Biological Function And Protein Structure Prediction Of ERK7 Gene In Toxoplasma Gondii

As the most ancient signal transduction pathway, mitogen-activated protein kinases（MAPKs）participate in many important biological and physiological processes such as proliferation,differentiation and apoptosis through phosphorylation and dephosphorylation, which also mediate the developments of stress, inflammatory and cancer. Extracellular signal-regulated kinases（ERKs） belong to the MAPK subfamily that exist in almost all the eukaryotic cells. As the reports of recent studies, two novel MAPK homologues（Tg MAPK1 and Tg ERK7） were identified in Toxoplasma gondii using sequence alignment methods. As the homologue of p38αMAPK, Tg MAPK1 plays an important role in the conversion of T. gondii from tachyzoites to bradyzoites in vitro. For Tg ERK7, very little is known especially about its biological function and protein structure, which were the research objectives in the present study.Part 1: Immune responses induced by p VAX/Tg ERK7 in BALB/c miceFive short peptides of Tg ERK7 protein（3-14 aa, 237-249 aa, 346-358 aa, 461-472 aa and647-658 aa） were determined using DNAStar 5.0, NCBI/BLAST and antigen profiler peptide tool software, and their specific antibodies with high titers（αA, αB, αC, αD and αE） were successfully obtained through peptide synthesis, conjugation with KLH and serial immunization.The normal biological activity of the obtained antibodies are confirmed by incubating with Triton X-permeated T. gondii GT1 parasites.The eukaryotic expression vector p VAX/Tg ERK7 was created and its immunogenicity was evaluated using BALB/c mice in the study. Our results indicated that lower levels of humoral immune response such as specific anti-T. gondii antibodies and cellular immune responses were both induced in BALB/c mice by p VAX/Tg ERK7 vector, whereas no significant difference were both detected in their survival periods through infection with 103 GT1 tachyzoites（10.8 ± 0.2 d）and 20 PRU cysts（19.4 ± 1.1 d） in comparison with that of controls（P>0.05）. In summary,p VAX/Tg ERK7 is unable to protect BALB/c mice against the acute and chronic T. gondii infections, though it can softly induce the humoral and cellular immune responses of BALB/c mice.Part 2: Immune responses of TgERK7 protein expressed in BL21 Escherichia coliTo further explore the unknown biological function, the open reading frame of Tg ERK7 gene was cloned according to the T. gondii GT1 reference sequence（Toxo DB: TGGT1₂33010）.p ET/Tg ERK7 vectors were further constructed and their expression in BL21 E. coli competent cells were stimulated using IPTG followed by identification and purification. Immune efficacy of the production against the Toxoplasma acute and chronic infections were evaluated using BALB/c mice. The data showed that levels of antibodies in sera collected from the immunized mice, CD3e+CD4+ T lymphocytes in spleen tissues and cytokines such as IFN-γ, TNF-α, IL2 significantly increased compared to that of the controls, indicating that Tg ERK7 protein and Freund’s adjuvant can both trigger the humoral and cellular immune responses of BALB/c mice.Additionally, only one kind of antibody（anti-peptide A） is specific for T. gondii infection which was speculated by comparing to that of other groups.The results of Toxoplasma infections showed that the survival periods of TgERK7protein-immunized BALB/c mice（13.8 ± 3.4 d） against wild-type GT1 parasites are significantly prolonged and the PRU cysts in the brains of the survival mice decrease, suggesting that Tg ERK7 protein is a potential vaccine candidate against T. gondii GT1 acute and PRU chronic infections. Furthermore, specific anti-Tg ERK7 antibody αA may play an important role in T.gondii GT1 infection by distinguishing the immune responses of BALB/c mice induced by p VAX/Tg ERK7 and Tg ERK7 protein.To further confirm our above deduction, high levels of specific anti-Toxoplasma antibodies（αA,αB, αC, αD and αE） were employed to interact with T. gondii GT1 tachyzoites in vitro; and the untreated parasites were used as negative control. Our results showed that the intracellular proliferation of αA-disposed parasites significantly decreased compared to that of the negative control and other groups（P<0.05）, suggesting that the infection process of T. gondii GT1 parasites are partly impeded by specific antibody αA. We thus concluded that Tg ERK7 protein is an important virulence factor for this parasite.Part 3: Creation, identification and virulence analysis of the knockout strainΔTg ERK7To further grope the functions of Tg ERK7 gene, the homologous recombinant vectorΔERK7-p Bluescript II SK（+） was created successfully through PCR amplification, restriction endonuclease digestion and T4 ligation. Part of Tg ERK7 locus was replaced with resistance gene Sh ble through electroporation into T. gondii GT1 tachyzoites and the knockout strain ΔTg ERK7 was finally obtained by selecting with phleomycin. Additionally, the identifications of specific PCR, Southern blotting and Western blotting suggested that partial deficiency can result in the global silence of Tg ERK7 gene.The results of virulence assessment indicated that the BALB/c mice challenged with 1×103ΔTg ERK7 parasites were all survival compared to that of wild-type GT1 tachyzoites（10.6 ± 0.3d）. Surprisingly, all the survival ΔTg ERK7-infected mice were also survival by challenging with the same dose of wild-type GT1, suggesting that Tg ERK7 gene and/or its products are important for the virulence of wild-type GT1 tachyzoites, which may participate in the pathopoiesia of this parasite.Moreover, levels of antibodies in sera, cytokines and NO in sera and peritoneal fluids of Toxoplasma-infected mice were measured. The results indicated that the overproduction of inflammatory-related cytokines such as IFN-γ, MCP-1 and IL6 leads to the acute death of wild-type GT1-infected mice. We also calculated the number of parasites in enterocoelia and spleen tissues using quantitative real-time PCR, and the data showed that the number ofΔTg ERK7 parasites in enterocoelia significantly decreased at 7 day post-challenge compared to that of wild-type GT1（P<0.05）, whereas no difference was detected in spleen tissues（P>0.05）,which suggested that Tg ERK7 is a novel virulence factor of wild-type GT1 and may participate in the intracellular proliferation of this parasite.Part 4: The biological function of Tg ERK7 gene in Toxoplasma gondiiTo provide essential experimental evidence and further confirm the obtained conclusions, the complementary vector p UC/SAG1/CAT/GRA1/SAG1/Tg ERK7/GRA1 was created based on the backbone of p UC19 vector followed by extracted, purified and repeatedly electroporated intoΔTg ERK7 parasites. The complementary strain p UC/Tg ERK7 was finally obtained throughchloramphenicol selection. Re-expression of Tg ERK7 protein in ΔTg ERK7 isolate was further confirmed by Western blotting and the intracellular proliferation analysis.The plaque, egress from host cells, intracellular proliferation, invasion, attachment and motility of all the obtained strains（ΔTg ERK7, p UC/Tg ERK7 and wild-type GT1） were examined and the subcellular localization of Tg ERK7 protein were detected using indirect immunofluorescence method. These results suggested that Tg ERK7 protein is an important factor mediating the invasion of T. gondii GT1 tachyzoites.Part 5: Structure prediction of Tg ERK7 protein in Toxoplasma gondiiIn the last part of our study, the secondary and tertiary structures of Tg ERK7 protein were successfully predicted and evaluated using the bioinformatics software such as PSI-PRED,DISOPRED, MEMSAT-SVM and SWISS-MODEL. Mingling the contents of biological function,subcellular localization and structure prediction of Tg ERK7 protein in the whole study, we speculated that some receptor of Tg ERK7 protein may exist in the surface of host cells and further mediate the invasion process of T. gondii.In summary, the present study demonstrated that Tg ERK7 protein is an important virulence factor that participates in the invasion of T. gondii GT1 parasites. Clearly, seeking the receptor/substrate and resolving the native conformation of Tg ERK7 protein will contribute to elucidate its regulation mechanism in this protozoan, which will be the focus of future researches.