Biological Functions Of 18 Protein Serine/Threonine Phosphatases(PSPs)of Toxoplasma Gondii

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects almost all warmblooded animals(including humans)and affects approximately one-third of the world’s human population and an incalculable number of animals,which has a serious impact on human health and animal husbandry,and causes significant economic loss.Protein kinases and protein phosphatases are mainly responsible for the precise regulation of the reversible phosphorylation in eukaryotic cells.The life cycles of apicomplexans are very complex,and their key life activities are precisely regulated by phosphorylation.Protein phosphatases are involved in the proliferation,tachyzoite-bradyzoite transformation and pathogenesis of T.gondii.However,compared with protein kinases,protein phosphatases have received less attention in the functional studies of T.gondii.In this study,18 genes in the protein serine/threonine phosphatase(PSPs)family of T.gondii were selected as the research objects,and gene editing technology and phosphoproteomics were used to explore their biological functions in vivo and in vitro.1.To characterize the contribution of these genes to the infectivity of T.gondii,localization and knock-out(KO)strains of type I RH strain deficient in the 17 PSP genes(PP5,PP7,EFPP,SLP,PPM3 F,PPM4,PPM5 A,PPM5B,PPM6,PPM8,PPM9,PPM12,PPM14,PPM18,CTD1,CTD2 and CTD3)were constructed by CRISPR-Cas9 mediated homologous recombination technology.The PSPs were detected in the cytoplasm(PP5,EFPP,PPM8,and CTD2),dense granules(SLP),nucleus(PPM4 and PPM9),inner membrane complex(PPM12),basal complex(CTD3)and apical pole(PP7)by indirect immunofluorescence assay(IFA).The remaining PSPs showed low or no expression in the RH strain,and their subcellular localization could not be detected by IFA.Plaque,replication,egress,and mouse virulence assays were performed on the knock-out strains of 17 PSPs,and no significant differences were observed between the 16 RHΔpsps strains and the wild-type strain(WT).However,RHΔpp7 showed significantly reduced plaque size and number(p<0.001),reduced invasion efficiency(p<0.05)and PV formation(p<0.001),as well as reduced virulence in mice(p<0.01)compared with WT strain.2.In view of the significant phenotypes of PP7 in RH strain and the significantly high expression of SLP in sexual stage observed from Toxo DB transcriptome datasets of T.gondii,we constructed the knock-out strains of PP7 and SLP in the T.gondii type II Pru strain to explore its role in chronic infection of T.gondii.Plaque assays showed that in the high dose of 5000 tachyzoites,the size and number of plaques were basically similar between the PruΔslp and WT strains,while the size and number of plaques formed by PruΔpp7 were significantly reduced(p<0.001).Likewise,in the low dose of 500 tachyzoites,the size and number of plaques formed by PruΔpp7 were significantly reduced compared to WT(p<0.001).Mice virulence experiments with high dose(5000 tachyzoites)showed that deletion of the slp gene in Pru strain did not reduce the virulence,while the depletion of pp7 significantly reduced the virulence of T.gondii compared to the WT strain(p<0.05).Meanwhile,the number of cysts in the brain of mice inoculated with PruΔpp7was significantly lower than that in mice inoculated with the PruΔslp and WT strains(p<0.05).When mice were inoculated with low-dose(500 tachyzoites)of PruΔpp7,no mortality was observed in inoculated mice(p<0.05),and the brain cyst burden was significantly lower than that in mice inoculated with the WT strain(p<0.001).3.We also studied the biological functions of the catalytic subunit PP6C of PP6.We successfully constructed the PP6C tagging strain,knock-out strain,complement strain and PP6C conserved motif mutant strains in T.gondii type I RH strain by CRISPR-Cas9 system.IFA showed that PP6C was located in the cytoplasm.Plaque assays showed that the size and number of plaques formed by RHΔpp6c were significantly lower than those of WT and RHΔpp6c-cp(p<0.001).RHΔpp6c exhibited asynchrony in division,deformity,and inability to form "rosette".Deletion of PP6C resulted in severe morphological defects,but did not affect the secretion of structures such as centrosomes,rhoptries,apicoplasts,glideassociated proteins,dense granules,mitochondria,preconical rings,micronemes and tubulins.We mutated six conserved motifs(GDIHG,GDYVDRG,RG,GNHE,HGG and H)of PP6C,respectively,and found that each motif plays a key role in the function of PP6C.Comparative phosphoproteomics revealed that cytoskeletal organization and membrane component proteins were highly enriched in the absence of PP6C,explaining the reasons for T.gondii body deformation and replication asynchrony.We selected three proteins(TGGT1＿254390,TGGT1＿252420 and TGGT1＿222400)among the differentially expressed proteins that were in a high fold phosphorylation state in the absence of PP6C by comparing phosphoproteomics.IFA showed that TGGT1＿254390,TGGT1＿252420 and TGGT1＿222400 were localized to the tachyzoite cytoplasm,plasma membrane and inner membrane complex,respectively.The plaque assays showed that the size and number of plaques formed by RHΔ222400 were significantly lower than those of RHΔ254390 and RHΔ252430(p<0.001).The virulence experiments showed knockout of PP6C attenuated the virulence of the RH strain,and inoculation with RHΔpp6c caused no death of mice.We further tested RHΔpp6c for inducing immune protection against acute and chronic infection with T.gondii.The results showed that inoculation of mice with RHΔpp6c induced significant immune protection against acute(p<0.001)infection with 1000 tachyzoites of type I RH and PYS strains,and chronic(p<0.001)infection with 20 cysts of type II Pru strain.In conclusion,the biological functions of 18 PSPs were explored in this study,and deletion of PP7 significantly reduced the growth capacity in vitro and virulence in vivo of T.gondii RH and Pru strains.As the catalytic subunit of PP6,knock-out of PP6C resulted in asynchronous replication and morphological changes in T.gondii,each conserved motif of PP6C plays a crucial role for its function,the potential downstream gene TGGT1＿222400 was identified by comparative phosphoproteomics,and the results showed that knock-out of TGGT1＿222400 significantly reduced the growth of T.gondii in vitro,RHΔpp6c can be considered as a candidate strain of live attenuated vaccine against T.gondii.These results lay a foundation for further in-depth study of the biological functions of other T.gondii PSPs and also provide new candidate live attenuated strains for the development of T.gondii vaccines.