Diversity in the oligomeric channel structure of the multidrug efflux pumps in Pseudomonas aeruginosa

MexAB-OprM, the multidrug efflux pump of Pseudomonas aeruginosa, contributes to the high resistance of this organism to a wide variety of antibiotics. To investigate the structure and function of OprM, the outer membrane channel of MexAB-OprM, we examined the oligomeric states of OprM and its homologues OprJ and OprN. These proteins were treated with crosslinking reagent after their reconstitution into liposome membranes. The crosslinked products indicated that OprM and OprN formed trimers, while OprJ unexpectedly appeared to form a tetramer. In order to test whether differences in oligomeric structure might be intimately related to channel function, we examined the channel-forming activity of these proteins by liposome swelling assay. However, no significant differences in channel characteristics were detected among OprM, OprJ, and OprN. We proposed the probable explanation for the diversity in the oligomeric structure of the channel proteins.     

Crystal structures of OprN and OprJ,outer membrane factors of multidrug tripartite efflux pumps of Pseudomonas aeruginosa

The genome of Pseudomonas aeruginosa encodes tripartite efflux pumps that extrude functionally and structurally dissimilar antibiotics from the bacterial cell. MexAB‐OprM, MexCD‐OprJ, MexEF‐OprN, and MexXY‐OprM are the main tripartite efflux pumps responsible for multidrug resistance in P. aeruginosa. The outer membrane factors OprN, OprJ, and OprM are essential components of functional tripartite efflux pumps. To elucidate the structural basis of multidrug resistance, we determined the crystal structures of OprN and OprJ. These structures revealed several features, including tri‐acylation of the N‐terminal cysteine, a small pore in the β‐barrel domain, and a tightly sealed gate in the α‐barrel domain. Despite the overall similarity of OprN, OprJ, and OprM, a comparison of their structures and electrostatic distributions revealed subtle differences at the periplasmic end of the α‐barrel domain. These results suggested that the overall structures of these outer membrane factors are specifically optimized for particular tripartite efflux pumps. Proteins 2016; 84:759–769. © 2016 Wiley Periodicals, Inc.     

Functional interaction sites of OprM with MexAB in the Pseudomonas aeruginosa multidrug efflux pump

Subunit-swapping between Pseudomonas aeruginosa MexAB-OprM and MexEF-OprN efflux pumps has shown that OprM can interact with MexEF to produce a functional efflux pump, but that OprN cannot functionally interact with MexAB. Taking advantage of this subunit selectivity, we carried out experiments using chimeric proteins composed of OprM and OprN to determine which regions of OprM are necessary for functional interaction with MexAB. We constructed two types of chimeric proteins: one with the N-terminal half of OprM and the C-terminal half of OprN (OprMN), and the second with these halves reversed (OprNM). Introduction of either of the chimeric protein genes into a mutant expressing MexEF alone restored the functionality of the efflux pump. However, expression of OprMN or OprNM in the presence of MexAB did not restore the pump functionality, indicating that the both the N- and C-terminal halves of OprM are necessary for a functional interaction with MexAB.     

The MexJK efflux pump of Pseudomonas aeruginosa requires OprM for antibiotic efflux but not for efflux of triclosan

Using the biocide triclosan as a selective agent, several triclosan-resistant mutants of a susceptible Pseudomonas aeruginosa strain were isolated. Cloning and characterization of a DNA fragment conferring triclosan resistance from one of these mutants revealed a hitherto uncharacterized efflux system of the resistance nodulation cell division (RND) family, which was named MexJK and which is encoded by the mexJK operon. Expression of this operon is negatively regulated by the product of mexL, a gene located upstream of and transcribed divergently from mexJK. The triclosan-resistant mutant contained a single nucleotide change in mexL, which caused an amino acid change in the putative helix-turn-helix domain of MexL. The MexL protein belongs to the TetR family of repressor proteins. The MexJK system effluxed tetracycline and erythromycin but only in the presence of the outer membrane protein channel OprM; OprJ and OprN did not function with MexJK. Triclosan efflux required neither of the outer membrane protein channels tested but necessitated the MexJ membrane fusion protein and the MexK inner membrane RND transporter. The results presented in this study suggest that MexJK may function as a two-component RND pump for triclosan efflux but must associate with OprM to form a tripartite antibiotic efflux system. Furthermore, the results confirm that triclosan is an excellent tool for the study of RND multidrug efflux systems and that this popular biocide therefore readily selects mutants which are cross-resistant with antibiotics.     

Contribution of multidrug efflux pumps to multiple antibiotic resistance in veterinary clinical isolates of Pseudomonas aeruginosa

The contribution of efflux pumps to multidrug resistance in 12 Pseudomonas aeruginosa isolates from various animal sources was assessed. Western immunoblot analyses demonstrated that all twelve isolates expressed significant levels of the MexAB OprM efflux system whereas two isolates simultaneously expressed the MexEF OprN or MexXY systems, respectively. One strain contained a single mutation in mexR, a regulator of mexAB-oprM expression, that did not adversely affect the MexR amino acid sequence, and three isolates contained the same, single base change in the mexA-mexR intergenic region. The MexXY-expressing strain contained two base substitutions in its mexZ regulatory gene which did not alter the MexR sequence.     

Inner membrane efflux components are responsible for beta-lactam specificity of multidrug efflux pumps in Pseudomonas aeruginosa.

A major feature of the MexAB-OprM multidrug efflux pump which distinguishes it from the MexCD-OprJ and MexEF-OprN multidrug efflux systems in Pseudomonas aeruginosa is its ability to export a wide variety of beta-lactam antibiotics. Given the periplasmic location of their targets it is feasible that beta-lactams exit the cell via the outer membrane OprM without interaction with MexA and MexB, though the latter appear to be necessary for OprM function. To test this, chimeric MexAB-OprJ and MexCD-OprM efflux pumps were reconstituted in delta mexCD delta oprM and delta mexAB delta oprJ strains, respectively, and the influence of the exchange of outer membrane components on substrate (i.e., beta-lactam) specificity was assessed. Both chimeric pumps were active in antibiotic efflux, as evidenced by their contributions to resistance to a variety of antimicrobial agents, although there was no change in resistance profiles relative to the native pumps, indicating that OprM is not the determining factor for the beta-lactam specificity of MexAB-OprM. Thus, one or both of inner membrane-associated proteins MexA and MexB are responsible for drug recognition, including recognition of beta-lactams.     

Assembly and channel opening of outer membrane protein in tripartite drug efflux pumps of Gram-negative bacteria

Gram-negative bacteria are capable of expelling diverse xenobiotic substances from within the cell by use of three-component efflux pumps in which the energy-activated inner membrane transporter is connected to the outer membrane channel protein via the membrane fusion protein. In this work, we describe the crystal structure of the membrane fusion protein MexA from the Pseudomonas aeruginosa MexAB-OprM pump in the hexameric ring arrangement. Electron microscopy study on the chimeric complex of MexA and the outer membrane protein OprM reveals that MexA makes a tip-to-tip interaction with OprM, which suggests a docking model for MexA and OprM. This docking model agrees well with genetic results and depicts detailed interactions. Opening of the OprM channel is accompanied by the simultaneous exposure of a protein structure resembling a six-bladed cogwheel, which intermeshes with the complementary cogwheel structure in the MexA hexamer. Taken together, we suggest an assembly and channel opening model for the MexAB-OprM pump. This study provides a better understanding of multidrug resistance in Gram-negative bacteria.     

The outer membrane component of the multidrug efflux pump from Pseudomonas aeruginosa may be a gated channel.

OprM, the outer membrane component of the MexAB-OprM multidrug efflux pump of Pseudomonas aeruginosa, has been assumed to facilitate the export of antibiotics across the outer membrane of this organism. Here we purified to homogeneity the OprM protein, reconstituted it into liposome membranes, and tested its channel activity by using the liposome swelling assay. It was demonstrated that OprM is a channel-forming protein and exhibits the channel property that amino acids diffuse more efficiently than saccharides. However, antibiotics showed no significant diffusion through the OprM channel in the liposome membrane, suggesting that OprM functions as a gated channel. We reasoned that the protease treatment may cause the disturbance of the gate structure of OprM. Hence, we treated OprM reconstituted in the membranes with alpha-chymotrypsin and examined its solute permeability. The results demonstrated that the protease treatment caused the opening of an OprM channel through which antibiotics were able to diffuse. To elucidate which cleavage is intimately related to the opening, we constructed mutant OprM proteins where the amino acid at the cleavage site was replaced with another amino acid. By examining the channel activity of these mutant proteins, it was shown that the proteolysis at tyrosine 185 and tyrosine 196 of OprM caused the channel opening. Furthermore, these residues were shown to face into the periplasmic space and interact with other component(s). We considered the possible opening mechanism of the OprM channel based on the structure of TolC, a homologue of OprM.     

Assembly of the MexAB-OprM multidrug pump of Pseudomonas aeruginosa: component interactions defined by the study of pump mutant suppressors

In an effort to identify key domains of the Pseudomonas aeruginosa MexAB-OprM drug efflux system involved in component interactions, extragenic suppressors of various inactivating mutations in individual pump constituents were isolated and studied. The multidrug hypersusceptibility of P. aeruginosa expressing MexB with a mutation in a region of the protein implicated in oligomerization (G220S) was suppressed by mutations in the alpha/beta domain of MexA. MexB(G220S) showed a reduced ability to bind MexA in vivo while representative MexA suppressors (V66M and V259F) restored the MexA-MexB interaction. Interestingly, these suppressors also restored resistance in P. aeruginosa expressing OprM proteins with mutations at the proximal (periplasmic) tip of OprM that is predicted to interact with MexB, suggesting that these suppressors generally overcame defects in MexA-MexB and MexB-OprM interaction. The multidrug hypersusceptibility arising from a mutation in the helical hairpin of MexA implicated in OprM interaction (V129M) was suppressed by mutations (T198I and F439I) in the periplasmic alpha-helical barrel of OprM. Again, the MexA mutation compromised an in vivo interaction with OprM that was restored by the T198I and F439I substitutions in OprM, consistent with the hairpin domain mediating MexA binding to this region of OprM. Interestingly, these OprM suppressor mutations restored multidrug resistance in P. aeruginosa expressing MexB(G220S). Finally, the oprM(T198I) suppressor mutation enhanced the yields of all three constituents of a MexA-MexB-OprM(T198I) pump as detected in whole-cell extracts. These data highlight the importance of MexA and interactions with this adapter in promoting MexAB-OprM pump assembly and in stabilizing the pump complex.     

Importance of the adaptor (membrane fusion) protein hairpin domain for the functionality of multidrug efflux pumps

Drug efflux pumps of Gram-negative bacteria are tripartite export machineries located in the bacterial envelopes contributing to multidrug resistance. Protein structures of all three components have been determined, but the exact interaction sites are still unknown. We could confirm that the hybrid system composed of Pseudomonas aeruginosa channel tunnel OprM and the Escherichia coli inner membrane complex, formed by adaptor protein (membrane fusion protein) AcrA and transporter AcrB of the resistance nodulation cell division (RND) family, is not functional. However, cross-linking experiments show that the hybrid exporter assembles. Exchange of the hairpin domain of AcrA with the corresponding hairpin from adaptor protein MexA of P. aeruginosa restored the functionality. This shows the importance of the MexA hairpin domain for the functional interaction with the OprM channel tunnel. On the basis of these results, we have modeled the interaction of the hairpin domain and the channel tunnel on a molecular level for AcrA and TolC as well as MexA and OprM, respectively. The model of two hairpin docking sites per TolC protomer corresponding with hexameric adaptor proteins was confirmed by disulfide cross-linking experiments. The role of this interaction for functional efflux pumps is discussed.     

Assignment of the outer-membrane-subunit-selective domain of the membrane fusion protein in the tripartite xenobiotic efflux pump of Pseudomonas aeruginosa

Early in vivo experiments revealed that the MexA-MexB dipartite pump unit of Pseudomonas aeruginosa conferred drug resistance to the cells, which expressed OprM, but not to the OprN-bearing cells. While the MexE-MexF unit interplayed with either the outer membrane subunits. Taking advantage of this subunit selectivity, we selected the MexA mutant that gained the ability to interplay with OprN. Four mutants have been isolated and all showed an amino acid substitution (Q116R) in the coiled-coil domain of MexA. The hybrid protein bearing the coiled-coil domain of MexA and the remainder domains from MexE retained the ability to interplay with OprM, but lost the functional interplay with OprN. These results established that the coiled-coil domain of the membrane fusion protein is responsible for selecting the compatible outer membrane subunit.     

Structure of reconstituted bacterial membrane efflux pump by cryo-electron tomography

Complexes of OprM and MexA, two proteins of the MexA-MexB-OprM multidrug efflux pump from Pseudomonasaeruginosa, an opportunistic Gram-negative bacterium, were reconstituted into proteoliposomes by detergent removal. Stacks of protein layers with a constant height of 21 nm, separated by lipid bilayers, were obtained at stoichiometry of 1:1 (w/w). Using cryo-electron microscopy and tomography, we showed that these protein layers were composed of MexA-OprM complexes self-assembled into regular arrays. Image processing of extracted sub-tomograms depicted the architecture of the bipartite complex sandwiched between two lipid bilayers, representing an environment close to that of the native whole pump (i.e. anchored between outer and inner membranes of P. aeruginosa). The MexA-OprM complex appeared as a cylindrical structure in which we were able to identify the OprM molecule and the MexA moiety. MexA molecules have a cylindrical shape prolonging the periplasmic helices of OprM, and widening near the lipid bilayer. The flared part is likely composed of two MexA domains adjacent to the lipid bilayer, although their precise organization was not reachable mainly due to their flexibility. Moreover, the intermembrane distance of 21 nm indicated that the height of the bipartite complex is larger than that of the tripartite AcrA-AcrB-TolC built-up model in which TolC and AcrB are docked into contact. We proposed a model of MexA-OprM taking into account features of previous models based on AcrA-AcrB-TolC and our structural results providing clues to a possible mechanism of tripartite system assembly.     

Novobiocin binding to NalD induces the expression of the MexAB‐OprM pump in Pseudomonas aeruginosa

NalD was reported to be the secondary repressor of the MexAB‐OprM multidrug efflux pump, the major system contributing to intrinsic multidrug resistance in Pseudomonas aeruginosa. Here, we show that novobiocin binds directly to NalD, which leads NalD to dissociate from the DNA promoter, and thus de‐represses the expression of the MexAB‐OprM pump. In addition, we have solved the crystal structure of NalD at a resolution of 2.90 Å. The structural alignment of NalD to its homologue TtgR reveals that the residues N129 and H167 in NalD are involved in its novobiocin‐binding ability. We have confirmed the function of these two amino acids by EMSA and plate assay. The results presented here highlight the importance and diversity of regulatory mechanism in bacterial antibiotic resistance, and provide further insight for novel antimicrobial development.     

Evaluation of a structural model of Pseudomonas aeruginosa outer membrane protein OprM, an efflux component involved in intrinsic antibiotic resistance

The outer membrane protein OprM of Pseudomonas aeruginosa is involved in intrinsic and mutational multiple-antibiotic resistance as part of two resistance-nodulation-division efflux systems. The crystal structure of TolC, a homologous protein in Escherichia coli, was recently published (V. Koronakis, A. Sharff, E. Koronakis, B. Luisl, and C. Hughes, Nature 405:914-919, 2000), demonstrating a distinctive architecture comprising outer membrane beta-barrel and periplasmic helical-barrel structures, which assemble differently from the common beta-barrel-only conformation of porins. Based on their sequence similarity, a similar content of alpha-helical and beta-sheet structure determined by circular dichroism spectroscopy, and our observation that OprM, like TolC, reconstitutes channels in planar bilayer membranes, OprM and TolC were considered to be structurally homologous, and a model of OprM was constructed by threading its sequence to the TolC crystal structure. Residues thought to be important for the TolC structure were conserved in space in this OprM model. Analyses of deletion mutants and previously isolated insertion mutants of OprM in the context of this model allowed us to propose roles for different protein domains. Our data indicate that the helical barrel of the protein is critical for both the function and the integrity of the protein, while a C-terminal domain localized around the equatorial plane of this helical barrel is dispensable. Extracellular loops appear to play a lesser role in substrate specificity for this efflux protein compared to classical porins, and there appears to be a correlation between the change in antimicrobial activity for OprM mutants and the pore size. Our model and channel formation studies support the "iris" mechanism of action for TolC and permit us now to form more focused hypotheses about the functional domains of OprM and its related family of efflux proteins.     

Differential impact of MexB mutations on substrate selectivity of the MexAB-OprM multidrug efflux pump of Pseudomonas aeruginosa

The integral inner membrane resistance-nodulation-division (RND) components of three-component RND-membrane fusion protein-outer membrane factor multidrug efflux systems define the substrate selectivity of these efflux systems. To gain a better understanding of what regions of these proteins are important for substrate recognition, a plasmid-borne mexB gene encoding the RND component of the MexAB-OprM multidrug efflux system of Pseudomonas aeruginosa was mutagenized in vitro by using hydroxylamine and mutations compromising the MexB contribution to antibiotic resistance identified in a DeltamexB strain. Of 100 mutants that expressed wild-type levels of MexB and showed increased susceptibility to one or more of carbenicillin, chloramphenicol, nalidixic acid, and novobiocin, the mexB genes of a representative 46 were sequenced, and 19 unique single mutations were identified. While the majority of mutations occurred within the large periplasmic loops between transmembrane segment 1 (TMS-1) and TMS-2 and between TMS-7 and TMS-8 of MexB, mutations were seen in the TMSs and in other periplasmic as well as cytoplasmic loops. By threading the MexB amino acid sequence through the crystal structure of the homologous RND transporter from Escherichia coli, AcrB, a three-dimensional model of a MexB trimer was obtained and the mutations were mapped to it. Unexpectedly, most mutations mapped to regions of MexB predicted to be involved in trimerization or interaction with MexA rather than to regions expected to contribute to substrate recognition. Intragenic second-site suppressor mutations that restored the activity of the G220S mutant version of MexB, which was compromised for resistance to all tested MexAB-OprM antimicrobial substrates, were recovered and mapped to the apparently distal portion of MexB that is implicated in OprM interaction. As the G220S mutation likely impacted trimerization, it appears that either proper assembly of the MexB trimer is necessary for OprM interaction or OprM association with an unstable MexB trimer might stabilize it, thereby restoring activity.     

Insertion mutagenesis and membrane topology model of the Pseudomonas aeruginosa outer membrane protein OprM

Pseudomonas aeruginosa OprM is a protein involved in multiple-antibiotic resistance as the outer membrane component for the MexA-MexB-OprM efflux system. Planar lipid bilayer experiments showed that OprM had channel-forming activity with an average single-channel conductance of only about 80 pS in 1 M KCl. The gene encoding OprM was subjected to insertion mutagenesis by cloning of a foreign epitope from the circumsporozoite form of the malarial parasite Plasmodium falciparum into 11 sites. In Escherichia coli, 8 of the 11 insertion mutant genes expressed proteins at levels comparable to those obtained with the wild-type gene and the inserted malarial epitopes were surface accessible as assessed by indirect immunofluorescence. When moved to a P. aeruginosa OprM-deficient strain, seven of the insertion mutant genes expressed proteins at variable levels comparable to that of wild-type OprM and three of these reconstituted MIC profiles resembling those of the wild-type protein, while the other mutant forms showed variable MIC results. Utilizing the data from these experiments, in conjunction with multiple sequence alignments and structure predictions, an OprM topology model with 16 beta strands was proposed.     

Membrane topology of the xenobiotic-exporting subunit, MexB, of the MexA,B-OprM extrusion pump in Pseudomonas aeruginosa

The MexA,B-OprM efflux pump assembly of Pseudomonas aeruginosa consists of two inner membrane proteins and one outer membrane protein. The cytoplasmic membrane protein, MexB, appears to function as the xenobiotic-exporting subunit, whereas the MexA and OprM proteins are supposed to function as the membrane fusion protein and the outer membrane channel protein, respectively. Computer-aided hydropathy analyses of MexB predicted the presence of up to 17 potential transmembrane segments. To verify the prediction, we analyzed the membrane topology of MexB using the alkaline phosphatase gene fusion method. We obtained the following unique characteristics. MexB bears 12 membrane spanning segments leaving both the amino and carboxyl termini in the cytoplasmic side of the inner membrane. Both the first and fourth periplasmic loops had very long hydrophilic domains containing 311 and 314 amino acid residues, respectively. This fact suggests that these loops may interact with other pump subunits, such as the membrane fusion protein MexA and the outer membrane protein OprM. Alignment of the amino- and the carboxyl-terminal halves of MexB showed a 30% homology and transmembrane segments 1, 2, 3, 4, 5, and 6 could be overlaid with the segments 7, 8, 9, 10, 11, and 12, respectively. This result suggested that the MexB has a 2-fold repeat that strengthen the experimentally determined topology model. This paper reports the structure of the pump subunit, MexB, of the MexA,B-OprM efflux pump assembly. This is the first time to verify the topology of the resistant-nodulation-division efflux pump protein.     

Crystal structure of the membrane fusion protein, MexA, of the multidrug transporter in Pseudomonas aeruginosa

The MexAB-OprM efflux pump of Pseudomonas aeruginosa is central to multidrug resistance of this organism, which infects immunocompromised hospital patients. The MexA, MexB, and OprM subunits were assumed to function as the membrane fusion protein, the body of the transporter, and the outer membrane channel protein, respectively. For better understanding of this important xenobiotic transporter, we show the x-ray crystallographic structure of MexA at a resolution of 2.40 A. The global MexA structure showed unforeseen new features with a spiral assembly of six and seven protomers that were joined together at one end by a pseudo 2-fold image. The protomer showed a new protein structure with a tandem arrangement consisting of at least three domains and presumably one more. The rod domain had a long hairpin of twisted coiled-coil that extended to one end. The second domain adjacent to the rod alpha-helical domain was globular and constructed by a cluster of eight short beta-sheets. The third domain located distal to the alpha-helical rod was globular and composed of seven short beta-sheets and one short alpha-helix. The 13-mer was shaped like a woven rattan cylinder with a large internal tubular space and widely opened flared ends. The 6-mer and 7-mer had a funnel-like structure consisting of a tubular rod at one side and a widely opened flared funnel top at the other side. Based on these results, we constructed a model of the MexAB-OprM pump assembly. The three pairs of MexA dimers interacted with the periplasmic alpha-barrel domain of OprM via the alpha-helical hairpin, the second domain interacted with both MexB and OprM at their contact site, and the third and disordered domains probably interacted with the distal domain of MexB. In this fashion, the MexA subunit connected MexB and OprM, indicating that MexA is the membrane bridge protein.