Calcium-Mediated Induction of Paradoxical Growth following Caspofungin Treatment Is Associated with Calcineurin Activation and Phosphorylation in Aspergillus fumigatus.
Calcium-Mediated Induction of Paradoxical Growth following Caspofungin Treatment Is Associated with Calcineurin Activation and Phosphorylation in Aspergillus fumigatus.
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DOI：
10.1128/aac.00263-15
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发表时间：
2015-08
影响因子：
4.9
通讯作者：
Steinbach WJ
中科院分区：
文献类型：
--
作者：
Juvvadi PR;Muñoz A;Lamoth F;Soderblom EJ;Moseley MA;Read ND;Steinbach WJ
棘球菌素抗真菌药物卡泊芬净在高浓度时可逆转烟曲霉的生长抑制，这一现象被称为“悖论效应”，而其他棘球菌素(米卡芬净和阿尼杜拉芬净)并不一致。以前对烟曲霉菌的研究表明，钙调神经磷酸酶在药物或遗传上的抑制后，这种矛盾的效应会消失，但其潜在的机制尚不清楚。在这里，我们利用了一个密码子优化的生物发光报告基因在烟曲霉中的表达系统，并表明卡泊芬净通过激活钙调蛋白-钙调神经磷酸酶信号通路，在真菌中诱导细胞内瞬时增加胞内游离钙([Ca+]c)，作为这一矛盾效应的初始触发。另一种无反常作用的棘球菌素米卡芬净可使细胞内[Ca~(2+)]_c升高，但反常生长浓度越高，[Ca~(2+)]_c升高越明显。用钙离子选择性螯合剂BAPTA[1，2-双(邻氨基苯氧基)乙烷-N，N，N‘，N’-四乙酸]或L类钙通道阻滞剂维拉帕米处理后，卡泊芬净介导的反常生长在野生型和棘球绦虫抗性株(EMFR-S678P)中均被取消。伴随着较高浓度卡泊芬净[Ca~(2+)]_c水平的升高，钙调蛋白和钙调神经磷酸酶基因表达增强。磷酸化蛋白质组学分析表明，钙调神经磷酸酶通过其丝氨酸富集区(SPRR)的磷酸化而激活，该区域以前被认为是调节菌丝生长的关键区域，只有在卡泊芬净的反常生长浓度下才能激活。我们的结果表明，与米卡芬净相反，高浓度卡泊芬净增加的[Ca~(2+)]c在转录和翻译后水平激活了钙调蛋白-钙调神经磷酸酶信号，最终导致了矛盾的真菌生长。
The echinocandin antifungal drug caspofungin at high concentrations reverses the growth inhibition of Aspergillus fumigatus, a phenomenon known as the “paradoxical effect,” which is not consistently observed with other echinocandins (micafungin and anidulafungin). Previous studies of A. fumigatus revealed the loss of the paradoxical effect following pharmacological or genetic inhibition of calcineurin, yet the underlying mechanism is poorly understood. Here, we utilized a codon-optimized bioluminescent Ca2+ reporter aequorin expression system in A. fumigatus and showed that caspofungin elicits a transient increase in cytosolic free Ca2+ ([Ca2+]c) in the fungus that acts as the initial trigger of the paradoxical effect by activating calmodulin-calcineurin signaling. While the increase in [Ca2+]c was also observed upon treatment with micafungin, another echinocandin without the paradoxical effect, a higher [Ca2+]c increase was noted with the paradoxical-growth concentration of caspofungin. Treatments with a Ca2+-selective chelator, BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid], or the L-type Ca2+ channel blocker verapamil abolished caspofungin-mediated paradoxical growth in both the wild-type and the echinocandin-resistant (EMFR-S678P) strains. Concomitant with increased [Ca2+]c levels at higher concentrations of caspofungin, calmodulin and calcineurin gene expression was enhanced. Phosphoproteomic analysis revealed that calcineurin is activated through phosphorylation at its serine-proline-rich region (SPRR), a domain previously shown to be essential for regulation of hyphal growth, only at a paradoxical-growth concentration of caspofungin. Our results indicate that as opposed to micafungin, the increased [Ca2+]c at high concentrations of caspofungin activates calmodulin-calcineurin signaling at both a transcriptional and a posttranslational level and ultimately leads to paradoxical fungal growth.