Construction Of Knockout Strains And Studies Of Basic Biological Functions Of 18 Novel Dense Granule Proteins (GRAs) Of Toxoplasma Gondii

Toxoplasma gondii is an important opportunistic pathogenic protozoon that can infect almost all thermostatic animals,including humans,resulting in zoonotic toxoplasmosis.About one third of the world’s population is chronically infected with T.gondii,which poses a serious threat to the health of immunocompromised patients,pregnant women,and pregnant animals.Thus,T.gondii infection is not conducive to the health of human and animals and the efficient development of animal husbandry.Dense granular proteins（GRAs）are very important secretory proteins of T.gondii,they regulate immune response and cell cycle of host cells by manipulation of host（cell）gene expression,signal pathway,and play important roles in the physiological processes such as protein transport and localization,nutrient uptake,the formation and stability maintenance of intravacuolar network（IVN）,egress and chronic infection.However,the function of many newly identified GRAs in T.gondii is still unknown.In this study,the basic biological functions of 18 novel GRAs in T.gondii were explored by CRISPR-Cas9 gene editing technology,transcriptomics,immunoprecipitation technology and other methods.1.To examine the localization of 15 novel GRAs（TGME49₃13440,TGME49＿247530,TGME49＿204320,TGME49₃15910,GME49＿266410,TGME49＿297900,TGME49＿267740,TGME49＿268970,TGME49＿279350,TGME49＿258462,TGME49＿214410,TGME49＿291630,TGME49＿248990,TGME49＿244530 and TGME49＿258458）in tachyzoites and bradyzoites,15 novel GRAs tagged strains were successfully constructed using CRISPR-Cas9 gene editing technology.Using immunofluorescence assay,we found that these 15 novel GRAs were localized at the parasitophorous vacuole（PV）,partly localized to PV membrane（PVM）in the tachyzoites and at the periphery of the bradyzoites-containing cysts.2.To determine the function of these novel GRAs,15 RHAgras strains were successfully constructed.Deletion of 13 novel GRAs（TGME49₃13440,TGME49＿247530,TGME49＿204320,TGME49＿297900,TGME49＿267740,TGME49＿268970,TGME49＿279350,TGME49＿258462,TGME49＿214410,TGME49＿291630,TGME49＿248990,TGME49＿244530 and TGME49＿258458）had no impact on the intracellular replication,egress and virulence of RH tachyzoites.Intriguingly,deletion of TGME49＿266410 and TGME49₃15910 in both RH and Pru strains decreased the parasite replication in vitro and attenuated virulence,and also reduced the cyst-forming ability of the Pru strain in mice during chronic infection.Comparison of the transcriptomic profiles of the 15 gra genes suggests that they may play roles in other life cycle stages and other genotypes of T.gondii.3.The C-terminal endogenous tagged strain,knockout strain and complemented strain were constructed to examine the role of a novel dense granule protein TGME49＿299780（designated as GRA76）.GRA76 was highly expressed and secreted mainly into the PV,and partly into the PVM mainly in tachyzoites.Intracellular replication and plaque assays showed that gra76 was important for the replication of RH and Pru strains in vitro but not for the invasion,egress and the trafficking of GRA16 and GRA24 to the host nucleus.Deleting gra76 remarkably attenuated the parasite virulence during acute and chronic infection and reduced the brain cyst burden in mice.RNA-Seq analysis revealed that the expression of genes involved in bradyzoite differentiation was increased in the mutant strain PruΔgra76.Immunization with PruΔgra76 partly protected mice from acute and chronic infection,demonstrating that PruΔgra76 could be used as a potential live-attenuated vaccine strain.4.The biological function of TGME49＿254000（named as GRA47）and TGME49＿272460（named GRA72）was also studied by the construction of epitope tagged strain,knockout strain and complemented strains.We found that GRA47 is a dense granular protein which is localized in PV and partly into the PVM in tachyzoites and in the cyst wall and matrix in bradyzoites.Deletion of gra47 significantly affect the permeability of PVM,resulting in aberrant morphology of PV and tachyzoites,severe growth deficient and remarkable attenuated virulence.However,loss of GRA47 had no influence on organelle structure and host mitochondria accumulation onto the PVM.Immunoprecipitation identified a GRA47interacting protein,GRA72.The localization of GRA72 was same as that of GRA47,and gra72 deletion also affects the PVM permeability,tachyzoite growth and in vivo virulence.Based on the results of co-localization assay and construction of double knockout strains of these four GRAs,it was speculated that GRA47 and GRA72 may form a molecular transport pore on PVM,or participate in the transport of GRA17 or GRA23,or play a synergistic role with GRA17 or GRA23 in maintaining the permeability of PVM.RHΔgra476 was confirmed to provide efficient protection against T.gondii infection.In conclusion,the basic biological functions of 18 novel GRAs were explored in this study.TGME49₃15910 and TGME49＿266410 are crucial to the growth of RH and Pru strains in vitro and virulence in vivo.GRA76 is a differentially expressed novel GRA in tachyzoites and bradyzoites.Knockout of gra76 in RH and Pru strains significantly affect the in vitro replication,the in vivo virulence and reduces the mice brain cyst burden.GRA76 is directly or indirectly involved in the conversion of tachyzoites to bradyzoites.Loss of GRA47 seriously affects the permeability of PVM,the morphology of tachyzoites,the growth ability in vitro and the virulence in vivo.GRA47-interacting protein GRA72 identified by Co-IP has the same subcellular localization with GRA47.GRA72 is also essential for PVM permeability,tachyzoite replication and virulence.GRA47 and GRA72 may form a molecular transport pore on PVM,or participate in the transport of GRA17 or GRA23,or play a synergistic role with GRA17 or GRA23 in maintaining the permeability of PVM.TGME49₃15910 and TGME49＿266410,GRA76,GRA47 and GRA72 are potential candidate genes for constructing attenuated T.gondii vaccine strains.These results improve the understanding of the mechanism of T.gondii maintaining the permeability of PVM,lay a foundation for further in-depth study of the biological function of GRAs,and provide new candidate genes for the development of anti-T.gondii vaccines.