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Efflux pumps of the resistance nodulation division (RND) superfamily, such as AcrB, make a major contribution to multidrug resistance in Gram-negative bacteria. The development of inhibitors of the RND pumps would improve the efficacy of current and next-generation antibiotics. To date, however, only one inhibitor has been cocrystallized with AcrB. Thus, in silico structure-based analysis is essential for elucidating the interaction between other inhibitors 7and the efflux pumps. In this work, we used computer docking and molecular dynamics simulations to study the interaction between AcrB and the compound MBX2319, a novel pyranopyridine efflux pump inhibitor with potent activity against RND efflux pumps of Enterobacteriaceae species, as well as other known inhibitors (D13-9001,1 -[ 1 -naphthylmethyl] -piperazine, andphenylalanylarginine-szlig;-naphthyl-amide) and the binding of doxorubicin to the efflux-defective F610A variant of AcrB. We also analyzed the binding of a substrate, minocycline, for comparison. Our results show that MBX2319 binds very tightly to the lower part of the distal pocket in the B protomer of AcrB, strongly interacting with the phenylalanines lining the hydrophobic trap, where the hydrophobic portion of Dl 3-9001 was found to bind by X-ray crystallography. Additionally, MBX2319 binds to AcrB in a manner that is similar to the way in which doxorubicin binds to the F610A variant of AcrB. In contrast, 1-(1-naphthylmethyl)-piperazine and phenylalanylarginine-β -naphthylamide appear to bind to somewhat different areas of the distal pocket in the B protomer of AcrB than does MBX2319. However, all inhibitors (except D13-9001) appear to distort the structure of the distal pocket, impairing the proper binding of substrates.

Transporters of the RND superfamily are well-known as the major drug efflux pumps of Gram-negative bacteria. However, they are widespread in organisms ranging from Archaea to Eukaryotes, and perform diverse functions. This review gives a brief overview of these diverse members of the superfamily with emphasis on their structure and functions., (Copyright © 2018 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

An experimental approach to detect the path a substrate takes through a complex membrane protein is described with emphasis on technical approach and theoretical considerations. The protocols for bacterial culture preparation, membrane protein purification, fluorescent assay standardization, data collection, and data analysis are provided. Useful software tools are recommended.

Transporters undergo large conformational changes in their functional cycle. RND (Resistance-Nodulation-Division) family efflux transporters usually exist as homotrimers, and each protomer was proposed to undergo a cycle of conformational changes in succession so that at any given time the trimer would contain three protomers of different conformations, the functionally rotating mechanism of transport. This mechanism implies that the inactivation of one protomer among three will inactivate the entire trimeric ensemble by blocking the functional rotation. We describe a biochemical approach to test this prediction by first producing a giant protein in which the three protomers of Escherichia coli AcrB efflux pump are covalently linked together through linker sequences, and then testing for its function by inactivation of a single protomer unit. Inactivation can be done permanently by mutating a residue involved in proton relay, or in "real time" by using a protein in which one protomer contains two Cys residues on both sides of the large cleft in the periplasmic domain and then by rapidly inactivating this protomer with a methanethiosulfonate cross-linker.

Background: The Resistance-Nodulation-Division (RND) family transporter AcrB plays a major role in the intrinsic and increased resistance of Escherichia coli to a large number of antibiotics. The distal binding pocket within this multidrug efflux transporter is very large, but the effort to define the roles of various residues facing this pocket through site-directed mutagenesis so far involved only the determination of minimal inhibitory concentrations of drugs in mutants., Methods: We measured in intact E. coli cells the kinetics of efflux of two substrates, nitrocefin (a cephalosporin) that is predicted mainly to bind to the upper, "groove" domain of the pocket, and L-alanyl-β-naphthylamide (Ala-Naph) that is likely to bind to the lower, "cave" domain, in a number of site-directed mutants of AcrB, where a hydrophobic or aromatic residue was changed into alanine., Results: The efflux of nitrocefin became attenuated by some mutations in the groove domain, such as I278A and F178A, but in some experiments a mutation in the cave domain, F628A produced a similar result. In some cases an increased value of K M was detected. The efflux of Ala-Naph was increased by mutations in the cave domain, such as F136A and I626A, but also by those in the groove domain (I277A, I278A, F178A). In most cases the increased V max values appeared to be responsible. F610A mutation had a profound effect on the efflux of both substrates, as reported earlier., Conclusions: Our data show for the first time effects of various substrate-binding pocket mutations on the kinetics of efflux of two substrates by the AcrB pump. They also confirm interactions between substrates and drugs predicted by MD simulation studies, and also reveal areas that need future research., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Klebsiella pneumoniae, one of the most important nosocomial pathogens, is becoming a major problem in health care because of its resistance to multiple antibiotics, including cephalosporins of the latest generation and, more recently, even carbapenems. This is largely due to the spread of plasmid-encoded extended-spectrum β-lactamases. However, antimicrobial agents must first penetrate the outer membrane barrier in order to reach their targets, and hydrophilic and charged β-lactams presumably diffuse through the porin channels. Unfortunately, the properties of K. pneumoniae porin channels are largely unknown. In this study, we made clean deletions of K. pneumoniae porin genes ompK35 and ompK36 and examined the antibiotic susceptibilities and diffusion rates of β-lactams. The results showed that OmpK35 and OmpK36 produced larger more permeable channels than their Escherichia coli homologs OmpF and OmpC; OmpK35 especially produced a diffusion channel of remarkably high permeability toward lipophilic (benzylpenicillin) and large (cefepime) compounds. These results were also confirmed by expressing various porins in an E. coli strain lacking major porins and the major multidrug efflux pump AcrAB. Our data explain why the development of drug resistance in K. pneumoniae is so often accompanied by the mutational loss of its porins, especially OmpK35, in addition to the various plasmid-carried genes of antibiotic resistance, because even hydrolysis by β-lactamases becomes inefficient in producing high levels of resistance if the bacterium continues to allow a rapid influx of β-lactams through its wide porin channels., Importance: In Gram-negative bacteria, drugs must first enter the outer membrane, usually through porin channels. Thus, the quantitative examination of influx rates is essential for the assessment of resistance mechanisms, yet no such studies exist for a very important nosocomial pathogen, Klebsiella pneumoniae We found that the larger channel porin of this organism, OmpK35, produces a significantly larger channel than its Escherichia coli homolog, OmpF. This makes unmodified K. pneumoniae strains more susceptible to relatively large antibiotics, such as the third- and fourth-generation cephalosporins. Also, even the acquisition of powerful β-lactamases is not likely to make them fully resistant in the presence of such an effective influx process, explaining why so many clinical isolates of this organism lack porins., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The Escherichia coli AcrAB-TolC efflux pump is the archetype of the resistance nodulation cell division (RND) exporters from Gram-negative bacteria. Overexpression of RND-type efflux pumps is a major factor in multidrug resistance (MDR), which makes these pumps important antibacterial drug discovery targets. We have recently developed novel pyranopyridine-based inhibitors of AcrB, which are orders of magnitude more powerful than the previously known inhibitors. However, further development of such inhibitors has been hindered by the lack of structural information for rational drug design. Although only the soluble, periplasmic part of AcrB binds and exports the ligands, the presence of the membrane-embedded domain in AcrB and its polyspecific binding behavior have made cocrystallization with drugs challenging. To overcome this obstacle, we have engineered and produced a soluble version of AcrB [AcrB periplasmic domain (AcrBper)], which is highly congruent in structure with the periplasmic part of the full-length protein, and is capable of binding substrates and potent inhibitors. Here, we describe the molecular basis for pyranopyridine-based inhibition of AcrB using a combination of cellular, X-ray crystallographic, and molecular dynamics (MD) simulations studies. The pyranopyridines bind within a phenylalanine-rich cage that branches from the deep binding pocket of AcrB, where they form extensive hydrophobic interactions. Moreover, the increasing potency of improved inhibitors correlates with the formation of a delicate protein- and water-mediated hydrogen bond network. These detailed insights provide a molecular platform for the development of novel combinational therapies using efflux pump inhibitors for combating multidrug resistant Gram-negative pathogens.

Efflux pumps of the resistance-nodulation division superfamily, such as AcrB, make a major contribution to multidrug resistance in Gram-negative bacteria. Inhibitors of such pumps would improve the efficacy of antibiotics, and ameliorate the crisis in health care caused by the prevalence of multidrug resistant Gram-negative pathogens. Phenylalanyl-arginine β-naphthylamide (PAβN), is a well-known inhibitor of AcrB and its homologs. However, its mechanism of inhibition is not clear. Because the hydrolysis of PAβN in Escherichia coli was nearly entirely dependent on an aminopeptidase, PepN, expression of PepN in periplasm allowed us to carry out a quantitative determination of PAβN efflux kinetics through the determination of its periplasmic concentrations by quantitation of the first hydrolysis product, phenylalanine, after a short period of treatment. We found that PAβN is efficiently pumped out by AcrB, with a sigmoidal kinetics. We also examined the behavior of PAβN homologs, Ala β-naphthylamide, Arg β-naphthylamide, and Phe β-naphthylamide, as substrates of AcrB and as modulators of nitrocefin efflux through AcrB. Furthermore, molecular dynamics simulations indicated that the mode of binding of these compounds to AcrB affects the modulatory activity on the efflux of other substrates. These results, and the finding that PAβN changes the nitrocefin kinetics into a sigmoidal one, suggested that PAβN inhibited the efflux of other drugs by binding to the bottom of the distal binding pocket, the so-called hydrophobic trap, and also by interfering with the binding of other drug substrates to the upper part of the binding pocket.

Small hydrophilic antibiotics traverse the outer membrane of Gram-negative bacteria through porin channels. Large lipophilic agents traverse the outer membrane through its bilayer, containing a majority of lipopolysaccharides in its outer leaflet. Genes controlled by the two-component regulatory system PhoPQ modify lipopolysaccharides. We isolate lipopolysaccharides from isogenic mutants of Salmonella sp., one lacking the modification, the other fully modified. These lipopolysaccharides were reconstituted as monolayers at the air-water interface, and their properties, as well as their interaction with a large lipophilic drug, novobiocin, was studied. X-ray reflectivity showed that the drug penetrated the monolayer of the unmodified lipopolysaccharides reaching the hydrophobic region, but was prevented from this penetration into the modified lipopolysaccharides. Results correlate with behavior of bacterial cells, which become resistant to antibiotics after PhoPQ-regulated modifications. Grazing incidence x-ray diffraction showed that novobiocin produced a striking increase in crystalline coherence length, and the size of the near-crystalline domains., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Unlabelled: The AcrB protein of Escherichia coli, together with TolC and AcrA, forms a contiguous envelope conduit for the capture and extrusion of diverse antibiotics and cellular metabolites. In this study, we sought to expand our knowledge of AcrB by conducting genetic and functional analyses. We began with an AcrB mutant bearing an F610A substitution in the drug binding pocket and obtained second-site substitutions that overcame the antibiotic hypersusceptibility phenotype conferred by the F610A mutation. Five of the seven unique single amino acid substitutions--Y49S, V127A, V127G, D153E, and G288C--mapped in the periplasmic porter domain of AcrB, with the D153E and G288C mutations mapping near and at the distal drug binding pocket, respectively. The other two substitutions--F453C and L486W--were mapped to transmembrane (TM) helices 5 and 6, respectively. The nitrocefin efflux kinetics data suggested that all periplasmic suppressors significantly restored nitrocefin binding affinity impaired by the F610A mutation. Surprisingly, despite increasing MICs of tested antibiotics and the efflux of N-phenyl-1-naphthylamine, the TM suppressors did not improve the nitrocefin efflux kinetics. These data suggest that the periplasmic substitutions act by influencing drug binding affinities for the distal binding pocket, whereas the TM substitutions may indirectly affect the conformational dynamics of the drug binding domain., Importance: The AcrB protein and its homologues confer multidrug resistance in many important human bacterial pathogens. A greater understanding of how these efflux pump proteins function will lead to the development of effective inhibitors against them. The research presented in this paper investigates drug binding pocket mutants of AcrB through the isolation and characterization of intragenic suppressor mutations that overcome the drug susceptibility phenotype of mutations affecting the drug binding pocket. The data reveal a remarkable structure-function plasticity of the AcrB protein pertaining to its drug efflux activity., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Recently we described a novel pyranopyridine inhibitor (MBX2319) of RND-type efflux pumps of the Enterobacteriaceae. MBX2319 (3,3-dimethyl-5-cyano-8-morpholino-6-(phenethylthio)-3,4-dihydro-1H-pyrano[3,4-c]pyridine) is structurally distinct from other known Gram-negative efflux pump inhibitors (EPIs), such as 1-(1-naphthylmethyl)-piperazine (NMP), phenylalanylarginine-β-naphthylamide (PAβN), D13-9001, and the pyridopyrimidine derivatives. Here, we report the synthesis and biological evaluation of 60 new analogs of MBX2319 that were designed to probe the structure activity relationships (SARs) of the pyranopyridine scaffold. The results of these studies produced a molecular activity map of the scaffold, which identifies regions that are critical to efflux inhibitory activities and those that can be modified to improve potency, metabolic stability and solubility. Several compounds, such as 22d-f, 22i and 22k, are significantly more effective than MBX2319 at potentiating the antibacterial activity of levofloxacin and piperacillin against Escherichia coli., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: Stroke-bypass transportation to the stroke center by paramedics is important to maximize the efficiency of intravenous tissue plasminogen activator (iv-tPA) therapy. To improve access to stroke thrombolysis, a citywide protocol was launched on January 2007 in Kawasaki City (population 1.4 million) using the Maria Prehospital Stroke Scale (MPSS), and quality assurance monitoring has been performed every 6 months. The aim was to identify whether the citywide quality assurance monitoring improves the process and outcome of iv-tPA therapy., Methods: All of the MPSS-based transportation data prospectively recorded by the Kawasaki City Fire Department and the associated clinical data in the 11 hospitals that accept stroke-bypass transfers were merged every 6 months for the quality assurance monitoring. Clinical indicators such as ambulance call-to-door time, onset-to-needle time, door-to-needle time, frequency of thrombolytic use, and outcome of thrombolytic therapy were analyzed. These clinical indicators were also compared between patients transferred on weekdays and on weekends., Results: A total of 2049 patients was registered from April 2009 to March 2013. Their mean age was 70.4 ± 13.2 (range, 24-98) years, and 64.3% were male. Ambulance call-to-door time decreased gradually from 37.5 ± 12.5 minutes to 33.9 ± 11.7 minutes over 4 years (P = .000, analysis of variance with the post hoc Dunnett test). Onset-to-needle time and door-to-needle time were similar over the 4 years. Good outcome (modified Rankin Scale score <2) after iv-tPA therapy increased from 24.1% to 35.3% (P = .045, 2010 vs. 2012). No deleterious effect of weekend admission was observed based on these clinical indicators., Conclusions: A citywide MPSS-based transportation protocol significantly decreased the delay in the ambulance call-to-door time. The implementation of standardized cross-institutional quality assurance programs for acute stroke therapy may improve the process and outcome of iv-tPA therapy in the community., (Copyright © 2015 National Stroke Association. Published by Elsevier Inc. All rights reserved.)

Acinetobacter baumannii contains RND-family efflux systems AdeABC and AdeIJK, which pump out a wide range of antimicrobial compounds, as judged from the MIC changes occurring upon deletion of the responsible genes. However, these studies may miss changes because of the high backgrounds generated by the remaining pumps and by β-lactamases, and it is unclear how the activities of these pumps compare quantitatively with those of the well-studied AcrAB-TolC system of Escherichia coli. We expressed adeABC and adeIJK of A. baumannii, as well as E. coli acrAB, in an E. coli host from which acrAB was deleted. The A. baumannii pumps were functional in E. coli, and the MIC changes that were observed largely confirmed the substrate range already reported, with important differences. Thus, the AdeABC system pumped out all β-lactams, an activity that was often missed in deletion studies. When the expression level of the pump genes was adjusted to a similar level for a comparison with AcrAB-TolC, we found that both A. baumannii efflux systems pumped out a wide range of compounds, but AdeABC was less effective than AcrAB-TolC in the extrusion of lipophilic β-lactams, novobiocin, and ethidium bromide, although it was more effective at tetracycline efflux. AdeIJK was remarkably more effective than a similar level of AcrAB-TolC in the efflux of β-lactams, novobiocin, and ethidium bromide, although it was less so in the efflux of erythromycin. These results thus allow us to compare these efflux systems on a quantitative basis, if we can assume that the heterologous systems are fully functional in the E. coli host., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

OmpF and OmpC porin channels are responsible for the passage of small hydrophilic solutes across the outer membrane of Escherichia coli. Although these channels are two of the most extensively studied porin channels, what had yet remained elusive was the reason why OmpC shows markedly lower permeability than OmpF, despite having little difference in its channel size. The OmpC channel, however, is known to contain a larger number of ionizable residues than the OmpF channel. In this study, we examined the channel property of OmpF and OmpC using the intact cell of E. coli, and we found that the permeability of several β-lactams and lactose through OmpC became increased to the level comparable with OmpF with up to 0.3 m salt that may increase the Debye-Hückel shielding or with 2% ethanol or 0.3 m urea that may perturb the short range ordering of water molecules. Replacing 10 pore-lining residues that show different ionization behavior between OmpC and OmpF led to substantial conversion of channel property with respect to their permeability and response to external salt concentration. We thus propose that the overall configuration of ionizable residues in the channel that may orient water molecules and the electrostatic profile of the channel play a decisive role in defining the channel property of the OmpC porin rather than its channel size., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)