Molecular determinants of transient and reversible induced up-regulation of CaCDR1 in azole susceptible clinical isolates of Candida albicans

Abstract

The present study examines the molecular mechanism underlying in vitro-induced resistance to FLC (fluconazole), KTC (ketaonazole), MCZ (miconazole) and CHX (cycloheximide) in AS (azole-susceptible) strains of Candida albicans when exposed to CaCDR1/CaCDR2 inducers like FPZ (fluphenazine) and steroids [PRG (progesterone) and β-EST (β-oestradiol)]. By employing spot and checkerboard titre assays, we provide evidence of an in vitro-induced antagonism between tested drugs and inducers, which was accompanied with a concomitant increase in CaCDR1 and CaCDR2 transcript levels. Notably, unlike AS isolates, parental WT (wild-type) and Δcdr2 null strains, Δcdr1 as well as Δcdr1/Δcdr2 nulls, when challenged with the inducers could not display antagonism. Our results validated by Northern blotting, reporter gene transcription and TRO (transcription run on) assays show that in vitro-induced antagonism between tested drugs and inducer in AS isolates was mainly due to a transient and reversible transcriptional activation of CaCDR1. Notwithstanding our earlier observation that consistent high transcript levels of CaCDR1 in clinical AR (azole-resistant) isolates were maintained due to the combination of its transcriptional activation and enhanced mRNA stability via elongated poly(A) tails, this study shows that transient and reversible transcriptional activation of CaCDR1 was the major determinant of induced antagonism in AS isolates. The distinct strategies between sustained (in AR isolates) and transiently induced resistance mechanisms (in AS isolates) adopted by Candida should become useful in improving therapeutic approaches.