對於每一種生物體的生長而言，鐵是一種必要的營養源。有些微生物寄生在人體，它們會在活體上繁殖並利用寄主做為營養來源，因而傷害寄主造成疾病。其中，調控體內鐵質的含量即是寄主對抗微生物感染的一種重要機制，微生物病原菌已經被研究出可利用各種不同的機制以便從寄主及其周邊環境中獲得鐵。此外，寄主體中的鐵質含量也是一個引發病原菌致病力的環境因子。白色念珠菌(Candida albicans)是一種伺機性的真菌病原菌，它通常是與寄主共生，然而，當寄主的免疫功能下降時，它可能導致危及生命的感染疾病。在白色念珠菌體中目前發現至少有三種不同的鐵攝取系統，其中包括嗜鐵蛋白(siderophore)吸收系統。 白色念珠菌可藉由細胞表面的運輸蛋白CaSit1運送嗜鐵蛋白進而攝取鐵質，CaSit1並且已被報導會參與白色念珠菌入侵到寄主組織之過程。CaSIT1基因的轉錄通常在低鐵環境中會大量表達；相反的，在高鐵的環境中則會受到抑制。本篇論文主要研究重點是藉由分析CaSIT1啟動子(promoter)的基因序列來研究CaSIT1的分子調控機制。經過一連串刪除啟動子區域的實驗，發掘了一段22個鹼基對的順式序列，參與抑制CaSIT1在高鐵環境中的表現。另兩段區域分別是27個和23個鹼基對，則參與CaSIT1基因在低鐵環境中的大量表達。此外，電泳遷移率檢驗(electrophoretic mobility shift assay)的實驗結果也明顯表明了有某些蛋白質可結合於所發現的鹼基對調控區域。最後，利用去氧核醣核酸親和沉澱法(DNA affinity precipitation assay)及液相層析儀質譜儀(LC-MS/MS)也分析、鑑別可能調控CaSIT1的那些DNA-鍵結蛋白質。本研究提供了一項幫助我們了解白色念珠菌致病力相關基因對鐵質反應的調控機制模式。

Iron is essential for the growth of almost every organism. Importantly, iron-withholding is also a mechanism for the host to against microbial infection. Microbial pathogens have developed different systems to acquire iron from the host and their surrounding environments. In addition, iron availability is shown to serve as a signal to induce the expression of virulence traits of pathogens. Candida albicans, an opportunistic fungal pathogen, usually presents as a commensal in the host. However, when the host becomes immunocompromised, it can cause life-threatening system infections. C. albicans harbors at least three different iron-uptake systems, including a siderophore uptake system mediated by a ferrichrome siderophore transporter Sit1. CaSit1 not only does uptake siderophore, but is also involved in C. albicans invasion to the host tissues. Transcription of the CaSIT1 gene is highly expressed at the low iron condition, while is repressed in high iron environment. The main focus of this study is to examine transcriptional regulation of CaSIT1 by analyzing the 5’UTR of CaSIT1 fused with a lacZ reporter gene. Serial deletion of the promoter region of CaSIT1 revealed a 22-bp cis-regulatory element that is responsible for gene repression at high-iron condition, while a 27-bp region and a 23-bp region are involved in gene activation under a low-iron condition. In addition, electrophoretic mobility shift assays were performed to examine proteins possibly interacting with these elements. The results suggest that there is a DNA binding protein represses CaSIT1 in high iron condition. Then, DNA affinity precipitation assay and LC-MS/MS were carried out to identify the protein which interacts with this cis-element. Together, this study provides an important insight for our understanding how C. albicans regulates its virulence-related gene in response to the host environments, such as iron availability.

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