Background: We have found that C. albicans azole-resistant strains overexpressing the multidrug transporter-encoding genes CDR1 and CDR2 also overexpress CaPDR16 which codes for a putative phosphatidylinositol transfer protein involved in azole resistance. This concomitant overexpression likely results from activating mutation(s) in a yet unknown transcriptional regulator controlling the expression of the three genes. We have undertaken a study of the CaPDR16 promoter to better understand this azole resistance regulatory pathway. Methods: Northern blotting was used to analyze the expression of CaPDR16, CDR1 and CDR2 under various conditions in experimental and clinical strains. The CaPDR16 promoter (-1,200 to -1 bp with respect to the ATG) was isolated from strain SC5314 and fused to the yEGFP reporter gene. This construction was integrated into C. albicans strains using the mycophenolic acid resistance marker MPAR and the promoter activity was quantified by fluorescence-activated cell sorting (FACS) analysis. Results: Our results show that fluphenazine, estradiol and rhodamine 6G induce the expression of CaPDR16, CDR1 and CDR2 in azole-susceptible but not in azole-resistant strains in which the three genes are already highly expressed. SC5314 cells carrying the CaPDR16p-yEGFP fusion displayed a low level of fluorescence which can be further induced by these three agents, demonstrating that the cloned CaPDR16 promoter fragment contains all the regulatory sequences necessary for its basal and inducible transcription. Finally, azole-resistant isolates carrying the CaPDR16p-yEGFP construction were strongly fluorescent, demonstrating that the CaPDR16 promoter is constitutively trans-activated in these cells. Conclusion: This system will allow the identification of the cis-acting sequences controlling CaPDR16 expression and eventually the trans-acting factor(s) responsible for the overexpression of the CaPDR16, CDR1 and CDR2 genes in azole-resistant clinical isolates.
Full conference title:
43rd Interscience Conference on Antimicrobial Agents
- ICAAC 43rd