Effects Of Monensin On Toxoplasma Gondii Gtna Expression And Metabolites Process In Host Cells

Toxoplasma gondii?T.gondii?is an opportunistic protozoan that is parasitic on nucleated cells.It has a wide range of intermediate hosts,and feline is the only definitive host.T.gondii infection can cause miscarriages,fetal malformations,and even stillbirths,but most common patients are clinical symptom negative.Anti-Toxoplasma vaccines are currently under development,and also there is currently no specific medicine for the treatment of toxoplasmosis.In general,the preferred anti-Toxoplasma drugs are pyrimethamine and sulfadiazine,but the side effects of sulfa drugs cannot be ignored.Therefore,screening a new type of drug with low toxicity,no side effects or low side effects has become the focus of current research.Monensin is an ionophore antibiotic used to treat coccidiosis in poultry and dairy cows.Studies have shown that monensin has anti-Toxoplasma activity,but its mechanism of action is not yet clear.We speculate that it may be because monensin has affected the metabolic pathway of T.gondii.This study evaluated the anti-T.gondii effects of monensin natrium,sulfadiazine and artemether through in vivo and in vitro infection with tachyzoites of T.gondii RH strain.In vitro experiment:toxicity and proliferation of human foreskin fibroblasts?HFF?were evaluated at three different drug concentrations by MTT assay;In vivo experiments:mice were infected with 5×104 tachyzoites of the RH strain.Four hours post infection,the mice were treated with the drugs for 5 days at three concentrations?high,medium and low doses?through gavage method.Survival rate of mice was recorded daily.In addition,the amount of tachyzoites in peritoneal fluid was counted daily,aiming to check whether the parasites recurred during withdrawal period,and to evaluate the in vivo anti-T.gondii effects of the three drugs.The in vitro experiments showed that monensin natrium significantly inhibited the proliferation of T.gondii,and the inhibition effect is better than that of artemether.The in vivo experiments showed that monensin natrium significantly increased the survival time of infected mice,followed by sulfadiazine and artemether.Both in vivo and in vitro experiments showed that monensin natrium can significantly inhibit the proliferation of T.gondii tachyzoites and prolong the survival time of mice.Tachyzoites of T.gondii RH strain were used to infect pig kidney cells?PK-15 cells?,and high-throughput sequencing?RNA-seq?revealed that 4868 genes were down-regulated and only 3 genes were up-regulated in T.gondii,Down-regulation indicates that monensin could inhibit expression of most T.gondii genes.The host cells had 1,346 genes up-regulated and 827 genes down-regulated,indicating that the host genes have also undergone significant changes.Monensin treatment appeared to adversely affect various key metabolic and cellular processes of T.gondii,such as in the endoplasmic reticulum,spliceosome,ribosome and protein processing.For host cells,chemokine signaling pathways related to the body's immune system,Fc-y-R-mediated phagocytosis,and T cell receptor signaling pathways were significantly enriched.The biological changes in these hosts may be caused by T.gondii infection.After infection of pig kidney cells?PK-15 cells?with tachyzoites of T.gondii RH strain,a total of 262 differential metabolite ions were identified in positive ion mode by LS-MS.Of which,22 were identified as up-regulated,15 were identified as down-regulated at 6 h;whereas 192 were up-regulated and 33 were down-regulated at 24 h.A total of 35 differential metabolite ions were identified in negative ion mode;of which,6 were up-regulated,5 down-regulated at 6 h,19 up-regulated and 5 down-regulated at 24 h.Most metabolic differences were up-regulated,which indicated that monensin up-regulated most metabolites.KEGG analysis showed that glycerophospholipid metabolism,linoleic acid metabolism pathways and metabolic pathways for amino acid synthesis were significantly enriched.Camphor ranked first in the enrichment of all metabolites,and it was found to be related to TRP channel-mediated inflammatory mediators and cancer pathways,and to some patients with epilepsy caused by T.gondii.These results could be used as a basis for the diagnosis of toxoplasmosis secondary infection to other diseases.In this study,by using of the CRISPR/Cas9-mediated homologous recombination technology,pyrimidine resistance cassette was inserted into the CDS 5' end of the three genes?TGME49₂49650,TGME49₂87040 and TGME49₃15310?,then terminate the transcriptions or expressions of the three genes.Through monoclonal screening and PCR identification,TGME49₂49650 and TGME49₂87040 gene knock-out strains were successfully obtained.The above research not only provides a reference for the subsequent research and clinical use of anti-T.gondii drugs,but also lays a foundation for the preliminary study of T.gondii vaccines.