DESCRIPTION: (provided by the applicant): The long-term objective of this grant application is to understand the molecular pathogenesis of Toxoplasma gondii infection. This proposal focuses on macrophage intracellular signaling pathways during T. gondii infection. Macrophages serve as important host cells during infection, despite the fact that they are a potent source of inflammatory mediators when activated by microbial stimuli such as bacterial lipopolysaccharide (LPS). Recently, we found that parasite infection of macrophages results in suppression of the NFkappaB-dependent cytokines TNF-alpha and IL- 12. The mechanism by which this is achieved is due to a parasite-induced blockade in IkcappaB degradation, the latter of which is required to enable NFkappaB nuclear translocation. The objective of this proposal is to examine this phenomenon in detail, and to examine the effect of infection on other signaling pathways and host cells. There are four specific aims to be addressed. 1, Determine the mechanism by which infection interferes with the LPS-triggered NFkappaB activation pathway, and determine which components of this signaling cascade are triggered by the parasite. This will be accomplished employing immunochemical techniques to evaluate IkappaB ubiquitination, and transfection experiments using dominant negative mutants of signaling molecules lying upstream of IkappaB phosphorylation. 2, Determine the involvement of MAPK family members in tachyzoite-induced blockade of TNF-alpha, IL- 12, and NO production. This will be achieved by a combination of phospho-specific immunoblotting, and activity assays on each of the major MAPK. 3, Determine whether parasite-induced inhibition occurs through decreased gene transcription or increased mRNA stability, using nuclear run-on assays and RPA, respectively. Bio-array technology will also be employed to identify other macrophage genes up- or down-regulated by Toxoplasma infection. 4, Determine whether macrophages remain LPS non-responsive when intracellular parasites are killed, and when cells internalize parasites by phagocytosis. Also, determine whether Toxoplasma inhibits the ability of dendritic cells and intestinal epithelial cells to respond to LPS. These objectives will be accomplished using confocal fluorescence microscopy and electrophoretic mobility shift assay to detect NFkappaB nuclear translocation. The health relatedness of this proposal is that Toxoplasma is a major opportunistic infection in immunocompromised patient populations, and a serious congenital infection. Understanding the molecular pathology of infection will lead to better control strategies for this and related microbial pathogens.
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