丝切蛋白Cofilin对烟曲霉细胞壁完整性信号通路和致病性的影响及调控机制研究

Invasive aspergillosis is difficult to be treated and has a high mortality. The primary pathogen of this disease is Aspergillus fumigatus. Its pathogenic mechanism is rather complicated and closely associated with its viability in diverse niches, stress resistance and host immune response, in which the cell wall integrity (CWI) signaling in A. fumigatus plays a predominant role to sense a wide range of cell wall perturbing conditions and orchestrate a respective response. As an actin depolymerization factor, cofilin is the key regulator on several physiological processes which accompanies cellular cytoskeleton rearrangement in mammalian cells. Intriguingly, our previous study showed that with distant genetic relationship with human cofilin, A. fumigatus cofilin is indispensable for viability and affects obviously the polysaccharide composition of cell wall and also the phosphorylation of the key gene, MpkA in CWI signalling. Based on these findings, we are eager to describe the expression and distribution of cofilin under stresses, investigate the influence of cofilin on stress resistance and metabolism of major polysaccharides of A. fumigatus, elucidate the molecular regulation mechanism on cell wall integrity signaling and analyze the effect of cofilin on A. fumigatus pathogenicity and host innate immune response in cells and animals. This study will demonstrate a novel regulation mechanism for fungal cell wall integrity signaling by close relationship with actin cytoskeleton, which would largely enrich our knowledge on molecular regulation of fungal cell wall integrity and its role in pathogenicity and could also offer important scientific evidences for discovering some potential targets of antifungal drugs.

侵袭性曲霉病治疗困难、致死率高。该病主要病原体烟曲霉的致病机制与其有效抵抗外界应激的能力有关，而细胞壁完整性（CWI）信号通路是这种应激信号调控的重要机制之一。Cofilin是一种肌动蛋白解聚因子，在哺乳动物细胞中调控许多涉及肌动蛋白骨架重排的重要生理过程，但其在烟曲霉中的功能尚不清楚。我们发现烟曲霉Cofilin是必需基因且与人同源蛋白亲缘关系较远，其表达改变可影响烟曲霉CWI通路中重要因子MpkA磷酸化和胞壁葡萄糖残基含量，这提示Cofilin可能参与调控CWI信号，但具体机制亟待阐明。基于此，我们渴望深入研究Cofilin影响烟曲霉细胞壁完整性应激、胞壁主要多糖组成的规律；解析其调控CWI信号通路的分子机制并探讨其对烟曲霉致病力的可能影响。项目将阐明肌动蛋白骨架蛋白调控烟曲霉CWI的新机制，有力丰富人们对该通路及其影响致病力的认识与理解，并为寻找可能的抗真菌药物靶点提供重要科学依据。

Cofilin是一种肌动蛋白解聚因子，在哺乳动物细胞中调控许多涉及肌动蛋白骨架重排的重要生理过程，但其在烟曲霉中的功能尚不清楚。我们发现烟曲霉Cofilin是必需基因且与人同源蛋白亲缘关系较远，其表达改变可影响烟曲霉细胞壁完整性（CWI）通路中重要因子MpkA磷酸化和胞壁葡萄糖残基含量，这提示Cofilin可能参与调控CWI信号，但具体机制亟待阐明。基于此，我们深入研究了Cofilin影响烟曲霉细胞壁完整性应激、胞壁主要多糖组成的规律；解析了其调控CWI信号通路的分子机制，并探讨了其对烟曲霉致病力的可能影响。结果显示，烟曲霉 cofilin是烟曲霉生存必需蛋白，其完全缺失导致菌株无法存活，而低表达严重损害烟曲霉极性生长和毒力。Cofilin低表达显著增强烟曲霉对SDS、DTT、H2O2和碱性pH的应激敏感性，但对细胞壁干扰剂包括法尼醇（FOH）、荧光白（CFW）和刚果红（CR），酸性pH的应激敏感性无明显变化。而烟曲霉cofilin过表达，对H2O2的敏感性是下降的，对细胞壁干扰剂、SDS、DTT、酸性pH、中性pH和碱性pH的应激敏感性均无明显变化。pH 9.0刺激时，烟曲霉cofilin蛋白分布无异常变化；但H2O2刺激时，cofilin蛋白分布发生改变，有聚集现象，主要向胞壁和顶端聚集，与细胞核分布无明显联系。烟曲霉cofilin表达的改变不仅显著影响烟曲霉中参与氧化和碱性pH应激的基因表达，而且显著影响SakA蛋白的激活。Cofilin条件性敲除株粘附和侵染细胞能力减弱，cofilin过表达株粘附细胞能力减弱，而侵染能力增强，这可能与cofilin过表达株在细胞内存活能力增强有关。我们还发现烟曲霉cofilin表达量改变通过影响表面多糖β-1,3-glucan合成酶Fksp、几丁质Chitin合成酶家族基因表达水平改变细胞壁成分，进而影响肺上皮细胞应对烟曲霉刺激时炎性因子MCP-1、IL-8和TNF-α的表达。cofilin低表达激活了烟曲霉CWI信号，但细胞壁合成酶基因表达不依赖MpkA的激活。此外，cofilin蛋白处于失活状态时显著抑制菌株生长，处于持续激活状态时不影响菌落形态、光镜下的结构以及菌株的生长速率。