ABSTRACT: Infection of RAW264.7 cells with RH?ku80 parasites or mock-infection for 24 hours To measure changes in gene expression induced in macropahges upon Toxoplasma infection, we infected RAW 264.7 macrophages in cell culture with RH?ku80 parasites or syringe-lysed human foreskin fibroblast monolayers (mock-infected). RNA was harvested 24 hours post infection. Cells were infected with Toxoplasma or mock-infected in vitro, in duplicate
Project description:Infection of RAW264.7 cells with RHΔku80 parasites or mock-infection for 24 hours To measure changes in gene expression induced in macropahges upon Toxoplasma infection, we infected RAW 264.7 macrophages in cell culture with RHΔku80 parasites or syringe-lysed human foreskin fibroblast monolayers (mock-infected). RNA was harvested 24 hours post infection.
Project description:The normally virulent type-I RH parasite is rendered avirulent when lacking ROP5. The avirulent phenotype is a consequence of interaction with the host innate immune system. We sought to understand if ROP5 alters host gene expression in order to escape host defenses. We saw no gene expression differences between host cells infected with wt (RH?ku80) or RH?ku80?rop5 parasites. We have included uninfected HFF samples that were harvested in parallel with the infected samples. Host gene expression in response to infection with Toxoplasma gondii. Two independent samples per sample type. Three sample types: HFF infected with RH?ku80, HFF infected with RH?ku80?rop5, and uninfected HFF.
Project description:The normally virulent type-I RH parasite is rendered avirulent when lacking ROP5. The avirulent phenotype is a consequence of interaction with the host innate immune system. We sought to understand if ROP5 alters host gene expression in order to escape host defenses. We saw no gene expression differences between host cells infected with wt (RH∆ku80) or RH∆ku80∆rop5 parasites. We have included uninfected HFF samples that were harvested in parallel with the infected samples.
Project description:Infection of RAW264.7 cells for 24 hours with 32 Toxoplasma Progeny from a Type II x Type III cross To measure changes in gene expression induced in macropahges upon Toxoplasma infection, we infected RAW 264.7 macrophages in cell culture with one of 32 Toxoplasma parasite progeny from a Type II x Type III cross. RNA was harvested 24 hours post infection. Cells were infected with Toxoplasma parasites, in vitro
Project description:Toxoplasma gondii is a ubiquitous protozoan pathogen able to infect both mammalian and avian hosts. Surprisingly, just three strains appear to account for the majority of isolates from Europe and N. America. To test the hypothesis that strain divergence might be driven by differences between mammalian and avian response to infection, we examine in vitro strain-dependent host responses in a representative avian host, the chicken. To identify parasite drivers of strain-dependent host response, QTL mapping was used; analysis revealed a locus on Toxoplasma chromosome VIIb. To determine whether this was the parasite gene ROP16, array analysis was performed on chicken embryonic fibroblasts infected with Type I parasites and ROP16-KO parasites (of a Type I background). Chicken embryonic fibroblasts were cultivated in vitro and infected with either Type I (RH) parasites or Type I ROP16-KO parasites; ROP16-dependent host transcriptional responses were then analyzed at 5 hours post-infection.
Project description:Pre-infection with Toxoplasma parasites inhibits induction of an interferon gamma responsive gene expression program. In this study we aimed to determine what role host negative regulatory proteins that are induced by Toxoplasma infection, such as SOCS-1, have in this inhibition. We used Affymetrix microarrays to analyze host cell transcription after pre-treatment with the protein synthesis inhibitor cyclohexamide (CHX), Toxoplasma infection, and subsequent interferon-gamma stimulation. Human foreskin fibroblasts (HFFs) were left untreated or treated with CHX for 40min. Cells were infected, or not, with type I (RH) Toxoplasma parasites, for one hour, and subsequently stimulated with human IFN-γ for one hour. Total RNA was isolated and hybridized to Affymetrix Human U133A 2.0 gene chips.
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:Toxoplasma gondii (T. gondii) is an obligate intracellular parasite that can infect almost all warm-blooded animals, causing serious public health problems. Lysine crotonylation (Kcr) is a newly discovered posttranslational modification (PTM), which has been proved that is relevant to procreation regulation, active transcription and cell signaling pathway. However, the biological functions of crotonylation have not yet been reported in macrophages infected with T. gondii. In our study, we performed a ChIP-seq analysis of porcine alveolar macrophages infected with T. gondii RH to explore the relationship of histone Kcr with T. gondii infection.