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Transcriptional Analysis Shows a Robust Host Response to Toxoplasma gondii during Early and Late Chronic Infection in Both Male and Female Mice.

ABSTRACT: The long-term host effects caused by the protozoan parasite Toxoplasma gondii are poorly understood. High-throughput RNA sequencing analysis previously determined that the host response in the brain was greater and more complex at 28 days than at 10?days postinfection. Here, we analyzed the host transcriptional profile of age- and sex-matched mice during very early (21?days), early (28?days), mid (3?months), and late (6?months) chronic infection. We found that a majority of the host genes which increase in abundance at day 21 postinfection are still increased at 6?months postinfection for both male and female mice. While most of the differentially expressed genes were similar between sexes, females had far fewer genes that were significantly less abundant, which may have led to the slightly increased cyst burden in males. Transcripts for C-X-C motif chemokine ligand 13 and a C-C motif chemokine receptor 2 (CCR2) were significantly higher in females than in males during infection. As T. gondii chronic infection and profilin (PRF) confer resistance to Listeria monocytogenes infection in a CCR2-dependent manner, the differences in CCR2 expression led us to retest the protection of PRF in both sexes. Male mice were nearly as effective as female mice at reducing the bacterial burden either with a chronic infection or when treated with PRF. These data show that most of the host genes differentially expressed in response to T. gondii infection are similar between males and females. While differences in transcript abundance exist between the sexes, the infection phenotypes tested here did not show significant differences.

SUBMITTER: Garfoot AL

PROVIDER: S-EPMC6479041 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Transcriptional Analysis Shows a Robust Host Response to <i>Toxoplasma gondii</i> during Early and Late Chronic Infection in Both Male and Female Mice.

Garfoot Andrew L AL Cervantes Patrick W PW Knoll Laura J LJ

Infection and immunity 20190301 5

The long-term host effects caused by the protozoan parasite <i>Toxoplasma gondii</i> are poorly understood. High-throughput RNA sequencing analysis previously determined that the host response in the brain was greater and more complex at 28 days than at 10 days postinfection. Here, we analyzed the host transcriptional profile of age- and sex-matched mice during very early (21 days), early (28 days), mid (3 months), and late (6 months) chronic infection. We found that a majority of the host genes ...[more]

PMID: 30858341

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Project description:Tools for tuning endogenous gene expression are key to determining the genetic basis of diverse cellular phenotypes. Although synthetic regulatable promoters are available in Toxoplasma, scalable methods for targeted and combinatorial downregulation of gene expression-like RNA interference-have yet to be developed. To investigate the feasibility of CRISPR-mediated transcriptional regulation, we examined the function of two catalytically inactive Cas9 (dCas9) orthologs, from Streptococcus pyogenes and Streptococcus thermophilus, in Toxoplasma. Following the addition of single-guide RNAs (sgRNAs) targeting the promoter and 5' untranslated region (UTR) of the surface antigen gene SAG1, we profiled changes in protein abundance of targeted genes by flow cytometry for transcriptional reporters and immunoblotting. We found that the dCas9 orthologs generated a range of target gene expression levels, and the degree of repression was durable and stably inherited. Therefore, S. pyogenes and S. thermophilus dCas9 can effectively produce intermediate levels of gene expression in Toxoplasma. The distinct sgRNA scaffold requirements of the two dCas9s permit their orthogonal use for simultaneous examination of two distinct loci through transcriptional modulation, labeling for microscopy-based studies, or other dCas9-based approaches. Taking advantage of newly available genomic transcription start site data, these tools will aid in the development of new loss-of-function screening approaches in Toxoplasma. IMPORTANCE Toxoplasma gondii is a ubiquitous intracellular parasite of humans and animals that causes life-threatening disease in immunocompromised patients, fetal abnormalities when contracted during gestation, and recurrent eye lesions in some patients. Despite its health implications, about half of the Toxoplasma genome still lacks functional annotation. A particularly powerful tool for the investigation of an organism's cell biology is the modulation of gene expression, which can produce the subtle phenotypes often required for informing gene function. In Toxoplasma, such tools have limited throughput and versatility. Here, we detail the adaptation of a new set of tools based on CRISPR-Cas9, which allows the targeted downregulation of gene expression in Toxoplasma. With its scalability and adaptability to diverse genomic loci, this approach has the potential to greatly accelerate the functional characterization of the Toxoplasma genome.

Dataset Information

Transcriptional Analysis Shows a Robust Host Response to Toxoplasma gondii during Early and Late Chronic Infection in Both Male and Female Mice.

Publications

Transcriptional Analysis Shows a Robust Host Response to <i>Toxoplasma gondii</i> during Early and Late Chronic Infection in Both Male and Female Mice.

Similar Datasets

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