Study of permissive/non-permissive T. gondii infections

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DESCRIPTION (provided by applicant): Toxoplasma gondii infects approximately 1.5 million human subjects annually, and is the third-leading cause of foodborne mortality in the US each year [1]. Infections are most often asymptomatic, yet can be dangerous to the unborn and to patients undergoing chemotherapy or organ transplant, and also to people with AIDS [2-4]. While this organism invades most nucleated host cells in vitro, it remains a paradox that necrotic plaques found in tissues of the brain are the primary pathology associated with severe or fatal encephalitis [1,5-7]. Tachyzoites invade neurons, microglia and astrocytes of the brain [8-11], but we have observed in mixed primary cultures that astrocytes limit parasite replication, when compared with the permissive microglia. These distinct host cell environments likely arise via activation of distinctly different biochemical and metabolic pathways in response to infection. It is evident that multiple host cell mRNAs in human fibroblasts are modulated following parasite infection [12, 41-42]. Here, we propose to characterize global host gene expression profiles using microarray technology, and to identify genes that are differentially expressed in response to parasite infection of the non-permissive astrocytes when compared with the replication-tolerant microglia. The identification of host cell-specific genes relevant to immune defense, metabolic, regulatory and signaling pathways modulated in response to tachyzoite infection will allow for focused, hypotheses driven molecular genetic experiments on individual or co-regulated groups of mRNAs defining the molecular correlates of these distinctly different host cell environments, and may provide the basis for potential development of an effective treatment against both acute toxoplasmosis and chronic disease. To this end, we will accomplish the following Specific AIMS: (1) isolate tachyzoite-infected astrocytes and microglia from primary murine brain cell cultures using flow cytometry and a high-speed cell sorter, and 2) compare gene expression in tachyzoite-infected and uninfected murine astrocytes and microglia using DNA microarrays.