In Gram-negative bacteria such as
Escherichia coli and
Pseudomonas aeruginosa, tripartite multidrug efflux systems extrude cytotoxic substances from the cell directly into the medium bypassing ...periplasm and the outer membrane. In
E. coli, the tripartite efflux system AcrA/AcrB/TolC is the pump that extrudes multiple antibiotics, dyes, bile salts and detergents. The inner membrane component AcrB, a member of the Resistance Nodulation cell Division (RND) family, is the major site for substrate recognition and energy transduction of the entire tripartite system. The drug/proton antiport processes in this secondary transporter are suggested to be spatially separated, a feature frequently observed for primary transporters like membrane-bound ATPases. The recently elucidated asymmetric structure of the AcrB trimer reveals three different monomer conformations proposed to represent consecutive states in a directional transport cycle. Each monomer shows a distinct tunnel system with entrances located at the boundary of the outer leaflet of the inner membrane and the periplasm through the periplasmic porter (pore) domain towards the funnel of the trimer and TolC. In one monomer a hydrophobic pocket is present which has been shown to bind the AcrB substrates minocyclin and doxorubicin. The energy conversion from the proton motive force into drug efflux includes proton binding in (and release from) the transmembrane part. The conformational changes observed within a triad of essential, titratable residues (D407/D408/K940) residing in the hydrophobic transmembrane domain appear to be transduced by transmembrane helix 8 and associated with the conformational changes seen in the periplasmic domain. From the asymmetric structure a possible peristaltic pump transport mechanism based on a functional rotation of the AcrB trimer has been postulated. The novel drug transport model combines the alternate access pump mechanism with the rotating site catalysis of F
1F
o ATPase as originally postulated by Jardetzky and Boyer, respectively, and suggests a working hypothesis for the transport mechanism of RND transporters in general.
Tissue-resident memory T (Trm) cells permanently localize to portals of pathogen entry, where they provide immediate protection against reinfection. To enforce tissue retention, Trm cells up-regulate ...CD69 and down-regulate molecules associated with tissue egress; however, a Trm-specific transcriptional regulator has not been identified. Here, we show that the transcription factor Hobit is specifically up-regulated in Trm cells and, together with related Blimp1, mediates the development of Trm cells in skin, gut, liver, and kidney in mice. The Hobit-Blimp1 transcriptional module is also required for other populations of tissue-resident lymphocytes, including natural killer T (NKT) cells and liver-resident NK cells, all of which share a common transcriptional program. Our results identify Hobit and Blimp1 as central regulators of this universal program that instructs tissue retention in diverse tissue-resident lymphocyte populations.
AcrB: a mean, keen, drug efflux machine Kobylka, Jessica; Kuth, Miriam S.; Müller, Reinke T. ...
Annals of the New York Academy of Sciences,
January 2020, Letnik:
1459, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Gram‐negative bacteria are intrinsically resistant against cytotoxic substances by means of their outer membrane and a network of multidrug efflux systems, acting in synergy. Efflux pumps from ...various superfamilies with broad substrate preferences sequester and pump drugs across the inner membrane to supply the highly polyspecific and powerful tripartite resistance–nodulation–cell division (RND) efflux pumps with compounds to be extruded across the outer membrane barrier. In Escherichia coli, the tripartite efflux system AcrAB–TolC is the archetype RND multiple drug efflux pump complex. The homotrimeric inner membrane component acriflavine resistance B (AcrB) is the drug specificity and energy transduction center for the drug/proton antiport process. Drugs are bound and expelled via a cycle of mainly three consecutive states in every protomer, constituting a flexible alternating access channel system. This review recapitulates the molecular basis of drug and inhibitor binding, including mechanistic insights into drug efflux by AcrB. It also summarizes 17 years of mutational analysis of the gene acrB, reporting the effect of every substitution on the ability of E. coli to confer resistance toward antibiotics (http://goethe.link/AcrBsubstitutions). We emphasize the functional robustness of AcrB toward single‐site substitutions and highlight regions that are more sensitive to perturbation.
This review recapitulates the molecular basis of drug and inhibitor binding, including mechanistic insights into drug efflux by the homotrimeric inner membrane component AcrB of the tripartite efflux system AcrAB–TolC. It also summarizes 17 years of mutational analysis of the gene acrB, reporting the effect of every substitution on the ability of E. coli to confer resistance toward antibiotics (http://goethe.link/AcrBsubstitutions). We emphasize the functional robustness of AcrB toward single‐site substitutions and highlight regions that are more sensitive to perturbation.
Abstract
Gram-negative bacteria maintain an intrinsic resistance mechanism against entry of noxious compounds by utilizing highly efficient efflux pumps. The
E. coli
AcrAB-TolC drug efflux pump ...contains the inner membrane H
+
/drug antiporter AcrB comprising three functionally interdependent protomers, cycling consecutively through the loose (L), tight (T) and open (O) state during cooperative catalysis. Here, we present 13 X-ray structures of AcrB in intermediate states of the transport cycle. Structure-based mutational analysis combined with drug susceptibility assays indicate that drugs are guided through dedicated transport channels toward the drug binding pockets. A co-structure obtained in the combined presence of erythromycin, linezolid, oxacillin and fusidic acid shows binding of fusidic acid deeply inside the T protomer transmembrane domain. Thiol cross-link substrate protection assays indicate that this transmembrane domain-binding site can also accommodate oxacillin or novobiocin but not erythromycin or linezolid. AcrB-mediated drug transport is suggested to be allosterically modulated in presence of multiple drugs.
Objectives To examine the risk of relapse and time to relapse after discontinuation of antidepressants in patients with anxiety disorder who responded to antidepressants, and to explore whether ...relapse risk is related to type of anxiety disorder, type of antidepressant, mode of discontinuation, duration of treatment and follow-up, comorbidity, and allowance of psychotherapy.Design Systematic review and meta-analyses of relapse prevention trials.Data sources PubMed, Cochrane, Embase, and clinical trial registers (from inception to July 2016).Study selection Eligible studies included patients with anxiety disorder who responded to antidepressants, randomised patients double blind to either continuing antidepressants or switching to placebo, and compared relapse rates or time to relapse.Data extraction Two independent raters selected studies and extracted data. Random effect models were used to estimate odds ratios for relapse, hazard ratios for time to relapse, and relapse prevalence per group. The effect of various categorical and continuous variables was explored with subgroup analyses and meta-regression analyses respectively. Bias was assessed using the Cochrane tool.Results The meta-analysis included 28 studies (n=5233) examining relapse with a maximum follow-up of one year. Across studies, risk of bias was considered low. Discontinuation increased the odds of relapse compared with continuing antidepressants (summary odds ratio 3.11, 95% confidence interval 2.48 to 3.89). Subgroup analyses and meta-regression analyses showed no statistical significance. Time to relapse (n=3002) was shorter when antidepressants were discontinued (summary hazard ratio 3.63, 2.58 to 5.10; n=11 studies). Summary relapse prevalences were 36.4% (30.8% to 42.1%; n=28 studies) for the placebo group and 16.4% (12.6% to 20.1%; n=28 studies) for the antidepressant group, but prevalence varied considerably across studies, most likely owing to differences in the length of follow-up. Dropout was higher in the placebo group (summary odds ratio 1.31, 1.06 to 1.63; n=27 studies).Conclusions Up to one year of follow-up, discontinuation of antidepressant treatment results in higher relapse rates among responders compared with treatment continuation. The lack of evidence after a one year period should not be interpreted as explicit advice to discontinue antidepressants after one year. Given the chronicity of anxiety disorders, treatment should be directed by long term considerations, including relapse prevalence, side effects, and patients’ preferences.
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.
Infections arising from multidrug-resistant pathogenic bacteria are spreading rapidly throughout the world and threaten to become untreatable. The origins of resistance are numerous and complex, but ...one underlying factor is the capacity of bacteria to rapidly export drugs through the intrinsic activity of efflux pumps. In this Review, we describe recent advances that have increased our understanding of the structures and molecular mechanisms of multidrug efflux pumps in bacteria. Clinical and laboratory data indicate that efflux pumps function not only in the drug extrusion process but also in virulence and the adaptive responses that contribute to antimicrobial resistance during infection. The emerging picture of the structure, function and regulation of efflux pumps suggests opportunities for countering their activities.
Tripartite efflux pumps and the related type 1 secretion systems (T1SSs) in Gram-negative organisms are diverse in function, energization, and structural organization. They form continuous conduits ...spanning both the inner and the outer membrane and are composed of three principal componentsthe energized inner membrane transporters (belonging to ABC, RND, and MFS families), the outer membrane factor channel-like proteins, and linking the two, the periplasmic adaptor proteins (PAPs), also known as the membrane fusion proteins (MFPs). In this review we summarize the recent advances in understanding of structural biology, function, and regulation of these systems, highlighting the previously undescribed role of PAPs in providing a common architectural scaffold across diverse families of transporters. Despite being built from a limited number of basic structural domains, these complexes present a staggering variety of architectures. While key insights have been derived from the RND transporter systems, a closer inspection of the operation and structural organization of different tripartite systems reveals unexpected analogies between them, including those formed around MFS- and ATP-driven transporters, suggesting that they operate around basic common principles. Based on that we are proposing a new integrated model of PAP-mediated communication within the conformational cycling of tripartite systems, which could be expanded to other types of assemblies.
•Structure-based mechanisms are available for five major classes of multi-drug transporters.•Three transporter classes form tripartite assemblies spanning the bacterial cell envelope.•The structure ...of a tripartite assembly provides mechanistic insight into these complex machines.•The different classes of transporters act as a system to transport a chemically broad range of drugs.
Cells from all domains of life encode energy-dependent trans-membrane transporters that can expel harmful substances including clinically applied therapeutic agents. As a collective body, these transporters perform as a super-system that confers tolerance to an enormous range of harmful compounds and consequently aid survival in hazardous environments. In the Gram-negative bacteria, some of these transporters serve as energy-transducing components of tripartite assemblies that actively efflux drugs and other harmful compounds, as well as deliver virulence agents across the entire cell envelope. We draw together recent structural and functional data to present the current models for the transport mechanisms for the main classes of multi-drug transporters and their higher-order assemblies.
Efflux pumps of the Resistance Nodulation Division (RND) superfamily play a major role in the intrinsic and acquired resistance of Gram-negative pathogens to antibiotics. Moreover, they are largely ...responsible for multi-drug resistance (MDR) phenomena in these bacteria. The last decade has seen a sharp increase in the number of experimental and computational studies aimed at understanding their functional mechanisms. Most of these studies focused on the RND drug/proton antiporter AcrB, part of the AcrAB-TolC efflux pump actively recognizing and expelling noxious agents from the interior of bacteria. These studies have been focused on the dynamical interactions between AcrB and its substrates and inhibitors, on the details of the proton translocation mechanisms, and on the way AcrB assembles with protein partners to build up a functional pump. In this review we summarize these advances focusing on the role of AcrB.
PDF
