A homologous overexpression system to study roles of drug transporters in Candida glabrata.
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.
['Kumari|Sonam|S|', 'Kumar|Mohit|M|', 'Khandelwal|Nitesh Kumar|NK|', 'Pandey|Ajay Kumar|AK|', 'Bhakt|Priyanka|P|', 'Kaur|Rupinder|R|', 'Prasad|Rajendra|R|', 'Gaur|Naseem A|NA|']
D018528:ATP-Binding Cassette Transporters D000935:Antifungal Agents D001692:Biological Transport D041221:Candida glabrata D017353:Gene Deletion D015966:Gene Expression Regulation, Fungal D007700:Kinetics D009154:Mutation
Candida glabrata ABC transporters GOF mutation expression plasmid azoles multi-deletion