Calcium signaling and cytoskeletal dynamics during polarized hyphal growth and infection by the human fungal pathogen Aspergillus fumigatus

世界范围内最常见的侵袭性霉菌感染是由曲霉引起的，90%以上的病例是由人类病原体烟曲霉引起的。在人类宿主肺部感染期间，烟曲霉通过信号转导级联反应直接环境，这是由受体、非蛋白信使、酶和转录因子网络传递的。Ca2+作为第二信使，在所有真核细胞的生长发育过程中起着重要的信号传导作用，是一种进化保守的、高度通用的细胞内信号分子。然而，令人惊讶的是，我们对真菌发病过程中的Ca2+信号知之甚少。当前提案的总体目标将是分析Ca2+信号在调节极化菌丝生长和细胞骨架动力学中的作用，这是烟曲霉致病性和成功感染所必需的。一个目标将是确定在极化菌丝生长和哺乳动物上皮细胞感染期间在Ca2+信号传导中起关键作用的蛋白质。各种Ca2+通道、-泵和-反转运蛋白将被定位和分析，以确定它们在生长菌丝尖端产生[Ca2+]c-尖峰中所起的作用。特异性的[Ca2+]c信号是否与时空发育过程有关，如极化生长和上皮细胞的感知和感染，将被研究。观察在上皮细胞层表面生长过程中菌丝中的[Ca2+]c，细胞骨架和运动蛋白动力学将进一步了解它们在感染过程中的作用。这些目标将解决使用尖端的实验技术，包括：突变和过表达分析；先进的活细胞成像使用共聚焦和TIRF显微镜；Ca2 +成像;活细胞中参与Ca2+信号、细胞骨架和分子马达的关键蛋白的定位和定量；哺乳动物上皮细胞感染试验。该研究的总体目标是对烟曲霉在极化生长和发病过程中Ca2+信号的复杂作用获得新的见解。由于真菌中的Ca2+信号/体内平衡机制与动物和植物中的Ca2+信号/体内平衡机制存在显著差异，因此该机制的各种成分有可能在未来为抗真菌药物提供新的靶点。

The most common invasive mould infection worldwide is caused by Aspergillus species, and over 90% of all cases are caused by the human pathogen Aspergillus fumigatus. During infection of the human host lung, A. fumigatus responds to the immediate environment via signal transduction cascades, which are conveyed by a network of receptors, non-protein messengers, enzymes and transcription factors. Ca2+, as a second messenger, plays a fundamental role in signaling during the growth and development of all eukaryotic cells and is an evolutionarily conserved, highly versatile intracellular signal molecular. However, surprisingly little is known about Ca2+-signalling during fungal pathogenesis.The overall objective of the current proposal will be to analyse the roles of Ca2+-signaling in regulating polarized hyphal growth and the cytoskeletal dynamics that are required for pathogenicity and successful infection by A. fumigatus. One aim will be to identify proteins that play key roles in Ca2+-signaling during polarized hyphal growth and infection of mammalian epithelial cells. Various Ca2+-channels, -pumps and -antiporters will be localized and analysed to determine the role they play in generating [Ca2+]c-spikes in growing hyphal tips. Whether specific [Ca2+]c-spiking signatures are associated with spatio-temporal developmental processes such as polarized growth and the sensing and infection of epithelial cells, will be investigated. Observing [Ca2+]c, cytoskeletal and motor protein dynamics in hyphae during growth over the surface of an epithelial cell layer will give further insights into their roles during the infection process. These aims will be addressed using cutting edge experimental techniques including: mutant and overexpression analyses; advanced live-cell imaging using confocal and TIRF microscopy; Ca2+-imaging; localization and quantification of key proteins involved in Ca2+-signaling, the cytoskeleton and molecular motors in living cells; and infection assays with mammalian epithelial cells.The overall objective of the proposed study will be to gain fundamentally new insights into the complex roles of Ca2+-signaling in A. fumigatus during polarized growth and pathogenesis. Because of significant differences between the Ca2+-signaling/ homeostatic machinery in fungi compared with that in animals and plants, various components of this machinery have potential in providing novel targets for antifungal drugs in the future.