Tripartite assembly of RND multidrug efflux pumps.
TL;DR: The reconstitution of native Pseudomonas aeruginosa MexAB–OprM and Escherichia coli AcrAB–TolC tripartite Resistance Nodulation and cell Division (RND) efflux systems in a lipid nanodisc system is presented.
Abstract: Tripartite multidrug efflux systems of Gram-negative bacteria are composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. They are assumed to form ducts inside the periplasm facilitating drug exit across the outer membrane. Here we present the reconstitution of native Pseudomonas aeruginosa MexAB-OprM and Escherichia coli AcrAB-TolC tripartite Resistance Nodulation and cell Division (RND) efflux systems in a lipid nanodisc system. Single-particle analysis by electron microscopy reveals the inner and outer membrane protein components linked together via the periplasmic adaptor protein. This intrinsic ability of the native components to self-assemble also leads to the formation of a stable interspecies AcrA-MexB-TolC complex suggesting a common mechanism of tripartite assembly. Projection structures of all three complexes emphasize the role of the periplasmic adaptor protein as part of the exit duct with no physical interaction between the inner and outer membrane components.
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TL;DR: The antibacterial mechanisms of NPs against bacteria and the factors that are involved are discussed and the limitations of current research are discussed.
Abstract: Nanoparticles (NPs) are increasingly used to target bacteria as an alternative to antibiotics. Nanotechnology may be particularly advantageous in treating bacterial infections. Examples include the utilization of NPs in antibacterial coatings for implantable devices and medicinal materials to prevent infection and promote wound healing, in antibiotic delivery systems to treat disease, in bacterial detection systems to generate microbial diagnostics, and in antibacterial vaccines to control bacterial infections. The antibacterial mechanisms of NPs are poorly understood, but the currently accepted mechanisms include oxidative stress induction, metal ion release, and non-oxidative mechanisms. The multiple simultaneous mechanisms of action against microbes would require multiple simultaneous gene mutations in the same bacterial cell for antibacterial resistance to develop; therefore, it is difficult for bacterial cells to become resistant to NPs. In this review, we discuss the antibacterial mechanisms of NPs against bacteria and the factors that are involved. The limitations of current research are also discussed.
2,178 citations
TL;DR: The mechanism of antibiotic resistance in P. aeruginosa is a recently characterized mechanism, which includes biofilm-mediated resistance and formation of multidrug-tolerant persister cells, and is responsible for recalcitrance and relapse of infections.
908 citations
TL;DR: This review attempted to combine a comprehensive list of various applications of nanodisc technology with systematic analysis of the most attractive features of this system and advantages provided by nanodISCs for structural and mechanistic studies of membrane proteins.
Abstract: Membrane proteins play a most important part in metabolism, signaling, cell motility, transport, development, and many other biochemical and biophysical processes which constitute fundamentals of life on the molecular level. Detailed understanding of these processes is necessary for the progress of life sciences and biomedical applications. Nanodiscs provide a new and powerful tool for a broad spectrum of biochemical and biophysical studies of membrane proteins and are commonly acknowledged as an optimal membrane mimetic system that provides control over size, composition, and specific functional modifications on the nanometer scale. In this review we attempted to combine a comprehensive list of various applications of nanodisc technology with systematic analysis of the most attractive features of this system and advantages provided by nanodiscs for structural and mechanistic studies of membrane proteins.
365 citations
TL;DR: The biological underpinnings of drug permeability and export using several prototypical influx and efflux systems are discussed, highlighting how new methods for the determination of antibacterial activities enable more careful quantitation and may provide a way forward for capturing and correlating the modes of action and kinetics of antibiotic uptake inside bacterial cells.
Abstract: Researchers, clinicians and governments all recognize antimicrobial resistance as a serious and growing threat worldwide. New antimicrobials are urgently needed, especially for infections caused by Gram-negative bacteria, whose cell envelopes are characterized by low permeability and often contain drug efflux systems. Individual bacteria and populations control their internal concentrations of antibiotics by regulating proteins involved in membrane permeability, such as porins or efflux pumps. Robust methods to quantify and visualize intrabacterial antibiotic concentrations have identified clear correlations between efflux activity and drug diffusion and accumulation in both susceptible and resistant strains, and have also clarified how certain chemical structures can affect drug entry and residence time within the cell. In this PERSPECTIVE, we discuss the biological underpinnings of drug permeability and export using several prototypical influx and efflux systems. We also highlight how new methods for the determination of antibacterial activities enable more careful quantitation and may provide us with a way forward for capturing and correlating the modes of action and kinetics of antibiotic uptake inside bacterial cells. Together, these advances will aid efforts to generate structurally improved molecules with better access and retention within bacteria, thereby reducing the emergence and spread of resistant strains and extending the clinical use of current antibiotics. A Perspective on unravelling the mechanisms of antibiotic penetration and efflux in Gram-negative bacteria.
206 citations
TL;DR: Different aspects of resistance and pathogenicity in P. aeruginosa, such as the role of outer membrane proteins, transcriptional regulators, efflux pumps, enzymes, and biofilms in antimicrobial resistance, are highlighted.
183 citations
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TL;DR: This review discusses the current knowledge on the molecular mechanisms involved in both types of resistance in bacteria.
Abstract: Large amounts of antibiotics used for human therapy, as well as for farm animals and even for fish in aquaculture, resulted in the selection of pathogenic bacteria resistant to multiple drugs. Multidrug resistance in bacteria may be generated by one of two mechanisms. First, these bacteria may accumulate multiple genes, each coding for resistance to a single drug, within a single cell. This accumulation occurs typically on resistance (R) plasmids. Second, multidrug resistance may also occur by the increased expression of genes that code for multidrug efflux pumps, extruding a wide range of drugs. This review discusses our current knowledge on the molecular mechanisms involved in both types of resistance.
1,331 citations
"Tripartite assembly of RND multidru..." refers background in this paper
...of the equatorial domain (2) and the tip of the a-barrel (3) protruding from...
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TL;DR: The structure of TolC is reported, revealing a distinctive and previously unknown fold that provides an explanation of how the cell cytosol is connected to the external environment during export, and suggests a general mechanism for the action of bacterial efflux pumps.
Abstract: Diverse molecules, from small antibacterial drugs to large protein toxins, are exported directly across both cell membranes of gram-negative bacteria. This export is brought about by the reversible interaction of substrate-specific inner-membrane proteins with an outer-membrane protein of the TolC family, thus bypassing the intervening periplasm. Here we report the 2.1-A crystal structure of TolC from Escherichia coli, revealing a distinctive and previously unknown fold. Three TolC protomers assemble to form a continuous, solvent-accessible conduit--a 'channel-tunnel' over 140 A long that spans both the outer membrane and periplasmic space. The periplasmic or proximal end of the tunnel is sealed by sets of coiled helices. We suggest these could be untwisted by an allosteric mechanism, mediated by protein-protein interactions, to open the tunnel. The structure provides an explanation of how the cell cytosol is connected to the external environment during export, and suggests a general mechanism for the action of bacterial efflux pumps.
1,066 citations
"Tripartite assembly of RND multidru..." refers background in this paper
...MexB exhibits the periplasmic part organized in two layers (funnel (4) and...
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TL;DR: This article highlights the recent progress obtained for organisms of clinical significance, together with methodological considerations for the characterization of MDR pumps, with particular focus on AcrAB-TolC and Mex pumps.
Abstract: The global emergence of multidrug-resistant Gram-negative bacteria is a growing threat to antibiotic therapy. The chromosomally encoded drug efflux mechanisms that are ubiquitous in these bacteria greatly contribute to antibiotic resistance and present a major challenge for antibiotic development. Multidrug pumps, particularly those represented by the clinically relevant AcrAB-TolC and Mex pumps of the resistance-nodulation-division (RND) superfamily, not only mediate intrinsic and acquired multidrug resistance (MDR) but also are involved in other functions, including the bacterial stress response and pathogenicity. Additionally, efflux pumps interact synergistically with other resistance mechanisms (e.g., with the outer membrane permeability barrier) to increase resistance levels. Since the discovery of RND pumps in the early 1990s, remarkable scientific and technological advances have allowed for an in-depth understanding of the structural and biochemical basis, substrate profiles, molecular regulation, and inhibition of MDR pumps. However, the development of clinically useful efflux pump inhibitors and/or new antibiotics that can bypass pump effects continues to be a challenge. Plasmid-borne efflux pump genes (including those for RND pumps) have increasingly been identified. This article highlights the recent progress obtained for organisms of clinical significance, together with methodological considerations for the characterization of MDR pumps.
1,016 citations
"Tripartite assembly of RND multidru..." refers background in this paper
...of the equatorial domain (2) and the tip of the a-barrel (3) protruding from...
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...Characteristic features are displayed: OprM b-barrel and ND (1); equatorial domain (2), tip of a barrel (30),...
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TL;DR: The results of this study provide an important structural characterization of self-assembled phospholipid bilayers and establish a framework for the design of soluble amphiphilic nanoparticles of controlled size.
Abstract: Using a recently described self-assembly process (Bayburt, T. H.; Grinkova, Y. V.; Sligar, S. G. Nano Letters 2002, 2, 853−856), we prepared soluble monodisperse discoidal lipid/protein particles with controlled size and composition, termed Nanodiscs, in which the fragment of dipalmitoylphosphatidylcholine (DPPC) bilayer is surrounded by a helical protein belt. We have customized the size of these particles by changing the length of the amphipathic helical part of this belt, termed membrane scaffold protein (MSP). Herein we describe the design of extended and truncated MSPs, the optimization of self-assembly for each of these proteins, and the structure and composition of the resulting Nanodiscs. We show that the length of the protein helix surrounding the lipid part of a Nanodisc determines the particle diameter, as measured by HPLC and small-angle X-ray scattering (SAXS). Using different scaffold proteins, we obtained Nanodiscs with the average size from 9.5 to 12.8 nm with a very narrow size distributi...
949 citations
TL;DR: In this article, the crystal structure of AcrB at 3.5 A resolution was determined, which implies that substrates translocated from the cell interior through the transmembrane region and from the periplasm through the vestibules are collected in the central cavity and then actively transported through the pore into the TolC tunnel.
Abstract: AcrB is a major multidrug exporter in Escherichia coli. It cooperates with a membrane fusion protein, AcrA, and an outer membrane channel, TolC. We have determined the crystal structure of AcrB at 3.5 A resolution. Three AcrB protomers are organized as a homotrimer in the shape of a jellyfish. Each protomer is composed of a transmembrane region 50 A thick and a 70 A protruding headpiece. The top of the headpiece opens like a funnel, where TolC might directly dock into AcrB. A pore formed by three alpha-helices connects the funnel with a central cavity located at the bottom of the headpiece. The cavity has three vestibules at the side of the headpiece which lead into the periplasm. In the transmembrane region, each protomer has twelve transmembrane alpha-helices. The structure implies that substrates translocated from the cell interior through the transmembrane region and from the periplasm through the vestibules are collected in the central cavity and then actively transported through the pore into the TolC tunnel.
927 citations