| ObjectiveToxoplasmosis is a world-wide parasitic zoonosis which causes pregnant abortion and fetal defects. It is one of the most important pathogens that cause the death of immunodeficient patients. However, all the drugs for treatment have severe side effects and no drug can eliminate the parasite in patients. The delayed death caused by the deficiency of apicoplast provides a new way for treatment of toxoplasmosis. This way could not be used as clinical treatment because of rare knowledgement of its mechanism. Therefore, study on the mechanism of delayed death is very important for prevention and treatment of toxoplasmosis.MethodsT. gondii RH strain tachyzoites were collected for RNA extraction. The ACP and FABZ coding genes were amplified by RT-PCR for construction of acp/pET28 and fabz/pET32 vectors respectively. The recombinant proteins were expressed in E. coil rostta strain and purified by Ni-NTA column and injected into rabbits to produce the polyclonal antibodies (pAbs). The expressions of ACP and FABZ in T. gondii were detected by the pAbs with Western blotting.T. gondii hxgpr- strain tachyzoites were collected for DNA extraction. The acp gene was amplified by PCR from genomic DNA and used for construction of pHX-ACP-GFP vector. The vector was transfected into tachyzoites by electroporation. The mutant tachyzoites were selected by mycophenolic acid and xanthine. The distributions of ACP-GFP were observed by Confocal microscope, and the expression of ACP-GFP was detected by anti-GFP tag pAb with Western blotting.Human foreskin fibroblast (HFF) cells were infected with 5×105 ACP-GFP mutant tachyzoites. After 24 h,1 mM clindamycin was added into culture medium. The tachyzoites were collected in 2 h,4 h and 6 h after adding the drug. The expressions of ACP and GRA1 coding mRNA were determined by Real-time PCR. The expressions of ACP and GRA1 were determined by Western blotting. The distributions of ACP-GFP in tachyzoite were observed under Confocal microscope.T. gondii TATi strain tachyzoites were collected for DNA extraction. The fabz gene was amplified by PCR from genomic DNA for construction of pTetO7-Sag1-FABZ-Ty-DHFR vector. The vector was transfected into TATi tachyzoite by electroporation. The mutant tachyzoites were selected by 1μg/ml pyrimethamine. The FABZ-Ty was detected by anti-Ty tag monoclonal antibody (mAb) with Western blotting. The tetracycline-inducible expression (TetO) system was induced by 1μg/ml anhydrotetracycline (ATc). When exposed to ATc for 24 h and 48 h, the expressions of FABZ were determined by Western blotting.HFF cells were infected with 5×105 TetO-FABZ tachyzoites.1μg/ml ATc in culture medium was added to induce the TetO system. When infected for 2 h,4 h,8 h,12 h,24 h and 48 h, the expressions of FABZ coding mRNA and the copies of uprt gene were determined by Real-time PCR. The tachyzoites/parasitophorous vacuoles in HFF cells were observed by Giemsa stain.ResultsThe 550 bp ACP and 700 bp FABZ coding genes were amplified from T. gondii cDNA. The results of PCR, restriction enzyme analysis and gene sequence indicated that acp/pET28 and fabz/pET32 had been constructed. The recombinant ACP and FABZ were expressed in E. coil rostta strain. The anti-ACP and anti-FABZ pAbs could detect the transit peptide type and mature type of ACP and FABZ in T. gondii.The acp gene of 1300 bp was amplified from the genomic DNA. The results of PCR, restriction enzyme analysis and gene sequence indicated that pHX-ACP-GFP had been constructed. A lot of tachyzoites were able to invade into HeLa cells after electric shock for once. The mycophenolic acid and xanthine in culture medium could kill the untransfected tachyzoites in 2-3 days. The punctiform and diffuse green fluorescences were distributed in apicoplast and endoplasmic reticulum respectively by Confocal microscope. The t-ACP and m-ACP could be detected by anti-GFP pAb with Western blotting.When exposed to 1μM clindamycin for 2 h and 4 h, the expressions of ACP coding mRNA had no significant change; for 6 h, the expression of ACP coding mRNA had a dramatic decrease. When exposed to clindamycin for 2 h, the expressions of t-ACP and m-ACP had no significant change; for 4 h, the expressions of m-ACP had significant decrease, but the t-ACP had no significant change; for 6 h, the expressions of t-ACP and m-ACP had dramatic decrease. When exposed to clindamycin for 2 h, the punctiform and diffuse green fluorescences were found in T. gondii at the same time by Confocal microscope; for 4 h, the intensity of punctiform green fluorescence had decrease, but the diffuse green fluorescence had no significant change; for 6 h, there was rare punctiform green fluorescence, and the intensity of diffuse green fluorescence had dramatic decrease.The length of 800 bp PCR production was amplified from TATi strain genomic DNA. The results of PCR, restriction enzyme analysis and gene sequence indicated that the pTetO7-Sag1-FABZ-Ty-DHFR vector had been constructed. The untransfected tachyzoites were killed by 1μg/ml pyrimethamine in 2-3 days. The expression of t-FABZ-Ty and m-FABZ-Ty were detected by anti-Ty tag mAb with Western blotting. The TetO system was induced when added 1μg/ml ATc in culture medium. After added ATc for 24 h and 48 h, the expressions of m-FABZ-Ty had a dramatic decrease.After exposed to ATc for 4 h, the expression of FABZ coding mRNA had a decrease compared with control group. When prolonged the exposure time, the expression of FABZ coding mRNA had significant decrease; for 48 h, the expression of FABZ coding mRNA was in very low level. When exposed to ATc for 2 h,4 h and 8 h, the numbers of tachyzoite in HFF cells had no significant difference compared with control group; for 12 h, the number of parasitophorous vacuole in ATc group was less than that of control group; for 24 h, in control group, HFF cells were destroyed by tachyzoites, and a small amount of free tachyzoites were found, but the number of parasitophorous vacuole increased and noHFF cells were destroyed in ATc group; for 48 h, there were a large number of parasitophorous vacuoles in HFF cells and some of HFF cells were destroyed in control group, while the number of parasitophorous vacuole increased in ATc group. When exposed to ATc for 2 h and 4 h, the copies of uprt gene had no significant difference between ATc group and control group; for 8 h and 12 h, the uprt gene copies of ATc group were lower than that of control group, but the difference had no significance; for 24 h and 48 h, the uprt gene copies of ATc group had a significant decrease compared with control group.ConclusionsThe transit peptide type and mature type of ACP and FABZ could be detected by anti-ACP and anti-FABZ pAbs which were produced by recombinant ACP and FABZ with Western blotting. This method is very useful for the apicoplast protein trafficking study. ACP, as an apicoplast label, can be used for study on nucleus gene coding apicoplast protein trafficking. HXGPRT is a perfect marker for T. gondii mutant construction because of the shorten time for screening mutant compared with other selection marker. It is convenient for study on T. gondii protein with the additional tag.Clindamycin could not directly interfere with the transcription and translate of nucleus gene coding apicoplast protein, but suppress the proliferation of apicoplast and decrease the number of apicoplast. Because of apicoplast defect, the apicoplast protein could not change to mature protein. The deficiency of apicoplast function leads to T. gondii delayed death.The TetO system can be used for study on essential gene function in T. gondii. This system does not need to knock out the essential gene which might cause T. gondii death. The TetO system can be induced by 1μg/ml ATc and have a significant suppression for T. gondii essential gene expression.When suppressed the expression of FABZ coding gene, T. gondii tachyzoites remain grow for some time. This phenomenon is accordance with the delayed death. Our results indicate that the defect of FASⅡmight be the reason for delayed death in T. gondii.
|
PDF Full Text Request