Ram Arthur F J, Klis Frans M
Institute of Biology Leiden, Leiden University, Molecular Microbiology, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands.
Nat Protoc. 2006;1(5):2253-6. doi: 10.1038/nprot.2006.397.
The fungal cell wall is an essential organelle and represents a considerable metabolic investment. Its macromolecular composition, molecular organization and thickness can vary greatly depending on environmental conditions. Its construction is also tightly controlled in space and time. Many genes are therefore involved in building the fungal cell wall. Here we present a simple approach for detecting these genes. The method is based on the observation that cell wall mutants are generally more sensitive to two related anionic dyes, Calcofluor white (CFW) and Congo red (CR), both of which interfere with the construction and stress response of the cell wall. CFW-based and CR-based susceptibility assays identify cell wall mutants not only in ascomycetous yeasts (such as Saccharomyces cerevisiae and Candida albicans) but also in mycelial ascomycetes (such as Aspergillus fumigatus and Aspergillus niger), basidiomycetous species (Cryptococcus neoformans) and probably also zygomycetous fungi. The protocol can be completed in 4-6 h (excluding the incubation time required for fungal growth).
真菌细胞壁是一种重要的细胞器，代表着相当大的代谢投入。其大分子组成、分子结构和厚度会因环境条件的不同而有很大差异。其构建在空间和时间上也受到严格控制。因此，许多基因参与了真菌细胞壁的构建。在此，我们提出一种检测这些基因的简单方法。该方法基于这样的观察结果：细胞壁突变体通常对两种相关的阴离子染料——荧光增白剂（CFW）和刚果红（CR）更为敏感，这两种染料都会干扰细胞壁的构建和应激反应。基于CFW和基于CR的敏感性测定不仅可以鉴定子囊菌酵母（如酿酒酵母和白色念珠菌）中的细胞壁突变体，还可以鉴定丝状子囊菌（如烟曲霉和黑曲霉）、担子菌（新型隐球菌）中的细胞壁突变体，可能还包括接合菌中的真菌。该实验方案可在4 - 6小时内完成（不包括真菌生长所需的培养时间）。
