A homologous overexpression system to study roles of drug transporters in Candida glabrata.
Candida glabrata
ABC transporters
GOF mutation
azoles
expression plasmid
multi-deletion
Journal
FEMS yeast research
ISSN: 1567-1364
Titre abrégé: FEMS Yeast Res
Pays: England
ID NLM: 101085384
Informations de publication
Date de publication:
01 06 2020
01 06 2020
Historique:
received:
24
04
2020
accepted:
02
06
2020
pubmed:
4
6
2020
medline:
9
7
2021
entrez:
4
6
2020
Statut:
ppublish
Résumé
Considering the relevance of drug transporters belonging to ABC and MFS superfamilies in pathogenic Candida species, there has always been a need to have an overexpression system where these membrane proteins for functional analysis could be expressed in a homologous background. We could address this unmet need by constructing a highly drug-susceptible Candida glabrata strain deleted in seven dominant ABC transporters genes such as CgSNQ2, CgAUS1, CgCDR1, CgPDH1, CgYCF1, CgYBT1 and CgYOR1 and introduced a GOF mutation in transcription factor (TF) CgPDR1 leading to a hyper-activation of CgCDR1 locus. The expression system was validated by overexpressing four GFP tagged ABC (CgCDR1, CgPDH1, CaCDR1 and ScPDR5) and an MFS (CgFLR1) transporters genes facilitated by an engineered expression plasmid to integrate at the CgCDR1 locus. The properly expressed and localized transporters were fully functional, as was revealed by their several-fold increased drug resistance, growth kinetics, localization studies and efflux activities. The present homologous system will facilitate in determining the role of an individual transporter for its substrate specificity, drug efflux, pathogenicity and virulence traits without the interference of other major transporters.
Identifiants
pubmed: 32490522
pii: 5850755
doi: 10.1093/femsyr/foaa032
pmc: PMC7611192
mid: EMS127438
pii:
doi:
Substances chimiques
ATP-Binding Cassette Transporters
0
Antifungal Agents
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : Wellcome Trust
Pays : United Kingdom
Organisme : DBT-Wellcome Trust India Alliance
ID : IA/S/15/1/501831
Pays : India
Informations de copyright
© FEMS 2020.
Références
Med Mycol. 2016 Jul 1;54(5):478-91
pubmed: 26782644
Front Microbiol. 2017 Jan 12;7:2173
pubmed: 28127295
Mol Microbiol. 2008 Apr;68(1):186-201
pubmed: 18312269
Mol Microbiol. 2006 Aug;61(3):704-22
pubmed: 16803598
J Biol Chem. 2011 Sep 30;286(39):34311-24
pubmed: 21832071
J Biol Chem. 1998 May 15;273(20):12612-22
pubmed: 9575223
Gene. 2007 Jan 15;386(1-2):63-72
pubmed: 17046176
Eukaryot Cell. 2007 Jul;6(7):1150-65
pubmed: 17513564
Microb Drug Resist. 1998 Fall;4(3):143-58
pubmed: 9818966
Antimicrob Agents Chemother. 2005 Feb;49(2):668-79
pubmed: 15673750
Antimicrob Agents Chemother. 2008 Feb;52(2):694-704
pubmed: 18056285
Front Microbiol. 2016 Dec 21;7:2045
pubmed: 28066366
PLoS One. 2015 May 07;10(5):e0126350
pubmed: 25951180
PLoS One. 2018 Aug 28;13(8):e0202993
pubmed: 30153284
Antimicrob Agents Chemother. 2011 May;55(5):1852-60
pubmed: 21321146
Eukaryot Cell. 2003 Dec;2(6):1361-75
pubmed: 14665469
Future Med Chem. 2016 Aug;8(12):1485-501
pubmed: 27463566
Biochim Biophys Acta. 2003 Feb 17;1610(1):11-22
pubmed: 12586375
Genes (Basel). 2018 Sep 19;9(9):
pubmed: 30235884
PLoS Pathog. 2009 Jan;5(1):e1000268
pubmed: 19148266
FEMS Yeast Res. 2015 Aug;15(5):fov042
pubmed: 26066553
FEBS J. 2015 Jun;282(11):2142-66
pubmed: 25772226
J Mol Biol. 2018 Mar 2;430(5):682-694
pubmed: 29341887