新生隐球菌粘附/基质蛋白Cfl1的分子功能研究

Cryptococcus neoformans is a ubiquitous human fungal pathogen, which claims about 600,000 deaths every year. This pathogen can undergo morphotype transition between the yeast form and the hyphal form. Such morphotype transition serves as a key biological mechanism ensuring its unusual success in coordinating natural adaptation and pathogenicity. Extracellular adhesion/matrix protein Cfl1 plays an important role in stimulating yeast-to-hypha transition through its bi-functionality. As morphological shifting occurs, some of the Cfl1 proteins attach the cell wall as a structurally novel adhesin while others, through shedding, are released into extracellular matrix as protein signal directing filamentation. However, it is enigmatic how the bi-functionality of Cfl1 is orchestrated and specialized during cryptococcal morphotype transition. Here, we propose to systematically explore the cryptococcal factors engaged in Cfl1’s bi-functionality using a high throughput genetic screen based on a fluorescence reporter system and the visualization of colony morphology. In addition, Cfl1’s orthologs are widely spread in the phylum Basidiomycota, likely representing a new family of fungal adhesin. Hence our study will also provide a mechanistic paradigm for this adhesin family.

新生隐球菌是重要的人类环境致病真菌，酵母-菌丝转换作为关键的适应性行为协调了该菌的生态性适应和致病性。细胞粘附/基质蛋白Cfl1对于该形态转换十分重要，并作为一个双功能分子，介导细胞粘附和细胞交流。由于Cfl1并不包括任何已知的功能结构域，暗示其可能采用独特的方式行使功能。本项目发展了一个依赖于荧光报告基因和菌落表型观察的多层次遗传筛选平台，并结合生物化学和细胞生物学等手段，揭示参与Cfl1剪切、细胞表面锚定及细胞粘附的隐球菌基因，旨在阐明Cfl1细胞粘附活性和剪切的关键分子机制，为开发隐球菌疫苗和发现新的药物靶点提供理论依据；此外，Cfl1的同源蛋白在担子菌门中广泛存在，本项目也将为该类蛋白的研究提供模板。

新生隐球菌是重要的人类病原真菌，每年导致数十万人死亡。有性生殖作为一个关键的毒力进化策略加速了高毒/药物抗性菌株在自然界中的涌现。Cfl1可作为一个新颖的胞外基质蛋白信号分子调控了有性生殖过程，但其作用机制以及上下游基因调控环路所知甚少。我们鉴定发现Cfl1包含了一个新颖的结构域SIGC，该结构域富含半胱氨酸，其缺失可导致其胞外调控活性的丧失，对其细胞粘附活性并未造成显著影响，而其粘附活性与荚膜的产生能力高度相关。此外，我们鉴定了Cfl1相关的胞外调控环路，发现一个群感效应因子Qsp1能够作为上游信号分子激活Cfl1编码基因的表达。通过建立多层次的遗传筛选策略，我们鉴定了响应Qsp1的关键转录因子Cqs2，发现Cqs2编码基因的缺失可阻断隐球菌细胞对Qsp1信号的响应，从而抑制了CFL1基因的激活，进而阻遏了有性生殖的发生。综上所述，我们的研究揭示了胞外基质蛋白信号分子Cfl1的信号途径及其分子功能机制，为微生物类似蛋白信号分子的研究提供了很好的范本，也为隐球菌致病机制的揭示提供了更多的参考。