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標題: | 熱帶念珠菌六個同源基因對菌絲及生物膜生成的影響探討 Investigation of six conserved transcriptional factors on the regulation of hyphal formation and biofilm development in Candida tropicalis |
作者: | Yu-Chia Chen 陳昱嘉 |
指導教授: | 林晉玄(Ching-Hsuan Lin) |
關鍵字: | 熱帶念珠菌,生物膜形成,yeast-hyphae 型態轉換,轉錄因子,白色念珠菌, Candida tropicalis,biofilm,yeast-hyphae transition,transcription factor,Candida albicans, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 念珠菌屬 (Candida species) 包括白色念珠菌 (Candida albicans) 及熱帶念珠菌 (Candida tropicalis) 皆屬於伺機性致病真菌,可以在不同的型態間轉換以適應變化的環境。其中 yeast-hyphae 的型態轉換對念珠菌的毒性、寄主免疫系統反應等形態特徵高度相關,也是念珠菌生物膜形成的重要步驟。生物膜是微生物在自然界中主要增生的方式,也是造成人體永久感染的主因之一。熱帶念珠菌是台灣僅次於白色念珠菌常見的念珠菌;儘管如此,對抗真菌藥物 fluconazole 耐受性菌株產生的速度卻比白色念珠菌快。因此本研究欲瞭解與熱帶念珠菌致病力高度相關的菌絲生成及生物膜形成的相關機制。過去研究顯示,白色念珠菌主要由包含 CaBcr1、 CaBrg1、 CaEfg1、 CaNdt80、 CaRob1與 CaTec1的六個轉錄因子組成生物膜生成控制網;除了 CaBcr1 之外的五個轉錄因子也參與菌絲生長的調控。因此,我們猜測熱帶念珠菌的六個同源轉錄因子在調控生物膜及菌絲生成同樣扮演重要角色。首先實驗構築熱帶念珠菌六個同源轉錄因子基因的突變株及互補株,觀察其生物膜及菌絲生成變化。六個基因突變株生物膜生成皆有缺失,顯示熱帶念珠菌六個同源基因皆能正向調控生物膜的形成;而除了 CtNdt80 抑制菌絲生成外,其餘五個轉錄因子皆促進菌絲生成。利用即時聚合酶鏈式反應分析六個基因在菌絲生成時的表現量,發現在最適菌絲生成條件下,只有 CtROB1 的表現量明顯上升;然而,與菌絲生成高度相關的基因 CtUME6 的表現量在熱帶念珠菌六個同源基因突變株中,皆顯著下降。為了探尋六個基因調控生物膜及菌絲生成的可能機制,我們將熱帶念珠菌六個同源基因分別送入白色念珠菌相對應的基因突變株內。在 Cabcr1、 Caefg1 或 Catec1 突變株分別表現 CtBCR1、 CtEFG1 或 CtTEC1,可以恢復生物膜及菌絲生成能力;而表現 CtBRG1 或 CtROB1 對 Cabrg1 或 Carob1 突變株的生物膜或菌絲生長能力沒有影響。CtNDT80 雖然無法回復 Candt80 突變株的生物膜生成缺失,但在 Spider 液態培養基刺激下,可觀察到菌絲的生長 [Candt80Δ(0%)、Candt80Δ::CtNDT801 (8.46%)];除此之外,在 Ctndt80 突變株中表現 CaNDT80,菌絲的生成比例反而會顯著下降 [ Ctndt80Δ(63.10%)、Ctndt80Δ::CaNDT801 (36.48%)]。根據上述實驗結果,本研究發現 C. tropicalis 及 C. albicans 的六個同源基因調控生物膜及菌絲生成具有保守性功能;然而,NDT80對菌絲的調控在兩物種間有所不同,暗示著菌種間演化出複雜的菌絲生成調控迴路,而使單一基因保守性功能無法彰顯。 Candida albicans and Candida tropicalis are opportunistic fungi, which can undergo morphological transition to adapt different environments. The yeast-hyphae transition is considered to be highly related to Candida properties in virulence, and host-fungus interactions. In addition, the transition from yeast to hyphae is an important step for Candida biofilms, which are a major growth form in natural environments and a leading cause of persistent infections. C. tropicalis is not only the 2nd common commensal pathogen among Candida species in Taiwan, but also develops resistant strains against fluconazole faster than C. albicans. It has been known that regulatory network composed of CaBcr1, CaBrg1, CaEfg1, CaNdt80, CaRob1 and CaTec1 is involved in biofilm development in C. albicans. Except for CaBcr1, other five genes hold the ability to regulate filamentous gowth as well. In this study, we speculated the functions of six homologous genes in C. tropicalis are the same as those genes in C. albicans. Six gene mutants and complementary strains were constructed in C. tropicalis to observe the differences of hyphae and biofilm formation. Six mutant strains were deficient in biofilm development, which means they positively regulate biofilm formation in C. tropicalis. Besides, while CtBcr1, CtBrg1, CtEfg1, CtRob1 and CtTec1 promoted hyphae formation in C. tropicalis, CtNdt80 inhibited filamentation. RT-qPCR showed that only CtROB1 was highly expressed during filamentation, but CtUME6 expression level, which is one of determining factors for hyphae formation, was downregulated in all six mutant genes. To understand the possible mechanisms of six genes in regulating biofilm and filamentation in C. tropicalis, we transformed C. tropicalis homologous genes into C. albicans mutans strains, separately. Biofilm and filament ability could be recovered by expression of CtBCR1, CtEFG1 and CtTEC1 in Cabcr1, Caefg1 and Catec1 mutants; however, there were no differences in biofilm and hyphae development when expressing CtBRG1 or CtROB1 in Cabrg1 or Carob1 mutants. CtNdt80 could not recover biofilm deficiency of Candt80 mutant; nevertheless, Candt80 mutant would form hyphae when expressing CtNDT80 in Spider medium [Candt80Δ(0%)、Candt80Δ::CtNDT80 (8.46%)]. Besides, the percetange of filaments became lower when CaNDT80 expressed in Ctndt80 mutant [ Ctndt80Δ(63.10%)、Ctndt80Δ::CaNDT80 (36.48%)]. Taken together, our study demonstrates that each single transcriptional gene hold the conserved function in biofilm and in filamental growth; however, the filament regulatory circuit of Ndt80 is different in two species, implying that species may have evolved a sophisticated regulatory network for the control of hyphae formation, leading to contrain or varnish over a single gene’s function. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/67518 |
DOI: | 10.6342/NTU201701794 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科技學系 |
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