Knocking down of the DHFR-TS gene in Toxoplasma gondii using siRNA and assessing the subsequences on toxoplasmosis in mice

Azimi-Resketi M., Eskandarian A., Ganjalikhani-Hakemi M., Zohrabi T.

Acta Tropica, vol.207, 2020 (SCI-Expanded) identifier identifier

  • Publication Type: Article / Article
  • Volume: 207
  • Publication Date: 2020
  • Doi Number: 10.1016/j.actatropica.2020.105488
  • Journal Name: Acta Tropica
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus, Academic Search Premier, Aerospace Database, Aquatic Science & Fisheries Abstracts (ASFA), BIOSIS, CAB Abstracts, Communication Abstracts, EMBASE, Geobase, MEDLINE, Metadex, Veterinary Science Database, Civil Engineering Abstracts
  • Keywords: Dihydrofolate reductase-thymidilate synthase, Sirna, Toxoplasma gondii, Toxoplasmosis
  • Istanbul Medipol University Affiliated: No


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.