Department of Medical Parasitology and Mycology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
Department of Medical Parasitology and Mycology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
Acta Trop. 2020 Jul;207:105488. doi: 10.1016/j.actatropica.2020.105488. Epub 2020 Apr 8.
Toxoplasma gondii (T. gondii), an obligatory intracellular parasite, is the etiologic agent of toxoplasmosis. Dihydrofolate reductase-thymidylate synthase (DHFR-TS) is one of the most important enzymes in toxoplasma folic acid cycle. Due to the emergence of resistance in RH strain of T. gondii against pyrimethamine that acts via DHFR-TS inhibition and also the crucial role of small interference RNA (siRNA) technology in gene silencing, we aimed to use siRNA to knock down DHFR-TS gene expression in T. gondii as a therapeutic target against toxoplasmosis in a mouse model. Based on the DHFR-TS gene sequence, siRNA was designed. The siRNAs were transfected into the parasites by electroporation. Total RNA was extracted using RNX-Plus kit. The viability of parasite was assessed by methylthiazole tetrazolium (MTT). The survival time of mice challenged with siRNA-treated T.gondii were compared to the control group infected with the same amount of wild-type tachyzoites. The viability of siRNA-embedded parasites was 70.7% (29.3% decreased) compared to the wild-type parasite as control (P = 0.0001). The transcription level of siRNA-transfected parasites was reduced to 17.4% (82.6% inhibition) (P = 0.016). The in vivo assessment showed that the mean survival time of the mice inoculated with modified parasites was increased about 2 days after the death of all mice in the control group. The designed siRNAs in the current study were able to silence the DHFR-TS gene efficiently. This silencing led to a decrease in viability of the parasites and an increase in the survival time of the parasites-treated mice.
刚地弓形虫（Toxoplasma gondii）是一种必需的细胞内寄生虫，是弓形体病的病原体。二氢叶酸还原酶-胸苷酸合酶（DHFR-TS）是刚地弓形虫叶酸循环中的最重要的酶之一。由于 RH 株弓形虫对作用于 DHFR-TS 抑制的嘧啶甲胺的耐药性的出现，以及小干扰 RNA（siRNA）技术在基因沉默中的关键作用，我们旨在使用 siRNA 敲低弓形虫中的 DHFR-TS 基因表达，作为一种治疗弓形虫病的方法。根据 DHFR-TS 基因序列，设计了 siRNA。通过电穿孔将 siRNA 转染到寄生虫中。使用 RNX-Plus 试剂盒提取总 RNA。通过甲基噻唑四唑（MTT）评估寄生虫的活力。用与相同数量的野生型速殖子感染的对照感染的 siRNA 处理的弓形虫感染的小鼠的存活时间进行比较。与作为对照的野生型寄生虫相比，siRNA 包埋寄生虫的活力为 70.7%（降低 29.3%）（P=0.0001）。转染 siRNA 的寄生虫的转录水平降低到 17.4%（抑制 82.6%）（P=0.016）。体内评估表明，与对照组所有小鼠死亡后相比，接种修饰寄生虫的小鼠的平均存活时间增加了约 2 天。在本研究中设计的 siRNAs 能够有效地沉默 DHFR-TS 基因。这种沉默导致寄生虫活力降低和寄生虫处理的小鼠存活时间增加。
