- 學位論文
白色念珠菌CDC4基因是菌絲生長的負調節者
Candida albicans CDC4 is a negative regulator of filamentous growth.
摘要
白色念珠菌 (Candida albicans) 是一種真菌病原菌，主要分布在腸胃道和泌尿道的黏膜表皮上，是正常的共生菌種，然而，在免疫功能不足或缺損的病人身上卻常會造成皮膚表面跟全身性的疾病，甚至於會有致死的現象，但儘管它對於人類疾病如此重要，到目前為止卻未能透徹了解，原因在於它本身特殊的生物特性，像它是屬於雙倍同源染色體，而且它並沒有一個完整的有性生殖世代交替，加上它的異常的密碼子選擇，這使得我們要對於白色念珠菌進行的遺傳操作上遇到許多阻礙，也大大的限制了它致病能力機制的研究。 由於細胞週期從G1進入S時期在出芽酵母菌(Saccharomyces cerevisiae)是細胞分化的關鍵時刻，無論是DNA的合成、紡錘極體的複製以及芽胞的形成都在此時決定，所以我們認為此時期對於白色念珠菌也極為重要。為了探討白色念珠菌細胞週期的G1到S轉變時期，我們以SCFCDC4 (Skp1-Cdc53-F-box protein)複合體中的CDC4為研究對象，因為它是出芽酵母菌細胞進入S時期所必須的且普遍存在於各類真核生物之中。於是，我們利用出芽酵母菌當作我們一個替代的系統，以cdc4 ts這個對溫度敏感的品系加以篩選，在限制溫度下挑選出可以挽救喪失Cdc4功能的白色念珠菌的遺傳組 DNA片段。而篩選出來的選殖片段能挽救帶有不同cdc4對偶基因突變的各種cdc4 ts突變品系，證實挑到了一個能替代S. cerevisiae CDC4 (ScCDC4)功能的白色念珠菌同源基因。接下來將我們挑到的基因做序列分析，並利用ScCDC4做比對，發現在F-box和八個WD-40 repeat這兩個ScCDC4的重要功能區域都有很高的相似性，因此，再次確認他們之間是結構的同源基因，因此，我們認定這個基因便是白色念珠菌的CDC4，命名為CaCDC4。 爲了確認CaCDC4在白色念珠菌扮演何種角色，接下來我們直接在白色念珠菌本身來觀察這個基因的功能。基於之前對出芽酵母菌CDC4的清楚認識，我們假定白色念珠菌的CaCDC4亦是一個必要基因且其雙倍同源染色體、無完整的有性生殖世代交替的生物特性，我們於是建構一白色念珠菌品系使一個CaCDC4對偶基因被破壞，而另一個CaCDC4對偶基因則是利用MET3啟動子來加以調控，當培養液含有甲硫胺酸及半胱氨酸時能遏止CaCDC4基因的表現。實驗結果有令人震驚的發現，當白色念珠菌細胞CaCDC4基因的表現被遏止時，仍能生長而形成菌落但以菌絲形態出現。因此，與ScCDC4 為必要基因扮演細胞週期G1進入S期的角色不同，CaCDC4並非一個必要基因，且其對於菌絲的生長扮演的是一個負調解者的角色。
並列摘要
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.