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Inactivation of phosphate transporters alters gene expression in T. gondii

ABSTRACT: Purpose:This study was aimed to profile the transcriptome changes of Toxoplasma after loss of phosphate transporters. For this purpose, phosphate transporter TgPiT and TgPT2 mutants were constructed by using direct knockout or conditional knockdown methods in RH or TATi parasite strain, respectively. The transcriptome changes were captured by RNA-seq analyses. Methods: Transcriptomic profiles of mutants (Δpit and iKD-PT2+ATc, respectively) and controls (RH and iKD-PT2-ATc,respectively) were generated by deep sequencing, in triplicate, using Illumina HiSeq xten. The sequence reads that passed quality filters were analyzed at the transcript level with TopHat (v2.1.1) followed by RSEM (v1.3.3). The differential expression genes were identified by DESeq2 (v1.24.0) analyses (mutant samples versus control samples). Results: Using an optimized data analysis workflow, More than 30 million paired-end reads were generated for each sample and about 90% of the clean reads were mapped to GT1 reference genome. RNA-seq data indicated deletion of TgPiT caused modest transcriptome changes in Toxoplasma. Within the 61 DEGs, several genes are typically increased during transition from tachyzoites to bradyzoites, were upregulated in the Δpit mutant, thus suggest stress response in Toxoplasma due to inactivation of TgPiT. While more than 900 genes (fold change ≥2 and adjusted p value <0.05) were significantly regulated in TgPT2 knockdown mutant. Enrichment of the differentially expressed genes using cluster of orthologous group indicate that these DEGs were enriched in pathways like genetic information processing (such as protein translation, transcription, RNA processing and modification, DNA replication and repair, chromatin structure regulation), nutrient transport and metabolism, signal transduction, intracellular trafficking, protein secretion and vesicular transport etc. 24 of 61 DEGs in Δpit strain were also found in DEGs of TgPT2 deficient parasites. The similar gene expression changes caused by inactivation of TgPiT and TgPT2 suggest that the two proteins have overlapping functions. Conclusions: Our study revealed the transcriptome changes in Toxoplasma due to phosphate transporters deficiency with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here provide the overall transcripts changes in TgPiT and TgPT2 defect strains, respectively. The results showed the unique and overlapping impacts of TgPiT and TgPT2 on Toxoplasma. These widen our understanding of phosphate requirements and potential transport pathway in Toxoplasma.

ORGANISM(S): Toxoplasma gondii

PROVIDER: GSE189677 | GEO | 2021/12/04

REPOSITORIES: GEO

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Project description:Toxoplasma gondii is a globally distributed obligate intracellular parasite which can cause zoonotic toxoplasmosis with great harms. The average death time of mice that infected with Toxoplasma gondii RH strain tachyzoites recovered from the liquid nitrogen was shortened after multiple generations. It has been reported that the parasite is in a state of static virulence during cryopreservation and the virulence of the protozoan parasite can be enhanced after continuous passages in hosts under laboratory conditions. However, no research has been conducted to elucidate its biological mechanism. Herein, we sequenced the T. gondii transcriptome using RNA-Seq technology and performed de novo assembly to investigated the virulence factors expression changes by comparing gene expression profiles between incipiently recovered and completely resuscitated tachyzoites. Transcriptome analysis identified 1,951 differentially expressed transcripts in infected liver, of which 1,752 were significantly downregulated and 199 upregulated. We identified many differentially expressed proteins and genes, including serine/threonine kinase, calnexin, myosin and microtubule-associated protein which have previously been reported to be either involved in cell adhesion, parasite gliding or participate in cell invasion. The great majority of the virulence factors including microneme proteins, rhoptry proteins and dense granule proteins were upregulated in fully recovered tachyzoites. The enhanced virulence of recovered Toxoplasma gondii RH strain from the liquid nitrogen is associated with the up-regulated expression of MICs, ROPs and GRAs. Our data will facilitate future genomic research and in-depth annotation of Toxoplasma gondii RH strain genomes. This study provides a profile of the candidate genes that are suspected to be involved with virulence enhancement of recovered Toxoplasma gondii RH strain tachyzoites. Many further studies should be carried out to confirm the function of the candidate genes. Moreover, the preliminary identification of genes and pathways exhibiting differential expression in complete resuscitation stage may further our general understanding of virulence enhancement in this parasite.

Project description:Purpose:This study was aimed to profile the transcriptome changes during Toxoplasma infection with porcine alveolar macrophages. for this purpose, Porcine alveolar macrophages (PAMs,3D4/21) were infected by Toxoplasma Me49 strain in vitro. The transcriptome changes were captured by dual RNA-seq analyses. Methods: Transcriptomic profiles of infection samples (Me49 infected PAMs) and controls (Me49 and PAMs, respectively) were generated by deep sequencing, in triplicate, using Illumina HiseqPE150. The sequence reads that passed quality filters were analyzed at the transcript level with TopHat (v2.0.12) followed by HTSeq (v0.6.1). The Cufflinks (v2.1.1) was used to construct and identify novel transcripts. qRT–PCR validation was performed using SYBR Green assays. Results: Using an optimized data analysis workflow, More than 30 million paired-end reads were generated for the Me49 controls and 93% of the clean reads were mapped to the Me49 reference genome. Nearly 2~3 times more reads were produced in the PAMs alone or infected PAMs samples. Among these, 60% to 80% of clean reads were mapped to the Sus Scrofa genome. In the infection groups, approximately 16% of total clean reads were aligned to the Me49 reference genome. RNA-seq data indicated the remarkable transcripts changes of both sides during infection process. More than 800 and 1800 genes (fold change ≥2 and adjusted p value <0.05) were significantly regulated in PAMS and Me49, respectively. Altered expression of 15 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. The differentially expressed genes in Toxoplasma were mainly related to metabolic process and macromolecule synthesis as reflected by enhanced activity of FASII pathway. Further analyses of differentially expressed genes in PAMs uncovered unique responses of porcine macrophages to Toxoplasma infection. These analyses provide novel insights into Toxoplasma interaction with it’s hosts. Conclusions: Our study represents the first detailed dual analyses of porcine alveolar macrophages and Me49 with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here provide the overall transcripts changes both PAMs and Me49. The results showed the unexpected responses of PAMs to Toxoplasma infection. These widen our understanding of interactions between Toxoplasma and it’s hosts.

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Inactivation of phosphate transporters alters gene expression in T. gondii

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