The parasite Toxoplasma gondii (T. gondii) causes devastating disease in individuals who are immune-compromised as a result of cancer, organ transplant, or AIDS. The life cycle of the parasite consists of sexual and asexual components with the asexual portion having two distinct interconvertible stages: the rapidly growing tachyzoite and the more slowly growing bradyzoite. Bradyzoites develop within cysts and lie dormant in the host undetected by the host immune system. In immune- compromised individuals, bradyzoites can become activated and revert to tachyzoites that grow uninhibited. Thus, it is important to understand how bradyzoites develop in order to analyze how they evade the immune system and also to develop treatments against T. gondii to augment cancer and AIDS therapies. Presently, the molecular mechanisms that regulate the interconversion between tachyzoites and bradyzoites are unknown. To identify genes required for bradyzoite development, DNA microarrays will be generated from normalized bradyzoite cDNA libraries. The microarrays will be probed with mRNA isolated from parasites induced to differentiate into bradyzoites with the addition of either high pH, high temperature, IFN-gamma, or atovaquone. Genes that are upregulated by all of these treatments will be considered candidate early bradyzoite genes and those that are amongst the earliest upregulated and display homology to either transcription factors or signaling molecule will be further examined. To assess the role of these genes in bradyzoite development, individual deletion mutant strains will be generated and assessed for effects on bradyzoite development in vitro and in vivo. In addition, these genes will be constitutively expressed in T. gondii to examine whether they are sufficient to stimulate bradyzoite development. In summary, these studies will identify genes whose products are critical in the conversion of tachyzoites to bradyzoites.
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