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Published in final edited form as: Curr Opin Microbiol. 2022 Jul 23;69:102179. doi: 10.1016/j.mib.2022.102179

Making sense of drug-efflux transporters in the physiological environment

Helen I Zgurskaya 1, Justyna W Adamiak 1, Inga V Leus 1
PMCID: PMC9942525  NIHMSID: NIHMS1870699  PMID: 35882103

Abstract

Bacterial drug-efflux transporters act synergistically with diffusion barriers of cellular membranes and other resistance mechanisms to protect cells from antibiotics and toxic metabolites. Their critical roles in clinical antibiotic and multidrug resistance are well established. In addition, a large body of evidence has been accumulated in support of their important contributions to bacterial growth and proliferation during infections. However, how these diverse functions of drug transporters are integrated at the level of bacterial cell physiology remains unclear. This opinion briefly summarizes the current understanding of substrate specificities and physiological roles of drug-efflux pumps from Resistance–Nodulation–Division (RND) superfamily of proteins in two ESKAPE pathogens Pseudomonas aeruginosa and Acinetobacter baumannii. Based on the analysis of phenotypic and transcriptomic studies in vitro and in vivo, we propose that RND pumps of Gram-negative bacteria fall into three categories: constitutively expressed, regulated, and silent. These three categories of efflux pumps participate in different physiological programs, which are not involved in the central metabolism and bacterial growth.

Introduction

Bacterial multidrug resistance is one of the leading public health threats of the 21st century [1]. Although different mechanisms are responsible for antibiotic resistance in clinical isolates, most of these mechanisms benefit dramatically from synergy with permeability barriers of bacterial cell envelopes [2]. These permeability barriers are established by drug transporters acting together with diffusion barriers of lipid membranes (Figure 1).

Figure 1.

Figure 1

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A schematic view of a Gram-negative cell envelopes with major fluxes of small compounds (C) and representative antibiotic-modifying enzymes shown. Recent studies led to a comprehensive kinetic model that integrates active drug efflux into contexts of various cell envelopes and resistance mechanisms [2]. The following heuristics have emerged: (i) drug transporters acting across the same membrane are additive to each other; (ii) transporters are synergistic with transporters in the other membrane; (iii) active efflux is synergistic with the limitations on transmembrane diffusion but only if the two fluxes occur across the same membrane; (iv) in mathematical terms, enzymatic inactivation of an antibiotic is no different from its active efflux and could be either additive or synergistic with efflux and transmembrane diffusion; (v) when active uptake is more efficient than efflux, the compound will be accumulated inside the cell [2].

Bacterial drug transporters are structurally diverse and belong to several protein families. A distinctive feature of drug transporters is that most of them are polyspecific and can reduce intracellular concentrations of multiple structurally unrelated compounds. This polyspecificity is a product of intrinsic structural features of drug transporters and their synergistic interactions with the substrate diffusion barriers [3]. The synergistic effects are important in all bacteria, but are especially strong in Gram-negative pathogens because of the presence of the outer membrane (OM) and efflux pumps acting across this membrane (Figure 1).

In Gram-negative bacteria, transporters belonging to the Resistance–Nodulation–Division (RND) superfamily of proteins assemble trans-envelope protein complexes spanning the inner and outer membranes and periplasm (Figure 2). Frequently overproduced in clinical isolates, RND-efflux pumps not only expel antibiotics from bacterial cells but are also tightly integrated into bacterial cell physiology. Changes in their expression are expected not only to affect the intracellular accumulation of various antibiotics and toxins, but also to rewire cell physiology to accommodate changes in efflux of various small molecules.

Figure 2.

Figure 2

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RND-type efflux transporters in P. aeruginosa and A. baumannii and their accessory proteins. Located in the inner membrane of Gram-negative bacteria, RND transporters associate with periplasmic membrane-fusion proteins and outer membrane-factor family channels to form trans-envelope drug-efflux pumps. The components of efflux pumps discussed in the text are indicated.

In this opinion, we summarize recent insights into complex interactions between RND-efflux pumps and bacterial physiology and the potential impact of these interactions on multidrug resistance in clinics. We use as examples Gram-negative P. aeruginosa and A. baumannii of the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, A. baumannii, P. aeruginosa and Enterobacter species) group, both are highly versatile multidrug-resistant pathogens.

Resistance–Nodulation–Division-efflux pumps of Pseudomonas aeruginosa

P. aeruginosa is believed to be ubiquitous in the environment, but the prevalence of this bacterium is higher in places linked to human activity [4]. Perhaps, there is a connection between the environment occurrence and the fact that P. aeruginosa is one of the major opportunistic pathogens and a leading cause of nosocomial infections [5]. The low- permeability OM and an arsenal of drug transporters, especially from the RND superfamily, are cited as responsible for the intrinsic multidrug resistance, whereas target mutations and enzymatic modifications further enable clinical resistance to specific antibiotic classes [5].

P. aeruginosa possesses many primary and secondary transporters. To date, 12 RND-efflux pumps have been characterized to some extent, out of which 11 pumps are capable of multidrug efflux (Figure 2) [6,7]. They can recognize and expel most classes of antibiotics, including macrolides, β-lactams, aminoglycosides, quinolones, dyes, and detergents. Six of the P. aeruginosa RND pumps are constitutively expressed under laboratory conditions [7]. Perhaps not surprising that these pumps differ by their substrate specificities and their involvement in growth physiology.

Roles in antibiotic resistance and cell physiology in vitro

MexAB–OprM is the most abundant pump and contributes significantly to intrinsic and clinical levels of drug resistance of P. aeruginosa. The expression of this pump does not change notably when cells are subjected to various stresses, typically associated with human infections (Figure 3a) [8]. The exceptions are nutritional downshift and nitrosative stress that repress the expression of MexAB–OprM by about 2-fold and the osmotic stress that increases its expression by the same extent. Studies that involved strains lacking or overproducing MexAB–OprM suggested that substrates of this efflux pump encompass structurally diverse compounds, including quorum-sensing signals [9,10]. However, transcriptomics and metabolomics studies showed that neither MexAB–OprM nor other RND pumps contribute to secretion of Pseudomonas quorum signal (PQS) molecules [7,11]. The efflux-deficient PΔ6 cells lacking six RND pumps including MexAB–OprM were found to secrete high levels of these signals [7].

Figure 3.

Figure 3

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Expression of P. aeruginosa RND-efflux pumps as reported in clinical samples [28] and in response to infection-related stresses [8]. The expression of first genes in the operons encoding periplasmic components is shown. As — acidic stress, Bs — bile stress, Hyp — hypoxia, Li — low ion, Nd — nutritional downshift, Ns — nitrosative stress, Oss — osmotic stress, Oxs — oxidative stress, Sp — stationary phase, Tm — temperature, Vic — virulenc- inducing conditions. The expression data for the indicated pumps were extracted from pathogenex.org [8] and expressed as log2 (fold change of expression at indicated condition vs. control). For in vivo data, log2(fold-change human vs. in vitro), log2(fold-change CF sputum vs. in vitro), and log2(fold-change soft tissue vs. in vitro) are plotted from [28].

Since PQS chelates ferric iron (Fe3+) in a fashion similar to the quinolobactin siderophore from P. fluorescens [12,13], its accumulation in PΔ6 cells leads to depletion of available iron, increased production of siderophores such as pyochelin and pyoverdine, and dramatic accumulation of chelated iron in the periplasm [7].

MexHI–OpmD is the most abundant pump in the stationary phase and biofilms of P. aeruginosa (Figure 3b). Inactivation of this pump leads to the loss of virulence, a reduction of quorum-sensing signaling molecules, and accumulation of the endogenously produced phenazine 5-methylphenazine-1-carboxylate [14,15]. The role of the pump in the phenazine metabolism is further underscored by findings that MexHI–OpmD controls intra- and extracellular concentrations of pyocyanin [16], a powerful P. aeruginosa toxin and virulence factor that inhibits the electron-transport chain and several other pathways [17,18]. The plasmid-borne overproduction of MexHI/OpmD protects cells from fluoroquinolones and several other antibiotics [16].

Homology and clustering analyses showed that MexHI–OpmD is the most closely related to MexVW (70% sequence similarity between MexI and MexW). Unlike MexAB–OprM and MexHI–OpmD, all components of which are encoded within the same operons, MexVW is an example of a pump lacking a gene encoding an outer membrane channel in its operon. It can function with OprM and OpmM and perhaps other channels [19]. Transcripts encoding this pump are also constitutively produced but more abundant in the exponential culture and are repressed in the stationary phase, during nutritional downshift, nitrosative, and osmotic stresses (Figure 3a). The overexpression of MexVW was reported to reduce susceptibility of P. aeruginosa to a variety of antibiotics, including chloramphenicol, fluoroquinolones, and macrolides [19]. However, its deletion does not generate notable changes to antibiotic susceptibilities and growth phenotypes [7].

Like MexVW, operons encoding MexJK and TriABC pumps do not contain genes encoding outer membrane channels and these pumps are dependent on the expression of OpmH encoded somewhere else on the chromosome. Both pumps are reported to have rather narrow substrate specificities. When overproduced, MexJK reduces susceptibility to tetracycline and triclosan, but can also protect against erythromycin in the OprM-dependent manner [20]. The expression of MexJK is strongly induced by exposure to bile salts and to a lesser extent by low iron and hypoxia, whereas nutritional downshift reduces its abundance (Figure 3b). TriABC is an unusual pump requiring two membrane-fusion proteins TriA and TriB for its activity and its overproduction leads to protection against triclosan and sodium dodecyl sulfate [21,22]. The expression of this pump remains largely unaffected by various stresses (Figure 3a).

Finally, transcripts of MuxABC–OpmB are consistently detected in growing P. aeruginosa cells. This pump requires two different RND subunits MuxB and MuxC for its function. The plasmid-borne overexpression of MuxABC–OpmB reduces the susceptibility of P. aeruginosa to aztreonam, macrolides, novobiocin, and tetracycline [23]. Surprisingly, its inactivation was also reported to lead to elevated production of β-lactamase with concurrent increased resistance to β-lactam antibiotics and reduced virulence in plants and insects [24]. In addition, inactivation of this pump affects specifically the susceptibility to novobiocin, reduces the amounts of pyoverdine, a coumarin-containing siderophore, and contributes to iron deficiency [7]. Hypoxia and elevated temperature and to a lesser extent low iron are conditions that increase the expression of MuxABC–OpmB, whereas nutritional downshift, stationary phase, and nitrosative stress are the strongest repressors (Figure 3b).

The remaining five RND pumps that are implicated into multidrug resistance are expressed at very low levels in both growing and stationary cells. Interestingly, the expression of these pumps also remains low under various stress conditions (Figure 3b-c). The exceptions are MexXY and MexPQ–OpmE, both of which are strongly, more than 3 and 20 times respectively, induced by the low iron stress (Figure 3b). To a lesser extent, MexXY is also induced by high temperature, whereas MexPQ–OpmE by hypoxia and nitrosative stresses. Uniquely, the overproduction of MexXY elevates resistance to aminoglycoside antibiotics along with several other classes of antibiotics [25]. The overproduction of MexPQ–OpmE in drug-hypersensitive cells elevates minimal inhibitory concentrations (MICs) of macrolides, fluoroquinolones, and other antibacterials [26].

Resistance–Nodulation–Division pumps that are overproduced in vivo

Several studies demonstrated that the physiology of P. aeruginosa during human infections is distinct from that in vitro. The risk of infection with P. aeruginosa is the highest in patients with a compromised immune system or a chronic lung disease, patients on breathing machines or catheters, and those with infection of wounds from surgery and burns [27]. In cystic fibrosis (CF) patients, these infections are often persistent and such patients receive multiple antibiotic treatments over years. The RNA-seq studies revealed that the expression of RND pumps is not only different from that in vitro, but also varies in human samples, CF sputum, and soft tissues (Figure 3) [28].

The expression of the constitutively expressed MexAB–OprM, TriABC, and MexVW remained unaffected or slightly repressed under all three in vivo conditions. Thus, physiological functions of these transporters are not in demand during various stresses and growth in human hosts (Figure 3a). In contrast, MexXY, MexPQ–OpmE, MexJK, MexGHI–OpmD and MuxABC–OpmB pumps, the expression of which is inducible by certain stresses, are all upregulated in vivo, albeit to a different degree and depending on the source (Figure 3b).

Finally, the expression levels of MexCD–OprJ, MexEF–OprN, and MexMN pumps, which are not inducible in vitro by changes in growth phases and by stresses, are affected by conditions in human samples (Figure 3c). Importantly, the overproduction of these pumps as well as that of MexXY in multidrug-resistant isolates is often linked to regulatory mutations, which are selected under antibiotic pressure [29]. Cells overproducing these pumps due to such mutations are also found to have pleiotropic phenotypes with significant fitness and virulence costs. Loss-of-function mutations in nfxB lead to upregulation of mexCD–oprJ expression and, consequently, increased resistance to fluoroquinolone antibiotics. Interestingly that in vitro, such nfxB mutants were impaired in all forms of motility as well as in the production of siderophores, rhamnolipid, secreted protease, and pyocyanin. Proteomic and metabolomic investigation showed that the exoproteome, endometabolome, and exometabolome of the nfxB mutant were all globally different than the wild type [30]. In particular, the enrichment of long-chain fatty acids suggested that they could be substrates of this pump. Another known regulatory gene, mexZ, encodes a negative regulator, and mutations in this gene lead to overexpression of MexXY [31,32]. In contrast, MexEF–OprN is positively regulated by the transcriptional activator MexT (LysR-type regulator) [33]. Mutations identified in clinical MexEF–OprN-overproducing strains affect either mexT or a gene, mexS, which encodes a presumed quinone oxidoreductase, MexS [33,34]. Unlike MexCD–OprJ, overexpression of MexEF–OprN does not cause a significant decrease in P. aeruginosa fitness in vitro, indicating the absence of a large metabolic burden [35]. However, HHQ (4-hydroxy-2-heptylquinoline), a direct precursor of PQS, was suggested to be a substrate of MexEF–OprN [36] and the overexpression of this RND transporter was found to reduce the production of PQS, as well as several PQS-regulated virulence factors such as pyocyanin and rhamnolipids [36,37].

Resistance–Nodulation–Division-efflux pumps of Acinetobacter baumannii

Acinetobacter spp. are ubiquitous in the environment, however, A. baumannii isolates are almost exclusively associated with clinical settings. This pathogen can cause a variety of severe nosocomial infections, including skin and soft-tissue infections, wound infections, urinary tract infections, and secondary meningitis. The infections with the highest mortality rates are ventilator-associated pneumonia and bloodstream infections [38].

Two major physiological advantages enabled the rapid spread of A. baumannii in clinics: high levels of acquired antibiotic resistance and its remarkable ability to persist in unfavorable conditions [39]. Unlike P. aeruginosa with its large arsenal of RND-efflux pumps, A. baumannii relies only on three AdeABC, AdeFGH, and AdeIJK. Other RND pumps (AdeDE, AdeXYZ, AbeD, ArpAB, and CzcABCD) [40-43], as well as drug transporters from other protein families, are being increasingly reported but mostly from in vitro-generated mutants and their relevance in clinical isolates remains unclear.

AdeIJK pump is constitutively expressed in various A. baumannii strains, provides intrinsic levels of antibiotic resistance, possesses a broadest substrate spectrum, and is rarely overproduced in clinical isolates. In all these properties as well as in its protein sequences, AdeIJK is the analog of P. aeruginosa’s MexAB–OprM. The similarity between the two pumps further extends into the lack of an upregulation in response to various virulence-related stresses (Figure 4). In addition, the absence of ppGpp [44] and the presence of human serum albumin [45] reduce the expression of this pump. The expression of AdeIJK is controlled by global regulator AdeN, a TetR-type negative regulator [46,47], but whether this regulator is responsible for the downregulation of adeIJK expression is unclear. The tight control of AdeIJK expression could be explained by findings that AdeIJK is critical for membrane-composition stability [47,48] and vital for lipid-homeostasis maintenance, specifically as a lipid-export mechanism [49].

Figure 4.

Figure 4

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Expression of A. baumannii RND -fflux pumps as reported in mice samples [64] and in response to infection-related stresses [8]. The expression of first genes in the operons encoding periplasmic components is shown. See Figure 3 for abbreviations.

AdeABC is the major contributor to the multi-drug resistance (MDR) phenotype and is reported to be overexpressed in many clinical isolates [50-52]. The substrate specificity of AdeABC is distinct from that of AdeIJK and is the most similar to that of MexXY–OprM in P. aeruginosa. Like MexXY–OprM, the overproduction of AdeABC leads to clinically significant levels of aminoglycoside resistance and several other antibiotics. There are also similarities in changes of AdeABC expression in response to various stresses. Like MexXY, the expression of AdeABC is induced by low iron and is repressed by nutritional downshift and stationary-phase transition (Figure 4). On the other hand, AdeABC is upregulated by hypoxia and downregulated by oxidative stress. The ppGpp synthetase [44], human serum albumin [45], and low iron conditions [53] also play a regulatory role for the adeABC operon. In addition, the adeABC operon is strongly induced by chlorhexidine [54] and different polyamines [55].

At least two two-component systems are involved in the positive regulation of AdeABC: AdeRS [51,56] and BaeSR [57]. Apparently, BaeSR also positively regulates adeIJK expression, but functional interactions between AdeRS and BaeSR remain unclear [57]. No cross-regulation was found between adeIJK and adeABC., as neither inactivation of adeB, adeIJK, nor both has a notable effect on the expression of each other and adeFGH [58]. However, the abundance of adeA transcript was found to be dramatically increased in adeB::T26 cells, suggesting its upregulation in response to the loss of AdeABC activity.

AdeFGH shows the narrowest substrate range [48] and is regulated by a LysR-type transcriptional repressor AdeL, encoded immediately upstream of the operon [59]. In P. aeruginosa, the closest analog of AdeFGH is MexEF–OprN pump. These two pumps share significant sequence homology, substrate preferences for chloramphenicol and trimethoprim, and negligible expression in vitro even under stress conditions (Figure 4). A bacterial genome-wide association study demonstrated that the adeFGH genes are highly conserved in the A. baumannii genome, but are largely absent from the transcriptome under laboratory growth conditions [60]. Like with MexEF–OprN, mutational or plasmid-borne overproduction of AdeFGH leads to fluoroquinolone resistance [47,48], whereas treatment with sublethal concentrations of the fluoroquinolone antibiotic levofloxacin has been shown to increase adeFGH expression [61].

Deletion of adeFGH has been shown to increase the expression of adeAB, but the inverse is not true, suggesting that cross-regulation occurs via an unknown mechanism in the one direction but not the other [48,58].

Integration of Resistance–Nodulation–Division pumps in A. baumannii physiology in vitro and in vivo

Overproduction of A. baumannii efflux pumps results in pleiotropic cell-envelope changes, including lipid composition of membranes [48,62]. In addition, overproduction of AdeABC or AdeIJK causes moderately reduced fitness in animal-infection models [63], reduced levels of pilus proteins, and decreased biofilm formation [47]. The overexpression of AdeFGH accelerates the synthesis and transport of acylated homoserine lactones during biofilm formation [61]. The expression adeABC, but not adeIJK or adeFGH, was found to be mildly, about 2-fold, upregulated during lung infection in mice (Figure 4)[64].

The phenotypic and RNA-seq studies of efflux-knockout mutants of MDR clinical isolates AYE and Ab5075, both overproducing AdeABC due to mutations, showed that the inactivation of AdeABC elicits a focused transcriptional response, which is different and additive to the loss of activity of AdeIJK [58]. Cells lacking adeABC, strongly downregulate genes involved in iron acquisition, competence, and motility [58]. In contrast, deletion of adeIJK leads to a broad transcriptomic response, including changes in pathways of lipid biosynthesis and turnover, as well as upregulation of motility genes [58]. In addition, deletions of adeAB and adeRS resulted in decreased biofilm formation in abiotic and biotic surfaces, and a decrease in virulence within A. baumannii [65].

Conclusions

The presented analysis shows that RND-efflux pumps of P. aeruginosa, A. baumannii, and likely other Gram-negative pathogens fall into three categories: 1) constitutively expressed in vitro and in vivo; 2) inducible by stresses, stationary phase, and during infections; 3) silent pumps activated by mutations in regulatory genes upon exposure to antibiotics or under currently unknown conditions. These three categories of efflux pumps participate in different physiological programs, which are not related to the central metabolism and bacterial growth. Different levels of expression of these pumps are critical to control small-molecule fluxes in cells, as seen from similar changes in expression of pumps from different categories in P. aeruginosa and A. baumannii. Direct and indirect effects on accumulation of signaling molecules, siderophores, and other metabolites are difficult to distinguish in studies of knockout and overproducer strains, and specific molecules transported by RND pumps remain for the most part illusive. Among the stress conditions, low iron, hypoxia, and bile salt exposure appears to upregulate expression of several P. aeruginosa RND-efflux pumps, whereas nutritional downshift and stationary phase downregulate pumps in both A. baumannii and P. aeruginosa.

Acknowledgements

This work was supported by National Institutes of Health, USA grant RO1-AI136799 and RO1-AI132836 to H.I.Z.

Footnotes

CRediT authorship contribution statement

Helen I. Zgurskaya: Writing – review & editing, Justyna Adamiak: Writing – review & editing, and Inga Leus: Writing – review & editing.

Conflict of interest statement

The authors declare no conflict of interest.

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