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Transcriptome analysis of mouse brain infected with Toxoplasma gondii.

ABSTRACT: Toxoplasma gondii is an obligate intracellular parasite that invades a wide range of vertebrate host cells. Chronic infections with T. gondii become established in the tissues of the central nervous system, where the parasites may directly or indirectly modulate neuronal function. However, the mechanisms underlying parasite-induced neuronal disorder in the brain remain unclear. This study evaluated host gene expression in mouse brain following infection with T. gondii. BALB/c mice were infected with the PLK strain, and after 32 days of infection, histopathological lesions in the frontal lobe were found to be more severe than in other areas of the brain. Total RNA extracted from infected and uninfected mouse brain samples was subjected to transcriptome analysis using RNA sequencing (RNA-seq). In the T. gondii-infected mice, 935 mouse brain genes were upregulated, whereas 12 genes were downregulated. GOstat analysis predicted that the upregulated genes were primarily involved in host immune responses and cell activation. Positive correlations were found between the numbers of parasites in the infected mouse brains and the expression levels of genes involved in host immune responses. In contrast, genes that had a negative correlation with parasite numbers were predicted to be involved in neurological functions, such as small-GTPase-mediated signal transduction and vesicle-mediated transport. Furthermore, differential gene expression was observed between mice exhibiting the clinical signs of toxoplasmosis and those that did not. Our findings may provide insights into the mechanisms underlying neurological changes during T. gondii infection.

SUBMITTER: Tanaka S

PROVIDER: S-EPMC3811780 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Transcriptome analysis of mouse brain infected with Toxoplasma gondii.

Tanaka Sachi S Nishimura Maki M Ihara Fumiaki F Yamagishi Junya J Suzuki Yutaka Y Nishikawa Yoshifumi Y

Infection and immunity 20130715 10

Toxoplasma gondii is an obligate intracellular parasite that invades a wide range of vertebrate host cells. Chronic infections with T. gondii become established in the tissues of the central nervous system, where the parasites may directly or indirectly modulate neuronal function. However, the mechanisms underlying parasite-induced neuronal disorder in the brain remain unclear. This study evaluated host gene expression in mouse brain following infection with T. gondii. BALB/c mice were infected ...[more]

PMID: 23856619

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Project description:BackgroundToxoplasmosis is a zoonotic parasitic disease caused by Toxoplasma gondii. Toxoplasma gondii infection of the lungs can lead to severe pneumonia. However, few studies have reported Toxoplasma pneumonia. Most reports were clinical cases due to the lack of a good disease model. Therefore, the molecular mechanisms, development, and pathological damage of Toxoplasma pneumonia remain unclear.MethodsA mouse model of Toxoplasma pneumonia was established by nasal infection with T. gondii. The model was evaluated using survival statistics, lung morphological observation, and lung pathology examination by hematoxylin and eosin (H&E) and Evans blue staining at 5 days post-infection (dpi). Total RNA was extracted from the lung tissues of C57BL/6 mice infected with T. gondii RH and TGME49 strains at 5 dpi. Total RNA was subjected to transcriptome analysis by RNA sequencing (RNA-seq) followed by quantitative real-time polymerase chain reaction (qRT-PCR) validation. Transcript enrichment analysis was performed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases to assess the biological relevance of differentially expressed transcripts (DETs).ResultsC57BL/6 mice infected with T. gondii via nasal delivery exhibited weight loss, ruffled fur, and respiratory crackles at 5 dpi. The clinical manifestations and lethality of RH strains were more evident than those of TGME49. H&E staining of lung tissue sections from mice infected with T. gondii at 5 dpi showed severe lymphocytic infiltration, pulmonary edema, and typical symptoms of pneumonia. We identified 3167 DETs and 1880 DETs in mice infected with the T. gondii RH and TGME49 strains, respectively, compared with the phosphate-buffered saline (PBS) control group at 5 dpi. GO and KEGG enrichment analyses of DETs showed that they were associated with the immune system and microbial infections. The innate immune, inflammatory signaling, cytokine-mediated signaling, and chemokine signaling pathways displayed high gene enrichment.ConclusionIn this study, we developed a new mouse model for Toxoplasma pneumonia. Transcriptome analysis helped to better understand the molecular mechanisms of the disease. These results provided DETs during acute T. gondii lung infection, which expanded our knowledge of host immune defenses and the pathogenesis of Toxoplasma pneumonia.

Project description:Central to the progression of cerebral toxoplasmosis is the interaction of Toxoplasma gondii with the blood-brain barrier (BBB) endothelial cells. In the present work, we tested the hypothesis that inhibition of Wnt pathway signalling by the monovalent ionophore monensin reduces the growth of T. gondii infecting human brain microvascular endothelial cells (hBMECs) or microglial cells. The anti-parasitic effect of monensin (a Wnt signalling inhibitor) on the in vitro growth of T. gondii tachyzoites was investigated using two methods (Sulforhodamine B staining and microscopic parasite counting). The monensin inhibited T. gondii growth (50% inhibitory concentration [IC50] = 0.61 ?M) with a selective index = 8.48 when tested against hBMECs (50% cytotoxic concentration [CC50] = 5.17 ?M). However, IC50 of monensin was 4.13 ?M with a SI = 13.82 when tested against microglia cells (CC50 = 57.08 ?M), suggesting less sensitivity of microglia cells to monensin treatment. The effect of T. gondii on the integrity of the BBB was assessed by the transendothelial electrical resistance (TEER) assay using an in vitro human BBB model. The results showed that T. gondii infection significantly decreased hBMECs' TEER resistance, which was rescued when cells were treated with 0.1 µM monensin, probably due to the anti-parasitic activity of monensin. We also investigated the host-targeted effects of 0.1 µM monensin on global gene expression in hBMECs with or without T. gondii infection. Treatment of hBMECs with monensin did not significantly influence the expression of genes involved in the Wnt signalling pathway, suggesting that although inhibition of the Wnt signalling pathway did not play a significant role in T. gondii infection of hBMECs, monensin was still effective in limiting the growth of T. gondii. On the contrary, monensin treatment downregulated pathways related to steroids, cholesterol and protein biosynthesis and their transport between endoplasmic reticulum and Golgi apparatus, and deregulated pathways related to cell cycle and DNA synthesis and repair mechanisms. These results provide new insight into the host-modulatory effect of monensin during T. gondii infection, which merits further investigation.

Project description:BackgroundToxoplasma gondii is a globally distributed protozoan parasite that establishes life-long asymptomatic infection in humans, often emerging as a life-threatening opportunistic pathogen during immunodeficiency. As an intracellular microbe, Toxoplasma establishes an intimate relationship with its host cell from the outset of infection. Macrophages are targets of infection and they are important in early innate immunity and possibly parasite dissemination throughout the host. Here, we employ an RNA-sequencing approach to identify host and parasite transcriptional responses during infection of mouse bone marrow-derived macrophages (BMDM). We incorporated into our analysis infection with the high virulence Type I RH strain and the low virulence Type II strain PTG. Because the well-known TLR-MyD88 signaling axis is likely of less importance in humans, we examined transcriptional responses in both MyD88+/+ and MyD88-/- BMDM. Long noncoding (lnc) RNA molecules are emerging as key regulators in infection and immunity, and were, therefore, included in our analysis.ResultsWe found significantly more host genes were differentially expressed in response to the highly virulent RH strain rather than with the less virulent PTG strain (335 versus 74 protein coding genes for RH and PTG, respectively). Enriched in these protein coding genes were subsets associated with the immune response as well as cell adhesion and migration. We identified 249 and 83 non-coding RNAs as differentially expressed during infection with RH and PTG strains, respectively. Although the majority of these are of unknown function, one conserved lncRNA termed mir17hg encodes the mir17 microRNA gene cluster that has been implicated in down-regulating host cell apoptosis during T. gondii infection. Only a minimal number of transcripts were differentially expressed between MyD88 knockout and wild type cells. However, several immune genes were among the differences. While transcripts for parasite secretory proteins were amongst the most highly expressed T. gondii genes during infection, no differentially expressed parasite genes were identified when comparing infection in MyD88 knockout and wild type host BMDM.ConclusionsThe large dataset presented here lays the groundwork for continued studies on both the MyD88-independent immune response and the function of lncRNAs during Toxoplasma gondii infection.

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Transcriptome analysis of mouse brain infected with Toxoplasma gondii.

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Transcriptome analysis of mouse brain infected with Toxoplasma gondii.

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