The Effect Of SPZ To Toxoplasma Gondii Before And After The Deletion Of ROP16 And The Preliminary Mechanism Of Its Resistance Against T.gondii

Toxoplasma is an obligate intracellular parasitic protozoon,can cause serious infectious in both animals and people with a high infection rate.ROP16 belongs to ROP2,is secreted by rhoptry of T.gondii.During T.gondii infection,ROP16 firstly is secreted into the cytoplasm of host cell,then gets to the cell nucleus,finally affects varies signal pathes.For example,ROP16 has an effect on STAT3/6,which plays a important role in immunoreaction.In previous study,we demonstrated that SPZ had a perfect protective effect against acute Toxoplasmosis in murine,which can protected 60% infected mouse from death.To further detect the special mechanism of SPZ against T.gondii besides the normal mechanisms which belonged to all the sulfonamides by inhibiting the synthesis of dihydrofolic acid,we studied the further effect of SPZ in a compound way,and build the ROP16 deletion and recompleted strains to research the biological function of ROP16 to verify the potential relationship between the effect of SPZ against T.gondii and the down-regulated host innate immune function of ROP16.Firstly we detected the treatment effect of compound SPZ against acute toxoplasmosis in murine.The results showed that SPZ,SDZ,lactate TMP,pyrimethamine,baicalin and glycyrrhiza could obviously inhibit the proliferation of T.gondii in vitro.The single dosage of SPZ(250mg/Kg and 200mg/Kg)could protect a portion of murine with acute toxoplasmosis from death(the survival rates were 50% and 25%)in vivo,while other five drugs could only prolong the survival times of mice in different degrees,and could not protect them from death.In the compound administration group(SPZ 200mg/Kg),SPZ with lactate TMP or pyrimethamine had no obvious synergistic effect,while SPZ-glycyrrhiza-baicalin could significantly improve the survival rate up to 40%.The results suggestted that the special therapeutic effect of SPZ was not only related to inhibiting the proliferation of T.gondii by interfering the metabolism of folic acid,but also due to other mechanisms to be found.Secondly,by building prokaryotic expression plasmids with vectors of p COLD1 and p GEX-6P-1,we expressed ROP16 both in whole length and divided parts.As a result,ROP16 wasn't soluble and all expressed in inclusion body.ELISA test showed that the expressed ROP16 could be recognized by dog positive serum,suggests the protein had good immunogenicity.Then immune mice with the purified protein to get the rat polyclonal antibodies of ROP16.By indirect ELISA test,the antibody titer up to 1:64000,which was high enough to meet the later use.Then we screened ROP16 deletion and complementary strains with the CRISPR/CAS9 system.PCR and western blot identified that we got ROP16 deletion and complementary strains successfully.Compared to the RH wild and complementary strains,the ROP16 deletion strains had a weak invade rate,terrified that ROP16 was a key factor related to parasite invasion.The level of cell factors IL-12 in mice serum infected with ROP16 knockout strains was higher than the RH strains infected group,suggested that ROP16 played an important role in down-regulating the host IL-12 expression.While there was no obvious difference of IL-5 and IFN-? in mice serum infected by RH and ROP16 deletion strains.The missing of ROP16 had no obvious effect on the lethality of mice infected by different parasites,suggested that ROP16 might also play a important role in reducing inflammatory reaction or other infection mechanism.Thirdly,to detect the new resistance mechanism of SPZ against T.gondii,we studied the microstructure of T.gondii using transmission electron microscope.Compared to the control group,most tachyzoites in the SPZ treatment group showed significantly impaired in the front area of rhoptry.And the level of IL-12 in mice serum infected by RH strains treated with SPZ was higher than that of the untreated group,the results showed that SPZ could make high levels of proinflammatory response factor IL-12 to assist the host to resist T.gondii.What's more,the effect of SPZ on T.gondii before and after knockout of ROP16 was observed.The mice separately infected by RH strain and ROP16 deleted strain were treated with SPZ.The results showed that low dose SPZ(150 mg/Kg)had no protective effects to RH strain infected mice,while the protection rate of ROP16 deleted strain infected mice up to 20%,indicating that the deleted gene of ROP16 relieved down-regulation natural immune function of ROP16 and increased the proinflammatory response of the host,which also enhanced the anti-T.gondii resistant effects of SPZ.With the increase concentration of SPZ,the protective rate of mice infected with RH strain raised gradually,together with the survival rate of ROP16 missing strain infected mice,the result revealed a scissors difference.This phenomenon indicated that SPZ may affect the down-rugulating natural immune function of ROP16 as well,thereby assisting the resistant effects of SPZ against T.gondii.In conclusion,the special resistance mechanism of SPZ against T.gondii in addition to inhibiting the proliferation of T.gondii by interfering the metabolism of folic acid,may also had a effect on the down-regulate innate immune function of ROP16,next prompted mice to produce high levels of proinflammatory response factor IL-12,then enhanced the resistance effect of SPZ against T.gondii.While the detail mechanism still need to be further explored.