Identification, Subcelluler Locatization And Analysis Of The Function Of TgPRMT5 In Toxoplasma Gondii

Background:Toxoplasma gondii is one of the single-celled intracellular parasites that can infect almost all warm blooded animal nucleated cells. It is estimated that a third of the worldwide population is under the threat of T. gondii and the serum positive rate in some area even can be as high as 80%.In addition, toxoplasmosis is also a clinically important opportunistic pathogen Chronic infection with latent bradyzoite cysts is asymptomatic in immunocompetent individuals; however, upon host immunosuppression such as cancer or AIDS patient, the parasite reconverts into its proliferative tachyzoite form, which causes severe tissue damage that can result in organ failure and death.Therefore, T. gondii is a widely distributed, serious zoonosis pathogen.T. gondii is also a opportunistic protozoan that can infect a wide range of nucleated cells and differentiate into bradyzoites within tissue cysts that remain latent. However, the current clinical drug for the treatment of toxoplasmosis, such as pyrimethamine, acetylspiramycin, trimethoprim-sulfamethoxazole and IFN-λ、 IL-2, has no effect on bradyzoites in cyst. Understanding the molecular mechanisms underpinning the conversion of these life stages may identify novel molecular targets for treatment. Translational control plays a critical role in the regulation of gene expression in most organisms. But few transcription factors can be found in apicomplexan phylum parasite genome for Plasmodium and toxoplasma, which indicates other regulation pathways exist in these top eukaryotes.Epigenetic gene regulation is an important way of gene regulation in eukaryotes.Chromatin can be fashioned to be transcriptionally repressive or permissive, dependent in part on posttranslational modifications made to histones, a phenomenon referred to as the histone code. It has been confirmed that T. gondii has a full set of coding histones and histone modification enzyme gene sequence under the scanning of T. gondii genome data. Kami. Kim has found that toxoplasma histone has many post-transcriptional modifications such as methylation modification and acetylated modification, etc through mass spectrometry research.Arginine methylation modification, a wide phenomenon in cytoplasm and nuclei of protein modification after translation, is becoming more and more important over the decades research. Protein arginine methyltransferases (PRMTs) are a family of enzymes that can methylate protein arginine residues. The PRMT family consists of protein Arg methyltransferases that are divided into two types,whereas the Type Ⅰ enzymes catalyse the formation of asymmetric dimethyl-Arg (ASDMA), the Type Ⅱ enzymes form symmetrical dimethyl-Arg (SDMA).T. gondii contains five putative PRMTs in its genome,TgPRMT1 can methylate Argonaute protein and H4R3, TgCARM1 can induce stage conversion to bradyzoite forms by modifying H3R17.PRMT5 belongs to the type Ⅱ arginine methyltransferase. It is reported that PRMT5 can msethylate H2A,H3,H4 to regulation gene transcription in mammals. Our research will identify the characteristics of PRMT5 and determine if PRMT 5 has an impact onT. gondii gene expression.objective:1. To amplify and sequence TgPRMT5 gene and analysis the expression of the gene between tachyzoite and bradyzoite2. To observe the subcellular location of TgPRMT5 between tachyzoite and bradyzoite3. To assay the metylation of TgPRMT5 in vivo and in vitro4. To observe influence of TgPRMT5 to toxoplasma goondiiMethods:1. Extract total RNA of toxoplasma gondii,the TgPRMT5 gene was amplify by RT-PCR and ligated into PEASY-Blunt to sequencing.compare the expression of the gene by RT-QPCR and western blot2.To observe the subcellular location of TgPRMT5 by IFA and western blot3.To verify toxoplasma histone whether was modified by symmetric dimethylarginine in vivo by western blot.4.To make use of HA tag to collect the TgPRMT5 complex by IP.MS analysis the complex and carry out methylation assay in vitro5.RT-PCR amplify the TgH3 and TgH4 gene and cloned to PET-32a(+).Transform the recombinant plasmids into BL(DE3) and induce by IPTQanalysis the expressed products by SDS-PAGE and western blot6. To purify the recombinant protein with Ni-NTA7.To constuct TUB-CAT-PRMT5 plasmid and electrotransfect to wild-type RH parasite,analysis the relative expression of TgPRMT5 by RT-QPCR8.To constuct Psagl:cas9-u6::sgPRMT5 plasmid and electrotransfect to wild-type RH parasite,pick the positive clone that the TgPRMT5 gene is knock out by PCRResults:1.We RT-PCR amplyfied the TgPRMT5 gene in RH parasite and Pru parasite.Compared with GTlparasite,it has 100% nucleic acid sequence homology in RH parasite and 99% nucleic acid identity in Pru parasite2.TgPRMT5 was detected throughout asexual life history.The expression has significant diference in tachyzoite and bradyzoite of the same gene-type parasite, it also showed evident difference in genotypes.3.Indirect immunofluorescence assay with anti-HA tag antibodies detected TgPRMT5 protein subcelluler location.we find that the protein mainly distributed in cytoplasm in tachyzoite,but in bradyzoite,it mainly located in nucleus.The western blot assay showed same results.4.we verified toxoplasma histone H4R3 and H3R26 locus existed symmetric dimethylarginine modification.5. We seccessfully constructed the plasmids PET-32a-TgH3 and PET-32a-TgH3 and induced and purified the recombinant protein with IPTG and Ni-NTA respectively.6.Endogenous TgPRMT5 complex was purified by Protein A/G Plus Agarose with HA-tag antibody and used for MS analysis and methylation assay.TgPRMT5 showed methylase activity towards either rTgH3 and rTgH4 or calf core histone in vitro. We have identified more than 100 proteins by MS analysis,they maby is substrate to TgPRMT5 or interact with it,such as H4.7.We seccessfully up-reguted the expression of TgPRMT5 in wild-type RH parasite by electrotransfect the plasmid TUB-CAT-PRMT5 and constuctd the Psag1:cas9-u6::sgPRMT5 plasmid to knock out the gene.We did not get the positive clone that TgPRMT5 is disrupted.Conclusion:We have determined the dynamic expression and cellular localization of TgPRMT5 in toxoplasma gondii.we also demonsrated that TgPRMT5 can methylate H4R3 and H3R26 in vivo and in vitro.This suggests that TgPRMT5 may function in regulation the proliferation or differentiation...