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

世界上最常见的侵袭性霉菌感染是由曲霉引起的，超过90%的病例是由人类病原体烟曲霉引起的。在人体肺部感染过程中，烟曲霉菌通过一系列的受体、非蛋白信使、酶和转录因子网络传递信号转导通路，从而对周围环境做出反应。钙离子作为第二信使，在真核细胞的生长发育过程中起着重要的信号转导作用，是一种进化上保守、用途广泛的胞内信号分子。然而，令人惊讶的是，人们对真菌致病过程中的钙信号转导知之甚少。本提案的总体目标将是分析钙信号在调节菌丝极化生长和细胞骨架动力学中的作用，这是致病和成功感染烟曲霉所必需的。其中一个目标是确定在哺乳动物上皮细胞的极化菌丝生长和感染过程中，在钙信号转导中起关键作用的蛋白质。不同的钙离子通道、泵和逆向转运蛋白将被定位和分析，以确定它们在生长的菌丝尖端产生[钙]c-峰所起的作用。具体的[Ca~(2+)]-c-峰信号是否与时空发育过程相关，如极化生长和上皮细胞的感知和感染，将被研究。观察菌丝在上皮细胞层表面生长过程中的[Ca~(2+)]c、细胞骨架和运动蛋白的动态变化，将进一步深入了解它们在感染过程中的作用。这些目标将通过尖端的实验技术来解决，包括：突变和过表达分析；使用共聚焦和TIRF显微镜的先进的活细胞成像；钙成像；参与钙信号的关键蛋白的定位和量化；活细胞中的细胞骨架和分子马达；以及哺乳动物上皮细胞的感染分析。拟议的研究的总体目标将是从根本上获得对钙信号在烟曲霉极化生长和发病过程中的复杂作用的新见解。由于真菌中的钙信号/稳态机制与动植物相比有很大的不同，这一机制的不同部分有可能为未来的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.