Isolation and characterization of VceC gain-of-function mutants that can function with the AcrAB multiple-drug-resistant efflux pump of Escherichia coli

VceC is the outer membrane component of the major facilitator (MF) VceAB-VceC multiple-drug-resistant (MDR) efflux pump of Vibrio cholerae. TolC is the outer membrane component of the resistance-nodulation-division AcrAB-TolC efflux pump of Escherichia coli. Although these proteins share little amino acid sequence identity, their crystal structures can be readily superimposed upon one another. In this study, we have asked if TolC and VceC are interchangeable for the functioning of the AcrAB and VceAB pumps. We have found that TolC can replace VceC to form a functional VceAB-TolC MDR pump, but VceC cannot replace TolC to form a functional AcrAB-VceC pump. However, we have been able to isolate gain-of-function (gof) VceC mutants which can functionally interface with AcrAB. These mutations map to four different amino acids located at the periplasmic tip of VceC. Chemical cross-linkage experiments indicate that both wild-type and gof mutant VceC can physically interact with the AcrAB complex, suggesting that these gof mutations are not affecting the recruitment of VceC to the AcrAB complex but rather its ability to functionally interface with the AcrAB pump.     

Assembly and channel opening in a bacterial drug efflux machine

Drugs and certain proteins are transported across the membranes of Gram-negative bacteria by energy-activated pumps. The outer membrane component of these pumps is a channel that opens from a sealed resting state during the transport process. We describe two crystal structures of the Escherichia coli outer membrane protein TolC in its partially open state. Opening is accompanied by the exposure of three shallow intraprotomer grooves in the TolC trimer, where our mutagenesis data identify a contact point with the periplasmic component of a drug efflux pump, AcrA. We suggest that the assembly of multidrug efflux pumps is accompanied by induced fit of TolC driven mainly by accommodation of the periplasmic component.     

Multidrug resistance mechanisms: drug efflux across two membranes

A set of multidrug efflux systems enables Gram-negative bacteria to survive in a hostile environment. This review focuses on the structural features and the mechanism of major efflux pumps of Gram-negative bacteria, which expel from the cells a remarkably broad range of antimicrobial compounds and produce the characteristic intrinsic resistance of these bacteria to antibiotics, detergents, dyes and organic solvents. Each efflux pump consists of three components: the inner membrane transporter, the outer membrane channel and the periplasmic lipoprotein. Similar to the multidrug transporters from eukaryotic cells and Gram-positive bacteria, the inner membrane transporters from Gram-negative bacteria recognize and expel their substrates often from within the phospholipid bilayer. This efflux occurs without drug accumulation in the periplasm, implying that substrates are pumped out across the two membranes directly into the medium. Recent data suggest that the molecular mechanism of the drug extrusion across a two-membrane envelope of Gram-negative bacteria may involve the formation of the membrane adhesion sites between the inner and the outer membranes. The periplasmic components of these pumps are proposed to cause a close membrane apposition as the complexes are assembled for the transport.     

Structure and mechanism of the tripartite CusCBA heavy-metal efflux complex

Gram-negative bacteria frequently expel toxic chemicals through tripartite efflux pumps that span both the inner and outer membranes. The three parts are the inner membrane, substrate-binding transporter (or pump); a periplasmic membrane fusion protein (MFP, or adaptor); and an outer membrane-anchored channel. The fusion protein connects the transporter to the channel within the periplasmic space. One such efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions. We previously described the crystal structures of both the inner membrane transporter CusA and the MFP CusB of Escherichia coli. We also determined the co-crystal structure of the CusBA adaptor-transporter efflux complex, showing that the transporter CusA, which is present as a trimer, interacts with six CusB protomers and that the periplasmic domain of CusA is involved in these interactions. Here, we summarize the structural information of these efflux proteins, and present the accumulated evidence that this efflux system uses methionine residues to bind and export Cu(I) and Ag(I). Genetic and structural analyses suggest that the CusA pump is capable of picking up the metal ions from both the periplasm and the cytoplasm. We propose a stepwise shuttle mechanism for this pump to export metal ions from the cell.     

Crystal Structure of the Periplasmic Component of a Tripartite Macrolide-Specific Efflux Pump

In Gram-negative bacteria, type I protein secretion systems and tripartite drug efflux pumps have a periplasmic membrane fusion protein (MFP) as an essential component. MFPs bridge the outer membrane factor and an inner membrane transporter, although the oligomeric state of MFPs remains unclear. The most characterized MFP AcrA connects the outer membrane factor TolC and the resistance-nodulation-division-type efflux transporter AcrB, which is a major multidrug efflux pump in Escherichia coli. MacA is the periplasmic MFP in the MacAB-TolC pump, where MacB was characterized as a macrolide-specific ATP-binding-cassette-type efflux transporter. Here, we report the crystal structure of E. coli MacA and the experimentally phased map of Actinobacillus actinomycetemcomitans MacA, which reveal a domain orientation of MacA different from that of AcrA. Notably, a hexameric assembly of MacA was found in both crystals, exhibiting a funnel-like structure with a central channel and a conical mouth. The hexameric MacA assembly was further confirmed by electron microscopy and functional studies in vitro and in vivo. The hexameric structure of MacA provides insight into the oligomeric state in the functional complex of the drug efflux pump and type I secretion system.     

Evaluation of a series of 2-napthamide derivatives as inhibitors of the drug efflux pump AcrB for the reversal of antimicrobial resistance

Drug efflux pumps confer multidrug resistance to dangerous pathogens which makes these pumps important drug targets. We have synthesised a novel series of compounds based on a 2-naphthamide pharmacore aimed at inhibiting the efflux pumps from Gram-negative bacteria. The archeatypical transporter AcrB from Escherichia coli was used as model efflux pump as AcrB is widely conserved throughout Gram-negative organisms. The compounds were tested for their antibacterial action, ability to potentiate the action of antibiotics and for their ability to inhibit Nile Red efflux by AcrB. None of the compounds were antimicrobial against E. coli wild type cells. Most of the compounds were able to inhibit Nile Red efflux indicating that they are substrates of the AcrB efflux pump. Three compounds were able to synergise with antibiotics and reverse resistance in the resistant phenotype. Compound A3, 4-(isopentyloxy)-2-naphthamide, reduced the MICs of erythromycin and chloramphenicol to the MIC levels of the drug sensitive strain that lacks an efflux pump. A3 had no effect on the MIC of the non-substrate rifampicin indicating that this compound acts specifically through the AcrB efflux pump. A3 also does not act through non-specific mechanisms such as outer membrane or inner membrane permeabilisation and is not cytotoxic against mammalian cell lines. Therefore, we have designed and synthesised a novel chemical compound with great potential to further optimisation as inhibitor of drug efflux pumps.     

Charged Amino Acids (R83, E567, D617, E625, R669, and K678) of CusA Are Required for Metal Ion Transport in the Cus Efflux System

Gram-negative bacteria expel various toxic chemicals via tripartite efflux pumps belonging to the resistance-nodulation-cell division superfamily. These pumps span both the inner and outer membranes of the cell. The three components of these tripartite systems are an inner-membrane, substrate-binding transporter (or pump); a periplasmic membrane fusion protein (or adaptor); and an outer-membrane-anchored channel. These three efflux proteins interact in the periplasmic space to form the three-part complexes. We previously presented the crystal structures of both the inner-membrane transporter CusA and membrane fusion protein CusB of the CusCBA tripartite efflux system from Escherichia coli. We also described the co-crystal structure of the CusBA adaptor-transporter, revealing that the trimeric CusA efflux pump assembles with six CusB protein molecules to form the complex CusB(6)-CusA(3). We here report three different conformers of the crystal structures of CusBA-Cu(I), suggesting a mechanism on how Cu(I) binding initiates a sequence of conformational transitions in the transport cycle. Genetic analysis and transport assays indicate that charged residues, in addition to the methionine pairs and clusters, are essential for extruding metal ions out of the cell.     

A data‐driven approach to modeling the tripartite structure of multidrug resistance efflux pumps

Many bacterial pathogens are becoming increasingly resistant to antibiotic treatments, and a detailed understanding of the molecular basis of antibiotic resistance is critical for the development of next‐generation approaches for combating bacterial infections. Studies focusing on pathogens have revealed the profile of resistance in these organisms to be due primarily to the presence of multidrug resistance efflux pumps: tripartite protein complexes which span the periplasm bridging the inner and outer membranes of Gram‐negative bacteria. An atomic‐level resolution tripartite structure remains imperative to advancing our understanding of the molecular mechanisms of pump function using both theoretical and experimental approaches. We develop a fast and consistent method for constructing tripartite structures which leverages existing data‐driven models and provide molecular modeling approaches for constructing tripartite structures of multidrug resistance efflux pumps. Our modeling studies reveal that conformational changes in the inner membrane component responsible for drug translocation have limited impact on the conformations of the other pump components, and that two distinct models derived from conflicting experimental data are both consistent with all currently available measurements. Additionally, we investigate putative drug translocation pathways via geometric simulations based on the available crystal structures of the inner membrane pump component, AcrB, bound to two drugs which occupy distinct binding sites: doxorubicin and linezolid. These simulations suggest that smaller drugs may enter the pump through a channel from the cytoplasmic leaflet of the inner membrane, while both smaller and larger drug molecules may enter through a vestibule accessible from the periplasm. Proteins 2015; 83:46–65. © 2014 Wiley Periodicals, Inc.     

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.     

A noncanonical chaperone interacts with drug efflux pumps during their assembly into bacterial outer membranes

Bacteria have membrane-spanning efflux pumps to secrete toxic compounds ranging from heavy metal ions to organic chemicals, including antibiotic drugs. The overall architecture of these efflux pumps is highly conserved: with an inner membrane energy-transducing subunit coupled via an adaptor protein to an outer membrane conduit subunit that enables toxic compounds to be expelled into the environment. Here, we map the distribution of efflux pumps across bacterial lineages to show these proteins are more widespread than previously recognised. Complex phylogenetics support the concept that gene cassettes encoding the subunits for these pumps are commonly acquired by horizontal gene transfer. Using TolC as a model protein, we demonstrate that assembly of conduit subunits into the outer membrane uses the chaperone TAM to physically organise the membrane-embedded staves of the conduit subunit of the efflux pump. The characteristics of this assembly pathway have impact for the acquisition of efflux pumps across bacterial species and for the development of new antimicrobial compounds that inhibit efflux pump function.

A crosslinking study reveals novel insights into how the chaperone TAM helps Gram-negative bacteria insert the drug efflux pump subunit TolC into their outer membrane. Bioinformatic analyses show that TolC-like proteins can be found in all LPS-containing bacteria, but also in some monodermic Firmicutes.     

Channel-tunnels: outer membrane components of type I secretion systems and multidrug efflux pumps of Gram-negative bacteria

For translocation across the cell envelope of Gram-negative bacteria, substances have to overcome two permeability barriers, the inner and outer membrane. Channel-tunnels are outer membrane proteins, which are central to two distinct export systems: the type I secretion system exporting proteins such as toxins or proteases, and efflux pumps discharging antibiotics, dyes, or heavy metals and thus mediating drug resistance. Protein secretion is driven by an inner membrane ATP-binding cassette (ABC) transporter while drug efflux occurs via an inner membrane proton antiporter. Both inner membrane transporters are associated with a periplasmic accessory protein that recruits an outer membrane channel-tunnel to form a functional export complex. Prototypes of these export systems are the hemolysin secretion system and the AcrAB/TolC drug efflux pump of Escherichia coli, which both employ TolC as an outer membrane component. Its remarkable conduit-like structure, protruding 100 ? into the periplasmic space, reveals how both systems are capable of transporting substrates across both membranes directly from the cytosol into the external environment. Proteins of the channel-tunnel family are widespread within Gram-negative bacteria. Their involvement in drug resistance and in secretion of pathogenic factors makes them an interesting system for further studies. Understanding the mechanism of the different export apparatus could help to develop new drugs, which block the efflux pumps or the secretion system. Electronic Publication     

Multiple antibiotic resistance and efflux

Multiple antibiotic resistance in bacteria was at first thought to be caused exclusively by the combination of several resistance genes, each coding for resistance to a single drug. More recently, it became clear that such phenotypes are often achieved by the activity of drug efflux pumps. Some of these efflux pumps exhibit an extremely wide specificity covering practically all antibiotics, chemotherapeutic agents, detergents, dyes, and other inhibitors, the exception perhaps being very hydrophilic compounds. Such efflux pumps work with exceptional efficiency in Gram-negative bacteria through their synergistic interaction with the outer membrane barrier. It is disturbing that the antibacterial agents of the most advanced type, which are unaffected by common resistance mechanisms, are precisely the compounds whose use appears to select for multidrug-resistant mutants that overproduce these efflux pumps of wide specificity.     

Conformational flexibility in the multidrug efflux system protein AcrA

Intrinsic resistance to multiple drugs in many gram-negative bacterial pathogens is conferred by resistance nodulation cell division efflux pumps, which are composed of three essential components as typified by the extensively characterized Escherichia coli AcrA-AcrB-TolC system. The inner membrane drug:proton antiporter AcrB and the outer membrane channel TolC export chemically diverse compounds out of the bacterial cell, and require the activity of the third component, the periplasmic protein AcrA. The crystal structures of AcrB and TolC have previously been determined, and we complete the molecular picture of the efflux system by presenting the structure of a stable fragment of AcrA. The AcrA fragment resembles the elongated sickle shape of its homolog Pseudomonas aeruginosa MexA, being composed of three domains: beta-barrel, lipoyl, and alpha-helical hairpin. Notably, unsuspected conformational flexibility in the alpha-helical hairpin domain of AcrA is observed, which has potential mechanistic significance in coupling between AcrA conformations and TolC channel opening.     

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.     

Inhibitors of efflux pumps in Gram-negative bacteria

In Gram-negative bacteria, efflux complexes, consisting of an inner-membrane pump, a periplasmic adaptor protein and outer-membrane channel, provide an efficient means for the export of structurally unrelated drugs, causing the multidrug-resistance phenotype. Resistance due to this antibiotic efflux is an increasing problem worldwide. A new molecular challenge is to combat this transport by searching for new molecules to block efflux and thus restore drug susceptibility to resistant clinical strains. Recent data shed new light on the structure and activity of the archetypal efflux pumps AcrAB-TolC and MexAB-OprM. Here, we describe recent insights into the molecular mechanisms of bacterial efflux pumps and their inhibitors. Current progress for the clinical use of efflux-pump inhibitors and new strategies to combat the drug-efflux mechanisms will be discussed.     

The Cus efflux system removes toxic ions via a methionine shuttle

Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes in the resistance-nodulation-cell division (RND) family to expel diverse toxic compounds from the cell. These efflux systems span the entire cell envelope to mediate the phenomenon of bacterial multidrug resistance. The three parts of the efflux complexes are: (1) a membrane fusion protein (MFP) connecting (2) a substrate-binding inner membrane transporter to (3) an outer membrane-anchored channel in the periplasmic space. One such efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions. We recently determined the crystal structures of both the inner membrane transporter CusA and MFP CusB of the CusCBA tripartite efflux system from E. coli. These are the first structures of the heavy-metal efflux (HME) subfamily of the RND efflux pumps. Here, we summarize the structural information of these two efflux proteins and present the accumulated evidence that this efflux system utilizes methionine residues to bind and export Cu(I)/Ag(I). Genetic and structural analyses suggest that the CusA pump is capable of picking up the metal ions from both the periplasm and cytoplasm. We propose a stepwise shuttle mechanism for this pump to extrude metal ions from the cell.     

Structural mechanisms of heavy-metal extrusion by the Cus efflux system

Resistance-nodulation-cell division (RND) superfamily efflux systems are responsible for the active transport of toxic compounds from the Gram-negative bacterial cell. These pumps typically assemble as tripartite complexes, spanning the inner and outer membranes of the cell envelope. In Escherichia coli, the CusC(F)BA complex, which exports copper(I) and silver(I) and mediates resistance to these two metal ions, is the only known RND transporter with a specificity for heavy metals. We have determined the crystal structures of both the inner membrane pump CusA and membrane fusion protein CusB, as well as the adaptor–transporter CusBA complex formed by these two efflux proteins. In addition, the crystal structures of the outer membrane channel CusC and the periplasmic metallochaperone CusF have been resolved. Based on these structures, the entire assembled model of the tripartite efflux system has been developed, and this efflux complex should be in the form of CusC₃–CusB₆–CusA₃. It has been shown that CusA utilizes methionine clusters to bind and export Cu(I) and Ag(I). This pump is likely to undergo a conformational change, and utilize a relay network of methionine clusters as well as conserved charged residues to extrude the metal ions from the bacterial cell.     

Funnel-like hexameric assembly of the periplasmic adapter protein in the tripartite multidrug efflux pump in gram-negative bacteria

Gram-negative bacteria expel diverse toxic chemicals through the tripartite efflux pumps spanning both the inner and outer membranes. The Escherichia coli AcrAB-TolC pump is the principal multidrug exporter that confers intrinsic drug tolerance to the bacteria. The inner membrane transporter AcrB requires the outer membrane factor TolC and the periplasmic adapter protein AcrA. However, it remains ambiguous how the three proteins are assembled. In this study, a hexameric model of the adapter protein was generated based on the propensity for trimerization of a dimeric unit, and this model was further validated by presenting its channel-forming property that determines the substrate specificity. Genetic, in vitro complementation, and electron microscopic studies provided evidence for the binding of the hexameric adapter protein to the outer membrane factor in an intermeshing cogwheel manner. Structural analyses suggested that the adapter covers the periplasmic region of the inner membrane transporter. Taken together, we propose an adapter bridging model for the assembly of the tripartite pump, where the adapter protein provides a bridging channel and induces the channel opening of the outer membrane factor in the intermeshing tip-to-tip manner.     

Synthesis and evaluation of inhibitors of bacterial drug efflux pumps of the major facilitator superfamily

Inhibitors of drug efflux pumps have great potential as pharmacological agents that restore the drug susceptibility of multidrug resistant bacterial pathogens. Most attention has been focused on the discovery of small molecules that inhibit the resistance nodulation division (RND) family drug efflux pumps in Gram-negative bacteria. The prototypical inhibitor of RND-family efflux pumps in Gram-negative bacteria is MC-207,110 (Phe-Arg-β-naphthylamide), a C-capped dipeptide. Here, we report that C-capped dipeptides inhibit two chloramphenicol-specific efflux pumps in Streptomyces coelicolor, a Gram-positive bacterium that is a relative of the human pathogen Mycobacterium tuberculosis. Diversity-oriented synthesis of a library of structurally related C-capped dipeptides via an Ugi four component reaction and screening of the resulting compounds resulted in the discovery of a compound that is threefold more potent as a suppressor of chloramphenicol resistance in S. coelicolor than MC-207,110. Since chloramphenicol resistance in S. coelicolor is mediated by major facilitator superfamily drug efflux pumps, our findings provide the first evidence that C-capped dipeptides can inhibit drug efflux pumps outside of the RND superfamily.     

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.