Bacterial Multidrug Efflux Pumps Research Articles

Role of bacterial multidrug efflux pumps during infection.

Multidrug efflux pumps are protein complexes located in the cell envelope that enable bacteria to expel, not only antibiotics, but also a wide array of molecules relevant for infection. Hence, they are important players in microbial pathogenesis. On the one hand, efflux pumps can extrude exogenous compounds, including host-produced antimicrobial molecules. Through this extrusion, pathogens can resist antimicrobial agents and evade host defenses. On the other hand, efflux pumps also have a role in the extrusion of endogenous compounds, such as bacterial intercommunication signaling molecules, virulence factors or metabolites. Therefore, efflux pumps are involved in the modulation of bacterial behavior and virulence, as well as in the maintenance of the bacterial homeostasis under different stresses found within the host. This review delves into the multifaceted roles that efflux pumps have, shedding light on their impact on bacterial virulence and their contribution to bacterial infection. These observations suggest that strategies targeting bacterial efflux pumps could both reinvigorate the efficacy of existing antibiotics and modulate the bacterial pathogenicity to the host. Thus, a comprehensive understanding of bacterial efflux pumps can be pivotal for the development of new effective strategies for the management of infectious diseases.

Insight into the AcrAB-TolC Complex Assembly Process Learned from Competition Studies.

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.

Physiological Functions of Bacterial “Multidrug” Efflux Pumps

Bacterial multidrug efflux pumps have come to prominence in human and veterinary pathogenesis because they help bacteria protect themselves against the antimicrobials used to overcome their infections. However, it is increasingly realized that many, probably most, such pumps have physiological roles that are distinct from protection of bacteria against antimicrobials administered by humans. Here we undertake a broad survey of the proteins involved, allied to detailed examples of their evolution, energetics, structures, chemical recognition, and molecular mechanisms, together with the experimental strategies that enable rapid and economical progress in understanding their true physiological roles. Once these roles are established, the knowledge can be harnessed to design more effective drugs, improve existing microbial production of drugs for clinical practice and of feedstocks for commercial exploitation, and even develop more sustainable biological processes that avoid, for example, utilization of petroleum.

Functional and Structural Roles of the Major Facilitator Superfamily Bacterial Multidrug Efflux Pumps.

Pathogenic microorganisms that are multidrug-resistant can pose severe clinical and public health concerns. In particular, bacterial multidrug efflux transporters of the major facilitator superfamily constitute a notable group of drug resistance mechanisms primarily because multidrug-resistant pathogens can become refractory to antimicrobial agents, thus resulting in potentially untreatable bacterial infections. The major facilitator superfamily is composed of thousands of solute transporters that are related in terms of their phylogenetic relationships, primary amino acid sequences, two- and three-dimensional structures, modes of energization (passive and secondary active), and in their mechanisms of solute and ion translocation across the membrane. The major facilitator superfamily is also composed of numerous families and sub-families of homologous transporters that are conserved across all living taxa, from bacteria to humans. Members of this superfamily share several classes of highly conserved amino acid sequence motifs that play essential mechanistic roles during transport. The structural and functional importance of multidrug efflux pumps that belong to the major facilitator family and that are harbored by Gram-negative and -positive bacterial pathogens are considered here.

PLGA microsphere-based composite hydrogel for dual delivery of ciprofloxacin and ginsenoside Rh2 to treat Staphylococcus aureus-induced skin infections

When antibiotic-resistant pathogenic bacteria pose a high threat to human health, bacterial multidrug efflux pumps become major contributors to the high-level antibiotic resistance in most microorganisms. Since traditional antibiotics are still indispensable currently, we report a dual drug delivery system to maximize the antibacterial efficacy of antibiotics by inhibiting efflux pumps in bacteria before their exposure to antibiotics. In this research, a microsphere/hydrogel composite was constructed from ciprofloxacin (Cip)-loaded poly (lactic-co-glycolic acid) (PLGA) microspheres and ginsenoside Rh2 (G-Rh2) dispersed thermo-sensitive hydrogel to treat skin infections. In vitro drug release studies indicated that while G-Rh2 in hydrogel presented a faster and short-term release manner to rapidly inhibit the NorA efflux pumps, Cip showed a sustained and long-term release behavior to provide a local high concentration gradient for facilitating drug percutaneous penetration. The combination of Cip and G-Rh2 demonstrated a high degree of synergism against both methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA), hence significantly improving their in vitro antibacterial activity and efficiency. Moreover, the antibacterial performance of the microsphere/hydrogel composite with a sequential release profile is superior to that of other formulations in mouse model of MRSA skin infections, indicating its great potential to treat antibiotic-resistant skin infections.

Computer simulations of the activity of RND efflux pumps

The putative mechanism by which bacterial RND-type multidrug efflux pumps recognize and transport their substrates is a complex and fascinating enigma of structural biology. How a single protein can recognize a huge number of unrelated compounds and transport them through one or just a few mechanisms is an amazing feature not yet completely unveiled. The appearance of cooperativity further complicates the understanding of structure-dynamics-activity relationships in these complex machineries. Experimental techniques may have limited access to the molecular determinants and to the energetics of key processes regulating the activity of these pumps. Computer simulations are a complementary approach that can help unveil these features and inspire new experiments. Here we review recent computational studies that addressed the various molecular processes regulating the activity of RND efflux pumps.

Study of the Expression of Bacterial Multidrug Efflux Pumps in Anaerobic Conditions.

Bacterial multidrug efflux pumps belong to a class of membrane transporter proteins that dedicate to the extrusion of a diverse range of substances out of cells including all classes of currently available antibiotics. They constitute an important mechanism of bacterial antibiotic and multidrug resistance. Since many ecological niches of bacteria and the infection foci in animal host display low oxygen tension under which condition bacterial pathogens undergo fundamental changes on their metabolic modes, it is necessary to study the expression profiles of drug efflux pumps under these physiologically and clinically relevant conditions. In this chapter, we first introduce procedures to culture bacteria under anaerobic conditions, which is achieved using screw-capped Pyrex culture tubes without agitation. We then introduce β-galactosidase activity assay using promoter-lacZ (encoding the β-galactosidase enzyme) fusion to measure the expression of efflux pumps at transcriptional level, and Western blot using chromosomal FLAG-tagged construct to examine the expression of these proteins at translational level. Applications of these gene expression studies to reveal the regulatory mechanisms of efflux genes expression as well as their physiological functions are also discussed.

Bacterial Multidrug Efflux Pumps of the Major Facilitator Superfamily as Targets for Modulation.

Causative agents of infectious disease that are multidrug resistant bacterial pathogens represent a serious public health concern due to the increasingly difficult nature of achieving efficacious clinical treatments. Of the various acquired and intrinsic antimicrobial agent resistance determinants, integral-membrane multidrug efflux pumps of the major facilitator superfamily constitute a major mechanism of bacterial resistance. The major facilitator superfamily (MFS) encompasses thousands of known related secondary active and passive solute transporters, including multidrug efflux pumps, from bacteria to humans. This review article addresses recent developments involving the targeting by various modulators of bacterial multidrug efflux pumps from the major facilitator superfamily. It is currently of tremendous interest to modulate bacterial multidrug efflux pumps in order to eventually restore the clinical efficacy of therapeutic agents against recalcitrant bacterial infections. Such MFS multidrug efflux pumps are good targets for modulation.

Current Advances in Developing Inhibitors of Bacterial Multidrug Efflux Pumps.

Antimicrobial resistance represents a significant challenge to future healthcare provision. An acronym ESKAPEE has been derived from the names of the organisms recognised as the major threats although there are a number of other organisms, notably Neisseria gonorrhoeae, that have become equally challenging to treat in the clinic. These pathogens are characterised by the ability to rapidly develop and/or acquire resistance mechanisms in response to exposure to different antimicrobial agents. A key part of the armoury of these pathogens is a series of efflux pumps, which effectively exclude or reduce the intracellular concentration of a large number of antibiotics, making the pathogens significantly more resistant. These efflux pumps are the topic of considerable interest, both from the perspective of basic understanding of efflux pump function, and its role in drug resistance but also as targets for the development of novel adjunct therapies. The necessity to overcome antimicrobial resistance has encouraged investigations into the characterisation of resistance-modifying efflux pump inhibitors to block the mechanisms of drug extrusion, thereby restoring antibacterial susceptibility and returning existing antibiotics into the clinic. A greater understanding of drug recognition and transport by multidrug efflux pumps is needed to develop clinically useful inhibitors, given the breadth of molecules that can be effluxed by these systems. This review discusses different bacterial EPIs originating from both natural source and chemical synthesis and examines the challenges to designing successful EPIs that can be useful against multidrug resistant bacteria.

Bacterial Multidrug Efflux Pumps: Much More Than Antibiotic Resistance Determinants.

Bacterial multidrug efflux pumps are antibiotic resistance determinants present in all microorganisms. With few exceptions, they are chromosomally encoded and present a conserved organization both at the genetic and at the protein levels. In addition, most, if not all, strains of a given bacterial species present the same chromosomally-encoded efflux pumps. Altogether this indicates that multidrug efflux pumps are ancient elements encoded in bacterial genomes long before the recent use of antibiotics for human and animal therapy. In this regard, it is worth mentioning that efflux pumps can extrude a wide range of substrates that include, besides antibiotics, heavy metals, organic pollutants, plant-produced compounds, quorum sensing signals or bacterial metabolites, among others. In the current review, we present information on the different functions that multidrug efflux pumps may have for the bacterial behaviour in different habitats as well as on their regulation by specific signals. Since, in addition to their function in non-clinical ecosystems, multidrug efflux pumps contribute to intrinsic, acquired, and phenotypic resistance of bacterial pathogens, the review also presents information on the search for inhibitors of multidrug efflux pumps, which are currently under development, in the aim of increasing the susceptibility of bacterial pathogens to antibiotics.

Bacterial Multidrug Efflux Pumps of the Major Facilitator Superfamily as Targets for Modulation

Bacterial Multidrug Efflux Pumps of the Major Facilitator Superfamily as Targets for Modulation

Multidrug efflux pumps in Staphylococcus aureus and their clinical implications.

Antibiotic resistance is rapidly spreading among bacteria such as Staphylococcus aureus, an opportunistic bacterial pathogen that causes a variety of diseases in humans. For the last two decades, bacterial multidrug efflux pumps have drawn attention due to their potential association with clinical multidrug resistance. Numerous researchers have demonstrated efflux-mediated resistance in vitro and in vivo and found novel multidrug transporters using advanced genomic information about bacteria. This article aims to provide a concise summary of multidrug efflux pumps and their important clinical implications, focusing on recent findings concerning S. aureus efflux pumps.

Bacterial multidrug efflux pumps: … and some suggestions on how they might work

One of the key mechanisms that confers drug resistance in bacteria involves the action of energyconsuming transporters that drive the efflux of toxic compounds from the cell. In the Gramnegative group of bacteria, which have a characteristic system of two lipid membranes, some of these transporters are the engine components of tripartite assemblies that span both membranes and the interstitial periplasm. These assemblies have been the focus of extensive structural and mechanistic studies for decades, and we provide a brief synopsis of the current understanding of how they are organized into functioning machines, how those machines might work in detail and the likely points in the machinery that could be targets for therapeutic intervention.

Homologs of the Acinetobacter baumannii AceI transporter represent a new family of bacterial multidrug efflux systems.

ABSTRACTMultidrug efflux systems are a major cause of resistance to antimicrobials in bacteria, including those pathogenic to humans, animals, and plants. These proteins are ubiquitous in these pathogens, and five families of bacterial multidrug efflux systems have been identified to date. By using transcriptomic and biochemical analyses, we recently identified the novel AceI (Acinetobacter chlorhexidine efflux) protein from Acinetobacter baumannii that conferred resistance to the biocide chlorhexidine, via an active efflux mechanism. Proteins homologous to AceI are encoded in the genomes of many other bacterial species and are particularly prominent within proteobacterial lineages. In this study, we expressed 23 homologs of AceI and examined their resistance and/or transport profiles. MIC analyses demonstrated that, like AceI, many of the homologs conferred resistance to chlorhexidine. Many of the AceI homologs conferred resistance to additional biocides, including benzalkonium, dequalinium, proflavine, and acriflavine. We conducted fluorimetric transport assays using the AceI homolog from Vibrio parahaemolyticus and confirmed that resistance to both proflavine and acriflavine was mediated by an active efflux mechanism. These results show that this group of AceI homologs represent a new family of bacterial multidrug efflux pumps, which we have designated the proteobacterial antimicrobial compound efflux (PACE) family of transport proteins.

Systems Level Study of Bacterial Multi-Drug Resistance from Efflux Machinery

Bacterial multi-drug resistance efflux pumps are complex molecular machines that expel multiple drugs and antibiotics. They also influence important genetic and cellular processes to confer additional drug resistance. We have developed a mathematical framework to integrate genetic and cellular processes to understand how efflux pumps confer resistance. Our model is validated against multiple lines of experimental evidence, such as time-kill behavior of wild-type cells, as well as mutant strains deficient in efflux pump or genetic regulation mechanisms. Our model exhibits quantitative agreement with the experiments and predicts specific, experimentally testable properties which are important for drug efflux. Also, we describe our current efforts to integrate molecular mechanisms from a data-driven structural model of the P. aeruginosa tripartite MexAB-OprM complex that was constructed based upon the crystal structures of the individual components MexA, MexB, and OprM, energetic calculations of the membrane-bound complex, and distance constraints between components derived from crosslinking studies.

Modulation of Bacterial Multidrug Resistance Efflux Pumps of the Major Facilitator Superfamily.

Bacterial infections pose a serious public health concern, especially when an infectious disease has a multidrug resistant causative agent. Such multidrug resistant bacteria can compromise the clinical utility of major chemotherapeutic antimicrobial agents. Drug and multidrug resistant bacteria harbor several distinct molecular mechanisms for resistance. Bacterial antimicrobial agent efflux pumps represent a major mechanism of clinical resistance. The major facilitator superfamily (MFS) is one of the largest groups of solute transporters to date and includes a significant number of bacterial drug and multidrug efflux pumps. We review recent work on the modulation of multidrug efflux pumps, paying special attention to those transporters belonging primarily to the MFS.

Repetitive methylene blue-mediated photoantimicrobial chemotherapy changes the susceptibility and expression of the outer membrane proteins of Pseudomonas aeruginosa

Since bacterial multidrug efflux pumps mediate intracellular photosensitizer methylene blue, a change in the expression alters the susceptibility to photoantimicrobial chemotherapy (PACT) of Pseudomonas aeruginosa, which may occur following repetitive sublethal challenges. We performed 10 consecutive, methylene blue-mediated PACT on one antibiotic-sensitive strain and three antibiotic-resistant strains of P. aeruginosa. Following each therapy, the surviving bacteria were collected for subsequent PACT. The susceptibility was compared for the pre- and the post-treated strains following repetitive PACT. To explore the existence of efflux pumps, one of the inhibitors, namely Phe-Arg β-naphthylamide dihydrochloride (PAβN 25 μg/ml), was added. Profiles of outer membrane proteins were obtained for the pre-treated and the post-treated strains. The susceptibility of PACT did not correlate with the antibiotic sensitivity. Following ten PACT, there was no significant change in susceptibility for three tested strains, except for one antibiotic-resistant strain, for which the 10th generation became less susceptible than the original one. With 2-D electrophoresis, a change in the expression of outer membrane proteins was observed. PAβN successfully increased the phototoxicity in all tested strains, especially the less PACT-susceptible 10th generation of the antibiotic-resistant strain. Following repetitive challenges, PACT had a consistent antimicrobial effect on three strains; however, one antibiotic-resistant strain, which was the most vulnerable to PACT, became more resistant after consecutive challenges. In addition, the post-PACT strain had different expression of outer membrane proteins, providing further evidence view that repetitive PACT with methylene blue could change the expression of efflux pumps.

Structure of an atypical periplasmic adaptor from a multidrug efflux pump of the spirochete Borrelia burgdorferi

Periplasmic adaptor proteins are essential components of bacterial tripartite multidrug efflux pumps. Here we report the 2.35Å resolution crystal structure of the BesA adaptor from the spirochete Borrelia burgdorferi solved using selenomethionine derivatized protein. BesA shows the archetypal linear, flexible, multi-domain architecture evident among proteobacteria and retains the lipoyl, β-barrel and membrane-proximal domains that interact with the periplasmic domains of the inner membrane transporter. However, it lacks the α-hairpin domain shown to establish extensive coiled-coil interactions with the periplasmic entrance helices of the outer membrane-anchored TolC exit duct. This has implications for the modelling of assembled tripartite efflux pumps.

Functionally Cloned pdrM from Streptococcus pneumoniae Encodes a Na+ Coupled Multidrug Efflux Pump

Multidrug efflux pumps play an important role as a self-defense system in bacteria. Bacterial multidrug efflux pumps are classified into five families based on structure and coupling energy: resistance−nodulation−cell division (RND), small multidrug resistance (SMR), major facilitator (MF), ATP binding cassette (ABC), and multidrug and toxic compounds extrusion (MATE). We cloned a gene encoding a MATE-type multidrug efflux pump from Streptococcus pneumoniae R6, and designated it pdrM. PdrM showed sequence similarity with NorM from Vibrio parahaemolyticus, YdhE from Escherichia coli, and other bacterial MATE-type multidrug efflux pumps. Heterologous expression of PdrM let to elevated resistance to several antibacterial agents, norfloxacin, acriflavine, and 4′,6-diamidino-2-phenylindole (DAPI) in E. coli KAM32 cells. PdrM effluxes acriflavine and DAPI in a Na+- or Li+-dependent manner. Moreover, Na+ efflux via PdrM was observed when acriflavine was added to Na+-loaded cells expressing pdrM. Therefore, we conclude that PdrM is a Na+/drug antiporter in S. pneumoniae. In addition to pdrM, we found another two genes, spr1756 and spr1877,that met the criteria of MATE-type by searching the S. pneumoniae genome database. However, cloned spr1756 and spr1877 did not elevate the MIC of any of the investigated drugs. mRNA expression of spr1756, spr1877, and pdrM was detected in S. pneumoniae R6 under laboratory growth conditions. Therefore, spr1756 and spr1877 are supposed to play physiological roles in this growth condition, but they may be unrelated to drug resistance.

Substrate Binding and Transport by a Bacterial Multidrug MFS Transporter

Bacterial multidrug efflux pumps confer resistance to a broad range of antibiotics, thus reducing the efficacy of clinical treatment of bacterial infection. EmrD is the only structurally-characterized representative of a drug:H+ antiporter of the major facilitator superfamily (MFS). However, the determinants of substrate binding and transport by EmrD remain unclear. Unlike other MFS transporters, EmrD contains no membrane-embedded acidic residues thought to be necessary for coupling drug:H+ antiport.