The Inhibitory Effect Of IFM-γ On Conversion From Bradyzoite To Tachyzoite Of Toxoplasma Gondii

Toxoplasma gondii is an obligate intracellular protozoan parasite that can infect most warm blooded animals, including birds and mammals, causing zoonotic toxoplasmosis. Toxoplasma infection generally does not have obvious clinical symptoms, most patients can carry tissue cysts for life and not exhibit any clinical symptoms. However, the infection can cause serious consequences in the crowd of some immune suppression, such as AIDS patients and organ transplant patients, by a change from chronic infection to acute infection. This opportunity pathogenicity of T.gondii is caused due to the mutual conversion between tachyzoite and bradyzoite. Transport stress can suppress immune function, causing decreased production of interferon-y (IFN-y), and often leads to the outbreak of some latent infection diseases.. IFN-y has a variety of immune regulatory function, and it can regulate the synthesis of a variety of parasiticidal effectors. IFN-y plays an important role in the anti-toxoplasma acute and chronic infection in the animal body. Therefore, we hypothesized that transport stress may induce reactivation of Toxoplasma chronic infection, causing acute toxoplasmosis, and the reason is probably due to the affect of IFN-y on the interconversion between tachyzoite and bradyzoite.In this study, firstly the inhibitory effect of IFN-y on the conversion from bradyzoite to tachyzoite was validated in vitro through the establishment of an interconversion cell model for tachyzoite-bradyzoite of T. gondii; secondly the reactivation of brain tissue cysts of toxoplasma chronic infection mice was induced by mimic transport stress, and then the inhibitory effect of IFN-y on the conversion from bradyzoite to tachyzoite was validated in vivo by using the interferon inducer PolyI:C under the same stress conditions, the work we have done includes the following aspects:(1) Establishment of an in vitro interconversion model for tachyzoite-bradyzoiteIn this study, the conversion from tachyzoite to bradyzoite of T.gondii RH strain was successfully induced by the alkaline condition of pH8.1and high temperature of43℃heat shock respectively. Parasites with cyst-like structure were observed both during the course of4days alkaline induction and48hours heat shock induction respectively. The expression of bradyzoite stage-specific gene BAG1and SAG2C can be detected by RT-PCR in both induction methods. The conversion process from tachyzoite to bradyzoite under the two induction condtions was monitored by Real-time PCR, the results showed that, under alkaline induction the relative expression levels of BAG1increased gradually at first and then decreased with the extension of induction time, and under high temperature induction the relative expression levels of BAG1increased gradually with the extension of induction time, indicating that more and more tachyzoites turned into bradyzoites in the tolerance range of host cells. In addition, the conversion from bradyzoite to tachyzoite of T.gondii PRU strain was successfully induced by the normal culture condition. Lots of parasites with rapid proliferation were observed after4days normal induction wtih inoculation of bradyzoites of PRU strain, and semi-quantitative RT-PCR showed the expression level of SAG1increased gradually whereas the expression level of BAG1became almost undetectable after lday normal induction.(2) In vitro inhibition of conversion from bradyzoite to tachyzoite by IFN-yThe impact of exogenous IFN-y on the parasite stage conversion was verified by using in vitro conversion model of bradyzoite to tachyzoite. The semi-quantitative RT-PCR results showed that, the expression of BAG1can both be detected in the cultures with the presence of IFN-y although alkaline-induced bradyzoites and the natural bradyzoites were cultured normally for2days, indicating that IFN-y can inhibit the conversion from bradyzoite to tachyzoite in a certain degree. In addition, IFN-y concentration gradient experiment showed that this inhibitory effect was related to the concentration of IFN-y, and the higher concentrations of IFN-y (80-160UI/mL) can effectively suppress the conversion from bradyzoite to tachyzoite in vitro.(3) In vivo inhibition of conversion from bradyzoite to tachyzoite by IFN-yChanges about the relative expression levels of IFN-y, IL-12, BAG1and SAG1in the brain tissue of chronic infection mice were detected by Real-time PCR before and after mimic transport stress. The results showed that, the relative expression levels of IFN-y, IL-12and BAG1were significantly decreased, while the relative expression levels of SAG1was significantly increased in the untreated stress group, suggesting that the conversion from bradyzoite to tachyzoite occurred in the brain and the mimic transport stress induced the reactivation of cysts. Conversely, in the PolyI:C pretreated stress group, the relative expression levels of IFN-y and IL-12decreased slightly but the differences were not significant, and the relative expression levels of BAG1and SAG1showed no significant changes too, indicating that the conversion from bradyzoite to tachyzoite was suppressed in vivo through the increase of IFN-y in the brain tissue by the interferon inducer-PolyI:C.The results proved that IFN-y plays a vital role in the host anti-Toxoplasma immunity, and will provide a theoretical basis for the prevention and control of the reactivation of Toxoplasma chronic infection under the conditions of transport stress.