不产孢不产黑烟曲霉特殊表型发生的分子机制研究

Invasive Aspergillus fumigatus infection is a disease with high mobidity and mortality rates. The abnormalities in sporulation and pigmentation can significantly alter the pathogenicity of A. fumigatus, so the mechanisms of asexual conidiation and pigment biosynthesis have drawn increasing attention. Our team recently isolated one nonsporulated white A. fumigatus called A1j (CBS126847). The infected patient was clinically cured and has no relapse for six years. Preliminary studies have shown that A1j exhibited dramatically reduced growth rates and virulence. Moreover, the genes related to conidiation and pigmentation were generally down-regulated in A1j compared to the standard strain. However, no mutations were found within those known key regulators of asexual conidiation. Thus, the molecular mechanisms of the unique nonsporulated and nonpigmented phenotype in A.fumigatus need to be clarified urgently. This project will focus on the genes which have significantly different expression levels and mutations compared to the standard strain. Among these genes, we have selected a series of genes through bioinformatics analysis. We will first analyze the roles of these genes in conidiation, pigmentation and virulence via knockout studies. Then we intend to explore the specific mechanisms of these new genes in conidiation and pigmentation based on the clues given by proteomics. Meanwhile, more clinically isolated A.fumigatus strains will be collected. We will explore the relationship between the functioning genes and the capabilities of conidiation, pigmentation and infection in those strains. This study is expected to discover new genes related to conidiation and pigmentation, which may work as diagnostic and prognostic biomarkers of A.fumigatus infections. We anticipate that this work will provide new drug targets for preventing and managing invasive aspergillosis from the source.

侵袭性烟曲霉感染临床发病率和死亡率高，而产孢产黑异常可明显改变烟曲霉致病力，其产孢产黑机制倍受关注。本项目组临床分离得完全不产孢的白色烟曲霉A1j（CBS126847），被感染患者临床治愈且随访6年未复发。初步研究显示，A1j生长缓慢，毒力下降，产孢产黑相关基因表达普遍下调，但已知的产孢关键基因均并无突变，其不产孢不产黑的分子机制亟待研究。本项目拟从不产孢不产黑烟曲霉A1j与标准株相比表达差异显著、存在突变、经生物信息学筛选所得的一系列基因入手，利用基因敲除技术分析突变基因在产孢、产黑、毒力等方面的功能，并通过蛋白质组学等方法探索功能基因导致产孢产黑缺陷的具体分子机制，同时进一步收集临床烟曲霉菌株，研究功能基因与烟曲霉产孢产黑能力及致病力的相关性。本研究有望发现新的产孢产黑相关基因，并可能作为烟曲霉感染新的临床诊断和预后评估的标志物，为从源头上防治侵袭性烟曲霉感染提供新的药物靶点。

侵袭性烟曲霉感染是一种高发病率和高死亡率的疾病。孢子形成和色素沉着的异常可以显着改变烟曲霉的致病性，因此无性分生孢子和色素生物合成的机制越来越受到关注。在米曲霉中，一种新的预测 bHLH 蛋白编码基因ecdR在无性发育中发挥作用。在此，我们通过测试 EcdR 是否影响该真菌的无性发育、黑色素合成和毒力调节来确定其在烟曲霉中的作用。为了探究ecdR基因在烟曲霉中的作用，我们通过基因敲除构建了ecdR缺陷型菌株，并选择RNA-seq和qRT-PCR等方法及表型分析检验该基因是否与产孢产黑相关。通过平板试验比较了ecdR缺陷菌株和野生型参照菌株对抗真菌药物及细胞壁破坏剂等的敏感性，并且利用免疫抑制小鼠模型测试了其毒力差异。结果发现：ecdR缺失突变体生长缓慢，在YAG琼脂上出现白色菌落。此外，ecdR缺失突变体表现出明显的孢子形成缺陷。与野生型相比，ecdR的缺失导致产孢中央调节途径brlA 、abaA和wetA 中基因的表达减少。qRT-PCR分析显示，在YAG琼脂上诱导无性发育8 h后，ΔecdR中pksP、arp1、arp2、abr1和abr2基因的表达显著低于WT。在渗透压条件下ecdR突变株恢复了少量色素沉着。ΔecdR 对不同浓度的 ITC 或 VRC 的敏感性明显低于 WT。但是对于 AMB，两者的敏感性没有显着差异。ΔecdR的唑类耐药可能与cdr1B的表达显着上调有关。在免疫功能低下的小鼠模型中，所有（100%）感染生理盐水的小鼠都存活到试验结束，但感染 WT 的小鼠中有 90% 在感染后的前 5 天死亡。与相同条件下ΔecdR 的 80% 存活率相比，差异显著。病灶呈多灶性肉芽肿伴坏死中心。与感染野生型烟曲霉的小鼠相比，感染ΔecdR的小鼠产生的真菌损伤更少且更小。结果表明ΔecdR的毒性低于野生型烟曲霉。.我们已经证明EcdR是正常无性发育所必需的，并且它调节中枢发育信号通路brlA 、abaA和wetA中的表达。此外，EcdR 在黑色素生成中起着至关重要的作用。我们还证实EcdR对致病性有负面影响。未来的研究将集中于阐明这种调节剂的强制过度表达是否对毒力和有益次级代谢物的产生有影响。其他研究还将提供进一步的见解，以确定其他与分生孢子调控有关的依赖于ecdR的基因。