Calcineurin is required forCandida glabrataPdr1 transcriptional activation

Abstract

AbstractFluconazole is the most commonly used antifungal today. A result of this has been the inevitable selection of fluconazole resistant organisms. This is an especially acute problem in the pathogenic yeastCandida glabrata. Elevated minimal inhibitory concentrations (MICs) for fluconazole inC. glabrataare frequently associated with substitution mutations within the Zn2Cys6 zinc cluster-containing transcription factor-encoding genePDR1. These mutant Pdr1 regulators drive constitutively high expression of target genes likeCDR1that encodes an ATP-binding cassette transporter thought to act as a drug efflux pump. Exposure ofC. glabratato fluconazole induced expression of both Pdr1 andCDR1, although little is known of the molecular basis underlying the upstream signals that trigger Pdr1 activation. Here, we show that the protein phosphatase calcineurin is required for fluconazole-dependent induction of Pdr1 transcriptional regulation. Calcineurin catalytic activity is required for normal Pdr1 regulation and a hyperactive form of this phosphatase can increase resistance to the echinocandin caspofungin but does not show a similar elevation for fluconazole resistance. Loss of calcineurin from strains expressing two different gain-of-function forms of Pdr1 also caused a decrease inCDR1expression and fluconazole resistance, demonstrating that even these hyperactive Pdr1 regulatory mutants cannot bypass the requirement for calcineurin. Our data implicate calcineurin activity as a link tying azole and echinocandin resistance together via the control of transcription factor activity.ImportanceWhile drug resistant microorganisms are a problem in treatment of all infectious disease, this is an especially acute problem with fungi due to the existence of only 3 classes of antifungal drugs, including the azole drug fluconazole. In the pathogenic yeastCandida glabrata, mutant forms of a transcription factor called Pdr1 are commonly associated with fluconazole resistance and poor clinical outcomes. Here we identify a protein phosphatase called calcineurin that is required for fluconazole-dependent induction of Pdr1 transcriptional activation and associated drug resistance. Gain-of-function mutant forms of Pdr1 still required the presence of calcineurin to confer normally elevated fluconazole resistance. Previous studies showed that calcineurin is required for resistance to the echinocandin class of antifungal drugs and our data demonstrate this protein phosphatase is also required for azole drug resistance. Calcineurin plays a central role in resistance to two of the three major classes of antifungal drugs inC. glabrata.