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Ntsogo Enguéné VY, Verchère A, Phan G, et al. Catch me if you can: a biotinylated proteoliposome affinity assay for the investigation of assembly of the MexA-MexB-OprM efflux pump from Pseudomonas aeruginosa. Front Microbiol. 2015;6:541doi: 10.3389/fmicb.2015.00541.
Ntsogo Enguéné, V. Y., Verchère, A., Phan, G., Broutin, I., & Picard, M. (2015). Catch me if you can: a biotinylated proteoliposome affinity assay for the investigation of assembly of the MexA-MexB-OprM efflux pump from Pseudomonas aeruginosa. Frontiers in microbiology, 6541. https://doi.org/10.3389/fmicb.2015.00541
Ntsogo Enguéné, Véronique Yvette, et al. "Catch me if you can: a biotinylated proteoliposome affinity assay for the investigation of assembly of the MexA-MexB-OprM efflux pump from Pseudomonas aeruginosa." Frontiers in microbiology vol. 6 (2015): 541. doi: https://doi.org/10.3389/fmicb.2015.00541
Ntsogo Enguéné VY, Verchère A, Phan G, Broutin I, Picard M. Catch me if you can: a biotinylated proteoliposome affinity assay for the investigation of assembly of the MexA-MexB-OprM efflux pump from Pseudomonas aeruginosa. Front Microbiol. 2015 Jun 02;6:541. doi: 10.3389/fmicb.2015.00541. eCollection 2015. PMID: 26082762; PMCID: PMC4451422.
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Front Microbiol. 2015 Jun 02;6:541. doi: 10.3389/fmicb.2015.00541. eCollection 2015.

Catch me if you can: a biotinylated proteoliposome affinity assay for the investigation of assembly of the MexA-MexB-OprM efflux pump from Pseudomonas aeruginosa.

Frontiers in microbiology

Véronique Yvette Ntsogo Enguéné, Alice Verchère, Gilles Phan, Isabelle Broutin, Martin Picard

Affiliations

  1. Laboratoire de Cristallographie et RMN Biologiques, Faculté de Pharmacie de Paris, UMR 8015 CNRS - Université Paris 089 Descartes , Paris, France.

PMID: 26082762 PMCID: PMC4451422 DOI: 10.3389/fmicb.2015.00541

Abstract

Efflux pumps are membrane transporters that actively extrude various substrates, leading to multidrug resistance (MDR). In this study, we have designed a new test that allows investigating the assembly of the MexA-MexB-OprM efflux pump from the Gram negative bacteria Pseudomonas aeruginosa. The method relies on the streptavidin-mediated pull-down of OprM proteoliposomes upon interaction with MexAB proteoliposomes containing a biotin function carried by lipids. We give clear evidence for the importance of MexA in promoting and stabilizing the assembly of the MexAB-OprM complex. In addition, we have investigated the effect of the role of the lipid anchor of MexA as well as the role of the proton motive force on the assembly and disassembly of the efflux pump. The assay presented here allows for an accurate investigation of the assembly with only tens of microgram of protein and could be adapted to 96 wells plates. Hence, this work provides a basis for the medium-high screening of efflux pump inhibitors (EPIs).

Keywords: efflux pump; macro-molecular assembly; membrane protein; multidrug resistance; proteoliposome

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