The cell and molecular biology of both Leishmania and Toxoplasma are investigated as models of intracellular parasitism. Emphasis is placed on characterizing both the biochemical functions and gene structure of their surface membrane and secreted proteins toward defining the roles of these constituents in parasite survival and development. A gene encoding the unique, trypanosomatid surface membrane enzyme, 3'-nucleotidase has been identified, cloned and fully sequenced from L. donovani. Similarly, full length genomic clones from an L. donovani secretory acid phosphatase gene family are being sequenced. The role of this enzyme within infected macrophages is also being investigated. Methods were devised for the continuous in vitro cultivation of large quantities of infectious amastigotes (Am-) from L. donovani and several other species. These Am- are being characterized with regard to their cell biology and biochemistry. At the molecular level, several genes have been identified which are uniquely or differentially expressed by Am- and these are being used as probes to study parasite gene-regulated differentiation and cell cycle development. Further, a portion of a gene for both a surface membrane 5'-nucleotidase and a """"""""secretory"""""""" chitinase have been identified and sequenced from PCR. Finally, a unique 5'-nucleotidase was identified in the surface membrane of Toxoplasma gondii tachyzoites and a PCR-generated probe is being used to identify the gene for this protein. The current results are of relevance toward the development of new diagnostic, chemotherapeutic and immunoprophylactic agents against these important human pathogens.
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