scholarly journals Reversal of the Drug Binding Pocket Defects of the AcrB Multidrug Efflux Pump Protein of Escherichia coli

2015 ◽  
Vol 197 (20) ◽  
pp. 3255-3264 ◽  
Author(s):  
Ketaki Soparkar ◽  
Alfred D. Kinana ◽  
Jon W. Weeks ◽  
Keith D. Morrison ◽  
Hiroshi Nikaido ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Efflux Pump ◽  
Conformational Dynamics ◽  
Binding Pocket ◽  
Drug Binding ◽  
Single Amino Acid ◽  
Multidrug Efflux Pump ◽  
Content Type ◽  
Isolation And Characterization ◽  
Efflux Kinetics

ABSTRACTThe AcrB protein ofEscherichia 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 ofN-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.IMPORTANCEThe 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.

scholarly journals Characterization and Molecular Determinants for β-Lactam Specificity of the Multidrug Efflux Pump AcrD from Salmonella typhimurium

Antibiotics ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1494
Author(s):  
Jenifer Cuesta Bernal ◽  
Jasmin El-Delik ◽  
Stephan Göttig ◽  
Klaas M. Pos
Keyword(s):  
Substrate Specificity ◽  
Salmonella Typhimurium ◽  
Efflux Pump ◽  
Binding Pocket ◽  
Salt Resistance ◽  
Drug Binding ◽  
Multidrug Efflux Pump ◽  
E Coli ◽  
Molecular Determinants ◽  
Groove Region

Gram-negative Tripartite Resistance Nodulation and cell Division (RND) superfamily efflux pumps confer various functions, including multidrug and bile salt resistance, quorum-sensing, virulence and can influence the rate of mutations on the chromosome. Multidrug RND efflux systems are often characterized by a wide substrate specificity. Similarly to many other RND efflux pump systems, AcrAD-TolC confers resistance toward SDS, novobiocin and deoxycholate. In contrast to the other pumps, however, it in addition confers resistance against aminoglycosides and dianionic β-lactams, such as sulbenicillin, aztreonam and carbenicillin. Here, we could show that AcrD from Salmonella typhimurium confers resistance toward several hitherto unreported AcrD substrates such as temocillin, dicloxacillin, cefazolin and fusidic acid. In order to address the molecular determinants of the S. typhimurium AcrD substrate specificity, we conducted substitution analyses in the putative access and deep binding pockets and in the TM1/TM2 groove region. The variants were tested in E. coli ΔacrBΔacrD against β-lactams oxacillin, carbenicillin, aztreonam and temocillin. Deep binding pocket variants N136A, D276A and Y327A; access pocket variant R625A; and variants with substitutions in the groove region between TM1 and TM2 conferred a sensitive phenotype and might, therefore, be involved in anionic β-lactam export. In contrast, lower susceptibilities were observed for E. coli cells harbouring deep binding pocket variants T139A, D176A, S180A, F609A, T611A and F627A and the TM1/TM2 groove variant I337A. This study provides the first insights of side chains involved in drug binding and transport for AcrD from S. typhimurium.

scholarly journals Multidrug Efflux Pump MdtBC of Escherichia coli Is Active Only as a B2C Heterotrimer

2009 ◽  
Vol 192 (5) ◽  
pp. 1377-1386 ◽  
Author(s):  
Hong-Suk Kim ◽  
Daniel Nagore ◽  
Hiroshi Nikaido
Keyword(s):  
Escherichia Coli ◽  
Efflux Pump ◽  
Proton Translocation ◽  
Drug Binding ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Wide Range ◽  
Resistance Nodulation Division ◽  
Covalently Linked ◽  
Made In

ABSTRACT RND (resistance-nodulation-division) family transporters in Gram-negative bacteria frequently pump out a wide range of inhibitors and often contribute to multidrug resistance to antibiotics and biocides. An archetypal RND pump of Escherichia coli, AcrB, is known to exist as a homotrimer, and this construction is essential for drug pumping through the functionally rotating mechanism. MdtBC, however, appears different because two pump genes coexist within a single operon, and genetic deletion data suggest that both pumps must be expressed in order for the drug efflux to occur. We have expressed the corresponding genes, with one of them in a His-tagged form. Copurification of MdtB and MdtC under these conditions showed that they form a complex, with an average stoichiometry of 2:1. Unequivocal evidence that only the trimer containing two B protomers and one C protomer is active was obtained by expressing all possible combinations of B and C in covalently linked forms. Finally, conversion into alanine of the residues, known to form a proton translocation pathway in AcrB, inactivated transport only when made in MdtB, not when made in MdtC, a result suggesting that MdtC plays a different role not directly involved in drug binding and extrusion.

scholarly journals Perturbed structural dynamics underlie inhibition and altered efflux of the multidrug resistance pump AcrB

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Eamonn Reading ◽  
Zainab Ahdash ◽  
Chiara Fais ◽  
Vito Ricci ◽  
Xuan Wang-Kan ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Structural Dynamics ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Binding Pocket ◽  
Drug Binding ◽  
Multidrug Efflux Pump ◽  
Transport Pathway ◽  
Efflux Pump Inhibitor ◽  
Wide Range

Abstract Resistance–nodulation–division efflux pumps play a key role in inherent and evolved multidrug resistance in bacteria. AcrB, a prototypical member of this protein family, extrudes a wide range of antimicrobial agents out of bacteria. Although high-resolution structures exist for AcrB, its conformational fluctuations and their putative role in function are largely unknown. Here, we determine these structural dynamics in the presence of substrates using hydrogen/deuterium exchange mass spectrometry, complemented by molecular dynamics simulations, and bacterial susceptibility studies. We show that an efflux pump inhibitor potentiates antibiotic activity by restraining drug-binding pocket dynamics, rather than preventing antibiotic binding. We also reveal that a drug-binding pocket substitution discovered within a multidrug resistant clinical isolate modifies the plasticity of the transport pathway, which could explain its altered substrate efflux. Our results provide insight into the molecular mechanism of drug export and inhibition of a major multidrug efflux pump and the directive role of its dynamics.

scholarly journals Interactions of a bacterial RND transporter with a transmembrane small protein in a lipid environment

2019 ◽  
Author(s):  
Dijun Du ◽  
Arthur Neuberger ◽  
Mona Wu Orr ◽  
Catherine E. Newman ◽  
Pin-Chia Hsu ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Drug Sensitivity ◽  
Efflux Pump ◽  
Binding Pocket ◽  
Drug Binding ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Small Protein ◽  
Rnd Transporter ◽  
Lipid Environment

AbstractThe small protein AcrZ in Escherichia coli interacts with the transmembrane portion of the multidrug efflux pump AcrB and increases the resistance of the bacterium to a subset of the antibiotic substrates of that transporter. It is not clear how the physical association of the two proteins selectively changes activity of the pump for defined substrates. Here, we report cryo-EM structures of AcrB and the AcrBZ complex in lipid environments, and comparisons suggest that conformational changes occur in the drug binding pocket as a result of AcrZ binding. Simulations indicate that cardiolipin preferentially interacts with the AcrBZ complex, due to increased contact surface, and we observe that the drug sensitivity of bacteria lacking AcrZ is exacerbated when combined with cardiolipin deficiency. Taken together, the data suggest that AcrZ and lipid cooperate to allosterically modulate the activity of AcrB. This mode of regulation by a small protein and lipid may occur for other membrane proteins.

scholarly journals Perturbed structural dynamics underlie inhibition and altered specificity of the multidrug efflux pump AcrB

2020 ◽  
Author(s):  
Eamonn Reading ◽  
Zainab Ahdash ◽  
Chiara Fais ◽  
Vito Ricci ◽  
Xuan Wang Kan ◽  
...  
Keyword(s):  
Structural Dynamics ◽  
Efflux Pump ◽  
Binding Pocket ◽  
Drug Binding ◽  
Multidrug Efflux Pump ◽  
Transport Pathway ◽  
Efflux Pump Inhibitor ◽  
Exchange Mass Spectrometry ◽  
Wide Range ◽  
Resistance Nodulation Division

AbstractResistance-nodulation-division (RND) efflux pumps play a key role in inherent and evolved multidrug-resistance (MDR) in bacteria. AcrB is the prototypical member of the RND family and acts to recognise and export a wide range of chemically distinct molecules out of bacteria, conferring resistance to a variety of antibiotics. Although high resolution structures exist for AcrB, its conformational fluctuations and their putative role in function are largely unknown, preventing a complete mechanistic understanding of efflux and inhibition. Here, we determine these structural dynamics in the presence of AcrB substrates using hydrogen/deuterium exchange mass spectrometry, complemented by molecular modelling, drug binding and bacterial susceptibility studies. We show that the well-studied efflux pump inhibitor phenylalanine-arginine-β-naphthylamide (PAβN) potentiates antibiotic activity by restraining drug-binding pocket dynamics, rather than preventing antibiotic binding. We also reveal that a drug-binding pocket substitution discovered within an MDR clinical isolate, AcrBG288D, modifies the plasticity of the transport pathway, which could explain its altered substrate specificity. Our results provide molecular insight into drug export and inhibition of a major MDR-conferring efflux pump and the important directive role of its dynamics.

scholarly journals Klebsiella pneumoniae Major Porins OmpK35 and OmpK36 Allow More Efficient Diffusion of β-Lactams than Their Escherichia coli Homologs OmpF and OmpC

2016 ◽  
Vol 198 (23) ◽  
pp. 3200-3208 ◽  
Author(s):  
Etsuko Sugawara ◽  
Seiji Kojima ◽  
Hiroshi Nikaido
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Outer Membrane ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Resistance Mechanisms ◽  
Fourth Generation ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Content Type

ABSTRACTKlebsiella 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 ofK. pneumoniaeporin channels are largely unknown. In this study, we made clean deletions ofK. pneumoniaeporin genesompK35andompK36and examined the antibiotic susceptibilities and diffusion rates of β-lactams. The results showed that OmpK35 and OmpK36 produced larger more permeable channels than theirEscherichia colihomologs 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 anE. colistrain lacking major porins and the major multidrug efflux pump AcrAB. Our data explain why the development of drug resistance inK. pneumoniaeis 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.IMPORTANCEIn 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 itsEscherichia colihomolog, OmpF. This makes unmodifiedK. pneumoniaestrains 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.

scholarly journals Mutations in Gene fusA1 as a Novel Mechanism of Aminoglycoside Resistance in Clinical Strains of Pseudomonas aeruginosa

2017 ◽  
Vol 62 (2) ◽  
Author(s):  
Arnaud Bolard ◽  
Patrick Plésiat ◽  
Katy Jeannot
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Efflux Pump ◽  
Elongation Factor ◽  
Genetic Alterations ◽  
Single Amino Acid ◽  
Multidrug Efflux Pump ◽  
Aminoglycoside Resistance ◽  
Content Type ◽  
Clinical Strains

ABSTRACT Resistance of clinical strains of Pseudomonas aeruginosa to aminoglycosides can result from production of transferable aminoglycoside-modifying enzymes, of 16S rRNA methylases, and/or mutational derepression of intrinsic multidrug efflux pump MexXY(OprM). We report here the characterization of a new type of mutant that is 4- to 8-fold more resistant to 2-deoxystreptamine derivatives (e.g., gentamicin, amikacin, and tobramycin) than the wild-type strain PAO1. The genetic alterations of three in vitro mutants were mapped on fusA1 and found to result in single amino acid substitutions in domains II, III, and V of elongation factor G (EF-G1A), a key component of translational machinery. Transfer of the mutated fusA1 alleles into PAO1 reproduced the resistance phenotype. Interestingly, fusA1 mutants with other amino acid changes in domains G, IV, and V of EF-G1A were identified among clinical strains with decreased susceptibility to aminoglycosides. Allelic-exchange experiments confirmed the relevance of these latter mutations and of three other previously reported alterations located in domains G and IV. Pump MexXY(OprM) partly contributed to the resistance conferred by the mutated EF-G1A variants and had additive effects on aminoglycoside MICs when mutationally upregulated. Altogether, our data demonstrate that cystic fibrosis (CF) and non-CF strains of P. aeruginosa can acquire a therapeutically significant resistance to important aminoglycosides via a new mechanism involving mutations in elongation factor EF-G1A.

Structural and functional consequences of single amino acid substitutions in the pyrimidine base binding pocket of Escherichia coli CMP kinase

FEBS Journal ◽  
2007 ◽  
Vol 274 (13) ◽  
pp. 3363-3373 ◽  
Author(s):  
Augustin Ofiteru ◽  
Nadia Bucurenci ◽  
Emil Alexov ◽  
Thomas Bertrand ◽  
Pierre Briozzo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Binding Pocket ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Pyrimidine Base ◽  
Functional Consequences

scholarly journals A Broad-Specificity Multidrug Efflux Pump Requiring a Pair of Homologous SMR-Type Proteins

2000 ◽  
Vol 182 (8) ◽  
pp. 2311-2313 ◽  
Author(s):  
Donald L. Jack ◽  
Michael L. Storms ◽  
Jason H. Tchieu ◽  
Ian T. Paulsen ◽  
Milton H. Saier
Keyword(s):  
Escherichia Coli ◽  
Efflux Pump ◽  
Multidrug Resistant ◽  
Drug Efflux ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Resistant Phenotype ◽  
Small Multidrug Resistance ◽  
Drug Efflux Pumps ◽  
Broad Specificity

ABSTRACT The Bacillus subtilis genome encodes seven homologues of the small multidrug resistance (SMR) family of drug efflux pumps. Six of these homologues are paired in three distinct operons, and coexpression in Escherichia coli of one such operon,ykkCD, but not expression of either ykkC orykkD alone, gives rise to a broad specificity, multidrug-resistant phenotype including resistance to cationic, anionic, and neutral drugs.

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