Gram-Negative Bacteria)
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25 pages, 465 KiB  
Review
Role of RND Efflux Pumps in Drug Resistance of Cystic Fibrosis Pathogens
by Viola Camilla Scoffone, Gabriele Trespidi, Giulia Barbieri, Samuele Irudal, Elena Perrin and Silvia Buroni
Antibiotics 2021, 10(7), 863; https://0-doi-org.brum.beds.ac.uk/10.3390/antibiotics10070863 - 15 Jul 2021
Cited by 19 | Viewed by 4014
Abstract
Drug resistance represents a great concern among people with cystic fibrosis (CF), due to the recurrent and prolonged antibiotic therapy they should often undergo. Among Multi Drug Resistance (MDR) determinants, Resistance-Nodulation-cell Division (RND) efflux pumps have been reported as the main contributors, due [...] Read more.
Drug resistance represents a great concern among people with cystic fibrosis (CF), due to the recurrent and prolonged antibiotic therapy they should often undergo. Among Multi Drug Resistance (MDR) determinants, Resistance-Nodulation-cell Division (RND) efflux pumps have been reported as the main contributors, due to their ability to extrude a wide variety of molecules out of the bacterial cell. In this review, we summarize the principal RND efflux pump families described in CF pathogens, focusing on the main Gram-negative bacterial species (Pseudomonas aeruginosa, Burkholderia cenocepacia, Achromobacter xylosoxidans, Stenotrophomonas maltophilia) for which a predominant role of RND pumps has been associated to MDR phenotypes. Full article
(This article belongs to the Special Issue The Structural and Functional Study of Efflux Pumps Belonging to the RND Transporters Family from Gram-Negative Bacteria)
32 pages, 4684 KiB  
Review
Ever-Adapting RND Efflux Pumps in Gram-Negative Multidrug-Resistant Pathogens: A Race against Time
by Martijn Zwama and Kunihiko Nishino
Antibiotics 2021, 10(7), 774; https://0-doi-org.brum.beds.ac.uk/10.3390/antibiotics10070774 - 25 Jun 2021
Cited by 42 | Viewed by 6290
Abstract
The rise in multidrug resistance (MDR) is one of the greatest threats to human health worldwide. MDR in bacterial pathogens is a major challenge in healthcare, as bacterial infections are becoming untreatable by commercially available antibiotics. One of the main causes of MDR [...] Read more.
The rise in multidrug resistance (MDR) is one of the greatest threats to human health worldwide. MDR in bacterial pathogens is a major challenge in healthcare, as bacterial infections are becoming untreatable by commercially available antibiotics. One of the main causes of MDR is the over-expression of intrinsic and acquired multidrug efflux pumps, belonging to the resistance-nodulation-division (RND) superfamily, which can efflux a wide range of structurally different antibiotics. Besides over-expression, however, recent amino acid substitutions within the pumps themselves—causing an increased drug efflux efficiency—are causing additional worry. In this review, we take a closer look at clinically, environmentally and laboratory-evolved Gram-negative bacterial strains and their decreased drug sensitivity as a result of mutations directly in the RND-type pumps themselves (from Escherichia coli, Salmonella enterica, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Acinetobacter baumannii and Legionella pneumophila). We also focus on the evolution of the efflux pumps by comparing hundreds of efflux pumps to determine where conservation is concentrated and where differences in amino acids can shed light on the broad and even broadening drug recognition. Knowledge of conservation, as well as of novel gain-of-function efflux pump mutations, is essential for the development of novel antibiotics and efflux pump inhibitors. Full article
(This article belongs to the Special Issue The Structural and Functional Study of Efflux Pumps Belonging to the RND Transporters Family from Gram-Negative Bacteria)
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