A systems biology approach to dissect the function of the antifungal protein AnAFPfor Aspergillus niger

该提案的总体目标是获得AnAFP对黑曲霉内源性功能的机制见解。关于丝状真菌中cys稳定的抗菌肽（AFPs）的生物学功能几乎一无所知。了解真菌afp的遗传、分子和进化基础对于了解其功能主要是防御还是与真菌生长发育有关具有重要意义。在我们之前的工作中，我们发现anafp的表达与自噬蛋白和自噬过程的表达相似，并且在营养菌丝的高度空泡化的区室中被选择性激活，该区室应该进行自噬以进行营养循环。鉴于AnAFP的膜活性潜力及其对黑曲霉菌微妙但致命的细胞壁应激活性，我们提出其基因表达的严格时空控制使AnAFP在营养饥饿（即自噬过程）期间履行重要的细胞内功能，从而有助于黑曲霉菌在这种危及生命的情况下存活。我们的工作假设也受到其他出版物的鼓舞，这些出版物表明，空心芽孢杆菌的无性孢子形成伴随着自溶和凋亡过程。这项提议的关键科学目标是验证这一假设。为了实现总体目标，我们将利用我们实验室建立的最先进的工具，包括系统生物学工具，合成生物学工具，用于生理表征的受控生物反应器培养，共聚焦荧光显微镜研究AnAFP定位和自噬过程中gfp标记的细胞器的命运，以及透射电子显微镜研究AFP暴露后黑曲霉的细胞超微结构。使用这种嵌入在四个工作包（WP）中的整体方法，将详细研究AnAFP对黑曲霉的作用，以实现以下四个目标：-揭示AnAFP对细胞存活和/或死亡的重要性-揭示AnAFP的细胞靶点并鉴定AnAFP相互作用蛋白-完善AnAFP共表达基因网络最终目标是了解AnAFP相关过程背后的机制，这些过程平衡细胞功能和细胞功能障碍，细胞存活和细胞死亡。了解确保在生存与死亡之间走钢丝的途径和过程，一方面将最终确定黑曲霉的“阿喀琉斯之踵”，从而为有针对性地干预这种真菌或来自曲霉属的相关致病真菌创造可能。另一方面，可以推断出在碳饥饿期间改善黑曲霉生长的新线索，这将提高该细胞工厂在工业发酵期间的生产率。

The overall objective of the proposal is to gain mechanistic insights into the endogenous function of AnAFP for A. niger. Almost nothing is known about the biological function of Cys-stabilized antimicrobial peptides (AFPs) from filamentous fungi. Understanding the genetic, molecular and evolutionary basis of AFPs from fungi is important for understanding whether their function is mainly defensive or associated with fungal growth and development. In our previous work we uncovered that anafp expression parallels with the expression of autophagic proteins and processes and is selectively activated in highly vacuolated compartments of the vegetative mycelium, supposed to undergo autophagy for nutrient recycling. In view of the membrane-active potential of AnAFP and its subtle but lethal cell-wall stress provoking activity on A. niger, we propose that the tight spatial and temporal control of its gene expression enables AnAFP to fulfil an important intracellular function during nutrient starvation, i.e. during autophagic processes, thus contributing to the survival of A. niger during this life-threatening condition. Our working hypothesis is also encouraged by publications from others who demonstrated that asexual sporulation of A. nidulans is accompanied by autolytic and apoptotic processes. The key scientific aim of this proposal is to test this hypothesis. To achieve the Overall objective, we will exploit state-of-the-art Tools which have been established in our lab for A. niger including systems biology tools, synthetic biology tools, controlled bioreactor cultivations for physiological characterizations, confocal fluorescence microscopy to study AnAFP localisation and the fate of GFP-labelled organelles during autophagy as well as transmission electron microscopy to study the cellular ultrastructure of A. niger upon AFP exposure. Using such a holistic approach, which is embedded in four work packages (WP), the role of AnAFP for A. niger will be studied in detail to achieve the following four objectives: -Gain mechanistic insights into the timely and quantitatively controlled expression of AnAFP -Unravel the importance of AnAFP for cell survival and / or cell death -Unravel cellular targets of AnAFP and identify AnAFP interacting proteins-Refine the AnAFP co-expression gene network The ultimate goal is in understanding the mechanisms behind AnAFP-related processes that balance cell function and cell dysfunction, cell survival and cell death. Understanding the pathways and processes ensuring the tightrope walk between survival and death will on the one Hand ultimately identify an 'Achilles heel' of A. niger thus creating the possibility for targeted intervention of this or related pathogenic fungi from the genus Aspergillus. On the other hand, new leads for improved growth of A. niger during carbon starvation can be deduced which will increase the productivity of this cell factory during industrial fermentation.