Moazeni Maryam, Khoramizadeh Mohammad Reza, Teimoori-Toolabi Ladan, Noorbakhsh Fatemeh, Rezaie Sassan
Division of Molecular Biology, Department of Medical Mycology & Parasitology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran..
Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran..
Acta Med Iran. 2014;52(1):9-14.
Efg1 transcription factor is believed to be the main regulator of hyphal formation under many different conditions. In addition, it is responsible for positive regulation of the expression of several hyphal-specific genes. SAP5, which encodes secreted aspartic proteinase, is one of the mentioned genes and is crucial for pathogenicity properties. In the present work we have established the experimental conditions for the use of siRNA in the diploid yeast Candida albicans in order to knock-down the EFG1 gene expression as well as the Efg1-dependent gene, SAP5. The 19-nucleotide siRNA was designed according to cDNA sequence of EFG1 gene in C. albicans and modified-PEG/LiAc method was applied for yeast transfection. To quantify the level of both EFG1 and SAP5 gene expression, the cognate mRNAs were measured in C. albicans by quantitative real-time RT-PCR and data was consequently analyzed by use of REST® software. Images taken by fluorescent microscopy method indicated the effectiveness of transfection. According to REST® software data analysis, expression of EFG1 gene decreased about 2.5-fold using 500 nM of siRNA. A 7-fold decrease in EFG1 gene expression was observed when applying 1 µM of siRNA (P<0.05). Consequently, the expression of SAP5 was significantly down-regulated both in yeast treated with 500 and 1000 nM of siRNA (P<0.05). In conclusion, post-transcriptional gene silencing (PTGS) is likely to be considered as a promising approach to discover new gene targets so as to design fungal-specific antifungal agents, and it is strongly possible that we are taking the right way to battle with C. albicans-associated infections.
Efg1转录因子被认为是许多不同条件下菌丝形成的主要调节因子。此外，它还负责对几个菌丝特异性基因的表达进行正向调控。编码分泌型天冬氨酸蛋白酶的SAP5就是上述基因之一，对致病性至关重要。在本研究中，我们建立了在二倍体酵母白色念珠菌中使用小干扰RNA（siRNA）的实验条件，以敲低EFG1基因表达以及Efg1依赖性基因SAP5。根据白色念珠菌EFG1基因的cDNA序列设计了19个核苷酸的siRNA，并采用改良的聚乙二醇/醋酸锂（PEG/LiAc）方法进行酵母转染。为了定量EFG1和SAP5基因的表达水平，通过定量实时逆转录聚合酶链反应（RT-PCR）在白色念珠菌中检测了相关mRNA，并使用REST®软件对数据进行分析。荧光显微镜法拍摄的图像表明转染是有效的。根据REST®软件的数据分析，使用500 nM的siRNA时，EFG1基因的表达下降了约2.5倍。当使用1 μM的siRNA时，观察到EFG1基因表达下降了7倍（P<0.05）。因此，在用500和1000 nM的siRNA处理的酵母中，SAP5的表达均显著下调（P<0.05）。总之，转录后基因沉默（PTGS）可能被认为是发现新基因靶点以设计真菌特异性抗真菌药物的一种有前景的方法，而且我们很有可能正走在对抗白色念珠菌相关感染的正确道路上。
