標題: 探討剔除CaENO1、CaREP5及CaREP6對白色念珠菌之影響
Effects of CaENO1, CaREP5 and CaREP6 Null Mutations in Candida albicans
作者: 柯惠菁
Ko, Hui-Ching
楊昀良
Yang, Yun-Liang
生物科技系所
關鍵字: 病原體;藥物感受性;發芽管形成;毒性;enolase;Regulator of Efflux Pump;pathogen;drug susceptibility;hyphal formation;virulence
公開日期: 2013
摘要: Enolase(2-phospho-D-glycerate hydrolase)是一個組成醣解途徑的酵素,在演化過程中具有高度保留性,在Candida albicans中,它是ENO1的基因產物。此基因(CaENO1)的表現受到調控致病力的EFG1的影響。因此,本研究利用SAT1 cassette進行基因剔除以了解CaENO1的相關功能,發現葡萄糖或果糖會抑制Caeno1/Caeno1同基因合子突變株的生長及發芽管之形成,突變株也無法在不含胺基酸的Difco-yeast nitrogen base培養基中生長。在相關的藥物感受性試驗中發現,Caeno1/Caeno1雙套基因突變株影響amphotericin B、miconazole和NaCl的感受性。因此CaENO1除了醣類代謝的機制,也涉及發芽管形成、藥物感受性與細胞間的離子滲透;另外,在小鼠體內試驗結果發現突變株失去致病力,這些研究結果將可能有助於於開創新一代的抗真菌製劑。 另外,先前實驗室經由在啤酒酵母菌(Saccharomyces cerevisiae)所進行的library screening發現在啤酒酵母菌中CaREP5(Regulator of Efflux Pump)和CaREP6能增加CDR1p-lacZ的β-galactosidase酵素活性。CDR1是一個抗藥基因,先前報導指出它也跟CaENO1一樣,涉及白色念珠菌的致病機制。本研究主要在白色念珠菌中針對CaREP5和CaREP6的基因遺傳學和功能性探討,希望藉此進一步了解抗藥性的調控機制。結果在測試的條件下,null突變株的表現型並無顯著改變。
Enolase (2-phospho-D-glycerate hydrolase) is an enzymatic component of the glycolytic pathway and has been well conserved throughout evolution. It is encoded by CaENO1 in Candida albicans, the most frequently isolated human fungal pathogen. The protein product can also be found on the cell surface and bind host plasminogen in association with tissue invasion. In order to understand the role other than that in glycolytic pathway, CaENO1 was subjected to mutagenesis analysis by the construction of null mutants via gene-replacement with the SAT1 flipping cassette. Strains lacking CaENO1 were not able to grow on glucose or fructose and it also failed to grow on Difco-yeast nitrogen base medium without amino acid. It was also observed that null mutations affected the susceptibility to amphotericin B and miconazole, in addition to the resistance to NaCl stress. Hence, CaENO1 was involved in drug susceptibility in addition to its role in carbon utilization. And it may also be involved in regulation of cell osmolarity or ion channels. Furthermore, the CaENO1 null mutant was avirulent when tested in a mouse model for systemic infection, and also exhibited defective hyphal formation. These results may help to design new and more effective antifungal agents. In addition, CaREP5 (Regulator of Efflux Pump) and CaREP6 were isolated from C. albicans genomic library due to its ability to increase the β-galactosidase activity of CDR1YM990348 promoter-lacZ fusion construct in Saccharomyces cerevisiae in the presence of miconazole. CDR1 is a drug resistance gene. And just like CaENO1, it is known to affect the pathogenesis of C. albicans. In this study, these genes were subjected to genetic and functional studies. And the results showed there were no significant differences in the phenotypes between the wild type and the Carep5/Carep5 or the Carep6/Carep6 under the conditions tested.
URI: http://140.113.39.130/cdrfb3/record/nctu/#GT079428811
http://hdl.handle.net/11536/74171
Appears in Collections:Thesis


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