BackgroundThe obligate intracellular parasite Toxoplasma gondii establishes a life-long chronic infection within any warm-blooded host. After ingestion of an encysted parasite, T. gondii disseminates throughout the body as a rapidly replicating form during acute infection. Over time and after stimulation of the host immune response, T. gondii differentiates into a slow growing, cyst form that is the hallmark of chronic infection. Global transcriptome analysis of both host and parasite during the establishment of chronic T. gondii infection has not yet been performed. Here, we conducted a dual RNA-seq analysis of T. gondii and its rodent host to better understand host and parasite responses during acute and chronic infection.ResultsWe obtained nearly one billion paired-end RNA sequences from the forebrains of uninfected, acutely and chronically infected mice, then aligned them to the genomic reference files of both T. gondii and Mus musculus. Gene ontology (GO) analysis of the 100 most highly expressed T. gondii genes showed less than half were shared between acute and chronic infection. The majority of the highly expressed genes common in both acute and chronic infection were involved in transcription and translation, underscoring that parasites in both stages are actively synthesizing proteins. Similarly, most of the T. gondii genes highly expressed during chronic infection were involved in metabolic processes, again highlighting the activity of the cyst stage at 28 days post-infection. Comparative analyses of host genes using uninfected forebrain revealed over twice as many immune regulatory genes were more abundant during chronic infection compared to acute. This demonstrates the influence of parasite development on host gene transcription as well as the influence of the host environment on parasite gene transcription.ConclusionsRNA-seq is a valuable tool to simultaneously analyze host and microbe transcriptomes. Our data shows that T. gondii is metabolically active and synthesizing proteins at 28 days post-infection and that a distinct subset of host genes associated with the immune response are more abundant specifically during chronic infection. These data suggest host and pathogen interplay is still present during chronic infection and provides novel T. gondii targets for future drug and vaccine development.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-806) contains supplementary material, which is available to authorized users.
Abstract. Saccharomyces cerevisiae has been used as a model for studying the regulation of protein N-myristoylation. MyristoylCoA:protein N-myristoyltransferase (Nmtlp), is essential for vegetative growth and uses myristoylCoA as its substrate. MyristoylCoA is produced by the fatty acid synthetase (Fas) complex and by cellular acylCoA synthetases. We have recently isolated three unlinked Fatty Acid Activation (FAA) genes encoding long chain acylCoA synthetases and have now recovered a fourth by genetic complementation. When Fas is active and NMT1 cells are grown on media containing a fermentable carbon source, none of the FAA genes is required for vegetative growth. When Fas is inactived by a specific inhibitor (cerulenin), NMT1 cells are not viable unless the media is supplemented with long chain fatty acids. Supplementation of cellular myristoylCoA pools through activation of imported myristate (C14:0) is predominantly a function of Faalp, although Faa4p contributes to this process. Cells with nmtl81p need larger pools of myristoylCoA because of the mutant enzyme's reduced affinity for this substrate. Faalp and Faa4p are required for maintaining the viability of nmt1-181 strains even when Fas is active. Overexpression of Faa2p can rescue nmtl-181 cells due to activation of an endogenous pool of C14:0. This pool appears to be derived in part from membrane phospholipids since overexpression of Plblp, a nonessential lysophospholipase/ phospholipase B, suppresses the temperature-sensitive growth arrest and C14:0 auxotrophy produced by nmt1-181.
Background BALB/c mice control infection with the obligate intracellular parasite Toxoplasma gondii and develop a latent chronic infection in the brain, as do immunocompetent humans. IFN-γ producing CD8+ T cells provide essential protection against T. gondii, but the epitopes recognized have so far remained elusive. Methods We employed caged MHC molecules to generate ~ 250 H-2Ld tetramers and distinguish T. gondii-specific CD8+ T cells in BALB/c mice. Results We identify two T. gondii specific H-2Ld-restricted T cell epitopes, one from dense granule protein GRA4 and the other from rhoptry protein ROP7. H-2Ld/GRA4 reactive T cells from multiple organ sources predominate 2 weeks after infection, while the reactivity of the H-2Ld/ROP7 T cells peaks 6–8 weeks after infection. BALB/c animals infected with T. gondii mutants defective in establishing a chronic infection show altered levels of antigen-specific T cells, depending on the T. gondii mutant used. Conclusions Our results shed light on the identity and the parasite stage-specificity of two CD8+ T cell epitopes recognized in the acute and chronic phase of infection with T. gondii.
Toxoplasma gondii has recently come under intense study as a model for intracellular parasitism because it has a number of properties that facilitate experimental manipulation. Attention is now being turned towards understanding the developmental biology of this complex parasite. The differentiation between the two asexual stages, the rapidly growing tachyzoites and the more slowly dividing, encysted bradyzoites, is of particular interest. Progression from the former to the latter is influenced by the host's immune response. This paper describes current progress on a number of research fronts, all aimed at understanding the triggers that push the tachyzoite–bradyzoite equilibrium in one or other direction and the changes that occur in gene expression (and ultimately metabolism and function). Chief among the techniques used for these studies are genetics and molecular genetics. Recent progress in these areas is described.
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