title: 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.
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).
authors.affiliation: Laboratoire de Cristallographie et RMN Biologiques, Faculté de Pharmacie de Paris, UMR 8015 CNRS - Université Paris 089 Descartes , Paris, France.
authors.forename: Véronique Yvette
authors.initials: VY
authors.lastname: Ntsogo Enguéné
authors.affiliation: Laboratoire de Cristallographie et RMN Biologiques, Faculté de Pharmacie de Paris, UMR 8015 CNRS - Université Paris 089 Descartes , Paris, France.
authors.forename: Alice
authors.initials: A
authors.lastname: Verchère
authors.affiliation: Laboratoire de Cristallographie et RMN Biologiques, Faculté de Pharmacie de Paris, UMR 8015 CNRS - Université Paris 089 Descartes , Paris, France.
authors.forename: Gilles
authors.initials: G
authors.lastname: Phan
authors.affiliation: Laboratoire de Cristallographie et RMN Biologiques, Faculté de Pharmacie de Paris, UMR 8015 CNRS - Université Paris 089 Descartes , Paris, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Broutin
authors.affiliation: Laboratoire de Cristallographie et RMN Biologiques, Faculté de Pharmacie de Paris, UMR 8015 CNRS - Université Paris 089 Descartes , Paris, France.
authors.forename: Martin
authors.initials: M
authors.lastname: Picard
keywords: efflux pump; macro-molecular assembly; membrane protein; multidrug resistance; proteoliposome