CRZ1, a target of the calcineurin pathway in Candida albicans

Calcineurin is a major player in calcium-dependent signal transduction pathways of eukaryotes. Calcineurin acts on transcription factors (e.g. CRZ1 in Saccharomyces cerevisiae) and governs the expression of genes in a species-dependent fashion. In Candida albicans, the calcineurin pathway is involved in tolerance to antifungal agents, cation homeostasis and virulence. However, the components of the calcineurin pathway are still poorly investigated in this yeast species. Taking S. cerevisiae as a model to reconstitute this pathway, two CRZ1-like genes, CRZ1 and CRZ2 (for calcineurin-responsive zinc finger 1 and 2 genes), were found with C(2)H(2) zinc finger domains. Only CRZ1 was able to restore the calcium hypersusceptibility of a S. cerevisiae crz1Delta mutant and to mediate calcium-dependent gene expression in this yeast species. Several experiments showed that CRZ1 was dependent on calcineurin in C. albicans: (i) phenotypic analysis of a crz1Delta/Delta mutant showed impaired growth as compared with the wild type in the presence of cations (Ca(2+), Mn(2+)) as does a mutant lacking calcineurin subunit A (cnaDelta/Delta) and (ii) a green fluorescent protein (GFP)-Crz1p fusion protein showed a calcium- and calcineurin-dependent nuclear localization. To further analyse the relationship between calcineurin and CRZ1, a comprehensive analysis of calcineurin/Crz1p-dependent gene expression following addition of Ca(2+) (200 mM) was performed. Among the expression of 264 genes altered by at least twofold, the upregulation of 60 genes was dependent on both calcineurin and CRZ1. Interestingly, a motif [5'-G(C/T)GGT-3'] with similarity to the target sequence of Crz1p (GNGGCG/TCA) from S. cerevisiae was identified as a putative regulatory sequence in the upstream regions of these calcineurin/Crz1p-dependent genes. However, additional experiments showed that calcineurin may have other targets in addition to CRZ1. First, CRZ1 was not involved in tolerance to antifungal agents (fluconazole, terbinafine) on the opposite to calcineurin. Second, CRZ1 was only moderately influencing virulence in a mice model of infection which is in sharp contrast to the strong avirulence of cnaDelta/Delta mutant in the same animal model. Even though this work establishes CRZ1 as a calcineurin target, further studies are needed to identify other calcineurin-dependent elements in C. albicans.