Silencing the Target Rhoptry Neck Protein (RON) Gene Responsible For the Invasion of Toxoplasma gondii with siRNA Transfection


YÜRÜK M., Aksoy T. , Sivcan E., Nergiz H.

MIKROBIYOLOJI BULTENI, cilt.53, ss.81-95, 2019 (SCI İndekslerine Giren Dergi) identifier identifier identifier

  • Cilt numarası: 53 Konu: 1
  • Basım Tarihi: 2019
  • Doi Numarası: 10.5578/mb.67701
  • Dergi Adı: MIKROBIYOLOJI BULTENI
  • Sayfa Sayıları: ss.81-95

Özet

Toxoplasma gondii is a coccidian protozoan that causes toxoplasmosis is a common disease in Turkey as well as all over the world. It causes various clinical symptoms depending on the immune system status, age, or location of the disease. There is an organelle called the apical complex at the anterior end of the parasite. Rhoptry Neck Proteins (RONs), a component of this organelle, play a critical role in the formation of "moving junction" and parasitophorous vacuoles during host cell invasion. On the other hand, interfering RNA (iRNA) treatment options developed in recent years have emerged. With small iRNAs (siRNA) it is also possible to treat and control parasitic diseases, too. From here it is thought to use this method against toxoplasmosis. Within the scope of the project, it is aimed to silence RON1 gene the target invasion molecules of T.gondii with siRNA transfection. In the study, the negative control group constitute HeLa cells, the positive control group constitute HeLa cells infected with T.gondii tachyzoites and the experimental group constitute HeLa cells infected with T.gondii tachyzoites after siRNA transfection were used. Samples were collected in each study group at 30 seconds, 1 minute, 5 minutes, 15 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 36 hours and 48 hours. In order to calculate the proportional change of the parasite loads between control groups and experimental groups, RNA and protein isolations were performed from these samples for real time polymerase chain reaction (qRt-PCR) and WB analyzes, respectively. The statistical difference between control groups and experimental groups was calculated. Significant difference in gene expression in experiments with siRNA1 (p< 0.0055), siRNA2 (p< 0.0003), siRNA3 (p<0.0001), siRNA4 (p< 0.0001), siRNA5 (p< 0.0001), siRNA6 (p< 0.0001), siRNA7 (p< 0.0182), siRNA9 (p< 0.0011) and siRNA10 (p< 0.0004) but there was no significant difference statistically in the experiment with siRNA8 (p< 0.4049) was detected. Thus, it has been detected that invasion was inhibited due to the lack of production of parasite antigen in the cell lysate belongs to experimental groups at WB assays with anti-T.gondii RON1 and total anti-T.gondii antibodies resulting in silencing of the RON1 gene. The suppression of the TgRON1 gene expression by this method is a promising step in the development of anti-toxoplasmosis vaccines and therapeutic agents.