Transcriptomic analysis by Next Generation Sequencing of mouse bone marrow derived macrophages (BMDMs) infected by Wild-Type and gra18 mutant strains of T. gondii.

Purpose: T. gondii is an obligate intracellular parasite that remodels host transcriptome during infection. How T. gondii provokes these changes is not fully understood. In the present study we found that GRA18 is secreted in the infected host cell and associates with the ß-catenin destruction complex to induce ß-catenin levels, notably in the host nuclei. Therefore, we hypothesized that GRA18 could modulate the expression of ß-catenin-dependent genes. To determine whether GRA18 induces changes in the host cell gene expression, we performed a transcriptomic analysis of BMDMs infected by Wild-Type and gra18 mutant strains of T. gondii. Methods: RNA profiles were generated from BMDMs (BALB/c mice) that were left uninfected or infected with the Pru ku80, Pru ku80 ?gra18, and Pru ku80 ?gra18, GRA18+++ strains (MOI = 6) for 18 hours and subsequently subjected or not to LPS stimulation for 4 hours.Total RNAs were extracted and purified using TRIzol (Invitrogen, Carlsbad, CA, USA) and RNeasy Plus Mini Kit (Qiagen). RNA-sequencing was performed by GENEWIZ (South Plainfield, NJ, USA). The RNA quality was checked with an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, California, USA) and Illumina TruSEQ RNA library prep and sequencing reagents were used following the manufacturer's recommendations (Illumina, San Diego, CA, USA). The samples were paired-end multiplex sequenced (2 × 125 bp) on the Illumina Hiseq 2500 platform and generated at least 70 million reads for each sample. The RNA-Seq reads (FASTQ) were processed and analyzed using the Lasergene Genomics Suite version 14 (DNASTAR, Madison, WI, USA) using default parameters. The paired-end reads were uploaded onto the SeqMan NGen (version 14, DNASTAR. Madison, WI, USA) platform for reference-based assembly using either the Mus musculus genome package (GRCm38.p3) or the Toxoplasma Type II ME49 strain (ToxoDB-24, ME49 genome) as reference template. The ArrayStar module (version 14, DNASTAR. Madison, WI, USA) was used for normalization, differential gene expression and statistical analysis of uniquely mapped paired-end reads using the default parameters. The expression data quantification and normalization were calculated using the RPKM (Reads Per Kilobase of transcript per Million mapped reads) normalization method. Conclusion: This approach identifies a specific set of genes that are regulated in a GRA18-dependent fashion in BMDM upon T. gondii infection. Particularly, GRA18 promotes the expression of chemokines Ccl17, Ccl22, and Ccl24. Overall design: A total of 8 samples were analyzed. The experiment contains two sets of data: in the absence of LPS stimulation and after LPS stimulation. Cells that were left uninfected (ui) and without LPS stimulation were used as controls.

Identifier
Source https://data.blue-cloud.org/search-details?step=~012605576767097915C31A6DF3D5DA1FD0FCD010171
Metadata Access https://data.blue-cloud.org/api/collections/605576767097915C31A6DF3D5DA1FD0FCD010171
Provenance
Instrument Illumina HiSeq 2500; ILLUMINA
Publisher Blue-Cloud Data Discovery & Access service; ELIXIR-ENA
Publication Year 2024
OpenAccess true
Contact blue-cloud-support(at)maris.nl
Representation
Discipline Marine Science
Temporal Point 2018-06-02T00:00:00Z