The Influence Of Fluridone On The Egression Of Toxoplasma Gondii And Its Host Cells

Background and ObjectiveToxoplasma gondii is an intracellular parasitic protozoa，whose lifecycle mainly include several steps，namely invading，reproducing，egressing and invading another host cells. Among them, the invasionmechanism of the parasite has been researched throughly, but its egressingfrom the host cells attracted less attention. The egression of T.gondii fromthe host cell is a continuous process, the rescent studies found that thisprocess required abscisic acid (ABA) as the start signaling molecules, andthen the protozoa secreted T. gondii perforin-like proteins (TgPLP1），andspeculated that T. gondii may have genes which are related to synthesize ofendogenous ABA. Our previous researches showed that some cells infectedwith T. gondii could be block in cell cycle and were caused to apoptosis.In this study，we chose signaling molecules which T. gondii dependentto egress as an entry point and ABA inhibitor fluridone as a means ofintervention. The culture cells of T. gondii were treated with fluridone, andexplored the effect and the molecular mechanisms involved of fluridone oncultured T. gondii in vitro，using morphology, molecular biology, cell biology and other research methods. Then, this study tried to find whetheror not there were an association between the egression of T. gondii and itshost cell cycle changes and apoptosis. Finaly, this study investigated theeffect of fluridone on mice infected with T.gondii.Methods1.Recovered T. gondii RH strain tachyzoites by inoculating parasitesinto maice, established the comitant culture models of hela cells withdifferent amounts of tachyzoites, the rate of infection of which were5:1,10:1,20:1respectively. The models were cultured for different times (24h,48h,72h,96h) and the proliferation of the parasites inside the cells wereobserved by Giemsa staining. Calculated the rate of infection to determinethe suitability rate and the culture time.2. Took the hela cells infected with tachyzoites treated withconcentration of PBS was50μM as control, hela cells infected withtachyzoites had been treated with different concentrations of flurodone(10μM、20μM、30μM、40μM、50μM). Observed the morphologic structuresof intracellular tachyzoites by Giemsa staining, counted the number ofparasitophorous vacuoles. The mRNA levels of ABA and TgPLP1of the T.gondii were detected by semi-quantitative RT-PCR， and the proteinexpression levels of TgPLP1were detected by SDS-PAGE electrophoresis.ELISA was applied to investigate the ABA levels in the culture solutions.3. After the treatment of fluoridone，the rates of growth inhibition of hela cells were investigated by the CCK-8method. Flow cytometry (FCM)was adopted to analyze the cell cycle distribution and apoptosis in the helacells infected with T. gondii tachyzoites at the fluridone concentration of30μM. The apoptosis of the infeted with T. gondii was also observed by theDNA Ladder method.4. The mice infected with T.gondii were treated with fluridone byintraperitoneal injection, and observed the survival time and the weightreduction rate of the mice. The modality of T.gondii in the intraperitonealcavity of mice was observed by microscope and electron microscope, andthe irradiation of T.gondii was evaluated by tissue slice printing.Results1. The parasitism and proliferation of T. gondii tachyzoites in helacells were observed by Giemsa staining and microscope with oil immersionlens. The infection rate of the cells was related to the infection ratio and thetime of culture. If the infection ratio was the same, the rate of infectionincreased with the cultue time increased; while the infection time was thesame, the rates of infection extended with the increase of the infection ratio.Choose the infection ratio of10:1, and the time of72h as the experimentalparameters.2. In the control group, the majority of cells were infected with T.gondii, the parasite proliferated， the morphology of most cells wasdamaged and there were some free tachyzoites. Compared with the control, T. gondii in the cells in the experimental group formed parasitophorousvacuoles the sum of which increased while the concentrations of fluoridoneincreased (P <0.05，except the concentration of50μM). The mRNA levelsof ABA and TgPLP1and the protein level of TgPLP1decreased with theincrease of the concentrations of fluoridone, and the ABA level in theculture solution increased significantly.3. The data of CCK-8shown that the proliferation inhibitory rate offluridone to hela cells infected by T. gondii drcreased (P<0.05). In the flowcytometric analysis, the percentage of Glphase of the hela cells wasdecreased, while the percentage of S and M cells was increasedsignificantly in the group treated with T. gondii (P<0.05). What’s more，thehela cells apoptosis also decreased in the group treated with fluridone(P<0.05).4. The symptom of toxoplasmosis in mice group treated with fluridoneappeared later and milder than that in the control group. The survivalduration was longer（P<0.05）and the extension rate was45%. The organsof the mice infected with T.gondii later and the degree of infection wasmild than the control.The most tachyzoites of T.gondii in the control groupwere in free forms, but most tachyzoites are intracellular parasites in micetreated with fluridone.Conclusions1. Fluoridone can inhibit the egression of the T. gondii tachyzoites in hela cells in vitro, which would be caused by the down-grade effection offluoridone on the ABA and TgPLP1.2. The egression of T. gondii tachyzoites was associated with theapoptosis of the host cells in vitro. The host cells proliferation have notbeen suppressed and the apoptosis have downgraded, which would becaused by the inhibitive effect of fluridone on egression of T. gondiitachyzoites.3. The fluridone could inhibit toxoplasmosis in mice，decreased T.gondii infectin of the mice and extended the life time of the mice.