title: In vitro transport activity of the fully assembled MexAB-OprM efflux pump from Pseudomonas aeruginosa.
abstract: Antibiotic resistance is a major public health issue and many bacteria responsible for human infections have now developed a variety of antibiotic resistance mechanisms. For instance, Pseudomonas aeruginosa, a disease-causing Gram-negative bacteria, is now resistant to almost every class of antibiotics. Much of this resistance is attributable to multidrug efflux pumps, which are tripartite membrane protein complexes that span both membranes and actively expel antibiotics. Here we report an in vitro procedure to monitor transport by the tripartite MexAB-OprM pump. By combining proteoliposomes containing the MexAB and OprM portions of the complex, we are able to assay energy-dependent substrate translocation in a system that mimics the dual-membrane architecture of Gram-negative bacteria. This assay facilitates the study of pump transport dynamics and could be used to screen pump inhibitors with potential clinical use in restoring therapeutic activity of old antibiotics.
authors.affiliation: Laboratoire de Cristallographie et RMN Biologiques, UMR 8015, CNRS, Université Paris Descartes, Faculté de Pharmacie de Paris, 4 Avenue de l'Observatoire, 75006 Paris, France.
authors.forename: Alice
authors.initials: A
authors.lastname: Verchère
authors.affiliation: Laboratoire de Cristallographie et RMN Biologiques, UMR 8015, CNRS, Université Paris Descartes, Faculté de Pharmacie de Paris, 4 Avenue de l'Observatoire, 75006 Paris, France.
authors.forename: Manuela
authors.initials: M
authors.lastname: Dezi
authors.affiliation: Laboratoire de physique statistique de l'École Normale Supérieure, UMR 8550, CNRS, Université Pierre et Marie Curie, 24 rue Lhomond, 75005 Paris, France.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Adrien
authors.affiliation: Laboratoire de Cristallographie et RMN Biologiques, UMR 8015, CNRS, Université Paris Descartes, Faculté de Pharmacie de Paris, 4 Avenue de l'Observatoire, 75006 Paris, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Broutin
authors.affiliation: Laboratoire de Cristallographie et RMN Biologiques, UMR 8015, CNRS, Université Paris Descartes, Faculté de Pharmacie de Paris, 4 Avenue de l'Observatoire, 75006 Paris, France.
authors.forename: Martin
authors.initials: M
authors.lastname: Picard
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D024881:Drug Resistance, Bacterial; D004996:Ethidium / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D026901:Membrane Transport Proteins / Q000378:metabolism*; D000077731:Meropenem; D011510:Proteolipids / Q000378:metabolism*; D011550:Pseudomonas aeruginosa; D013845:Thienamycins / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D005456:Fluorescent Dyes; D026901:Membrane Transport Proteins; C118598:MexA protein, Pseudomonas aeruginosa; C118597:MexB protein, Pseudomonas aeruginosa; C402263:OprM protein, Pseudomonas aeruginosa; D011510:Proteolipids; D013845:Thienamycins; C020485:proteoliposomes; D004996:Ethidium; D000077731:Meropenem