The Development Of An In Vitro Interconversion System For Tachyzoite-bradyzoite Of Toxoplasma Gondii And Preliminary Study Of The Function Of BAG1 During Parasite Differentiation

Background:Toxoplasma gondii is a widespread obligate intracellular protozoan parasite which could infect all species of mammals (including human) and is responsible for toxoplasmosis. It is considered be one of the most ubiquitously distributed and widespread parasites with a worldwide seroprevalence of up to 30% in humans. Toxoplasma gondii is an opportunistic parasite:in an immunocompetent host infected by Toxoplasma gondii, the disease is generally asymptomatic. Parasites can escape immune attack and form cysts which are able to persist in the body of a host for a few months, several years or even a life time. However, as far as immunocompromised or immunodeficiency individuals are concerned, such as patients suffering from neoplastic disease, organ transplantation and AIDS patients et al, T. gondii tachyzoites could multiply rapidly and produces necrotic lesions that may result in serious damage such as toxoplasmic encephalitis, toxoplasmic retinochoroiditis, even 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. During recent years, owing to the increasing number of AIDS patients and pet-keepers, the infection of T. gondii becomes a severe problem to public health.The interconversion between tachyzoite and bradyzoite is the key to pathogenesis of toxoplasmosis. The mechanism about the interconversion is still unknown. There are not specific medicine for toxoplasmosis. Drugs, such as pyrimethamine, acetyl spiramycin,et al, are widely used to treat toxoplasmosis, but the drugs can only kill or depress tachyzoite without affecting bradyzoites in the tissue cyst. Reactivation of bradyzoite could result in serious damage of patients. The study on the mechanism of the interconversion has become a hot spot, which is important for the development of drugs and vaccines. It is also very significant to develop an in vitro interconversion system for tachyzoite-bradyzoite of Toxoplasma gondii. After a long-term exploration, scientists have found that changing the cell culture conditions--the physical and chemical (such as medium temperature, pH, or denial of nutrients) and biological (eg, by adding IFN-γor NO) conditions can increase the efficacy of bradyzoite development in vitro. However, there is still a certain gap about the transformation between changing the external conditions and maintaining natural conditions. The transformation study is more significant under natural conditions, so the conditions are largely maintained to set up the interconversing system from tachzoites to bradyzoites of T.gondii in COS-7 cells.Bradyzoite antigen (BAG) 1 is a bradyzoite-specific protein and it is expressed in the earlier period of the interconversion from tachzoites to bradyzoites of T.gondii.and is a main protein of the mature cysts, but the function of the BAG1 gene remains unclear in the interconversion. Recently, with the development in reverse genetics based on RNAi and transgenic techniques, it provides a new way to analyze gene function from gene variation. In this thesis, the vector pHANA-hairpin/BAG1 containing the GFP gene driven by the SAG1 promoter of T. gondii and targeted BAG1 gene is constructed and electroporated into tachyzoites of T. gondii RH strain. Finally a transgenic strain of Toxoplasma gondii is obtained successfully. The construction of the RNAi vector contains inverted repeat sequences of the BAG1 gene. During the transcription process, the inverted repeat sequences will get folded and form Hairpin dsRNA by intramolecular hybridization. HpRNAs eventually form siRNA under the influence of the Dicer enzyme. The siRNA will combine the endogenous BAG1 mRNA and interfere the function of BAG1 gene. Recombinant T. gondii expressing green fluorescent protein (GFP) under control of the SAG1 promoter are generated, which works at the tachyzoite stage, but decreases or disappeare at the bradyzoite stage. Thus, the conversion state of Toxoplasma gondii in terms of the expression of green fluorescence is determined and the function of the BAG1 gene in the process of the differentiation from tachyzoite to bradyzoite is further illuminated.Objective:1. To develop the in vitro interconversing system from tachzoites to bradyzoites of Toxoplasma gondii.2. To construct the transgenic strain of Toxoplasma gondii.3. Targeted knock down the endogenous bradyzoite-specific gene, BAG1, and analyze its effect on cyst formation.Methods:1. Tachyzoites of T. gondii RH strain were inoculated into the peritoneal cavity of mouse, the peritoneal fluid was collected at the 3-4 day after the inoculation. 2. According to the ratio of 1:10, the purified tachyzoites were inoculated into COS-7 cell layer at the point which the grown COS-7 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% CO2.3. The shape of the cultured cells and parasites were observed and their total RNAs were extracted on 1 to 6 days post inoculation respectively. The expression of tachyzoite-specific protein (SAG1) and bradyzoite-specific proteins (BAG1 and SAG2C) were identified by RT-PCR.4. The vector pHANA-hairpin/BAG1 containing the GFP gene driven by the SAG1 promoter of T. gondii and targeted BAG1 gene was electroporated into tachyzoites of T.gondii RH strain. Individual positive clones were screened by limited dilution.5. The COS-7 cells were inoculated with the purified transgenic tachyzoites of T.gondii RH strain and cultured under certain condition.The stage conversion from tachzoites to bradyzoites of T.gondii was observed and their total RNAs were extracted on 1 to 6 days post inoculation respectively. The expression of tachyzoite-specific protein (SAG1) and bradyzoite-specific proteins (BAG1 and SAG2C) were identified by RT-PCR. Optimize expression conditions and purify expression products.6. The COS-7 cells were inoculated with the purified transgenic tachyzoites and tachyzoites of T.gondii RH strain and cultured under certain condition. The encapsulation-like structures were calculated from transgenic parasites and parasites in COS-7 cell on 1 to 6 days through a fluorescence microscope. Then data collection and statistical analysis are carried out.Results:1. As time went by, the number of parasites in COS-7 cell increased gradually but the proliferation rate decreased. Intracellular tachyzoites kept breeding in budding mode and binary fission, which led to the changes on arrangement of the parasites in the cells from bigeminal curved, rose-shaped, or cluster-shaped, semi-circular shape to round encapsulation-like structure. These series of changes indicated that the tachyzoites could gradually be transformed into bradyzoites. The expressions of the tachyzoite-specific SAG1 gene could be detected from the first day to the 6th day. The expression of the bradyzoite-specific BAG1 gene was detected since the second day of post inoculation and SAG2C gene was detected since the fifth day. Changing the culture condition, the bradyzoites were able to be gradually transformed into tachyzoites as well.2. Within 24 hours after the electroporation, the transfected tachyzoites of T.gondii expressing green fluorescence were observed with a fluorescence microscope.3. As time went by, the number of the purified transgenic tachyzoites in COS-7 cell increased gradually but the proliferation rate decreased. Intracellular tachyzoites kept breeding in budding mode and binary fission, which caused the changes on arrangement of the parasites in the cells from bigeminal curved, rose-shaped, or cluster-shaped, semi-circular shape to round encapsulation-like structure. This series of changes indicated that the tachyzoites could gradually transform into bradyzoites. With time going by, Fluorescence intensity reduced gradually. The expressions of the tachyzoite-specific SAG1 gene could be detected from the first day to the 6th day. The expression of the bradyzoite-specific BAG1 gene had been detected since the fifth day of post inoculation and SAG2C gene had been detected since the fifth day. Targeted disruption of the bradyzoite-specific gene BAG1 could delay the expression of the bradyzoite-specific BAG1 gene rather than stop tissue cyst formation in Toxoplasma gondii.4,The number of encapsulation-like structure from the transgenic parasites and parasites has statistical significance. (F=53.263, P<0.001). The data states that the encapsulation-like structure from the transgenic parasites appear later and remain less in number compared with ordinary Toxoplasma gondii.Conclusion:1. An in vitro interconversing system from tachzoites to bradyzoites of T. gondii is developed successfully in present work, which sets up a good basis for further study on the mechanism of interconversion.2. A transgenic strain of Toxoplasma gondii is obtained successfully.3. Targeted disruption of the bradyzoite-specific gene BAG1 could not stop tissue cyst formation in Toxoplasma gondii.