Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Mouse bone marrow-derived macrophages infected with T. gondii (Type I, RH strain) for 6 hr

ABSTRACT: Toxoplasma gondii is a globally distributed parasite pathogen that infects virtually all warm-blooded animals. A hallmark of immunity to acute infection is the production of IFN-γ and IL-12, followed by a protective T cell response that is critical for parasite control. Naïve T cell activation requires both TCR stimulation and the engagement of costimulatory receptors. Because of their important function in activating T cells, the expression of co-stimulatory ligands is believed to be under tight control. The molecular mechanisms governing their induction during microbial stimulation, however, are not well understood. We found that all three strains of T. gondii (Types I, II, and III) up-regulated the expression of B7-2, but not B7-1, on the surface of mouse bone marrow-derived macrophages. This induction occurred at the transcriptional level, required active parasite invasion, and was not dependent on MyD88 or TRIF. Genome-wide transcriptional analysis comparing infected and uninfected macrophages revealed the activation of MAPK signaling in infected cells. Using specific inhibitors against MAPKs, we determined that parasite-induced B7-2 is dependent on JNK, but not ERK or p38 signaling. We also observed that T. gondii-induced B7-2 expression on human peripheral blood monocytes is dependent on JNK signaling, indicating that a common mechanism of B7-2 regulation by T. gondii may exist in both humans and mice. We used microarrays to examine genome-wide changes in host cell gene expression during T. gondii infection at an early time point (6 hpi). BMdM were mock-treated or infected with T. gondii (Type I, RH strain) at an MOI of 2 and cultured for 6 hours. Biological triplicates were performed. Total RNA was harvested and used for cDNA synthesis, labeling, and hybridization to Affymetrix mouse 430 2.0 expression arrays.

ORGANISM(S): Mus musculus

SUBMITTER: Melissa Lodoen

PROVIDER: E-GEOD-27972 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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:Intracellular pathogens including the apicomplexan and opportunistic parasite Toxoplasma gondii profoundly modify their host cells in order to establish infection. We have shown previously that intracellular T. gondii inhibit up-regulation of regulatory and effector functions in murine macrophages (MΦ) stimulated with interferon (IFN)-γ, which is the cytokine crucial for controlling the parasites’ replication. Using genome-wide transcriptome analysis we show herein that infection with T. gondii leads to global unresponsiveness of murine macrophages to IFN-γ. More than 61% and 89% of the transcripts, which were induced or repressed by IFN-γ in non-infected MΦ, respectively, were not altered after stimulation of T. gondii-infected cells with IFN-γ. These genes are involved in a variety of biological processes, which are mostly but not exclusively related to immune responses. Analyses of the underlying mechanisms revealed that IFN-γ-triggered nuclear translocation of STAT1 still occurred in Toxoplasma-infected MΦ. However, STAT1 bound aberrantly to oligonucleotides containing the IFN-γ-responsive gamma-activated site (GAS) consensus sequence. Conversely, IFN-γ did not induce formation of active GAS-STAT1 complexes in nuclear extracts from infected MΦ. Mass spectrometry of protein complexes bound to GAS oligonucleotides showed that T. gondii-infected MΦ are unable to recruit non-muscle actin to IFN-γ-responsive DNA sequences, which appeared to be independent of stimulation with IFN-γ and of STAT1 binding. IFN-γ-induced recruitment of BRG-1 and acetylation of core histones at the IFN-γ-regulated CIITA promoter IV, but not β-actin was diminished by >90% in Toxoplasma-infected MΦ as compared to non-infected control cells. Remarkably, treatment with histone deacetylase inhibitors restored the ability of infected macrophages to express the IFN-γ regulated genes H2-A/E and CIITA. Taken together, these results indicate that Toxoplasma-infected MΦ are unable to respond to IFN-γ due to disturbed chromatin remodelling, but can be rescued using histone deacetylase inhibitors. Comparison of 4 different RNA pools with a 2-Color-Loop Design including 10 microarrays: [1] T. gondii infected and IFN-gamma treated, [2] T. gondii infected and untreated, [3] Non-infected and IFN-gamma treated, and [4] Non-infected and untreated.

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Mouse bone marrow-derived macrophages infected with T. gondii (Type I, RH strain) for 6 hr

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