Mechanism Research Of The Calmodulin During Toxoplasma Gondii Invasion

Toxoplasma gondii,as the model organism of apicomplexa parasites,is a rigid intracellular protozoan.T.gondii can infect one third population in worldwide,leading to severe zoonosis toxoplasmosis in humans and animals,especially on individuals and newborns with low immune condition.Due to its complex mechanism of invasion,T.gondii has a wide range of hosts.Calcium ion can regulate related vital activities of T.gondii as an important second messenger in cell,in particular with the parasite invasion and escape processes.A variety of Ca2+-related proteins play an important role on the Ca2+ signaling pathway,but little is known about these Ca2+-related proteins and the relationship among them.To deeply discover their functions in the Ca2+ signaling pathway and their interactions can further explain the invasion mechanism of Toxoplasma gondii,and provide the basis for research on pathogenesis and drug target design in toxoplasmosis.In this study,we used CRISPR / Cas9 and Cre-Lox P system to knockout Ca M in RH?HX strain,then comparing the difference of ability of replication and invasion between Ca M gene knockout strain and RH ? HX strain.The interaction protein of Ca M was screened by Bio ID technique in order to discover more functional proteins in Ca2+signaling pathway at the same time.(1)Prokaryotic expression of Ca M protein and preparation of polyclonal antibodyFirstly,we constructed p GEX-KG-Ca M prokaryotic expression plasmid which can express in BL21(DE3)smoothly,then immunized animal with the purificated protein.Collecting animal sera after immunization,enzyme-linked immunosorbent assay was used to detect antibody potency.The results revealed that the polyclonal antibody has a higher level of antibody titer.(2)Construction the RH?HX-Lox P-Ca M strainCre-Lox P system was performed in conditional knockout of Ca M gene.First,we constructed a CRISPR plasmid p SAG1 :: Cas9-U6 :: sg Ca M which could recognize the Ca M locus,and contained a homologously recombination template plasmid p UC19-Ca M-YFP.Then the homologous fragments and plasmid p SAG1 :: Cas9-U6 ::sg Ca M were transferred into the tachyzoites of RH ? HX strain.After screening by xanthine and mycophenolic acid and limited dilution,single clone was selected.Theresults indicated that the RH?HX-Lox P-Ca M strain was successfully obtained by the method of PCR amplification and IFA.(3)phenotype analysis of Ca M knockout strainsOn the basis of the Ca M conditional knockout strain,Ca M gene was deleted by transferring of a plasmid pmin-e GFP-Cre which can express Cre-splicing enzyme.Ca M deficient strain and RH ? HX strain were simultaneously conducted in intracellular replication and invasion experiments.The results showed that there was no significant difference in the replication ability between the deletion strain and RH ? HX strain.However,the invading ability of the deletion strain was obviously declining,indicating the invasive ability of deficient strain was significantly lower than wild strain.(4)Screening interaction proteins with Ca M by Bio ID techniqueThe genetically engineered strain RH?HX-Bir A*-Ca M and the original strain RH?HX were labeled with biotin,the result showed that some specific proteins labeled with biotin were detected by IFA and western-blot after 24 hours.In order to further identify these proteins,we analysed by mass spectrometry.Twenty-five specific proteins were obtained from this experiment.According to function and localization of these proteins what we have identified,there is possibility that the inner membrane complex(IMC)and fructose 1,6-diphosphate aldolase can interact with Ca M,but the specific relationship between them also need to be verified by further experimental.In general,the Cre-Lox P conditional knockout system and CRISPR / Cas9 system were used to knockout the Ca M gene in RH? HX strain.Comparing with Ca M gene deletion strain and RH?HX strain,we find that the deletion strain is defective in the invasion ability,but no significant difference in intracellular replication ability.At the same time,25 specific proteins were screened by Bio ID and LC-MS/MS.The research above on function of Ca M may provide a theoretical basis for the study of Toxoplasma gondii invasion mechanism.