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Purification and characterization of Cdr1, the drug-efflux pump conferring azole resistance in Candida species.
Pata, Jorgaq; Moreno, Alexis; Wiseman, Benjamin; Magnard, Sandrine; Lehlali, Idriss; Dujardin, Marie; Banerjee, Atanu; Högbom, Martin; Boumendjel, Ahcène; Chaptal, Vincent; Prasad, Rajendra; Falson, Pierre.
Afiliação
  • Pata J; Drug Resistance & Membrane Proteins Group, CNRS-Lyon 1 University Laboratory UMR 5086, IBCP, 69367, CEDEX Lyon 07, France.
  • Moreno A; Drug Resistance & Membrane Proteins Group, CNRS-Lyon 1 University Laboratory UMR 5086, IBCP, 69367, CEDEX Lyon 07, France; CALIXAR, 60 Avenue Rockefeller, Lyon, France.
  • Wiseman B; Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, Stockholm, Sweden.
  • Magnard S; Drug Resistance & Membrane Proteins Group, CNRS-Lyon 1 University Laboratory UMR 5086, IBCP, 69367, CEDEX Lyon 07, France.
  • Lehlali I; Drug Resistance & Membrane Proteins Group, CNRS-Lyon 1 University Laboratory UMR 5086, IBCP, 69367, CEDEX Lyon 07, France.
  • Dujardin M; CALIXAR, 60 Avenue Rockefeller, Lyon, France.
  • Banerjee A; Amity Institute of Biotechnology and Amity Institute of Integrative Sciences and Health, Amity University Haryana, Gurgaon, India.
  • Högbom M; Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, Stockholm, Sweden.
  • Boumendjel A; Univ. Grenoble Alpes, INSERM, LRB, 38000, Grenoble, France.
  • Chaptal V; Drug Resistance & Membrane Proteins Group, CNRS-Lyon 1 University Laboratory UMR 5086, IBCP, 69367, CEDEX Lyon 07, France.
  • Prasad R; Amity Institute of Biotechnology and Amity Institute of Integrative Sciences and Health, Amity University Haryana, Gurgaon, India.
  • Falson P; Drug Resistance & Membrane Proteins Group, CNRS-Lyon 1 University Laboratory UMR 5086, IBCP, 69367, CEDEX Lyon 07, France. Electronic address: pierre.falson@ibcp.fr.
Biochimie ; 220: 167-178, 2024 May.
Article em En | MEDLINE | ID: mdl-38158037
ABSTRACT
Candida albicans and C. glabrata express exporters of the ATP-binding cassette (ABC) superfamily and address them to their plasma membrane to expel azole antifungals, which cancels out their action and allows the yeast to become multidrug resistant (MDR). In a way to understand this mechanism of defense, we describe the purification and characterization of Cdr1, the membrane ABC exporter mainly responsible for such phenotype in both species. Cdr1 proteins were functionally expressed in the baker yeast, tagged at their C-terminal end with either a His-tag for the glabrata version, cgCdr1-His, or a green fluorescent protein (GFP) preceded by a proteolytic cleavage site for the albicans version, caCdr1-P-GFP. A membrane Cdr1-enriched fraction was then prepared to assay several detergents and stabilizers, probing their level of extraction and the ATPase activity of the proteins as a functional marker. Immobilized metal-affinity and size-exclusion chromatographies (IMAC, SEC) were then carried out to isolate homogenous samples. Overall, our data show that although topologically and phylogenetically close, both proteins display quite distinct behaviors during the extraction and purification steps, and qualify cgCdr1 as a good candidate to characterize this type of proteins for developing future inhibitors of their azole antifungal efflux activity.
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Texto completo: 1 Base de dados: MEDLINE Assunto principal: Proteínas de Membrana Transportadoras / Azóis / Candida albicans / Proteínas Fúngicas / Farmacorresistência Fúngica / Antifúngicos Idioma: En Ano de publicação: 2024 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Proteínas de Membrana Transportadoras / Azóis / Candida albicans / Proteínas Fúngicas / Farmacorresistência Fúngica / Antifúngicos Idioma: En Ano de publicação: 2024 Tipo de documento: Article