Structure, function and inhibition of RND efflux pumps in Gram-negative bacteria: an update

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Structure, function and inhibition of RND efflux pumps in Gram-negative bacteria: an update Jessica MA Blair and Laura JV Piddock Resistance nodulation division efflux systems have a major role in both intrinsic and acquired multi-drug resistance in Gramnegative bacteria. The recent structure of an assembled tripartite system, AcrAB-TolC, revealed that AcrB is docked onto TolC, which remains in an open state once part of the assembled complex and three AcrA molecules complete the structure. This is in contrast to data for the MexAB-OprM system of P. aeruginosa that, depending on pH, has between two and six MexA molecules per OprM trimer. RND systems are also important for pathogenicity of several bacteria and for Salmonellae lacking components of AcrAB-TolC, expression of known virulence determinants were significantly altered. The importance of these systems in both MDR and pathogenicity has made RND systems the target of new drugs aimed at inhibiting their function. The wealth of new structural and functional data will inform rational drug design. Address Antimicrobial Agents Research Group, School of Immunity and Infection, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK Corresponding author: Piddock, Laura JV ([email protected])

Current Opinion in Microbiology 2009, 12:512–519 This review comes from a themed issue on Antimicrobials Edited by Christopher Walsh and Gerry Wright Available online 5th August 2009 1369-5274/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2009.07.003

Introduction The resistance nodulation division (RND) of efflux pumps are found ubiquitously throughout the Bacteria, Archaea and Eukaryotes. In Gram-negative bacteria they are situated within the inner membrane and function in complex with two other proteins, an outer membrane channel and a periplasmic adaptor protein, to form a tripartite efflux pump spanning both the inner and outer membrane (Figure 1). These multi-protein complexes transport a wide variety of substrates including antibiotics, dyes, detergents and host derived molecules from the periplasm to the extra-cellular space.

Impact of RND pumps on MDR Active efflux by RND systems plays an important role in the innate resistance of Gram-negative bacteria to Current Opinion in Microbiology 2009, 12:512–519

multiple classes of structurally distinct antimicrobials, including those that are clinically relevant. Lack of functional RND components renders the bacterium susceptible to these agents, whereas for example, overexpression of AcrB in laboratory mutants of Escherichia coli or Salmonella enterica confers multi-drug resistance (MDR) [1–4]. Overexpression of RND systems in clinical isolates is also associated with MDR [5]. In addition, MDR bacteria can over-produce more than one efflux system simultaneously; for instance, of 450 clinical isolates of ticarcillin resistant Pseudomonas aeruginosa 28% of isolates simultaneously overexpressed MexAB-OprM and MexXY [6]. Similarly, expression of acrA, acrB, acrE and acrF and efflux pump encoding genes of other classes was increased in both laboratory selected and clinical fluoroquinolone resistant S. Typhimurium [7]. Enhanced efflux is also implicated in rapidly emerging clinical resistance to new antimicrobials such as tigecycline, for which overexpression of AcrAB was detected in E. coli and Enterobacter cloacae [8,9]. Acriflavine and ethidium bromide are known substrates of AcrAB-TolC. However, a recent model for the transport of these dyes showed that the single component transporters EmrE and MdfA are required for the intrinsic resistance of E. coli to both [10]. These plasma membrane proteins transport the dyes from the cytoplasm to the periplasm where they are captured by AcrAB-TolC and transported across the outer membrane [10].

Structure RND pump structure

The crystal structures of the two of the best studied RND transporters, AcrB of E. coli and MexB of P. aeruginosa, have been solved [11–13]. AcrB and MexB are closely related with 69% identity and 83% similarity and their structures share many common features [5,13]. Three identical monomers of AcrB and MexB form an integral membrane protein complex within the cytoplasmic membrane [11,13] (Figure 2a). Each monomer has a transmembrane domain composed of 12 membrane-spanning helices and a large periplasmic domain folded in a complex manner to give the pore or porter domain [12–14]. The monomers exist in one of three conformations and conformational cycling is responsible for the pumping mechanism of AcrB [12–16]. Biochemical evidence to support this hypothesis has emerged recently. Seeger et al. [16] introduced constraining di-sulfide bonds between subdomains of AcrB; these inhibited movement and pump function and increased susceptibility to toxic www.sciencedirect.com

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Figure 1

Schematic diagram of a tripartite RND system, AcrAB-TolC of E. coli. In Gram-negative bacteria RND pumps (e.g. AcrB) are situated in the inner membrane and function in a complex with two other proteins, an outer membrane channel (e.g. TolC) and a periplasmic adaptor protein (e.g. AcrA). From [90].

compounds. This effect was reversed after exposure to the reducing agent dithiothreitol, removing the constraints [16]. Takatsuka and Nikaido [17] took the novel approach of creating a single ‘giant gene’ containing three acrB sequences connected by short linker sequences enabling inactivation of a single protomer in the trimeric complex. Inactivation of one protomer abolished the activity of the whole complex confirming that the AcrB trimer is the functional unit and providing strong evidence for the functionally rotating hypothesis [17]. Efflux by RND pumps is driven by the proton motive force. The proton translocation site of AcrB is a triplet of charged amino acid residues, Asp 407, Asp 408 and Lys 940 found on the fourth and tenth trans-membrane domains, in a spatially separate location from the substrate binding pocket [14,18,19]. The trans-membrane domains of AcrB and MexB are particularly well conserved with almost all residues being identical, including this amino acid triplet involved in proton translocation suggesting that the mechanism may be common to all RND systems [13]. The periplasmic domain is partially responsible for the substrate specificity of RND pumps [20–24]. Chimeric www.sciencedirect.com

studies in which sections of this domain from E. coli AcrB were replaced with the highly similar sections from AcrD showed that the substrate specificity of the chimeric protein resembled that of AcrD, not AcrB [25,26]. Furthermore, a single point mutation at Val-610 of E. coli YhiV, also within the periplasmic domain of this RND pump also significantly altered substrate specificity [27]. The substrate binding pocket of AcrB is rich in aromatic residues, particularly phenylalanines, that interact with the structurally varied substrates [12,14]. AcrB has been crystallised in complex with bile acid; the binding site for this natural substrate is in a similar position to that for other published complexes including ethidium bromide, ciprofloxacin and PAbN [21,28]. Site directed mutagenesis was used to show that mutation of different phenylalanine residues within the binding pocket had variable effects on susceptibility to substrates and highlighted the F610 residue as having a particularly significant and broad impact on susceptibility [29]. Conversely, a substitution at the AcrB 616 residue was found to be particularly relevant for macrolide resistance [30] and a serine 715 substitution had a specific effect on bile resistance [28]. Redundancy of phenylalanines in the pocket may partially account for the varied consequences of substituting different residues. Some substrates are still accommodated in a mutated binding pocket while other larger substrates, such as the macrolides, are unable to adapt to changes caused by a single substitution [29]. The docking domain, which interacts with the outer membrane channel, is highly conserved between AcrB and MexB although the two corresponding outer membrane proteins (TolC and OprM, respectively) cannot complement the other’s function suggesting that it is the periplasmic component that is responsible for the specificity of the component interactions [13,26]. A protrusion from the docking region into the adjacent RND monomer holds the three monomers together and possibly stabilises the complex [13,31]. Periplasmic adaptor protein structure

The periplasmic adaptor proteins (PAP) (formerly described as membrane fusion proteins) AcrA from E. coli and MexA from P. aeruginosa share 62% sequence identity and 73% similarity but cannot complement each other’s function [26]. Partial crystal structures of these PAPs revealed three distinct domains: a b-barrel, a central lipoyl domain and an a-helical coiled coil hairpin [32–35]. However, these structures lacked the 130 residues forming the N and C termini of the proteins. The structures of PAPs AcrA and MexA was completed in 2009 revealing that the N terminus (residues 13–27) and C terminus (residues 262–339) together form a membrane proximal (MP) domain that is a compact b-roll linked to the bbarrel domain by a b-ribbon linker (Figure 2b) [36]. This domain is highly conserved between the two Current Opinion in Microbiology 2009, 12:512–519

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Figure 2

The individual completed structures of MexB, MexA and OprM. The crystal structure of the individual components of MexAB-OprM have been solved. (a) MexB is a homotrimer in which each subunit adopts a different conformation. This structure is highly homologous to that of AcrB. From [13]. (b) The complete structure of MexA showing the four domains: the a-helical hairpin in blue, the lipoyl domain in green, the b-barrel in yellow and the newly modelled membrane proximal domain in orange. The blue dotted line represents the 12 residues of the N-terminal strand and the blue atom is the N-terminal cysteine with its lipid tail. (c) OprM is also a homotrimer forming a pore in the membrane with a diameter of 6–8 A˚. From [46].

proteins and the similarity between this MP domain and the b-barrel domain suggests that the structure may have arisen owing to domain duplication [36]. Another study has confirmed the importance of the C-terminal region of AcrA [37]. Conformational flexibility has been revealed in the structure of both AcrA [33,34] and MexA [38]. Conformational changes in AcrA were detected in vitro in response to acidic pH, similar to that present in the periplasm [34], and subsequently crystallisation revealed four different AcrA conformations [33]. This flexibility comes from the hinges between the lipoyl domain and the a-helical hairpin and b-barrel domains. Further flexibility was revealed upon completion of the crystal structure of MexA. The orientation of the newly modelled MP domain was found to differ by up to 858 in relation to the rest of the protein owing to conformational change in the b-linker [36]. The surface of AcrB, which associates with AcrA is relatively stable and does not undergo large conformational changes previously associated with the functionally rotating mechanism [14,31]. On the periplasmic surface the largest conformational change comes from the PC2 domain that is spatially separated from the AcrA attachment site suggesting that once docked, the conformational change required of the PAP would be limited [36]. Current Opinion in Microbiology 2009, 12:512–519

Until recently the stoichiometry of RND system components in vivo was unclear [39]. Following the completion of the AcrA crystal structure, evidence from modelling with the complete structures of the three components and validated by cross-linking at pH 7.5 has supported a stoichiometry of 3 AcrB:3 AcrA:3 TolC (Figure 3) [36]. This is consistent with predictions based upon known structures and predicting the most energetically favourable stoichiometric composition [40]. However, this is in contrast with recent data from Reffay et al. concerning the stoichiometry of MexA suggesting variation between two MexA molecules per OprM trimer at pH 7.5 to six MexA molecules per OprM trimer at pH 5.5 [41]. Two putative RND systems have been shown to contain two different PAPs. TriABC-OpmH of P. aeruginosa has TriA and TriB that are both required for efflux pump function [42] while ZrpADBC of Serratia sp., includes ZrpA and ZrpD of which only the latter is essential for pump function [43]. Outer membrane channel

The structures of several outer membrane proteins that associate with RND efflux systems have been solved [44–46] (Figure 2c). TolC is trimeric and each protomer has a 40 A˚ long b-barrel domain anchored to the outer membrane and a 100 A˚ long a-helical domain consisting of 12 coiled coils that project into the periplasm. Once www.sciencedirect.com

Structure, function and inhibition of RND efflux pumps in Gram-negative bacteria: an update Blair and Piddock 515

Figure 3

overall structure despite low sequence identity and lack of conservation of many functionally important residues between all members of this family [45]. Previous models of TolC opening involved the relaxation of the tightly packed helices that obstruct the pore entrance, allowing the helices to rearrange in an ‘iris like’ movement [48]. More recently, molecular dynamics simulations have suggested that the iris opening analogy to describe TolC opening may be too simplistic and that an additional twisting motion in the upper portion of the periplasmic tunnel leads to a peristaltic action that may aid movement of the substrate along the tunnel [49]. Recent modelling based on extensive site-specific crosslinking, suggested that the interaction between TolC and AcrA was most favourable with TolC in its open state [36]. Symmons et al. [36] proposed that once AcrA is bound to AcrB, TolC is recruited to the complex causing TolC to open and remain in a constitutively open state, allowing the high-throughput transport displayed by this system. In this model, TolC closure is not required because substrates can pass straight from AcrB to TolC without leakage into the periplasm as the connection between the two components is successfully stabilised by AcrA [36] (Figure 3). A fourth component?

Crystal structures of endogenous AcrB complexed with a novel transmembrane protein, termed YajC, have been reported predicting that AcrB formed a complex with YajC once the AcrAB-TolC efflux complex had assembled [50]. This induces a functionally significant rotation of the porter domain of AcrB relative to the transmembrane domain. Deletion of YajC resulted in only a modest increase in susceptibility to ampicillin and nafcillin. Tornroth-Horsefield [50] also suggested that the N-terminus of YajC could interact with AcrA, transmitting the rotation in AcrB to TolC, facilitating the opening of the channel.

Other functions of RND efflux systems The assembled tri-partite efflux pump AcrAB-TolC. From MF Symmons, V Koronakis, personal communication. The assembled structure of AcrAB-TolC with 3 AcrA:3 AcrB:3 TolC ratio. The AcrB subunits are shown in shades blue, TolC subunits in orange/yellow and AcrA is shown in green. Membrane exposed surfaces and the TolC equatorial domain are shown grey. This model, based on extensive site directed crosslinking data, shows TolC, in its open conformation, is directly docked onto AcrB [36].

assembled the monomers form an outer membrane pore and a periplasmic tunnel that projects into the periplasm and narrows to a closed end approximately 2 A˚ in diameter [44,47]. TolC, OprM and VceC have a similar www.sciencedirect.com

In addition to an established role in MDR, RND efflux pumps also contribute to the basic biology of several pathogens. RND systems are required for the virulence of several species as mutants lacking functional RND efflux pumps are attenuated [1,51–60]. Partly, the contribution of these efflux systems to pathogenicity is explained by their involvement in export of host derived substrates such as bile salts, fatty acids and steroid hormones thereby enabling bacteria to survive within the hostile environment of the host [28,61–63]. AcrAB from E. coli has a far greater affinity for bile acids than for antibiotics suggesting that AcrB is better adapted for the efflux of bile acids [64,65]. Furthermore, expression of acrAB in Salmonella Typhimurium and acrD, acrE, emrK, mdtA and mdtE of E. coli are induced by the presence of Current Opinion in Microbiology 2009, 12:512–519

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indole or bile, both of which are present in the human intestine, allowing these toxic compounds to be exported permitting bacterial survival within the host [66,67]. Another class of host derived molecules that are substrates of RND efflux pumps are the antimicrobial peptides (AMPs) [68–70]. The human AMP LL-37 and the porcine protegrin-1 are substrates of the MtrCDE complex of Neisseria gonorrhoeae leading to the hypothesis that efflux may have evolved as a mechanism to circumvent the innate antimicrobial defences of the host [71]. Furthermore, mutation of mtrR, which represses mtrCDE transcription, led not only to increased resistance to antimicrobials including host derived peptides but also to an increase in fitness in the in vivo mouse model of infection suggesting that resistance to AMPs could contribute to virulence [71–73]. AMPs may not be substrates of all RND pumps or may have a species-specific role as resistance to several AMPs was not mediated by AcrAB of E. coli or MexAB of P. aeruginosa [74]. In support of the hypothesis that MDR efflux pumps are involved in survival in the hostile host environment, during macrophage infection by Salmonella the major facilitator superfamily (MFS) genes emrAB and the RND pump genes mdsABC and mdtABC were increased in expression [75,76]. This suggests a role for these genes during infection. However, expression of acrAB, acrEF and tolC were unaffected [75].

Inactivation of acrA, acrB or tolC had different effects on the transcriptome suggesting that the attenuation of virulence in strains lacking AcrA, AcrB or TolC could be mediated by different mechanisms. P. aeruginosa lacking the MexAB-OprM efflux system had a reduced ability to invade Madin-Darby Canine Kidney (MDCK) cells and invasiveness was restored not only by complementation of the disrupted genes but also by addition of culture supernatant from MDCK cells infected with wild-type P. aeruginosa. This suggests that the MexAB-OprM system exports a factor, present in the supernatant, that is able to ameliorate the virulence defect in the mutant [54]. It has also been suggested that AcrAB and its homologues export potentially toxic cellular metabolites, although evidence for this hypothesis is limited [79–81]. A novel physiological role for AcrB in E. coli in contactdependent growth inhibition (CDI) has been proposed that is independent of both AcrA and TolC [82]. CDI is the regulation of growth via cell to cell contact and significant numbers of mutants with resistance to CDI showed mutations in acrB or bamA, the latter of which is a member of the Omp85 family of essential outer membrane proteins. The specific role of AcrB in this process has not been elucidated but it may be a downstream target for the CDI signal [82]. Inhibition of efflux

Strains lacking functional RND pump components have an impaired ability to cause infection even when host derived molecules are absent. For Salmonellae the attenuation due to lack of TolC has been associated with reduced production of Salmonella Pathogenicity Island (SPI-1) genes [77,78]. Likewise, inactivation of acrB led to reduced expression of SPI-1 genes and its effectors and other pathogenicity related genes [78]. Webber et al. [78] also showed that lack of functional AcrA was associated with decreased expression of genes found in SPI-2.

The role of RND systems in innate and acquired MDR and virulence makes these proteins attractive targets for development of combination therapy involving an antibiotic administered with an inhibitor of efflux. In this way it is hoped that the clinical effectiveness of the remaining agents with activity against Gram-negative pathogens can be preserved. A recent review by Page`s and Amaral discusses in detail the most recent advances in efflux pump inhibitors (EPIs) so here we focus only on recent data for RND efflux pumps [83].

Figure 4

The structures of PAbN and chlorpromazine. The chemical structure of (a) PAbN and (b) chlorpromazine. Current Opinion in Microbiology 2009, 12:512–519

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Structure, function and inhibition of RND efflux pumps in Gram-negative bacteria: an update Blair and Piddock 517

PAbN was the first molecule identified as an EPI against P. aeruginosa MexB but is now thought to active against many more RND systems (Figure 4a) [84,85]. This molecule is a competitive efflux pump inhibitor that is preferentially pumped out by MexB. This means that less antibiotic is exported so intracellular accumulation is higher and toxicity is reached at lower concentrations leading to restoration of antibiotic sensitivity. PAbN is also thought to target C. jejuni CmeABC as addition increased susceptibility to bile and and oral administration to chickens 2–4 days post-inoculation significantly reduced C. jejuni colonisation [86]. The anti-psychotic phenothiazine drugs have also emerged as possible EPIs with one member, chlorpromazine, showing EPI-like activity for Salmonella Typhimurium, Burkholderia pseudomallei and E. coli (Figure 4b) [87–89]. Bailey et al. [88] showed that chlorpromazine reduced expression of acrB suggesting that the EPI-like activity was due to inhibition of AcrB production rather than interaction with the AcrB protein [88]. The wealth of new structural data including the publication of the first complete structure of a tripartite RND pump complex has provided an unprecedented insight into the function of these systems. These data will inform future rational design of antimicrobials targeted against these efflux systems.

Acknowledgements We are very grateful to Martyn Symmons for helpful discussions and providing the diagram of the complete AcrAB-TolC system (Figure 3) and for the edited MexA structure (Figure 2b) that were presented in a different format to those in [36]. We also thank Vassillis Koronakis. We are also grateful to Klaas Pos, Markus Gru¨tter and Atsushi Nakagawa for permission to reproduce Figures 1 and 2a,c, respectively.

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest

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transmembrane drug-efflux pump from Gram-negative bacteria. FEBS Lett 2004, 578:5-9. 41. Reffay M, Gambin Y, Benabdelhak H, Phan G, Taulier N, Ducruix A, Hodges RS, Urbach W: Tracking membrane protein association in model membranes. PLoS ONE 2009, 4:e5035. 42. Mima T, Joshi S, Gomez-Escalada M, Schweizer HP: Identification and characterization of TriABC-OpmH, a triclosan efflux pump of Pseudomonas aeruginosa requiring two membrane fusion proteins. J Bacteriol 2007, 189:7600-7609. 43. Gristwood T, Fineran PC, Everson L, Salmond GPC: PigZ, a TetR/ AcrR family repressor, modulates secondary metabolism via the expression of a putative four-component resistancenodulation-cell-division efflux pump, ZrpADBC, in Serratia sp. ATCC 39006. Mol Microbiol 2008, 69:418-435. 44. Koronakis V, Sharff A, Koronakis E, Luisi B, Hughes C: Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Nature 2000, 405:914-919. 45. Federici L, Du D, Walas F, Matsumura H, Fernandez-Recio J, McKeegan KS, Borges-Walmsley MI, Luisi BF, Walmsley AR: The crystal structure of the outer membrane protein VceC from the bacterial pathogen Vibrio cholerae at 1.8 A˚ resolution. J Biol Chem 2005, 280:15307-15314. 46. Akama H, Kanemaki M, Yoshimura M, Tsukihara T, Kashiwagi T, Yoneyama H, Narita S-i, Nakagawa A, Nakae T: Crystal structure of the drug discharge outer membrane protein, OprM, of Pseudomonas aeruginosa: dual modes of membrane anchoring and occluded cavity end. J Biol Chem 2004, 279:52816-52819. 47. Koronakis V, Eswaran J, Hughes C: Structure and function of TolC: the bacterial exit duct for proteins and drugs. Annu Rev Biochem 2004, 73:467-489. 48. Andersen C, Koronakis E, Bokma E, Eswaran J, Humphreys D, Hughes C, Koronakis V: Transition to the open state of the TolC periplasmic tunnel entrance. Proc Natl Acad Sci U S A 2002, 99:11103-11108. 49. Vaccaro L, Scott KA, Sansom MSP: Gating at both ends and  breathing in the middle: conformational dynamics of TolC. Biophys J 2008, 95:5681-5691. This paper describes the molecular dynamics of TolC and suggests an additional twisting motion in the upper portion of the periplasmic tunnel leading to a peristaltic action that may aid movement of the substrate along the tunnel. 50. Tornroth-Horsefield S, Gourdon P, Horsefield R, Brive L, Yamamoto N, Mori H, Snijder A, Neutze R: Crystal structure of AcrB in complex with a single transmembrane subunit reveals another twist. Structure 2007, 15:1663-1673. 51. Piddock LJV: Multidrug-resistance efflux pumps not just for resistance. Nat Rev Micro 2006, 4:629-636. 52. Buckley AM, Webber MA, Cooles S, Randall LP, La Ragione RM, Woodward MJ, Piddock LJV: The AcrAB-TolC efflux system of Salmonella enterica serovar Typhimurium plays a role in pathogenesis. Cell Microbiol 2006, 8:847-856. 53. Burse A, Weingart H, Ullrich MS: The phytoalexin-inducible multidrug efflux pump AcrAB contributes to virulence in the fire blight pathogen, Erwinia amylovora. Mol Plant Microbe Interact 2004, 17:43-54. 54. Hirakata Y, Srikumar R, Poole K, Gotoh N, Suematsu T, Kohno S, Kamihira S, Hancock REW, Speert DP: Multidrug efflux systems play an important role in the invasiveness of Pseudomonas aeruginosa. J Exp Med 2002, 196:109-118. 55. Jerse AE, Sharma ND, Simms AN, Crow ET, Snyder LA, Shafer WM: A gonococcal efflux pump system enhances bacterial survival in a female mouse model of genital tract infection. Infect Immun 2003, 71:5576-5582. 56. Lin J, Sahin O, Michel LO, Zhang Q: Critical role of multidrug efflux pump cmeabc in bile resistance and in vivo colonization of Campylobacter jejuni. Infect Immun 2003, 71:4250-4259. www.sciencedirect.com

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