Table_3_The Pheromone Module SteC-MkkB-MpkB-SteD-HamE Regulates Development, Stress Responses and Secondary Metabolism in Aspergillus fumigatus.DOCX

In order for eukaryotes to efficiently detect and respond to environmental stimuli, a myriad of protein signaling pathways are utilized. An example of highly conserved signaling pathways in eukaryotes are the mitogen-activated protein kinase (MAPK) pathways. In fungi, MAPK pathways have been shown to regulate a diverse array of biological processes, such as asexual and sexual development, stress responses and the production of secondary metabolites (SMs). In the model fungus Aspergillus nidulans, a MAPK pathway known as the pheromone module is utilized to regulate both development and SM production. This signaling cascade consists of the three kinases SteC, MkkB, and MpkB, as well as the SteD adaptor protein and the HamE scaffold. In this study, homologs of each of these proteins have been identified in the opportunistic human pathogen A. fumigatus. By performing epitope tagging and mass spectrometry experiments, we have shown that these proteins form a pentameric complex, similar to what is observed in A. nidulans. This complex has been shown to assemble in the cytoplasm and MpkB enters the nucleus, where it would presumably interact with various transcription factors. Pheromone module mutant strains exhibit drastic reductions in asexual sporulation, vegetative growth rate and production of SMs, such as gliotoxin. Mutants also display increased sensitivity to cell wall and oxidative stress agents. Overall, these data provide evidence of the existence of a conserved MAP kinase signaling pathway in Aspergillus species and suggest that this pathway is critical for the regulation of fungal development and secondary metabolism.

为使真核生物（eukaryotes）能够高效感知并响应环境刺激，生物体需利用大量蛋白质信号通路。真核生物中高度保守的信号通路之一为丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）通路。在真菌中，已有研究表明MAPK通路可调控多样的生物学过程，包括无性与有性发育、应激响应以及次级代谢产物（secondary metabolites，SMs）的合成。在模式真菌构巢曲霉（Aspergillus nidulans）中，一类被称为信息素模块的MAPK通路被用于调控发育与次级代谢产物合成。该信号级联反应包含三种激酶SteC、MkkB与MpkB，以及接头蛋白SteD与支架蛋白HamE。本研究在机会致病人类病原体烟曲霉（A. fumigatus）中鉴定出了上述所有蛋白的同源蛋白。通过表位标签（epitope tagging）与质谱（mass spectrometry）实验，我们证实这些蛋白可形成五聚体复合物，其组装模式与构巢曲霉中所观察到的一致。该复合物在细胞质中组装，而MpkB可进入细胞核，推测其会与各类转录因子相互作用。信息素模块突变菌株的无性产孢能力、营养生长速率以及胶霉毒素（gliotoxin）等次级代谢产物的产量均出现显著下降。同时，突变菌株对细胞壁应激与氧化应激试剂的敏感性有所升高。综上，本研究数据证实了曲霉属物种中存在保守的MAP激酶信号通路，并表明该通路对于调控真菌发育与次级代谢至关重要。