Comparative studies of Toxoplasma gondii transcriptomes: insights into stage conversion based on gene expression profiling and alternative splicing

Abstract

BackgroundToxoplasma 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.ResultsWe 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.ConclusionThis 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.Electronic supplementary materialThe online version of this article (10.1186/s13071-018-2983-5) contains supplementary material, which is available to authorized users.

Potent Tetrahydroquinolone Eliminates Apicomplexan Parasites

“…In the future, with formulation and pharmacokinetics of JAG21 optimized, it will be of interest to determine whether JAG21 can eliminate these organisms and any residual structures as in Figure 6C, or whether adding synergistic compounds such as atovaquone ( Figure 4B) or antisense effective against these upregulated molecular targets, such as kinases, ATPases, AP2s (Figure 6D and Supplemental Table 2), or a newly recognized bradyzoite master regulator of differentiation might be effective alone or might be synergistic with JAG21 against this RPS13, as well as the conventional recognized tachyzoite and bradyzoite life cycle stages. Chen et al reported in the transcriptomes of established bradyzoite in vivo cysts that EIF2kinase of stressed parasites is present (Chen et al, 2018), but we have not found other overlap of Chen's transciptome with P cynomogli or RPS13 transcriptomes. Others have described EIF2kinase and stress granules only in transitioning or extracellular parasites (Watts et al, 2015).…”

Metabolic interactions between Toxoplasma gondii and its host

“…Similar limitations apply to conclusions based on transcriptomic and proteomic data. While these approaches are powerful to functionally associate genes and resolve differences of physiological states of the parasite under different conditions, such as host environments 64 and stage conversion states 75 , 76 , functional implications on the phenotype level and mechanistic insights are constrained by functional gene annotations. Combined multi-omics approaches might provide insights beyond the metabolic dimension of host–parasite interactions in the future 77 .…”

Alternative Splicing in Apicomplexan Parasites

“…Due to contamination with mosquito RNA, they only managed to sequence a relatively small amount of ookinete-specific RNA (0.7 million reads compared to ∼5 million reads for RNA-seq analyses of other apicomplexan stages), resulting in identification of only 15 additional alternatively spliced transcripts in this stage. A similar study of Toxoplasma also found different alternative splice events in proliferating tachyzoite stages (42 genes with alternative splicing detected using a de novo assembly detection method) compared to persistent bradyzoite stages (65 genes with alternative splicing) (43). However, as with the other work described above, this study examined splicing independently at each stage and did not test for statistically differential splicing between stages, which must be a priority for future multistage studies of splicing in Apicomplexa.…”