ABSTRACT

Cryptococcus neoformans is a pathogenic fungus which causes millions of deaths and infections, especially threatening immunocompromised individuals. During the development of new drugs, the ubiquitination has been found to play an important role in the regulation of the virulence and cell cycle of this fungus. Based on this mechanism, ubiquitination-related mutant strains exhibiting cell cycle arrest have been established for drug development for the fungus. However, flow cytometry detection of the cell cycle in fungi is generally difficult because the thick cell wall and capsule of fungi generally contribute to a nonspecific signal of cytometry. In this study, an improved method, derived from Saccharomyces cerevisiae assays, is developed to specifically stain C. neoformans, in whose cell cycle the G1 and G2 peaks are separated enough to be allowed for cell cycle analysis. As a result, the improved method facilitates the detection of the alterations in the cell cycle of C. neoformans with a mutation that results in cell cycle arrest, which distinctly delays the cell division of C. neoformans. Thus, the improved method reported here provides detailed technical information regarding assays on C. neoformans and, more importantly, offers a solution for assessing the cell cycle in other fungi in the future.

Abbreviation: PI: propidium iodide

 抽象的

新型隐球菌是一种致病真菌，可导致数百万人死亡和感染，尤其威胁免疫功能低下的个体。在新药开发过程中，人们发现泛素化在调节该真菌的毒力和细胞周期中发挥着重要作用。基于这种机制，已经建立了表现出细胞周期停滞的泛素化相关突变菌株，用于该真菌的药物开发。然而，真菌细胞周期的流式细胞术检测通常很困难，因为真菌厚的细胞壁和荚膜通常会导致细胞计数的非特异性信号。在这项研究中，开发了一种源自酿酒酵母测定的改进方法，用于特异性染色新型隐球菌，在其细胞周期中，G1 和 G2 峰分离得足以进行细胞周期分析。因此，改进的方法有助于检测具有导致细胞周期停滞的突变的新型隐球菌细胞周期的改变，这明显延迟了新型隐球菌的细胞分裂。因此，本文报道的改进方法提供了有关新型隐球菌测定的详细技术信息，更重要的是，为将来评估其他真菌的细胞周期提供了解决方案。

缩写： PI：碘化丙啶