Karababa Mahir, Valentino Emilio, Pardini Giacomo, Coste Alix T, Bille Jacques, Sanglard Dominique
Institute of Microbiology, University Hospital Lausanne, CH-1011 Lausanne, Switzerland.
Mol Microbiol. 2006 Mar;59(5):1429-51. doi: 10.1111/j.1365-2958.2005.05037.x.
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.
钙调神经磷酸酶是真核生物钙依赖信号转导途径中的主要参与者。钙调神经磷酸酶作用于转录因子（如酿酒酵母中的CRZ1），并以物种依赖的方式调控基因表达。在白色念珠菌中，钙调神经磷酸酶途径参与抗真菌药物耐受性、阳离子稳态和毒力的调节。然而，在这种酵母中，钙调神经磷酸酶途径的组成成分仍未得到充分研究。以酿酒酵母为模型重建该途径时，发现了两个具有C(2)H(2)锌指结构域的CRZ1样基因，即CRZ1和CRZ2（分别为钙调神经磷酸酶反应性锌指1和2基因）。只有CRZ1能够恢复酿酒酵母crz1Delta突变体对钙的超敏感性，并介导该酵母中钙依赖的基因表达。多项实验表明，在白色念珠菌中CRZ1依赖于钙调神经磷酸酶：(i) crz1Delta/Delta突变体的表型分析显示，与野生型相比，在存在阳离子（Ca(2+)、Mn(2+)）的情况下其生长受损，缺乏钙调神经磷酸酶亚基A的突变体（cnaDelta/Delta）也是如此；(ii) 绿色荧光蛋白（GFP）-Crz1p融合蛋白显示出钙和钙调神经磷酸酶依赖的核定位。为了进一步分析钙调神经磷酸酶与CRZ1之间的关系，在添加Ca(2+)（200 mM）后对钙调神经磷酸酶/Crz1p依赖的基因表达进行了全面分析。在至少两倍变化的264个基因的表达中，60个基因的上调依赖于钙调神经磷酸酶和CRZ1。有趣的是，在这些钙调神经磷酸酶/Crz1p依赖基因的上游区域，发现了一个与酿酒酵母Crz1p的靶序列（GNGGCG/TCA）相似的基序[5'-G(C/T)GGT-3']，被确定为推定的调控序列。然而，额外的实验表明，除了CRZ1外，钙调神经磷酸酶可能还有其他靶点。首先，与钙调神经磷酸酶相反，CRZ1不参与抗真菌药物（氟康唑、特比萘芬）耐受性。其次，在感染小鼠模型中，CRZ1仅适度影响毒力，这与同一动物模型中cnaDelta/Delta突变体的强无毒性形成鲜明对比。尽管这项工作将CRZ1确立为钙调神经磷酸酶的靶点，但仍需要进一步研究以确定白色念珠菌中其他钙调神经磷酸酶依赖元件。
