RESUMO
Candida albicans is an important human fungal pathogen but its study has been hampered for being a natural diploid that lacks a complete sexual cycle. Gene knock-out and essential gene repression are used to study gene function in C. albicans. To effectively study essential genes in wild-type C. albicans, we took advantage of the compatible effects of the antibiotics hygromycin B and nourseothricin, the recyclable CaSAT1-flipper and the tetracycline-repressible (Tet-off) system. To allow deleting two alleles simultaneously, we created a cassette with a C. albicans HygB resistance gene (CaHygB) flanked with the FLP recombinase target sites that can be operated alongside the CaSAT1-flipper. Additionally, to enable conditionally switching off essential genes, we created a CaHygB-based Tet-off cassette that consisted of the CaTDH3 promoter, which is used for the constitutive expression of the tetracycline-regulated transactivator and a tetracycline response operator. To validate the new systems, all strains were constructed based on the wild-type strain and selected by the two dominant selectable markers, CaHygB and CaSAT1. The C. albicans general transcriptional activator CaGCN4 and its negative regulator CaPCL5 genes were targeted for gene deletion, and the essential cyclin-dependent kinase CaPHO85 gene was placed under the Tet-off system. Cagcn4, Capcl5, the conditional Tet-off CaPHO85 mutants, and mutants bearing two out of the three mutations were generated. By subjecting the mutants to various stress conditions, the functional relationship of the genes was revealed. This new system can efficiently delete genes and conditionally switch off essential genes in wild-type C. albicans to assess functional interaction between genes.
