Development of tetracycline regulatable expression systems to facilitate the study of functional interaction between genes in Candida albicans

Autor: Wei-Chung Lai, 賴威仲
Jazyk: en_US
Rok vydání: 2015
Druh dokumentu: 學位論文 ; thesis
Popis: 104
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 two alleles to be deleted simultaneously, we created a cassette carrying the C. albicans HygB resistance gene (CaHygB) flanked with FLP recombinase target sites operated by a CaSAT1-flipper. Additionally, to enable conditionally switching off essential genes, we created a CaHygB-based Tet-off cassette that consisted of a tetracycline response operator and a CaTDH3 promoter, which is used for the constitutive expression of the tetracycline-regulated transactivator. 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. C. albicans genes of general transcriptional activator CaGCN4 and its negative regulator CaPCL5 were targeted for gene deletion, and the essential cyclin-dependent kinase CaPHO85 gene was placed under the control of 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. Besides gene inactivation, the tools that enable gene overexpression are an opposite way to determine the role of a gene. The tetracycline-inducible (Tet-on) vector of pTET25M was incorporated with the mini Ura-blaster carrying the recyclable CaURA3 marker and the multiple cloning sites flanked the GFP coding sequence for cloning. Through doxycycline-induced expression of GFP as a reporter, a dose- and time-dependent regulation of the pTET25M-based system was validated. Application of pTET25M derivatives confirmed that (1) the Tet-on with 6xHis-tagged CaCdc10p and GFP-tagged septin made proteins detectable and their localization visually tractable and (2) the FLAG-tagged assorted domains of CaCdc4p under Tet-on control together with the full-length of CaCDC4 repressed condition revealed the involvement of the domains in morphogenesis. The bimolecular fluorescence complementation (BiFC) is an approach for visualization of protein-protein interaction. To apply BiFC approach in Candida albicans, the Tet-on-based vectors pWTN1 and pWTN2 were created. Both vectors carrying a hygromycin B resistant marker (CaHygB) were found to be compatible with the original Tet-on vector pNIM1 carrying a nourseothricin resistant marker (CaSAT1). By using GFP and yEmRFP (mCherry) as reporters, the two complementary Tet-on systems appeared to (1) operate dose-dependently and synergistically with doxycycline in both yeast and filamentous form of C. albicans and (2) work functionally as fusion proteins as demonstrated by the residing of CaCdc11p-GFP and RFP-CaCdc10p fusions at the ring of septum of C. albicans. Importantly, the observation that the N-terminal (amino acids 1-159) and C-terminal (amino acids 160-237) yEmRFP fused with CaCdc42p (CaCdc42p-N) and CaRdi1p (C-CaRdi1p), whose interaction is known to require for polarized growth, were reconstituted as a fluorescent complex verified the applicability of the BiFC. Establishment of the Tet-on based BiFC system in C. albicans would facilitate functional study and high-throughput screening of protein-protein interaction in C. albicans.
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