Comparative Study Of MicroRNAs Between Bradyzoites And Tachyzoites Of Prugniaud Strain Of Toxoplasma Gondii

Toxoplasma gondii has a very complex life cycle and a wide range of intermediate host including human being. And the biological reason for the opportunitic pathopoiesis of this parasite is the conversion between tachyzoites and bradyzoites. In the course of transition,various changes occurred in the morphous and physiology of parasites, including the variance on surface, metobolism and gene expression. These transformations are usually modulated on transcriptional level. However there is the apparent lack, in apicomplexan parasites, of large families of recognizable specific transcription factors （TFs） operating in other eukaryotes [5]. While the previous research revealed that the proportion of transcription in TFs would become more and more among the whole transcription with the advancing in biology, taking examples for 3.4% in yeasts,4.2% in nematode,5.5% in drosophila and 8% in human being. The disproportionation of huge changes in gene expression and the number of TFs between the inter-conversion reminded that there must be alternative strategies existed to modulate the many phases of the Toxoplasma complex life cycle. Will miroRNA be one of the ways to regulate the gene expression in Toxoplasma gondii?Quite intriguingly, the genome sequence of T. gondii revealed the existence of Dicer, AGO, and RdRp homologues that appeared to haveplant/fungal （Dicer/RdRp） and metazoan （AGO） signatures.The first time in the world, Braun et al. illustrated the 14 miRNA families in tachyzoites of RH strain of Toxoplasma gondii in 2010. Wang et al comparatively studied small RNAs in Toxoplasma gondii of distinct genotypes in 2012.17 conserved miRNAs were discovered and 339 novel miRNAs in 3 classical Tg.genome which is actually 57 miRNAs.155 miRNAs upregulated in tachyzoites of RH strain,20 miRNAs upregulated in tachyzoites of ME49 strain and 6 miRNAs equally expressed in the two. Comprehensive analysis of miRNAs discovered in T.gondii, the conserved miRNAs between different genotypes have a special expressing method by switching arms of pre-miRNA, and the expression level is distinct with the variation of host cells and environments.It is inferred that miRNAs is possibly a important modulator of gene expression in T.gondii. While the number, type and the role in tachyzoite and bradyzoite of same genotype is poorly known. So the study of miroRNA on Toxoplasma gondii may uncover the mystery of this parasite, which is promising to be a new genetic tool and drug target for controlling and curing Toxoplasmosis. The study will also give the origination of miRNA a new explanation.Objective1. The establishment of animal model acutely and chronically infected by Toxoplasma gondii2. Purification of tachyzoites and bradyzoites and the extraction of their total RNA.3. Solexa sequencing of small RNAs from tachyzoites and bradyzoites and the bioinformatical analysis of the role of sRNAs in the modulation of gene expression between the conversion of tachyzoites and bradyzoites.Methods1. The establishment of animal model chronically infected by Toxoplasma gondii2. Collection and purification of cysts the brains of mice chronically infected by Toxoplasma gondii purifie density-gradient centrifugation on Ficoll.3. The rupture of cysts wall and the extraction of total RNA from bradyzoites4. The establishment of animal model acutely infected by Toxoplasma gondii5. The culture and purification of tachyozite in vitro and the ex extraction of total RNA from tachyzoites6. The establishment of small RNAs library from tachyzoites and bradyzoites, highthroughout sequencing and the bioinformatic analysis of the two small RNAs libray.6.1 Date cleaning of the primary sequences.6.2 Genetic a of the sequencing small RNAs from tachyzoites and bradyzoits.6.3 Statistics and length distribution of the sequencing small RNAs from tachyzoites and bradyzoits.6.4 Analysis of common and specific sequences between two samples6.5 Annotation of the sequencing small RNAs from tachyzoites and bradyzoits.6.6 Reprediction of the identified miRNA in the reference of Tg.genome and the differencial expression of repredicted miRNAs.6.6.1 Reprediction of the identified miRNA in the reference of Tg.genome by the Mireap in BGI.6.6.2 RNAfold software were used to analyse the minimum free energy6.6.3 The differential expression analysis of qualified known miRNA6.7 Prediction of novel miRNA and its secondary structure by Mireap from unannotated sRNAs and the differential expression analysis of novel miRNA.6.8 Characteristics of novel miRNAs.6.9 Analysis of conserved miRNAs of Toxoplasma gondii in PRU strain.6.10 Target prediction for novel miRNAs6.11 GO enrichment analysis and KEGG pathway analysis6.12 The conserved miRNAs in strains of Toxoplasma gondiiResults1. About 50% mice chronically infected T.gondii survived after the acute period of disease. The animal model will be successfully established after 30 days pi.2.11,000 cysts were purified from brain of 60 chroniclly infected mice by T.gondii of PRU strain.3.1 Trizol regent cannot fully permeate through the cyst wall and clearage the bradyzoits in cysts.3.20.25% trypsin were used to digest the cyst wall for 1 minute at room temperature. And the vitality of released bradyoites were verified by vital staining and dead body dyeing.3.3 Total RNA were extracted from bradyzoites by Trizol regent.4. The acutely T.gondii infected animal model is successfully established. And the bradyzoites were activated into tachyzoite in vivo.5. Tachyzoites collected in vivo would infected HFF cell for 6-7 days in vitro. And tachyzoites were purified and total RNA extracted.6. Establishing sRNA libraries, high throuth-out sequencing and bioinformatic analysis of tachyzoites and bradyzoits.6.19206309 and 9873245 clean reads were obtained from tachyzoites and bradyzoite of PRU-T.gondii （Table.6-1）. Their length distributions dominated in 21 nt and 20 nt respectively.6.2 Genetic a of the sequencing small RNAs from tachyzoites and bradyzoits.6.2.1507371 reads,5.51% reads in tachyzoite sRNA library, matched to human genome only,which was deleted in the following analysis. And 52.4% reads belong to the ToxoDB.6.2.2 3173753 reads,32.15%reads in bradyzoite sRNA library, matched to mouse genome only,which was deleted in the following analysis. And 51.67% reads belong to the ToxoDB.6.3 The length distribution of the two samples ranges from 18 nt to 23nt, and dominated in 21 nt and 20 nt respectively.6.4 In the sRNA libray of tachyzoite,94.13% （511692） of the total uniques are tachyzoite-specific reads, while 93.05%（452710 unique-reads） of that are bradyzoite-specific reads. The common unique reads between the two samples are 31914 reads.6.5Proportion of the known miRNA, rRNA, tRNA, snRNA and noRNA is similar in the two samples.6.6 Two known miRNAs similar to hsa-mir-4463 and cin-mir--4183-5p were discovered in tachyzoites and bradyzoites. Both the two up-regulated in bradyzoites.6.7 Novel miRNA discovered in tachyzoites and bradyzoites of PRU.T.gondii214 novel miRNAs in bradyzoites and 24 novel miRNA in tachyzoites were discovered in bradyzoites and tachyzoites. In the 13 common novel miRNAs expressed in the two samples （Fig 6-18）,9 of them upregulated in bradyzoites, while the other 4 miRNAs equally expressed （Fig 6-19）About 50.41% and 58.5% of the novel miRNAs in tachyzoites and bradyzoites have a 5’"A"bias in the first nucleotide （Fig 6-20）. According to genomic locations, T. gondii MIRNAs were categorized into six types:UTR, intronic, intergenic, mix regions, exon, and 2-3 kinds in the 3 classical genomes （Fig 6-21）.84 MIRNA located in exons,7 MIRNA in mix region, and 71 miRNAs have 2-3 kinds of location. A great number of miRNA has counts less than 20 （Fig 6-22）. Only 57 miRNAs have miR* （Fig 6-23）. Majoriy of the miRNAs have 3 pre-miRNA predicted in the 3 T.gondii genomes.But a few miRNAs has more than 3 precursors such as novel_mir₁22（16 precursors）, novel_mir₄0 （9 precursors）, novel_mir₁65 （8 precursors）.6.8 Analysis of conserved miRNAs in PRU.Tgondii. special conserved miRNA6.9 Target gene prediction of the two samples 234138 predicted target genes of 24 novel miRNAs in tachyzoites and 2114530 predicted target genes of 214 novel miRNAs in bradyzoites were obtained.6.10 General analysis of GO terms and KEGG pathways of target genes 7 GO fuction terms were significantly abundant in the target genes of tachyzoites’ miRNAs. （i） hydrolase activity, acting on acid anhydrides; （ii） hydrolase activity, acting on acid anhydrides, in phosphorus-containing anhydrides; （iii） pyrophosphatase activity; （iv） ATPase activity, coupled; （v） peptidase activity; （vi） peptidase activity, acting on L-amino acid peptides; and （vii） catalytic activity.6.11 Conserved miRNAs in T.gondii of different strains.6 conserved miRNAs in different strains of T.gondii were analysed. Their distinct expression methods and level were observed as well.Conclusion1. The first revelation of the small RNAs from tachyzoites and bradyzoites of same genotype.2.2 known miRNAs and 225 novel miRNAs in PRU.Tgondii were discovered and majority of them have a differenial expression in the two samples.3.7 GO fuction terms were significantly abundant in the target genes of tachyzoites’ miRNAs, which surpports that the miRNAs in tachyzoite repress the catabolism of energy and material of itself.4. comprehensively analysis of the conserved miRNAs in T.gondii suggests that miRNA is of great possibility to play a role in the conversion between tachyzoites and bradyzoites.