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Distinct strains of Toxoplasma gondii feature divergent transcriptomes regardless of developmental stage.

ABSTRACT: Using high through-put RNA sequencing, we assayed the transcriptomes of three different strains of Toxoplasma gondii representing three common genotypes under both in vitro tachyzoite and in vitro bradyzoite-inducing alkaline stress culture conditions. Strikingly, the differences in transcriptional profiles between the strains, RH, PLK, and CTG, is much greater than differences between tachyzoites and alkaline stressed in vitro bradyzoites. With an FDR of 10%, we identified 241 genes differentially expressed between CTG tachyzoites and in vitro bradyzoites, including 5 putative AP2 transcription factors. We also observed a close association between cell cycle regulated genes and differentiation. By Gene Set Enrichment Analysis (GSEA), there are a number of KEGG pathways associated with the in vitro bradyzoite transcriptomes of PLK and CTG, including pyrimidine metabolism and DNA replication. These functions are likely associated with cell-cycle arrest. When comparing mRNA levels between strains, we identified 1,526 genes that were differentially expressed regardless of culture-condition as well as 846 differentially expressed only in bradyzoites and 542 differentially expressed only in tachyzoites between at least two strains. Using GSEA, we identified that ribosomal proteins were expressed at significantly higher levels in the CTG strain than in either the RH or PLK strains. This association holds true regardless of life cycle stage.

SUBMITTER: Croken MM

PROVIDER: S-EPMC4230917 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Distinct strains of Toxoplasma gondii feature divergent transcriptomes regardless of developmental stage.

Croken Matthew McKnight MM Ma Yanfen Y Markillie Lye Meng LM Taylor Ronald C RC Orr Galya G Weiss Louis M LM Kim Kami K

PloS one 20141113 11

Using high through-put RNA sequencing, we assayed the transcriptomes of three different strains of Toxoplasma gondii representing three common genotypes under both in vitro tachyzoite and in vitro bradyzoite-inducing alkaline stress culture conditions. Strikingly, the differences in transcriptional profiles between the strains, RH, PLK, and CTG, is much greater than differences between tachyzoites and alkaline stressed in vitro bradyzoites. With an FDR of 10%, we identified 241 genes differentia ...[more]

PMID: 25393307

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Project description:tRNAs are critical linker molecules that couple mRNA to protein translation and their availability is a limiting factor for translation. tRNA transcription and the composition of the tRNA pool are both dynamically regulated in response to environmental cues. Recent work suggests that they also play an important role in regulating cell growth and differentiation. Toxoplasma gondii is a highly adaptable, early eukaryotic protozoan that is able to replicate in virtually any nucleated cell and many different host organisms, thus requiring fine-tuning of protein expression. A key feature of parasite pathogenesis is the ability to differentiate between fast growing tachyzoite and slow growing bradyzoite cyst forms, in response to stress e.g. nutrient starvation. Differentiation is accompanied by changes to the mRNA transcriptome and proteome but little is known about translational regulation. In this study, we used comparative genomics to catalogue the repertoire of tRNA genes in T. gondii and their genomic distribution. Interestingly, many tRNAs occur in regulatory regions and intronic sequences of protein-coding genes, which correlates with increased gene expression. To characterize the tRNA pool of T. gondii during stress and differentiation, we surveyed levels of precursor tRNA molecules in the tRNA population by small RNA-seq in three divergent parasite strains with varying bradyzoite differentiation competencies, cultured in normal or bradyzoite differentiation-inducing (pH shock) conditions. Almost all tRNA genes are constitutively transcribed in T. gondii tachyzoites and transcription of individual tRNA genes differs significantly between strains. The abundance of individual tRNAs in the tRNA pool reflects proteomic codon usage, suggesting that tRNA levels are tightly controlled according to the demands of translation. Together, this work suggests that modulation of the tRNA pool is a mechanism by which the protein landscape in T. gondii is regulated in different strains and developmental stages.

Project description:BACKGROUND:Toxoplasma gondii is one of the most important apicomplexan parasites and infects one-third of the human population worldwide. Transformation between the tachyzoite and bradyzoite stages in the intermediate host is central to chronic infection and life-long risk. There have been some transcriptome studies on T. gondii; however, we are still early in our understanding of the kinds and levels of gene expression that occur during the conversion between stages. RESULTS:We used high-throughput RNA-sequencing data to assemble transcripts using genome-based and de novo strategies. The expression-level analysis of 6996 T. gondii genes showed that over half (3986) were significantly differentially expressed during stage conversion, whereas 2205 genes were upregulated, and 1778 genes were downregulated in tachyzoites compared with bradyzoites. Several important gene families were expressed at relatively high levels. Comprehensive functional annotation and gene ontology analysis revealed that stress response-related genes are important for survival of bradyzoites in immune-competent hosts. We compared Trinity-based de novo and genome-based strategies, and found that the de novo assembly strategy compensated for the defects of the genome-based strategy by filtering out several transcripts with low expression or those unannotated on the genome. We also found some inaccuracies in the ToxoDB gene models. In addition, our analysis revealed that alternative splicing can be differentially regulated in response to life-cycle change. In depth analysis revealed a 20-nt, AG-rich sequence, alternative splicing locus from alt_acceptor motif search in tachyzoite. CONCLUSION:This study represents the first large-scale effort to sequence the transcriptome of bradyzoites from T. gondii tissue cysts. Our data provide a comparative view of the tachyzoite and bradyzoite transcriptomes to allow a more complete dissection of all the molecular regulation mechanisms during stage conversions. A better understanding of the processes regulating stage conversion may guide targeted interventions to disrupt the transmission of T. gondii.

Project description:Toxoplasma gondii has evolved a number of strategies to evade immune responses in its many hosts. Previous genetic mapping of crosses between clonal type 1, 2, and 3 strains of T. gondii, which are prevalent in Europe and North America, identified two rhoptry proteins, ROP5 and ROP18, that function together to block innate immune mechanisms activated by interferon gamma (IFNg) in murine hosts. However, the contribution of these and other virulence factors in more genetically divergent South American strains is unknown. Here we utilized a cross between the intermediately virulent North American type 2 ME49 strain and the highly virulent South American type 10 VAND strain to map the genetic basis for differences in virulence in the mouse. Quantitative trait locus (QTL) analysis of this new cross identified one peak that spanned the ROP5 locus on chromosome XII. CRISPR-Cas9 mediated deletion of all copies of ROP5 in the VAND strain rendered it avirulent and complementation confirmed that ROP5 is the major virulence factor accounting for differences between type 2 and type 10 strains. To extend these observations to other virulent South American strains representing distinct genetic populations, we knocked out ROP5 in type 8 TgCtBr5 and type 4 TgCtBr18 strains, resulting in complete loss of virulence in both backgrounds. Consistent with this, polymorphisms that show strong signatures of positive selection in ROP5 were shown to correspond to regions known to interface with host immunity factors. Because ROP5 and ROP18 function together to resist innate immune mechanisms, and a significant interaction between them was identified in a two-locus scan, we also assessed the role of ROP18 in the virulence of South American strains. Deletion of ROP18 in South American type 4, 8, and 10 strains resulted in complete attenuation in contrast to a partial loss of virulence seen for ROP18 knockouts in previously described type 1 parasites. These data show that ROP5 and ROP18 are conserved virulence factors in genetically diverse strains from North and South America, suggesting they evolved to resist innate immune defenses in ancestral T. gondii strains, and they have subsequently diversified under positive selection.

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Distinct strains of Toxoplasma gondii feature divergent transcriptomes regardless of developmental stage.

Publications

Distinct strains of Toxoplasma gondii feature divergent transcriptomes regardless of developmental stage.

Similar Datasets

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