Jorgaq Pata , Alexis Moreno , Benjamin Wiseman , Sandrine Magnard , Idriss Lehlali , Marie Dujardin , Atanu Banerjee , Martin Högbom , Ahcène Boumendjel , Vincent Chaptal , Rajendra Prasad , Pierre Falson
{"title":"Purification and characterization of Cdr1, the drug-efflux pump conferring azole resistance in Candida species","authors":"Jorgaq Pata , Alexis Moreno , Benjamin Wiseman , Sandrine Magnard , Idriss Lehlali , Marie Dujardin , Atanu Banerjee , Martin Högbom , Ahcène Boumendjel , Vincent Chaptal , Rajendra Prasad , Pierre Falson","doi":"10.1016/j.biochi.2023.12.007","DOIUrl":null,"url":null,"abstract":"<div><p><span><em>Candida albicans</em></span> and <em>C. glabrata</em><span> 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 </span><em>glabrata</em> version, <em>cg</em><span>Cdr1-His, or a green fluorescent protein (GFP) preceded by a proteolytic cleavage site for the </span><em>albicans</em> version, <em>ca</em><span>Cdr1-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 </span><em>cg</em>Cdr1 as a good candidate to characterize this type of proteins for developing future inhibitors of their azole antifungal efflux activity.</p></div>","PeriodicalId":251,"journal":{"name":"Biochimie","volume":null,"pages":null},"PeriodicalIF":3.3000,"publicationDate":"2023-12-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochimie","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0300908423003310","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
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.
期刊介绍:
Biochimie publishes original research articles, short communications, review articles, graphical reviews, mini-reviews, and hypotheses in the broad areas of biology, including biochemistry, enzymology, molecular and cell biology, metabolic regulation, genetics, immunology, microbiology, structural biology, genomics, proteomics, and molecular mechanisms of disease. Biochimie publishes exclusively in English.
Articles are subject to peer review, and must satisfy the requirements of originality, high scientific integrity and general interest to a broad range of readers. Submissions that are judged to be of sound scientific and technical quality but do not fully satisfy the requirements for publication in Biochimie may benefit from a transfer service to a more suitable journal within the same subject area.