Toxoplasmosis is an important opportunistic infection in AIDS patients. A key feature of T. gondii pathogenesis is the ability of the tachyzoite form of the parasite to use the host cells it invades as incubators for proliferation. Recently an important aspect of this incubation program has emerged for several protozoan pathogens, including T. gondii: the ability of the parasite to promote host cell survival (inhibition of apoptosis). It is unknown how the parasite prevents host cell death. In host macrophages, the problem is compounded by the fact that T. gondii invasion takes place without activation of the anti-apoptotic NF-kB pathway that is normally stimulated by exposure of this cell type to pathogens. Three questions need to be addressed: Is there an in vivo death pathway, generated by host defense, whose initiation or execution is prevented by the intracellular parasite? What is the mechanism by which death is prevented? Finally, how does the parasite benefit from this process? The long-term objectives of this study are to answer these three questions. T. gondii-inhibited apoptosis has been described in vitro using parasitized cell lines and exogenous apoptotic stimuli. Results presented here show in vivo that parasitized macrophages are preferentially spared from apoptosis in a mouse model of acute toxoplasmosis, indicating that the parasite-mediated protection is a naturally occurring pathophysiologic mechanism. This study will develop a convergence between this in vivo model and mechanistic analysis of T. gondii-regulated apoptosis in cultured macrophages. Specific components of the host animal inflammatory response will be manipulated using inhibitors and gene-targeted mice to assess the initiating factors for macrophage apoptosis that is susceptible to parasite regulation. Expression and blockade of apoptotic regulators will be studied in vitro and in vivo to determine the mechanism of parasite-mediated protection. Adoptive transfer assays will be used to link the in vitro and in vivo studies. Finally cytotoxic T lymphocytes will be exploited as a tool to distinguish parasite-regulated apoptosis from parasite-regulated death per se with respect to effects on T. gondii expansion in vivo. ? ? ?
