Transcriptomics of Toxoplasma gondii sporulating oocysts

Toxoplasma gondii is an apicomplexan parasite that approximately infects one third of the human population worldwide. When sporulated oocysts that are shed in the faeces of infected felids are accidentally ingested through contaminated food or water, sporozoites can infect the intestine of the intermediate host. Upon this infection, the fast replicating tachyzoites then disseminate overall the body and can cross the blood-brain, blood-retina or the blood-placenta barrier. Eventually, immune pressure will lead to differentiation into the slow replicating bradyzoites that are contained within tissue cysts for life. Despite the importance of sporulation for transmission, the process is not yet understood. We therefore exploited Illumina Next-Generation RNA-Sequencing to analyse the transcriptome of oocysts in the process of sporulation and compared it to unsporulated and sporulated oocysts. We thus identified genes that are highly specific to the sporulation process and that may be involved in the formation of sporozoites. Overall design: Oocysts were enriched from cat faeces using sucrose flotation and CsCl gradient. Unsporulated were preserved in QIAzol at -80 °C until RNA extraction. To initiate sporulation, oocysts were re-suspended in 2% sulphuric acid. Sporulating oocysts were harvested 8 h after re-suspension in sulphuric acid and stored in QIAzol at -80 °C. Fully sporulated oocysts were harvested 8 days after initiation of sporulation and were stored as the samples above. Total RNA was extracted, enriched for mRNA and subjected to RNA-Sequencing using the Illumina HiSeq 4000 system. Differential gene expression was assessed by DESeq2.