Adhesion of Toxoplasma gondii tachyzoite-infected vehicle leukocytes to capillary endothelial cells triggers timely parasite egression.
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ABSTRACT:
SUBMITTER: Baba M
PROVIDER: S-EPMC5515940 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:PRMT1 is thought to be responsible for the majority of PRMT activity in Toxoplasma gondii, but its exact function is unknown. We generated T. gondii mutants lacking PRMT1 (∆prmt1) by deletion of the PRMT1 gene. ∆prmt1 parasites exhibit morphological defects during cell division and grow slowly, and this phenotype reverses in the complemented strain ∆prmt::PRMT1mRFP. PRMT1 localizes primarily in the cytoplasm with enrichment at the centrosome, and the strain lacking PRMT1 is unable to segregate progeny accurately. Unlike wild-type and complemented parasites, ∆prmt1 parasites have abnormal daughter buds, perturbed centrosome stoichiometry, and loss of synchronous replication. Whole genome expression profiling demonstrated differences in expression of cell cycle regulated genes in ∆prmt1 relative to the complemented ∆prmt1::PRMT1mRFP and parental wild-type strains, but these changes did not correlate with a specific block in cell cycle. Although PRMT1’s primary biological function was previously proposed to be methylation of histones, our genetic studies suggest that the most critical function of PRMT1 is within the centrosome as a regulator of daughter cell counting to assure the proper replication of the parasite.
2015-09-18 | GSE73177 | GEO
Project description:Toxoplasma gondii is a highly successful parasite that infects approximately one-third of the human population and can cause fatal disease in immunocompromised individuals. Systemic parasite dissemination to organs such as the brain and eye is critical to pathogenesis. T. gondii can disseminate via the circulation, and both intracellular and extracellular modes of transport have been proposed. However, the processes by which extracellular tachyzoites adhere to and migrate across vascular endothelium under the conditions of rapidly flowing blood remain unknown. We used microfluidics and time-lapse fluorescence microscopy to examine the interactions between extracellular T. gondii and primary human endothelial cells under conditions of physiologic shear stress. Remarkably, tachyzoites adhered to and glided on human vascular endothelium under shear stress conditions. Compared to static conditions, shear stress enhanced T. gondii helical gliding, resulting in a significantly greater displacement, and increased the percentage of tachyzoites that invaded or migrated across the endothelium. The intensity of the shear forces (from 0.5 to 10 dynes/cm(2)) influenced both initial and sustained adhesion to endothelium. By examining tachyzoites deficient in the T. gondii adhesion protein MIC2, we found that MIC2 contributed to initial adhesion but was not required for adhesion strengthening. These data suggest that under fluidic conditions, T. gondii adhesion to endothelium may be mediated by a multistep cascade of interactions that is governed by unique combinations of adhesion molecules. This work provides novel information about tachyzoite interactions with vascular endothelium and contributes to our understanding of T. gondii dissemination in the infected host. IMPORTANCE Toxoplasma gondii is a global parasite pathogen that can cause fatal disease in immunocompromised individuals. An unresolved question is how the parasites circulate in the body to tissues to cause disease. T. gondii parasites are found in the bloodstream of infected animals and patients, and they have been shown to adhere to and cross the endothelial cells that line blood vessel walls. To investigate these interactions, we devised a microfluidic system to visualize parasites interacting with vascular endothelium under conditions similar to those found in the bloodstream. Interestingly, parasite migration was significantly influenced by the mechanical force of shear flow. Furthermore, we identified a role for the parasite surface protein MIC2 in the initial phase of adhesion. Our study is the first to document T. gondii interactions with endothelium under shear stress conditions and provides a foundation for future studies on the molecules that mediate parasite interaction with the vasculature.
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Project description:Toxoplasma gondii induces a potent IL-12 response early in infection that results in IFN-?-dependent control of parasite growth. It was previously shown that T. gondii soluble tachyzoite antigen (STAg) injected 48 hr before intraperitoneal infection reduces lipoxin A4 and 5-lipoxygenase (5-LO)-dependent systemic IL-12 and IFN-? production as well as hepatic immunopathology. This study investigated the ability of STAg-pretreatment to control the fatal intestinal pathology that develops in C57BL/6 mice orally infected with 100 T. gondii cysts. STAg-pretreatment prolonged the animals' survival by decreasing tissue parasitism and pathology, mainly in the ilea. Protection was associated with decreases in the systemic IFN-? levels and IFN-? and TNF message levels in the ilea and with increased TGF-? production in this tissue, but protection was independent of 5-LO and IL-4. STAg-pretreatment decreased CD4(+) T cell, NK cell, CD11b(+) monocyte and CD11b(+)CD11c(+) dendritic cell numbers in the lamina propria and increased CD8(+) T cells in the intestinal epithelial compartment. In parallel, decreases were observed in iNOS and IL-17 expression in this organ. These results demonstrate that pretreatment with STAg can induce the recruitment of protective CD8(+) T cells to the intraepithelial compartment and decrease proinflammatory immune mechanisms that promote intestinal pathology in T. gondii infection.
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Project description:PRMT1 is thought to be responsible for the majority of PRMT activity in Toxoplasma gondii, but its exact function is unknown. We generated T. gondii mutants lacking PRMT1 (∆prmt1) by deletion of the PRMT1 gene. ∆prmt1 parasites exhibit morphological defects during cell division and grow slowly, and this phenotype reverses in the complemented strain ∆prmt::PRMT1mRFP. PRMT1 localizes primarily in the cytoplasm with enrichment at the centrosome, and the strain lacking PRMT1 is unable to segregate progeny accurately. Unlike wild-type and complemented parasites, ∆prmt1 parasites have abnormal daughter buds, perturbed centrosome stoichiometry, and loss of synchronous replication. Whole genome expression profiling demonstrated differences in expression of cell cycle regulated genes in ∆prmt1 relative to the complemented ∆prmt1::PRMT1mRFP and parental wild-type strains, but these changes did not correlate with a specific block in cell cycle. Although PRMT1’s primary biological function was previously proposed to be methylation of histones, our genetic studies suggest that the most critical function of PRMT1 is within the centrosome as a regulator of daughter cell counting to assure the proper replication of the parasite. RNA samples were isolated in triplicates from RH-hxgprt parent strain (W), PRMT1 knockout (K) strain and PRMT1 knockout strain complemented with RFP-tagged PRMT1 protein (C). Parasites were grown for 32h at 37C. Samples were hybridized to the Toxoplasma gondii Affymetrix microarray (ToxoGeneChip: http://ancillary.toxodb.org/docs/Array-Tutorial.html). Hybridization data was preprocessed with Robust Multi-array Average (RMA) and normalized using per chip and per gene median polishing and analyzed using the software package GeneSpring GX (Agilent Technologies).
2015-09-18 | E-GEOD-73177 | biostudies-arrayexpress
Project description:Toxoplasmosis is a major health issue worldwide especially for immune-deficient individuals and the offspring of newly infected mothers. It is caused by a unicellular intracellular parasite called Toxoplasma gondii. Although the drugs commonly used to treat toxoplasmosis are efficient, they present serious side effects and adverse events are common. Therefore, there is a need for the discovery of new compounds with potent anti-T. gondii activity. We have tested compounds designed to target enzymes that are involved in the epigenetic regulation of gene expression. Among the most active compounds, we identified a HDAC inhibitor that shows an IC50 of around 30 nM with a selectivity index of more than 100. MC1742 is active at inhibiting the growth of the parasite in vitro but also at preventing the consequences of the acute disease in vivo. This compound induces a hyper-acetylation of histones while acetylated tubulin level remains unchanged. After MC1742 treatment, the parasite expression profile is profoundly changed with the activation of genes preferentially expressed in the sexual stages that are normally repressed at the tachyzoite stage. These findings suggest that this compound disturbs the T. gondii gene expression program, inducing parasite death.
2024-05-22 | GSE175919 | GEO
Project description:Apicomplexan parasites replicate by varied and unusual processes where the typically eukaryotic expansion of cellular components and chromosome cycle are coordinated with the biosynthesis of parasite-specific structures essential for transmission.Here we describe the global cell cycle transcriptome of the tachyzoite stage of Toxoplasma gondii. In dividing tachyzoites, more than a third of the mRNAs exhibit significant cyclical profiles whose timing correlates with biosynthetic events that unfold during daughter parasite formation. These 2,833 mRNAs have a bimodal organization with peak expression occurring in one of two transcriptional waves that are bounded by the transition into S phase and cell cycle exit following cytokinesis. The G1-subtranscriptome is enriched for genes required for basal biosynthetic and metabolic functions, similar to most eukaryotes, while the S/M-subtranscriptome is characterized by the uniquely apicomplexan requirements of parasite maturation, development of specialized organelles, and egress of infectious daughter cells. Two dozen AP2 transcription factors form a series through the tachyzoite cycle with successive sharp peaks of protein expression in the same timeframes as their mRNA patterns, indicating that the mechanisms responsible for the timing of protein delivery might be mediated by AP2 domains with different promoter recognition specificities.Underlying each of the major events in apicomplexan cell cycles, and many more subordinate actions, are dynamic changes in parasite gene expression. The mechanisms responsible for cyclical gene expression timing are likely crucial to the efficiency of parasite replication and may provide new avenues for interfering with parasite growth.
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Project description:Toxoplasma gondii is an intracellular parasite with a significant impact on human health, especially in cases where individuals are immunocompromised (e.g., due to HIV/AIDS). In Europe and North America only a few clonal genotypes appear to be responsible for the vast majority of Toxoplasma infections, and these clonotypes have been intensely studied to identify strain-specific phenotypes that may play a role in the manifestation of more severe disease. To identify and genetically map strain-specific differences in gene expression, we have carried out expression quantitative trait locus (eQTL) analysis on Toxoplasma gene expression phenotypes using spotted cDNA microarrays. This led to the identification of 16 Toxoplasma genes that had significant and mappable strain-specific variation in hybridization intensity. While the analysis should identify both cis and trans-mapping hybridization profiles, we only identified loci with strain-specific hybridization differences that are most likely due to differences in the locus itself (i.e., cis-mapping). Interestingly, a larger number of these cis-mapping genes than would be expected by chance encode either confirmed or predicted secreted proteins, many of which are known to localize to the specialized secretory organelles characteristic of members of the phylum Apicomplexa. For 6 of the cis-mapping loci we determined if the strain-specific hybridization differences were due to true transcriptional differences or rather strain-specific differences in hybridization efficiency because of extreme polymorphism and/or deletion, and we found examples of both scenarios. Keywords: eQTL mapping; virulence; Toxoplasma gondii
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Project description:The opportunistic apicomplexan parasite Toxoplasma gondii damages fetuses in utero and threatens immunocompromised individuals. The toxicity associated with standard antitoxoplasmal therapies, which target the folate pathway, underscores the importance of examining alternative pharmacological strategies. Parasitic protozoa cannot synthesize purines de novo; consequently, targeting purine salvage enzymes is a plausible pharmacological strategy. Several enzymes critical to purine metabolism have been studied in T. gondii, but IMP dehydrogenase (IMPDH), which catalyzes the conversion of IMP to XMP, has yet to be characterized. Thus, we have cloned the gene encoding this enzyme in T. gondii. Northern blot analysis shows that two IMPDH transcripts are present in T. gondii tachyzoites. The larger transcript contains an open reading frame of 1,656 nucleotides whose deduced protein sequence consists of 551 amino acids (TgIMPDH). The shorter transcript is an alternative splice product that generates a 371-amino-acid protein lacking the active-site flap (TgIMPDH-S). When TgIMPDH is expressed as a recombinant protein fused to a FLAG tag, the fusion protein localizes to the parasite cytoplasm. Immunoprecipitation with anti-FLAG was employed to purify recombinant TgIMPDH, which converts IMP to XMP as expected. Mycophenolic acid is an uncompetitive inhibitor relative to NAD+, with a intercept inhibition constant (Kii) of 0.03+/-0.004 microM. Tiazofurin and its seleno analog were not inhibitory to the purified enzyme, but adenine dinucleotide analogs such as TAD and the nonhydrolyzable beta-methylene derivatives of TAD or SAD were inhibitory, with Kii values 13- to 60-fold higher than that of mycophenolic acid.
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Project description:Epidemiological and experimental studies have shown an inverse relationship between infections with certain parasites and a reduced incidence of allergic diseases. We and others have shown that infection with Toxoplasma gondii prevents the development of allergy in mice. To establish whether this beneficial effect could be recapitulated by soluble products of this parasite, we tested an extract derived from T. gondii tachyzoites. Immunization of BALB/c mice with tachyzoites lysate antigen (TLA) elicited mixed Th1/Th2 responses. When TLA was applied together with the sensitizing ovalbumin (OVA), the development of allergic airway inflammation was reduced, with decreased airway hyperresponsiveness associated with reduced peribronchial and perivascular cellular infiltration, reduced production of OVA-specific Th2 cytokines in lungs and spleens and reduced levels of serum OVA-specific IgG1 as well as IgE-dependent basophil degranulation. Of note, TLA retained its immunomodulatory properties, inducing high levels of IL-6, TNF?, IL-10 and IL-12p70 in bone marrow-derived dendritic cells after heat-inactivation or proteinase K-treatment for disruption of proteins, but not after sodium metaperiodate-treatment that degrades carbohydrate structures, suggesting that carbohydrates may play a role in immunomodulatory properties of TLA. Here we show that extracts derived from parasites may replicate the benefits of parasitic infection, offering new therapies for immune-mediated disorders.
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