| Toxoplasma gondii is a serious zoonotic parasite that causes disease in humans and a variety of economically important animal species. Up to one-third of the world's population is seropositive for T. gondii. Although the majority of infections are asymptomatic, cervical lymphadenopathy or ocular disease can be present in some patients. In immunocompromised patients, reactivation of latent disease can cause life-threatening encephalitis. T. gondii is also an important cause of congenital birth defects, and the infected fetuses may exhibit permanent neurological damage, with or without hydrocephalus, as well as chorioretinitis with visual impairment. To date, sulfonamides and pyrimethamine are the most widely used drugs for the treatment of toxoplasmosis in humans. Although the combination of the two drugs is the first choice for most clinical conditions, they usually fail to efficiently eradicate infection and, as a result, nearly 50% of the patients do not respond well to this therapy. Moreover, this drug regimen does not kill Toxoplasma tissue cysts and can induce significant side-effects in patients. Thus, the discovery of new therapeutic targets and the development of new drugs are needed. As T. gondii cannot synthesize purines de novo, it has to salvage them from the environment through two available pathways that are defined by the enzymes hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) and adenosine kinase (AK), respectively. These features of purine metabolism in T. gondii make the two key enzymes attractive targets for therapy. Although RNA interference (RNAi) holds substantial promise for both drug development and basic research, there is little information concerning RNAi in T. gondii. The purpose of this study is to elucidate whether siRNA and long dsRNA transcribled in vitro can effectively work in T. gondii and to determine the replicative rate of the parasites when the genes encoding AK and HXGPRT are inhibited simultaneously using siRNA or long dsRNA.1. Decreased levels of mRNA and enzyme activity in long dsRN-treated parasitesThe level of AK mRNA did not decrease significantly in parasites treated with 8μg or 12μg of AK dsRNA as compared with the mock electroporation 24 hrs after treatment, while the mRNA levels in parasites treated with 4μg of AK dsRNA were 34±13% that of the calibrators at the same timepoint (P < 0.05). Twenty-four hours following electroporation, a significant decrease in the levels of HXGPRT mRNA was also observed in parasites treated with 4μg of HXGPRT dsRNA, with the mRNA levels decreasing to 33±16% that of the calibrators (P < 0.05). No significant decreases in the levels of HXGPRT mRNA were found in parasites electroporated with 8μg or 12μg of HXGPRT dsRNA. LacZ dsRNA, which was used as negative control, did not decrease AK or HXGPRT mRNA levels after electroporation into parasites at concentrations varying from 4μg to 12μg.We further confirmed the inhibitory effects of dsRNAs transcribed in vitro on AK and HXGPRT activity by measuring their protein levels. Forty-eight hours after treatment, the parasites electroporated with 4μg of AK dsRNA exhibited significantly lower AK activity compared with mock electroporated parasites, and the relative value obtained for [3H] -adenosine incorporations was 0.56±0.15 (P < 0.05). Similarly, HXGPRT activity in parasites treated with 4μg of HXGPRT dsRNA decreased to 0.59±0.02 that of the mock electroporation (P < 0.05). We observed, however, that concentrations of AK or HXGPRT dsRNA ranging from 8μg to 12μg did not inhibit AK or HXGPRT expression 24hrs and 48 hrs following treatment. The dsRNA homologous to the lacZ gene had no effect on AK or HXGPRT activity at various timepoints following electroporation at concentrations ranging from 4μg to 12μg.2. Inhibition of parasite replication following simultaneous treatment with dsRNA of AK and HXGPRTTo further observe the effect of dsRNA treatment on T. gondii proliferation, we simultaneously targeted the genes encoding AK and HXGPRT in T. gondii for inhibition using dsRNA. After electroporation with 4μg of AK and HXGPRT dsRNA, the parasites were inoculated onto the HFF monolayer. Parasites treated with dsRNA continued to replicate, but they divided more slowly than wild type parasites. For example, the average doubling for mock- and dsRNA-treated parasites was 2.37±0.91 and 1.85±0.91(P < 0.05), respectively, 24 hrs post-inoculation. The average doubling for dsRNA-treated parasites at 30 hrs and 42 hrs was 2.62±1.00 and 4.27±1.07, respectively, significantly lower than the values obtained for mock-treated parasites at the same timepoints (P < 0.05).3. Decreased levels of mRNA and enzyme activity in siRNA-treated parasitesIn three designed AK 25nt siRNAs, only two siRNAs, namely siRNA786 and siRNA1200, worked effectively in T. gondii. siRNA786 were electroporated into parasites with concentration varying from 1μM to 4μM. At 24 hrs following electroporation, significant decrease in levels of AK mRNA was observed in parasites treated with 2μM siRNA786 and with 4μM siRNA786. In parasites treated with 2μM siRNA786, the mRNA levels decreased to 0.59±0.08 of calibrators (P < 0.05), while in parasites treated with 4μM siRNA786 the mRNA levels decreased to 0.47±0.13 (P < 0.05), and we found no decreased change of AK mRNA in parasites treated with 1μM siRNA786. The relative levels of AK mRNA in parasites electroporated 4μM siRNA1200 decreased to 0.51±0.19 of mock electroporation (P < 0.05). We further confirmed the inhibitory effects of siRNAs on AK activity as a measure of protein level. When 2μM or 4μM siRNA786 was electroporated into the parasites, the treated parasites exhibited significantly lower AK activity compared with the parasites of the mock electroporation after 24hrs of treatment, and the relative values of [3H] -adenosine incorporations in parasites treated with 2μM and 4μM siRNA786 decreased to 0.45±0.19 and 0.39±0.11, respectively. The inhibitory effect on AK activity of 4μM siRNA1200 was similar to that of SiRNA786, and the relative value was 0.39±0.07 compared with those of the mock electroporation. We observed no decreased effect of the scrambled siRNA on the AK activity. In three designed HXGPRT 21nt siRNAs, only one siRNA, namely siRNA415 worked effectively in T. gondii. At 24hrs post-infection, the relative value of HXGPRT mRNA levels and HXGPRT activity decreased to 0.36±0.04 and 0.51±0.03, respectively.4. Inhibition of parasite replication following simultaneous treatment with siRNA of AK and HXGPRTTo further observe the effect of siRNA treatment on T. gondii proliferation, we simultaneously targeted the genes encoding AK and HXGPRT in T. gondii for inhibition using siRNA. After electroporation with 4μM of siRNA786 and siRNA415, the parasites were inoculated onto the HFF monolayer. The average doubling for siRNA-treated, SDZ-treated and mock-treated parasites was 1.64±0.95, 1.04±0.88 and 2.32±0.90, respectively, 24 hrs post-inoculation. When compared with mock-treated parasites, the rate of replication for siRNA-treated and SDZ-treated was significantly slowed (P < 0.05). At 40hrs after infection, the average doubling for siRNA-treated and SDZ-treated was 3.00±1.04 and 2.56±1.04,respectivly, and significantly lower than the values obtained for mock-treated parasites at the same timepoints (P < 0.05).In order to determine the virulence of parasites, mice were arbitrarily assigned to four treatment groups, comprising seven animals each. The mice infected with mock-treated parasites died within 8 days p.i., with a mean survival time 7.14±0.37 days. The mean survival time of SDZ-treated and siRNA-treated mice was 8.85±1.34 days and 8.14±1.34 days, respectively, and there are no siginicant differences as compared with animals infected with mock-treated parasites. However, SDZ & siRNA-treated mice with a mean survival time 10.83±4.67 days, displayed a siginificnat increase when compared with animals infected with mock-treated parasites (P < 0.05).In conclusion, both 21nt and 25nt siRNAs and long dsRNA transcribled in vitro can work effectively to knockdown the expression of target genes in T. gondii. When the genes encoding AK and HXGPRT were inhibited simultaneously using siRNAs or dsRNA, the rate of replication was significantly slowed.
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