Antimicrobial resistance is a current major challenge in chemotherapy and infection control. The ability of bacterial and eukaryotic cells to recognize and pump toxic compounds from within the cell to the environment before they reach their targets is one of the important mechanisms contributing to this phenomenon. Drug efflux pumps are membrane transport proteins that require energy to export substrates and can be selective for a specific drug or poly-specific that can export multiple structurally diverse drug compounds. These proteins can be classified into seven groups based on protein sequence homology, energy source and overall structure. Extensive studies on efflux proteins have resulted in a wealth of knowledge that has made possible in-depth understanding of the structures and mechanisms of action, substrate profiles, regulation and possible inhibition of many clinically important efflux pumps. This review focuses on describing known families of drug efflux pumps using examples that are well characterized structurally and/or biochemically.

Introduction

Antimicrobial resistance (AMR) is an increasingly serious global public health problem, where existing antibiotics are losing their effects and are no longer clinically useful. In 2014, WHO reported very high rates of resistance in common pathogenic bacteria such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus, where many of these organisms exhibit simultaneous resistance to multiple antimicrobials, a phenotype that is referred to as multidrug resistance (MDR) and can be attributable to a single or multiple resistance mechanisms [1]. These mechanisms that bacteria exploit to resist the toxic effects of antimicrobials can involve enzymatic modification or destruction of antimicrobials, target modification that moderates the affinity of target proteins for antimicrobials, and limitation of drug access to cellular targets mediated by both reduced influx and increased efflux of antimicrobials. Drug efflux, defined as recognition and expulsion of toxic compounds from within the cell to the external medium before they reach their targets, represents a major mechanism of drug resistance [2].

Drug efflux mechanisms involve energy-dependent efflux transport proteins, which are located in the cytoplasmic membrane (CM). They can be grouped into the following seven families/superfamilies based on protein sequence homology, overall protein structure, energy source (ATP hydrolysis or ion electrochemical gradient), substrate specificity and other properties. These families are: (i) the ATP-binding cassette (ABC) superfamily, members of which are primary active transporters that use the free energy of ATP hydrolysis to energize efflux; (ii) the resistance-nodulation division (RND) superfamily; (iii) the major facilitator superfamily (MFS); (iv) the multidrug and toxic compound extrusion (MATE) family; (v) the small multidrug resistance (SMR) family; (vi) the proteobacterial antimicrobial compound efflux (PACE) family and (vii) the p-aminobenzoyl-glutamate transporter (AbgT) family (Figure 1). The latter six groups are secondary active transporters that couple efflux with ion electrochemical membrane gradient as an energy source [35].

Schematic illustration of the seven families/superfamilies of multidrug transport proteins

Figure 1
Schematic illustration of the seven families/superfamilies of multidrug transport proteins

Each group is shown by a distinct shape and highlighted with a different colour. Where available, a crystal structure of the functional unit or monomer of a representative protein of each group is shown in a similar colour to the corresponding diagram. Symmetric structures in proteins are visualized with different colours but in the same category of colours that matches the diagram’s colours. Two crystal structures of the transporter contain a substrate that is represented as a green sphere. The CM and OM of Gram-negative bacteria are illustrated. The ABC superfamily (cyan rectangle), uses free energy of ATP hydrolysis catalysed by the NBDs of the transporter. The crystal structure of S. aureus Sav1866 (pdb: 2HYD), composed of a dimer (green and cyan), contains two TMDs and two NBDs and representing a functional unit of the ABC transporter is shown. Five singlet system secondary active transporter families/superfamilies: MFS, MATE, PACE, SMR and AbgT are shown by oval, rectangle, circle, chord and parallelogram shapes respectively. Crystal structures of the E. coli MFS MdfA (pdb: 4ZOW), V. cholerae MATE NorM (NorM_VC) (pdb: 3MKT), E. coli SMR EmrE (pdb: 3B5D) and N. gonorrhoeae AbgT MtrF (pdb: 4R1I) representative transport proteins are shown. A diagram containing four TMS representing the PACE family AceI transporter is also shown. The multicomponent RND superfamily system is shown by a diagram containing the CM-bound, MFP and OMF components, each highlighted by a different colour. Crystal structures of the E. coli AcrB (pdb: 2DRD) representing the integral CM protein, AcrA (pdb: 2F1M) the MFP and TolC (pdb: 2VDE) the OMF of an RND efflux system, are shown.

Figure 1
Schematic illustration of the seven families/superfamilies of multidrug transport proteins

Each group is shown by a distinct shape and highlighted with a different colour. Where available, a crystal structure of the functional unit or monomer of a representative protein of each group is shown in a similar colour to the corresponding diagram. Symmetric structures in proteins are visualized with different colours but in the same category of colours that matches the diagram’s colours. Two crystal structures of the transporter contain a substrate that is represented as a green sphere. The CM and OM of Gram-negative bacteria are illustrated. The ABC superfamily (cyan rectangle), uses free energy of ATP hydrolysis catalysed by the NBDs of the transporter. The crystal structure of S. aureus Sav1866 (pdb: 2HYD), composed of a dimer (green and cyan), contains two TMDs and two NBDs and representing a functional unit of the ABC transporter is shown. Five singlet system secondary active transporter families/superfamilies: MFS, MATE, PACE, SMR and AbgT are shown by oval, rectangle, circle, chord and parallelogram shapes respectively. Crystal structures of the E. coli MFS MdfA (pdb: 4ZOW), V. cholerae MATE NorM (NorM_VC) (pdb: 3MKT), E. coli SMR EmrE (pdb: 3B5D) and N. gonorrhoeae AbgT MtrF (pdb: 4R1I) representative transport proteins are shown. A diagram containing four TMS representing the PACE family AceI transporter is also shown. The multicomponent RND superfamily system is shown by a diagram containing the CM-bound, MFP and OMF components, each highlighted by a different colour. Crystal structures of the E. coli AcrB (pdb: 2DRD) representing the integral CM protein, AcrA (pdb: 2F1M) the MFP and TolC (pdb: 2VDE) the OMF of an RND efflux system, are shown.

Drug efflux pumps are present in all bacterial species and other organisms. In bacteria, they play a key role not only in drug resistance but also in the movement of pathogenicity factors, e.g. by modulation of biofilm formation and possibly other physiological processes. They can also interact synergistically with other AMR mechanisms, the cumulative effects of which can result in AMR of clinical importance. Many efflux transporters can accommodate an extremely wide range of antimicrobial compounds (MDR efflux pumps), whereas others only export a specific substrate and its closely related derivatives. Both types are found in Gram-positive and Gram-negative bacteria and can contribute to clinical resistance. This review focuses on describing the known MDR export systems in bacteria and their affect on AMR.

The ABC superfamily

ABC transporters represent one of the largest superfamilies of proteins known in prokaryotes and eukaryotes involving both importers and exporters. Members of this superfamily translocate a wide range of substrates including sugars, amino acids, lipids and antimicrobial compounds. This substrate diversity reflects the numerous physiological roles that ABC transporters play in the cell, e.g. nutrient uptake, removal of waste and toxic products and export of cellular components such as cell wall polysaccharides [6]. LmrA from Lactobacillus lactis is a well-described ABC MDR efflux pump that exports a wide variety of substrates including antibiotics, toxic ions, lipids, antimicrobial peptides and antiseptics [7]. The E. coli MacB protein, another ABC MDR efflux pump, which are similar to RND pumps (described in the next section), functions together with its cognate membrane fusion protein (MFP) MacA and the outer membrane (OM) channel TolC, to confer resistance to macrolides [8]. The orthologous MacAB pump of Neisseria gonorrhoeae has also been shown to export multiple substrates including macrolides. Clinical isolates of N. gonorrhoeae, such as H041, have shown extensive resistance to extended-spectrum cephalosporins and the macrolide azithromycin and inactivation of MacAB renders the bacterium clinically susceptible to azithromycin according to MIC breakpoints indicating a key role of this pump in azithromycin resistance [9].

Solving the crystal structure of the S. aureus Sav1866 protein, which can transport structurally unrelated substrates such as ethidium, tetraphenylphosphonium and Hoechst 33342, marked one of the major steps in understanding ABC transporters at a structural and functional level (Figure 1) [10]. ABC transporters have a basic organization of two TMDs that contain the substrate translocation pathway and two nucleotide-binding domains (NBDs), which bind and hydrolyse ATP [10]. NBDs are highly conserved among ABC superfamily proteins and contain several motifs characteristic of this group of transporters. These include: the Walker A and B motifs, present in many nucleotide-binding proteins; motif C, specific to the ABC superfamily; and the stacking aromatic D-, H- and Q-loops and conserved sequences in loop regions that communicate conformational changes from the NBDs to the TMDs [6,11]. The functional unit of mammalian ABC transporters is a dimer, with each monomer containing two domains (TMD and NBD) contained in a single polypeptide. In bacterial transporters, however, each monomer is considered as a half transporter that is formed by the fusion of one TMD and one NBD, which are formed by separate polypeptides. In most ABC transporters, a functional transport complex is a homodimer, with each monomer containing six α-helical transmembrane segments (TMS) spanning the CM and 12 TMS in total per functional unit. Most ABC transporters follow this two-times-six TMS paradigm, albeit exceptions exist [12]. The structure of Sav1866 showed for the first time that TMS and NBD domains are connected via transmembrane helices and nucleotide binding is associated with dimerization [10].

A general model, referred to as conformational coupling, was proposed as the functional mechanism of ABC transporters. In this model, ATP-dependent conformational changes in the NBDs drive rearrangements of the TMS that enforces the link between transport and ATP hydrolysis and results in a transport mechanism often referred to as ‘alternating access’ [13]. This mechanism explains how the transport protein undergoes conformational changes, in which a central binding site located within a TMD is exposed alternately to the inside (inward-facing) or the outside (outward-facing) of the cell, but never simultaneously to both sides allowing the binding site alternating accessibility to each side of the membrane [13]. The current structural evidence obtained from several bacterial ABC transporters including Sav1866 [10], the lipid A and lipopolysaccharide transporter MsbA [14], BmrA from Bacillus subtilis and McjD from E. coli, indicate that these ABC exporters adopt an inward-facing conformation in the absence of nucleotide or when bound to ADP, and that they progress via an ATP-bound occluded transition state into the ATP-bound outward-facing conformation. Finally, the transporter is reset to the inward-facing conformation by dissociation of the NBD dimer mediated by ATP hydrolysis [13].

The RND superfamily

The RND superfamily represents a large ubiquitous group of efflux proteins that are found in prokaryotes, eukaryotes and archaea [15]. Of the eight families within this superfamily, one – the hydrophobic amphiphilic efflux 1 (HAE1) family, contains well-characterized, Gram-negative bacterial-specific RND transporters [15]. RND efflux pumps exhibit extensive topological similarity, usually with 12 TMS and two large hydrophilic periplasmic domains between TMS 1 and 2, and TMS 7 and 8 [15]. The N-terminal half of these RND transporters shares homology with the C-terminal half, suggesting an internal gene duplication event. RND integral membrane proteins function as part of a three-component system that includes two other proteins; the periplasmic adaptor protein belonging to the MFP family and the OM channel, belonging to the OM factor (OMF) family, that together form a functional tripartite efflux complex spanning the CM, the periplasm and the OM (Figure 1) [16]. RND transporters exhibit specificity mainly for hydrophobic and amphiphilic compounds, catalysing export of a wide spectrum of compounds including detergents, antibiotics, dyes, bile salts, fatty acids, biocides, isoflavonoids and aliphatic and aromatic organic solvents and capture their substrates preferentially from the periplasm [15].

The biochemically and structurally well-characterized E. coli AcrB protein is the prototype of RND efflux pumps. AcrB exports a large group of substrates including most antibacterial compounds. The X-ray crystal structure of AcrB without bound substrate was first reported in 2002, revealing a homotrimer [17]. Each monomer contained a large periplasmic domain and a TMD organized into 12 TMS, in addition to many subdomains and other structural features [17]. Further crystal structures of AcrB solved by three laboratories in 2006–2007 significantly advanced the understanding of the structure and function of RND pumps, in particular that each monomer has a unique conformation slightly different from that of its neighbours [1820]. These three conformations correspond to three functional stages of the protein i.e. access, binding and extrusion that describe substrate binding and efflux by AcrB.

AcrB co-crystallized with the substrates minocycline and doxorubicin identified a multisite drug binding (distal) pocket deep within the pore domain [18] and substrate molecules were only found in one monomer, called the binding monomer. The asymmetric crystal structure suggested that drugs are exported by a three-step functionally rotating mechanism, in which each monomer cycles through sequential conformations and substrates undergo ordered binding change [18]. Within the pore domain, crystal structures of AcrB bound to rifampicin and erythromycin identified an additional drug binding pocket [21] (the proximal pocket), which is hypothesized to accommodate and bind large substrates such as rifampicin. These two intramolecular substrate binding pockets are separated by a ‘switch loop’ that swings during conformational change from access to binding stages allowing substrates to move from the proximal site to the distal pocket [22]. Three entrances lead to and merge in the proximal pocket. Interestingly, it was shown that the hydrophobic parts of many compounds that can inhibit RND proteins (efflux pump inhibitors; EPIs), tightly bound to a site in the lower part of distal binding pocket known as the ‘hydrophobic trap’, thereby, distorting the shape of the rest of the pocket resulting in inhibition of efflux of typical substrates of AcrB [23]. A third drug-binding area was identified in the central cavity, from structures of AcrB bound to substrates such as ethidium. However, the significance of this drug-binding site could not be ascertained by site-directed mutagenesis [24,25]. It is believed that multisite multiple drug binding in AcrB follows the same principles elucidated by crystallographic studies of QacR, the soluble regulator of the multidrug efflux protein QacA [26]. The multisite-drug-oscillation hypothesis, which explains recognition of drugs with low-binding affinity to specific sites in the AcrB, is another possible multidrug recognition mechanism [27].

The energy requirements for substrate translocation originate from the proton motive force (PMF) [28], where four essential AcrB charged residues form a transmembrane proton-relay network involved in proton translocation [29]. A conformational cycling mechanism involving two remote alternating access sites within each AcrB monomer, one in the TMD for proton translocation and one for substrate translocation in the periplasmic domain, has been proposed [30].

AcrB operates as part of a tripartite system including AcrA and TolC (Figure 1); however, the stoichiometry of these three components in the functional complex is controversial. Based on individual crystal structures and cross-linking experiments, the docking stoichiometry of AcrB3–AcrA3–TolC3 is a possible model of the tripartite assembly [27]. This model is challenged by electron microscopic images of the AcrAB–TolC complex obtained by Du et al. [31] as well as electron microscopic images of the native AcrAB–TolC, the MexAB–OprM and the interspecies AcrA–MexB–TolC complexes in nanodiscs [32]. These images indicate no direct interaction between the integral CM protein and the OM channel and suggested a 3:6:3 stoichiometry. It was previously shown that the small 49 amino acid AcrZ protein of E. coli interacts with AcrB to enhance the ability of the AcrAB–TolC pump system to export certain classes of substrates [33].

Expression of acrAB and tolC in E. coli is regulated at both local and global levels. The acrAB operon and the tolC gene are chromosomally encoded, and although in separate locations, their expression is often regulated by common mechanisms, involving repressors, activators, two-component systems and small RNAs. The acrR gene is divergently transcribed from acrAB and directly represses acrAB and its own expression [34]. Belonging to the TetR/QacR family of transcriptional regulators, AcrR functions as a dimeric two-domain molecule, in which the N-terminal region comprises the helix-turn-helix containing DNA-binding domain and the C-terminal region forms the dimerization and multidrug-binding domain [35,36]. AcrR, however, does not completely inhibit the expression of acrAB, allowing constitutive expression of the transporter and thus intrinsic MDR. Two other proteins are also involved in the regulation of acrAB. AcrS, a direct repressor of the AcrEF pump, also represses expression of other multidrug transporter genes including acrAB [37]. The quorum-sensing receptor SdiA, positively controls expression of acrAB likely by acting as a minor activator [38]. Furthermore, in terms of activation, three global regulators, MarA, SoxS and Rob also positively control expression of acrAB and tolC [3942]. In addition, the two-component system EvgAS regulates expression of both acrAB and tolC, and a second two-component system, PhoPQ, regulates expression of tolC [43]. Furthermore, the small RNA RyeB, has been shown to be involved in controlling acrAB expression. Multiple regulatory systems controlling expression of RND pumps at local and global levels facilitate adaptation of host strains to diverse conditions [44].

There is overwhelming evidence that overexpression of RND pumps (and some exporters from other families) is significantly associated with MDR in antibiotic-resistant bacteria commonly isolated from humans and animals, where it can lead to resistance levels for multiple antibiotics at clinically significant levels. Many studies have shown this in MDR clinical isolates of E. coli, K. pneumoniae, Salmonella enterica serovar Typhimurium, P. aeruginosa, Campylobacter jejuni, Acinetobacter baumannii and other Gram-negative bacteria [4548]. For example, P. aeruginosa that contains several RND pumps including MexAB-OprM, MexXY-OprM, MexCD-OprJ and MexEF-OprN, can export antibiotics used in current clinical settings such as fluoroquinolones, tetracycline, chloramphenicol and some β-lactams [45]. In addition to these, an interesting study was recently performed in clinical isolates of N. gonorrhoeae that were extensively resistant to third generation cephalosporins (including ceftriaxone) and the macrolide azithromycin [9]. It was found that even in the presence of other mechanisms of drug resistance, deletion of the main RND pump, MtrCDE, from a number of these strains significantly reduced the MICs for many antibiotics (in particular ceftriaxone and azithromycin), below their clinical breakpoints [9].

The MFS

MFS transporters are also efflux proteins that are found ubiquitously in the membranes of all types of living organisms and compose the largest superfamily of secondary transporters. These proteins utilize the power in electrochemical gradients of either protons or sodium ions to transport amino acids, sugars, peptides and drugs, among others and can act as symporters, uniporters or antiporters. This superfamily is currently composed of 82 recognized subfamilies [49,50]. Many drug and multidrug transporters are classified within the MFS superfamily, with three drug/H+ antiporter (DHA) families i.e. DHA1, DHA2 and DHA3, comprised exclusively of efflux transporters [5,51]. DHA1 and DHA3 families contain transport proteins that display 12 TMS and include several well-characterized MDR transporters such as MdfA and EmrD of E. coli and NorA of S. aureus. In contrast, members of the DHA2 family such as QacA/QacB and EmrB, contain 14 TMS [52]. It is believed that the most abundant MFS transporters i.e. 12-TMS proteins, have arisen from a single 2-TMS hairpin that triplicated, giving rise to a protein with 6 TMS that through a duplication event resulted in the formation of 12-TMS proteins [49]. Several superfamily and family specific amino acid sequences (motifs) within MFS transporters have been identified [5]. High conservation of these sequences/motifs within MFS members indicates that they are possibly involved in structural or functional roles. Motif A is known as the MFS-specific motif and structural analysis of the MFS transporter YjaR has demonstrated that residues contained in motif A sense and respond to protonation inside the central cavity [53].

Analyses of MFS transporters, including structures of MDR exporters EmrD and MdfA, have resulted in the following postulations: (i) MFS transporters generally function as monomers; (ii) they have a common structural architecture called the MFS-fold which comprises two domains, each consisting of bundles of six consecutive TMS organized into a pair of inverted ‘3 + 3’ repeats; (iii) the N- and C-domains are related by a pseudo two-fold symmetry axis in the membrane plane and are linked by a long cytoplasmic loop or by two TMS and (iv) a membrane-embedded central cavity is present between these two domains, thus forming the substrate-transport pathway. In EmrD, this central cavity is quite large and surrounded by hydrophobic and aromatic side chains. This is in line with the more recent MdfA structure (Figure 1) that also possesses a central cavity of hydrophobic residues. In EmrD, the loops connecting TMS 4 and 5, and TMS 10 and 11 protrude into the cytoplasm and it is believed that they play a role in substrate recognition and capture [54].

Alternating access is the widely accepted mechanism of MFS substrate translocation, similar to that proposed for other transport proteins. Analyses of crystal structures of MFS transporters in multiple conformations (inward-open, occluded and outward-open) have enabled the elucidation of details of alternating access and energy-coupling mechanisms of MFS substrate transport translocation. These analyses suggested that the translocation pathway of the substrate follows the interface between the two six-helices, with the substrate binding pocket located halfway into the membrane [55]. A rocker-switch type of movement mechanism involving successive and concerted conformational changes of the two halves of the protein among inward-open, occluded and outward-open states alternately generate a pathway of access to either surface of the membrane. The transport coupling of MFS proteins occurs through sequential binding/releasing of substrate/counter-ion and this varies among family members [54,55].

Multiple MFS efflux transporters can be found in Gram-negative bacteria. In E. coli K-12, for example 70 MFS proteins have been identified, 15 of which may be considered drug efflux transporters. Some of these are present as tripartite systems, as in a similar fashion to the RND systems they use their cognate adaptor protein and the TolC channel. In E. coli for example, EmrB (occurring with the cognate adaptor EmrA) and EmrY (with EmrK) are believed to be involved in the efflux of uncouplers and other substrates. In contrast, MdfA is a singlet pump that has been studied extensively [44]. MdfA is known to confer resistance to a variety of structurally distinct cationic and zwitterionic lipophilic compounds, as well as to a number of electroneutral antibiotics of clinical importance [56]. MDR E. coli clinical strains have been shown to overexpress MdfA from plasmids and MdfA orthologues have been identified in several pathogenic bacterial species [56,57]. Recent structures of MdfA in complex with three different ligands identified and confirmed residues responsive to substrate binding in addition to confirming the recently ascribed role of D34 in substrate binding through protonation [57]. The plasmid encoded QacA MDR transporter is the best characterized 14-TMS MFS member. QacA specifies resistance to a range of structurally dissimilar organic cations, including monovalent cations, such as ethidium, benzalkonium and cetrimide and divalent cations, such as chlorhexidine, pentamidine and dequalinium (Figure 2). Interestingly, the highly homologous QacB can only efflux monovalent compounds; these two proteins differ by only seven residues [52,5861].

Representative substrates of the staphylococcal multidrug efflux protein QacA

Figure 2
Representative substrates of the staphylococcal multidrug efflux protein QacA

QacA mediates resistance to an extensive range of monovalent and divalent cationic lipophilic compounds, which can be broadly classified as dyes, quaternary ammonium compounds, biguanidines, diamidines and guanylhydrazones. Chemical structures of a few examples of QacA substrates are displayed to illustrate the diversity of structures recognized, which have been identified as substrates of other pump systems. Six monovalent and five divalent cationic compounds are shown at the top and bottom of the picture respectively. Positive charges on each molecule are highlighted in red.

Figure 2
Representative substrates of the staphylococcal multidrug efflux protein QacA

QacA mediates resistance to an extensive range of monovalent and divalent cationic lipophilic compounds, which can be broadly classified as dyes, quaternary ammonium compounds, biguanidines, diamidines and guanylhydrazones. Chemical structures of a few examples of QacA substrates are displayed to illustrate the diversity of structures recognized, which have been identified as substrates of other pump systems. Six monovalent and five divalent cationic compounds are shown at the top and bottom of the picture respectively. Positive charges on each molecule are highlighted in red.

Recently, a new mechanism of efflux by secondary transporters was described for MdfA[62]. Unlike QacA that can export both monovalent and divalent cationic substrates, MdfA was previously known to export only neutral or monovalent substrates by a mechanism involving the exchange of a single proton. However, it was shown later that by including an additional acidic residue, MdfA can catalyse efflux of divalent cations that have a unique architecture: two charged moieties that must be separated by a long linker [62]. Examples of these substrates, chlorhexidine and dequalinium, are shown in Figure 2. These two MFS transporters export this type of divalent cationic substrate in two successive transport cycles, where each cationic moiety is transported as if it were a separate substrate and involves the exchange of two protons for one substrate molecule. It was proposed that secondary transporters can adopt a processive-like mode of action, thus expanding the substrate spectrum of multidrug transporters [62].

The MATE family

Over 1000 transport proteins are members of the MATE family and can be allocated into three subfamilies, which are further subdivided into 14 subfamilies [63]. These transporters use the membrane gradient of Na+, H+ or both, as an energy source for driving export of cationic compounds although the details of coupling mechanism is not fully understood yet. The NorM antiporter from Vibrio parahaemolyticus (NorM_VP) was the first MATE protein identified [64]. When expressed in E. coli, NorM_VP confers resistance to norfloxacin and ciprofloxacin, as well as structurally unrelated compounds like ethidium, kanamycin and streptomycin [64]. Other MATE members, including the N. gonorrhoeae NorM (NorM_NG), E. coli YdhE and S. aureus MepA proteins have been well characterized, with extensive substrate profiles [65,66].

Most proteins of the MATE family consist of 400–550 amino acids, with 12 TMS. They share ~45% similarity suggesting that they have common structural and functional features. NorM from Vibrio cholerae (NorM_VC) was the first member of the MATE family whose crystal structures were solved with and without substrate (rubidium) (Figure 1). These crystal structures showed that the membrane spanning protein is composed of two bundles of 6-TMS (TMS 1–6 and TMS 7–12) forming a large internal cavity that is open to the extracellular space (outward facing) [67]. These two halves have an intramolecular two-fold symmetry, suggesting that they have arisen due to a gene duplication. A cytoplasmic loop, also seen in the MFS transporters, connects these two halves. Two substrate passages, one from each side of the protein, are opened to the outer leaflet of the CM. In addition, a monocation-binding site was identified in the internal cavity suggesting that the outward facing conformation favours high affinity binding of monocations such as Na+ and low affinity binding for substrates. The crystal structures of the Na+-coupled NorM_NG in complex with three substrates, as well as Cs+ (a Na+ congener), were all solved in extracellular facing and drug-bound states [68]. These structures identified a multidrug-binding cavity containing four negatively charged residues and details of rearrangement of some TMS upon Na+-induced drug export [68]. Structures and functional analyses of a MATE transporter from Pyrococcus furiosus in two distinct apo-form conformations and complexed with a norfloxacin derivative and three peptide inhibitors, showed that the protonation of Asp41 in the N-terminal part of the protein induces the bending of TMS 1, which in turn collapses the N-terminal part cavity, thereby extruding the substrate drug to the extracellular space [69].

The SMR family

The SMR family of transporters contain the smallest membrane transport proteins. This family can be divided into three subfamilies; the small multidrug pumps (SMP), paired small multidrug resistance pumps (PSMRs) and suppressors of groEL mutations (SUGs). Phylogenetic analysis of SMR family proteins identified two phylogenetic clusters, with only one of these containing proteins able to catalyse drug export [70]. This largest cluster is composed of the SMP proteins including QacC from S. aureus and EmrE from E. coli, two of the best characterized SMR family proteins.

SMR family proteins are highly hydrophobic and typically contain 100–150 amino acids constrained in four TMS. They are proton-coupled transporters that can confer low-level resistance to a variety of antimicrobial agents, including a number of biocides in addition to toxic lipophilic cations that function as DNA-intercalating agents. Additionally, SMR family proteins display regions of conservation with other larger transporters in the drug/metabolite efflux family [70]. The functional unit of SMP subfamily proteins is believed to be a homodimer. In contrast, members of the PSMR subfamily require simultaneous expression of two SMR gene copies co-located within the same operon that form a heteroligomer that can then actively extrude compounds. The most well-characterized PSMR proteins are EbrAB from B. subtilis. Structural and functional studies of this system have shown that these proteins form a heteroligomer where the two proteins are in opposite orientations [71].

The E. coli EmrE SMP protein is the prototype of all SMR proteins. It has been subjected to numerous studies by multiple laboratories analysing the biochemical and structural complexities of this protein. A number of structural approaches have been applied to EmrE including both solution and solid-state NMR, cryo-EM, spin-labelling EPR and X-ray diffraction. This 110-residue protein pumps out positively charged aromatic compounds such as ethidium, acriflavine, methyl viologen, benzalkonium, tetraphenylphosphonium, tetracycline, erythromycin and sulfadiazine. Like PSMR proteins, EmrE functions as a dimer, however, the organization of EmrE monomers in the membrane has been hotly contested. Current consensus is that the dual-topology protein EmrE has an asymmetrical arrangement where each protein monomer is in an antiparallel orientation. This dimeric EmrE exchanges two protons/substrate/transport cycle [72]. Site-directed mutagenic studies have shown that the antiport of drug and proton relies on a highly conserved negatively charged glutamate residue in the first TMS of SMR family proteins.

Substrate binding has been proposed to occur at the monomer–monomer interface, at the centre of the protein, from the inner leaflet of the CM and results in the movement of at least one TMS [73]. Similar to that determined for membrane transporters from other superfamilies (see above), the single site alternating access model has also been proposed for the mechanism of SMR proteins [74]. Thus, each state in the transport cycle is only open to one side of the membrane (outward-facing conformation) and only when a substrate or two protons are bound, does the protein interconvert to an inward-facing state.

The PACE and AbgT families

Recent studies have identified two new secondary active drug transporter families; the PACE family and the antimetabolite transporters forming the AbgT family (Figure 1). The first transport protein of the PACE family, AceI (Figure 1), was identified during a transcriptomic study in A. baumannii after a shock treatment with the biocide chlorhexidine was applied [75]. Although annotated as a hypothetical membrane protein, studies revealed that AceI conferred resistance to chlorhexidine via an active efflux mechanism. Bioinformatic analyses and subsequent functional assays have identified AceI homologues in a large number of species and the substrates of these transporters have been expanded to other synthetic biocides including benzalkonium, proflavine, acriflavine and dequalinium. PACE family members are small membrane proteins of ~150 residues and contain two tandem bacterial transmembrane pair (BTP; Pfam accession number PF05232) domains. To date, there are more than 750 protein sequences containing this domain listed in the Pfam database.

The latest family of MDR transporters is the AbgT family that is exemplified by the MtrF MDR protein of N. gonorrhoeae (Figure 1). Normally, this family contains proteins that act as importers, such as the AbgT (YdaH) protein of E. coli [76] that may be involved in the uptake of p-aminobenzoyl-glutamate, a precursor of folic acid biosynthesis [77,78]. Required for high-level hydrophobic AMR in N. gonorrhoeae, the MtrF protein was the first efflux transporter described in the AbgT family and acts in conjunction with the MtrCDE RND efflux system [79]. The YdaH protein of Alcanivorax borkumensis, a marine bacterium that uses oil hydrocarbons as its exclusive source of carbon and energy, was also recently identified to be a member of the AbgT family [4]. Crystal structures of both the N. gonorrhoeae MtrF and the A. borkumensis YdaH determined these two transporters assemble as dimers with architectures different from all other families of transporters. In both the proteins, the dimer forms a bowl-shaped structure with a solvent-filled basin extending from the cytoplasm midway through the membrane bilayer. Each monomer of MtrF and YdaH contains nine TMS and two hairpins. Further structural analyses combined with biochemical studies implied that both the MtrF and YdaH proteins are efflux pumps capable of producing resistance by exporting sulfonamide antimetabolites [4].

Control of efflux pumps

The flexible nature of drug efflux pump regulation enables bacteria to fine-tune expression of their systems to adapt to diverse environments [44]. This adaptation can occur either via conferring AMR or contributing to the bacterium’s ability to invade and survive in host cells or specific host environments. As described above, the regulation of efflux pump expression can be controlled by local and global transcriptional regulators, modulators and two-component regulatory systems that involve different regulatory cascades and small RNA molecules. Mutations in the genes encoding these trans-acting regulatory elements can alter the expression levels of the efflux pumps sometimes with negative clinical outcomes [9,48]. For example in E. coli and other Enterobacteriaceae, mutations inactivating the local repressor acrR of acrAB, result in overexpression of this efflux pump and subsequent MDR [8082]. Expression of AcrAB can also be increased by mutating marR and soxR as this can result in the enhanced production of the global activators, MarA and SoxS respectively, that also act on the AcrAB system.

Mutations in the regulatory sequences such as promoters can also increase the expression levels of efflux pumps and give rise to resistance with clinical importance. Called cis-acting regulatory elements, these important mechanisms of resistance are commonly identified in pathogenic bacteria. For example in clinical N. gonorrhoeae isolates, four cis-acting elements have been found associated with increased expression of the MtrCDE efflux system and MDR. These elements include: (i) a single bp deletion or a dinucleotide insertion within an inverted repeat in the mtrR promoter; (ii) a point mutation, located at 120-nts upstream of the mtrC start codon and (iii) a 153-bp insertion mutation in the mtrRmtrCDE intervening region that is called Correia element [83]. Similarly, promoter mutations have been identified for the S. aureus NorA MFS MDR efflux pump, that result in overexpression of norA in clinical strains. Additionally, clinical isolates of Candida albicans have been isolated that are azole resistant due to constitutively high-level expression of the ABC superfamily CDR1 and CDR2 pumps; a drug-responsive element sequence within their promoters was identified [84].

Since the discovery of efflux pump-mediated AMR, the development of EPIs as an effective strategy for combating AMR has been considered. These EPIs could promote the return of certain antibiotics into clinical practice and may also broaden the spectrum of antibiotics to cover Gram-negative bacteria that are naturally resistant to those antibiotics that act on Gram-positive bacteria [85]. It has been shown that EPIs significantly decrease the frequency of development of AMR mutants. In addition, inhibition of efflux would also have extra benefits of decreasing bacterial resistance to mediators of the innate immune response that are also substrates of efflux pumps and attenuating virulence of Gram-negative bacteria that is partly mediated by biofilm formation [86]. Many EPIs have been identified and characterized, including verapamil, reserpine, PAβN, NMP, D13-9001 and MBX2319. Some of these inhibitors are specific for an efflux pump whereas others inhibit the efflux activity of multiple proteins. Unfortunately, a number of EPIs have been shown to possess acute toxicity and prolonged accumulation in tissue cells [87]. Subsequently, no EPI has reached clinical use yet. This shows that the development of universal or even specific EPIs and/or drugs that bypass efflux pumps is not without problems. These challenges include: conversion of a lead compound to a new drug; formulation of effective combinational therapy; clinical trials; difficulties that are related to the nature of a molecule that can be used as an antibiotic or inhibitor in terms of physical and chemical properties; toxicity for humans and animals and identifying a molecule that can inhibit such flexible multispecific binding sites. Advances in science and technology have made it possible to identify and characterize many AMR-related efflux transporters and in particular develop an in-depth understanding of the structure and mechanisms of action of many of them as well as their interaction with a wide range of substrates. However, further progress in analyses of efflux transporters especially MDR-related systems is required to enable rational design of drugs or inhibitors in the future that could overcome to the current limitations.

Summary

  • MDR efflux pumps are active transporters that mediate resistance to a broad range of structurally diverse drugs and are of increasing concern in chemotherapy of infections and cancer.

  • Efflux pumps are widespread among bacteria and eukaryotes including those that are pathogens and can be grouped into the seven categories.

  • Typical membrane structure (topology) of efflux pumps comprises 4, 6, 12 or 14 transmembrane spanning helices.

  • Alternating access is the common mechanism used by many efflux systems to translocate substrates.

  • Multidrug efflux pumps utilize one or more drug-binding pockets with multiple drug-binding sites to recognize and transport structurally diverse substrates.

  • Regulation of efflux pump expression is typically controlled by the participation of several local and global transcriptional regulators, modulators and two-component regulatory systems that involve different regulatory pathways and mutations in expression control elements, such as promoters, also play a role.

  • Several EPIs have been identified, however, none have progressed to clinical use yet.

  • In-depth understanding of interaction of efflux pumps with antibiotics and efflux inhibitors will enable rational drug and inhibitor design.

Funding

This work was supported by the National Health and Medical Research Council Australia Project Grant [grant number 1002670]; a Flinders Foundation Seeding Grant; a Weizmann-Australia Joint Research Program; and a Australian Government Research Training Program Scholarship (to M.C.). .

Competing interests

The authors declare that there are no competing interests associated with the manuscript.

Abbreviations

     
  • ABC

    ATP-binding cassette

  •  
  • AMR

    antimicrobial resistance

  •  
  • ATP

    adenosine tri-phosphate

  •  
  • CM

    cytoplasmic membrane

  •  
  • DHA

    drug/H+ antiporter

  •  
  • EPI

    efflux pump inhibitor

  •  
  • MATE

    multidrug and toxic compound extrusion

  •  
  • MDR

    multidrug resistance

  •  
  • MFP

    membrane fusion protein

  •  
  • MFS

    major facilitator superfamily

  •  
  • NBD

    nucleotide-binding domain

  •  
  • NMP

    NMP1-(1-naphthylmethyl)-piperazine

  •  
  • OM

    outer membrane

  •  
  • OMF

    OM factor

  •  
  • PAβN

    phenylalanylarginine-β-naphthylamide

  •  
  • PACE

    proteobacterial antimicrobial compound efflux

  •  
  • PDB

    protein data bank

  •  
  • PSMR

    paired small multidrug resistance pump

  •  
  • RND

    resistance-nodulation division

  •  
  • SMP

    small multidrug pump

  •  
  • SMR

    small multidrug resistance

  •  
  • TMD

    transmembrane domain

  •  
  • TMS

    transmembrane segment

  •  
  • WHO

    world health organization

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