The protozoan pathogen Toxoplasma gondii is a significant cause of disease in humans and in domestic animals. T. gondii can cause acute and often fatal opportunistic infection in patients undergoing immunosuppressive therapy. AIDS patients with a previously latent infection may develop toxoplasmic encephalitis which is a significant cause of morbidity and mortality. IN intermediate host, T. gondii exists in two forms, the tachyzoite and the bradyzoite. During the acute phase of infection, the invasive tachyzoite form predominates and is responsible for tissue destruction. Tachyzoites switch to the bradyzoite form enclosed within tissue cysts which persist during the latent infection even in healthy individuals and patients undergoing current anti-T gondii drug therapies. Reactivation of a previously latent infection resulting from cyst rupture in the brain or other tissue leads to the reemergence of the tachyzoite form and is thought to be the major cause of active toxoplasmosis in AIDS patients. Since tissue cysts are involved in the maintenance of the latent infection and are the source of parasites in reactivation, it is important to study the mechanisms of interconversion between the tachyzoite and bradyzoite stages. In order to study the regulation of stage specific gene expression during conversion, several stage-specific genes were cloned including two with strong homology to lactate dehydrogenase (LDH) from other species. One LDH gene is bradyzoite specific and the other is tachzoite specific. TO study the mechanisms of the stage-specific regulation of these LDH genes, the following approaches will be taken : (1) The profiles of expression of LDH polypeptide will be studied during interconversion of bradyzoites and tachyzoites using in vitro models of differentiation, (2) the transcriptional activity of the LDH2 gene will be studied during stage conversion, (3) the promoter and DNA sequence domains responsible for the developmental transcriptional activation and regulation of the LDH2 gene will be identified by deletional and mutational analysis, and (4) the functional significance of the bradyzoite-specifically expressed LDH isoform will be examined by generating null mutants by gene replacement. Gene replacement studies of the bradyzoite LDH gene will performed to determine its importance to cyst development. Characterization of the DNA regulatory domains in the LDH promoters involved in their development. Characterization of the DNA regulatory domains in the LDH promoters involved in their developmental expression might lead to the isolation of the factors that bind these domains. These studies of stage-specific gene expression might lead to the identification of potential target for anti-T gondii drug design aimed at blocking cyst development or reactivation.
