Studies Of The Basic Functions And Immunoprotective Effect Of Toxoplasma Gondii Calcium-dependent Protein Kinases

Toxoplasma gondii,an important protozoan parasite,infects almost all warm-blooded animals and humans and causes serious health problem to animals and humans.Although Toxoplasma infection in health individuals does not display obvious clinical symptoms,it may cause severe consequences in immunocompromised patients,such as AIDS patients,and pregnant women.Furthermore,primary infection during pregnancy can cause severe neonatal malformations,miscarriage,blindness,chorioretinitis,hydrocephalus and mental retardation in their fetus.However,there were no available drugs to effectively eliminate the Toxoplasma cysts.Calcium dependent protein kinases?CDPKs?,the key mediators of signaling in Toxoplasma,have been considered as the promising drug targets for chemotherapy due to their essential roles in Toxoplasma and the absence from the genomes of mammals.Toxoplasma gondii contains 14annotated CDPKs and only three of them were studied in detail.Thus,the objectives of the present study were to investigate the functions of Toxoplasma CDPKs and evaluate whether they can be used as drug targets against Toxoplasma infection.Firstly,multiple studies have demonstrated that plant CDPKs regulate abiotic and biotic stress responses,including salt,temperature and drought stresses.In oder to elucidate whether the functions of Toxoplasma CDPKs are similar to that in plant CDPKs,we carried out a comprehensive expression analysis of CDPKs based on previously published microarrays datasets.Furthermore,we also examined the expression patterns of Toxoplasma CDPK genes under alkali,acid,low temperature and high temperature conditions.Our results showed that most Toxoplasma CDPKs exhibited different expression levels at different developmental stages and may regulate stress responses.Secondly,the objective of the third part of this study was to study the functions of six CDPKs?CDPK4,CDPK4A,CDPK5,CDPK6,CDPK8,and CDPK9?in Toxoplasma to assess whether they are suitable as potential drug targets.CRISPR-Cas9 system were used to disrupt six CDPK genes successfully by insertion of DHFR*at the guide RNA-targeted region in the six endogenous CDPK loci and successfully obtained the six knockout CDPK strains.The biological characteristics of the six strains were evaluated by plaque assays,invasion,replication,egress,and virulence assays,respectively.The results indicated that there was no significant difference between the six?CDPK strains and the wild-type strain in the lytic cycle including invasion,replication and egress,and virulence in mice.In conclusion,the six CDPKs are not essential for Toxoplasma lytic cycle and also not virulence factors for mice,suggesting that the six CDPKs may have functional redundancy.Thirdly,in order to study the functional redundancy among the six CDPKs,eight double gene knockouts were generated using the CRISPR-Cas9 systems to elevate whether these CDPK genes have redundant functions during the parasite's lytic cycle in vitro and in mice in vivo.We used?CDPK8 or?CDPK4A as the background strains to delete CDPK4,CDPK5,CDPK6 and CDPK9successfully by insertion of the Ble amplicons in the coding sequence.The biological phenotypes of the double gene knockouts were tested by plaque assays,invasion,egress,replication,and virulence assays,respectively.However,these results showed that there was no significant difference between these double gene knockouts and the wild-type RH strain in growth defect in vitro and in the mouse model in vivo,suggesting that these CDPKs may participate in other functions in Toxoplasma or have some functions in other developmental stage?s?or in special hosts.Fourthly,in order to determine the immunogenicity of Toxoplasma CDPK2 in BABL/c mice,the eukaryotic plasmid pVAX-CDPK2 was constructed and this plasmid was successfully expressed in HEK293T cells.BABL/c mice were intramuscularly immunized with pVAX-CDPK2and followed by intraperitoneal injection with the highly virulent Toxoplasma RH strain.Immunization with p VAX-CDPK2 induced specific Th1-biased immune responses,with high IgG antibody titers and increased T cells.The levels of Th1-type cytokine?IFN-?and IL-12?and Th2-type cytokine?IL-10?immunized pVAX-CDPK2 were significantly increased.After challenge with 10³ RH tachyzoites,the immunized mice survived significantly longer compared to the control mice.Finally,in T.gondii,CDPK2 deficiency causes unchecked starch accumulation and death of chronic-stage parasites,suggesting that CDPK2 mutants can be used as a live attenuated vaccine.We evaluated the ability of PRU?CDPK2 to induce long-term immunity against acute,chronic and congenital toxoplasmosis in Kunming mice.Kunming mice vaccinated with PRU?CDPK2elicited strong Th1-type humoral and cellular responses with a higher level of serum IgG titers,and increased cytokines levels of IFN-?,IL-12,IL-2,and IL-10.Mice vaccinated with PRU?CDPK2 were resistant to subsequent challenge with 10³ tachyzoites of type I RH and two ToxoDB#9 strains?PYS and TgC7?.For the chronic infection,vaccinated mice challenged with 20PRU cysts exhibited significant reduction of brain cyst compared to naive infected mice.Kunming mice vaccinated with PRU?CDPK2 elicited strong Th1-type humoral and cellular responses with a higher level of serum IgG titers,and increased cytokines levels of IFN-?,IL-12,IL-2,and IL-10.Mice vaccinated with PRU?CDPK2 were resistant to subsequent challenge with 10³ tachyzoites of type I RH and two ToxoDB#9 strains?PYS and TgC7?.For the chronic infection,vaccinated mice challenged with 20 PRU cysts exhibited significant reduction of brain cyst compared to naive infected mice.In the model of congenital toxoplasmosis,mice vaccinated with PRU?CDPK2 that were infected?RH or PRU cysts?during pregnancy significantly increased the survival rates of pups and also reduced the number of infected pups and the brain cysts loads in the pups.Taken together,our results showed that PRU?CDPK2 is an effective vaccine against acute,chronic and congenital toxoplasmosis in Kunming mice.In conclusion,we present a genome-wide expression analysis of CDPK genes in T.gondii for the first time,and the mRNA levels change with abiotic and biotic stresses,suggesting their functional roles in these processes and demonstrated six CDPKs?CDPK4,CDPK4A,CDPK5,CDPK6,CDPK8 and CDPK9?are not essential for T.gondii lytic cycle and also not virulence factors for mice,suggesting that the six CDPKs may participate in other functions in T.gondii.In addition,we also evaluated the protective immunity of PVAX-CDPK2 and PRU?CDPK2,and our results showed that PVAX-CDPK2 and PRU?CDPK2 are effective vaccines against Toxoplasma gondii infection in mice.