2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38599We have been interested in studying the control mechanism leading to morphological changes of Candida. albicans as these changes are critical in pathogenesis. We have used the budding yeast Saccharomyces cerevisiae as a comparative system to analyze the relationship between cell cycle and morphogenesis in C. albicans due to their similarity both in the mode of mitotic division cycle and morphogenesis. We have focused our study on C. albicans at the G1 phase of the cell cycle as it is the point where cell makes decisions on a particular developmental path for diverse morphological states. We have confirmed conservation of cell cycle at the G1 phase between S. cerevisiae and C. albicans by identification of functional homologues of CDC28, G1 cyclins from C. albicans. We have recently isolated C. albicans homologues of S. cerevisiae CDC4 and CDC7, which require G1-S transition and S phase, respectively. Unlike its S. cerevisiae counterpart, C. albicans CDC4 (CaCDC4) is nonessential and acts as a negative regulator of filament. C. albicans CDC7 (CaCDC7) encoding a protein serine/threonine kinase shares structural similarity with its evolutionary counterparts with conserved function for initiation of DNA replication. Surprisingly, our preliminary results suggest that contrasting to S. cerevisiae CDC7 being essential and required for initiation of DNA replication, CaCDC7 appears to be nonessential and in its absence is critical for pseudohyphal growth in C. albicans. We postulate that CaCDC7 is a key morphological switch in C. albicans and propose a three-year project to validate this hypothesis. Three specific aims are to be completed. Firstly, we will verify that CaCDC7 is nonessential and regulates pseudohyphal growth negatively in C. albicans. We will determine the function of CaCDC7 by characterizing strains of CaCDC7 homozygous null or overexpressing CaCDC7 for phenotypes. Secondly, we intend to determine the importance of kinase activity and the possible requirement of the regulatory subunit of C. albicans Dbf4 (CaDbf4) for the function of CaCdc7. The kinase deficient CaCDC7 mutant will be analyzed in vitro and in vivo. The physical interaction of CaCdc7 with CaDbf4 will be established by either yeast two-hybrid or co-immunoprecipitation. The functional dependency of CaCDC7 with CaDBF4 will be determined by strains with CaCdc7 overexpressed and CaDbf4 depleted or vise versa for phenotypes. Thirdly, we will determine CaCDC7 associated regulation during morphogenesis. Epistasis analysis of CaCDC7 with others genes relevant to morphogenesis or environmental cues for filaments will be established. Genes that are in direct association with the function of CaCDC7 will be identified by either yeast two-hybrid or affinity purification using CaCdc7 as bait. The success in three-year proposal-funding will allow us to fill the gap of knowledge in the function of CDC7, being uniquely as a morphological switch in C. albicans, and how this morphological control is achieved. This should facilitate our further understanding of how the genetic reorganization specific to C. albicans is evolved to allow coordination of the cell cycle to morphological states unique to C. albicans.我們對研究白色念珠菌型態變化之調控有莫大之興趣因其形態之改變被認為與致 病性有關聯。由於其有絲分裂模式及型態形成的相似性，我們利用出芽酵母菌作為一比 較系統研究白色念珠菌細胞周期及型態形成的關係。我們將研究白色念珠菌重點放在其 細胞周期的G1因此點是細胞決定該往那條分化路徑進行形成不同型態的關鍵點。我們 由分離出芽酵母菌CDC28及G1 cyclins的白色念珠菌同源基因證實有絲分裂細胞周期 在出芽酵母菌及白色念珠菌是高度保留的。 最近我們也成功地分離出分別在G1-S轉換期及S期所需之出芽酵母菌CDC4及 CDC7的白色念珠菌同源基因。與出芽酵母菌CDC4不同的是，白色念珠菌CDC4 (CflCDC4)並¥^ 一必要基因且扮演菌絲生長負調節者的角色。C. albicans CDC7 (CaCDC7) 編碼一種與演化同源基因產物結構相似之絲氨酸/酥胺酸蛋白質激酶具高度保留控制 DNA複製起始之功能。非常意外的是，我們初步的實驗結果顯示與出芽酵母菌CDC7 為必要基因且調控DNA複製形成強烈對比的是，CaCDC7為一非必要基因且在其不存 在時能造成假菌絲之生長。 我們提出CaCDC7是白色念珠菌型態形成的主要開關的假說而提出的三年計劃 驗證此假說。將完成三個目標。首先，我將提出證據證實CaCDC7為一非必要基因且扮 演假菌絲生長負調節者的角色。我們將藉白色念珠菌同質無效CaCDC7及CaCDC7過 量表達突變株之外表型決定CaCDC7之功能。其次，我們將決定蛋白質激酶活性及可能 存在調節次單元CaDbf4對CaCdc7功能的重要性。CaCDC7蛋白質激酶缺失之突變株 將於體外及體内進行分析。利用酵母菌雙雜合及免疫沉澱決定CaCdc7與CaDbf4的交 互作用。利用CaCdc7過量表達而CaDbf4耗盡或反而行之決定兩者功能的依賴性。最 後，我們將決定在型態形成上與CaCDC7有關的調節。將建立CaCDC7與其他跟型態 形成基因及促進絲狀生成環境因子間之上位性關係。基因與CaCDC7有直接功能上的關 係將以CaCdc7為餌利用酵母菌雙雜合及親合力純化法分離。成功獲得三年計畫經費將 能順利執行此研究而了解有關新穎CaCDC7在型態形成的功能及其如何控制型態轉 變。並將有助於進一步了解白色念珠菌如何衍生特有的遺傳重新組織使其具獨特細胞周期及型態形成協調方式。細胞周期CDC7絲氨酸酥胺酸蛋白質激酶白色念珠菌型態形成cell cycleCDC7Candida albicansprotein serinethreonine kinasemorphogenesisProject