| The protozoan parasite Toxoplasma gondii is an obligate intracellular parasite.In the life cycle of T.gondii,tachyzoites are responsible for the acute infection in intemmediate hosts,and asexual development is the only form of reproduction in this infectious stage.Repeated endodyogeny is the form of tachyzoite to multiply,in which the parent parasites divide into two progeny.The parasites can reproduce continuously if not defended by an effective immune response.Cancer is induced by abnormal activation of the proto-oncogene.Whether exist some correlations between the constant propagation of T.gondii and the uncontrolled cancer cells.Histones are mall,conservative proteins that accounts for 50%of the total weight of chromatin and can modify the information contained in the DNA.Therefore.the present study of T.gondii histones is mainly focused on epigenetic direction,and few studies have been reported on other functional studies of histones.In this study,the oncogene Tyrosine kinase protein(TKL),Ras family protein(Ras),Rab18/RabC font-family small GTPase(Rab18),Rab43,Eukaryotic transcription Initiation 5A(eIF-5A)were selected to study the effects on T.gondii.After knocking out of the oncogenes through CRISPR/Cas9,no parasite was survival,which indicated that these oncogenes were necessary genes for the growth of T.gondii.Therefore,in subsequent experiments,we conducted further functional studies on Rab18,Rab43 and eIF-5A.In addition,we also analyzed the effects of T.gondii H3 and H4 on the functions of murine macrophages.This study is of great significance to elucidate the reproductive mechanism of T.gondii,and provides a reference for the study of the relationship between the parasite and the host.1 Generation of oncogene knockout strains of Toxoplasma gondiiIn this study,Toxoplasma gondii(T.gondii)Tyrosine kinase protein(TKL),Ras family protein(Ras),Rab18/RabC font-family small GTPase(Rab 18),Rab43,Eukaryotic transcription Initiation 5A(eIF-5A)and control gene Calcium dependent protein kinase 3(CDPK3)targeting CRISPR plasmids were constructed by changing the sgRNA of the plasmid pSAGl-CAS9-TgU6-sgRNA(UPRT)to the specific sgRNAs through Q5 Site-Directed Mutagenesis Kit.The homology template plasmids for deleting the target sequences were generated by flanking the regions 5' and 3' of each gene coding regions surrounding the pyrimethamine-resistant DHFR*cassette using Clon Express(?)MultiS one step cloning kit.After the identification of PCR and restriction enzyme digestion,all plasmids were given the correct target band.Electroporation method was used to introduce the CRISPR plasmids along with the disrupting plasmids into the parasites.After screening by 1 ?M Pyrimethamine,genomic DNA was extracted and identified by PCR.The knockout experiments were performed independently five times,only CDPK3 was identified as positive.Thus,it can be concluded that TKL,Ras,Rab18,Rab43 and eIF-5A are necessary genes for the growth of T.gondii.2 The effects of Toxoplasma gondii eIF-5A on murine macrophages and parasites reproductionIn this study,the recombinant protein of Toxoplasma gondii(T.gondii)eIF-5A(rTgeIF-5A)was obtained,and the effects of the rTgeIF-5A on the functions of macrophages was analyzed by incubating the rTgeIF-5A with murine macrophages.The results showed that eIF-5A protein could promote the proliferation,phagocytosis and apoptosis of macrophages,and induce the secretion of NO,IL-6 and TNF--? in macrophages.However,the TLR4 level of macrophages and chemotaxis was inhibited.In the previous study,it was indicated that eIF-5A was necessary in the growth of T.gondiii.Furthermore,RNA interference technology was used to analyze the function of eIF-5A.The results of plaque and reproduction assay showed that eIF-5A could decrease the breeding rate of T.gondiii in vitro.Adhesion and invasive experiments further demonstrated that eIF-5A enables the parasite to establish a firm clasp on the host cell and commit to invasion.Finally,in the virulence experiment the survival time of the mice was prolonged after eIF-5A disturbance.In conclusion,eIF-5A is a necessary gene for T.gondiii,which can affect the host's response in vitro,and plays an important role in the reproduction of T.gondiii.3 The effects of Toxoplasma gondii eIF-5A on the proteome of the parasitesThe purpose of this chapter is to identify the proteins regulated by Toxoplasma gondii eIF-5A.The eIF-5A specific siRNA was transfected into T.gondii RH strains,and after incubation for 24 hours,the parasite was collected and the protein was extracted.Proteome analysis was performed using Isobaric tags for relative and absolute quantitation(ITRAQ)technology.The results showed that a total of 1391 peptide segments and 581 proteins were identified.Among the 581 identified proteins,the expressions of 359 proteins were changed after eIF-5A disturbance,among which 223 were down-regulated and 136 were up-regulated.Through the analysis of the results,eIF-5A can regulate the expression of multiple genes and participate in the regulation of important signaling pathway.Some of the proteins related to the invasion and virulence which were secreted by three special secretory organelles,microneme,rhoptry and dense granules were included in the down-regulated proteins.Therefore,it is possible to further explain the preliminary experimental conclusion that eIF-5A is the essential gene of T.gondii,and is involved in the invasion and reproduction of parasites.4 The effects of Toxoplasma gondii Rab43 on parasites reproductionRab43 is an essential gene for the growth of T.gondii.To continue the study,RNA interference technology was used to study the function of Rab43,and it was indicated that after the interference of Rab43,the adhesion and invasion ability of T.gondii were significantly decreased.The plaque assay showed that.after the interference of Rab43 the formation of plaque slowed down indicated the replication was inhibited.In addition,it was found that after the interference of Rab43 the numbers of parasites in the vacuole was much fewer than the control group.Finally,in the virulence study,the survival time of the mice was significantly prolonged after the interference of Rab43.Taken together,Rab43 plays an important role in the reproduction of parasites in vivo and in vitro.5 The effects of TgCDPK3 on the reproduction of Toxoplasma gondiiIn previous study,the gene deletion strain of Toxoplasma gondii CDPK3 was obtained using CRISPR/Cas9 technology.In this study,the effects on the reproductive function of Toxoplasma gondii were studied in vivo and in vitro.It was found that the absence of CDPK3 gene had little effect on the propagation of T.gondii in vitro.The effect of CDPK3 in mice was analyzed and the results showed that the survival time of the mice infected with CDPK3 knock out parasites was prolonged.The results of adhesion and invasion experiments showed that,after CDPK3 knock out,the parasite's adhesion and invasion ability to host cells decreased.Taken together,CDPK3 gene deletion can slow the reproduction of T.gondii through the influence of adhesion and invasion.6 The effects of Toxoplasma gondii Rab18 on the functions of murine macrophageIn the present study,the effects of Toxoplasma gondii Rab18(TgRabl8)on the function of macrophages was analyzed.The recombinant TgRab 18 was co-incubated with Ana-1 cells,and immunofluorescence technique was used to verify the binding ability of rTgRabl8 to Ana-1 cells.The effects of rTgRab18 on apoptosis,phagocytosis,toll-like receptor 4(TLR4)expression level and cytokine secretion were detected by flow cytometry.The results showed that rTgRab 18 can inhibit the proliferation level,chemokines and expression levels of the cell surface TLR4 level through combining with murine macrophages.In addition,it indicated that rTgRabl8 could significantly promote the apoptosis and phagocytic ability of macrophages.After incubating with rTgRabl8,the secretion of tumor necrosis factor a(TNF-?),interleukin-6(IL-6)and nitric oxide(NO)levels were significantly increased.The above results showed that T.gondii Rabl8 could affect the functions of murine macrophages in vitro.7 The effects of Toxoplasma gondii Histone3 on the functions of murine macrophages.In this study,the prokaryotic expression plasmid pet-3 2a(+)-TgH3 of was constructed,and rTgH3 protein was obtained after IPTG induction and purification.The analysis of TgH3 sequence shows that it is highly conservative.Western blotting results showed that anti-TgH3 polyclonal antibodies and anti-T.gondii were able to identify total soluble proteins and rTgH3 proteins of T.gondii.Immunofluorescence experimental results showed that TgH3 could combine with murine macrophage.After incubation with rTgH3,the level of toll-like receptor 4(TLR4)of macrophages was decreased.In addition,the chemotactic and proliferative effects of macrophages are inhibited.However,rTgH3 can enhance the phagocytosis of macrophages,promote cell apoptosis and cell secretion of nitric oxide(NO),interleukin-6(IL-6)and tumor necrosis factor a(TNF-?).To sum up,rTgH3 can affect some functions of murine macrophages in vitro.8 The effects of Toxoplasma gondii Histone4 on the functions of murine macrophagesToxoplasma gondii(T.gondii)is an obligate intracellular protozoa that can infect almost all nucleated cells.Histone proteins and DNA fonn the nucleosomes,which are the fundamental building blocks of eukaryotic chromatin.Histone 4 is an essential component of a histone octamer.In the present study,T.gondii histone 4(TgH4)was cloned and the regulation effect of TgH4 on murine macrophages was characterized.The results showed that TgH4 was highly conserved in structure.Western blotting outcomes suggested that the anti-TgH4 antibody and anti-T.gondii antibody can identify the total soluble protein of T.gondii tachyzoites and recombinant TgH4(rTgH4)protein,respectively.Immunofluorescence assay showed that TgH4 was a binding protein of macrophages.Following incubation with rTgH4,the Toll-like receptor 4 level of the macrophages was downregulated.Meanwhile,chemotaxis and the proliferation of macrophages were inhibited.However,rTgH4 can promote phagocytosis,apoptosis,and the secretion of nitric oxide,interleukin-6,and tumor necrosis factor-a of macrophages.Just 80 ?g/mL rTgH4 can significantly elevate the secretion of interleukin-10 and interleukin-1?(p<0.05 and p<0.01).Viewed together,these outcomes indicated that rTgH4 can affect some functions of murine macrophages in vitro. |
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