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A pseudouridine synthase homologue is critical to cellular differentiation in Toxoplasma gondii.

ABSTRACT: Toxoplasma gondii is a haploid protozoan parasite infecting about one in seven people in the United States. Key to the worldwide prevalence of T. gondii is its ability to establish a lifelong, chronic infection by evading the immune system, and central to this is the developmental switch between the two asexual forms, tachyzoites and bradyzoites. A library of mutants defective in tachyzoite-to-bradyzoite differentiation (Tbd(-)) was created through insertional mutagenesis. This library contains mutants that, compared to the wild type, are between 20% and 74% as efficient at stage conversion. Two mutants, TBD5 and TBD8, with disruptions in a gene encoding a putative pseudouridine synthase, PUS1, were identified. The disruption in TBD8 is in the 5' end of the PUS1 gene and appears to produce a null allele with a 50% defect in differentiation. This is about the same switch efficiency as obtained with an engineered pus1 deletion mutant (Deltapus1). The insertion in TBD5 is within the PUS1 coding region, and this appears to result in a more extreme phenotype of only approximately 10% switch efficiency. Complementation of TBD8 with the genomic PUS1 allele restored wild-type differentiation efficiency. Infection of mice with pus1 mutant strains results in increased mortality during the acute phase and higher cyst burdens during the chronic infection, demonstrating an aberrant differentiation phenotype in vivo due to PUS1 disruption. Our results suggest a surprising and important role for RNA modification in this biological process.

SUBMITTER: Anderson MZ

PROVIDER: S-EPMC2653242 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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A pseudouridine synthase homologue is critical to cellular differentiation in Toxoplasma gondii.

Anderson Matthew Z MZ Brewer Jeremy J Singh Upinder U Boothroyd John C JC

Eukaryotic cell 20090105 3

Toxoplasma gondii is a haploid protozoan parasite infecting about one in seven people in the United States. Key to the worldwide prevalence of T. gondii is its ability to establish a lifelong, chronic infection by evading the immune system, and central to this is the developmental switch between the two asexual forms, tachyzoites and bradyzoites. A library of mutants defective in tachyzoite-to-bradyzoite differentiation (Tbd(-)) was created through insertional mutagenesis. This library contains ...[more]

PMID: 19124578

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Project description:Toxoplasmosis, which is one of the most widespread zoonoses worldwide, has a high incidence and infection can result in severe disease in humans and livestock. Citrate synthase (CS) is a component of nearly all living cells that plays a vital role in the citric acid cycle, which is the central metabolic pathway of aerobic organisms. In the present study, the citrate synthase I gene of Toxoplasma gondii (T. gondii) (TgCSI) was cloned and characterized. The TgCSI gene had an open reading frame of 1665 bp nucleotides encoding a 555 amino acid protein with a molecular weight of 60 kDa. Using western blotting assay, the recombinant protein was successfully recognized by the sera of rats experimentally infected with T. gondii, while the native protein in the T. gondii tachyzoites was detected in sera from rats immunized with the recombinant protein of TgCSI. Binding of the protein to murine macrophages was confirmed by immuno fluorescence assay. Following incubation of macrophages with rTgCSI, the rTgCSI protein was found to have a dual function, with low concentrations (5-10 μg/mL) enhancing phagocytosis and high levels (80 μg/mL) inhibiting phagocytosis. Investigation of murine macrophage apoptosis illustrated that 5 μg/mL rTgCSI protein can significantly induce early apoptosis and late stage apoptosis (*p < 0.05), while 10 μg/mL rTgCSI protein significantly induced early apoptosis, but had no effect on late stage of apoptosis (**p < 0.01), and 80 μg/mL rTgCSI protein inhibited late stage apoptosis of macrophages (*p < 0.05). Cytokine detection revealed that the secretion of interleukin-10, interleukin-1β, transforming growth factor-β1 and tumor necrosis factor-α of macrophages increased after the cells were incubated with all concentration of rTgCSI, with the exception that 5 μg/mL rTgCSI had no effect on the secretion of interleukin-10 and interleukin-1β. However, secretion of NO and cell proliferation of the macrophages were substantially reduced. Taken together, these results suggested that TgCSI can affect the immune functions of murine macrophages by binding to the cells in vitro.

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A pseudouridine synthase homologue is critical to cellular differentiation in Toxoplasma gondii.

Publications

A pseudouridine synthase homologue is critical to cellular differentiation in Toxoplasma gondii.

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