The Screening And Identification Of Differentially Expressed Proteins In Conversion From Bradyzoite To Tachyzoite Of Toxoplasma Gondii Under Transport Stress

Toxoplasma gondii is an obligate intracellular protozoa, which can causes severe zoonotic toxoplasmosis. T. gondii can infect nearly all warm-blooded vertebrates, including poultry, livestock, companion animal and human. In immunocompetent individuals, T.gondii is forced to form semidormancy cyst reside in host brain,muscle and eyes for life time. In this case, T.gondii infection does not show obvious clinical symptoms. However, when host immune system is suppressed, T.gondii cyst can reactivate from bradyzoite to tachyzoite, resulting in acute infection. Thus, the conversion between T.gondii bradyzoite and tachyzoite plays crucial roles in pathogenic progress. Previous studies have showed that, transport stress induces resurgence of diseases by suppressing host immune system. So we hypothesize that transport stress induces opportunistic pathogen Toxoplasma gondii conversion from bradyzoite to tachyzoite.Stage conversion between bradyzoite and tachyzoite is associated with differentially expressed molecules, incluing stage-specific proteins, stage-specific enzymes and heat shock proteins. The differentially expressed proteins play a major role in regulation of T.gondii growth and metabolism, which stimulates our interest greatly.In this study,firstly we vertified the activation of cyst induced by transport stress through an animal model establishing for T.gondii bradyzoite and tachyzoite conversion; Secondly, we discovered the expression level of IFN-γ, IL-1α, IL-4, IL-10, IL-12 and M-CSF decreased remarkablely in mouse serum under transport stress by cytokine antibody array detection; Thirdly, we identified 47 Mus musculus proteins and 15 T.gondii proteins in conversion from bradyzoite to tachyzoite through Two-dimensional Electrophoresis(2DE) and Mass Spectrometry(MS) technology. Our work including the following aspects:(1) Establishment of animal model for conversion from T.gondii bradyzoite to tachyzoiteIn this study, conversion from T.gondii bradyzoite to tachyzoite is successfully induced by transport stress. Three days after stress, the tachyzoite-specific gene SAG1 and bradyzoite-specific gene BAG1 m RNA level in T.gondii chronic infection mouse brain is monitored by Real-time PCR. The results showed that, the SAG1 m RNA relative expression level increased and BAG1 m RNA level decreased after transport stress, indicating that transport stress induces activation of cyst from bradyzoite to tachyzoite.(2) Transport stress influence T.gondii chronic infection mouse immune systemWe use cytokine antibody array to detect cytokine changes in mouse serum after different transport stress times through animal model for T.gondii conversion. Results showed that, as transport stress time goes, the expression level of IFN-γ, IL-1α, IL-4, IL-10, IL-12 and M-CSF decreased gradually, and significantly reduced three days after stress. In conclusion, transport stress can suppress host immune system.(3) Identification of differentially expressed proteins in conversion from T.gondii bradyzoite to tachyzoiteWe extracted total protein of purified cysts collecting from T.gondii chronic infection mouse after different transport stress times. Then Two-dimensional Electrophoresis(2DE) and Mass Spectrometry(MS) technology were used to screen and identify differentially expressed proteins in bradyzoite and tachyzoite conversion. Consequencely, we successfully identified 47 Mus musculus proteins and 15 T.gondii proteins. Then we use Real-time PCR to detect five identified genes ’ m RN A level in chronic infection mouse brain. Results showed that, all five T.gondii genes Actin, MIC13, GRA9, GRA1 and ENO1 m RNA relative expression level reduced after transport stress, which is consistent with 2DE and MS results.To conclude, our study verified stress suppressed host immune system through transport stress animal model and identified 47 Mus musculus proteins and 15 T.gondii proteins in conversion from T.gondii bradyzoite to tachyzoite, which provide theoretical basis for illumination of T.gondii bradyzoite and tachyzoite conversion mechanism.