The Cloning,Expression,Cellular Localization And Primary Functional Analysis Of Puf2,A Member Of The Puf Family RNA Binding Proteins From Toxoplasma Gondii

1.BackgroundThe obligate intracellular parasite Toxoplasma gondii is able to infect almost all the nucleated cells besides the erythrocytes in the host,and it may invade brain,lymph nodes,eyes,liver,heart,lung,muscle and other tissues and organs,causing zoonotic parasitic diseases,such as Toxoplasma encephalitis,Toxoplasma eye diseases etc.In general popuation,when the toxoplasma gondii tachyzoites invade into the host,it may converse into bradyzoites in the formation called cyst with weaker ability of infection,staying in the brain,muscle and retina and other cells and making the host becoming chronic infected.When the host suffering from AIDS,malignant tumors and some immune impaired stage,the cyst can be activated into tachyzoites,which may proliferate rapidly causing tissue damage,local inflammation,even death.Therefore,the conversion between tachyzoite and bradyzoite is the central part of the pathogenicity of Toxoplasma Gondii.Whereas its essence of the biological basis or mechanism in acute and chronic infection transformation is the changes in the transcription,post-transcriptional,translation,or change in the level of post-translational modifications,with the result of a different protein specificity expression.The right time,quantity and position of the correct gene expression in a life are all tightly supported by the transcription,multi-level control post-transcription and so on.Compared with the transcriptional regulation,translational regulation of gene expression in the cells can respond more quickly to external stimulation.Puf proteins,a type of RNA binding protein,have been studied in recent years from yeast to humans.Puf proteins can specificly bind proteins and RNA targets,leading them degradation or inhibited.The Puf family RNA-binding proteins(RBPs)modulate mRNA expression in a wide variety of eukaryotic species.Puf family was named after the two first-characterized proteins Pumilio in Drosophila melanogaster and fem-3 binding factor(FBF)in Caenorhabditis elegans.Puf proteins execute translation control by binding to specific ribonucleotide sequences called Puf-binding element(PBE),which typically reside in the 3’ untranslated region(3’ UTR)of target mRNAs.The signature feature of the Puf proteins is that they have a highly conserved core RNA-binding domain,referred to as the Puf domain,which always contains eight copies of a similar α-helical repeat flanked by one imperfect pseudo-repeat at each end.According to a number of studies,Puf proteins can regulate a variety progresses of eukaryotic cells,such as stem cell maintenance,biosynthetic organelles,the occurence of oocyte,neuronal function and the formation of memories etc.Remarkably,Muller’s studies in Plasmodium Falciparum Puf2 genetic revealed that the gametophyte differentiation and male gametophyte/female gametophyte proportion can be promoted after the Puf2 of plasmodium falciparum and plasmodium berghei have been knockout.Further,in the Plasmodium Berghei,after Puf2 gene being knockout,the sporozoite in mosquito’s salivary glands will convert into the similar form of the early stage in the liver in advance.All these studies reveal that Puf2 proteins in Apicomplexa can adjust a molecular mechanism of mRNA translation.As a member in Apicomplexa,do the Puf2 proteins exist in Toxoplasma gondii or what kind of function do they play in the bradyzoite and tachyzoite conversion?In this paper,we performed molecular characterization of TgPuf2 in T.gondii and determined its expression,cellular localization.2.Objective1)To detect whether RH strainToxoplasma gondii has Puf2 in the mRNA level or not;2)To observate the expression of Puf2 in Toxoplasma gondii RH strain bradyzoite and tachyzoite in the mRNA level;3)To observate the cell localization of TgPuf2 protein in RH strain tachyzoites;4)The primary functional analysis of TgPuf2.3.Methods1)In order to detect whether there is TgPuf2 in the RH strain Toxoplasma Gondii,according to the gene sequence in Toxo DB database GT1 strains of Toxoplasma Gondii Puf2(TGGT1-122890),the specific primers P1 were designed,and the RH strain cDNA was used as a template for amplification.2)Stage-specific expression of the TgPuf2 in tachyzoite and bradyzoite(induced by alkaline-stress pH 8.2)were detected by RT-qPCR.3)A Toxoplasma clone stably expressing TgPuf2 tagged at its C-terminus with the 3X hemagglutinin(HA)epitope was generated by targeting the endogenous TgPuf2locus using homologous recombination.RH genomic DNA was used to amplify a 1.5-kb fragment of the Puf2 3’end using the specific primers P2.This Puf2 fragment was inserted into the pLIC-HAx3-DHFRTs endogenous tagging vector such that the TgPuf2 coding sequence was fused in frame with the epitope coding region.The pLIC-Puf2HAx3-DHFRTs construction was confirmed by sequencing.RHAHXAKu80 parasite strains were transfected with pLIC-Puf2HAx3-DHFRTs plasmid,and after overnight growth in HFF,parasite cultures were selected with1.0μMpyrimethamine.Drug-resistant parasites were cloned by limiting dilution and screened by Western blot and immunofluorescence assays(IFA)for expression of HA-tagged TgPuf2.4)To investigate whether TgPuf2 has RNA binding activity,the putative Puf domain of TgPuf2(PCR using the specific primers P3 were expressed in a bacterial expression system.The TgPuf2 Puf domain was expressed as a fusion to the carboxyl-terminus of histidine(His).The His-tagged rTgPuf2 was affinity-purified and verificated by immunoblotting with the anti-His antibody.5)Using Electrophoretic Mobility Shift Assay(EMSA)detecting the RNA binding function of the Puf domain in the recombination protein rTgPuf2-His.6)The NO.C2 TgPuf2-HA genetically modified Toxoplasma Gondii were cultrured.An d the collected tachyzoites were used for the Immunoprecipitation experiment,then th e total RNA was extracted from the precipitate for mass analysis in order to find the RNA species which would combine with Puf2.4.Results1)In the mRNA level,TgPuf2 was found in RH strain of Toxoplasma Gondii using RT-PCR.2)Comparison of the expression of TgPuf2 in tachyzoites and bradyzoites from mRNA levels,the RT-qPCR resluts show the expression of bradyzoite is lower than tachyzoite.3)Successful tagging of the endogenous TgPuf2 protein was confirmed by IF A and Western blot.Two clones with the HA tag integrated at the TgPuf2 locus were selected for protein expression analysis.Western blot using the anti-HA antibodies detected a specific protein band of 206kDa,consistent with the predicted size of the TgPuf2-HA fusion protein,whereas this protein was not detected in the control RHΔHXΔKu80 parasites.4)IFA with anti-HA antibodies detected TgPuf2 protein in the cytoplasm,consistent with its function in translation control.5)The recombinant Puf domain of TgPufl showed high purity,and can be used in subsequent experiments like RNA binding experiments.5.ConclusionsTgPuf2 appears to be different expression levels in the tachyzoite and bradyzoite,suggesting that TgPuf2 may have some function in regulating the proliferation or/and differentiation that are important in providing parasites with the ability to respond rapidly to changes in environmental conditions.This study provides a starting point for elucidating the function of TgPuf2 during the parasite development.