In vitro transport activity of the fully assembled MexAB-OprM efflux pump from Pseudomonas aeruginosa

Verchère, Alice; Dezi, Manuela; Adrien, Vladimir; Broutin, Isabelle; Picard, Martin

doi:10.1038/ncomms7890

Cited by 48 publications

(48 citation statements)

References 28 publications

(28 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…OprM (respectively homologous of AcrA and TolC in Pseudomonas aeruginosa) further corroborates our hypothesis (56). Moreover, an analysis of the hydrophilic character of the internal surfaces of AcrA and TolC in the recently published structure of the complete AcrABZ-TolC assembly (57) confirmed these findings ( Figure S4).…”

Section: Biological Implicationssupporting

confidence: 88%

“…Moreover, an analysis of the hydrophilic character of the internal surfaces of AcrA and TolC in the recently published structure of the complete AcrABZ-TolC assembly (57) confirmed these findings ( Figure S4). Therefore, it is plausible that upon crossing the Gate, the substrate will wander within an environment that would drive it out (56).…”

Section: Biological Implicationsmentioning

confidence: 99%

See 1 more Smart Citation

Water-mediated interactions enable smooth substrate transport in a bacterial efflux pump

Vargiu

Ramaswamy

Malvacio

et al. 2017

Preprint

View full text Add to dashboard Cite

Efflux pumps of the Resistance-Nodulation-cell Division superfamily confer multi-drug resistance to Gram-negative bacteria. AcrB of Escherichia coli is a paradigm model of these polyspecific transporters. The molecular determinants and the energetics of the functional rotation mechanism proposed for the export of substrates by this protein have not yet been unveiled. To this aim, we implemented an original protocol that allows mimicking substrate transport in silico. We show that the conformational changes occurring in AcrB enable the formation of a layer of structured waters on the surface of the substrate transport channel. This, in turn, allows for a fairly constant hydration of the substrate that facilitates its diffusion.Our findings reveal a new molecular mechanism of transport in polyspecific systems, whereby waters contribute by screening potentially strong substrate-protein interactions. The mechanistic understanding of a fundamental process related to multi-drug transport provided here could help rationalizing the behavior of other polyspecific systems.. CC-BY 4.0 International license not peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was . http://dx.doi.org/10.1101/182683 doi: bioRxiv preprint first posted online 2 Multi-Drug Resistant (MDR) pathogens represent one of the most pressing health concerns of the XXI Century due to their ability to elude the action of most (in some instances all) antibiotics (1-4). A special family of membrane transport proteins, the so-called efflux pumps, plays a major role in conferring MDR by shuttling a broad spectrum of chemically unrelated cytotoxic molecules out of bacteria (5-9). Polyspecificity and partial overlap among the substrate specificities of different pumps are striking properties of these efflux machineries (10, 11), making them a key survival tool of bacteria.The efflux systems of the Resistance Nodulation-cell Division (RND) superfamily, which span the entire periplasm connecting the inner and the outer membranes, are mainly involved in the onset of MDR in Gram-negative bacteria (5, 12-14). The AcrABZ-TolC efflux pump of Escherichia coli is the paradigm model and the most studied RND efflux pump. It is composed of the outer membrane efflux duct TolC, the inner membrane-anchored adaptor protein AcrA, the small transmembrane protein AcrZ and the inner membrane RND protein AcrB (15). The lattermost is a drug/H + antiporter fuelled by the proton gradient across the inner membrane and involved in the recognition and translocation of a very broad range of compounds (16).The multi-drug recognition capabilities and the postulated efflux mechanism of RND transporters are linked through an intriguing structural puzzle, which raised the question of how these proteins achieve their special features. An important step in this direction was made with the publication of the structure of AcrB ( Figure 1A Successively, a second conformational change from T to O (supposed to be the energyrequi...

show abstract

Section: Biological Implicationssupporting

confidence: 88%

Section: Biological Implicationsmentioning

confidence: 99%

Water-mediated interactions enable smooth substrate transport in a bacterial efflux pump

Vargiu

Ramaswamy

Malvacio

et al. 2017

Preprint

View full text Add to dashboard Cite

show abstract

“…MexA is essential for the assembly of the MexAB-OprM efflux pump and for in vivo transport (62). The identified premature termination codons reside in helix 2 or the disordered C-terminal domain (63). Truncations in helix 2 should result in nonfunctional proteins, but truncations of the C-terminal end could result in functional protein variants that affect the architecture of the tripartite efflux pump and modify the OprM-mediated stimulation of the MexB pump (62).…”

Section: Resultsmentioning

confidence: 99%

Molecular Epidemiology of Mutations in Antimicrobial Resistance Loci of Pseudomonas aeruginosa Isolates from Airways of Cystic Fibrosis Patients

Greipel

Fischer

Klockgether

et al. 2016

Antimicrob Agents Chemother

View full text Add to dashboard Cite

bThe chronic airway infections with Pseudomonas aeruginosa in people with cystic fibrosis (CF) are treated with aerosolized antibiotics, oral fluoroquinolones, and/or intravenous combination therapy with aminoglycosides and ␤-lactam antibiotics. An international strain collection of 361 P. aeruginosa isolates from 258 CF patients seen at 30 CF clinics was examined for mutations in 17 antimicrobial susceptibility and resistance loci that had been identified as hot spots of mutation by genome sequencing of serial isolates from a single CF clinic. Combinatorial amplicon sequencing of pooled PCR products identified 1,112 sequence variants that were not present in the genomes of representative strains of the 20 most common clones of the global P. aeruginosa population. A high frequency of singular coding variants was seen in spuE, mexA, gyrA, rpoB, fusA1, mexZ, mexY, oprD, ampD, parR, parS, and envZ (amgS), reflecting the pressure upon P. aeruginosa in lungs of CF patients to generate novel protein variants. The proportion of nonneutral amino acid exchanges was high. Of the 17 loci, mexA, mexZ, and pagL were most frequently affected by independent stop mutations. Private and de novo mutations seem to play a pivotal role in the response of P. aeruginosa populations to the antimicrobial load and the individual CF host. C ystic fibrosis (CF) is a severe autosomal recessive trait that is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) (1). CFTR dysfunction affects many organs, but lung disease is responsible for the vast majority of morbidity and mortality in people with CF. The basic defect in CF predisposes to viral and bacterial airway infections (2). The most prevalent airway pathogen in adolescents and adults with CF is Pseudomonas aeruginosa (3, 4). The chronic airway colonization with P. aeruginosa has been associated with a rapid decline in lung function, heightened lung inflammation, and worse prognosis (1).Antipseudomonal chemotherapy in CF comprises intravenous combination therapy with ␤-lactam antibiotics and aminoglycosides, oral fluoroquinolones, immunomodulatory treatment with azithromycin and aerosolized antibiotics such as tobramycin, aztreonam, or colistin (4-8). The high antimicrobial pressure exerted on the microorganism, which can persist and diversify in the patient's airways for decades, drives the emergence of drug resistance phenotypes (9, 10). Typical targets are elements of replication, transcription, and translation, the cell wall, and transport and its regulation (9-11).During ongoing studies on the long-term microevolution of P. aeruginosa in lungs of 12 CF patients, we have identified 17 loci to be repetitively hit by mutations. These loci are known from the literature to be associated with the emergence of drug resistance in P. aeruginosa (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Hence, we became interested to know if and to what extent the global P. aeruginosa population in lungs of patients (CF lungs) carries mutations in these ge...

show abstract

“…Monitoring the activity of tripartite efflux pump from Gram negative bacteria is very challenging because they span the double membrane, and transport occurs from one environment to an other (see schematic representation Figure 1a). In the past, we have designed procedures to mimic efflux by reconstituting the respective protein partners in lipid vesicles and monitor in vitro transport through a reconstituted pump [20][21][22][23] . Following similar lines, we decided to set up a membrane-based system capable of mimicking the MacAB TolC tripartite complex.…”

Section: Overall Principle Of the Assaymentioning

confidence: 99%

Quantum dot probes for the quantitative study of drug transport by the MacAB TolC efflux pump in lipid scaffolds

Souabni

Santos

Cece

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

ABC tripartite efflux pumps are macromolecular membrane protein machineries that expel a large variety of drugs and export virulence factors from Gram negative bacteria. Using a lipid scaffold mimicking the two-membrane environment of the transporter and designing spectroscopic conditions allowing the monitoring of both ATP hydrolysis and substrate transport in real time, we show that MacAB-TolC accommodates transport and energy consumption with high coupling efficiency.

show abstract

In vitro transport activity of the fully assembled MexAB-OprM efflux pump from Pseudomonas aeruginosa

Cited by 48 publications

References 28 publications

Water-mediated interactions enable smooth substrate transport in a bacterial efflux pump

Water-mediated interactions enable smooth substrate transport in a bacterial efflux pump

Molecular Epidemiology of Mutations in Antimicrobial Resistance Loci of Pseudomonas aeruginosa Isolates from Airways of Cystic Fibrosis Patients

Quantum dot probes for the quantitative study of drug transport by the MacAB TolC efflux pump in lipid scaffolds

Contact Info

Product

Resources

About