Human and Toxoplasma gondii Genetics and Cellular/Molecular Interactions
Toxoplasma gondii is the most common parasitic infection worldwide. Thirty percent of all persons throughout the world are infected. Different T. gondii strains are associated with different virulence degrees and produce different flavors of the disease in the human host. T. gondii has the capacity to modulate host signaling cascades and remodel its host cells’ transcriptomes and proteomes profoundly. Thousands of genes in fibroblasts and mouse macrophages, including those involved in inflammation, metabolism, apoptosis, growth, and differentiation are affected. Among the host molecules modulated by T. gondii are microRNAs (miRNAs). MiRNAs are small endogenous noncoding RNAs which negatively regulate gene expression. They accomplish this through binding to mRNA and inducing their translational cleavage, repression, or accelerated decay. Each miRNA regulates tens to hundreds of genes serving as master switches. MiRNAs also are being recognized to be biomarkers of infection and tissue destruction even before clinical symptoms appear. They are often tissue and life cycle stage specific. MiRNAs control critical processes in mammalian cells to re-shape the parasite’s cellular environment and therefore constitute potential molecular therapeutic targets. T. gondii regulates its host’s miRNAs and has miRNAs important in its own gene regulation. In human foreskin fibroblasts (HFF) T. gondii upregulates HFF mir-155, mir-106b~25, and mir-17~92 but down regulates let7 [1, 2]. MiRNAs are transcriptionally regulated by NF-kappaB which is modulated by T. gondii GRA15 . T. gondii modulates p38 MAPkinase which phosphorylates AGO influencing human miRNAs . It has also been shown that RH type I parasites regulate 155 miRNAs and ME49 type II parasites regulate 20 miRNAs .
During infection T. gondii parasites not only alter the transcriptional profile of the host cells but also modulate host-signaling cascades at the protein level by secreting a battery of kinase and pseudokinase proteins into the host cell potentially modifying the phosphorylation status and function of many host proteins. For example the T. gondii kinase ROP18 together with the pseudokinase co-factor ROP5 are able to phosphorylate the interferon-gamma-inducible immunity-related GTPases (IRG proteins) involved in host resistance to T. gondii .
In that context, the goal of this project is to infect relevant human host cells (monomacs, neuronal stem cells and differentiated neurons) with parasites of differing lineages to generate transcriptional mRNA and miRNA profiles. We will also utilize laser capture of encysted parasites within neuronal cells in human brain and eye tissue to characterize mRNA transcriptomes of parasites, host and contiguous cells in situ. These datasets will allow the characterization of how genetically different parasites that cause distinct types of human toxoplasmosis alter the expression of protein-encoding and miRNA-encoding genes in both the human host and the parasite. Finally, we will carry out a small phosphoproteomics study to identify host proteins whose phosphorylation state changes during T. gondii invasion.
MiRNA-seq and mRNA-seq will be carried out from the same RNA sample thereby integrating the mRNA and miRNA “sequenomes” of infected human cells most relevant to human toxoplasmosis. MiRNA expression data will be integrated with mRNA expression data to identify miRNA–mRNA functional pairs. This likely will clarify these interactions relevant to disease. MiRNA-seq, in conjunction with mRNA-seq is a key part of this project as understanding miRNAs produced by T. gondii-infected human cells relevant to human diseases will be valuable for those working in the field.
The transcriptomics and phosphoproteomics data generated by this project will allow the identification of novel host and parasite genes and pathways that play a key role during infection. This will provide the bases for new lines of research for the development of new therapies, anti-parasitic medicines and vaccines.
White Paper Access
The initial white paper submitted can be downloaded here. Since white papers are not always approved exactly as submitted, this document may not exactly describe the final form of the project. Please contact firstname.lastname@example.org if you have any questions.
This project has been funded in whole or part with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services under contract numbers N01-AI30071 and/or HHSN272200900007C.