This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Toxoplasma gondii is a major opportunistic pathogen of the central nervous system, that causes significant disease in AIDS patients and other immunocompromised individuals, such as transplant recipients and cancer patients undergoing chemotherapy. In these individuals infection is due to reactivation of a latent infection in the brain and results in severe and often fatal necrotizing encephalitis. Cytokines play an important role in the regulation of T. gondii in the central nervous system and interferon-gamma (IFNv) is the main cytokine controlling replication of T. gondii in the brain and in other tissues. The mechanism of IFNy inhibition in murine astrocytes and macrophages is partially dependent upon the IFNv-induced response protein, IGTP. Natural host-defense mechanisms are influenced by the parasite, which is known to substantially alter host cell transcription, and there is clear evidence for active parasite intervention in pathways affecting host cell apoptosis and the cytokine response. The biochemical outcome of this tug-of-war influences the establishment of a host cell environment that either supports or is hostile to parasite growth and development. The host cell components and the factors by which the parasite manipulates the host cell environment are not understood, but these mechanisms appear to vary extensively between strains that express a wide range of growth and virulent phenotypes. Astrocytes are an important host cell for T. gondii in the brain and an important IFNy-activated effector cell, mediating resistance to T. gondii in the brain. As such, the astrocyte model provides an opportunity to understand host-defense and parasite survival mechanisms in a clinically relevant cell type. We will take a comprehensive approach to characterize the changes in host mRNA and protein expression and protein post-translational modification that occur in primary astrocytes obtained from animals that are exposed in vitro to Toxoplasma infection, under conditions where protection is afforded by IFNy stimulation in this project. We will define the IFNy response in astrocytes and explore how these host cell changes are altered by parasites of distinct genetic lineage and virulent phenotypes. The three specific aims of this proposal are: 1) Transcriptional analysis of the host cell response to T. gondii infection in IFNy stimulated vs. unstimulated astrocytes, 2) Proteomic analysis of the host cell response to T. gondii infection in IFNy stimulated vs. unstimulated astrocytes and 3) Development of a screen for T. gondii survival mutants subjected to IFNy stimulation in astrocytes.
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