Dissection of regulatory elements in the promoter of Candida albicans virulence gene FTR1

白色念珠菌致病基因FTR1啟動子調控區域之分析研究

黃思璇(Szu-Hsuan Huang)

指導教授 ： 藍忠昱

國立清華大學/生命科學院/分子與細胞生物研究所/碩士(2010年)

摘要

中文摘要
白色念珠球菌 (Candida albicans)是人體內最常見的真菌共生菌。當宿主的免疫功能下降時，它會轉變成為病源菌，可能導致危及生命的全身性感染疾病。對於絕大多數生物體而言，鐵是一種必需的營養源。然而，於宿主體內，鐵極少以游離態存在，微生物可獲取之鐵含量極低。為了在此環境下生存，白色念珠球菌必須和宿主及其他微生物共同競爭鐵。研究發現白色念珠球菌已發展出許多不同的鐵攝取機制(iron-uptake systems)，同時也被證實鐵攝取機制和其致病力有關，其中包含表現細胞膜上高親和性鐵通透酶Ftr1(high-affinity iron permease)。在老鼠實驗中證實，當剃除白色念珠球菌FTR1基因，會降低其致病力。FTR1基因也被發現只在低鐵的環境下會大量轉錄，然而，此基因被鐵調控的分子機制仍是未知。本篇論文主要在於分析FTR1基因啟動子(promoter)的基因序列及反式調節因子(trans-factors)，進而探討FTR1基因的分子調控機制。實驗發現兩段受鐵調控的順式調節因子(cis-elements)，命名為IRE1及IRE2，參與調控FTR1基因在低鐵環境下大量表現。利用電腦序列分析發現，IRE1及IRE2分別擁有NRE及CCAAT兩種已知的順式調節因子序列存在。透過電泳遷移率檢驗(electrophoretic mobility shift assay)和定點突變分析(site-directed mutagenesis)，在低鐵環境下，CCAAT結合因子(CCAAT-binding factor)及Nrg1可以直接辨識並結合FTR1啟動子上的特定序列。綜合而言，本研究幫助我們了解白色念珠球菌致病力相關基因對宿主環境反應的調控機制。

關鍵字

白色念珠球菌 ； 高親和性鐵通透酶 ； 啟動子

並列摘要

Abstract
Candida albicans is the most common fungus in humans. It can become a pathogen and cause systemic infection in immunocompromised persons. Iron is an essential nutrient for almost every organism. However, free iron is highly limiting within the host. In order to acquire iron and survive under such an iron-restricted condition, C. albicans must compete with their host and other microorganisms. Moreover, previous studies have demonstrated that possession of specialized iron-uptake systems of C. albicans and some of them are related to its virulence. One is the FTR1 gene, which encodes a transmembrane high-affinity iron permease; cells lacking of FTR1 attenuate C. albicans virulence in a mouse model of systemic infection. Interestingly, the expression of FTR1 gene is highly induced under iron-limited environment. However, the molecular mechanism of FTR1 gene regulation in response to iron availability is mostly unknown. In this work, the main focus is to dissect the cis-regulatory elements and trans-factors that are involved in FTR1 gene regulation. Two cis-regulatory elements, IRE1 and IRE2, were revealed in the upstream promoter region of FTR1 and they were responsible for FTR1 gene activation at the low-iron condition. Using computational analysis, NRE and CCAAT box were found to exist within the IRE1 and IRE2 elements. EMSA and site-directed mutagenesis were undertaken to demonstrate that CCAAT-binding factors (CBF) and Nrg1 can directly bind to the regulatory sequences of FTR1 promoter under the low-iron condition. Taken together, this work provides important insight into regulation of C. albicans virulence-related gene in response to the host environment.

參考文獻

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