The Antifungal Activity of the Penicillium chrysogenum Protein PAF Disrupts Calcium Homeostasis in Neurospora crassa
The Antifungal Activity of the Penicillium chrysogenum Protein PAF Disrupts Calcium Homeostasis in Neurospora crassa
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DOI：
10.1128/ec.00050-10
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发表时间：
2010-09-01
期刊：
影响因子：
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通讯作者：
Marx, Florentine
中科院分区：
文献类型：
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作者：
Binder, Ulrike;Chu, Meiling;Marx, Florentine
产黄青霉的抗真菌蛋白PAF对多种丝状真菌表现出生长抑制活性。这项研究的证据表明，钙信号/动态平衡的破坏在PAF作为生长抑制物的机制基础上起着重要作用。添加高钙浓度的生长介质可拮抗PAF对PAF敏感霉菌的毒力。通过使用表达密码子优化的aequorin的粗糙脉孢霉转基因菌株，PAF发现PAF能显著增加细胞内游离钙([Ca+](C))的静息水平。在PAF存在的情况下，机械扰动或低渗休克刺激引起的细胞内钙信号明显改变。钙离子选择性螯合剂BAPTA[双(氨基苯氧基)乙烷-N，N，N‘，N’-四乙酸]在生长抑制实验中可改善PAF的毒性，并拮抗PAF对细胞内钙平衡的影响。这些结果表明，细胞外钙离子是PAF诱导这些效应的主要来源。L类钙通道阻滞剂地尔硫卓以类似于房颤的方式破坏钙稳态。地尔硫卓与PAF联合应用，可增强细胞生长抑制作用，增强细胞内钙信号对外界刺激的反应。值得注意的是，PAF和地尔硫卓都提高了静息状态下的[Ca~(2+)](C)水平。然而，对粗枝线虫DeltaCCH-1缺失株的实验表明，L型钙通道CCH-1不是PAF诱导的[Ca~(2+)](C)静息水平升高的原因。这项研究首次证明了丝状子囊菌中的一种抗真菌蛋白扰乱了真菌的钙稳态，并为PAF的作用模式提供了重要的新见解。
The antifungal protein PAF from Penicillium chrysogenum exhibits growth-inhibitory activity against a broad range of filamentous fungi. Evidence from this study suggests that disruption of Ca2+ signaling/homeostasis plays an important role in the mechanistic basis of PAF as a growth inhibitor. Supplementation of the growth medium with high Ca2+ concentrations counteracted PAF toxicity toward PAF-sensitive molds. By using a transgenic Neurospora crassa strain expressing codon-optimized aequorin, PAF was found to cause a significant increase in the resting level of cytosolic free Ca2+ ([Ca2+](c)). The Ca2+ signatures in response to stimulation by mechanical perturbation or hypo-osmotic shock were significantly changed in the presence of PAF. BAPTA [bis-(aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid], a Ca2+ selective chelator, ameliorated the PAF toxicity in growth inhibition assays and counteracted PAF induced perturbation of Ca2+ homeostasis. These results indicate that extracellular Ca2+ was the major source of these PAF-induced effects. The L-type Ca2+ channel blocker diltiazem disrupted Ca2+ homeostasis in a similar manner to PAF. Diltiazem in combination with PAF acted additively in enhancing growth inhibition and accentuating the change in Ca2+ signatures in response to external stimuli. Notably, both PAF and diltiazem increased the [Ca2+](c) resting level. However, experiments with an aequorin-expressing Delta cch-1 deletion strain of N. crassa indicated that the L-type Ca2+ channel CCH-1 was not responsible for the observed PAF-induced elevation of the [Ca2+](c) resting level. This study is the first demonstration of the perturbation of fungal Ca2+ homeostasis by an antifungal protein from a filamentous ascomycete and provides important new insights into the mode of action of PAF.