| Summary: | 碩士 === 中山醫學大學 === 口腔醫學研究所 === 94 === Candida albicans is an opportunistic fungal pathogen found in the normal gastrointestinal flora of most healthy humans. However, in immunocompromised patients, it often causes superficial and systemic infections. In its severity, death can occur. Despite the importance of C. albicans to human disease, comprehensive understanding the biology and pathogenesis of C. albicans remains largely illusive due to the diploid nature of the organism, together with its lack of a known sexual phase and unusual codon usage.
We have been interested in studying the event governing G1 to S phase transition of the cell cycle in C. albicans. Being essential for entering S phase and universally present in species currently examined, the Skp1-Cdc53-F-box protein SCFCDC4 has been sought to study. By using budding yeast Saccharomyces cerevisiae as a surrogate system, we have isolated a multicopy suppressor clone of budding yeast cdc4ts under restrictive temperature. It appeared that the clone could rescue several cdc4ts with different CDC4 mutations, suggesting that the isolated clone contains a C. albicans functional homolog of S. cerevisiae CDC4 (ScCDC4). The analysis of DNA sequence revealed that the gene encodes a protein with a F-box and eight repeats of WD-40, which is structurally homologous to ScCdc4 protein. Consequently, we concluded that this gene is C. albicans CDC4, named CaCDC4.
We are interested in examining the function of CaCDC4 directly in C. albicans. On the basis of its ability to replace ScCDC4, we assume that CaCDC4 is also an essential gene in C. albicans. We have therefore constructed a C. albicans strain with one CaCDC4 disrupted and the other under MET3 promoter control that is methionine/cycteine repressible. Surprisingly, under repressed condition, cells of this C. albicans strain were able to proliferate and to form colony with a filamentous morphology. Hence, contrast to ScCDC4 being essential for G1 to S phase transition, CaCDC4 appeared to be nonessential and required for negatively regulating the filamentous growth.
In the future, we will dissect the control mechanism of morphogenesis in C. albicans determined by CaCDC4. Firstly, epistasis of CaCDC4 with others genes or environmental cues for filaments will be established to determine the importance of the CaCDC4 as a negative regulator. Secondly, yeast two-hybrid will be use to identify CaCdc4 associated proteins, including members of SCF complex, regulators, and targets. Analysis of interaction between CaCdc4 and its associated proteins will reveal the role of CaCdc4 in the network of regulation in filamentous growth.
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