Synergistic effects of putative Ca²⁺-binding sites of calmodulin in fungal development, temperature stress and virulence of <i>Aspergillus fumigatus</i>

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 <i>in vivo</i> 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, <i>Aspergillus fumigatus</i>. Site-directed mutations of calmodulin revealed that a single Ca²⁺-binding site mutation had no significant effect on <i>A. fumigatus</i> 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 <i>Af</i>CaM, demonstrating that Ca²⁺-binding sites of calmodulin are required for calcium signalling and its major transcription factor CrzA. Importantly, <i>in situ</i> mutations for four Ca²⁺-binding sites of calmodulin resulted in an almost complete loss of virulence in the <i>Galleria mellonella</i> 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 <i>A. fumigatus</i>, which enhances our understanding of calmodulin biological functions in cells of opportunistic fungal pathogens.在致病真菌中，钙调神经磷酸酶（calcium-calmodulin-dependent serine-threonine-specific phosphatase calcineurin）参与形态发生与致病过程。因此，钙调神经磷酸酶及其紧密关联的蛋白复合物成为极具潜力的抗真菌药物靶点。然而，目前关于钙调蛋白（calmodulin, CaM）的体内（in vivo）钙结合位点与其在调控应激反应、形态发生及致病机制中的功能之间的关联，相关认知仍十分有限。本研究证实，在人类致病真菌烟曲霉（Aspergillus fumigatus）中，钙调蛋白对于菌丝生长、产孢及致病力均不可或缺。对钙调蛋白进行定点突变后发现，单个钙结合位点突变对烟曲霉的菌丝发育无显著影响，但多个钙结合位点突变则表现出协同效应，尤其在42℃培养时更为明显，这表明钙调蛋白响应温度应激的功能依赖于其钙结合位点。蛋白质免疫印迹（Western blotting）实验结果显示，钙结合位点突变会产生大量降解的钙调蛋白片段，提示钙结合位点的缺失会降低蛋白稳定性。此外，正常的细胞内钙稳态以及转录因子CrzA的核转位均依赖于烟曲霉钙调蛋白（AfCaM）的钙结合位点，这表明钙调蛋白的钙结合位点对于钙信号通路及其核心转录因子CrzA的功能至关重要。尤为关键的是，对钙调蛋白的四个钙结合位点进行原位（in situ）突变后，在大蜡螟（Galleria mellonella）幼虫感染模型中，菌株的致病力几乎完全丧失。本研究进一步阐明了烟曲霉中钙调蛋白潜在钙结合位点在形态发生与致病力中的功能特征，加深了我们对机会致病性真菌细胞内钙调蛋白生物学功能的理解。