Strategies for bypassing the membrane barrier in multidrug resistant Gram-negative bacteria

In Gram-negative bacteria, the envelope is a sophisticated barrier protecting the cell against external toxic compounds. Membrane transporters, e.g., porins or efflux pumps, are main filters regulating the internal accumulation of various hydrophilic molecules. Regarding bacterial susceptibility towards antibacterial agents, membrane permeability is part of the early bacterial defense. The bacterium manages the translocation process, influx and efflux, to control the intracellular concentration of various molecules. Antibiotics and biocides are substrates of these mechanisms and the continuing emergence of multidrug resistant isolates is a growing worldwide health concern. Different strategies could be proposed to bypass the bacterial membrane barrier, comprising influx and efflux mechanisms, in order to restore the activity of antibiotics against resistant bacteria.     

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.     

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.     

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.     

Making a beta-barrel: assembly of outer membrane proteins in Gram-negative bacteria

The outer membrane (OM) of Gram-negative bacteria is an essential organelle that serves as a selective permeability barrier by keeping toxic compounds out of the cell while allowing vital nutrients in. How the OM and its constituent lipid and protein components are assembled remains an area of active research. In this review, we describe our current understanding of how outer membrane proteins (OMPs) are delivered to and then assembled in the OM of the model Gram-negative organism Escherichia coli.     

A small molecule that disrupts S. Typhimurium membrane voltage without cell lysis reduces bacterial colonization of mice

As pathogenic bacteria become increasingly resistant to antibiotics, antimicrobials with mechanisms of action distinct from current clinical antibiotics are needed. Gram-negative bacteria pose a particular problem because they defend themselves against chemicals with a minimally permeable outer membrane and with efflux pumps. During infection, innate immune defense molecules increase bacterial vulnerability to chemicals by permeabilizing the outer membrane and occupying efflux pumps. Therefore, screens for compounds that reduce bacterial colonization of mammalian cells have the potential to reveal unexplored therapeutic avenues. Here we describe a new small molecule, D66, that prevents the survival of a human Gram-negative pathogen in macrophages. D66 inhibits bacterial growth under conditions wherein the bacterial outer membrane or efflux pumps are compromised, but not in standard microbiological media. The compound disrupts voltage across the bacterial inner membrane at concentrations that do not permeabilize the inner membrane or lyse cells. Selection for bacterial clones resistant to D66 activity suggested that outer membrane integrity and efflux are the two major bacterial defense mechanisms against this compound. Treatment of mammalian cells with D66 does not permeabilize the mammalian cell membrane but does cause stress, as revealed by hyperpolarization of mitochondrial membranes. Nevertheless, the compound is tolerated in mice and reduces bacterial tissue load. These data suggest that the inner membrane could be a viable target for anti-Gram-negative antimicrobials, and that disruption of bacterial membrane voltage without lysis is sufficient to enable clearance from the host.     

The role of the TolC family in protein transport and multidrug efflux. From stereochemical certainty to mechanistic hypothesis.

Gram-negative bacteria are enveloped by a system of two membranes, and they use specialized multicomponent, energy-driven pumps to transport molecules directly across this double-layered partition from the cell interior to the extra-cellular environment. One component of these pumps is embedded in the outer-membrane, and the paradigm for its structure and function is the TolC protein from Escherichia coli. A common component of a wide variety of efflux pumps, TolC and its homologues are involved in the export of chemically diverse molecules ranging from large protein toxins, such as alpha-hemolysin, to small toxic compounds, such as antibiotics. TolC family members thus play important roles in conferring pathogenic bacteria with both virulence and multidrug resistance. These pumps assemble reversibly in a transient process that brings together TolC or its homologue, an inner-membrane-associated periplasmic component, an integral inner-membrane translocase and the substrate itself. TolC can associate in this fashion with a variety of different partners to participate in the transport of diverse substrates. We review here the structure and function of TolC and the other components of the efflux/transport 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.     

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.     

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.     

Breaching the Barrier: Quantifying Antibiotic Permeability across Gram-negative Bacterial Membranes

The double-membrane cell envelope of Gram-negative bacteria is a sophisticated barrier that facilitates the uptake of nutrients and protects the organism from toxic compounds. An antibiotic molecule must find its way through the negatively charged lipopolysaccharide layer on the outer surface, pass through either a porin or the hydrophobic layer of the outer membrane, then traverse the hydrophilic peptidoglycan layer only to find another hydrophobic lipid bilayer before it finally enters the cytoplasm, where it typically finds its target. This complex uptake pathway with very different physico-chemical properties is one reason that Gram-negative are intrinsically protected against multiple classes of antibiotic-like molecules, and is likely the main reason that in vitro target-based screening programs have failed to deliver novel antibiotics for these organisms. Due to the lack of general methods available for quantifying the flux of drugs into the cell, little is known about permeation rates, transport pathways and accumulation at the target sites for particular molecules. Here we summarize the current tools available for measuring antibiotic uptake across the different compartments of Gram-negative bacteria.     

Interaction between the α-barrel tip of Vibrio vulnificus TolC homologs and AcrA implies the adapter bridging model

The AcrAB-TolC multidrug efflux pump confers resistance to Escherichia coli against many antibiotics and toxic compounds. The TolC protein is an outer membrane factor that participates in the formation of type I secretion systems. The genome of Vibrio vulnificus encodes two proteins homologous to the E. coli TolC, designated TolCV1 and TolCV2. Here, we show that both TolCV1 and TolCV2 partially complement the E. coli TolC function and physically interact with the membrane fusion protein AcrA, a component of the E. coli AcrAB-TolC efflux pump. Using site-directed mutational analyses and an in vivo cross-linking assay, we demonstrated that the α-barrel tip region of TolC homologs plays a critical role in the formation of functional AcrAB-TolC efflux pumps. Our findings suggest the adapter bridging model as a general assembly mechanism for tripartite drug efflux pumps in Gram-negative bacteria.     

Lpp positions peptidoglycan at the AcrA-TolC interface in the AcrAB-TolC multidrug efflux pump

The multidrug efflux pumps of Gram-negative bacteria are a class of complexes that span the periplasm, coupling both the inner and outer membranes to expel toxic molecules. The best-characterized example of these tripartite pumps is the AcrAB-TolC complex of Escherichia coli. However, how the complex interacts with the peptidoglycan (PG) cell wall, which is anchored to the outer membrane (OM) by Braun’s lipoprotein (Lpp), is still largely unknown. In this work, we present molecular dynamics simulations of a complete, atomistic model of the AcrAB-TolC complex with the inner membrane, OM, and PG layers all present. We find that the PG localizes to the junction of AcrA and TolC, in agreement with recent cryo-tomography data. Free-energy calculations reveal that the positioning of PG is determined by the length and conformation of multiple Lpp copies anchoring it to the OM. The distance between the PG and OM measured in cryo-electron microscopy images of wild-type E. coli also agrees with the simulation-derived spacing. Sequence analysis of AcrA suggests a conserved role for interactions with PG in the assembly and stabilization of efflux pumps, one that may extend to other trans-envelope complexes as well.     

Lipopolysaccharide biogenesis and transport at the outer membrane of Gram-negative bacteria

The outer membrane (OM) of Gram-negative bacteria is an asymmetric lipid bilayer containing a unique glycolipid, lipopolysaccharide (LPS) in its outer leaflet. LPS molecules confer to the OM peculiar permeability barrier properties enabling Gram-negative bacteria to exclude many toxic compounds, including clinically useful antibiotics, and to survive harsh environments. Transport of LPS poses several problems to the cells due to the amphipatic nature of this molecule. In this review we summarize the current knowledge on the LPS transport machinery, discuss the challenges associated with this process and present the solutions that bacterial cells have evolved to address the problem of LPS transport and assembly at the cell surface. Finally, we discuss how knowledge on LPS biogenesis can be translated for the development of novel antimicrobial therapies. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.     

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.     

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     

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 Forward Chemical Screen Identifies Antibiotic Adjuvants in Escherichia coli

Multi-drug-resistant infections caused by Gram-negative pathogens are rapidly increasing, highlighting the need for new chemotherapies. Unlike Gram-positive bacteria, where many different chemical classes of antibiotics show efficacy, Gram-negatives are intrinsically insensitive to many antimicrobials including the macrolides, rifamycins, and aminocoumarins, despite intracellular targets that are susceptible to these drugs. The basis for this insensitivity is the presence of the impermeant outer membrane of Gram-negative bacteria in addition to the expression of pumps and porins that reduce intracellular concentrations of many molecules. Compounds that sensitize Gram-negative cells to "Gram-positive antibiotics", antibiotic adjuvants, offer an orthogonal approach to addressing the crisis of multi-drug-resistant Gram-negative pathogens. We performed a forward chemical genetic screen of 30,000 small molecules designed to identify such antibiotic adjuvants of the aminocoumarin antibiotic novobiocin in Escherichia coli. Four compounds from this screen were shown to be synergistic with novobiocin including inhibitors of the bacterial cytoskeleton protein MreB, cell wall biosynthesis enzymes, and DNA synthesis. All of these molecules were associated with altered cell shape and small molecule permeability, suggesting a unifying mechanism for these antibiotic adjuvants. The potential exists to expand this approach as a means to develop novel combination therapies for the treatment of infections caused by Gram-negative pathogens.     

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.     

Current mechanistic understanding of intermembrane lipid trafficking important for maintenance of bacterial outer membrane lipid asymmetry

The outer membrane (OM) of Gram-negative bacteria exhibits unique lipid asymmetry that makes it an effective permeability barrier against toxic molecules, including antibiotics. Central to the maintenance of OM lipid asymmetry is the OmpC-Mla (maintenance of lipid asymmetry) system, which mediates the retrograde transport of phospholipids from the outer leaflet of the OM to the inner membrane. The molecular mechanism(s) of this lipid trafficking process is not fully understood; however, recent advances in structural elucidations and biochemical reconstitutions have provided detailed new insights. Here, we present an integrated understanding of how the OmpC-Mla system transports mislocalized phospholipids across the bacterial cell envelope.