GSE116539
Transcriptome sequencing of WT and chloroquine resistance transporter (TgCRT) ortholog-deficient Toxoplasma parasites
Purpose: TgCRT spans the membrane of the digestive vacuole of Toxoplasma parasites. The deletion of TgCRT causes the swelling of the digestive vacuole, which could interfere with the physiology and function of the VAC. The goals of this study are to compare transcriptome profiling (RNA-Seq) of WT and TgCRT-deletion strains to understand what strategies Toxoplasma takes to regulate the size and morphology of the digestive vacuole to help parasite disseminate infection. Methods: WT and TgCRT-deficient parasites were grown in human foreskin fibroblast cells for 2 days. The parasites were syringed, filter-purified, and resuspended in ice-cold PBS buffer. A trizol-based method was used to extract the total RNA from both strains. Total RNA was subjected to cDNA library construction using Illumina TruSeq RNA sample prep kit and Hi-Seq transcriptome sequencing. Results: Approximately 20 million sequence reads per sample were mapped to the genome of Toxoplasma GT1 strain (release 34). The differential gene analysis identified 102 genes with a fold-change =1.5 and FDR <0.05. Conclusions: Our study found that the ?crt parasites reduce the transcript levels of several endolysosomal proteases, which could alleviate the proteolytic activities within the VAC to minimize the swelling. In addition, the RNA-Seq analysis also suggests that the parasites may activate their signaling pathways to manage the stress caused by the swelled VAC and increase other transporters to regulate the morphology of the digestive vacuole. Grant title: Regulation of proteolytic activity within a digestive vacuole in Toxoplasma gondii, the most common pathogen causing infectious posterior uveitis in infants and children Name of the funding source: Knights Templar Eye Foundation Principal Investigator: Dr. Zhicheng Dou Overall design: Total RNAs of WT and ?crt parasites were converted to cDNA libraries that were subjected to Illumina HiSeq transcriptome sequencing. The assay was replicated in duplicate.