Establishment Of The Model About Tachyzoite-bradyzoite Interconversion Of Toxoplasma Gondii In Vitro And Preliminary Study On Inducible RNAi

Toxoplasma gondii is a widespread obligate intracellular protozoan parasite which could invade almost any nucleated cell and result in toxoplasmosis of amphixenosis. Toxoplasma gondii is an opportunistic parasite, in most humans infected with Toxoplasma gondii, the disease is asymptomatic. However, in immunocompromised or immunodeficiency individuals, such as patients with tumor, AIDS patients, et al, T. gondii tachyzoites could multiply rapidly and cause disseminated lesion in the body, even result in death of the patients. Toxoplasma gondii could be transmitted vertically to foetus through placenta and cause premature birth, abortion, fetal death, abnormity or a baby with developmental malformation. The infection of T. gondii becomes a severe problem of public health.The interconversion between tachyzoite and bradyzoite is the key of pathogenesis of toxoplasmosis. Tachyzoites could cause acute infection, and bradyzoites in the tissue cyst could product chronic disease, the mechanism about the interconversion was not known. There were not specific medicine to toxoplasmosis, the drugs, such as pyrimethamine, acetyl spiramycin, et al, are used widely to treat toxoplasmosis, but the drugs only kill or depress tachyzoite, do not affect bradyzoites in the tissue cyst. Reactivation of bradyzoite could result in serious damage to patients. It has become a hot spot and a difficult problem about the mechanism of the interconversion.Last years, with the development in reverse genetics based on RNAi and transgenic techniques, it provides a new way to analyze gene function from gene variation. After we comprehend the status about the study of tachyzoite-bradyzoite interconversion and the obstacles in the work, we would apply the methods and principles of transgenesis, RNAi and reverse genetics to establish the model of tachyzoite-bradyzoite interconversion of Toxoplasma gondii RH strain in vitro, to clone the genes of difference display in the earlier period of the interconversion, to construct a RNAi vector system with inverted repeat sequences, which was drived by T. gondii heat shock protein 70 (HSP70) promotor, and to set up a technical platform to analyse the gene function of T. gondii. We would focus on the genes which expressed in the earlier period of the differentiation from tachyzoite to bradyzoite, and illuminate the function in the differentiation through the research.1. Establishment of the model about tachyzoite - bradyzoite interconversion of T. gondii in vitroTachyzoites of T. gondii RH strain were inoculated into the peritoneal cavity of mouse, the peritoneal fluid was collected at 3-4 days after the inoculation, and passed sequentially through a No. 4.5 needle to release parasities from the host cells, the tachyzoites were purified through a 3.0 um pore-size filter, purity coefficient could arrive at above 98%.According to the ratio of 1:10, the purified tachyzoites were inoculated into NIH3T3 cell layer at the point which the grown NIH3T3 cell layer occupied 60%-70% of the culture surface, using DMEM supplemented with 5% new-born calf serum, the cells were cultured at 37℃, in 5% CO₂. The tachyzoite could multiply rapidly, and disrupt the considerable host cells. They were harvested at 5-6 days after the inoculation and applied to the next cultivation or the experimental study.To induce tachyzoite transformation into bradyzoite with the alkaline culture media, the purified tachyzoites were inoculated into NIH3T3 cell layer at the ratio of 1:10, 4h after infection, the cell layer was washed softly with DMEM at pH8.1 to remove tachyzoites which did not invade into the cells, then the media was replace with DMEM supplemented with 2.5% new-born calf serum, pH8.1. The cultures were maintained at 37℃in air to avoid pH variation due to CO₂. At 96h after infection, many round cysts were observed in the kytoplasm of NIH3T3, strong refraction were also observed from cyst wall. Total RNA was extracted from the cultures, BAG1 mRNA was detected by RT-PCR. A 700bp band was obtained through electrophoresis. The PCR product was cloned into T-vector, and sequenced. The sequencing result of the cloned gene has the homology of 99.3% to the sequence of published BAG1 mRNA, and has no introns. So the bradyzoites had been induced successfully. In order to monitor proceeding of transformation, a bottle of cells was taken out at 0h, 24h, 48h, 72h, 96h respectively after the induction, and total RNA was extracted. The RT-PCR products ofβ-tubulin gene were taken as the control, with demi-quantitate RT-PCR, the products of BAG1 mRNA were raised following times of the induction, the results could indicate more bradyzoite formation with longer times of the induction.In order to induce the transformation of bradyzoite into tachyzoite, the induced bradyzoites by alkaline pH8.1 were recovered in culture media of pH7.2 and cultivated in normal culture conditions. A few tachyzoites were seen in the media at 24h after the induction, and the tachyzoites began to multiply greatly and disrupt host cells, so new NIH3T3 cells would be added in the cultures for further culture.To explore heat shock treatment on the differentiation from tachyzoite to bradyzoite of T. gondii. The NIH3T3 cell layers were cultured in the incubator at 37℃, 39℃, 41℃, 43℃, 5% CO₂ for 3h, then the purified tachyzoites were inoculated into NIH3T3 cell layer at the ratio of 1:10, 4h after infection, the cell layer was washed softly with pre-heat DMEM to remove tachyzoites which did not invade into the cells, then the cultivation went on at 37℃, 39℃, 41℃, 43℃, 5% CO₂ for 48h. The cultures were collected, and total RNA was extracted for RT-PCR. In conclusion, in the condition of 39℃, tachyzoite could multiply more rapidly and could not convert into bradyzoites. In the condition of 43℃, NIH3T3 cells couldn't grow, the tachyzoites could not convert into bradyzoites with lacking stable circumstances in host cells. In the condition of 41℃, it had been proved that the tachyzoites could differentiate into bradyzoites.2. The construction of the inherited and inducible RNAi vector system of T. gondii1)The construction of the vector with the gene of green fluorescent protein(GFP) drived by the SAG1 promotor of T. gondii: 5'UTR promotor sequences of SAG1 gene (SAG1/5UTR),3'UTR sequences of SAG1 gene (SAG1/3UTR), and the coding sequences of GFP gene (eGFP), were cloned by PCR respectively. The PCR products were digested by the corresponding enzymes and ligatd into the intermedial vectors. Finally, the vector, pBSK-SAG1/5UTR-eGFP-SAG1/3UTR (pBSK-SAG1/GFP), with the GFP gene drived by the SAG1 promotor of T. gondii, was constructed successfully, and the results of sequencing were correct.2)The construction of the inducible RNAi vector with inverted repeat: 5'UTR promotor sequences of HSP70 gene (HSP70/5UTR),3'UTR sequences of HSP70 gene (HSP70/3UTR), and the intron C sequence ofβ-tubulin gene of T. gondii, were cloned by PCR respectively. The PCR products were digested by the corresponding enzymes and ligatd into the intermedial vectors. Finally, the inducible RNAi vector, pBSK-HSP70/5UTR-IntronC-HSP70/3UTR, with the HSP70 gene promotor as a promotor, the intron C sequence ofβ-tubulin gene as intervening sequence, 3'UTR sequences of HSP70 gene as transcription stop signals, was constructed successfully, and the results of sequencing were correct.3)The construction of the inherited and inducible RNAi vector system of T. gondii: The fragment of SAG1/5UTR-eGFP-SAG1/3UTR in pBSK-SAG1/GFP vector was cloned into the vector of pBSK-HSP70/5UTR-IntronC-HSP70/3UTR to construct pBSK-GFP-Hairpin vector, then the fragment of GFP-Hairpin in pBSK-GFP-Hairpin vector was cloned into pHANA-0.5 vector. Finally, the vector pHANA-hairpin was constructed successfully, and the results of sequencing were correct.4)The construction of RNAi vector targeted SAG1 gene: The sequences and reversing sequences of SAG1 gene of T. gondii were cloned by PCR, The PCR products were digested by the corresponding enzymes and ligatd into the vectors of pHANA-hairpin. Finally, the RNAi vector targeted SAG1 gene, pHANA-hairpin/SAG1, was constructed successfully, and the results of sequencing were correct.5)The construction of RNAi vector targeted BAG1 gene: The sequences and reversing sequences of BAG1 gene of Toxoplasma gondii were cloned by PCR. The PCR products were digested by the corresponding enzymes and ligatd into the vectors of pHANA-hairpin. Finally, the RNAi vector targeted BAG1 gene, pHANA-hairpin/BAG1, was constructed successfully, and the results of sequencing were correct.3.The construction of transgenic strains of T. gondii and preliminary study on RNAiThe plasmid pHANA-hairpin/SAG1 or pHANA-hairpin/BAG1 was electroporated respectively into RH strain tachyzoites of T. gondii to construct transgenic toxoplasma. 1×10⁷ Toxoplasma gondii tachyzoites and 10μg plasmid DNA were resuspended in 800 ul of cytomix buffer. The entire mixture was transferred to an electroporation cuvette(4-mm gap) and exposed to an electric pulse with an electroporator in the high- capacitance mode, the charging voltage set to 0.4 kV, electric capacity set 800 uF, the pulse length was 6-9msec. The transgenic toxoplasma was cultured for 24h, and observed through fluorescence inverted microscope. The green fluorescent light was seen in the two strains of transgenic toxoplasma. The total RNA of the two strains was extracted and used for RT-PCR. RT-PCR product of the GFP gene could been detected, and it demonstrated the plasmids were electroporated into the tachyzoites successfully.To induce tachyzoite transformation into bradyzoite, the two strains of transgenic Toxoplasma gondii were cultured with the alkaline culture media. after 96h induction, the green fluorescent light was seen in the two strains of transgenic toxoplasma. The RT-PCR products of the SAG1, BAG1 and GFP gene could been detected. Several reasons could be for the phenomenon: 1. some transgenic toxoplasma tachyzoites could not converted to bradyzoites. 2. some induced transgenic toxoplasma were in the middle stage between tachyzoite and bradyzoite, and could not form mature bradyzoites and cysts. 3. SAG1 gene or BAG1 gene targeted by RNAi could affect the proceed of the conversion, and the induced transgenic toxoplasma tachyzoites could remain the stage of tachyzoite. These problems should be invested further.