PDF(Pubmed)
Abstract：
In pathogenic fungi, calcium-calmodulin-dependent serine-threonine-specific phosphatase calcineurin is involved in morphogenesis and virulence. Therefore, calcineurin and its tightly related protein complexes are attractive antifungal drug targets. However, there is limited knowledge available on the relationship between in vivo Ca2+-binding sites of calmodulin (CaM) and its functions in regulating stress responses, morphogenesis, and pathogenesis. In the current study, we demonstrated that calmodulin is required for hyphal growth, conidiation, and virulence in the human fungal pathogen, Aspergillus fumigatus. Site-directed mutations of calmodulin revealed that a single Ca2+-binding site mutation had no significant effect on A. fumigatus hyphal development, but multiple Ca2+-binding site mutations exhibited synergistic effects, especially when cultured at 42 °C, indicating that calmodulin function in response to temperature stress depends on its Ca2+-binding sites. Western blotting implied that mutations in Ca2+-binding sites caused highly degraded calmodulin fragments, suggesting that the loss of Ca2+-binding sites results in reduced protein stability. Moreover, normal intracellular calcium homeostasis and the nuclear translocation of the transcriptional factor CrzA are dependent on Ca2+-binding sites of AfCaM, demonstrating that Ca2+-binding sites of calmodulin are required for calcium signalling and its major transcription factor CrzA. Importantly, in situ mutations for four Ca2+-binding sites of calmodulin resulted in an almost complete loss of virulence in the Galleria mellonella wax moth model. This study shed more light on the functional characterization of putative calcium-binding sites of calmodulin in the morphogenesis and virulence of A. fumigatus, which enhances our understanding of calmodulin biological functions in cells of opportunistic fungal pathogens.
摘要：
在病原真菌中，钙-钙调蛋白依赖性丝氨酸-苏氨酸特异性磷酸酶钙调磷酸酶参与形态发生和毒力。因此,钙调神经磷酸酶及其紧密相关的蛋白质复合物是有吸引力的抗真菌药物靶标。然而,关于钙调蛋白（CaM）的体内Ca2结合位点与其调节应激反应的功能之间的关系的知识有限，形态发生,和发病机制。在目前的研究中,我们证明了菌丝生长需要钙调蛋白，分生孢子，和人类真菌病原体的毒力，烟曲霉。钙调蛋白的定点突变表明，单个Ca2+结合位点突变对烟曲霉菌丝发育没有显著影响，但多个Ca2+结合位点突变表现出协同作用，尤其是在42℃培养时，表明钙调蛋白响应温度胁迫的功能取决于其Ca2结合位点。蛋白质印迹表明，Ca2+结合位点的突变导致钙调蛋白片段高度降解，这表明Ca2+结合位点的丢失导致蛋白质稳定性降低。此外，正常的细胞内钙稳态和转录因子CrzA的核易位取决于AfCaM的Ca2结合位点，证明钙调蛋白的Ca2结合位点是钙信号及其主要转录因子CrzA所必需的。重要的是,钙调蛋白的四个Ca2结合位点的原位突变导致Galleriamelonella蜡蛾模型中毒力几乎完全丧失。这项研究进一步阐明了钙调蛋白在烟曲霉的形态发生和毒力中推定的钙结合位点的功能特征，这增强了我们对机会性真菌病原体细胞中钙调蛋白生物学功能的理解。
