标题: | 探讨剔除CaENO1、CaREP5及CaREP6对白色念珠菌之影响 Effects of CaENO1, CaREP5 and CaREP6 Null Mutations in Candida albicans |
作者: | 柯惠菁 Ko, Hui-Ching 杨昀良 Yang, Yun-Liang 生物科技系所 |
关键字: | 病原体;药物感受性;发芽管形成;毒性;enolase;Regulator of Efflux Pump;pathogen;drug susceptibility;hyphal formation;virulence |
公开日期: | 2013 |
摘要: | Enolase(2-phospho-D-glycerate hydrolase)是一个组成醣解途径的酵素,在演化过程中具有高度保留性,在Candida albicans中,它是ENO1的基因产物。此基因(CaENO1)的表现受到调控致病力的EFG1的影响。因此,本研究利用SAT1 cassette进行基因剔除以了解CaENO1的相关功能,发现葡萄糖或果糖会抑制Caeno1/Caeno1同基因合子突变株的生长及发芽管之形成,突变株也无法在不含胺基酸的Difco-yeast nitrogen base培养基中生长。在相关的药物感受性试验中发现,Caeno1/Caeno1双套基因突变株影响amphotericin B、miconazole和NaCl的感受性。因此CaENO1除了醣类代谢的机制,也涉及发芽管形成、药物感受性与细胞间的离子渗透;另外,在小鼠体内试验结果发现突变株失去致病力,这些研究结果将可能有助于于开创新一代的抗真菌制剂。 另外,先前实验室经由在啤酒酵母菌(Saccharomyces cerevisiae)所进行的library screening发现在啤酒酵母菌中CaREP5(Regulator of Efflux Pump)和CaREP6能增加CDR1p-lacZ的β-galactosidase酵素活性。CDR1是一个抗药基因,先前报导指出它也跟CaENO1一样,涉及白色念珠菌的致病机制。本研究主要在白色念珠菌中针对CaREP5和CaREP6的基因遗传学和功能性探讨,希望藉此进一步了解抗药性的调控机制。结果在测试的条件下,null突变株的表现型并无显着改变。 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 |
显示于类别: | Thesis |
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