条件致病菌烟曲霉中铁平衡功能基因的筛选和分子调控机制研究

Iron is an essential element for most organisms, as it is a cofactor in key metabolic processes such as nucleotide biosynthesis or energy production. Although iron is the second most abundant metal on earth, its bioavailability is very low in an aerobic environment because iron is mostly present as insoluble ferric hydroxides. This task is particularly challenging for pathogenic microorganisms, since iron is tightly bound to host proteins such as transferrin or lactoferrin to maintain homeostasis and to restrict access by pathogens through by innate nutritional defense. Therefore, iron acquisition during infection of a human host is a challenge that must be surmounted by every successful pathogenic microorganisms. On the other hand, an excess of iron within cells can be deleterious on account of catalysis of cell-damaging reactive oxygen species (ROS) via Haber Weiss/Fenton reactions. Therefore, all organisms especially the pathogenic microorganisms have developed highly regulated strategies to regulate the iron homeostasis. It has been reported that the key regulators of iron homeostasis in pathogenic microorganisms also played important roles in virulence. However, our knowledge of the molecular regulation mechanisms of the iron homeostasis in pathogenic microorganisms is rather limited. Aspergillus fumigatus is the most common cause of airborne fungal infections worldwide. The most serious disease caused by A. fumigatus is invasive aspergillosis (IA) when susceptible individuals in especial immunosuppressed patients inhale conidia. To identify the iron homeostasis related genes in A. fumigatus, a random mutagenesis library constructed by Agrobacterium tumefaciens-mediated transformation (ATMT) was screened. The resulting mutant showed serious growth defects under iron-depleted conditions while almost identical phenotypes with wild type stain in iron-replete conditions. The target gene was then cloned by thermal asymmetric interlaced PCR and the bioinformatics analysis showed that it encoded a predicted Zn(2)Cys(6) DNA-binding protein with function uncharacterized in fungi. Due to its function in A. fumigatus iron homeostasis, the protein is referred to as IhrA (Iron Homeostasis Regulator A). The study will focus on the roles of IhrA on the iron homeostasis in A.fumigatus through analyzing the genetic functions of IhrA in the iron uptake and utilization, studying the regulation relationships between IhrA and the conserved transcription factors in fungal iron homeostasis, HapX and SreA. We will further screen and confirm the key candidate genes downstream of IhrA and the interaction proteins which formed complexes with IhrA in the regulation of iron homeostasis in A. fumigatus. Those results will deepen our understanding on the iron homeostasis regulatory mechanisms in pathogenic fungi and provide the theory bases to control the aspergillosis.

铁是生物体必需元素之一。由于人体内铁元素多以微生物不能直接利用的铁蛋白形式存在，所以微生物能够从宿主体内成功获取铁是其成为病原菌的关键。另一方面，胞内铁过量会对细胞产生毒害作用。因此，病原菌会进化出一套严格的铁平衡调控机制来维持其在宿主体内的生长。研究表明，病原菌中参与铁平衡的关键调节基因同时也与其致病力密切相关。然而，目前对铁平衡调控机制的认识还非常有限。本项目以引起侵袭性曲霉病的主要病原菌烟曲霉为材料，通过对T-DNA随机插入突变体库的筛选，获得了一个烟曲霉中铁平衡的关键调控因子IhrA。进一步的工作将围绕铁平衡的分子调控机理这一主题，研究IhrA在烟曲霉中对铁吸收和利用的功能，筛选和鉴定IhrA直接调控的铁平衡相关基因和与IhrA协同作用的蛋白，阐明IhrA在烟曲霉中铁平衡的分子调控机理。本项目的开展将会加深我们对病原菌铁平衡调控机制的认识，为防治曲霉感染提供新的思路。

铁是生物体必需元素之一。由于人体内铁元素多以微生物不能直接利用的铁蛋白形式存在，所以微生物能够从宿主体内成功获取铁是其成为病原菌的关键。另一方面，胞内铁过量会对细胞产生毒害作用。因此，病原菌会进化出一套严格的铁平衡调控机制来维持其在宿主体内的生长。本项目以引起侵袭性曲霉病的主要病原菌烟曲霉为材料，通过对T-DNA随机插入突变体库的筛选获得低铁敏感的突变菌株，通过TAIL-PCR等技术成功克隆了铁平衡调控关键基因ihrA。IhrA为一个含有锌指结构（Zn2Cys6）的转录因子，irhA的缺失会显著影响与铁载体(TAFC)合成、转运相关基因的转录以及铁载体的合成，从而影响烟曲霉在低铁环境下的生长，并降低烟曲霉的致病力。在irhA缺失菌的基础上敲除烟曲霉中调节铁平衡的转录因子hapX会加剧irhA缺失菌株低铁敏感的表型。在irhA 缺失菌上敲除负责铁离子吸收的转录因子sreA则会恢复irhA对低铁敏感表型，说明irhA与烟曲霉保守的调节铁离子平衡的转录因子hapX和sreA存在遗传调控关系。我们进一步通过染色质免疫共沉淀-测序技术（CHIP-Seq）结合凝胶阻滞实验(electrophoretic mobility shift assays, EMSA)发现irhA能够直接结合在hapX启动子保守区域模块(CCGN4CGG)，直接调控hapX的表达。除了hapX外，IrhA直接调控的靶基因还包括铁载体合成、转运相关基因(sidC, sidI, sidA, mirB和sitT)和编码位于线粒体的铁离子转运通道基因mrsA。鉴于mrsA在烟曲霉中的功能未知，我们对其进行了研究。mrsA缺失会导致sreA的转录明显下调，而受SreA抑制的和铁离子吸收相关的基因如sidA，mirB，ftrA以及fetC的转录明显上调，从而造成胞内铁离子调控发生紊乱。此外，通过对T-DNA随机插入突变体库的筛选发现位于线粒体上负责转运焦磷酸硫氨素的通道蛋白TpcA也参与胞内铁离子的平衡，可能是通过影响线粒体能量代谢进而影响烟曲霉的低铁适应能力和致病力。本项目研究加深了我们对病原真菌铁平衡调控机制的认识，为防治曲霉感染提供了新靶点和新思路。