title: Transcriptional activation and increased mRNA stability contribute to overexpression of CDR1 in azole-resistant Candida albicans.
abstract: Many azole-resistant (AR) clinical isolates of Candida albicans display increased expression of the drug transporters CDR1 and CDR2. In this study, we evaluate the molecular mechanisms that contribute to the maintenance of constitutively high CDR1 transcript levels in two matched pairs of azole-susceptible (AS) and AR clinical isolates of C. albicans. To address this, we use reporter constructs of GFP and lacZ fused either to the CDR1 promoter (P CDR1-GFP/lacZ; transcriptional fusion) or to the CDR1 open reading frame (P CDR1-CDR1-GFP/lacZ; translational fusion) integrated at the native CDR1 locus. It is observed that expression of the two reporter genes as a transcriptional fusion in the AR isolates is higher than that in matched AS isolates. However, the difference in the reporter activity between the AS and AR isolates is even greater for the translational fusions, indicating that the sequences within the CDR1 coding region also contribute to its increased expression in AR isolates. Further analysis of these observations by transcription run-on assays demonstrated a approximately 5- to 7-fold difference in the transcription initiation rates for the AR isolates from those for their respective matched AS isolates. Measurement of mRNA stability showed that the half-life of CDR1 mRNA in the AR isolates was threefold higher than that in the corresponding AS isolates. Our results demonstrate that both increased CDR1 transcription and enhanced CDR1 mRNA stability contribute to the overexpression of CDR1 in AR C. albicans isolates.
authors.affiliation: Membrane Biology, School of Life Sciences, Jawaharlal Nehru University, JNU Campus, New Mehrauli Road, New Delhi 110067, India.
authors.forename: Raman
authors.initials: R
authors.lastname: Manoharlal
authors.forename: Naseem Akhtar
authors.initials: NA
authors.lastname: Gaur
authors.forename: Sneh Lata
authors.initials: SL
authors.lastname: Panwar
authors.forename: Joachim
authors.initials: J
authors.lastname: Morschhäuser
authors.forename: Rajendra
authors.initials: R
authors.lastname: Prasad
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology; D001393:Azoles / Q000494:pharmacology; D002176:Candida albicans / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D025141:Drug Resistance, Fungal; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D020871:RNA Stability; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation
chemical_list: D000935:Antifungal Agents; D001393:Azoles; C094693:CDR1 protein, Candida albicans; D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger